0163-769X/00/$03.00/0 Endocrine Reviews 21(2): 115–137 Copyright © 2000 by The Endocrine Society Printed in U.S.A. Birth and Death of Bone Cells: Basic Regulatory Mechanisms and Implications for the Pathogenesis and Treatment of Osteoporosis STAVROS C. MANOLAGAS Division of Endocrinology & Metabolism, Center for Osteoporosis & Metabolic Bone Diseases, University of Arkansas for Medical Sciences, and the Central Arkansas Veterans Healthcare System, Little Rock, Arkansas 72205, USA ABSTRACT The adult skeleton regenerates by temporary cellular structures that comprise teams of juxtaposed osteoclasts and osteoblasts and replace periodically old bone with new. A considerable body of evidence accumulated during the last decade has shown that the rate of genesis of these two highly specialized cell types, as well as the prevalence of their apoptosis, is essential for the maintenance of bone homeostasis; and that common metabolic bone disorders such as osteoporosis result largely from a derangement in the birth or death of these cells. The purpose of this article is 3-fold: 1) to review the role and the molecular mechanism of action of regulatory molecules, such as cytokines and hormones, in osteoclast and osteoblast birth and apoptosis; 2) to review the evidence for the contribution of changes in bone cell birth or death to the pathogenesis of the most common forms of osteoporosis; and 3) to highlight the implications of bone cell birth and death for a better understanding of the mechanism of action and efficacy of present and future pharmacotherapeutic agents for osteoporosis. (Endocrine Reviews 21: 115–137, 2000) I. Introduction II. Physiological Bone Regeneration A. Remodeling by the basic multicellular unit (BMU) III. Osteoblastogenesis and Osteoclastogenesis A. Growth factors and their antagonists B. Cytokines C. Systemic hormones D. Adhesion molecules IV. Reciprocal Relationship Between Osteoblastogenesis and Adipogenesis V. Serial and Parallel Models of Osteoblast and Osteoclast Development VI. Function of the Mature Cells A. Osteoblasts B. Osteocytes C. Lining cells D. Osteoclasts VII. Death of Bone Cells by Apoptosis VIII. Regulation of Bone Cell Proliferation and Activity IX. Pathogenesis of Osteoporosis A. Sex steroid deficiency B. Senescence C. Glucocorticoid excess X. Pharmacotherapeutic Implications of Osteoblast and Osteocyte Apoptosis A. Intermittent PTH administration B. Bisphosphonates and calcitonin C. Novel pharmacotherapeutic strategies XI. Summary and Conclusions I. Introduction And Athena lavished a marvelous splendor on the prince so that all the people gazed in wonder as he came forward. The elders making way as he took his father’s seat. The first to speak was an old lord, Aegyptius, stooped with age, who knew the world by heart. Homer, the Odyssey: translation by Robert Fagles L OSS OF height (stooping), Dowager’s hump, and kyphosis are some of the most visible signs of old age in humans. The primary reason for these involutional changes is a progressive loss of bone mass that affects the axial (primarily trabecular) as well as the appendicular (primarily cortical) skeleton. Loss of bone mass, along with microarchitectural deterioration of the skeleton, leads to enhanced bone fragility and increased fractures—the bone disease known as osteoporosis (1). Both men and women start losing bone in their 40s. However, women experience a rapid phase of loss during the first 5–10 yr after menopause, due to the loss of estrogen (2). In men this phase is obscure, since there is only a slow and progressive decline in sex steroid production; hence, the loss of bone in men is linear and slower (3). In addition to losing bone faster at the early postmenopausal years, women also accumulate less skeletal mass than men during growth, particularly in puberty, resulting in smaller bones with thinner cortices and smaller diameter. Consequently, the incidence of bone fractures is 2- to 3-fold higher in women as compared with men (4). In addition to sex steroid deficiency and the aging process Address reprint requests to: Stavros C. Manolagas, M.D., Ph.D., Center for Osteoporosis & Metabolic Bone Diseases, Division of Endocrinology & Metabolism, University of Arkansas Medical School, 4301 West Markam Street, Little Rock, Arkansas 72205 USA. E-mail: [email protected] *Research from the author’s laboratory described in this review was supported by the NIH (P01-AG13918, R01-AR43003), the Department of Veterans Affairs (merit grant and a research enhancement award program, REAP), and the 1999 Allied Signal Award for Research on Aging. 115 116 MANOLAGAS itself, loss of bone mass is accentuated when several other conditions are present. The most common are chronic glucocorticoid excess (5), particularly its iatrogenic form, hyperthyroidism as well as inappropriately high T4 replacement, alcoholism, prolonged immobilization, gastrointestinal disorders, hypercalciuria, some types of malignancy, and cigarette smoking (6). Bone loss and eventually fractures are the hallmarks of osteoporosis, regardless of the underlying cause or causes. The bone loss associated with normal aging in women has been divided into two phases: one that is due to menopause and one that is due to aging and affects men as well (7, 8). In elderly women these two phases eventually overlap, making it difficult to distinguish the effect of sex steroid deficiency from the effect of the aging process itself. The effect of the aging process itself is also frequently obscured because of secondary hyperparathyroidism (9), resulting from impaired calcium absorption from the intestine with advancing age (⬎75 yr old). The bone loss that is due to glucocorticoid excess shares several features with the bone loss due to senescence, but also has unique features of its own. Nonetheless, as is the case with the other types of bone loss, the heterogeneity of the underlying conditions, some of which (e.g., postmenopausal state, rheumatoid arthritis, etc.) independently contribute to skeletal deterioration, can distort the clinical and histological picture (10). Irrespective of the overlap, it is important to recognize that the pathogenetic mechanisms are quite distinct in the various forms of osteoporosis and that sex hormone deficiency and aging have independent effects. During the last few years, there have been significant advances in our understanding of the pathogenetic mechanisms responsible for the bone loss associated with sex steroid deficiency, old age, and glucocorticoid excess. All these conditions do not cause loss of bone mass by turning on a completely new process. Instead, they cause a derangement in the normal process of bone regeneration. Therefore, to understand the pathogenesis of osteoporosis and rationalize its treatment, one must first appreciate the basic principles of physiological bone regeneration. II. Physiological Bone Regeneration The skeleton is a highly specialized and dynamic organ that undergoes continuous regeneration. It consists of highly specialized cells, mineralized and unmineralized connective tissue matrix, and spaces that include the bone marrow cavity, vascular canals, canaliculi, and lacunae. During development and growth, the skeleton is sculpted to achieve its shape and size by the removal of bone from one site and deposition at a different one; this process is called modeling. Once the skeleton has reached maturity, regeneration continues in the form of a periodic replacement of old bone with new at the same location (11). This process is called remodeling and is responsible for the complete regeneration of the adult skeleton every 10 yr. The purpose of remodeling in the adult skeleton is not entirely clear, although in bones that are load bearing, remodeling most likely serves to repair fatigue damage and to prevent excessive aging and its consequences. Hence, the most likely purpose of bone remodeling is to Vol. 21, No. 2 prevent accumulation of old bone. Remodeling, with positive balance, does occur in the growing skeleton as well. Its purpose, quite different from those proposed for the adult skeleton, is to expand the marrow cavity while increasing trabecular thickness (12). A. Remodeling by the basic multicellular unit (BMU) Removal of bone (resorption) is the task of osteoclasts. Formation of new bone is the task of osteoblasts. Bone resorption and bone formation, however, are not separate, independently regulated processes. In the uninjured adult skeleton, all osteoclasts and osteoblasts belong to a unique temporary structure, known as a basic multicellular unit or BMU (13). Although during modeling one cannot distinguish anatomical units analogous to BMU per se, sculpting of the growing skeleton requires spatial and temporal orchestration of the destination of osteoblasts and osteoclasts, albeit with different rules and coordinates to those operating in the BMU of the remodeling skeleton. The BMU, approximately 1–2 mm long and 0.2– 0.4 mm wide, comprises a team of osteoclasts in the front, a team of osteoblasts in the rear, a central vascular capillary, a nerve supply, and associated connective tissue (13). In healthy human adults, 3– 4 million BMUs are initiated per year and about 1 million are operating at any moment (Table 1). Each BMU begins at a particular place and time (origination) and advances toward a target, which is a region of bone in need of replacement, and for a variable distance beyond its target (progression) and eventually comes to rest (termination) (10). In cortical bone, the BMU travels through the bone, excavating and replacing a tunnel. In cancellous bone, the BMU moves across the trabecular surface, excavating and replacing a trench. In both situations, the cellular components of the BMUs maintain a well orchestrated spatial and temporal relationship with each other. Osteoclasts adhere to bone and subsequently remove it by acidification and proteolytic digestion. As the BMU advances, osteoclasts leave the resorption site and osteoblasts move in to cover the excavated area and begin the process of new bone formation by secreting osteoid, which is eventually mineralized into new bone. The lifespan of the BMU is 6 –9 months; much longer than the lifespan of its executive cells (Table 1). Therefore, continuous supply of new osteoclasts and osteoblasts from their respective progenitors in the bone marrow is essential for the origination of BMUs and their progression on the bone surTABLE 1. Vital statistics of adult bone remodelinga ● ● ● ● ● ● Lifespan of BMU ⬃6 –9 months Speed ⬃25 m/day Bone volume replaced by a single BMU ⬃0.025 mm3 Lifespan of osteoclasts ⬃2 weeks Lifespan of osteoblasts (active) ⬃3 months Interval between successive remodeling events at the same location ⬃2–5 years. ● Rate of turnover of whole skeleton ⬃10% per yearb a From A. Michael Parfitt (13) The 10% per year approximation for the entire skeleton is based on an average 4% turnover per year in cortical bone, which represents roughly 75% of the entire skeleton; and an average 28% turnover per year in trabecular bone, which represents roughly 25% of the skeleton (0.75 ⫻ 4 ⫽ 3 and 0.25 ⫻ 28 ⫽ 7; 3 ⫹ 7 ⫽ 10). b April, 2000 BIRTH AND DEATH OF BONE CELLS face. Consequently, the balance between the supply of new cells and their lifespan are key determinants of the number of either cell type in the BMU and the work performed by each type of cells and are critical for the maintenance of bone homeostasis. III. Osteoblastogenesis and Osteoclastogenesis Both osteoblasts and osteoclasts are derived from precursors originating in the bone marrow. The precursors of osteoblasts are multipotent mesenchymal stem cells, which also give rise to bone marrow stromal cells, chondrocytes, muscle cells, and adipocytes (14 –16), whereas the precursors of osteoclasts are hematopoietic cells of the monocyte/macrophage lineage (17, 18). Long before these cells could be cultured, the existence of multipotent mesenchymal stem cells was suspected (19), based on the evidence that fibroblastic colonies formed in cultures of adherent bone marrow cells can differentiate, under the appropriate stimuli, into each of the above mentioned cells; these progenitors were named colony forming unit fibroblasts (CFU-F). When CFU-F are cultured in the presence of ␤-glycerophosphate and ascorbic acid, the majority of the colonies form a mineralized bone nodule; these bone-forming colonies are known as CFU-osteoblast (CFU-OB) (16). Osteoblast progenitors may originate not only from stromal mesenchymal progenitors of the marrow, but also pericytes — mesenchymal cells adherent to the endothelial layer of vessels (20). Whereas osteoclast precursors reach bone from the circulation, osteoblast precursors most likely reach bone by migration of progenitors from neighboring connective tissues. The development and differentiation of osteoblasts and osteoclasts are controlled by growth factors and cytokines produced in the bone marrow microenvironment as well as adhesion molecules that mediate cell-cell and cell-matrix interactions. Several systemic hormones as well as mechanical signals also exert potent effects on osteoclast and osteoblast development and differentiation. Although many details remain to be established concerning the operation of this network, a few themes have emerged (21). First, several of the growth factors and cytokines control each other’s production in a cascade fashion and, in some instances, form positive and negative feedback loops. Second, there is extensive functional redundancy among them. Third, some of the same factors are capable of influencing the differentiation of both osteoblasts and osteoclasts. Fourth, systemic hormones influence the process of osteoblast and osteoclast formation via their ability to control the production and/or action of local mediators. A. Growth factors and their antagonists The only factors capable of initiating osteoblastogenesis from uncommitted progenitors are bone morphogenetic proteins (BMPs) (22). BMPs have been long implicated in skeletal development during embryonic life and fracture healing. More recently, it has become apparent that BMPs, and in particular BMP-2 and – 4, also initiate the commitment of mesenchymal precursors of the adult bone marrow to the osteoblastic lineage (23). BMPs stimulate the transcription of 117 the gene encoding an osteoblast-specific transcription factor, known as osteoblast specific factor 2 (Osf2) or core binding factor a1 (Cbfa1), hereafter referred to as Cbfa1 (24). In turn, Cbfa1 activates osteoblast-specific genes such as osteopontin, bone sialoprotein, type I collagen, and osteocalcin. The importance of Cbfa1 for osteoblasts has been highlighted by the evidence that knockout of the Cbfa1 gene in mice prevents osteoblast development (25, 26). In addition to Cbfa1, BMP-4 induces a homeobox-containing gene, distal-less 5(Dlx5), which also seems to act as a transcription factor, probably as a heterodimer with another homeobox-containing protein (Msx2). Like Cbfa1, Dlx5 regulates the expression of osteoblast-specific genes such as osteocalcin and alkaline phosphatase, as well as mineralization (27–29). Other factors such as transforming growth factor ␤ (TGF␤), platelet-derived growth factor (PDGF), insulin-like growth factors (IGFs), and members of the fibroblast growth factor (FGF) family can all stimulate osteoblast differentiation (30, 31). However, whereas TGF␤, PDGF, FGF, and IGFs are able to influence the replication and differentiation of committed osteoblast progenitors toward the osteoblastic lineage, they cannot induce osteoblast differentiation from uncommitted progenitor cells. In addition to growth factors, bone cells produce proteins that modulate the activity of growth factors either by binding to them and thereby preventing interaction with their receptors, by competing for the same receptors, or by promoting the activity of a particular factor. For example, osteoblasts produce several IGF-binding proteins (IGFBPs). Of these, IGFBP-4 binds to IGF and blocks its action, whereas IGFBP-5 promotes the stimulatory effects of IGF on osteoblasts (30). During the last few years, several proteins able to antagonize BMP action have also been discovered. Of them, noggin, chordin, and cerberus were initially found in the Spemann organizer of the Xenopus embryo and shown to be essential for neuronal or head development (32–35). Noggin and chordin inhibit the action of BMPs by binding directly and with high affinity with the latter proteins (36, 37). Such binding is highly specific for BMP-2 and 4, as noggin binds BMP-7 with very low affinity and does not bind TGF␤ or IGF-I. Addition of human recombinant noggin to bone marrow cell cultures from normal adult mice inhibits not only osteoblast, but also osteoclast, formation, and these effects can be reversed by exogenous BMP-2 (23). Consistent with this evidence, BMP-2 and -4 and BMP-2/4 receptor transcripts and proteins are found in bone marrow cultures and in bone marrow-derived stromal/osteoblastic cell lines, as well as in murine adult whole bone. Noggin expression has also been documented in all these cell preparations. These findings indicate that BMP-2 and -4 are expressed in the bone marrow in postnatal life and serve to maintain the continuous supply of osteoblasts. B. Cytokines Since the early stages of hematopoiesis and osteoclastogenesis proceed along identical pathways, it is not surprising that a large group of cytokines and colony-stimulating factors that are involved in hematopoiesis also affect osteoclast development (38). This group includes the interleukins IL-1, 118 MANOLAGAS IL-3, IL-6, IL-11, leukemia inhibitory factor (LIF), oncostatin M (OSM), ciliary neurotropic factor (CNTF), tumor necrosis factor (TNF), granulocyte macrophage-colony stimulating factor (GM-CSF), M-CSF, and c-kit ligand. As opposed to the above mentioned cytokines that stimulate osteoclast development, IL-4, IL-10, IL-18, and interferon-␥ inhibit osteoclast development. In the case of IL-18 the effect is mediated through GM-CSF (39). IL-6 has attracted particular attention because of evidence that it plays a pathogenetic role in several disease states characterized by accelerated bone remodeling and excessive focal or systemic bone resorption (40). IL-6 is produced at high levels by cells of the stromal/osteoblastic lineage in response to stimulation by a variety of other cytokines and growth factors such as IL-1, TNF, TGF␤, PDGF, and IGF-II (41– 43). Binding of IL-6 or other members of the same cytokine family (IL-11, LIF, OSM) to cytokine-specific cell surface receptors (in the case of IL-6, the IL-6R␣) causes recruitment and homo- or heterodimerization of the signal transducing protein gp130, which is then tyrosine phosphorylated by members of the Janus family of tyrosine kinases (JAKs) (44). This event results in tyrosine phosphorylation of several downstream signaling molecules, including members of the signal transducers and activators of transcription (STAT) family of transcription factors (45, 46). Phosphorylated STATs, in turn, undergo homo- and heterodimerization and translocate to the nucleus where they activate cytokineresponsive gene transcription (47). The ␣-subunit of the IL-6 receptor also exists in a soluble form (sIL-6R), but unlike most soluble cytokine receptors, it functions as an agonist by binding to IL-6 and then interacting with membrane-associated gp130 to stimulate JAK/STAT signaling (44). On the other hand, the soluble form of gp130 blocks IL-6 action (48). Alone or in concert with other agents, IL-6 stimulates osteoclastogenesis and promotes bone resorption. The cells that mediate the actions of the IL-6 type cytokines on osteoclast formation appear to be the stromal/osteoblastic cells, as stimulation of IL-6R␣ expression on these cells allows them to support osteoclast formation in response to IL-6 (49). These findings indicate that the osteoclastogenic property of IL-6 depends not only on its ability to act directly on hematopoietic osteoclast progenitors, but also on the activation of gp130 signaling in the stromal/osteoblastic cells that provide essential support for osteoclast formation. STAT3 activation in stromal/osteoblastic cells is essential for gp130-mediated osteoclast formation (50). Despite the effects of IL-6 on osteoclastogenesis in experimental in vitro systems, IL-6 is not required for osteoclastogenesis in vivo under normal physiological conditions. In fact, osteoclast formation is unaffected in sex steroid-replete mice treated with a neutralizing anti-IL-6 antibody, or in IL-6-deficient mice (51, 52). The most likely explanation for this is that the ␣-subunit of the IL-6 receptor in bone is a limiting factor, and that both a change in the receptor and the cytokine are required for the IL-6mediated increased osteoclastogenesis, seen in pathological states. IL-6 type cytokines are capable of influencing the differentiation of osteoblasts as well. Thus, receptors for these cytokines are expressed on a variety of stromal/osteoblastic cells, and ligand binding induces progression toward a more Vol. 21, No. 2 mature osteoblast phenotype, characterized by increased alkaline phosphatase and osteocalcin expression, and a concomitant decrease in proliferation (53, 54). Moreover, IL-6 type cytokines stimulate the development of osteoblasts from noncommitted embryonic fibroblasts obtained from 12-day-old murine fetuses (55). Consistent with the in vitro evidence, several in vivo studies have demonstrated increased bone formation in transgenic mice overexpressing OSM or LIF (56, 57). TGF␤ is another example of a factor affecting both bone formation and bone resorption (58). Thus, in addition to its ability to stimulate osteoblast differentiation, TGF␤ increases bone resorption by stimulating osteoclast formation. Injection of TGF␤ into the subcutaneous tissue that overlies the calvaria of adult mice causes increased bone resorption accompanied by the development of unusually large osteoclasts, as well as increased bone formation. The effects of TGF␤ might be mediated by other cytokines involved in osteoclastogenesis as TGF␤ can stimulate their production. Mice lacking the TGF␤1 gene due to targeted disruption exhibit excessive production of inflammatory cells, suggesting that this growth factor normally operates to suppress hematopoiesis (59). C. Systemic hormones The two principal hormones of the calcium homeostatic system, namely PTH and l,25-dihydroxyvitamin D3 [1,25(OH)2D3], are potent stimulators of osteoclast formation (17, 60). The ability of these hormones to stimulate osteoclast development and to regulate calcium absorption and excretion from the intestine and kidney, respectively, are the key elements of extracellular calcium homeostasis. Calcitonin, the third of the classical bone-regulating hormones, inhibits osteoclast development and activity and promotes osteoclast apoptosis. Although the antiresorptive properties of calcitonin have been exploited in the management of bone diseases with increased resorption, the role of this hormone in bone physiology in humans, if any, remains questionable (61– 63). PTH, PTH-related peptide, and 1,25-(OH)2D3 stimulate the production of IL-6 and IL-11 by stromal/osteoblastic cells (49, 64 – 66). Several other hormones, including estrogen, androgen, glucocorticoids, and T4, exert potent regulatory influences on the development of osteoclasts and osteoblasts by regulating the production and/or action of several cytokines (21, 64, 67– 69). D. Adhesion molecules In addition to autocrine, paracrine, and endocrine signals, cell-cell and cell-matrix interactions are also required for the development of osteoclasts and osteoblasts (70 –72). Such interactions are mediated by proteins expressed on the surface of these cells and are responsible for contact between osteoclast precursors with stromal/osteoblastic cells and facilitation of the action of paracrine factors anchored to the surface of cells that are required for bone cell development. Adhesion molecules are also involved in the migration of osteoblast and osteoclast progenitors from the bone marrow to sites of bone remodeling as well as the cellular polarization April, 2000 BIRTH AND DEATH OF BONE CELLS of osteoclasts and the initiation and cessation of osteoclastic bone resorption. More important, for the purpose of this review, adhesion molecules play a role in the control of osteoblast and osteoclast development and apoptosis (73–77). The list of adhesion molecules that influence bone cell development and function includes the integrins, particularly ␣v␤3 and ␣2␤1, selectins, and cadherins, as well as a family of transmembrane proteins containing a disintegrin and metalloprotease domain (ADAMS). Each of these proteins recognizes distinct ligands. For example, some integrins recognize a specific amino acid sequence (RGD) present in collagen, fibronectin, osteopontin, thrombospondin, bone sialoprotein, and vitronectin (78). IV. Reciprocal Relationship Between Osteoblastogenesis and Adipogenesis The cells that comprise the bone marrow stroma can serve several diverse functions including support of hematopoiesis and osteoclastogenesis, fat accumulation, and bone formation (79). This functional adaptation is apparently accomplished by the plasticity of some of the stem cell progeny as exemplified by the ability of stromal cells to convert between the osteoblast and adipocyte phenotype. Thus, a stromal cell type known as the Westen-Bainton cell exhibits PTH receptors and high alkaline phosphatase activity and gives rise to osteoblasts during fetal development and in hyperparathyroidism. On the other hand, when marrow hematopoietic activity is reduced using chemotherapeutic agents, these cells convert into adipocytes and can support myeloid cell production (80 – 84). Further, adipocytes isolated by limiting dilution from cultures of rabbit bone marrow can form bone in diffusion chamber implants (85). Conversely, addition of certain fatty acids to cultures of osteoblastic cells causes them to differentiate into adipocyte-like cells (86). It is likely that interconversion of stromal cells among phenotypes, as well as commitment to a particular lineage with suppression of alternative phenotypes, is governed by specific transcription factors. Indeed, Cbfa1 is required for commitment of mesenchymal progenitors to the osteoblast lineage. Mice that are deficient in this factor lack osteoblasts and mineralized bone matrix (26); and expression of Cbfa1 in fibroblastic cells induces transcription of osteoblastspecific genes (24). On the other hand, CCAAT/enhancer binding protein ␣ (C/EBP␣), C/EBP␤, and C/EBP␦, as well as peroxisome proliferator activated receptor ␥1 (PPAR␥1) and PPAR␥2 orchestrate adipocyte differentiation (87–90). Introduction of C/EBP␣ in fibroblastic cells induces adipocyte differentiation (91, 92), and transfection of fibroblastic cells with PPAR␥2 and subsequent activation with an appropriate ligand causes the development of adipocytes (93). Using clonal cell lines isolated from the murine bone marrow, it has been demonstrated that PPAR␥2 can convert stromal cells from a plastic osteoblastic phenotype that reversibly expresses adipocyte characteristics to terminally differentiated adipocytes. Moreover, PPAR␥2 suppresses the expression of Cbfa1 and thereby osteoblast-specific genes (94). Similar to the inhibitory effect of PPAR␥2 on the osteoblast phenotype, the combination of PPAR␥ and C/EBP␣ 119 suppresses the muscle cell phenotype when transfected into G8 myoblastic cells (95). Taken together, these findings strongly suggest that PPAR␥2 plays a hierarchically dominant role in the determination of the fate of mesenchymal progenitors, due to its ability to inhibit the expression of other lineage-specific transcription factors. Studies with a clonal cell line (2T3) suggest that BMP-2 induces osteoblast or adipocyte differentiation in mesenchymal precursors, depending on whether the BMP receptor type IA or IB is activated. Therefore, BMP receptors may also play a critical role in both the specification and reciprocal differentiation of osteoblast and adipocyte progenitors (96). V. Serial and Parallel Models of Osteoblast and Osteoclast Development Even though millions of small packets of bone are constantly remodeled, bone mass is preserved thanks to a remarkably tight balance between the amount of bone resorbed and formed during each cycle of remodeling. In any established BMU, bone resorption and formation are happening at the same time; new osteoblasts assemble only at sites where osteoclasts have recently completed resorption, a phenomenon referred to as coupling, and formation begins to occur while resorption advances. The end result is a new packet of bone, either a cylindrical osteon or Haversian system, or a plate-like hemiosteon, that has replaced the older bone that was removed (97). As the BMU advances, cells are successively recruited at each new cross-sectional location. Osteoblasts do not arrive until the osteoclasts have moved on. However, during the longitudinal progression of the BMU as a whole, new osteoclasts and new osteoblasts are needed simultaneously, although not at the same location. Two models of osteoblast recruitment, a serial and a parallel (Fig. 1), can explain the distinction between the cross-sectional and longitudinal events during BMU progression (38). According to the serial model, factors released from resorbed bone or the local increase in mechanical strain resulting from bone resorption, stimulate osteoblast precursor cell proliferation and differentiation (98 –100). According to the parallel model, osteoblast and osteoclast precursor proliferation and differentiation occur concurrently in response to whatever signal conveys the need for initiation of new BMUs, and whatever hormone prolongs their progression (10, 38). With either model, new osteoblasts must be directed to the right location. Concurrent osteoblast and osteoclast production makes teleological sense as at least one of the means of maintaining a balance between bone formation and resorption under normal conditions. In support of the existence of a parallel pathway of osteoblast and osteoclast formation, it is well established that osteoclasts cannot be formed in vitro unless appropriate stromal cells, analogous to the bone marrow stromal cells that support hematopoiesis, are present to provide essential support. The precise phenotype of the cells that support osteoclast development remains unknown, but they are clearly related to both the osteoblast and the bone marrow stromal/adipocytic lineages (101–104). Interestingly, bone marrow-derived cells with both osteoblastic and adi- 120 MANOLAGAS Vol. 21, No. 2 FIG. 1. Serial and parallel models of osteoblast and osteoclast development. For explanation, please see text. PreOC, Preosteoclast; GF, growth factors released from the matrix of resorbed bone; preOB, osteoblast progenitors. The expressions of RANK ligand and RANK on preosteoblasts and preosteoclasts, respectively, are depicted to indicate their critical contribution in osteoclastogenesis and thereby the dependency of osteoclastogenesis on preosteoblastic cells. pogenic characteristics support the formation of osteoclasts, but marrow-derived cells that exhibit a purely osteoblastic or adipocytic phenotype do not (94). More to the point, noggin, a BMP antagonist, blocks not only osteoblastogenesis but also osteoclastogenesis in murine bone marrow cultures, indicating that commitment of mesenchymal progenitors to the osteoblastic lineage is prerequisite for osteoclastogenesis (23). This evidence suggests that the early less differentiated progeny of common mesenchymal progenitors of the osteoblastic and adipocytic lineage can support osteoclast development, but more differentiated cells that have committed to either the osteoblast or the adipocyte pathway lose this property. It is possible, but as yet untested, that the cells that provide support for osteoclast development are a distinct progeny of mesenchymal progenitors, which displays permanently a phenotype with mixed adipocytic/osteoblastic characteristics, but never progresses to a terminally differentiated osteoblast or adipocyte. For convenience and lack of a better term, the cells that support osteoclast development are frequently referred to as stromal/osteoblastic to indicate their similarities to both bone marrow stromal cells and osteoblasts. In full agreement with the in vitro evidence for the dependency of osteoclast development on support by cells related to osteoblasts, mice lacking osteoblasts due to Cbfa1 deficiency also lack osteoclasts (26). In addition, marrow cells from SAMP6 mice, a strain with defective osteoblastogenesis, exhibit decreased osteoclastogenesis (105) and do not exhibit the expected increase in osteoclastogenesis nor do they lose bone after loss of sex steroids (106). The molecular mechanism of the dependency of osteoclastogenesis on cells of the mesenchymal lineage has been elucidated during the last 2 yr with the discovery of three proteins involved in the TNF signaling pathway (reviewed in Ref. 107). Two of these proteins are membrane-bound cytokine-like molecules: the receptor activator of nuclear factor-B (NF-B) (RANK) and the RANK-ligand. Other names used in the literature for RANK are osteoprotegerin ligand (OPG-L) and TRANCE. RANK is expressed in hematopoietic osteoclast progenitors, while RANK-ligand is expressed in committed preosteoblastic cells and T lymphocytes (108 – 110). RANK-ligand binds to RANK with high affinity. This interaction is essential and, together with M-CSF, sufficient for osteoclastogenesis. 1,25-(OH)2D3, PTH, PTHrP, gp130 activating cytokines (e.g., IL-6, IL-11), and IL-1 induce the expression of the RANK-ligand in stromal/osteoblastic cells (50, 107). Osteoprotegerin (OPG), the third of the three proteins, unlike the other two, is a secreted disulfide-linked dimeric glycoprotein. A hydrophobic leader peptide and three and one-half TNF receptor-like cysteine-rich pseudorepeats characterize the amino terminus of this protein. Unlike other members of the TNF receptor family, OPG does not posses a transmembrane domain. OPG has very potent inhibitory effects on osteoclastogenesis and bone resorption in vitro and in vivo (111). Consistent with an important role of OPG in the regulation of osteoclast formation, OPG transgenic mice develop osteopetrosis, whereas OPG knockout mice exhibit severe osteoporosis (112). The antiosteoclastogenic property of OPG is due to its ability to act as a decoy by binding to RANK-ligand and blocking the RANK-ligand/ RANK interaction. In addition to skeletal metabolism, the RANK/RANK-ligand/OPG circuit may regulate several other biological systems. Indeed, OPG is produced by many tissues other than bone, including skin, liver, stomach, intestine, lung, heart, kidney, and placenta as well as hematopoietic and immune organs. Consistent with this, mice deficient in RANK-ligand completely lacked lymph nodes as well as osteoclasts (113). Moreover, OPG is also a receptor for the cytotoxic ligand TRAIL (TNF-related apoptosis-inducing ligand) to which it binds with high affinity and inhibits TRAIL-mediated apoptosis in lymphocytes (114) and also regulates antigen presentation and T cell activation (115). Osteoblastic cells and T lymphocytes, the two cell types that express high levels of RANK-ligand, are also the two cell types that express high levels of the osteoblast-specific transcription factor Cbfa1 (24). More intriguingly, both the murine and human RANK-ligand genes contain two functional Cbfa1 sites, and mutation of these sites abrogates the tran- April, 2000 BIRTH AND DEATH OF BONE CELLS scriptional activity of the RANK-ligand gene promoter (116). Therefore, the cell-specific expression of RANK-ligand in cells of the stromal/osteoblastic lineage and concurrent differentiation of osteoblasts and osteoclasts might be dictated, at least in part, by interaction between an osteoblast-specific transcription factor and RANK-ligand. BMP 2 and -4 stimulate Cbfa1 expression. Based on these lines of evidence, it has been postulated that the molecular underpinnings of the control of the rate of bone regeneration and the concurrent production of osteoclasts and osteoblasts could well be a BMP3Cbfa13RANK-ligand gene expression cascade in cells of the bone marrow stromal/osteoblastic lineage (117, 118). According to this hypothesis, BMPs may provide the tonic baseline control of both processes, and thereby the rate of bone remodeling, upon which other inputs (e.g., biomechanical, hormonal, etc.) operate. Studies with transgenic and knockout animal models as well as models with spontaneous genetic mutations have identified at least three transcription factors that are required for osteoclast differentiation: PU-1, fos, and NF-b. A review of the precise role of these factors is beyond the scope of this article, but the reader is referred to a recent excellent review of the topic (119). VI. Function of the Mature Cells A. Osteoblasts The fully differentiated osteoblasts produce and secrete proteins that constitute the bone matrix (120). The matrix is subsequently mineralized under the control of the same cells. A major product of the bone-forming osteoblast is type I collagen. This polymeric protein is initially secreted in the form of a precursor, which contains peptide extensions at both the amino-terminal and carboxyl ends of the molecule. The propeptides are proteolytically removed. Further extracellular processing results in mature three-chained type I collagen molecules, which then assemble themselves into a collagen fibril. Individual collagen molecules become interconnected by the formation of pyridinoline cross-links, which are unique to bone. Bone-forming osteoblasts synthesize a number of other proteins that are incorporated into the bone matrix, including osteocalcin and osteonectin, which constitute 40% to 50% of the noncollagenous proteins of bone. Mice deficient in osteocalcin develop a phenotype marked by higher bone mass and improved bone quality, suggesting that osteocalcin functions normally to limit bone formation without compromising mineralization (121). Conversely, mice deficient in osteonectin exhibit decreased osteoclast and osteoblast numbers and bone remodeling and profound osteopenia, suggesting that, under normal conditions, this protein may play a role in the birth or survival of these cells (122). Other osteoblast-derived proteins include glycosaminoglycans, which are attached to one of two small core proteins: PGI (or biglycan) and decorin; the latter has been implicated in the regulation of collagen fibrillogenesis. A number of other minor proteins such as osteopontin, bone sialoprotein, fibronectin, vitronectin, and thrombospondin serve as attachment factors that interact with integrins. In addition to being the cells that produce the osteoid 121 matrix, mature osteoblasts are essential for its mineralization, the process of deposition of hydroxyapatite (123, 124). Osteoblasts are thought to regulate the local concentrations of calcium and phosphate in such a way as to promote the formation of hydroxyapatite. In view of the highly ordered, well aligned, collagen fibrils complexed with the noncollagenous proteins formed by the osteoblast in lamellar bone, it is likely that mineralization proceeds in association with, and perhaps governed by, the heteropolymeric matrix fibrils themselves. Osteoblasts express relatively high amounts of alkaline phosphatase, which is anchored to the external surface of the plasma membrane. Alkaline phosphatase has been long thought to play a role in bone mineralization. Consistent with this, deficiency of alkaline phosphatase due to genetic defects leads to hypophosphatasia, a condition characterized by defective bone mineralization (125). However, the precise mechanism of mineralization and the exact role of alkaline phosphatase in this process remain unclear. Bone mineralization lags behind matrix production and, in remodeling sites in the adult bone, occurs at a distance of 8 –10 m from the osteoblast. Matrix synthesis determines the volume of bone but not its density. Mineralization of the matrix increases the density of bone by displacing water, but does not alter its volume. B. Osteocytes Some osteoblasts are eventually buried within lacunae of mineralized matrix. These cells are termed osteocytes and are characterized by a striking stellate morphology, reminiscent of the dendritic network of the nervous system (126, 127). Osteocytes are the most abundant cell type in bone: there are 10 times as many osteocytes as osteoblasts. Osteocytes are regularly spaced throughout the mineralized matrix and communicate with each other and with cells on the bone surface via multiple extensions of their plasma membrane that run along the canaliculi; osteoblasts, in turn, communicate with cells of the bone marrow stroma which extend cellular projections onto endothelial cells inside the sinusoids. Thus, a syncytium extends from the entombed osteocytes all the way to the vessel wall (128) (Fig. 2). As a consequence, the strategic location of osteocytes makes them excellent candidates for mechanosensory cells able to detect the need for bone augmentation or reduction during functional adaptation of the skeleton, and the need for repair of microdamage, and in both cases to transmit signals leading to the appropriate response; albeit this remains hypothetical (129). Osteocytes evidently sense changes in interstitial fluid flow through canaliculi (produced by mechanical forces) and detect changes in the levels of hormones, such as estrogen and glucocorticoids, that influence their survival and that circulate in the same fluid (130 –132). Therefore, disruption of the osteocyte network is likely to increase bone fragility. C. Lining cells The surface of normal quiescent bone (i.e., bone that is not undergoing remodeling) is covered by a 1–2-m thick 122 MANOLAGAS Vol. 21, No. 2 FIG. 2. Functional syncytium comprising osteocytes, osteoblasts, bone marrow stromal cells, and endothelial cells. [Adapted from G. Marotti and reproduced with the permission of the Editor of Journal of Clinical Investigation 104: 1363–1374, 1999 (219). layer of unmineralized collagen matrix on top of which there is a layer of flat and elongated cells. These cells are called lining cells and are descendents of osteoblasts (13). Conversion of osteoblasts to lining cells represents one of the fates of osteoblasts that have completed their bone forming function; another being entombment into the matrix as osteocytes. Osteoclasts cannot attach to the unmineralized collagenous layer that covers the surface of normal bone. Therefore, other cells, perhaps the lining cells, secrete collagenase, which removes this matrix before osteoclasts can attach to bone. It has been proposed that targeting of osteoclast precursors to a specific location on bone depends on a “homing” signal given by lining cells; and that lining cells are instructed to do so by osteocytes, the only bone cells that can sense the need for remodeling at a specific time and place (133). vacuolar H⫹-ATPase) located in the ruffled border membrane. The protein components of the matrix, mainly collagen, are degraded by matrix metalloproteinases, and cathepsins K, B, and L are secreted by the osteoclast into the area of bone resorption (134). The degraded bone matrix components are endocytosed along the ruffled border within the resorption lacunae and then transcytosed to the membrane area opposite the bone, where they are released (135, 136). Another feature of osteoclasts is the presence of high amounts of the phosphohydrolase enzyme, tartrate-resistant acid phosphatase (TRAPase). This feature is commonly used for the detection of osteoclasts in bone specimens (137). Mice deficient in TRAPase exhibit a mild osteopetrotic phenotype (due to an intrinsic defect of osteoclastic resorptive activity) and defective mineralization of the cartilage in developing bones (138). D. Osteoclasts Mature osteoclasts are usually large (50 to 100 m diameter) multinucleated cells with abundant mitochondria, numerous lysosomes, and free ribosomes. Their most remarkable morphological feature is the ruffled border, a complex system of finger-shaped projections of the membrane, the function of which is to mediate the resorption of the calcified bone matrix (17, 123). This structure is completely surrounded by another specialized area, called the clear zone. The cytoplasm in the clear zone area has a uniform appearance and contains bundles of actin-like filaments. The clear zone delineates the area of attachment of the osteoclast to the bone surface and seals off a distinct area of the bone surface that lies immediately underneath the osteoclast and which eventually will be excavated. The ability of the clear zone to seal off this area of bone surface allows the formation of a microenvironment suitable for the operation of the resorptive apparatus. The mineral component of the matrix is dissolved in the acidic environment of the resorption site, which is created by the action of an ATP-driven proton pump (the so-called VII. Death of Bone Cells by Apoptosis The average lifespan of human osteoclasts is about 2 weeks, while the average lifespan of osteoblasts is 3 months (Table 1). After osteoclasts have eroded to a particular distance, either from the central axis in cortical bone or to a particular depth from the surface in cancellous bone, they die and are quickly removed by phagocytes (139). The majority (65%) of the osteoblasts that originally assembled at the remodeling site also die (140). The remaining are converted to lining cells that cover quiescent bone surfaces or are entombed within the mineralized matrix as osteocytes (Fig. 3A). Both osteoclasts and osteoblasts die by apoptosis, or programmed cell death, a process common to several regenerating tissues (141). As in other tissues, bone cells undergoing apoptosis are recognized by condensation of chromatin, the degradation of DNA into oligonucleosome-sized fragments, and the formation of plasma and nuclear membrane blebs (Fig. 4). Eventually the cell breaks apart to form so-called apoptotic bodies. Osteoblast apoptosis explains the fact that 50 –70% April, 2000 BIRTH AND DEATH OF BONE CELLS 123 FIG. 3. Osteoblast apoptosis and its implications. A, The average life span of a matrix forming osteoblast (⬃200 h in the mouse) is indicated by the continuous line. The process of apoptosis represents only a small fraction of this time period. The alternative two fates of osteoblasts are to become lining cells or osteocytes. The fraction of osteoblasts that undergo apoptosis in vivo (fApoptosis) can be estimated from a bone biopsy specimen. The duration of the apoptosis phase that can be observed in the specimen (tApop) depends on the sensitivity of the detection method. For example, in the case of the TUNEL technique (without CuSO4 enhancement), the TUNEL-labeled phase of apoptosis is estimated to be approximately 2 h. In a steady state, the fraction of cells at a particular stage is the same as the corresponding fraction of time spent in that stage. Assuming an apoptosis detection time of 2 h and a 200-h life span, a prevalence of TUNEL positive osteoblasts in the biopsy of 0.005 indicates that half of the osteoblasts die by apoptosis. B, A change in the timing and extent of osteoblast apoptosis (fApoptosis) from 50% to zero should increase the number of osteoblasts present at the site of bone formation and thereby the work output, i.e., the amount of bone formed by a given team of matrix-forming osteoblasts. It will also lead to an increase in the density of osteocyte apoptosis, as illustrated by the example shown. of the osteoblasts initially present at the remodeling site of human bone cannot be accounted for after enumeration of lining cells and osteocytes (142). Moreover, the frequency of osteoblast apoptosis in vivo is such that changes in its timing and extent could have a significant impact in the number of osteoblasts present at the site of bone formation (130). Osteocytes are long-lived but not immortal cells; some die by apoptosis (132, 143, 144). Osteocyte apoptosis could be of importance to the origination and/or progression of the BMU. Indeed, osteocytes are the only cells in bone that can sense the need for remodeling at a specific time and place. Moreover, osteocytes are in direct physical contact with lining cells on the bone surface, and targeting of osteoclast precursors to a specific location on bone depends on a “homing” signal given by lining cells (133). The same growth factors and cytokines that stimulate osteoclast and osteoblast development can also influence their apoptosis. For example, TGF␤ promotes osteoclast apoptosis while it inhibits osteoblast apoptosis. IL-6 type cytokines have antiapoptotic effects on animal and human osteoblastic cells (and at least in vitro they antagonize proapoptotic effects of glucocorticoids) as well as on osteoclasts and their progenitors (54, 140, 145, 146). VIII. Regulation of Bone Cell Proliferation and Activity A large body of literature suggests that growth factors, cytokines, hormones, and drugs regulate the proliferation of 124 MANOLAGAS Vol. 21, No. 2 FIG. 4. Two osteoblasts undergoing apoptosis in a section of murine cancellous bone (TUNEL staining with toluidine blue counterstain, ⫻630). Apoptotic osteoblasts (shown in brown) are adjacent to an intact osteoblast (shown in blue), on the surface of a trabecula occupying the right lower portion of the picture in which two intact (blue stained) osteocytes are also seen. Apoptotic osteoblasts display nuclear condensation and fragmentation. [Photomicrograph provided by Robert S. Weinstein, M.D., University of Arkansas for Medical Sciences.] committed cells or the biosynthetic and functional activity of the differentiated osteoblasts and osteoclasts. Hence, in addition to cell number, alterations in the functional activity of individual cells, i.e., cell vigor, may contribute to changes in the rate of bone resorption and formation. However, because of the inherent difficulty in demonstrating changes in individual cell vigor in vivo, the vast majority of such literature and its conclusions rely heavily, if not exclusively, on in vitro experimentation. A detailed discussion of this work is beyond the scope of this review, and the reader is referred to other articles (30, 147–151). Nonetheless, some general aspects merit discussion here, as they are important for putting the significance of birth rate and apoptosis into a broader perspective. In regenerating tissues, the initial commitment of a stem cell progeny is followed by amplification with several or many rounds of cell division. In most tissues, division of stem cells is infrequent, and almost all of the divisions that produce the final population of differentiated cells occur in the so-called transit compartment (152). This notion is obscured by the frequent practice of showing linear diagrams representing the transition from one cell type to another and ignoring completely the amplification during the transition. In general, terminally differentiated cells do not divide, and an osteoblast, defined as a cell making bone matrix, and osteoclast, as a cell resorbing bone, are in this category. Therefore, the in vivo relevance of much of the in vitro evidence on the regulation of osteoblastic or osteoclastic cell proliferation, using established cell lines or primary cultures of isolated cells, must be largely confined to changes in this transit compartment. Irrespective of whether a given regulatory factor, be that a cytokine or a hormone, influences the initial commitment of a stem cell, or the subsequent amplification of its progeny, or both, the end result is a change in the rate of production and therefore the number of cells available for the execution of the biological task. For the sake of simplicity, the terms birth, rate of birth, osteoblastogen- esis, or osteoclastogenesis, as used in this article, implicitly combine commitment and amplification. The in vivo relevance of numerous reports of in vitro observations of changes in the biosynthetic activity of osteoblastic cells, e.g., changes in the level of expression of osteocalcin or alkaline phosphatase in response to a given agent, is also a matter of conjecture. Most likely, given the nature of commonly used in vitro cell models which, by and large, represent preterminally differentiated cells, such observations might be more relevant to postcommitment differentiation events, than to altered activity of the fully differentiated cell. Moreover, even if some agents can alter the function of terminally differentiated cells in short-term cultures in vitro, heretofore there is no evidence that short-term change in the rate of collagen production or bone matrix digestion, for example, are ultimately translated into differences in the amounts of bone matrix formed or resorbed. In the bone literature there is considerable ambiguity when using the term “activation.” It is important that one distinguishes between activation as a switch from an off state to an on state, and activation as modulation of activity of an already active cell. Morphological evidence summarized elsewhere does not support the notion that completely inactive osteoclasts are waiting for a stimulus to make them active (117). However, this evidence does not address the issue of whether there are variations in the rate of bone matrix dissolution by individual osteoclasts after they started work. Studies with the widely used bone slice pit bioassay have shown that several regulatory factors can cause a decrease or increase in the resorptive ability of individual osteoclasts (153–155). However, it is not clear to what extent these observations reflect a change in cell vigor as opposed to a change in the precariously short lifespan of osteoclasts in vitro. In estrogen deficiency, individual osteoclasts are seemingly more “active” as they dig deeper resorption cavities often leading to trabecular perforation, but it has been con- April, 2000 BIRTH AND DEATH OF BONE CELLS vincingly argued that this is due to delayed apoptosis (133). In Paget’s disease osteoclasts are certainly far more aggressive than normal, perhaps as a result of their uniquely large size and number of nuclei (156). Today, the most compelling evidence in support of the notion that the vigor of individual osteoclasts may not always be maximal comes from in vitro as well as in vivo experiments with soluble RANK-ligand (157). Specifically, it has been found that RANK-ligand acts on mature rat osteoclasts in vitro to stimulate more frequent cycles of resorption and induce rearrangements of the cytoskeleton. Moreover, intravenous administration of RANKligand to mice elevates the circulating concentration of ionized calcium within 1 h. RANK-ligand has potent antiapoptotic effects on cultured osteoclasts (Ref. 158 and William Boyle, personal communication). Therefore, definitive conclusions regarding RANK-ligand’s ability to modulate osteoclast vigor will have to await dissection of the contribution of its effects on osteoclast survival in vivo. Similar to osteoclasts, the bone-forming ability of osteoblasts may not be always maximal, as suggested by the evidence that PTH can rapidly enhance bone formation when administered by subcutaneous injections to rats (159). To conclude this section, it is intuitive that the amount of bone resorbed or formed by a team of osteoclasts and osteoblasts should be a function of the total cell number as well as the vigor of individual cells. However, whereas cell number can be quantified on bone sections from animals and humans with conventional histomorphometric techniques, quantification of individual cell vigor cannot. This situation makes it difficult to judge at present whether cell vigor is or is not a critical component in the pathogenesis of abnormal skeletal regeneration in common acquired metabolic bone diseases such as postmenopausal, senile, or steroid-induced osteoporosis. For this reason and space limitations, changes in bone cell vigor or other potential mechanisms of osteoporosis resulting from changes in extraskeletal tissues, for example altered calcium absorption or excretion, will not be discussed in the following section. This also reflects the author’s intention to focus on the dynamics of bone cell number, rather than a dismissal of other mechanisms. 125 IX. Pathogenesis Of Osteoporosis From the brief discussion of the principles of physiological bone regeneration and the role of osteoblasts and osteoclasts in the process, it is obvious that the rate of supply of new osteoblasts and osteoclasts and the timing of the death of these cells by apoptosis are critical determinants of the initiation of new BMUs and/or extension or shortening of the lifetime of existing ones. Recent advances in our understanding of the pathogenesis of the various forms of osteoporosis have confirmed this truism by revealing that over- or undersupply of these cells relative to the need for remodeling are the fundamental problems in all these conditions (160) (Table 2). A. Sex steroid deficiency The mechanism of action of sex steroids on the skeleton is not fully understood. At menopause (or after castration in men), the rate of bone remodeling increases precipitously. This fact may be explained by evidence, derived primarily from studies in mice, that loss of sex steroids up-regulates the formation of osteoclasts and osteoblasts in the marrow by up-regulating the production and action of cytokines that are responsible for osteoclastogenesis and osteoblastogenesis (21, 161, 162). Indeed, both estrogen and androgen suppress the production of IL-6, as well as the expression of the two subunits of the IL-6 receptor, IL-6R␣ and gp130, in cells of the bone marrow stromal/osteoblastic lineage (40, 163). Suppression of IL-6 production by estrogen or selective estrogen receptor modulators (SERMs), such as raloxifene, does not require direct binding of the estrogen receptor to DNA. Instead, it is due to protein-protein interaction between the estrogen receptor and transcription factors such as NF-K␤ and C/EBP. This mechanism provides a model that best fits current understanding of the molecular pharmacology of estrogen and SERMs (164). Consistent with the suppressive effect of sex steroids on IL-6 and its receptor, several, albeit not all, studies have shown that the level of expression of IL-6, as well as IL-6R␣ and gp130, is elevated in estrogen- TABLE 2. Cellular changes and their culprits in the three most common causes of bone loss Sex steroid deficiency Senescence Glucocorticoid excess a Cellular changes Probable culprits 1Osteoblastogenesis 1Osteoclastogenesisa 1Lifespan of osteoclasts 2Lifespan of osteoblasts 2Lifespan of osteocytes 2Osteoblastogenesisb 2Osteoclastogenesis 1Adipogenesis 2Lifespan of osteocytes 2Osteoblastogenesis 2Osteoclastogenesis 1Adipogenesis 1Lifespan of osteoclastsc 2Lifespan of osteoblasts 2Lifespan of osteocytes Increased IL-6; TNF; IL-1RI/IL-RII MCSF; decreased TGF␤; OPG Loss of pro- and antiapoptotic effects of sex steroids, respectively Increased PPAR␥2, PGJ2, noggin; Deceased IL-11, IGFs Decreased Cbfal and TGF␤ R1; and BMP-2 and IGF1 action Increased PPAR␥2 Decreased Bcl-2/BAX ratio Oversupply of osteoclasts relative to the need for remodeling. Undersupply of osteoblasts relative to the need for cavity repair. c Osteoclast numbers may transiently increase in the earlier stages of steroid therapy, without an increase in osteoclastogenesis, indicating increased lifespan. b 126 MANOLAGAS deficient mice and rats as well as in humans, in the bone marrow and in the peripheral blood (165–168). Furthermore, neutralization of IL-6 with antibodies or knockout of the IL-6 gene in mice prevents the expected cellular changes in the marrow and in trabecular bone sections and protects the mice from bone loss after loss of sex steroids (51, 67). Consistent with its pathogenetic role in the bone loss caused by loss of sex steroids, IL-6 seems to play a similar role in several other conditions associated with increased bone resorption as evidenced by increased local or systemic production of IL-6 and the IL-6 receptor in patients with multiple myeloma, Paget’s disease, rheumatoid arthritis, Gorham-Stout or disappearing bone disease, hyperthyroidism, primary and secondary hyperparathyroidism, as well as McCune Albright Syndrome (66, 68, 169 –174). In line with the fact that loss of sex steroids increases the rate of bone remodeling, in addition to up-regulating osteoclastogenesis, loss of sex steroids increases the number of osteoblast progenitors in the murine bone marrow. These changes are temporally associated with increased bone formation and parallel the increased osteoclastogenesis and bone resorption (175). As IL-6 type cytokines can stimulate osteoblast development and differentiation (54, 55, 146), increased sensitivity to IL-6 and other members of this cytokine’s family may account also for the increased osteoblast formation that follows the loss of gonadal function. In view of the fact that mesenchymal cell differentiation and osteoclastogenesis are tightly linked, stimulation of mesenchymal cell differentiation toward the osteoblastic lineage after sex steroid loss may be the first event that ensues after the hormonal change, and increased osteoclastogenesis and bone loss might be downstream consequences of this change (106). In addition to IL-6, estrogen also suppress TNF and M-CSF (176, 177), and estrogen loss may increase the sensitivity of osteoclasts to IL-1 by increasing the ratio of the IL-1RI over the IL-1 receptor antagonist (IL-RII) (178). As in the case of IL-6, the effects of estrogen on TNF and M-CSF are mediated via protein-protein interactions between the estrogen receptor and other transcription factors. In agreement with the evidence that IL-1 and TNF play a role in the bone loss caused by loss of estrogen, administration of IL-1RA and/or a TNF soluble receptor ameliorates the bone loss caused by ovariectomy in rats and mice (179 –181). Because of the interdependent nature of the production of IL-1, IL-6, and TNF, a significant increase in one of them may amplify, in a cascade fashion, the effect of the others (161). Interestingly, recent in vitro studies with human osteoblastic cells indicate that OPG production is stimulated by estrogen, suggesting that this cytokine may also play an important role in the antiosteoclastogenic (antiresorptive) action of estrogen on bone (182). Increased remodeling, resulting from up-regulation of osteoblastogenesis and osteoclastogenesis, alone can cause a transient acceleration of bone mineral loss because bone resorption is faster than bone formation, and new bone is less dense than older bone. However, in addition to increased bone remodeling, loss of sex steroids leads to a qualitative abnormality: osteoclasts erode deeper than normal cavities (133, 183). In this manner, sex steroid deficiency leads to the removal of some cancellous elements entirely; the remainder Vol. 21, No. 2 are more widely separated and less well connected. An equivalent amount of cancellous bone distributed as widely separated, disconnected, thick trabeculae is biomechanically less competent than when arranged as more numerous, connected, thin trabeculae. Concurrent loss of cortical bone occurs by enlargement and coalescence of subendocortical spaces, a process due to deeper penetration of endocortical osteoclasts. This deeper erosion can be now explained by evidence that estrogen acts on mature osteoclasts to promote their apoptosis; consequently, loss of estrogen leads to prolongation of the lifespan of osteoclasts (133). Specifically, estrogen promotes osteoclast apoptosis in vitro and in vivo by 2- to 3-fold, an effect seemingly mediated by TGF␤ (139). In direct contrast to their proapoptotic effects on osteoclasts, estrogen (as well as androgen) exerts antiapoptotic effects on osteoblasts and osteocytes; consequently, loss of estrogen or androgen leads to shorter lifespan of osteoblasts and osteocytes (184). Extension of the working life of the bone-resorbing cells and simultaneous shortening of the working life of the boneforming cells, can explain the imbalance between bone resorption and formation that ensues after loss of sex steroids. Furthermore, the increase in osteocyte apoptosis could further weaken the skeleton by impairment of the osteocytecanalicular mechanosensory network. The increase in bone remodeling that occurs with estrogen deficiency would partly replace some of the nonviable osteocytes in cancellous bone, but cortical apoptotic osteocytes might accumulate because of their anatomic isolation from scavenger cells and the need for extensive degradation to small molecules to dispose of the osteocytes through the narrow canaliculi. Hence, the accumulation of apoptotic osteocytes caused by loss of estrogen, or glucocorticoid excess (130), could increase bone fragility even before significant loss of bone mass, because of the impaired detection of microdamage and repair of substandard bone. In conclusion, the increased rate of bone remodeling in estrogen deficiency may be due to increased production of both osteoclasts and osteoblasts, and the imbalance between bone resorption and formation is due to an extension of the working lifespan of the osteoclast and shortening of the working lifespan of the osteoblast. Moreover, a delay of osteoclast apoptosis seems responsible for the deeper resorption cavities and thereby the trabecular perforation associated with estrogen deficiency. Clinical observations of decreased bone mass in a male with mutant estrogen receptor (185), and increased bone mass after treatment with estrogen in two males with P-450 aromatase deficiency (186, 187), have raised the possibility that estrogen derived by peripheral aromatization of androgens is critical for the maintenance of bone mass in men as well as in women (188). However, in all three cases, the decreased bone mass in young males with estrogen deficiency in the face of androgen sufficiency could be due to failure in achieving peak bone mass from defects occurring during development or growth, not to loss of bone mass, as it is the case with common forms of osteoporosis. In addition, individuals with complete androgen insensitivity, due to mutations in the androgen receptor gene on the X chromosome and increased testosterone and estrogen production, April, 2000 BIRTH AND DEATH OF BONE CELLS have decreased bone mass, in spite of the elevated estrogen levels (189). Moreover, androgens, including nonaromatizable ones, have identical effects to those of estrogen on the biosynthetic activity and the birth as well as the death of bone cells in vitro and in vivo, at least in rodents (67, 106, 190). It is therefore more likely that both estrogen and androgen are important for the maintenance of bone mass in the adult male skeleton. B. Senescence The amount of bone formed during each remodeling cycle decreases with age in both sexes. This is indicated by a consistent histological feature of the osteopenia that occurs during aging, namely a decrease in wall thickness, especially in trabecular bone (191–193). Wall thickness is a measure of the amount of bone formed in a remodeling packet of cells and is determined by the number or activity of osteoblasts at the remodeling site. Studies measuring bone turnover by histomorphometry (194), or indirectly by circulating markers (195–197), have suggested that in aging women, even in extreme old age, bone turnover is most likely increased by secondary hyperparathyroidism or by the continuing effect of estrogen deficiency. Increased turnover and reduced wall thickness are not inconsistent, as the former is the result of increased activation frequency, and the decreased wall thickness—an index of decreased bone formation by osteoblasts—in senescence is local and relative to the demand created by resorption. Changes in the birth of bone cells in the bone marrow provide a mechanistic explanation for the contribution of senescence to bone loss, independently from sex steroid deficiency. Specifically, using SAMP6 mice, a murine model of age-related osteopenia (but sufficient in sex steroids and with intact reproductive function), a tight association among reduced number of osteoblast progenitors, decreased bone formation, and decreased bone mass has been established (105). Decreased osteoblastogenesis with advancing age has been confirmed in the human bone marrow (198, 199). Importantly, the decrease in osteoblastogenesis is accompanied by increased adipogenesis and myelopoiesis, as well as decreased osteoclastogenesis, the latter most likely caused by a reduction in the stromal/osteoblastic cells that support osteoclast formation (105, 200). This suggests that in aging there must be changes in the expression of genes that favor the differentiation of multipotent mesenchymal stem cells toward adipocytes at the expense of osteoblasts. The evidence that PPAR␥2 induces the terminal differentiation of marrow cells with both osteoblastic and adipocytic characteristics to adipocytes, and simultaneously suppresses Cbfa1 expression and terminal differentiation to osteoblasts (94), raises the possibility that increased expression of PPAR␥2 or its ligands, e.g., PGJ2, may be some of the culprits responsible for the reciprocal change between adipogenesis and osteoblastogenesis with advancing age (Table 2). Uptake of oxidized low-density lipoproteins (LDL) play an important role in foam cell formation and the pathogenesis of atherosclerosis. Two of the major components of oxidized LDL, 9-hydroxy-9,11-octadecadienoic acid (HODE) and 13- 127 HODE, are endogenous ligands and activators of PPAR␥ (201), and PPAR␥ promotes monocyte/macrophage differentiation and uptake of oxidized LDL (202). Taken together with these findings, the evidence that activated PPAR␥2 promotes adipocyte differentiation at the expense of osteoblastogenesis in the bone marrow by suppressing Cbfa1 (94) suggests a mechanistic link among dietary fat/lipoproteins, bone marrow stromal cell differentiation, osteoporosis, and atherogenesis. In support of the existence of such a link, activation of PPAR␥ by thiazolidinediones or oxidatively modified LDL inhibits osteoblastogenesis of bone marrowderived stromal cells in vitro (94, 203, 204). Moreover, high fatty acid content in rabbit serum or high fat diet of mice for 4 months decreases osteogenic cell differentiation in ex vivo bone marrow cell cultures (86, 204). These new advances may explain clinical observations that atherosclerosis and osteoporosis coexist (205, 206). Quantitative trait loci (QTLs) analysis of osteopenia-associated loci using closely related mouse strains have mapped five loci to regions of chromosomes 2, 7, 11, and 16 (207). Association of these same loci with bone mineral density has been reproduced in crosses of different recombinant-inbred mouse strains (208, 209). Such recurrent appearance of QTL, especially in crosses involving distantly related strains, implies that polymorphism at these loci may be favored by evolution and might underlie variation in peak bone density among humans. Intriguingly, of the more than 12 genes affecting bone homeostasis that were localized near these QTLs, 2 are prostaglandin synthases, a third is the BMP-2/4 antagonist noggin, a fourth is the proapoptotic protein bax, and the fifth is IL-11. Hence, the transcription factor PPAR␥2 and its ligand, PGJ2, noggin, and IL-11 are potentially responsible for the decreased osteoblastogenesis with advancing age. This contention is supported by the evidence that the reciprocal relationship between decreased osteoblastogenesis and increased adipogenesis in the SAMP6 mouse may be explained by a change in the expression of PPAR␥ or its ligands in early mesenchymal progenitors; that BMP-2/4, in balance with noggin, may determine the tonic baseline control of the rate of osteoblastogenesis; and that IL-11 is a potent inhibitor of adipogenesis, which stimulates osteoblast differentiation and the expression of which is reduced in SAMP6 mice. In addition to these factors, growth factors such as IGFs have also been implicated in the bone loss associated with senescence (210, 211). Irrespective of the identity of the precise mediator, the reciprocal change between adipogenesis and osteoblastogenesis can explain the association of decreased relative bone formation and the resulting osteopenia with the increased adiposity of the marrow seen with advancing age in animals and humans (105, 142, 212–215). C. Glucocorticoid excess The cardinal histological features of glucocorticoid-induced osteoporosis are decreased bone formation rate, decreased wall thickness of trabeculae (a strong indication of decreased work output by osteoblasts), and in situ death of portions of bone. Increased bone resorption, decreased osteoblast proliferation and biosynthetic activity, and sex-steroid deficiency, as well as hyperparathyroidism resulting from decreased intestinal cal- 128 MANOLAGAS cium absorption and hypercalciuria due to defective vitamin D metabolism, have all been proposed as mechanisms for the loss of bone that ensues with glucocorticoid excess (216). The decreased bone formation and osteonecrosis can now be explained by evidence that glucocorticoid excess has a suppressive effect on osteoblastogenesis in the bone marrow and also promotes the apoptosis of osteoblasts and osteocytes (118). Indeed, mice receiving glucocorticoids for 4 weeks, a period equivalent to ⬃3– 4 yr in humans, exhibit decreased bone mineral density associated with a decrease in the number of osteoblast, as well as osteoclast, progenitors in the bone marrow and a dramatic reduction in cancellous bone area and in trabecular width compared with placebo controls. These changes are associated with a significant reduction in osteoid area and a decrease in the rates of mineral apposition and bone formation. More strikingly, glucocorticoid administration to mice causes a 3-fold increase in the prevalence of osteoblast apoptosis in vertebrae and induced apoptosis in 28% of the osteocytes in metaphyseal cortical bone. Nevertheless, even though there is a significant correlation between the severity of the bone loss and the extent of reduction in bone formation, some of the bone loss may be due to an early increase in bone resorption as evidenced by an early increase in osteoclast perimeter of vertebral cancellous bone after 7 days of steroid treatment. In vivo studies with mice from this author’s group show that at this early time point (7 days after glucocorticoid administration) osteoclastogenesis in ex vivo bone marrow cultures is decreased by half, while the number of osteoclasts in bone sections doubles (Robert Weinstein, personal communication), suggesting that an early effect of glucocorticoid excess might be increased osteoclast survival. In vitro studies by others, on the other hand, have shown that glucocorticoids inhibit OPG and concurrently stimulate the expression of RANK-ligand in human osteoblastic, primary, and immortalized bone marrow stromal cells (217). Taken together, these lines of evidence suggest that the initial rapid phase of bone loss with glucocorticoid treatment could be due to an extension of the lifespan of preexisting osteoclasts, mediated by RANKligand (117). The same histomorphometric changes that have been found in mice after a 4-week treatment with steroids have been confirmed in biopsies from patients receiving long-term glucocorticoid therapy. Moreover, as in mice, an increase in osteoblast and osteocyte apoptosis is found in human biopsies. Compared with osteoblast apoptosis, apoptotic osteocytes are far more prevalent, at least in metaphyseal cortices, probably because of the anatomical isolation of osteocytes from scavenger cells. Consistent with these findings, glucocorticoids promote osteoblast and osteocyte apoptosis in vitro (218, 219). Decreased production of osteoclasts can explain the reduction in bone turnover with chronic glucocorticoid excess, whereas decreased production and apoptosis of osteoblasts can explain the decline in bone formation and trabecular width. Accumulation of apoptotic osteocytes may also explain the so-called “osteonecrosis,” also known as aseptic or avascular necrosis, another manifestation of steroid-induced osteoporosis that causes collapse of the femoral head in as many as 25% of patients (220). This contention is supported by evidence that whole femoral heads obtained Vol. 21, No. 2 from patients with glucocorticoid-induced osteoporosis exhibit abundant apoptotic osteocytes adjacent to the subchondral fracture crescent (221). Glucocorticoid-induced osteocyte apoptosis, a cumulative and unrepairable defect, could uniquely disrupt the proposed mechanosensory role of the osteocyte network and thus promote collapse of the femoral head. At this time, the mediators of the cellular changes caused by glucocorticoid excess are only a matter of conjecture. Nonetheless, glucocorticoids directly suppress BMP-2 and Cbfa1-2—two critical factors for osteoblastogenesis—and may also decrease the production of IGFs while they stimulate the transcriptional activity of PPAR␥2 (222–225) (Table 2). X. Pharmacotherapeutic Implications of Osteoblast and Osteocyte Apoptosis Estrogen replacement therapy (ERT), various bisphosphonates (e.g., alendronate), the SERM raloxifene, calcitonin, and sodium fluoride, as well as calcium and vitamin D, are approved modalities for the prevention and treatment of bone loss, irrespective of its cause. Decreased osteoclast progenitor development and/or decreased osteoclast recruitment and promotion of apoptosis of mature osteoclasts leading to a slowing of the rate of bone remodeling are thought to be the main mechanisms of the so-called “antiresorptive” agents estrogen, bisphosphonates, SERMs, and calcitonin. Sodium fluoride has anabolic properties, but its therapeutic range is very narrow. Calcium and vitamin D are rarely sufficient on their own, but they are considered a very useful supplementation in any regimen for osteoporosis. A. Intermittent PTH administration The ideal therapy for osteoporosis, especially in elderly patients who already have advanced bone loss, would be an anabolic agent that will increase bone mass by rebuilding bone. It is well established that daily injections of low doses of PTH—an agent better known for its role in calcium homeostasis—increases bone mass in animals and humans (226 –231) as does the PTH-related protein (PTHrP), the only other known ligand of the PTH receptor (232). Indeed, although constant, high levels of PTH cause increased bone resorption and osteitis fibrosa cystica, low and intermittent doses of PTH, too small to affect serum calcium concentrations, promote bone formation and increase bone mineral density at the lumbar spine and hip. This so-called anabolic effect can be now explained by evidence that PTH increases the life span of mature osteoblasts in vivo by reducing the prevalence of their apoptosis from 1.7–2.2% to as little as 0.1– 0.4% rather than by affecting the generation of new osteoblasts (218). The antiapoptotic effect of PTH in mice was sufficient to account for the increase in bone mass and was confirmed in vitro using rodent and human osteoblasts and osteocytes. Like PTH, PGE inhibits periosteal cell apoptosis via cAMP-dependent stimulation of sphingosine kinase (233). Interestingly, whereas PTH inhibits apoptosis in cells overexpressing Gs, an activator of adenylate cyclase, PTH stimulates apoptosis via G protein-coupled receptors in cells April, 2000 BIRTH AND DEATH OF BONE CELLS overexpressing Gq (an activator of JNK and calcium signaling), suggesting that the antiapoptotic effects of PTH are mediated by signals transduced through the Gs pathway (234). Osteocytes in the newly made lamellar cancellous bone in the mice receiving daily PTH injections were closer together and more numerous than those found in the animals receiving vehicle alone (218). The closely spaced, more numerous osteocytes are the predictable consequence of protecting osteoblasts from apoptosis (Fig. 3B). The antiapoptotic effect of PTH on osteoblasts as well as osteocytes has been confirmed in vitro using primary bone cell cultures and established cell lines. Elucidation of this mechanism provides for the first time proof that inhibition of osteoblast apoptosis may represent a novel therapeutic strategy for augmenting bone mass. Be that as it may, several alternative mechanisms, including activation of lining cells, have been proposed, and they may also contribute to the anabolic effect of PTH (235–237). Nonetheless, lining cells cover at least 3 times more surface than osteoblasts. Therefore, conversion of lining cells to boneforming osteoblasts alone would be insufficient to cover the increased cancellous bone area observed in rats and to account for the expanded bone perimeter and the increased osteocyte number and density observed with PTH treatment in mice (218). Daily subcutaneous injections of PTH are safe and effective in the treatment of patients with corticosteroid-induced osteoporosis (230). The elucidation of the importance of osteoblast and osteocyte apoptosis in the mechanism of glucocorticoid-induced osteoporosis, and the elucidation of the importance of preventing apoptosis in the anabolic effects of PTH on bone, readily explain how PTH can be such an effective therapy in this condition. Hence, PTH and perhaps future PTH mimetics represent, for the first time, pathophysiology-based, i.e., rational as opposed to empirical, pharmacotherapies for osteopenias, in particular, those in which osteoblast progenitor formation is suppressed. In any case, future studies to assess the antifracture efficacy of these compounds will be needed before their effectiveness for the management of osteoporosis can be established. B. Bisphosphonates and calcitonin Bisphosphonates, stable analogs of pyrophosphate, and calcitonin are potent inhibitors of bone resorption and effective therapies for the management of osteoporosis and other diseases characterized by bone loss (238, 239). The main mechanism of the antiresorptive actions of these agents is decreased development of osteoclast progenitors, decreased osteoclast recruitment, and promotion of apoptosis of mature osteoclasts leading to a slowing rate of bone remodeling (133, 240 –242). Nonetheless, the antifracture efficacy of these agents is disproportional to their effect on bone mass (243), suggesting an additional effect on bone strength unrelated to effects on bone resorption. Moreover, long-term treatment of human and nonhuman primates with bisphosphonates has been shown to increase wall thickness, an index of increased osteoblast numbers or activity (244 –246), raising the possibility that they may not only inhibit bone resorption, but may 129 also have a positive effect on bone formation. An explanation for this evidence is now provided by studies demonstrating that bisphosphonates such as etidronate, alendronate, pamidronate, olpadronate, or amino-olpadronate (IG9402, a bisphosphonate that lacks antiresorptive activity), as well as calcitonin have antiapoptotic effects on osteoblasts and osteocytes (219). These effects are associated with a rapid increase in the phosphorylated fraction of extracellular regulated signal kinases (ERKs) and are blocked by specific inhibitors of ERK activation. In agreement with the in vitro results, alendronate abolishes the increase in the prevalence of vertebral, cancellous bone osteocyte and osteoblasts apoptosis induced by administration of prednisolone in mice. These findings raise for the first time the possibility that increased survival of osteoblasts and osteocytes may both contribute to the efficacy of bisphosphonates and calcitonin in the management of disease states due to loss of bone. If both “antiresorptive” and “anabolic” agents [e.g., intermittent PTH] prevent osteoblast and osteocyte apoptosis, why is increased formation so much more apparent in the case of the “anabolic” agents? The discussion of physiological bone regeneration in the beginning of this review article provides an answer. Bone formation occurs only on sites of previous osteoclastic bone resorption, i.e., on sites undergoing remodeling. Each remodeling cycle is a transaction that, once consummated, is irrevocable. Therefore, agents with antiapoptotic properties that do not have antiresorptive/antiremodeling properties, i.e., they do not decrease the number of remodeling units, are expected to rebuild more bone and therefore increase the overall bone mass, because of the greater number of profitable transactions. Hence, by decreasing the prevalence of osteoblast apoptosis, agents with pure antiapoptotic properties, such as intermittent PTH, can expand the pool of mature osteoblasts at sites of new bone formation and allow these cells more time to make bone, to a much greater degree than the antiresorptive agents that also slow remodeling. However, in the case of either class of agents, upholding the osteocyte-canalicular network by preventing osteocyte apoptosis, should contribute to antifracture efficacy, over and above that resulting from their effects on bone mass (Fig. 5). Therefore, the distinction between antiresorptive and anabolic agents may be more apparent than real when it comes to antifracture efficacy. C. Novel pharmacotherapeutic strategies Based on the understanding of the role of growth factors on osteoblast development, proliferation, and differentiation, several of them (e.g., GH, the insulin-like growth factors I and II, TGF␤, BMPs, and FGF) have been advocated as potential future therapeutic agents for the management of bone loss (247, 248). However, with the exception of BMPs, which may be of value in local augmentation of bone mass and acceleration of fracture healing, none of them has been shown to be efficacious (let alone safe or convenient and practical) for the management of common metabolic bone disorders such as osteoporosis. The recent elucidation of the mechanism of the anabolic effects of PTH, and specifically the demonstration of increased work output of a cell pop- 130 MANOLAGAS Vol. 21, No. 2 FIG. 5. Hypothetical model of the implications of the effects of antiresorptive (i.e., antiremodeling) vs. non-antiresorptive agents on prolonging the life span of mature osteoblasts and osteocytes. For explanation, please see text. ulation by suppressing apoptosis to augment tissue mass, points to an entirely new avenue for future drug discovery. Indeed, in addition to PTH and PTH mimetics, which by virtue of their peptidic nature carry the inconvenience of daily injections, one can for the first time envisage nonpeptide inhibitors of proapoptotic pathways in osteoblasts and osteocytes as therapeutic agents for osteopenias and especially those in which osteoblast progenitors are low, such as age-related and glucocorticoid-induced osteoporosis. As discussed in Section VIII in this review, estrogen and androgen deficiency increase osteoblast and osteocyte apoptosis in humans, rats, and mice; and these changes have been shown to be reversed by replacement therapy, at least in mice (132, 144, 184, 190). In full agreement with these in vivo observations, 17␤-estradiol inhibits osteoblast and osteocyte apoptosis in vitro. The antiapoptotic effect of 17␤estradiol on osteoblasts and osteocytes require the presence of the estrogen receptor-␣ or -␤ (249). Nonetheless, unlike the classical mechanism of estrogen receptor action that involves direct or indirect interaction with the transcriptional apparatus, the estrogen receptor-dependent antiapoptotic effect of 17␤-estradiol is due to rapid (within 5 min) phosphorylation of ERKs (250). Moreover, the antiapoptotic effect of 17␤-estradiol can be reproduced by 17␣-estradiol, a compound thought of as an inactive analog of 17␤-estradiol, as well as a membrane-impermeable conjugate of 17␤-estradiol with BSA (17␤E2-BSA). Numerous effects of estrogen have been observed over the last few years in a variety of cell types, including osteoblasts, the rapidity of which makes a genomic mechanism of action unlikely (251–256). Many of these rapid actions have been attributed to the ability of estrogen to act at the cell membrane on a membrane-associated estrogen receptor (257–260). The antiapoptotic effects of estrogen on osteoblasts and osteocytes fall into this category of “nongenomic” actions. Based on this, the term “activators of non-genomic estrogen-like signaling” (ANGELS), has been coined for compounds that mimic the nongenomic effects of estrogen, but have reduced classical estrogenic actions (261). A paradigm of such agents is the synthetic compound estratriene-3-ol, which has decreased transcriptional activity as compared with 17␤-estradiol (262, 263), is a potent neuroprotective compound (264 –266), and does exhibit potent antiapoptotic effects on osteoblasts and osteocytes in vitro. In support of the hypothesis that ANGELS can be used as a novel, advantageous mode of therapy for the augmentation of bone mass and/or fracture prevention in diseases characterized by low bone mass and increased fragility, preliminary evidence indicates that estratriene-3-ol increases BMD and bone strength in both estrogen-replete and estrogen-deficient mice (261). In view of this preclinical finding and the evidence that androgen (190), as well as estrogen, have antiapoptotic effects on osteoblasts and osteocytes, one is encouraged to think that estrogenic, androgenic, or even nonsteroidal compounds that can activate antiapoptotic, but not antiremodeling, signals on osteoblasts and osteocytes, are candidates for future osteoporosis treatments that, unlike existing ones that prevent or retard bone loss, may augment bone mass. XI. Summary and Conclusions In 1995, it was proposed that “changes in the numbers of bone cells, rather than changes in the activity of individual cells, form the pathogenetic basis of osteoporosis”; and that “excessive osteoclastogenesis and inadequate osteoblastogenesis are responsible for the mismatch between the formation and resorption of bone in postmenopausal and agerelated osteopenia” (21). Since then, this paradigm shift of thinking has led to important new discoveries that, along with several other independent breakthroughs, refine the concept of “cell number” and broaden its relevance to the physiology and pathophysiology of bone at large. Moreover, these discoveries provide a new landscape for critical reevaluation of our current thinking about therapeutic strategies for bone diseases. Indeed, it is now clear that bone cells must be continually replaced, and the number present depends not only on their birth rate, which reflects the frequency of cell division of the appropriate precursor cell, but also on the life span, which most likely reflects the timing of death by apoptosis. The process of replacement of osteoblasts and osteoclasts is tightly coordinated and orchestrated at the April, 2000 BIRTH AND DEATH OF BONE CELLS early progenitor level. Changes in the birth rate and/or apoptosis of bone cells may account for previously unexplained bone diseases, such as the osteoporosis caused by sex steroid deficiency, old age, and glucocorticoid excess. Moreover, attenuation of the rate of apoptosis of osteoblastic cells may be a key mechanism for the effects of anabolic agents, such as PTH. Proof of the principle that the work performed by a cell population can be increased by suppression of apoptosis provides clues for the development of novel pharmacotherapeutic strategies for pathological conditions such as osteoporosis in which tissue mass diminution has compromised functional integrity. Nevertheless, changes in cell birth and death, as well as other mechanisms including changes in bone cell activity, need to be investigated in humans more extensively before definitive conclusions on the pathogenesis of the various causes of bone loss and the development of osteoporosis can be reached. Acknowledgments The author is grateful to A. Michael Parfitt, Robert L. Jilka, Robert S. Weinstein, Teresita Bellido, Charles A. O’Brien, Robert S. Reis, Paula Roberson, Beata Lecka-Czernik, Etsuko Abe, Donald L. Bodenner, and Stavroula Kousteni for many insightful discussions, sharing of ideas, and contribution of research findings during the preparation of the manuscript; and to Tonya Smith for secretarial assistance. References 1. Riggs BL, Melton III LJ 1986 Involutional osteoporosis. N Engl J Med 314:1676 –1686 2. Lindsay R 1988 Sex steroids in the pathogenesis and prevention of osteoporosis. In: Riggs BL (ed) Osteoporosis: Etiology, Diagnosis and Management. Raven Press, New York, pp 333–358 3. Melton III LJ, Atkinson EJ, O’Connor MK, O’Fallon WM, Riggs BL 1998 Bone density and fracture risk in men. J Bone Miner Res 13:1915–1923 4. Orwoll ES, Klein RF 1995 Osteoporosis in men. Endocr Rev 16: 87–116 5. Reid IR 1998 Glucocorticoid effects on bone. J Clin Endocrinol Metab 83:1860 –1862 6. Cummings SR, Nevitt MC, Browner WS, Stone K, Fox KM, Ensrud KE, Cauley J, Black D, Vogt TM 1995 Risk factors for hip fracture in white women. Study of Osteoporotic Fractures Research Group. N Engl J Med 332:767–773 7. Riggs BL, Melton III LJ 1983 Evidence for two distinct syndromes of involutional osteoporosis. Am J Med 75:899 –901 8. Parfitt AM 1992 The two-stage concept of bone loss revisited. Triangle 31:99 –110 9. Silverberg SJ, Fitzpatrick LA, Bilezikian JP 1995 Hyperparathyroidism. In: Becker KL (ed) Principles and Practice of Endocrinology and Metabolism. JB Lippincott, Philadelphia, pp 512–519 10. Parfitt AM 1996 Skeletal heterogeneity and the purposes of bone remodeling: Implications for the understanding of osteoporosis. In: Marcus R, Feldman D, Kelsey J (eds) Osteoporosis. Academic Press, San Diego, CA, pp 315–329 11. Frost HM 1973 Bone Remodeling and Its Relationship to Metabolic Bone Disease. Charles C. Thomas, Springfield, MA 12. Parfitt AM, Rauch F, Travers R, Glorieux F 1999 A new model of cancellous bone growth. J Bone Miner Res S206 (Abstract) 13. Parfitt AM 1994 Osteonal and hemi-osteonal remodeling: the spatial and temporal framework for signal traffic in adult human bone. J Cell Biochem 55:273–286 14. Friedenstein AJ, Chailakhjan RK, Latsinik NV, Panasyuk AF, Keiliss-Borok IV 1974 Stromal cells responsible for transferring the microenvironment of the hemopoietic tissues. Cloning in vitro and retransplantation in vivo. Transplantation 17:331–340 131 15. Owen M 1985 Lineage of osteogenic cells and their relationship to the stromal system. In: Peck WA (ed) Bone and Mineral Research. Elsevier, Amsterdam, vol 3:1–25 16. Triffitt JT 1996 The stem cell of the osteoblast. In: Bilezikian JP, Raisz LG, Rodan GA (eds) Principles of Bone Biology. Academic Press, San Diego, CA, pp 39 –50 17. Roodman GD 1996 Advances in bone biology: the osteoclast. Endocr Rev 17:308 –332 18. Suda T, Takahashi N, Martin TJ 1992 Modulation of osteoclast differentiation. Endocr Rev 13:66 – 80 19. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S, Marshak DR 1999 Multilineage potential of adult human mesenchymal stem cells. Science 284:143–147 20. Schor AM, Canfield AE, Sutton AB, Arciniegas E, Allen TD 1995 Pericyte differentiation. Clin Orthop 313:81–91 21. Manolagas SC, Jilka RL 1995 Bone marrow, cytokines, and bone remodeling— emerging insights into the pathophysiology of osteoporosis. N Engl J Med 332:305–311 22. Rosen V, Cox K, Hattersley G 1996 Bone morphogenetic proteins. In: Bilezikian JP, Raisz LG, Rodan GA (eds) Principles of Bone Biology. Academic Press, San Diego, CA, pp 661– 671 23. Abe E, Yamamoto M, Taguchi Y, Lecka-Czernik B, Economides AN, Stahl N, Jilka RL, Manolagas SC 2000 Essential requirement of BMPs 2/4 for both osteoblast and osteoclast formation in bone marrow cultures from adult mice: antagonism by noggin. J Bone Miner Res 15:663– 673 24. Ducy P, Zhang R, Geoffroy V, Ridall AL, Karsenty G 1997 Osf2/ Cbfa1: a transcriptional activator of osteoblast differentiation. Cell 89:747–754 25. Gao YH, Shinki T, Yuasa T, Kataoka-Enomoto H, Komori T, Suda T, Yamaguchi A 1998 Potential role of Cbfa1, an essential transcriptional factor for osteoblast differentiation, in osteoclastogenesis: regulation of mRNA expression of osteoclast differentiation factor (ODF). Biochem Biophys Res Commun 252:697–702 26. Komori T, Yagi H, Nomura S, Yamaguchi A, Sasaki K, Deguchi K, Shimizu Y, Bronson RT, Gao Y-H, Inada M, Sato M, Okamoto R, Kitamura Y, Yoshiki S, Kishimoto T 1997 Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts. Cell 89:755–764 27. Ryoo HM, Hoffmann HM, Beumer T, Frenkel B, Towler DA, Stein GS, Stein JL, Van Wijnen AJ, Lian JB 1997 Stage-specific expression of Dlx-5 during osteoblast differentiation: involvement in regulation of osteocalcin gene expression. Mol Endocrinol 11: 1681–1694 28. Newberry EP, Latifi T, Towler DA 1998 Reciprocal regulation of osteocalcin transcription by the homeodomain proteins Msx2 and Dlx5. Biochemistry 37:16360 –16368 29. Miyama K, Yamada G, Yamamoto TS, Takagi C, Miyado K, Sakai M, Ueno N, Shibuya H 1999 A BMP-inducible gene, dlx5, regulates osteoblast differentiation and mesoderm induction. Dev Biol 208: 123–133 30. Canalis E 1996 Skeletal Growth Factors. In: Marcus R, Feldman D, Kelsey J (eds) Osteoporosis. Academic Press, San Diego, CA, pp 261–279 31. Bonewald LF, Dallas SL 1994 Role of active and latent transforming growth factor ␤ in bone formation. J Cell Biochem 55:350 –357 32. Piccolo S, Sasai Y, Lu B, De Robertis EM 1996 Dorsoventral patterning in Xenopus: inhibition of ventral signals by direct binding of chordin to BMP-4. Cell 86:589 –598 33. Holley SA, Neul JL, Attisano L, Wrana JL, Sasai Y, O’Connor MB, De Robertis EM, Ferguson EL 1996 The Xenopus dorsalizing factor noggin ventralizes Drosophila embryos by preventing DPP from activating its receptor. Cell 86:607– 617 34. Valenzuela DM, Economides AN, Rojas E, Lamb TM, Nunez L, Jones P, Ip NY, Espinosa III R, Brannan CI, Gilbert DJ, Copeland NG, Jenkins NA, Beau MML, Harland RM, Yancopoulos GD 1995 Identification of mammalian noggin and its expression in the adult nervous system. J Neurosci 15:6077– 6084 35. Bouwmeester T, Kim S, Sasai Y, Lu B, De Robertis EM 1996 Cerberus is a head-inducing secreted factor expressed in the anterior endoderm of Spemann’s organizer. Nature 382:595– 601 36. Zimmerman LB, De Jesús-Escobar JM, Harland RM 1996 The 132 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. MANOLAGAS Spemann organizer signal noggin binds and inactivates bone morphogenetic protein 4. Cell 86:599 – 606 Re’em-Kalma Y, Lamb T, Frank D 1995 Competition between noggin and bone morphogenic protein 4 activities may regulate dorsalization during Xenopus development. Proc Natl Acad Sci USA 92:12141–12145 Manolagas SC, Jilka RL, Bellido T, O’Brien CA, Parfitt AM 1996 Interleukin-6-type cytokines and their receptors. In: Bilezikian JP, Raisz LG, Rodan GA (eds) Principles of Bone Biology. Academic Press, San Diego, CA, pp 701–713 Udagawa N, Horwood NJ, Elliott J, Mackay A, Owens J, Okamura H, Kurimoto M, Chambers TJ, Martin TJ, Gillespie MT 1997 Interleukin-18 (interferon-␥-inducing factor) is produced by osteoblasts and acts via granulocyte/macrophage colony-stimulating factor and not via interferon-␥ to inhibit osteoclast formation. J Exp Med 185:1005–1012 Manolagas SC 1998 The role of IL-6 type cytokines and their receptors in bone. Ann NY Acad Sci 840:194 –204 Girasole G, Jilka RL, Passeri G, Boswell S, Boder G, Williams DC, Manolagas SC 1992 17␤-Estradiol inhibits interleukin-6 production by bone marrow-derived stromal cells and osteoblasts in vitro: a potential mechanism for the antiosteoporotic effect of estrogens. J Clin Invest 89:883– 891 Franchimont N, Canalis E 1995 Platelet-derived growth factor stimulates the synthesis of interleukin-6 in cells of the osteoblast lineage. Endocrinology 136:5469 –5475 Franchimont N, Durant D, Rydziel S, Canalis E 1999 Plateletderived growth factor induces interleukin-6 transcription in osteoblasts through the activator protein-1 complex and activating transcription factor-2. J Biol Chem 274:6783– 6789 Stahl N, Yancopoulos GD 1993 The alphas, betas, and kinases of cytokine receptor complexes. Cell 74:587–590 Stahl N, Boulton TG, Farruggella T, Ip NY, Davis S, Witthuhn BA, Quelle FW, Silvennoinen O, Barbieri G, Pellegrini S, Ihle JN, Yancopoulos GD 1994 Association and activation of Jak-Tyk kinases by CNTF-LIF- OSM-IL-6 ␤ receptor components. Science 263:92–95 Boulton TG, Stahl N, Yancopoulos D 1994 Ciliary neurotrophic factor/leukemia inhibitory factor/interleukin 6/oncostatin M family of cytokines induces tyrosine phosphorylation of a common set of proteins overlapping those induced by other cytokines and growth factors. J Biol Chem 269:11648 –11655 Darnell Jr JE 1997 STATs and gene regulation. Science 277:1630 – 1635 Narazaki M, Yasukawa K, Saito T, Ohsugi Y, Fukui H, Koishihara Y, Yancopoulos GD, Taga T, Kishimoto T 1993 Soluble forms of the interleukin-6 signal-transducing receptor component gp130 in human serum possessing a potential to inhibit signals through membrane-anchored gp130. Blood 82:1120 –1126 Udagawa N, Takahashi N, Katagiri T, Tamura T, Wada S, Findlay DM, Martin TJ, Hirota H, Tada T, Kishimoto T, Suda T 1995 Interleukin (IL)-6 induction of osteoclast differentiation depends on IL-6 receptors expressed on osteoblastic cells but not on osteoclast progenitors. J Exp Med 182:1461–1468 O’Brien CA, Gubrij I, Lin S-C, Saylors RL, Manolagas SC 1999 STAT3 activation in stromal/osteoblastic cells is required for induction of the receptor activator of NF-kB ligand and stimulation of osteoclastogenesis by gp130-utilizing cytokines or interleukin-1 but not 1,25-dihydroxyvitamin D3 or parathyroid hormone. J Biol Chem 274:19301–19308 Jilka RL, Hangoc G, Girasole G, Passeri G, Williams DC, Abrams JS, Boyce B, Broxmeyer H, Manolagas SC 1992 Increased osteoclast development after estrogen loss: mediation by interleukin-6. Science 257:88 –91 Poli V, Balena R, Fattori E, Markatos A, Yamamoto A, Tanaka H, Ciliberto G, Rodan GA, Costantini F 1994 Interleukin-6 deficient mice are protected from bone loss caused by estrogen depletion. EMBO J 13:1189 –1196 Bellido T, Stahl N, Farruggella TJ, Borba V, Yancopoulos GD, Manolagas SC 1996 Detection of receptors for interleukin-6, interleukin-11, leukemia inhibitory factor, oncostatin M, and ciliary neurotrophic factor in bone marrow stromal/osteoblastic cells. J Clin Invest 97:431– 437 Vol. 21, No. 2 54. Bellido T, Borba VZ, Roberson P, Manolagas SC 1997 Activation of the Janus kinase/STAT (signal transducer and activator of transcription) signal transduction pathway by interleukin-6-type cytokines promotes osteoblast differentiation. Endocrinology 138:3666 –3676 55. Taguchi Y, Yamamoto M, Yamate T, Lin SC, Mocharla H, DeTogni P, Nakayama N, Boyce BF, Abe E, Manolagas SC 1998 Interleukin-6-type cytokines stimulate mesenchymal progenitor differentiation toward the osteoblastic lineage. Proc Assoc Am Physicians 110:559 –574 56. Malik N, Haugen HS, Modrell B, Shoyab M, Clegg CH 1995 Developmental abnormalities in mice transgenic for bovine oncostatin M. Mol Cell Biol 15:2349 –2358 57. Ware CB, Horowitz MC, Renshaw BR, Hunt JS, Liggitt D, Koblar SA, Gliniak BC, McKenna HJ, Papayannopoulou T, Thoma B 1995 Targeted disruption of the low-affinity leukemia inhibitory factor receptor gene causes placental, skeletal, neural and metabolic defects and results in perinatal death. Development 121:1283–1299 58. Bonewald L 1996 Transforming growth factor-␤. In: Bilezikian JP, Raisz L, Rodan GA (eds) Principles of Bone Biology. Academic Press, San Diego, CA, pp 647– 659 59. Kulkarni AB, Karlsson S 1993 Transforming growth factor-␤1 knockout mice: a mutation in one cytokine gene causes a dramatic inflammatory disease. Am J Pathol 143:3–9 60. Suda T, Udagawa N, Takahashi N 1996 Cells of bone: osteoclast generation. In: Bilezikian JP, Raisz L, Rodan GA (eds) Principals of Bone Biology. Academic Press, San Diego, CA, pp 87–102 61. Rico H, Hernandez ER, Revilla M, Gómez-Castresana F 1992 Salmon calcitonin reduces vertebral fracture rate in postmenopausal crush fracture syndrome. Bone Miner 16:131–138 62. Ikegame M, Rakopoulos M, Zhou H, Houssami S, Martin TJ, Moseley JM, Findlay DM 1995 Calcitonin receptor isoforms in mouse and rat osteoclasts. J Bone Miner Res 10:59 – 65 63. Martin TJ, Udagawa N 1998 Hormonal regulation of osteoclast function. Trends Endocrinol Metab 9:6 –12 64. Girasole G, Passeri G, Jilka RL, Manolagas SC 1994 Interleukin11: a new cytokine critical for osteoclast development. J Clin Invest 93:1516 –1524 65. Greenfield EM, Shaw SM, Gornik SA, Banks MA 1995 Adenyl cyclase and interleukin 6 are downstream effectors of parathyroid hormone resulting in stimulation of bone resorption. J Clin Invest 96:1238 –1244 66. Grey A, Mitnick MA, Masiukiewicz U, Sun BH, Rudikoff S, Jilka RL, Manolagas SC, Insogna K 1999 A role of interleukin-6 in parathyroid hormone-induced bone resorption in vivo. Endocrinology 140:4683– 4690 67. Bellido T, Jilka RL, Boyce BF, Girasole G, Broxmeyer H, Dalrymple SA, Murray R, Manolagas SC 1995 Regulation of interleukin-6, osteoclastogenesis and bone mass by androgens: the role of the androgen receptor. J Clin Invest 95:2886 –2895 68. Lakatos P, Foldes J, Horvath C, Kiss L, Tatrai A, Takacs I, Tarjan G, Stern PH 1997 Serum interleukin-6 and bone metabolism in patients with thyroid function disorders. J Clin Endocrinol Metab 82:78 – 81 69. Ray A, LaForge KS, Sehgal PB 1990 On the mechanism for efficient repression of the interleukin-6 promoter by glucocorticoids: Enhancer, TATA box, and RNA start site (Inr motif) occlusion. Mol Cell Biol 10:5736 –5746 70. Grzesik WJ, Gehron Robey P 1994 Bone matrix RGD glycoproteins: immunolocalization and interaction with human primary osteoblastic bone cells in vitro. J Bone Miner Res 9:487– 496 71. Rodan GA 1995 Osteopontin overview. Ann NY Acad Sci 760:1–5 72. Horton MA, Townsend P, Nesbitt S 1996 Cell surface attachment molecules in bone. In: Bilezikian JP, Raisz LG, Rodan GA (eds) Principles of Bone Biology. Academic Press, San Diego, CA, pp 217–230 73. Yamate T, Mocharla H, Taguchi Y, Igietseme JU, Manolagas SC, Abe E 1997 Osteopontin expression by osteoclast and osteoblast progenitors in the murine bone marrow: demonstration of its requirement for osteoclastogenesis and its increase after ovariectomy. Endocrinology 138:3047–3055 74. Takeuchi Y, Suzawa M, Kikuchi T, Nishida E, Fujita T, Matsumoto T 1997 Differentiation and transforming growth factor-␤ re- April, 2000 75. 76. 77. 78. 79. 80. 81. 82. 83. 84. 85. 86. 87. 88. 89. 90. 91. 92. 93. 94. 95. BIRTH AND DEATH OF BONE CELLS ceptor down-regulation by collagen-␣2␤1 integrin interaction is mediated by focal adhesion kinase and its downstream signals in murine osteoblastic cells. J Biol Chem 272:29309 –29316 Zhao W, Krane S 1998 Inability of collagenase to cleave type I collagen in vivo is associated with osteocyte apoptosis. Bone 23:S185 (Abstract) Globus RK, Doty SB, Lull JC, Holmuhamedov E, Humphries MJ, Damsky CH 1998 Fibronectin is a survival factor for differentiated osteoblasts. J Cell Sci 111:1385–1393 Xiao G, Wang D, Benson MD, Karsenty G, Franceschi RT 1998 Role of the alpha2-integrin in osteoblast-specific gene expression and activation of the osf2 transcription factor. J Biol Chem 273: 32988 –32994 Ross FP, Chappel J, Alvarez JI, Sander D, Butler WT, FarachCarson MC, Mintz KA, Robey PG, Teitelbaum SL, Cheresh DA 1993 Interactions between the bone matrix proteins osteopontin and bone sialoprotein and the osteoclast integrin ␣v␤3 potentiate bone resorption. J Biol Chem 268:9901–9907 Owen M, Friedenstein AJ 1988 Stromal stem cells: marrowderived osteogenic precursors. Ciba Found Symp 136:42– 60 Westen H, Bainton DF 1979 Association of alkaline-phosphatasepositive reticulum cell in bone marrow with granulocyte precursors. J Exp Med 150:919 –937 Weiss L 1988 Bone marrow. In: Weiss L (ed) Cell and Tissue Biology. Urban and Schwarzenberg, Baltimore, pp 469 – 478 Bianco P, Costantini M, Dearden LC, Bonucci E 1988 Alkaline phosphatase positive precursors of adipocytes in the human bone marrow. Br J Haematol 68:401– 403 Rouleau MF, Mitchell J, Goltzman D 1988 In vivo distribution of parathyroid hormone receptors in bone: evidence that a predominant osseous target cell is not the mature osteoblast. Endocrinology 123:187–192 Bianco P, Bradbeer JN, Riminucci M, Boyde A 1993 Marrow stromal (Western-Bainton) cells: identification, morphometry, confocal imaging and changes in disease. Bone 14:315–320 Bennett JH, Joyner CJ, Triffitt JT, Owen ME 1991 Adipocytic cells cultured from marrow have osteogenic potential. J Cell Sci 99: 131–139 Diascro Jr DD, Vogel RL, Johnson TE, Witherup KM, Pitzenberger SM, Rutledge SJ, Prescott DJ, Rodan GA, Schmidt A 1998 High fatty acid content in rabbit serum is responsible for the differentiation of osteoblasts into adipocyte-like cells. J Bone Miner Res 13:96 –106 Yeh WC, Cao Z, Classon M, McKnight SL 1995 Cascade regulation of terminal adipocyte differentiation by three members of the C/EBP family of leucine zipper proteins. Genes Dev 9:168 –181 Spiegelman BM, Flier JS 1996 Adipogenesis and obesity: rounding out the big picture. Cell 87:377–389 Wu Z, Bucher NL, Farmer SR 1996 Induction of peroxisome proliferator-activated receptor ␥ during the conversion of 3T3 fibroblasts into adipocytes is mediated by C/EBP␤, C/EBP␦, and glucocorticoids. Mol Cell Biol 16:4128 – 4136 Shao D, Lazar MA 1997 Peroxisome proliferator activated receptor ␥, CCAAT/enhancer-binding protein ␣, and cell cycle status regulate the commitment to adipocyte differentiation. J Biol Chem 272:21473–21478 Samuelsson L, Stromberg K, Vikman K, Bjursell G, Enerback S 1991 The CCAAT/enhancer binding protein and its role in adipocyte differentiation: evidence for direct involvement in terminal adipocyte development. EMBO J 10:3787–3793 Freytag SO, Paielli DL, Gilbert JD 1994 Ectopic expression of the CCAAT/enhancer-binding protein ␣ promotes the adipogenic program in a variety of mouse fibroblastic cells. Genes Dev 8:1654 – 1663 Tontonoz P, Hu E, Spiegelman BM 1994 Stimulation of adipogenesis in fibroblasts by PPAR ␥ 2, a lipid-activated transcription factor. Cell 79:1147–1156 Lecka-Czernik B, Gubrij I, Moerman E, Kajkenova O, Lipschitz D, Manolagas S, Jilka RL 1999 Inhibition of Osf2/Cbfa1 expression and terminal osteoblast differentiation by PPAR ␥2. J Cell Biochem 74:357–371 Hu E, Tontonoz P, Spiegelman BM 1995 Transdifferentiation of 96. 97. 98. 99. 100. 101. 102. 103. 104. 105. 106. 107. 108. 109. 110. 111. 133 myoblasts by the adipogenic transcription factors PPAR ␥ and C/EBP ␣. Proc Natl Acad Sci USA 92:9856 –9860 Chen D, Ji X, Harris MA, Feng JQ, Karsenty G, Celeste AJ, Rosen V, Mundy GR, Harris SE 1998 Differential roles for bone morphogenetic protein (BMP) receptor type IB and IA in differentiation and specification of mesenchymal precursor cells to osteoblast and adipocyte lineages. J Cell Biol 142:295–305 Ott SM 1996 Theoretical and methodological approach. In: Bilezikian JP, Raisz LG, Rodan GA (eds) Principles of Bone Biology. Academic Press, San Diego, CA pp 231–241 Mohan S, Baylink DJ 1991 The role of IGF-1 in the coupling of bone formation to resorption. In: Spencer EM (ed) Modern Concepts of Insulin-Like Growth Factors. Elsevier, New York, pp 169 –184 Bonewald LF, Wakefield L, Oreffo ROC, Escobedo A, Twardzik DR, Mundy GR 1991 Latent forms of transforming growth factor-␤ (TGF␤) derived from bone cultures: identification of a naturally occurring 100-kDa complex with similarity to recombinant latent TGF␤. Mol Endocrinol 5:741–751 Rodan GA 1996 Coupling of bone resorption and formation during bone remodeling. In: Marcus R, Feldman D, Kelsey J (eds) Osteoporosis. Academic Press, San Diego, CA, pp 289 –299 Corral DA, Amling M, Priemel M, Loyer E, Fuchs S, Ducy P, Baron R, Karsenty G 1998 Dissociation between bone resorption and bone formation in osteopenic transgenic mice. Proc Natl Acad Sci USA 95:13835–13840 Udagawa N, Takahashi N, Akatsu T, Sasaki T, Yamaguchi A, Kodama H, Martin TJ, Suda T 1989 The bone marrow-derived stromal cell lines MC3T3–G2/PA6 and ST2 support osteoclast-like cell differentiation in cocultures with mouse spleen cells. Endocrinology 125:1805–1813 Dorheim MA, Sullivan M, Dandapani V, Wu X, Hudson J, Segarini PR, Rosen DM, Aulthouse AL, Gimble JM 1993 Osteoblastic gene expression during adipogenesis in hematopoietic supporting murine bone marrow stromal cells. J Cell Physiol 154:317–328 Kelly KA, Tanaka S, Baron R, Gimble JM 1998 Murine bone marrow stromally derived BMS2 adipocytes support differentiation and function of osteoclast-like cells in vitro. Endocrinology 139:2092–2101 Jilka RL, Weinstein RS, Takahashi K, Parfitt AM, Manolagas SC 1996 Linkage of decreased bone mass with impaired osteoblastogenesis in a murine model of accelerated senescence. J Clin Invest 97:1732–1740 Weinstein RS, Jilka RL, Parfitt AM, Manolagas SC 1997 The effects of androgen deficiency on murine bone remodeling and bone mineral density are mediated via cells of the osteoblastic lineage. Endocrinology 138:4013– 4021 Suda T, Takahashi N, Udagawa N, Jimi E, Gillespie MT, Martin TJ 1999 Modulation of osteoclast differentiation and function by the new members of the tumor necrosis factor receptor and ligand families. Endocr Rev 20:345–357 Anderson DM, Maraskovsky E, Billingsley WL, Dougall WC, Tometsko ME, Roux ER, Teepe MC, DuBose RF, Cosman D, Galibert L 1997 A homologue of the TNF receptor and its ligand enhance T-cell growth and dendritic-cell function. Nature 390: 175–179 Lacey DL, Timms E, Tan HL, Kelley MJ, Dunstan CR, Burgess T, Elliott R, Colombero A, Elliott G, Scully S, Hsu H, Sullivan J, Hawkins N, Davy E, Capparelli C, Eli A, Qian YX, Kaufman S, Sarosi I, Shalhoub V, Senaldi G, Guo J, Delaney J, Boyle WJ 1998 Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell 93:165–176 Yasuda H, Shima N, Nakagawa N, Yamaguchi K, Kinoshaki M, Mochizuki S, Tomoyasu A, Yano K, Goto M, Murakami A, Tsuda E, Morinaga T, Higashio K, Udagawa N, Takahashi N, Suda T 1998 Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL. Proc Natl Acad Sci USA 95:3597–3602 Simonet WS, Lacey DL, Dunstan CR, Kelley M, Chang MS, Luthy R, Nguyen HQ, Wooden S, Bennett L, Boone T, Shimamoto G, DeRose M, Elliott R, Colombero A, Tan HL, Trail G, Sullivan J, Davy E, Bucay N, Renshaw-Gegg L, Hughes TM, Hill D, Pattison W, Campbell P, Boyle WJ 1997 Osteoprotegerin: a novel secreted protein involved in the regulation of bone density. Cell 89:309 –319 134 MANOLAGAS 112. Bucay N, Sarosi I, Dunstan CR, Morony S, Tarpley J, Capparelli C, Scully S, Tan HL, Xu WL, Lacey DL, Boyle WJ, Simonet WS 1998 Osteoprotegerin-deficient mice develop early onset osteoporosis and arterial calcification. Genes Dev 12:1260 –1268 113. Kong YY, Yoshida H, Sarosi I, Tan HL, Timms E, Capparelli C, Morony S, Oliveira-dos-Santos AJ, Van G, Itie A, Khoo W, Wakeham A, Dunstan CR, Lacey DL, Mak TW, Boyle WJ, Penninger JM 1999 OPGL is a key regulator of osteoclastogenesis, lymphocyte development and lymph-node organogenesis. Nature 397:315–323 114. Degli-Esposti M 1999 To die or not to die–the quest of the TRAIL receptors. J Leukoc Biol 65:535–542 115. Emery JG, McDonnell P, Burke MB, Deen KC, Lyn S, Silverman C, Dul E, Appelbaum ER, Eichman C, DiPrinzio R, Dodds RA, James IE, Rosenberg M, Lee JC, Young PR 1998 Osteoprotegerin is a receptor for the cytotoxic ligand TRAIL. J Biol Chem 273: 14363–14367 116. O’Brien CA, Farrar N, Manolagas SC 1998 Identification of an OSF-2 binding site in the murine RANKL/OPGL gene promoter: a potential link between osteoblastogenesis and osteoclastogenesis. Bone 23:S149 (Abstract) 117. Manolagas SC 1999 Cell number vs. cell vigor–what really matters to a regenerating skeleton? Endocrinology 140:4377– 4381 118. Manolagas SC, Weinstein RS 1999 New developments in the pathogenesis and treatment of steroid-induced osteoporosis. J Bone Miner Res 14:1061–1066 119. Reddy SV, Roodman GD 1998 Control of osteoclast differentiation. Crit Rev Eukaryot Gene Expr 8:1–17 120. Robey PG, Boskey AL 1995 The biochemistry of bone. In: Marcus R, Feldman D, Bilezikian JP, Kelsey J (eds) Osteoporosis. Academic Press, New York, pp 95–183 121. Ducy P, Desbois C, Boyce B, Pinero G, Story B, Dunstan C, Smith E, Bonadio J, Goldstein S, Gundberg C, Bradley A, Karsenty G 1996 Increased bone formation in osteocalcin-deficient mice. Nature 382:448 – 452 122. Delany A, Amling M, Priemel M, Delling G, Howe C, Baron R, Canalis E 1998 Osteonectin-null mice develop severe osteopenia. Bone 23:S199 (Abstract) 123. Boskey AL 1998 Biomineralization: conflicts, challenges, and opportunities. J Cell Biochem Suppl 30 –31:83–91 124. Boskey AL 1996 Matrix proteins and mineralization: an overview. Connect Tissue Res 35:357–363 125. Whyte MP 1994 Hypophosphatasia and the role of alkaline phosphatase in skeletal mineralization. Endocr Rev 15:439 – 461 126. Marotti G, Cane V, Palazzini S, Palumbo C 1990 Structure-function relationships in the osteocyte. Ital J Miner Electro Metab 4:93–106 127. Nijweide PJ, Burger EH, Klein-Nulend J, van der Pluijm G 1996 The Osteocyte. In: Bilezikian JP, Raisz LG, Rodan GA (eds) Principles of Bone Biology. Academic Press, San Diego, CA, pp 115–126 128. Marotti G 1996 The structure of bone tissues and the cellular control of their deposition. Ital J Anat Embryol 101:25–79 129. Aarden EM, Burger EH, Nijweide PJ 1994 Function of osteocytes in bone. J Cell Biochem 55:287–299 130. Weinstein RS, Jilka RL, Parfitt AM, Manolagas SC 1998 Inhibition of osteoblastogenesis and promotion of apoptosis of osteoblasts and osteocytes by glucocorticoids: potential mechanisms of their deleterious effects on bone. J Clin Invest 102:274 –282 131. Tomkinson A, Gevers EF, Wit JM, Reeve J, Noble BS 1998 The role of estrogen in the control of rat osteocyte apoptosis. J Bone Miner Res 13:1243–1250 132. Tomkinson A, Reeve J, Shaw RW, Noble BS 1997 The death of osteocytes via apoptosis accompanies estrogen withdrawal in human bone. J Clin Endocrinol Metab 82:3128 –3135 133. Parfitt AM, Mundy GR, Roodman GD, Hughes DE, Boyce BF 1996 A new model for the regulation of bone resorption, with particular reference to the effects of bisphosphonates. J Bone Miner Res 11:150 –159 134. Bossard MJ, Tomaszek TA, Thompson SK, Amegadzie BY, Hanning CR, Jones C, Kurdyla JT, McNulty DE, Drake FH, Gowen M, Levy MA 1996 Proteolytic activity of human osteoclast cathepsin K— expression, purification, activation, and substrate identification. J Biol Chem 271:12517–12524 Vol. 21, No. 2 135. Nesbitt SA, Horton MA 1997 Trafficking of matrix collagens through bone-resorbing osteoclasts. Science 276:266 –269 136. Salo J, Lehenkari P, Mulari M, Metsikkö K, Väänänen HK 1997 Removal of osteoclast bone resorption products by transcytosis. Science 276:270 –273 137. Udagawa N, Takahashi N, Akatsu T, Tanaka H, Sasaki T, Nishihara T, Koga T, Martin TJ, Suda T 1990 Origin of osteoclasts: mature monocytes and macrophages are capable of differentiating into osteoclasts under a suitable microenvironment prepared by bone marrow-derived stromal cells. Proc Natl Acad Sci USA 87: 7260 –7264 138. Hayman AR, Jones SJ, Boyde A, Foster D, Colledge WH, Carlton MB, Evans MJ, Cox TM 1996 Mice lacking tartrate-resistant acid phosphatase (Acp 5) have disrupted endochondral ossification and mild osteopetrosis. Development 122:3151–3162 139. Hughes DE, Dai A, Tiffee JC, Li HH, Mundy GR, Boyce BF 1996 Estrogen promotes apoptosis of murine osteoclasts mediated by TGF-␤. Nat Med 2:1132–1136 140. Jilka RL, Weinstein RS, Bellido T, Parfitt AM, Manolagas SC 1998 Osteoblast programmed cell death (apoptosis): modulation by growth factors and cytokines. J Bone Miner Res 13:793– 802 141. Steller H 1995 Mechanisms and genes of cellular suicide. Science 267:1445–1449 142. Parfitt AM 1990 Bone-forming cells in clinical conditions. In: Hall BK (ed) Bone. The Osteoblast and Osteocyte. Telford Press and CRC Press, Boca Raton, FL, vol 1:351– 429 143. Frost HM 1960 In vivo osteocyte death. J Bone Joint Surg [Am] 42:138 –143 144. Noble BS, Stevens H, Loveridge N, Reeve J 1997 Identification of apoptotic changes in osteocytes in normal and pathological human bone. Bone 20:273–282 145. Hughes DE, Boyce BF 1997 Apoptosis in bone physiology and disease. Mol Pathol 50:132–137 146. Bellido T, O’Brien CA, Roberson PK, Manolagas SC 1998 Transcriptional activation of the p21WAF1,CIP1,SDI1 gene by interleukin-6 type cytokines—a prerequisite for their pro-differentiating and anti-apoptotic effects on human osteoblastic cells. J Biol Chem 273:21137–21144 147. Quarles LD, Siddhanti SR 1996 Guanine nucleotide bindingprotein coupled signaling pathway regulation of osteoblast-mediated bone formation (editorial). J Bone Miner Res 11:1375–1383 148. Teitelbaum SL, Tondravi MM, Ross FP 1996 Osteoclast biology. In: Marcus R, Feldman D, Kelsey J (eds) Osteoporosis. Academic Press, San Diego, CA, pp 61–94 149. Suda T, Nakamura I, Jimi E, Takahashi N 1997 Regulation of osteoclast function. J Bone Miner Res 12:869 – 879 150. Lian JB, Stein GS, Stein JL, Van Wijnen AJ 1998 Osteocalcin gene promoter: unlocking the secrets for regulation of osteoblast growth and differentiation. J Cell Biochem Suppl 30 –31:62–72 151. Greenfield EM, Bi Y, Miyauchi A 1999 Regulation of osteoclast activity. Life Sci 65:1087–1102 152. Lajtha L 1983 Stem cell concepts. In: Potten CS (ed) Stem Cells— Their Identification and Characterisation. Churchill Livingston, New York, pp 1–11 153. Chambers TJ, Magnus CJ 1982 Calcitonin alters behavior of isolated osteoclasts. J Pathol 136:27–39 154. Fuller K, Wong B, Fox S, Choi Y, Chambers TJ 1998 TRANCE is necessary and sufficient for osteoblast-mediated activation of bone resorption in osteoclasts. J Exp Med 188:997–1001 155. Wani MR, Fuller K, Kim NS, Choi Y, Chambers T 1999 Prostaglandin E2 cooperates with TRANCE in osteoclast induction from hemopoietic precursors: synergistic activation of differentiation, cell spreading, and fusion. Endocrinology 140:1927–1935 156. Reid IR, Nicholson GC, Weinstein RS, Hosking DJ, Cundy T, Kotowicz MA, Murphy WAJ, Yeap S, Dufresne S, Lombardi A, Musliner TA, Thompson DE, Yates AJ 1996 Biochemical and radiologic improvement in Paget’s disease of bone treated with alendronate: a randomized, placebo-controlled trial. Am J Med 101: 341–348 157. Burgess TL, Qian YX, Kaufman S, Ring BD, Van G, Capparelli C, Kelley M, Hsu HL, Boyle WJ, Dunstan CR, Hu S, Lacey DL 1999 The ligand for osteoprotegerin (OPGL) directly activates mature osteoclasts. J Cell Biol 145:527–538 April, 2000 BIRTH AND DEATH OF BONE CELLS 158. Lean J, Matsuo K, Wong B, Wagner EF, Choi Y, Chambers T 1999 Infection with TRANCE-expressing retrovirus for the generation of osteoclastic cell lines. Bone 23:S165 (Abstract) 159. Dobnig H, Turner RT 1997 The effects of programmed administration of human parathyroid hormone fragment (1–34) on bone histomorphometry and serum chemistry in rats. Endocrinology 138:4607– 4612 160. Manolagas SC 1998 Cellular and molecular mechanisms of osteoporosis. Aging Clin Exp Res 10:182–190 161. Jilka RL 1998 Cytokines, bone remodeling, and estrogen deficiency: a 1998 update. Bone 23:75– 81 162. Pacifici R 1998 Cytokines, estrogen, and postmenopausal osteoporosis—the second decade. Endocrinology 139:2659 –2661 163. Lin SC, Yamate T, Taguchi Y, Borba VZ, Girasole G, O’Brien CA, Bellido T, Abe E, Manolagas SC 1997 Regulation of the gp80 and gp130 subunits of the IL-6 receptor by sex steroids in the murine bone marrow. J Clin Invest 100:1980 –1990 164. McDonnell DP, Norris JD 1997 Analysis of the molecular pharmacology of estrogen receptor agonists and antagonists provides insights into the mechanism of action of estrogen in bone. Osteoporos Int 7 Suppl 1:S29 –S34 165. Miyaura C, Kusano K, Masuzawa T, Chaki O, Onoe Y, Aoyagi M, Sasaki T, Tamura T, Koishihara Y, Ohsugi Y, Suda T 1995 Endogenous bone-resorbing factors in estrogen deficiency: cooperative effects of IL-1 and IL-6. J Bone Miner Res 10:1365–1373 166. Bismar H, Diel I, Ziegler R, Pfeilschifter J 1995 Increased cytokine secretion by human bone marrow cells after menopause or discontinuation of estrogen replacement. J Clin Endocrinol Metab 80:3351–3355 167. Cheleuitte D, Mizuno S, Glowacki J 1998 In vitro secretion of cytokines by human bone marrow: effects of age and estrogen status. J Clin Endocrinol Metab 83:2043–2051 168. Kassem M, Khosla S, Spelsberg TC, Riggs BL 1996 Cytokine production in the bone marrow microenvironment: failure to demonstrate estrogen regulation in early postmenopausal women. J Clin Endocrinol Metab 81:513–518 169. Roodman GD, Kurihara N, Ohsaki Y, Kukita A, Hosking D, Demulder A, Smith JF, Singer FR 1992 Interleukin 6. A potential autocrine/paracrine factor in Paget’s disease of bone. J Clin Invest 89:46 –52 170. Klein B, Wijdenes J, Zhang X-G, Jourdan M, Boiron J-M, Brochier J, Liautard J, Merlin M, Clement C, Morel-Fournier B, Lu Z-Y, Mannoni P, Sany J, Bataille R 1991 Murine anti-interleukin-6 monoclonal antibody therapy for a patient with plasma cell leukemia. Blood 78:1198 –1204 171. Kotake S, Sato K, Kim KJ, Takahashi N, Udagawa N, Nakamura I, Yamaguchi A, Kishimoto T, Suda T, Kashiwazaki S 1996 Interleukin-6 and soluble interleukin-6 receptors in the synovial fluids from rheumatoid arthritis patients are responsible for osteoclast-like cell formation. J Bone Miner Res 11:88 –95 172. Takayanagi H, Juji T, Miyazaki T, Iizuka H, Takahashi T, Isshiki M, Okada M, Tanaka Y, Koshihara Y, Oda H, Kurokawa T, Nakamura K, Tanaka S 1999 Suppression of arthritic bone destruction by adenovirus-mediated csk gene transfer to synoviocytes and osteoclasts. J Clin Invest 104:137–146 173. Siddiqi A, Monson JP, Wood DF, Besser GM, Burrin JM 1999 Serum cytokines in thyrotoxicosis. J Clin Endocrinol Metab 84: 435– 439 174. Yamamoto T, Ozono K, Kasayama S, Yoh K, Hiroshima K, Takagi M, Matsumoto S, Michigami T, Yamaoka K, Kishimoto T, Okada S 1996 Increased IL-6 production by cells isolated from the fibrous bone dysplasia tissues in patients with McCune-Albright syndrome. J Clin Invest 98:30 –35 175. Jilka RL, Takahashi K, Munshi M, Williams DC, Roberson PK, Manolagas SC 1998 Loss of estrogen upregulates osteoblastogenesis in the murine bone marrow: evidence for autonomy from factors released during bone resorption. J Clin Invest 101:1942–1950 176. Srivastava S, Neale WM, Kimble RB, Rizzo M, Zahner M, Milbrandt J, Patrick RF, Pacifici R 1998 Estrogen blocks M-CSF gene expression and osteoclast formation by regulating phosphorylation of egr-1 and its interaction with Sp-1. J Clin Invest 102:1850 –1859 177. Srivastava S, Weitzmann MN, Cenci S, Ross FP, Adler S, Pacifici R 1999 Estrogen decreases TNF gene expression by blocking JNK 178. 179. 180. 181. 182. 183. 184. 185. 186. 187. 188. 189. 190. 191. 192. 193. 194. 195. 196. 197. 135 activity and the resulting production of c-Jun and JunD. J Clin Invest 104:503–513 Sunyer T, Lewis J, Collin-Osdoby P, Osdoby P 1999 Estrogen’s bone-protective effects may involve differential IL-1 receptor regulation in human osteoclast-like cells. J Clin Invest 103:1409 –1418 Kimble RB, Matayoshi AB, Vannice JL, Kung VT, Williams C, Pacifici R 1995 Simultaneous block of interleukin-1 and tumor necrosis factor is required to completely prevent bone loss in the early postovariectomy period. Endocrinology 136:3054 –3061 Kitazawa R, Kimble RB, Vannice JL, Kung VT, Pacifici R 1994 Interleukin-1 receptor antagonist and tumor necrosis factor binding protein decrease osteoclast formation and bone resorption in ovariectomized mice. J Clin Invest 94:2397–2406 Ammann P, Rizzoli R, Bonjour JP, Bourrin S, Meyer JM, Vassalli P, Garcia I 1997 Transgenic mice expressing soluble tumor necrosis factor-receptor are protected against bone loss caused by estrogen deficiency. J Clin Invest 99:1699 –1703 Hofbauer LC, Khosla S, Dunstan CR, Lacey DL, Spelsberg TC, Riggs BL 1999 Estrogen stimulates gene expression and protein production of osteoprotegerin in human osteoblastic cells. Endocrinology 140:4367– 4370 Eriksen EF, Langdahl B, Vesterby A, Rungby J, Kassem M 1999 Hormone replacement therapy prevents osteoclastic hyperactivity: a histomorphometric study in early postmenopausal women. J Bone Miner Res 14:1217–1221 Manolagas SC, Weinstein RS, Bellido T, Bodenner DL 1999 Opposite effects of estrogen on the life span of osteoblasts/osteocytes vs. osteoclasts in vivo and in vitro: an explanation of the imbalance between formation and resorption in estrogen deficiency. J Bone Miner Res 14:S169 (Abstract) Smith EP, Boyd J, Frank GR, Takahashi H, Cohen RM, Specker B, Williams TC, Lubahn DB, Korach KS 1994 Estrogen resistance caused by a mutation in the estrogen-receptor gene in a man. N Engl J Med 331:1056 –1061 Carani C, Qin K, Simoni M, Faustini-Fustini M, Serpente S, Boyd J, Korach KS, Simpson ER 1997 Effect of testosterone and estradiol in a man with aromatase deficiency. N Engl J Med 337:91–95 Bilezikian JP, Morishima A, Bell J, Grumbach MM 1998 Increased bone mass as a result of estrogen therapy in a man with aromatase deficiency. N Engl J Med 339:599 – 603 Riggs BL, Khosla S, Melton III LJ 1998 Unitary model of osteoporosis revisited. J Bone Miner Res 13:1954 –1955 Schwartz BD, Zhu YS, Cordero J, Imperato-McGinley J, 5␣Reductase deficiency and complete androgen insenstitivity: natural models to suggest a direct role for androgens on bone density in men. Program of the 81st Annual Meeting of The Endocrine Society, San Diego, CA, 1999, p 93 (Abstract) Weinstein RS, Bellido T, Chambers TM, Crawford JA, Swain FL, Han L, Manolagas SC 1999 Like estrogen, androgen exert potent and direct anti-apoptotic effects on osteoblasts and osteocytes in vivo and in vitro. J Bone Miner Res 14:S451 (Abstract) Lips P, Courpron P, Meunier PJ 1978 Mean wall thickness of trabecular bone packets in the human iliac crest: changes with age. Calcif Tissue Res 26:13–17 Parfitt AM, Villanueva AR, Foldes J, Rao DS 1995 Relations between histologic indices of bone formation: implications for the pathogenesis of spinal osteoporosis. J Bone Miner Res 10:466 – 473 Parfitt AM, Han ZH, Palnitkar S, Rao DS, Shih MS, Nelson D 1997 Effects of ethnicity and age or menopause on osteoblast function, bone mineralization, and osteoid accumulation in iliac bone. J Bone Miner Res 12:1864 –1873 Eastell R, Delmas PD, Hodgson SF, Eriksen EF, Mann KG, Riggs BL 1988 Bone formation rate in older normal women: concurrent assessment with bone histomorphometry, calcium kinetics, and biochemical markers. J Clin Endocrinol Metab 67:741–748 Melton LJ, Khosla S, Atkinson EJ, O’Fallon WM, Riggs BL 1997 Relationship of bone turnover to bone density and fractures. J Bone Miner Res 12:1083–1091 Garnero P, Hausherr E, Chapuy MC, Marcelli C, Grandjean H, Muller C, Cormier C, Breart G, Meunier PJ, Delmas PD 1996 Markers of bone resorption predict hip fracture in elderly women: the EPIDOS Prospective Study. J Bone Miner Res 11:1531–1538 Garnero P, Sornay-Rendu E, Chapuy MC, Delmas PD 1996 In- 136 198. 199. 200. 201. 202. 203. 204. 205. 206. 207. 208. 209. 210. 211. 212. 213. 214. 215. 216. 217. MANOLAGAS creased bone turnover in late postmenopausal women is a major determinant of osteoporosis. J Bone Miner Res 11:337–349 D’ippolito G, Schiller PC, Ricordi C, Roos BA, Howard GA 1999 Age-related osteogenic potential of mesenchymal stromal stem cells from human vertebral bone marrow. J Bone Miner Res 14: 1115–1122 Mueller S, Glowacki J 1999 The effect of age on the osteogenic potential of human bone marrow stromal cells. J Bone Miner Res 14:S354 (Abstract) Kajkenova O, Lecka-Czernik B, Gubrij I, Hauser SP, Takahashi K, Parfitt AM, Jilka RL, Manolagas SC, Lipschitz DA 1997 Increased adipogenesis and myelopoiesis in the bone marrow of SAMP6, a murine model of defective osteoblastogenesis and low turnover osteopenia. J Bone Miner Res 12:1772–1779 Nagy L, Tontonoz P, Alvarez JG, Chen H, Evans RM 1998 Oxidized LDL regulates macrophage gene expression through ligand activation of PPAR␥. Cell 93:229 –240 Tontonoz P, Nagy L, Alvarez JG, Thomazy VA, Evans RM 1998 PPAR␥ promotes monocyte/macrophage differentiation and uptake of oxidized LDL. Cell 93:241–252 Nuttall ME, Patton AJ, Olivera DL, Nadeau DP, Gowen M 1998 Human trabecular bone cells are able to express both osteoblastic and adipocytic phenotype: implications for osteopenic disorders. J Bone Miner Res 13:371–382 Parhami F, Jackson SM, Tintut Y, Le V, Balucan JP, Territo M, Demer LL 1999 Atherogenic diet and minimally oxidized low density lipoprotein inhibit osteogenic and promote adipogenic differentiation of marrow stromal cells. J Bone Miner Res 14:2067–2078 Banks LM, Lees B, MacSweeney JE, Stevenson JC 1994 Effect of degenerative spinal and aortic calcification on bone density measurements in post-menopausal women: links between osteoporosis and cardiovascular disease? Eur J Clin Invest 24:813– 817 Laroche M, Pouilles JM, Ribot C, Bendayan P, Bernard J, Boccalon H, Mazzaferro S 1994 Comparison of the bone mineral content of the lower limbs in men with ischaemic atherosclerotic disease. Clin Rheumatol 13:611– 614 Shmookler Reis RJ, Benes H, McClure T, Zheng W, Weinstein RS, Shelton R, Jilka RL, Manolagas SC 1999 Genetic mapping of loci conferring osteopenia using closely-related mouse strains. J Bone Miner Res 14:S141 (Abstract) Klein RF, Mitchell SR, Phillips TJ, Belknap JK, Orwoll ES 1998 Quantitative trait loci affecting peak bone mineral density in mice. J Bone Miner Res 13:1648 –1656 Shimizu M, Higuchi K, Bennett B, Xia C, Tsuboyama T, Kasai S, Chiba T, Fujisawa H, Kogishi K, Kitado H, Kimoto M, Takeda N, Matsushita M, Okumura H, Serikawa T, Nakamura T, Johnson TE, Hosokawa M 1999 Identification of peak bone mass QTL in a spontaneously osteoporotic mouse strain. Mamm Genome 10:81– 87 Mohan S, Baylink DJ 1997 Serum insulin-like growth factor binding protein (IGFBP)-4 and IGFBP-5 levels in aging and ageassociated diseases. Endocrine 7:87–91 Rajaram S, Baylink DJ, Mohan S 1997 Insulin-like growth factorbinding proteins in serum and other biological fluids: regulation and functions. Endocr Rev 18:801– 831 Rozman C, Feliu E, Berga L, Reverter JC, Climent C, Ferran MJ 1989 Age-related variations of fat tissue fraction in normal human bone marrow depend both on size and number of adipocytes: a stereological study. Exp Hematol 17:34 –37 Gimble JM, Robinson CE, Wu X, Kelley KA 1996 The function of adipocytes in the bone marrow stroma: an update. Bone 19:421– 428 Tavassoli M 1989 Fatty involution of marrow and the role of adipose tissue in hemopoiesis. In: Tavassoli M (ed) Handbook of the Hematopoietic Microenvironment. Humana Press, Clifton, NJ, pp 157–187 Kajkenova O, Gubrij I, Hauser SP, Takahashi K, Jilka RL, Manolagas SC, Lipschitz DA 1995 Increased hematopoiesis accompanies reduced osteoblastogenesis in the senescence-accelerated mouse (SAM-P/6). J Bone Miner Res 10[Suppl 1]:S431 (Abstract) Fitzpatrick LA 1994 Glucocorticoid-induced osteoporosis. In: Marcus R (ed) Osteoporosis. Blackwell Science, Boston, pp 202–226 Hofbauer LC, Gori F, Riggs BL, Lacey DL, Dunstan CR, Spelsberg TC, Khosla S 1999 Stimulation of osteoprotegerin ligand and inhibi- 218. 219. 220. 221. 222. 223. 224. 225. 226. 227. 228. 229. 230. 231. 232. 233. 234. 235. 236. 237. 238. 239. Vol. 21, No. 2 tion of osteoprotegerin production by glucocorticoids in human osteoblastic lineage cells: potential paracrine mechanisms of glucocorticoid-induced osteoporosis. Endocrinology 140:4382– 4389 Jilka RL, Weinstein RS, Bellido T, Roberson P, Parfitt AM, Manolagas SC 1999 Increased bone formation by prevention of osteoblast apoptosis with PTH. J Clin Invest 104:439 – 446 Plotkin L, Weinstein RS, Parfitt AM, Roberson PK, Manolagas SC, Bellido T 1999 Prevention of osteocyte and osteoblasts apoptosis by bisphosphonates and calcitonin. J Clin Invest 104:1363– 1374 Mankin HJ 1992 Nontraumatic necrosis of bone (osteonecrosis). N Engl J Med 326:1473–1479 Weinstein RS, Nicholas RW, Kirchner JR, Crawford JA, Skinner RA, Swain FL, Manolagas SC 1998 Anatomic juxtaposition of apoptotic osteocytes and avascular necrosis in femurs from patients with glucocorticoid-excess. Bone 23:S461 (Abstract) Chang DJ, Ji C, Kim KK, Casinghino S, McCarthy TL, Centrella M 1998 Reduction in transforming growth factor beta receptor I expression and transcription factor CBFa1 on bone cells by glucocorticoid. J Biol Chem 273:4892– 4896 Centrella M, Rosen V, Wozney JM, Casinghino SR, McCarthy TL 1997 Opposing effects by glucocorticoid and bone morphogenetic protein-2 in fetal rat bone cell cultures. J Cell Biochem 67:528 –540 Canalis E 1998 Inhibitory actions of glucocorticoids on skeletal growth. Is local insulin-like growth factor I to blame? Endocrinology 139:3041–3042 Shi XM, Chang ZJ, Blair HC, McDonald JM, Cao X 1998 Glucocorticoids induce adipogenesis of stromal cells by transcriptionally activating PPARy2. Bone 23:S454 (Abstract) Dempster DW, Cosman F, Parisien M, Shen V 1993 Anabolic actions of parathyroid hormone on bone. Endocr Rev 14:690 –709 Tam CS, Heersche JNM, Murray TM, Parsons JA 1982 Parathyroid hormone stimulates the bone apposition rate independently of its resorptive action: differential effects of intermittent and continuous administration. Endocrinology 110:506 –512 Reeve J, Meunier PJ, Parsons JA, Bernat M, Bijvoet OL, Courpron P, Edouard C, Klenerman L, Neer RM, Renier JC, Slovik D, Vismans FJ, Potts JTJ 1980 Anabolic effect of human parathyroid hormone fragment on trabecular bone in involutional osteoporosis: a multicentre trial. Br Med J 280:1340 –1344 Finkelstein JS, Klibanski A, Arnold AL, Toth TL, Hornstein MD, Neer RM 1998 Prevention of estrogen deficiency-related bone loss with human parathyroid hormone-(1–34): a randomized controlled trial. JAMA 280:1067–1073 Lane NE, Sanchez S, Modin GW, Genant HK, Pierini E, Arnaud C 1998 Parathyroid hormone treatment can reverse corticosteroidinduced osteoporosis. J Clin Invest 102:1627–1633 Reeve J, Hesp R, Williams D, Hulme P, Klenerman L, Zanelli JM, Darby AJ, Tregear GW, Parsons JA 1976 Anabolic effect of low doses of a fragment of human parathyroid hormone on the skeleton in postmenopausal osteoporosis. Lancet 1:1035–1038 Stewart AF 1996 PTHrP(1–36) as a skeletal anabolic agent for the treatment of osteoporosis. Bone 19:303–306 Machwate M, Rodan SB, Rodan GA, Harada SI 1998 Sphingosine kinase mediates cyclic AMP suppression of apoptosis in rat periosteal cells. Mol Pharmacol 54:70 –77 Turner PR, Bencsik M, Malecz N, Christakos S, Nissenson RA 1998 Apoptosis mediated by the PTH/PTHrP receptor: role of JNK and calcium signaling pathways. Bone 23:S155 (Abstract) Leaffer D, Sweeney M, Kellerman LA, Avnur Z, Krstenansky JL, Vickery BH, Caulfield JP 1995 Modulation of osteogenic cell ultrastructure by RS-23581, an analog of human parathyroid hormone (PTH)-related peptide-(1–34), and bovine PTH-(1–34). Endocrinology 136:3624 –3631 Dobnig H, Turner RT 1995 Evidence that intermittent treatment with parathyroid hormone increases bone formation in adult rats by activation of bone lining cells. Endocrinology 136:3632–3638 Hock JM 1999 Stemming bone loss by suppressing apoptosis. J Clin Invest 104:371–373 Papapoulos SE 1996 Bisphosphonates: pharmacology and use in the treatment of osteoporosis. In: Marcus R, Feldman D, Kelsey J (eds) Osteoporosis. Academic Press, San Diego, CA, pp 1209 –1234 Azria M, Avioli LV 1996 Calcitonin. In: Bilezikian JP, Raisz LG, April, 2000 240. 241. 242. 243. 244. 245. 246. 247. 248. 249. 250. 251. 252. BIRTH AND DEATH OF BONE CELLS Rodan GA (eds) Principles of Bone Biology. Academic Press, San Diego, CA, pp 1083–1097 Hughes DE, Wright KR, Uy HL, Sasaki A, Yoneda T, Roodman GD, Mundy GR, Boyce BF 1995 Bisphosphonates promote apoptosis in murine osteoclasts in vitro and in vivo. J Bone Miner Res 10:1478 –1487 Fisher JE, Rogers MJ, Halasy JM, Luckman SP, Hughes DE, Masarachia PJ, Wesolowski G, Russell RGG, Rodan GA, Reszka AA 1999 Alendronate mechanism of action: geranylgeraniol, an intermediate in the mevalonate pathway, prevents inhibition of osteoclast formation, bone resorption, and kinase activation in vitro. Proc Natl Acad Sci USA 96:133–138 Fleisch H 1998 Bisphosphonates: mechanisms of action. Endocr Rev 19:80 –100 Cummings S, Black D, Vogt TM 1996 Changes in BMD substantially underestimate the anti-fracture effects of alendronate and other antiresorptive drugs. J Bone Miner Res 11:S102 (Abstract) Chavassieux PM, Arlot ME, Reda C, Wei L, Yates AJ, Meunier PJ 1997 Histomorphometric assessment of the long-term effects of alendronate on bone quality and remodeling in patients with osteoporosis. J Clin Invest 100:1475–1480 Balena R, Toolan BC, Shea M, Markatos A, Myers ER, Lee SC, Opas EE, Seedor JG, Klein H, Frankenfield D, Quartuccio H, Fioravanti C, Clair J, Brown E, Hayes WC, Rodan GA 1993 The effects of 2-year treatment with the aminobisphosphonate alendronate on bone metabolism, bone histomorphometry, and bone strength in ovariectomized nonhuman primates. J Clin Invest 92: 2577–2586 Storm T, Steiniche T, Thamsborg G, Melsen F 1993 Changes in bone histomorphometry after long-term treatment with intermittent, cyclic etidronate for postmenopausal osteoporosis. J Bone Miner Res 8:199 –208 Mohan S, Baylink D 1996 Therapeutic potential of TGF-beta, BMP, and FGF in the treatment of bone loss. In: Bilezikian JP, Raisz LG, Rodan GA (eds) Principles of Bone Biology, pp 1111–1123 Rosen C, Wuster C 1996 Growth hormone, insulin-like growth factors. In: Marcus R, Feldman D, Kelsey J (eds) Osteoporosis. Academic Press, San Diego, CA, pp 1313–1333 Bodenner DL, Yamamoto M, Kozlowski M, Manolagas SC 1999 Essential requirement of the estrogen receptor alpha or beta for (non-genomic) anti-apoptotic effects of estrogen. J Bone Miner Res 14:S227 (Abstract) Bellido T, Plotkin LI, Han L, Manolagas SC 1999 Estrogen inhibit apoptosis of osteoblasts and osteocytes through rapid (non-genomic) activation of extracellular signal-regulated kinases (ERKs). J Bone Miner Res 14:S342 (Abstract) Lieberherr M, Grosse B, Kachkache M, Balsan S 1993 Cell signaling and estrogens in female rat osteoblasts: a possible involvement of unconventional nonnuclear receptors. J Bone Miner Res 8:1365–1376 Endoh H, Sasaki H, Maruyama K, Takeyama K, Waga I, Shimizu 253. 254. 255. 256. 257. 258. 259. 260. 261. 262. 263. 264. 265. 266. T, Kato S, Kawashima H 1997 Rapid activation of MAP kinase by estrogen in the bone cell line. Biochem Biophys Res Commun 235:99 –102 Aronica SM, Kraus WL, Katzenellenbogen BS 1994 Estrogen action via the cAMP signaling pathway: stimulation of adenylate cyclase and cAMP-regulated gene transcription. Proc Natl Acad Sci USA 91:8517– 8521 McEwen BS, Alves SE 1999 Estrogen actions in the central nervous system. Endocr Rev 20:279 –307 Chen Z, Yuhanna IS, Galcheva-Gargova Z, Karas RH, Mendelsohn ME, Shaul PW 1999 Estrogen receptor alpha mediates the nongenomic activation of endothelial nitric oxide synthase by estrogen. J Clin Invest 103:401– 406 Castoria G, Barone MV, Di Domenico M, Bilancio A, Ametrano D, Migliaccio A, Auricchio F 1999 Non-transcriptional action of oestradiol and progestin triggers DNA synthesis. EMBO J 18:2500 – 2510 Pietras RJ, Szego CM 1977 Specific binding sites for oestrogen at the outer surfaces of isolated endometrial cells. Nature 265:69 –72 Pappas TC, Gametchu B, Watson CS 1995 Membrane estrogen receptors identified by multiple antibody labeling and impededligand binding. FASEB J 9:404 – 410 Razandi M, Pedram A, Greene GL, Levin ER 1999 Cell membrane and nuclear estrogen receptors (ERs) originate from a single transcript: Studies of ER␣ and ER␤ expressed in Chinese hamster ovary cells. Mol Endocrinol 13:307–319 Norfleet AM, Thomas ML, Gametchu B, Watson CS 1999 Estrogen receptor-alpha detected on the plasma membrane of aldehydefixed GH3/B6/F10 rat pituitary tumor cells by enzyme-linked immunocytochemistry. Endocrinology 140:3805–3814 Manolagas SC, Weinstein RS, Bellido T, Bodenner DL, Jilka RL, Parfitt AM 1999 Activators of non-genomic estrogen-like signalling (ANGELS): a novel class of small molecules with bone anabolic properties. J Bone Miner Res 14:S180 (Abstract) VanderKuur JA, Hafner MS, Christman JK, Brooks SC 1993 Effects of estradiol-17␤ analogues on activation of estrogen response element regulated chloramphenicol acetyltransferase expression. Biochemistry 32:7016 –7021 Christman JK, Nehls S, Polin L, Brooks SC 1995 Relationship between estrogen structure and conformational changes in estrogen receptor/DNA complexes. J Steroid Biochem Mol Biol 54: 201–210 Green PS, Gridley KE, Simpkins JW 1998 Nuclear estrogen receptor-independent neuroprotection by estratrienes: a novel interaction with glutathione. Neuroscience 84:7–10 Green PS, Gordon K, Simpkins JW 1997 Phenolic A ring requirement for the neuroprotective effects of steroids. J Steroid Biochem Mol Biol 63:229 –235 Shi J, Zhang YQ, Simpkins JW 1997 Effects of 17␤-estradiol on glucose transporter 1 expression and endothelial cell survival following focal ischemia in the rats. Exp Brain Res 117:200 –206 STADY II-International Symposium on Signal Transduction in Health and Disease September 12–15, 2000 Tel Aviv, Israel For further information, contact: Professor Zvi Naor Department of Biochemistry Tel Aviv University Tel Aviv, Israel Telephone: 972-3-640-9032/641-7057 Fax: 972-3-640-6834 E-mail: [email protected] or [email protected] 137 IBMS BoneKEy. 2011 February;8(2):74-83 http://www.bonekey-ibms.org/cgi/content/full/ibmske;8/2/74 doi: 10.1138/20110493 PERSPECTIVES The Osteoclast Cytoskeleton: How Does It Work? Steven L. Teitelbaum and Wei Zou Washington University School of Medicine, St. Louis, Missouri, USA Abstract The capacity of the osteoclast to resorb bone is distinctive, as is the cell’s appearance. Both characteristics reflect cytoskeletal organization that yields structures such as the sealing zone and ruffled border. The unique nature of these organelles and their dependence upon contact with bone have been appreciated for some time but insights into the mechanisms by which they are generated come from more recent studies. These insights include the role of integrins, particularly αvβ3, in cytoskeletal organization and the canonical signaling pathway they activate. Investigators now appreciate that the sealing zone isolates the resorptive microenvironment from the general extracellular space, permitting secretion of matrixdegrading molecules on the bone surface. Thus, the osteoclast is a secretory cell that depends upon polarization of exocytic vesicles to the bone-apposed plasma membrane into which they insert under the aegis of vesicle/membrane fusion proteins. This process focally expands and convolutes the plasmalemma included within the sealing zone, eventuating in formation of the ruffled border. Many of these events are now better understood and are the focus of this Perspective. IBMS BoneKEy. 2011 February;8(2):74-83. 2011 International Bone & Mineral Society Isolating the Microenvironment Resorptive Osteoclasts are polykaryons and members of the macrophage lineage with the unique capacity to degrade the inorganic and organic matrices of bone. If excessive, the bone resorptive activity of osteoclasts causes osteoporosis. Conversely, the osteoclast initiates remodeling that likely removes structurally compromised bone, thereby maintaining mechanical integrity (1). Skeletal health, therefore, requires optimal osteoclast function. Resorption is initiated by attachment of osteoclasts to bone. They then develop a compartment between their plasma membrane and the bone surface into which the cells transport matrix-degrading + molecules including H and Cl , which, in concert, demineralize the target bone, and cathepsin K, which degrades the exposed collagen fibers and associated proteins. To isolate this resorptive microenvironment from the general extracellular space, osteoclasts reorganize their cytoskeleton to generate an encompassing, actin-rich, gasket-like, sealing zone. A single osteoclast, being a large cell, enjoys multiple contacts with bone and therefore generates numerous sealing zones and resorptive microenvironments. When most other cells attach to matrix they generate focal adhesions. These stable structures contain integrins and signaling and cytoskeletal molecules that, upon contact with matrix, promote formation of actin stress fibers. Consistent with the lack of actin stress fibers in mammalian osteoclasts, they lack focal adhesions but in their place develop podosomes (2;3). These punctuate structures contain an F-actin core and a peripheral “cloud” of a loose network of radial actin cables (4-7). In contrast to focal adhesions, podosomes are transient but mediate substrate adhesion and thus formation of the resorptive microenvironment. Most past studies of osteoclast podosomes utilized cells resident on plastic or glass. In these circumstances, the punctuate structures initially appear in clusters but ultimately coalesce, first into an 74 Copyright 2011 International Bone & Mineral Society IBMS BoneKEy. 2011 February;8(2):74-83 http://www.bonekey-ibms.org/cgi/content/full/ibmske;8/2/74 doi: 10.1138/20110493 intracytoplasmic actin ring and then a peripheral actin belt (4). The physiological relevance of this observation was challenged by the absence of apparent podosomes or an actin belt in non-stressed osteoclasts on bone (4). While the relevance of the peripheral belt is not established, it is clear that, like the actin ring, the sealing zone of bone-residing osteoclasts also consists of podosome-containing structural units (8). Microtubules Are Important Depending upon their state of acetylation, microtubules in osteoclasts are transient or polymerized and relatively stable (9-11). Unlike actin ring formation by glass-residing cells, sealing zone generation in boneresorbing osteoclasts is characterized by microtubule acetylation, again illustrating the influence of substrate on the cell’s cytoskeleton (10). The histone deacetylase HDAC6 depolymerizes tubulin, thereby destabilizing microtubules. Cbl proteins compete with the deacetylase for tubulin binding and thus promote polymerization (12). While destabilizing microtubules in other cells (13), RhoA appears to activate HDAC6 in glassgenerated osteoclasts, suggesting the GTPase negatively regulates the cell. When on bone, however, osteoclasts in which RhoA is inhibited lose their apical-basal polarity and, consequently, are incapable of optimal resorption (6). Furthermore, RhoA promotes actin ring and podosome formation and osteoclast motility (6;14). When osteoclasts contact bone, RhoA is activated and localizes to the cytoskeleton (15;16), The fact that RhoA activation is diminished, in osteoclasts lacking αvβ3, establishes that the GTPase is regulated by the integrin (3). Because of the conflicting phenotypes of osteoclasts on plastic or bone, the impact of active RhoA on stability of their microtubules remains unknown. degrading bone and its absence indicates the cell is not doing so. Reflecting multiple contacts with bone and attendant sealing zones, ruffled borders are also numerous in a given osteoclast. This complex enfolding of the plasma membrane, unique to the osteoclast, abuts and extends into the resorptive space. It is surrounded by the sealing zone and is the venue by which the cell secretes matrixdegrading molecules on the bone surface (17). It is therefore the resorptive organelle and is absent or deranged in many forms of osteopetrosis. As osteoclast resorption alternates with migration, the ruffled border is a transient structure. The ruffled border, which forms only upon contact with mineralized substrate, is initiated by transport of cathepsin K – and/or + H ATPase – and Cl channel-bearing vesicles to the bone-apposed plasma membrane (17), likely under the aegis of GTPases such as Rabs 7, 9 and 3 (18-22). While unproven, studies in chicken osteoclasts raise the possibility that ruffled border-forming vesicles may polarize to the resorptive surface via microtubules (23). The polarized vesicles fuse with the boneapposed plasma membrane, to increase its complexity, via a process mirroring exocytosis (17). Similar to that occurring in the context of neurotransmitter exocytosis, vesicle/plasmalemma fusion is regulated by v- (vesicular) and t- (target) SNAREs (soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein (SNAP) receptors) (24). The Ruffled Border Is King Ruffled border formation requires a synaptotagmin (Syt) linking the vesicle and target plasma membrane. Fifteen Syt isoforms have been identified in mammalian cells but Syt VII generates ruffled borders and is essential for bone degradation (17). Syt VII also enables osteoblasts to secrete bone matrix proteins and as such, both resorption and formation are repressed in Syt VII-deficient mice. The ruffled border is the morphological sine qua non of the resorbing osteoclast as only its presence assures that the cell is Autophagy is a cellular degradative process by which cells recycle organelles and longlived proteins. Autophagosomes, which are 75 Copyright 2011 International Bone & Mineral Society IBMS BoneKEy. 2011 February;8(2):74-83 http://www.bonekey-ibms.org/cgi/content/full/ibmske;8/2/74 doi: 10.1138/20110493 double-membrane bound vesicles, envelop and then deliver cellular components to lysosomes for degradation. While the process promotes survival in starved or stressed cells as well as maintenance of organelle quality (25-27), recent evidence indicates it may also participate in regulated exocytosis (28). In fact, Atg5, Atg7 and LC3β autophagy proteins, representing the two ubiquitin-like conjugation systems, are important for generation of the osteoclast ruffled border and the secretory function of osteoclasts both in vitro and in vivo. Thus, osteoclasts lacking these proteins do not efficiently polarize cathepsin K to the resorptive microenvironment and are incapable of optimal bone resorption (29). Integrin Activity Starts It All Skeletal resorption requires osteoclast-bone recognition that is mediated by α/β heterodimers known as integrins. β1containing heterodimers probably participate in the process but αv/β3 is the key integrin regulating skeletal degradation (30). While the αv subunit is constitutively expressed throughout osteoclastogenesis, the associated β chains alter with differentiation. Specifically, immature osteoclast precursors, in the form of bone marrow macrophages, express abundant αvβ5 and little αvβ3. As the cells commit to the osteoclast phenotype, the magnitude of expression of the two heterodimers reverses (31). Hence, αv/β3 is a relatively specific marker of osteoclastogenesis. This integrin is liganded by the amino acids Arg-Gly-Asp (RGD), a motif present in bone proteins such as osteopontin and bone sialoprotein. Small molecules mimicking this sequence suppress osteoclast activity and are candidate anti-resorptive drugs (32-34). In keeping with its governance of osteoclast function, absence of αv/β3, globally and conditionally in osteoclasts, increases bone mass and protects against estrogendeficient osteoporosis (35;36). Reflecting the integrin’s role in cytoskeletal organization, αv/β3 deficiency yields deranged ruffled borders, failure of cell spreading and suboptimal bone resorption (35). Consequently, β3(-/-) mice are hypocalcemic and osteoclast number is substantially increased in these animals, likely reflecting secondary hyperparathyroidism and an abundance of osteoclastogenic cytokines in the marrow (35;37;38). However, in contrast, absence of αv/β3 diminishes the abundance of the polykaryons in vitro (33). As differentiation, apoptosis and precursor proliferation are not compromised, a reasonable hypothesis holds that the paucity of osteoclasts, in culture, reflects cytoskeletal dysfunction, and specifically impairment of migration necessary for cell fusion. αv/β3 signaling in osteoclasts is initiated by changing the integrin’s conformation from a low to a high affinity state by outside-in or inside-out activation (3;39). Outside-in activation is characterized by integrin clustering, thereby increasing avidity and affinity. Inside-out activation is an indirect event wherein signals emanating from liganded growth factor or cytokine receptors target the integrin’s intracellular region, changing its conformation and consequently, that of the extracellular domain (40). As will be discussed, the adaptor protein, talin, is essential for inside-out αv/β3 activation in osteoclasts and its absence arrests bone resorption. Resorption is a cyclical event wherein a portion of the osteoclast migrates to a candidate bone resorptive site and forms an actin ring and ruffled border. Following matrix degradation, the cell detaches and reinitiates the cycle. Prior to bone recognition, the integrin is predominantly in a low affinity state and confined to podosomes within the sealing zone (3;41). Activated αv/β3 leaves the podosome and transits to lamellipodia that mediate motility, compromised in the absence of the integrin (3). During resorption, the heterodimer appears in the ruffled membrane (3;41). Integrins serve as attachment molecules but their intracellular transmission of matrixderived signals is at least as important. For example, αv/β3 substrate robustly activates ERKs in wild-type osteoclastic cells but not those lacking the integrin (3). Since 76 Copyright 2011 International Bone & Mineral Society IBMS BoneKEy. 2011 February;8(2):74-83 http://www.bonekey-ibms.org/cgi/content/full/ibmske;8/2/74 doi: 10.1138/20110493 activation of these MAP kinases typically stimulates proliferation, their absence in β3 knockout osteoclasts may contribute to their reduced numbers in vitro. How Does αv/β 3 Do It? In 1991, Soriano et al. determined that c-src deletion eventuates in severe osteopetrosis due to osteoclast dysfunction (42). Interestingly, c-src-deficient mice, like those lacking αv/β3, have increased numbers of osteoclasts that fail to organize their cytoskeleton. c-src is a tyrosine kinase and an adaptor protein and both functions are necessary for optimal cytoskeletal organization (43). Because c-srcand αv/β3-deficient osteoclasts share qualitatively similar cytoskeletal features, the kinase presents as a mediator of integrin signaling. In fact, under the aegis of phospholipase Cγ (PLCγ2) (44), c-src binds directly to the β3 subunit in the bone resorptive cell, and we have found this to be a constitutive event (45). Others, however, propose that αv/β3 occupancy phosphorylates the focal adhesion kinase family member, Pyk2, which recruits c-src to the integrin (44;46). αv/β3-associated c-src phosphorylates c-Cbl which, in turn, inhibits c-src’s activity (3;47). Regardless of the mechanism of association, c-src activation requires integrin occupancy and again, signaling via PLCγ2. Activated c-src prompts podosomal disassembly, most probably by phosphorylating cortactin (48;49). Hence, podosomes are more abundant in c-src(-/-) than wild-type osteoclasts and the mutant cells are less motile. In keeping with Pyk2 regulating the cell’s cytoskeleton, osteoclasts lacking the kinase are unable to generate normal sealing zones on bone (11). The cytoskeletal effects of Pyk2, however, may reflect its promotion of tubulin acetylation. Syk is another non-receptor tyrosine kinase mediating αv/β3 signaling in osteoclasts. Upon integrin occupancy it binds the β3 cytoplasmic domain close to c-src, which activates it (45). Syk is also negatively regulated by the ubiquitinating activity of cCbl (50;51). The ITAM-bearing adaptors, Dap 12 and FcRγ, are expressed by osteoclasts and their combined, but not individual deletion prompts severe osteopetrosis (45;52-54). While deletion of both co-stimulatory molecules is reported to arrest osteoclastogenesis (55), we find such is not the case (56), suggesting their resorptive abnormality reflects deranged osteoclast function but not generation. The same obtains regarding osteoblast-mediated generation of osteoclasts lacking Dap12, with or without FcRγ. These mutant cells form in normal numbers but fail to organize their cytoskeleton or resorb bone. Among the most dramatic consequences of this dysfunction is the inability of Dap12(-/-) osteoclasts to migrate through a layer of osteoblasts, required to attach to a candidate resorptive bone surface (56;57). Thus the dominant role of ITAM proteins in the osteoclast appears to be cytoskeletal organization and not differentiation (56;58). Syk-mediated organization of the osteoclast cytoskeleton involves Vav3. This guanine nucleotide exchange factor (GEF) is uniquely expressed in abundance in the cell and activated upon αv/β3 occupancy in a SLP-76-dependent manner (15;59). Vavs transit cytoskeleton-organizing Rho GTPases from their inactive GDP- to their active GTP-associated conformation. Thus, Vav3(-/-) osteoclasts are dysfunctional and the mice from which they are derived are osteopetrotic. Vavs are Rac GEFs and it is therefore not surprising that this Rho GTPase regulates the osteoclast cytoskeleton in an αv/β3dependent manner (60;61). The two isoforms expressed in osteoclasts, Rac1 and Rac2, are mutually compensatory (62). Effective deletion of both, however, produces severe osteopetrosis in which osteoclasts fail to organize their cytoskeleton. Absence of the related Rho family GTPase, cdc42, also causes osteopetrosis but in this circumstance the dominant mechanism is arrested osteoclast 77 Copyright 2011 International Bone & Mineral Society IBMS BoneKEy. 2011 February;8(2):74-83 http://www.bonekey-ibms.org/cgi/content/full/ibmske;8/2/74 doi: 10.1138/20110493 Fig. 1. Proposed mechanism organizing the cytoskeleton of resorbing osteoclasts. 1). M-CSF occupying its receptor, c-fms, stimulates inside-out αvβ3 activation by inducing talin association with the β3 cytoplasmic domain that binds c-src constitutively. 2). Clustering of the integrin by RGD ligand increases avidity as well as affinity by outside-in activation. The liganded integrin activates c-src as evidenced by Y416 phosphorylation. Activated c-src tyrosine phosphorylates ITAM proteins that recruit Syk to the integrin by binding Syk-SH2 domains. c-src activates β3-associated Syk that phosphorylates Vav3 in the context of SLP-76. Vav3 then shuttles Rac-GDP to its activated GTP-associated state. 3). Rac-GTP prompts association of lysosome-derived secretory vesicles with microtubules (MTs) that deliver them to the boneapposed plasma membrane into which they insert under the influence of Syt VII and LC3. Rac-GTP and MTs also promote sealing zone (SZ) formation. Secretory vesicle fusion focally expands the plasma membrane forming the ruffled border and eventuating in discharge of cathepsin K (CTK) and HCl into the resorptive microenvironment. recruitment due to inhibited precursor proliferation and accelerated apoptosis of the mature polykaryon (63). M-CSF Helps αv/β3 RANK ligand (RANKL) and M-CSF are the requisite osteoclastogenic cytokines but each also promotes the resorptive activity of the mature polykaryon. In the case of MCSF, the cytokine interacting with its receptor, c-fms, stimulates a signaling pathway remarkably similar to that induced by αv/β3, thereby organizing the cytoskeleton (37;58;64). The means by which M-CSF structures the osteoclast cytoskeleton may, therefore, be independent of the integrin, or alternatively, represent inside-out αv/β3 activation. In fact, M-CSF transits αv/β3 from its default low affinity to its high affinity conformation by inducing talin binding to the β3 cytoplasmic domain (39). Absence of talin, in osteoclast precursors, does not arrest differentiation but blocks substrate adherence and motility. The impaired function of talin-deficient osteoclasts results in a 5-fold increase in the bone mass of mutant mice. Interestingly, the osteopetrotic phenotype of mice with talin (-/-) osteoclasts is more severe than of those 78 Copyright 2011 International Bone & Mineral Society IBMS BoneKEy. 2011 February;8(2):74-83 http://www.bonekey-ibms.org/cgi/content/full/ibmske;8/2/74 doi: 10.1138/20110493 lacking αv/β3, which likely represents arrest of compensatory integrins, particularly those bearing β1 (30;35). Conclusion The magnitude of bone resorption reflects osteoclast number and function of the individual cell, the latter dependent upon cytoskeletal organization. The osteoclast cytoskeleton is a unique structure whose conversion to its active state depends upon contact with mineralized matrix (Fig. 1). These extracellular signals, which polarize the resorptive machinery to the bone-cell interface, are transmitted intracellularly by integrins dominated by αv/β3. In conjunction with M-CSF-stimulated inside-out activation, a canonical signaling pathway emanates from the αv/β3 integrin. Occupancy of the heterodimer phosphorylates constitutively associated c-src which in turn targets Dap 12. The ITAM’s phosphotyrosines serve to recruit Syk to the β3 cytoplasmic domain where it is also phosphorylated by c-src. Utilizing SLP-76, Syk activates Vav3, eventuating in formation of Rac-GTP and organization of the resorptive cytoskeleton. Given current concerns regarding longacting anti-resorptive agents, such as bisphosphonates, short-acting counterparts are in demand. Delineating the molecular mechanism by which osteoclasts organize their cytoskeleton to degrade bone has provided an array of candidate therapeutic targets. Conflict of Interest: None reported. Peer Review: This article has been peer-reviewed. References 1. Teitelbaum SL. Bone resorption by osteoclasts. Science. 2000 Sep 1;289(5484):1504-8. 2. Marchisio PC, Bergui L, Corbascio GC, Cremona O, D'Urso N, Schena M, Tesio L, Caligaris-Cappio F. Vinculin, talin, and integrins are localized at specific adhesion sites of malignant B lymphocytes. Blood. 1988 Aug;72(2):830-3. 3. Faccio R, Novack DV, Zallone A, Ross FP, Teitelbaum SL. Dynamic changes in the osteoclast cytoskeleton in response to growth factors and cell attachment are controlled by beta3 integrin. J Cell Biol. 2003 Aug 4;162(3):499-509. 4. Saltel F, Chabadel A, Bonnelye E, Jurdic P. Actin cytoskeletal organisation in osteoclasts: a model to decipher transmigration and matrix degradation. Eur J Cell Biol. 2008 Sep;87(8-9):45968. 5. Chabadel A, Bañon-Rodríguez I, Cluet D, Rudkin BB, Wehrle-Haller B, Genot E, Jurdic P, Anton IM, Saltel F. CD44 and beta3 integrin organize two functionally distinct actin-based domains in osteoclasts. Mol Biol Cell. 2007 Dec;18(12):4899-910. 6. Saltel F, Destaing O, Bard F, Eichert D, Jurdic P. Apatite-mediated actin dynamics in resorbing osteoclasts. Mol Biol Cell. 2004 Dec;15(12):5231-41. 7. Destaing O, Sanjay A, Itzstein C, Horne WC, Toomre D, De Camilli P, Baron R. The tyrosine kinase activity of c-Src regulates actin dynamics and organization of podosomes in osteoclasts. Mol Biol Cell. 2008 Jan;19(1):394-404. 8. Luxenburg C, Geblinger D, Klein E, Anderson K, Hanein D, Geiger B, Addadi L. The architecture of the adhesive apparatus of cultured osteoclasts: from podosome formation to sealing zone assembly. PLoS ONE. 2007 Jan 31;2(1):e179. 9. Waterman-Storer CM, Salmon E. Positive feedback interactions between microtubule and actin dynamics during cell motility. Curr Opin Cell Biol. 1999 Feb;11(1):61-7. 10. Destaing O, Saltel F, Gilquin B, Chabadel A, Khochbin S, Ory S, Jurdic P. A novel Rho-mDia2-HDAC6 pathway controls podosome patterning through 79 Copyright 2011 International Bone & Mineral Society IBMS BoneKEy. 2011 February;8(2):74-83 http://www.bonekey-ibms.org/cgi/content/full/ibmske;8/2/74 doi: 10.1138/20110493 microtubule acetylation in osteoclasts. J Cell Sci. 2005 Jul 1;118(Pt 13):2901-11. and osteoblast secretion. Dev Cell. 2008 Jun;14(6):914-25. 11. Gil-Henn H, Destaing O, Sims NA, Aoki K, Alles N, Neff L, Sanjay A, Bruzzaniti A, De Camilli P, Baron R, Schlessinger J. Defective microtubule-dependent podosome organization in osteoclasts leads to increased bone density in Pyk2(-/-) mice. J Cell Biol. 2007 Sep 10; 178(6):1053-64. 18. Abu-Amer Y, Teitelbaum SL, Chappel JC, Schlesinger P, Ross FP. Expression and regulation of RAB3 proteins in osteoclasts and their precursors. J Bone Miner Res. 1999 Nov;14(11):1855-60. 12. Purev E, Neff L, Horne WC, Baron R. cCbl and Cbl-b act redundantly to protect osteoclasts from apoptosis and to displace HDAC6 from beta-tubulin, stabilizing microtubules and podosomes. Mol Biol Cell. 2009 Sep;20(18):4021-30. 13. Cook TA, Nagasaki T, Gundersen GG. Rho guanosine triphosphatase mediates the selective stabilization of microtubules induced by lysophosphatidic acid. J Cell Biol. 1998 Apr 6;141(1):175-85. 14. Chellaiah MA, Soga N, Swanson S, McAllister S, Alvarez U, Wang D, Dowdy SF, Hruska KA. Rho-A is critical for osteoclast podosome organization, motility, and bone resorption. J Biol Chem. 2000 Apr 21;275(16):119932002. 15. Faccio R, Teitelbaum SL, Fujikawa K, Chappel J, Zallone A, Tybulewicz VL, Ross FP, Swat W. Vav3 regulates osteoclast function and bone mass. Nat Med. 2005 Mar;11(3):284-90. 16. Lakkakorpi PT, Wesolowski G, Zimolo Z, Rodan GA, Rodan SB. Phosphatidylinositol 3-kinase association with the osteoclast cytoskeleton, and its involvement in osteoclast attachment and spreading. Exp Cell Res. 1997 Dec 15;237(2):296306. 17. Zhao H, Ito Y, Chappel J, Andrews NW, Teitelbaum SL, Ross FP. Synaptotagmin VII regulates bone remodeling by modulating osteoclast 19. Zhao H, Laitala-Leinonen T, Parikka V, Väänänen HK. Downregulation of small GTPase Rab7 impairs osteoclast polarization and bone resorption. J Biol Chem. 2001 Oct 19;276(42):39295-302. 20. Van Wesenbeeck L, Odgren PR, Coxon FP, Frattini A, Moens P, Perdu B, MacKay CA, Van Hul E, Timmermans JP, Vanhoenacker F, Jacobs R, Peruzzi B, Teti A, Helfrich MH, Rogers MJ, Villa A, Van Hul W. Involvement of PLEKHM1 in osteoclastic vesicular transport and osteopetrosis in incisors absent rats and humans. J Clin Invest. 2007 Apr;117(4):919-30. 21. Pavlos NJ, Xu J, Riedel D, Yeoh JS, Teitelbaum SL, Papadimitriou JM, Jahn R, Ross FP, Zheng MH. Rab3D regulates a novel vesicular trafficking pathway that is required for osteoclastic bone resorption. Mol Cell Biol. 2005 Jun;25(12):5253-69. 22. Zhao H, Ettala O, Väänänen HK. Intracellular membrane trafficking pathways in bone-resorbing osteoclasts revealed by cloning and subcellular localization studies of small GTPbinding rab proteins. Biochem Biophys Res Commun. 2002 May 10;293(3):1060-5. 23. Abu-Amer Y, Ross FP, Schlesinger P, Tondravi MM, Teitelbaum SL. Substrate recognition by osteoclast precursors induces C-src/microtubule association. J Cell Biol. 1997 Apr 7;137(1):247-58. 24. Jahn R, Scheller RH. SNAREs--engines for membrane fusion. Nat Rev Mol Cell Biol. 2006 Sep;7(9):631-43. 80 Copyright 2011 International Bone & Mineral Society IBMS BoneKEy. 2011 February;8(2):74-83 http://www.bonekey-ibms.org/cgi/content/full/ibmske;8/2/74 doi: 10.1138/20110493 25. Mizushima N, Yoshimori T, Levine B. Methods in mammalian autophagy research. Cell. 2010 Feb 5;140(3):31326. 26. Virgin HW, Levine B. Autophagy genes in immunity. Nat Immunol. 2009 May;10(5):461-70. 27. Levine B, Kroemer G. Autophagy in the pathogenesis of disease. Cell. 2008 Jan 11;132(1):27-42. 28. Cadwell K, Liu JY, Brown SL, Miyoshi H, Loh J, Lennerz JK, Kishi C, Kc W, Carrero JA, Hunt S, Stone CD, Brunt EM, Xavier RJ, Sleckman BP, Li E, Mizushima N, Stappenbeck TS, Virgin HW 4th. A key role for autophagy and the autophagy gene Atg16L1 in mouse and human intestinal Paneth cells. Nature. 2008 Nov 13;456(7219):259-63. 29. DeSelm C, Miller B, Zou W, Virgin H, Teitelbaum S. Autophagy related proteins mediate osteoclast function in vitro and in vivo. J Bone Miner Res. 2010;25(Suppl 1). [Abstract] 30. Helfrich MH, Nesbitt SA, Lakkakorpi PT, Barnes MJ, Bodary SC, Shankar G, Mason WT, Mendrick DL, Väänänen HK, Horton MA. Beta 1 integrins and osteoclast function: involvement in collagen recognition and bone resorption. Bone. 1996 Oct;19(4):31728. 31. Inoue M, Ross FP, Erdmann JM, AbuAmer Y, Wei S, Teitelbaum SL. Tumor necrosis factor alpha regulates alpha(v)beta5 integrin expression by osteoclast precursors in vitro and in vivo. Endocrinology. 2000 Jan;141(1):284-90. 32. Yamamoto M, Fisher JE, Gentile M, Seedor JG, Leu CT, Rodan SB, Rodan GA. The integrin ligand echistatin prevents bone loss in ovariectomized mice and rats. Endocrinology. 1998 Mar;139(3):1411-9. 33. Engleman VW, Nickols GA, Ross FP, Horton MA, Griggs DW, Settle SL, Ruminski PG, Teitelbaum SL. A peptidomimetic antagonist of the alpha(v)beta3 integrin inhibits bone resorption in vitro and prevents osteoporosis in vivo. J Clin Invest. 1997 May 1;99(9):2284-92. 34. Murphy MG, Cerchio K, Stoch SA, Gottesdiener K, Wu M, Recker R; L000845704 Study Group. Effect of L000845704, an alphaVbeta3 integrin antagonist, on markers of bone turnover and bone mineral density in postmenopausal osteoporotic women. J Clin Endocrinol Metab. 2005 Apr;90(4):2022-8. 35. McHugh KP, Hodivala-Dilke K, Zheng MH, Namba N, Lam J, Novack D, Feng X, Ross FP, Hynes RO, Teitelbaum SL. Mice lacking beta3 integrins are osteosclerotic because of dysfunctional osteoclasts. J Clin Invest. 2000 Feb;105(4):433-40. 36. Zhao H, Kitaura H, Sands MS, Ross FP, Teitelbaum SL, Novack DV. Critical role of beta3 integrin in experimental postmenopausal osteoporosis. J Bone Miner Res. 2005 Dec;20(12):2116-23. 37. Faccio R, Takeshita S, Zallone A, Ross FP, Teitelbaum SL. c-Fms and the alphavbeta3 integrin collaborate during osteoclast differentiation. J Clin Invest. 2003 Mar;111(5):749-58. 38. Weir EC, Lowik CW, Paliwal I, Insogna KL. Colony stimulating factor-1 plays a role in osteoclast formation and function in bone resorption induced by parathyroid hormone and parathyroid hormone-related protein. J Bone Miner Res. 1996 Oct;11(10):1474-81. 39. Zou W, Petrich B, Monkley S, Critchley D, Ginsberg M, Teitelbaum S. Talin is critical for osteoclast function by mediating inside-out integrin activation. J Bone Miner Res. 2010;25(Suppl 1). [Abstract] 81 Copyright 2011 International Bone & Mineral Society IBMS BoneKEy. 2011 February;8(2):74-83 http://www.bonekey-ibms.org/cgi/content/full/ibmske;8/2/74 doi: 10.1138/20110493 40. Shattil SJ, Kim C, Ginsberg MH. The final steps of integrin activation: the end game. Nat Rev Mol Cell Biol. 2010 Apr;11(4):288-300. 41. Faccio R, Grano M, Colucci S, Villa A, Giannelli G, Quaranta V, Zallone A. Localization and possible role of two different alpha v beta 3 integrin conformations in resting and resorbing osteoclasts. J Cell Sci. 2002 Jul 15;115(Pt 14):2919-29. 42. Soriano P, Montgomery C, Geske R, Bradley A. Targeted disruption of the csrc proto-oncogene leads to osteopetrosis in mice. Cell. 1991 Feb 22;64(4):693-702. 43. Schwartzberg PL, Xing L, Hoffmann O, Lowell CA, Garrett L, Boyce BF, Varmus HE. Rescue of osteoclast function by transgenic expression of kinasedeficient Src in src-/- mutant mice. Genes Dev. 1997 Nov 1;11(21):283544. 44. Epple H, Cremasco V, Zhang K, Mao D, Longmore GD, Faccio R. Phospholipase Cgamma2 modulates integrin signaling in the osteoclast by affecting the localization and activation of Src kinase. Mol Cell Biol. 2008 Jun;28(11):3610-22. 45. Zou W, Kitaura H, Reeve J, Long F, Tybulewicz VL, Shattil SJ, Ginsberg MH, Ross FP, Teitelbaum SL. Syk, c-Src, the alphavbeta3 integrin, and ITAM immunoreceptors, in concert, regulate osteoclastic bone resorption. J Cell Biol. 2007 Mar 12;176(6):877-88. 46. Sanjay A, Houghton A, Neff L, DiDomenico E, Bardelay C, Antoine E, Levy J, Gailit J, Bowtell D, Horne WC, Baron R. Cbl associates with Pyk2 and Src to regulate Src kinase activity, alpha(v)beta3 integrin-mediated signaling, cell adhesion, and osteoclast motility. J Cell Biol. 2001 Jan 8;152(1):181-95. 47. Duong LT, Rodan GA. PYK2 is an adhesion kinase in macrophages, localized in podosomes and activated by beta(2)-integrin ligation. Cell Motil Cytoskeleton. 2000 Nov;47(3):174-88. 48. Wu H, Reynolds AB, Kanner SB, Vines RR, Parsons JT. Identification and characterization of a novel cytoskeletonassociated pp60src substrate. Mol Cell Biol. 1991 Oct;11(10):5113-24. 49. Tehrani S, Faccio R, Chandrasekar I, Ross FP, Cooper JA. Cortactin has an essential and specific role in osteoclast actin assembly. Mol Biol Cell. 2006 Jul;17(7):2882-95. 50. Shao Y, Yang C, Elly C, Liu YC. Differential regulation of the B cell receptor-mediated signaling by the E3 ubiquitin ligase Cbl. J Biol Chem. 2004 Oct 15;279(42):43646-53. 51. Zou W, Reeve JL, Zhao H, Ross FP, Teitelbaum SL. Syk tyrosine 317 negatively regulates osteoclast function via the ubiquitin-protein isopeptide ligase activity of Cbl. J Biol Chem. 2009 Jul 10;284(28):18833-9. 52. Faccio R, Zou W, Colaianni G, Teitelbaum SL, Ross FP. High dose MCSF partially rescues the Dap12-/osteoclast phenotype. J Cell Biochem. 2003 Dec 1;90(5):871-83. 53. Mócsai A, Humphrey MB, Van Ziffle JA, Hu Y, Burghardt A, Spusta SC, Majumdar S, Lanier LL, Lowell CA, Nakamura MC. The immunomodulatory adapter proteins DAP12 and Fc receptor gamma-chain (FcRgamma) regulate development of functional osteoclasts through the Syk tyrosine kinase. Proc Natl Acad Sci U S A. 2004 Apr 20;101(16):6158-63. 54. Tomasello E, Vivier E. KARAP/DAP12/TYROBP: three names and a multiplicity of biological functions. Eur J Immunol. 2005 Jun;35(6):1670-7. 55. Koga T, Inui M, Inoue K, Kim S, Suematsu A, Kobayashi E, Iwata T, Ohnishi H, Matozaki T, Kodama T, 82 Copyright 2011 International Bone & Mineral Society IBMS BoneKEy. 2011 February;8(2):74-83 http://www.bonekey-ibms.org/cgi/content/full/ibmske;8/2/74 doi: 10.1138/20110493 Taniguchi T, Takayanagi H, Takai T. Costimulatory signals mediated by the ITAM motif cooperate with RANKL for bone homeostasis. Nature. 2004 Apr 15;428(6984):758-63. 56. Zou W, Zhu T, Craft CS, Broekelmann TJ, Mecham RP, Teitelbaum SL. Cytoskeletal dysfunction dominates in DAP12-deficient osteoclasts. J Cell Sci. 2010 Sep 1;123(Pt 17):2955-63. 57. Saltel F, Chabadel A, Zhao Y, LafageProust MH, Clézardin P, Jurdic P, Bonnelye E. Transmigration: a new property of mature multinucleated osteoclasts. J Bone Miner Res. 2006 Dec;21(12):1913-23. 58. Zou W, Reeve JL, Liu Y, Teitelbaum SL, Ross FP. DAP12 couples c-Fms activation to the osteoclast cytoskeleton by recruitment of Syk. Mol Cell. 2008 Aug 8;31(3):422-31. 59. Reeve JL, Zou W, Liu Y, Maltzman JS, Ross FP, Teitelbaum SL. SLP-76 couples Syk to the osteoclast cytoskeleton. J Immunol. 2009 Aug 1;183(3):1804-12. 60. Razzouk S, Lieberherr M, Cournot G. Rac-GTPase, osteoclast cytoskeleton and bone resorption. Eur J Cell Biol. 1999 Apr;78(4):249-55. 61. Hall A. G proteins and small GTPases: distant relatives keep in touch. Science. 1998 Jun 26;280(5372):2074-5. 62. Croke M, Ross F, Teitelbaum S. Rac deletion in osteoclasts causes severe osteopetrosis. J Bone Miner Res. 2010;25(Suppl 1). [Abstract] 63. Ito Y, Teitelbaum SL, Zou W, Zheng Y, Johnson JF, Chappel J, Ross FP, Zhao H. Cdc42 regulates bone modeling and remodeling in mice by modulating RANKL/M-CSF signaling and osteoclast polarization. J Clin Invest. 2010 Jun 1;120(6):1981-93. 64. Ross FP, Teitelbaum SL. alphavbeta3 and macrophage colony-stimulating factor: partners in osteoclast biology. Immunol Rev. 2005 Dec;208:88-105. 83 Copyright 2011 International Bone & Mineral Society R E V I E W Osteoclast Activity and Subtypes as a Function of Physiology and Pathology—Implications for Future Treatments of Osteoporosis K. Henriksen, J. Bollerslev, V. Everts, and M. A. Karsdal Nordic Bioscience A/S (K.H., M.A.K.), DK-2730 Herlev, Denmark; Section of Endocrinology (J.B.), Department of Medicine, Rikshospitalet, Oslo University Hospital and the University of Oslo, 0450 Oslo, Norway; and Department Oral Cell Biology (V.E.), Academic Centre of Dentistry Amsterdam, University of Amsterdam and VU University Amsterdam, Research Institute Move, 1066 EA Amsterdam, The Netherlands Osteoclasts have traditionally been associated exclusively with catabolic functions that are a prerequisite for bone resorption. However, emerging data suggest that osteoclasts also carry out functions that are important for optimal bone formation and bone quality. Moreover, recent findings indicate that osteoclasts have different subtypes depending on their location, genotype, and possibly in response to drug intervention. The aim of the current review is to describe the subtypes of osteoclasts in four different settings: 1) physiological, in relation to turnover of different bone types; 2) pathological, as exemplified by monogenomic disorders; 3) pathological, as identified by different disorders; and 4) in drug-induced situations. The profiles of these subtypes strongly suggest that these osteoclasts belong to a heterogeneous cell population, namely, a diverse macrophage-associated cell type with bone catabolic and anabolic functions that are dependent on both local and systemic parameters. Further insight into these osteoclast subtypes may be important for understanding cell– cell communication in the bone microenvironment, treatment effects, and ultimately bone quality. (Endocrine Reviews 32: 31– 63, 2011) I. II. III. IV. Introduction Bone Remodeling The Classical Osteoclast Osteoclast Subtypes in Physiological Situations A. Endochondral vs. intramembranous bone osteoclasts B. Chondroclasts C. Osteoclasts involved in targeted and stochastic remodeling D. Trabecular and cortical osteoclasts E. Diurnal variation in osteoclasts or osteoclast activity? V. Osteoclast Subtypes in Pathological Situations A. Osteoporotic osteoclasts B. Changes in osteoclast activities with increasing bone matrix age C. Osteoclast-rich osteopetrosis D. Osteoclast-poor osteopetrosis E. Pycnodysostotic osteoclasts ISSN Print 0021-972X ISSN Online 1945-7197 Printed in U.S.A. Copyright © 2011 by The Endocrine Society doi: 10.1210/er.2010-0006 Received March 19, 2010. Accepted August 10, 2010. First Published Online September 17, 2010 F. Other diseases characterized by increased osteoclast activity VI. Drug-Induced Osteoclast Subtypes A. Existing drugs B. Future treatments VII. The Bone Anabolic Effects of the Osteoclasts VIII. Conclusions and Future Perspectives I. Introduction steoclasts are multinucleated bone-resorbing cells that are unique in their ability to degrade mineralized matrices, such as bone and calcified cartilage (1). Osteoclasts have for a long time been considered boneresorbing “machines,” yet some years ago it was demonstrated that not all osteoclasts are the same and that careful elucidation of the osteoclast subtype may prove O Abbreviations: AGE, Advanced glycation end-product; BMD, bone mineral density; CAII, carbonic anhydrase II; ClC-7, chloride channel 7; CT-1, cardiotrophin-1; CTX, C-terminal crosslinked telopeptide of type I collagen; ER, estrogen receptor; GLP, glucagon-like peptide; HRT, hormone replacement therapy; ICTP, carboxyterminal peptide of type I collagen; MMP, matrix metalloproteinase; ONJ, osteonecrosis of the jaw; OPG, osteoprotegerin; RA, rheumatoid arthritis; RANK, receptor activator of nuclear factor B; RANKL, RANK ligand; SERM, selective estrogen receptor modulator; TRACP, tartrate-resistant acid phosphatase; V-ATPase, vacuolar type ATPase. Endocrine Reviews, February 2011, 32(1):31– 63 edrv.endojournals.org 31 32 Henriksen et al. Osteoclast Subtypes beneficial (1– 4). As illustrated in Table 1, under normal circumstances, osteoclasts are influenced by a complex combination of systemic hormones and local mediators present in the different bones. Under pathological conditions, such as cessation of estrogen production or inflammatory conditions, additional cytokines are present. Even the bone type and age may influence the phenotype of the osteoclast (2, 5–7). Finally and importantly, different classes of drugs used for treatment of osteoporosis and other diseases also influence the osteoclasts significantly. The aim of this review is to provide a thorough description of the complex nature of osteoclasts under healthy and diseased states and to describe their modulation by drugs that have been approved for use or are under development. The paper will also emphasize the role of osteoclasts in initiating bone formation, a recently discovered activity of these cells that has gained much attention (3, 8 –13). II. Bone Remodeling Bone remodeling is required for optimal control of calcium homeostasis and strength of the bones and is essential for the continued maintenance of a healthy skeleton (14). Bone remodeling is performed by three cell types: 1) the osteoclasts, which are the sole cells in the body possessing the ability to degrade both the inorganic calcium matrix and the organic collagen matrix; 2) the osteoblasts, which are the bone-forming cells; and 3) the osteocytes, which appear to regulate the activity of both osteoclasts and osteoblasts (14, 15). In healthy adults, under normal circumstances, bone resorption is always followed by an equal degree of bone formation, a tightly balanced process referred to as coupling (9, 16). The modulation of activities of the cells involved in the remodeling cycle was recently described in detail (15). Coupling was initially discovered in the 1960s when Frost and co-workers (17, 18) demonstrated that osteoblasts filled the resorption pits created by osteoclasts in more than 97% of the cases (17–21). Since then, coupling has been understood as a coordinated and balanced induction of osteoblastic bone formation in response to prior bone resorption (19). Uncoupling occurs when the balance between resorption and formation is disrupted, which often leads to pathological situations such as osteoporosis or osteopetrosis (3, 9, 14, 22, 23). However, uncoupling also occurs under physiological conditions, i.e., during skeletal growth in children, where bone formation exceeds bone resorption (10). Hypogonadal osteoporosis is usually caused by a decrease or loss of sex steroid production, which results in accelerated osteoclastogenesis and bone resorption (24) Endocrine Reviews, February 2011, 32(1):31– 63 that cannot be completely countered by an increase in bone formation. This results in low bone mass, in deterioration of the microarchitecture of the skeleton, and often in fractures (25). Osteoporotic fractures are associated with increased morbidity and mortality and give rise to a significant public health problem (24). Osteopetrosis, on the other hand, is a rare, inherited disease in various species including man, which was originally identified by Albers-Schönberg in 1904 (26). In the majority of cases, it is caused by defective resorption by the osteoclasts, resulting in high bone mass with poor bone quality and increased fracture frequency due to defective bone remodeling (1, 26, 27). However, recent studies also characterized patients with osteopetrosis due to dysfunctional osteoclastogenesis either directly affecting the osteoclast precursors or indirectly through the osteoblasts, and thus the phenotype was caused by the absence of osteoclasts, rather than inactivity of these cells (28 –31). Interestingly, the studies of osteopetrotic patients have indicated that the presence of osteoclasts, but not their activity, is essential for bone formation, indicating that some aspects of the coupling principle should be revised (1, 3). Because hypogonadal osteoporosis is associated with increased numbers and activity of osteoclasts (16), most treatments developed so far, such as bisphosphonates and hormone replacement therapy (HRT)/selective estrogen receptor modulators (SERMs), have focused on eliminating or reducing the number of osteoclasts and thereby reducing bone resorption (32). These treatments are associated with secondary decreases in bone formation due to the coupled nature of the bone remodeling process, which naturally limits their efficacy (3, 24). However, as seen in the osteopetrotic syndromes, there are indications that bone resorption and bone formation can be dissociated, and from recent studies it appears that the osteoclast itself, whether it is a physiological, pathological, or druginduced subtype, is highly important for a secondary effect on bone formation (1, 3). In this review, we describe differences in osteoclast activity and subtypes in relation to physiology, pathophysiology, and medication, with special attention to coupling in the bone remodeling process. Ultimately, this review highlights potential directions for new treatment modalities. III. The Classical Osteoclast Osteoclastogenesis is a complex process requiring both the correct extracellular stimuli and the correct cellular molecules to interact without impediment (22, 33). Osteoclasts arise from hematopoietic stem cells that, in the presence of Endocrine Reviews, February 2011, 32(1):31– 63 edrv.endojournals.org 33 TABLE 1. A simplified summary of osteoclast phenotypes as a function of physiology, pathology, and drugs, also indicating areas of osteoclast biology that are not well-understood Osteoclast no. Bone resorption Classical osteoclast Physiology Targeted Bone formation Resorptive process Acid Cat K MMP ⫹⫹⫹ ⫹⫹⫹ ⫹/⫺ Balanced Balanced Normal Normal Recruitment to specific areas increased Not clear Normal Normal Normal Normal Normal Increased ⫹⫹ ? ? Not clear Increased Decreased Normal Normal Normal ⫹ ⫹⫹ ⫹ ⫹ ⫹ ⫹ ? ⫹⫹ ? ⫹ ⫹ ⫺ ? ⫹/⫺ ? ⫹⫹ ⫺ ⫹ Normal Normal Normal Normal ⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹ ⫺ ⫺ Increased Increased ⫹ ⫹ ⫹⫹ ⫺ Age OC-rich OP Increased Greatly increased Increased Decreased ⫹⫹ ⫺ ⫹⫹ ⫺ ? ⫺ OC-poor OP Pycnodysostosis No osteoclasts Unchanged/increased osteoclast size Greatly increased at local sites Decreased Decreased ⫺ ⫹ ⫺ ⫺ ⫺ ⫹⫹⫹ ⫹⫹ ⫹ ⫹⫹⫹ Greatly increased at local sites Greatly increased at local sites Not known ⫹ ⫹ ⫹⫹⫹ Increased at local sites ⫹ ⫹ ⫹⫹⫹ Drug-induced BPs Reduced Decreased ⫺ ⫺ ? HRT/SERMs Reduced Decreased ⫺ ⫺ ? Calcitonin Unchanged Decreased ⫺ ⫺ ? PTH Increased/unchanged Decreased ⫹⫹ ⫹⫹ ? Strontium ranelate GCs Denosumab Unchanged Unchanged/increased Greatly reduced Decreased? Unchanged/increased Decreased ⫺ ⫹ ⫺ ⫺ ⫹ ⫺ ? ? ⫺ Cat K inhibitors Unchanged Decreased ⫹ ⫺ ⫹⫹⫹ GLP-2 Unchanged Decreased ⫺ ⫺ ? Acidification inhibitors Increased Decreased ⫺ ⫺ ⫺ Stochastic Night Day Chondroclast Endochondral Intramembraneous Trabecular Cortical Pathology Osteoporosis Paget’s RA Lytic metastases Increased at local sites Not clear Minor up-regulation Minor down-regulation Balanced Balanced Not balanced, opposite side of bone than resorption Balanced Balanced Increased, but less than resorption Decreased Increased according to increased OC number Decreased? Not clear Increased locally, but does not compensate resorption Not known Increased locally, but does not compensate resorption Decreased secondary to resorption Decreased secondary to resorption Not changed or minor decrease Increased, but only temporarily Increased Decreased strongly Decreased secondary to resorption Decreased secondary to resorption Not changed, but long term effects are not known Increased, but so far only in animal models The table shows the subtype of osteoclasts, the number of osteoclasts, the effect on bone resorption, which part of the resorption machinery that is active/affected, and the effect on bone formation. OC, Osteoclast; OP, osteopetrosis; Cat K, cathepsin K; BPs, bisphosphonates; GCs, glucocorticoids; MMP, matrix metalloproteinase; RA, rheumatoid arthritis; PTH, parathyroid hormone; HRT, hormone replacement therapy; SERMs, selective estrogen receptor modulators; GLP-2, glucagon-like peptide 2. 34 Henriksen et al. Osteoclast Subtypes Endocrine Reviews, February 2011, 32(1):31– 63 FIG. 1. Schematic illustration of the molecules involved in osteoclastogenesis and function. (See Refs. 1, 33, and 42). NFB, nuclear factor B; TRAF6, TNF receptor-associated factor 6; nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1; PYK2, Proline-rich Tyrosine Kinase 2; MitF, microphthalmia-associated transcription factor; ClC-7; Chloride Channel 7; PLEKHM1, pleckstrin homology domain containing, family M (with RUN domain) member 1; osteopetrosis associated transmembrane protein 1; CA2, carbonic anhydrase II; AE2, anion exchanger 2. receptor activator of nuclear factor B (RANK) ligand (RANKL) and macrophage-colony stimulating factor, undergo differentiation and fusion resulting in large multinucleated cells characterized by expression of a series of osteoclast markers, such as tartrate-resistant acid phosphatase (TRACP), matrix metalloproteinase (MMP)-9, cathepsin K, carbonic anhydrase II (CAII), the a3 subunit of the vacuolar [H⫹]-ATPase, chloride channel 7 (ClC-7), osteopetrosis-associated transmembrane protein 1, and the calcitonin receptor (34 – 41). Osteoclastogenesis and the molecules involved in this process are summarized in Fig. 1, but are not discussed in any further detail because several excellent reviews have been published recently on this topic (1, 22, 33, 42). Polarization and formation of the sealing zone, which is a specialized ring structure containing a high number of ␤-actin filaments, are the next steps in the life span of an osteoclast (43, 44). These processes require the ␣v␤3 integrin and the intracellular signal transducers c-src, Syk, and proline-rich tyrosine kinase 2, as well as the microphthalmia transcription factor (33, 45, 46), which appears to be an important regulator of osteoclastic gene transcription (47– 49). The final step of osteoclastogenesis is the activation of resorption, a process that is characterized by the formation of a ruffled border that is an intensely convoluted membrane present inside the sealing zone (43, 44). The formation of the ruffled border is not well-characterized; however, the signaling molecule Rab7 is required (50). Bone resorption takes place at the ruffled border localized at the apical side of the osteoclasts, and it can be divided into two processes, namely acid secretion and pro- teolysis, although these processes likely occur at the same time (44, 51). Bone resorption is initiated by active secretion of protons through a vacuolar type ATPase (V-ATPase) and passive transport of chloride through a chloride channel (52, 53). The secretion of hydrochloric acid leads to dissolution of the inorganic matrix of the bones (54). The osteoclastic V-ATPase is functionally specific and contains the a3 subunit, and accordingly, loss of a3 leads to osteopetrosis (37, 55–57). In both mice and man, the chloride channel ClC-7, has been shown to mediate chloride transport, thereby ensuring the electrochemical balance required for intense acidification (Fig. 2) (8, 39, 58). Recent data showed that ClC-7 functions as a proton-chloride antiporter (59, 60). To generate the necessary levels of H⫹ and Cl⫺, the enzyme CAII catalyzes conversion of CO2 and H2O into H2CO3, which ionizes into H⫹ and HCO3⫺ (35), thereby providing the protons for the V-ATPase (27). Meanwhile, basolateral exchange of HCO3⫺ ions for Cl⫺ by anion exchanger 2 (61– 63) provides Cl⫺ ions required for the intense acidification occurring in the resorption lacuna. Interestingly, long bones differ from flat bones with respect to the molecular nature of the acidification machinery (62). Proteolysis of the type I collagen matrix in bones is mainly mediated by the cysteine proteinase, cathepsin K. This enzyme is active at low pH in the resorption lacuna (Fig. 2) (64 – 68). The neutral MMPs also appear to play a minor role during organic matrix degradation; however, the exact role of MMPs is still being investigated (69) and is highly dependent on the bone type (38, 70, 71). The resorbed material is removed from the resorption pit by Endocrine Reviews, February 2011, 32(1):31– 63 edrv.endojournals.org 35 FIG. 2. Top, Schematic illustration of the differences between acid secretion and proteolysis during osteoclastic bone resorption, illustrating that the collagen matrix is removed by proteolysis after acidification. Bottom, Mutations/knockout in genes/proteins involved in bone resorption, phenotypes, and effect on osteoclasts. Ae2, Anion exchanger 2; a3 V-ATPase, a3 subunit of the osteoclast-specific V-ATPase. ClC-7, chloride channel 7; OSTM1, osteopetrosis associated transmembrane protein 1; CA2, carbonic anhydrase 2; MMP-9, matrix metalloproteinase 9. uptake and transcytosis through the osteoclast (72, 73). After completing resorption, osteoclasts either undergo apoptosis or perform a further round of resorption (44) (Table 1). In summary, the osteoclasts are highly specialized for both dissolution of the inorganic matrix and degradation of the organic matrix of the bones. These highly polarized cells are characterized by a unique set of membrane-bound molecules that ensure an efficient resorption of bone and other mineralized tissues. This complex machinery may be affected by a range of important parameters in physiology and pathology, and importantly in drug-induced situations that are important to identify and advance osteoclast research and biology. IV. Osteoclast Subtypes in Physiological Situations Osteoclast activities are essential for development, as well as remodeling of bone in response to aging and stress (6, 14, 15). Under normal physiological circumstances, the osteoclasts can be categorized into subgroups depending on the matrix on which they are positioned, the time of day, and the type of remodeling in which they participate. These different groups of osteoclasts have provided key information on skeletal maintenance. A. Endochondral vs. intramembranous bone osteoclasts Anatomically, two types of bones are present in the body, the long bones (e.g., the femur and tibia) and the 36 Henriksen et al. Osteoclast Subtypes flat bones (e.g., the calvarium), with the main difference between these two types of bone being their development (74). Studies also indicate that osteoclasts on these two types of bones are functionally different with respect to both acid secretion and proteases involved in degradation (2). Evidence that differences between resorption of flat and long bone exist was presented in 1999 (4). However, indications that even the acidification process is different have been published only recently (62). Data from mice deficient in the bicarbonate-anion exchanger Ae2 (Slc4a2) have shown that it is essential for bone resorption in long bones (61, 63), whereas it is not involved in bone resorption in calvariae (62), showing that distinct acid transport mechanisms are present in different subsets of osteoclasts. With respect to acid secretion into the resorption lacunae, it is presently not known whether any differences exist, although the absence of calvarial thickening in patients with defective ClC-7 strongly suggests that ClC-7 is not involved in resorption of the flat bones (75). Extensive research into the proteolytic processes involved in resorption of flat and long bones clearly demonstrates that the proteolytic processes involved in degradation of these two types of bone matrix are distinct (4, 71). Osteoclasts in flat bones preferentially appear to engage in MMP-mediated bone resorption, although cathepsin L seems to be involved, too. Osteoclasts in long bones primarily depend on cysteine proteinases, in particular cathepsin K (4, 71). TRACP also appears to be involved in bone resorption, and more so in calvarial bone (76 –78). When osteoclasts generated from human peripheral blood are seeded on cortical bone, they primarily depend on cathepsin K, whereas when cathepsin K activity is blocked, there is some compensatory bone resorption mediated by MMPs (69). How these osteoclasts behave on bone substrates other than cortical bone is presently not known (Table 1). Data suggesting that the bone matrix could play a role in the control of osteoclastic activities were presented in a study showing compositional differences between long bone and flat bone matrices, including differences in the presence of putative cysteine proteinase inhibitors (79). The functional significance of these data still remains to be fully elucidated, although they clearly illustrate the importance of understanding how a given context affects the osteoclasts. B. Chondroclasts It has long been discussed whether chondroclasts are a “real” cell type or whether they simply are osteoclasts that reside on cartilage instead of bone (77, 80 – 82). Chondroclasts are mainly important in endochondral bone development, and in addition there is some evidence that Endocrine Reviews, February 2011, 32(1):31– 63 chondroclasts may also play a role in both rheumatoid arthritis (RA) and osteoarthritis (83– 85). The term chondroclast derives from the localization of these cells on calcified cartilage as seen in the expanding growth plates during endochondral ossification (80, 86). For their formation, these cells are dependent on the presence of macrophage-colony stimulating factor and RANKL, as are bone-resorbing osteoclasts (87–90). Most of the evidence for the functionality of chondroclasts is derived from studies of the longitudinal growth of long bones, i.e., metatarsals and tibias isolated from mouse embryos (38, 80, 91–93). First, bone/cartilage resorption in these models is still dependent on acid secretion as evidenced by mice with mutations in the acid secretion process, i.e., ClC-7-deficient and a3 V-ATPase-deficient mice, as well as mice unable to form sealing zones, i.e., c-src-deficient mice, in which the massive bones mainly consist of calcified cartilage due to the defective resorption process (56, 94, 95). Similar findings have been noted in the corresponding human disease(s) (27, 96, 97). Interestingly, the ruffled border is less prominent in the chondroclasts than osteoclasts, potentially suggesting that lower levels of acid secretion are required for dissolution of this matrix (98, 99). The main difference between chondroclasts and osteoclasts is in the profiles of enzymes necessary for tissue degradation. Resorption by chondroclasts does not appear to depend as much on cathepsin K as does resorption by osteoclasts. Cathepsin K-deficient mice show no evidence of calcified cartilage in the marrow cavity of long bones, indicating that the removal of calcified cartilage during endochondral ossification occurs, although there are indications that the process is delayed (65, 100). Of importance is the observation of a massive compensation by MMPs in the absence of cathepsin K, which obscures the interpretation of data from cathepsin K-deficient systems (69, 70, 101–103). Finally, one study has indicated that TRACP is activated and secreted at the ruffled border in cathepsin K-deficient osteoclasts, and only in cells that play a role in the removal of calcified cartilage (104) (Table 1). Although chondroclasts are involved in the degradation of a different matrix than osteoclasts, an interesting observation is that bone formation is tightly coupled to resorption of the mineralized matrix, as has clearly been demonstrated in studies of endochondral ossification (86). It is more likely that bone formation is coupled to chondroclast numbers because release of molecules from degradation of cartilage, which in composition is far from bone, would be expected to be different from molecules released during bone resorption; however, this has never been studied in detail. Endocrine Reviews, February 2011, 32(1):31– 63 edrv.endojournals.org 37 FIG. 3. Schematic illustration of stochastic vs. targeted remodeling. The figure illustrates the local nature of targeted remodeling, which is activated at specific sites after the formation of microcracks and leads to removal of the microcrack and restoration of the damaged bone. Stochastic remodeling, on the other hand, is of a systemic nature and is activated by low calcium levels in the circulation leading to PTH release. Other hormones, such as vitamin D3 (VitD3), and potentially calcitonin also play roles in stochastic remodeling. It appears that there are two levels of calcium homeostasis— one mediated by osteocytes independent of osteoclasts, and one including the osteoclasts—although the balance between these two ways of releasing calcium still remains to be fully understood. C. Osteoclasts involved in targeted and stochastic remodeling Two different modes of remodeling have been proposed: targeted and stochastic. Targeted bone remodeling takes place at specific sites, whereas stochastic remodeling occurs more randomly (19, 105, 106). The first type is primarily performed to replace microdamaged bone and thus to maintain the load-bearing capacity of the skeleton. The second type of remodeling appears random with respect to localization, although it may be involved in maintaining integrity of the bones, independent of damage. This process is hormonally regulated (105, 107). The balance between these two modes of bone remodeling has not been fully elucidated yet, but studies in dogs indicate that approximately 30% of all remodeling is targeted and the remaining 70% is stochastic (105). With respect to the osteoclasts mediating these two types of remodeling, most studies have focused on how targeted remodeling is controlled. It appears that the bone matrix contains signals regulating the activity of the osteoclasts (108). A recent study demonstrated that aged bones were more readily resorbed than young bones, thus supporting the hypothesis that the bone matrix composition influences remodeling rates (7). Furthermore, areas of microdamage, which are character- ized by high numbers of apoptotic osteocytes, are preferentially and rapidly degraded by osteoclasts. This further supports the possibility that changes in the bone matrix and the balance between live and dead osteocytes determine which areas will be remodeled (108). Finally, a recent study demonstrated that targeted ablation of osteocytes led to a dramatic increase in osteoclast activity (109) (Table 1). These data indicate that death of osteocytes is a key point in induction of osteoclast activity (Fig. 3). Taken together with the finding that bones of different age lead to different levels of osteoclastogenesis (7), it appears that osteoclast functionality is at least partially controlled by osteocyte-derived molecules, which are sequestered in the bone matrix. Stochastic remodeling, while occurring at random sites, is centrally regulated by hormones such as PTH, vitamin D3, and potentially calcitonin, and its main role is the regulation of calcium homeostasis (110 –112). It has even been questioned to what extent this process depends on the presence of osteoclasts because patients with nonfunctional osteoclasts have normal calcium homeostasis (110 – 112). Yet, when they are calcium-deprived, osteopetrotic patients fail to correct their calcium levels, indicating that osteoclasts, which are either absent or nonfunctional in 38 Henriksen et al. Osteoclast Subtypes these patients, do play a role in calcium homeostasis, and thus stochastic remodeling (Fig. 3) (113). In summary, targeted remodeling is beginning to be understood in detail, and it is a tightly regulated and coupled process involving osteocytes, osteoclasts, and cells of the osteoblast lineage. On the other hand, stochastic remodeling and the role it plays in calcium homeostasis are still not very well understood, although there are indications that there is a level of regulation by the activity of osteoclasts. D. Trabecular and cortical osteoclasts Bone remodeling does not occur with the same frequency in cortical and trabecular bone. Every year, 25% of the trabecular bone matrix, but only 4% of the cortical matrix, is remodeled (114). Interestingly, most in vitro osteoclast experiments are based on cortical bone (or dentine) substrates, which are either slowly remodeled or not remodeled at all (7, 102, 114 –116). Studies have shown that bones endogenously contain signals regulating osteoclastogenesis and resorption and that these signals appear to be related to the age of the bone (7, 117) (Table 1). Thus, an interesting question is whether osteoclasts themselves are indeed different when derived from different matrices or whether the difference is matrix related. Furthermore, systemic regulation is likely to be involved in controlling which bones are resorbed to some extent. These data also correlate with evidence indicating that remodeling of different bone compartments can be either primarily targeted, such as in the cortex, or primarily stochastic, as seen in some parts of trabecular bone (106). A further understanding of this could provide directions for the development of novel drugs producing optimal benefit at the sites where it is most needed, i.e., leading to a better fracture reduction than that presently obtained. E. Diurnal variation in osteoclasts or osteoclast activity? Bone resorption markers measured in serum may be interpreted as indicating the net result of all osteoclast subtypes and activity levels at one particular time. A wide range of factors, known and unknown, may influence the interpretation (15). Diurnal variation is a well-established and important parameter of bone turnover. Postprandially, bone resorption decreases by approximately 50% compared with that of fasting individuals, but during the night, bone resorption increases to an equally large degree (118 –120). Several investigations have demonstrated that the circadian variation in bone resorption is induced in part by food intake (121–123), which, at least partially, involves the peptide hormone glucagon-like peptide (GLP) 2 (124). Interestingly, the osteoclast number does not appear to depend on the time of day, further emphasizing differences between osteoclast Endocrine Reviews, February 2011, 32(1):31– 63 number and activity (32) (Table 1). An interesting aspect of this is that targeting nocturnal resorptive activity appears to lead to inhibition of bone resorption, whereas not attenuating bone formation (125–127), thereby highlighting an interesting prospect of reducing bone resorption in a specific, nocturnal manner. In summary, studies of osteoclasts under different physiological conditions, such as those listed above, have highlighted the heterogeneity of these cells. Furthermore, these studies highlighted the importance of the balance between bone resorption and bone formation, a tightly regulated phenomenon that rarely is disturbed under physiological conditions. Finally, how the heterogeneity of the osteoclasts affects bone formation is presently not well understood, but a further understanding of this process could help optimal treatment of diseases involving alterations in bone remodeling. V. Osteoclast Subtypes in Pathological Situations Changes in osteoclast activity and number have been detected in several diseases, ranging from illnesses involving excessive bone resorption, such as osteoporosis and Paget’s disease; to those involving secondary activation of osteoclasts, such as osteolytic metastases and RA; to diseases involving defective osteoclast differentiation and/or function, such as osteopetrosis. These different types of diseases have shed important light on osteoclastic function with respect to obtaining the right type of treatment. They have also shed light on a very central aspect in bone biology, the coupling principle. The coupling principle describes the phenomenon that bone formation follows bone resorption, which leads to a complete restoration of the bone removed during bone resorption (17). A. Osteoporotic osteoclasts 1. Changes in osteoclastogenic potential in osteoporosis An important aspect of osteoporosis is whether the number of osteoclast precursors in the circulation increases, and, if so, whether the osteoclastogenic potential of these cells is increased. Eghbali-Fatourechi et al. (128) showed that the overall number of cells expressing RANKL is increased in postmenopausal women compared with premenopausal or estrogen-treated women, clearly indicating that the bone marrow microenvironment, including stromal, T, and B cells, changes in a proosteoclastic direction when estrogen is reduced. These data were supported by a recent study from the same authors showing that bone marrow cells isolated from estrogentreated or control postmenopausal women displayed Endocrine Reviews, February 2011, 32(1):31– 63 reduced osteoclastogenic potential (129) (Table 1). Estrogen was shown to have a dual mode of action—the first leading to overall lower RANKL expression by bone marrow cells, and the second reducing the osteoclastogenic response to RANKL (130). Interestingly, aging of mice was also shown to increase the osteoclastogenic potential of bone marrow cells, both by upregulation of RANKL production and by increasing precursor sensitivity to RANKL (131). In vitro studies of the changes in cellular activity of osteoclasts from osteoporosis patients are limited, but these have indicated both an accelerated osteoclastogenesis and resorption (132, 133). Furthermore, a key cell in the up-regulation of osteoclastogenesis appears to be the T cell, which responds to lowered estrogen levels by increasing RANKL production (134). The main issue with all the studies of osteoporotic osteoclasts and their precursors is the use of mixed cell populations, which clouds the interpretation of the results, and therefore these aspects of osteoclastic function still require further investigation. Furthermore, with the recent publication of the possibility of assessing the “anabolic potential” of osteoclasts (11, 13), it would be of interest to investigate the anabolic capacity of osteoporotic osteoclasts and thus shed light on the imbalance between bone resorption and bone formation in osteoporosis. 2. A direct role for sex steroids on osteoclasts The role of estrogen on cells belonging to the osteoclastic lineage has been studied extensively, with several findings indicating that estrogen suppresses osteoclastogenesis but not the resorptive activity of mature osteoclasts (135– 137). Androgens, such as dihydrotestosterone, exhibit similar effects to estrogen on osteoclasts in vitro, although this has not been studied in great detail (138). Finally, a recent study using mice deficient for the estrogen receptor (ER)-␣, specifically in mature osteoclasts, showed bone loss in female, but not male, mice (139). This demonstrated that estrogen likely plays a direct role in bone resorption by even mature osteoclasts (139). Although the authors used the cathepsin K promoter to ensure specific knock-down of the ER-␣ in osteoclasts, cathepsin K is also expressed in preosteoclasts, albeit to a lower extent (139). More studies are needed to investigate the role of ER-␣ in mature osteoclasts specifically. Interestingly, osteoclasts in different bone sites preferentially express different ERs, with cortical osteoclasts mainly expressing ER-␣ and trabecular osteoclasts mainly expressing ER-␤ (140), whereas from a functional point of view ER-␣ appears to be more relevant for trabecular, not cortical, bone (141). Furthermore, the expression pattern also differs between mature and differentiating osteoclasts; ER-␣ is mainly expressed in immature cells, and ER-␤ is present at all stages edrv.endojournals.org 39 of osteoclastogenesis (137). Again, there appear to be different osteoclastic subtypes, which also appear to be relevant in the context of bone loss rates in different bone compartments during osteoporosis (114). As indicated above, an important point is the difference between genders (139). In mice, the gender-based difference between cortical apposition and endocortical resorption that becomes more apparent with increasing age might be explained by differences in ER expression (142). With respect to changes in the osteoclasts after menopause, a couple of studies have clearly demonstrated that bone resorption, as well as bone formation, increases in women after menopause (143–145), and these changes become more explicit in high- and low-turnover patients (145). Although bone formation increases as a function of the increased resorption, it does not match bone resorption, thereby illustrating the importance of understanding the interplay between osteoclasts and osteoblasts in detail. B. Changes in osteoclast activities with increasing bone matrix age Numerous studies have investigated the control of osteoclast activity as a function of changes in biochemical properties of the bone matrix. Aging leads to accumulation of different biochemical modifications of the bone matrix, such as advanced glycation end-products (AGEs), homocysteine, increased calcium concentration, as well as some modifications of the collagen matrix (146). Recent studies have indicated that these modifications of the bone matrix itself actually modulate the activity of the osteoclasts to a certain extent (7, 117). Homocysteine, which accumulates in bone and in circulation with age, was shown to activate osteoclastogenesis and bone resorption (147) (Table 1). AGEs are modifications of proteins that accumulate in various tissues with age, and they have been implicated in the pathology of osteoporosis (146, 148). Some evidence indicating a direct regulation of osteoclast activity by AGEs has been published, but these studies are contradictory. One study shows activation of resorption by AGE-modified proteins (149), whereas the other study shows the opposite (117); however, quite different techniques were used. Interestingly, AGEs are accumulated in diabetes, and they have been speculated to be involved in the increased fracture rates observed in patients with this disease (150, 151). Another intriguing finding is the induction of apoptosis in osteoblasts by AGEs (152), which potentially could play a role in the imbalance between osteoclast and osteoblast function during osteoporosis and aging. These findings are all preliminary in nature, and they await confirmation from independent research groups. However, once again they illustrate the heterogeneity of osteoclasts, 40 Henriksen et al. Osteoclast Subtypes in this case as a function of matrix age, and the importance of understanding this phenomenon. C. Osteoclast-rich osteopetrosis The most frequently occurring forms of osteopetrosis are those caused by mutations in either the a3 subunit of the V-ATPase, ClC-7, or osteopetrosis-associated transmembrane protein 1. Osteoclasts from patients with mutations in these genes or proteins and from knockout mice have been studied quite extensively (8, 39, 56, 58, 95, 153–156). Microscopic analyses of cells from patients with defective acid secretion by osteoclasts indicated defective ruffled border formation, but also accumulation of material in vesicles, indicating hampered transcytosis (157). Apart from confirming the defective acid secretion and thereby bone resorption, when either ClC-7 or the a3 subunit is mutated (8, 39, 56, 58, 95, 153, 154), these studies also shed light on important aspects of bone remodeling. In vitro studies indicate that osteoclasts with impaired acid secretion have higher survival rates than cells with normal secretion, due to the reduced release of proapoptotic signals during resorption (159). This observation correlates well with the high numbers of osteoclasts observed in vivo in this group of patients, as well as with findings in mice with attenuated acidification of the resorption lacunae (95, 97) (Table 1). Furthermore, significantly increased resorbed areas are seen during impaired acid secretion, but the resorption pits are shallow, indicating a disturbed activity of the osteoclasts (97). More importantly, these studies highlighted that bone formation in these patients is ongoing—a process that appears to be correlated to the increased number of osteoclasts rather than bone resorption (96, 154, 160, 161). These findings contrast with the classical perception that bone formation always follows bone resorption in a tightly coordinated manner and illustrate the importance of the actual presence of osteoclasts to maintain bone formation. D. Osteoclast-poor osteopetrosis Several murine forms of osteoclast-poor osteopetrosis have been described in the literature (1, 42). In general, whereas the mutations express a pronounced osteopetrotic phenotype and few or no osteoclasts are present, the phenotypes are less severe than the phenotypes of the different osteoclast-rich osteopetrotic mutations (1). These data strongly suggest that the osteoclasts are indeed involved in the production of anabolic signals for bone formation (3, 15). Studies of mice deficient in c-src and c-fos, a key molecule involved in ruffled border formation and a key signal transducer for osteoclastogenesis, clearly demonstrated Endocrine Reviews, February 2011, 32(1):31– 63 that osteopetrosis was due to nonfunctional osteoclasts or the absence of osteoclasts, respectively (162, 163). Interestingly, these two groups of mice have opposing phenotypes with respect to bone formation. The osteoclast-rich c-src knockouts have increased bone formation (164), and the osteoclast-poor c-fos knockouts have decreased bone formation (165). The anabolic effects of PTH are present in the c-src⫺/⫺ mice but are blunted in the c-fos⫺/⫺ mice (166), indicating that osteoclasts are central for bone formation (Table 1). Two recent studies identified mutations in the genes for RANK and RANKL as the causes of osteopetrosis in a novel group of patients (28, 29). No indications of osteoclasts were found in these patients (28, 29), which is consistent with previous observations in mice deficient in both RANKL and RANK (87, 90). Patients with mutations in either RANKL or RANK have a pronounced osteopetrotic phenotype and classical histological hallmarks of osteopetrosis including unresorbed primary spongiosa. However, although limited data have been published, the osteopetrotic phenotype appears to be less severe than the one observed in the osteoclast-rich forms (1). Thus, mutations within the RANK/RANKL/osteoprotegerin (OPG) system can lead to osteoclast-poor osteopetrosis with low bone formation in mice and men. Interestingly, alterations in osteoblast function, such as changes in the production of RANKL and OPG, may have the same effect. Stabilizing osteoblastic ␤-catenin in transgenic mice, thus mimicking constitutive activation of the canonical Wnt signaling pathway, was followed by an up-regulation of OPG in relation to RANKL (31, 167). As expected, the mice developed osteoclast-poor osteopetrosis with failure of tooth eruption, a classical phenomenon in murine osteopetrosis. Mutations within LRP5 related to the Wnt signaling pathway have underscored the fundamental importance of this pathway for regulation of bone mass. The osteoporosis pseudoglioma syndrome was found to be caused by loss of function mutations in the gene for LRP5 (168). In contrast, mutations affecting the first propeller of the coreceptor, presumed to be followed by chronic activation of the Wnt pathway, were found in various forms of monogenic human osteosclerotic phenotypes (169). Among these, autosomal dominant osteopetrosis type 1 has been well characterized clinically, biochemically, histomorphometrically, and biomechanically (75). Autosomal dominant osteopetrosis type 1 is an osteoclast-poor osteopetrotic phenotype with increased biomechanical competence and no low-energy fractures. Osteoclast profiles are markedly decreased (97), bone formation seems to be normal, and OPG levels in the circulation increased (170). However, when investigating os- Endocrine Reviews, February 2011, 32(1):31– 63 teoclasts ex vivo from these patients, they express normal bone resorptive capacity (30). In summary, osteoclast-poor osteopetrosis can arise in murine mutations/transgenics or humans when the OPG/ RANKL/RANK system is affected directly or indirectly. These findings underscore this cytokine system as a key regulator of osteoclastogenesis. Moreover, the phenotypes seem to be less affected than the osteoclast-rich forms, the reason for which is so far unresolved, although there are indications that reductions in bone formation are involved (1, 28, 29). E. Pycnodysostotic osteoclasts An interesting subtype of osteoclasts with defective bone resorption is observed in patients with pycnodysostosis. Pycnodysostosis is caused by loss of function or loss of expression mutations in the cysteine proteinase cathepsin K, which in humans causes dwarfism and poor bone quality due to defective remodeling of the bones (36, 171– 173). Few studies examining the phenotype of pycnodysostotic osteoclasts have been published. Microscopic analyses of the osteoclasts have shown significantly increased amounts of demineralized collagen matrix in the resorption pit, but also inside the osteoclasts, indicating disturbed resorption and trafficking of resorbed components (174, 175). A study of biochemical markers of bone turnover showed that C-terminal crosslinked telopeptide of type I collagen (CTX-I) release was reduced, whereas production of the MMP-generated type I collagen fragment carboxyterminal telopeptide of type I collagen (ICTP) was increased (67) (Table 1). Several studies of cathepsin Kdeficient mice have been published, and whereas they confirm that cathepsin K is essential for degradation of the organic matrix in bone (65, 66, 176), there are also several differences between the human and mouse phenotypes (103). Furthermore, in cathepsin K-deficient mice, bone formation parameters are highly increased (100). These findings have not been replicated in pycnodysostosis patients in whom the bone matrix is disordered (177), and a clinical case study indicated that anabolic response to PTH was absent (174). Two recently published clinical studies have shown that whereas bone resorption markers are strongly reduced, bone formation is also suppressed in women treated with the cathepsin K inhibitor odanacatib (178). In a monkey study monitoring bone formation by histomorphometry reductions in bone formation, rates were shown in the trabecular bone compartment, whereas bone formation was increased in the cortical compartment (179 –181). In conclusion, cathepsin K mediates cleavage of type I collagen in the resorption lacunae, but its secondary effects on bone formation are bone type-dependent and still need to be investigated further. edrv.endojournals.org 41 F. Other diseases characterized by increased osteoclast activity Apart from hypogonadal osteoporosis, several diseases are characterized by accelerated osteoclastogenesis and function. Although the etiology of these diseases is different, there are interesting overlaps and discrepancies that provide highly useful information about osteoclastic function and secondary effects on bone formation under different circumstances (182). 1. Pagetic osteoclasts Paget’s disease is a late-onset disease that is quite common in the elderly Caucasian population, where it affects approximately 3% of individuals (182). The disease is characterized by focal increases in osteoclast numbers, nuclearity, and size, which leads to localized bone destruction, although surrounding osteoblasts also are activated (183) (Table 1). The identified causes of the disease include mutations in four different genes, TNFRSF11A, TNFRSF11B, VCP, and SQSTM1 (182, 184 –186). These genes encode RANK, OPG, p97, and p62, all of which are involved in the regulation of osteoclastogenesis. The mutations all result in different subtypes of Paget’s (182, 184 –186). These mutations render the osteoclast precursors more sensitive to RANKL stimulation, resulting in a higher number of osteoclasts, and potentially also explaining the presence of giant osteoclasts (185, 187, 188). Interestingly, a recent study in mice indicated that the most common mutation in p62 does not make the osteoclasts Pagetic alone, although it sensitizes them to other yet-tobe-described causes of Paget’s (189, 190). In Paget’s patients, biochemical markers of both bone resorption and bone formation are increased, showing an overall increase in bone turnover at the affected sites. However, bone resorption clearly exceeds bone formation (182). Whether the osteoclasts in Paget’s behave differently from those in healthy individuals during bone resorption is presently unknown. In particular, it is not known whether osteoclasts in Paget’s require acidification to resorb bone, or whether cathepsin K is the main protease, although answers to these questions might be of value in the development of new therapies for Paget’s. Furthermore, an explanation for the localized nature of Pagetic lesions has still not been found. Even under the extreme circumstances seen in Pagetic lesions, bone formation is coupled to osteoclastic parameters, although whether this is due to increased osteoclast numbers or activities is not known. Moreover, in this case a treatment type eliminating the activity of both types of cells is most likely to be preferred because the increase in bone formation occurring is part of the pathology, and likely will provide no benefit for the bones if maintained. Thus, bisphosphonate, which strongly attenuated overall bone 42 Henriksen et al. Osteoclast Subtypes turnover, appears to be highly relevant in the context of Paget’s disease (191). 2. Osteolytic osteoclasts Several forms of cancer can metastasize to bone and form osteolytic metastases (192–196). Once the cancer has reached the bone, tumor and bone interact in a vicious cycle in which tumor-secreted factors, such as PTHrP, stimulate bone cells, which in turn release growth factors and cytokines that promote further tumor cell growth (192, 197). The activation of osteoclastogenesis induced by tumor cells has been shown to involve a switch in the RANKL/OPG ratio favoring osteoclastogenesis and activation, leading to release of the tumorigenic factor TGF-␤, and thereby inducing the vicious cycle (198). As a function of the increased numbers of osteoclasts and accelerated bone resorption, a marked up-regulation of osteoblast activities is also observed (199, 200). The activity of osteoclasts in metastases has been monitored closely using biochemical markers of bone turnover (199, 200), and these studies have indicated that bone resorption by tumor-induced osteoclasts to some extent depends on MMP activity, rather than cathepsin K, because the type I collagen fragment ICTP is released in high amounts (199, 201) (Table 1). Animal models of breast cancer bone metastases are to some extent sensitive to inhibitors of both cathepsin K and MMPs (101, 202–204); however, clinical data for MMP inhibitors have been disappointing (205, 206). An interesting question is whether these agents, to be effective, have to inhibit MMPs before the tumors actually metastasize. For cathepsin K inhibitors, the data indicate a beneficial effect on the release of the bone resorption marker N-terminal crosslinked peptide of type I collagen, and an increase in ICTP levels (204). However, further information is needed to draw reliable conclusions on the usefulness of cathepsin K inhibitors for metastatic bone disease. In contrast, treatments ablating both osteoclasts and the increased osteoblast activity, such as denosumab and bisphosphonates, reduce the destructive capacity of the metastasis and, importantly, the afflicted pain. However, they do not appear to affect the cancer cells (192, 207–210), although there are some indications that the bisphosphonates affect the life span of the cancer cells as well as reducing osteolysis (211). In summary, from a treatment point of view, there are several similarities between Paget’s and osteolytic metastases. The optimal approach appears to involve a strategy of reducing overall bone turnover toward the normal range, such as with the use of denosumab or bisphosphonates An intriguing possibility would be to target only the areas undergoing destruction, but whether this is feasible is presently not known. Endocrine Reviews, February 2011, 32(1):31– 63 3. Arthritic osteoclasts Later stages of RA are characterized by massive bone destruction caused by osteoclasts (212, 213). However, there are several indications that these osteoclasts are not classical bone-resorbing osteoclasts but include cells that degrade calcified cartilage (83, 214) (Table 1). Several studies have indicated that TNF-␣ at least partially drives osteoclastogenesis in RA (215), as exemplified by mice overexpressing human TNF-␣ with massive joint destruction including bone erosion (216). Furthermore, TNF-␣neutralizing antibodies, such as infliximab, or soluble TNF-␣ receptor antagonists, such as etanercept, provide amelioration of RA in humans (217, 218). Apart from TNF-␣, RANKL is, not surprisingly, a crucial factor in osteoclastogenesis during RA (87), and mice deficient in RANKL are protected against bone, but not cartilage, erosion (219). Treatment with OPG of mice with collageninduced arthritis also leads to amelioration of bone destruction, while having a markedly lower effect on cartilage degradation (220). In addition, a study in which TNF-␣ overexpressing mice were crossed with mice deficient in c-fos (i.e., deficient in osteoclasts), showed no bone destruction but clear evidence of cartilage destruction (221). Furthermore, human clinical trials using denosumab have demonstrated that the RANKL/RANK axis is a key player in RA and that inhibition of RANKL signaling may provide a useful treatment option (222). Finally, more recent evidence has indicated that IL-1␣ and IL-1␤ both play a partial role in bone resorption and cartilage degradation (223). Anakinra, which is a soluble IL-1 receptor antagonist, is also used for treatment of RA, although it appears to be less effective than the TNF-␣ inhibitors (217). In addition, tocilizumab (anti-interleukin-6 receptor inhibitor) has shown promise in preventing RA progression through an effect including a reduction in osteoclast numbers (158, 324). Because osteoclasts play a significant role in RA, bisphosphonates appear to be an attractive treatment option. Early evidence has indicated that zolendronate may be useful (224), although this has not been fully established yet (225). Furthermore, interpretation of the effects of bisphosphonates in RA is often clouded by glucocorticoid treatment of the same patients because glucocorticoids are associated with rapid systemic bone loss, independent of RA (226, 227). However, in both collageninduced arthritis in rats and in the TNF-␣ transgenic mouse model, zolendronate was effective in reducing both bone and cartilage destruction (228, 229). The bone resorption process in RA is still not completely understood despite several studies into the molecular mechanisms. The role of cathepsin K has been extensively studied, and the data are somewhat conflicting Endocrine Reviews, February 2011, 32(1):31– 63 (230 –232). Overexpression of cathepsin K has been shown to accelerate joint destruction in mice (231), and overexpression of cathepsin K has been observed in humans with RA (233, 234). However, studies in the TNF-␣ overexpression model crossed with cathepsin K-deficient mice showed that cathepsin K plays only a marginal role in bone resorption in RA (232), a finding supported by a case study showing severe arthritis in a pycnodysostotic patient (235), although there are still controversies with respect to the role of cathepsin K in RA (236). Other cathepsins have not been explored in detail, and their expression patterns do not indicate a particular effect on osteoclast function in RA (237). Under some circumstances, MMPs also play a role in bone resorption (2, 81). Studies showing that the MMPderived collagen type I fragment, ICTP, is increased in RA could indicate that osteoclasts used MMPs to digest matrix under these circumstances (238, 239). Infliximab treatment has been shown to reduce ICTP levels, as well as osteoclast numbers (240), further indicating that osteoclasts utilize MMP-mediated bone degradation in RA. However, a direct link between the production of ICTP and osteoclasts has not been demonstrated yet. Whether acid secretion by osteoclasts is needed for bone destruction in RA is also not clear. Because bone destruction is likely to occur as a result of MMP activity, the need for acidification may be reduced when compared with “classical” bone resorption (69), although this is still not fully understood. Another possibility is that MMPmediated collagen type I degradation is mediated by another cell type, although this still remains to be clarified. A case study of arthritis in a case of autosomal dominant osteopetrosis type II (241) showed a lack of bone degradation, whereas cartilage degradation was abundant, thereby mimicking the situation seen in osteoclast-deficient systems (221) and indicating that bone resorption in RA depends fully on acid secretion. In summary, development of severe RA involves osteoclasts, and a reduction of bone resorption by these cells is desired. This may be obtained through inhibition of inflammation and thereby bone and cartilage destruction, as seen with anti-TNF-␣ therapy. Alternatively, therapies such as denosumab that target the osteoclasts directly may also be useful, although these fail to eliminate inflammation and only partially prevent cartilage degradation (220). The optimal therapy could be a combination of antiinflammatory and antiosteoclastic measures, although this is presently not known. In summary, studies of osteoclasts under pathological circumstances have highlighted some important phenomena. First, osteoclasts themselves, not just their resorptive activity, mediate bone formation and therefore perform an edrv.endojournals.org 43 important secondary role in bone remodeling, which is of importance when developing novel treatments for osteoporosis (15). Second, excessive and local activation of osteoclasts occurs in several diseases, and interestingly the osteoclasts appear to switch subtype with respect to their resorption machinery. These findings highlight the importance of characterizing the function of osteoclasts under pathological circumstances to optimize treatment strategies. VI. Drug-Induced Osteoclast Subtypes A. Existing drugs Several antiresorptive drugs for the treatment of osteoporosis, as well as glucocorticoids and PTH treatment, are known to alter osteoclasts in various ways. These drugs all provide critical information on osteoclast function, and furthermore, they have also played a great role in illustrating the interplay between osteoclasts and osteoblasts, as will be described in the following section. 1. Bisphosphonates Bisphosphonates have long been associated with induction of apoptosis in osteoclasts, and the mechanism of action underlying the apoptotic effect depends on whether or not the bisphosphonates contain nitrogen (242). Both classes of bisphosphonates bind to the bone matrix and are taken up by the osteoclast during bone resorption. The simple bisphosphonates are metabolized into toxic ATP analogs, thereby inducing osteoclast apoptosis in vitro (242). The nitrogen-containing bisphosphonates exert their function by inhibiting the mevalonate pathway, which leads to the generation of an ATP analog known to induce apoptosis in osteoclasts in vitro (242). The antiresorptive potency of the nitrogen-containing bisphosphonates in vivo is controlled by mineral binding affinity and by their ability to inhibit the mevalonate pathway (242). Although in vitro data clearly show that bisphosphonates induce apoptosis, analyses of osteoclast numbers in iliac crest biopsies failed to show a reduction in the number of osteoclasts when patients were treated with bisphosphonates (243–245). On the other hand, bisphosphonates reduce systemic levels of TRACP 5b and cathepsin K, both markers of osteoclast number (32, 246 –248), potentially indicating that osteoclasts undergo systemic apoptosis, which correlates well with the expected effects of bisphosphonates (242) (Table 1). Other studies have shown that when bisphosphonate therapy continues for more than 1 yr, the number of circulating osteoclast precursors is reduced, and these reductions are speculated to be related to reduced serum RANKL levels (249, 250). 44 Henriksen et al. Osteoclast Subtypes A recent study of biopsies from alendronate-treated patients showed the presence of giant hypernucleated, detached, and frequently apoptotic osteoclasts, and the number of these abnormal osteoclasts correlated with the cumulative dose of bisphosphonate (251). Although interesting, the biological implications of this finding are not clear yet. One potential explanation for the discrepancies in scoring osteoclasts in the iliac crest biopsies is the very low number of osteoclasts observed in general. Recent reports have also debated the clinical relevance of studying iliac crest biopsies because they are from non-weight-bearing bones and these are different from weight-bearing bones (252–254), and in general more data are needed to draw a final conclusion on the osteoclastic response to bisphosphonates. On the other hand, the effect on reduction of bone resorption measured both by biochemical markers and by bone histomorphometry (activation frequency) confirms a potent reduction in bone resorption, and the level of reduction is often down to the lower range of premenopausal levels, although this depends heavily on the efficacy of the individual bisphosphonate (242, 243, 245, 255–258). With respect to secondary effects on bone formation, measurement of biochemical markers of bone turnover shows a marked reduction in bone formation markers, and the effects are maintained throughout the treatment period, although this again is dependent on the individual bisphosphonate (242, 243, 245, 255–258). Biopsy studies have confirmed that bone formation is reduced when compared with placebo, and although the reduction in bone formation rates is dependent on the individual bisphosphonate, the data indicate that bone formation is not completely suppressed but is reduced to the lower postmenopausal levels (243, 245, 258). The FLEX study (Fracture Intervention Trial Long-term Extension), although showing continued reductions in vertebral fractures, increase in bone mineral density (BMD), and reduction of bone turnover markers with alendronate, did not show a significant reduction in bone formation rates when comparing patients stopping alendronate to patients continuing treatment; however, the numbers of biopsies were low (259). All in all, there is no doubt about the fracture-preventing effects of bisphosphonates; however, knowledge of the effect of bisphosphonates on osteoclasts in vivo is quite limited. Apoptosis of the osteoclasts most likely explains the reduction in bone resorption. Furthermore, although the extent of the secondary reduction in bone formation is still discussed, it appears to be clinically relevant, and it most likely is the explanation for the attenuation of the BMD increase seen after the first year of treatment. Endocrine Reviews, February 2011, 32(1):31– 63 a. Osteonecrosis of the jaw (ONJ) and bisphosphonates. Bisphosphonate therapy, especially in the case of malignancy-induced bone loss, has been connected to the occurrence of ONJ, mainly due to the ability of bisphosphonates to strongly suppress bone turnover (260 –262). Although the probability of ONJ is very low for the dosing regimens used for treatment of osteoporosis, there has still been a lot of debate about whether ONJ is the result of the massive suppression of bone turnover in the jaw (262). Interestingly, alveolar bone of the jaw is very similar to bone matrix in the long bone, i.e., it contains the classical cell types as well as the lamellar structure (263). Furthermore, bone remodeling occurs normally in alveolar bone, although the rate of remodeling has been estimated to be up to 10-fold higher than the corresponding rate in long bones (263–266). In ONJ, the number of osteoclasts has been investigated, and it appears that the osteoclasts are absent from the lesions (267, 268), although opposing evidence also exists (269) and thus more studies are needed. It has been speculated that massive suppression of osteoclast function, and thus bone turnover, in this highturnover compartment is what causes ONJ to occur; however, there are several other factors involved, such as tooth extraction or infections, and the overall causality is still not clear (262). One point of particular interest is whether this phenomenon is specific for bisphosphonates or whether it will happen with other very potent and long-lived antiresorptives; however, this is presently not known. 2. Selective estrogen receptor modulators/hormone replacement therapy Because cessation of estrogen production is a major cause of osteoporosis (3, 14) and both estrogen and SERMs are used for treatment of osteoporosis, several studies have been conducted to clarify their effect on osteoclasts. Estrogen has been shown to exert direct antiosteoclastic effects at several stages of osteoclastic differentiation and function, namely osteoclastogenesis, resorption, and apoptosis. Direct inhibition of the formation of multinucleated osteoclasts is thought to be caused by suppression of RANKL-induced c-Jun and basal c-Jun N-terminal kinase activity in osteoclast precursor cells (135, 136). In nonpurified osteoclast-precursor systems, estrogen was found to inhibit osteoclastic differentiation in a human system (270), possibly via down-regulation of the ␣v␤3 integrin (271). Two studies of estrogen have been conducted using CD14⫹ osteoclast precursors. As mentioned earlier, one study showed significant inhibition of osteoclastogenesis (137), whereas the other showed no direct effect on osteoclast precursors (272). To date, there is no explanation for this discrepancy. Endocrine Reviews, February 2011, 32(1):31– 63 Studies of the effects of SERMs on osteoclasts have shown that tamoxifen inhibits osteoclastogenesis directly, whereas raloxifene and ospemifene only inhibited osteoclasts through up-regulation of the expression of OPG by osteoblasts (273). Although early studies showed an effect of raloxifene on osteoclastogenesis, these were conducted using mixed cell populations and therefore most likely reflect the increase in OPG (274). Mature osteoclasts have also been shown to respond directly to estrogen (275, 276). These studies showed that both the activity and the production of the lysosomal enzymes are down-regulated by estrogen (277, 278), possibly explaining the reduction in resorption by the downregulation of cathepsin K and TRACP (36, 66, 77, 279) (Table 1). In summary, in vitro data clearly demonstrate that estrogen and SERMs reduce osteoclast numbers via inhibition of osteoclastogenesis, and potential effects on bone resorption and apoptosis might add to the in vivo effect. Although some studies of osteoclasts in patients treated with either HRT or SERMs have been conducted, the effects of both estrogen and SERMs on bone remodeling indices based on histomorphometry are quite modest (280 –284). Overall, these studies show a reduction in activation, frequency, and depth of resorption, as well as— where detectable—a small decrease in osteoclast numbers. Reduced bone formation rates were also observed, confirming the coupled nature of inhibition mediated by estrogen and SERMs (280 –284). These data are corroborated by biochemical markers of bone turnover, which clearly demonstrated a coupled reduction in bone resorption and bone formation (32, 285–287), and furthermore explain the plateau effect observed in BMD measurements after 1 yr (285). In summary, many of the numerous studies of the in vitro mode of action of HRT and SERMs show a reduction in osteoclastogenesis. In alignment, in vivo studies of these therapies on osteoclasts confirm that osteoclastogenesis is lower than in the untreated population, and importantly, these also confirm the secondary decrease in bone formation. 3. Calcitonin Calcitonin is a natural peptide hormone produced by parafollicular cells (C cells) of the thyroid gland. Calcitonin possesses potent antiresorptive effects (288), and binding of calcitonin to the calcitonin receptor on osteoclasts induces a rapid change in the cytoskeletal structure of the osteoclasts in vitro, which in turn leads to a reduction in bone resorption without inducing apoptosis of the cells (102, 289, 290). Calcitonin in either an injectable or a nasal form has been approved for treatment of osteoporosis; however, because it only prevents about 35% of edrv.endojournals.org 45 vertebral fractures, most likely due to low exposure, the clinical usefulness is limited (122). Recent studies have indicated that a recently developed oral formulation of salmon calcitonin will lead to improved efficacy because it has been optimized with respect to pharmacokinetic and pharmacodynamic properties. This has led to a 10-fold higher exposure and thereby a greater reduction in bone resorption parameters. Thus, this agent will most likely provide improved efficacy in preventing fractures (291), and although it remains to be proven in long-term clinical trials, the phase II data are promising (127). The mode of action of oral calcitonin is a transient suppression of the nocturnal rise in bone resorption obtained by giving the treatment at the right time of day—in the evening (292), which results in a reduction in bone resorption, but no effect or very modest secondary effects on bone formation (127) (Table 1). These findings are further supported by other clinical studies showing that calcitonin may inhibit bone resorption without affecting bone formation, a finding observed independent of administration route (293–296). There are histological indications that calcitonin attenuates ruffled border formation by osteoclasts (296 –298), and this appears to be the mode of action underlying the antiresorptive effects of calcitonin in vivo, thereby elaborating on the previously described transient reduction in bone resorption (292). Studies of mice lacking the calcitonin receptor indicated that bone formation was increased, and thus that calcitonin is a suppressor of bone formation (299, 300). These studies were conducted mainly in young mice. A recent study in mice deficient in the calcitonin receptor specifically in osteoclasts failed to reproduce this finding (301, 302). However, considering the very modest, or nonexistent, suppression of bone formation in patients treated with calcitonin, the mice data appear of low relevance in the clinical setting (127, 293–296). Further studies are needed to understand this potential dissociation of bone resorption and bone formation. It may be that this dissociation occurs because calcitonin disappears quickly from the circulation and thus is a completely reversible treatment (122). An interesting question is whether calcitonin treatment may result in better bone quality than potent antiresorptives due to the lack of effect on bone formation and the lower suppression of bone resorption, which is expected to lead to a slow, yet prolonged increase in BMD (6, 303). 4. Parathyroid hormone Although PTH does not appear to affect osteoclasts directly because these cells do not appear to express the PTH receptor, PTH nonetheless affects osteoclast function on many different levels (10). In vitro studies of the 46 Henriksen et al. Osteoclast Subtypes effects of PTH on osteoclasts all show that PTH induces osteoclastogenesis and that induction of a transient RANKL expression is essential for this effect (10). However, PTH has mainly been studied in relation to its powerful anabolic effects on osteoblasts (10). Intermittent dosing of PTH in human subjects results in a marked increase in bone formation markers, and secondarily in activation of bone resorption through increased RANKL expression (9, 304, 305). Bone histomorphometric and biochemical marker studies confirm the increase in bone turnover (306 –308) (Table 1). The anabolic mode of action of PTH has been debated extensively. Studies show that PTH directly activates bone formation by osteoblasts when given intermittently (309, 310). In mouse models that are either deficient in osteoclasts or deficient in bone resorption, data suggest that the anabolic effect of PTH is dependent on the presence of mature osteoclasts, but not on their activity (165, 166, 311). Furthermore, initial clinical trials combining alendronate and PTH showed that alendronate blunted the anabolic effect of PTH (312, 313), and there were indications that even pretreatment with alendronate led to a blunting of the PTH response (314). On the other hand, animal studies indicate that PTH can be combined with a bisphosphonate (315, 316), but, as noted by Johnston et al. (315), there are marked differences in the doses of PTH used in rodents and in humans. Collectively, PTH exerts marked regulation of bone turnover (15), including the activation of osteoclasts. The potential anabolic role of osteoclasts and, especially, how to achieve the right osteoclast subtype are debated intensely. Future studies will most likely explain this complex interplay between bone cells and thus guide the right combination of PTH and antiresorptive. 5. Strontium ranelate Strontium ranelate is approved for treatment of osteoporosis, albeit only in Europe, through its ability to reduce fracture risk in patients (317–321). The mode of action has been studied extensively, and yet it is not fully clear exactly how it works in vivo. Bone biopsies have been investigated, and these indicated small increases in bone formation and mineralization rates but no changes in bone resorption or osteoclast parameters, thus indicating that strontium ranelate stimulates novel bone formation (322). These data were supported by analysis of biochemical markers of bone turnover demonstrating increased bone formation (308, 323), while also showing a modest decrease in bone resorption markers (323, 325). In vitro studies support the hypothesis that strontium ranelate has a dual effect, namely inhibition of bone resorption while stimulating bone formation (326 –329). Endocrine Reviews, February 2011, 32(1):31– 63 Furthermore, strontium has also been shown to increase OPG expression by osteoblasts (330). In summary, strontium ranelate is a very interesting molecule with respect to effects on osteoclasts, and several lines of in vitro evidence indicate that it reduces osteoclast function (326). However, the relevance of the effect on osteoclasts is still debated, and thus the overall effects of this “uncoupling” molecule are still not fully understood. 6. Glucocorticoids Glucocorticoids are used to overcome inflammatory conditions, such as inflammatory bowel diseases and RA (331). Glucocorticoid use is associated with severe bone loss due to strongly attenuated bone formation (332). This attenuation of bone formation leads to a rapid acceleration in the number of fractures in glucocorticoid-treated patients (332), especially in trabecular bone compartments such as vertebrae (333). Glucocorticoid treatment is the most common cause of secondary osteoporosis (333), and thus patients on glucocorticoids are often treated with antiresorptives (334). In vivo, glucocorticoids inhibit osteoblastogenesis, the generation of bone-forming osteoblasts, and promote apoptosis of osteoblasts and osteocytes, which is consistent with the well-known inhibition of bone formation (335). In contrast, the cellular effects of glucocorticoids on osteoclasts are a subject of controversy. In vivo, the effects appear to fall into two categories, one being a short-lived acceleration of osteoclastogenesis and bone resorption, whereas the other is a reduction in osteoclast numbers, which is not well-characterized with respect to exposure time to glucocorticoids (335–340) (Table 1). Interestingly, a study by Kim et al. (340) showed that the detrimental effect of glucocorticoids on bone formation was absent when the glucocorticoid receptor was ablated specifically in osteoclasts in mice. In vitro studies of glucocorticoids are often conducted in the presence of contaminating cells, and because glucocorticoid treatment also promotes RANKL and reduces OPG expression in osteoblasts, it is unclear exactly to what extent they influence the osteoclasts (341, 342). Two recent studies showed that glucocorticoid treatment hyperactivated osteoclasts and thus suggests that glucocorticoids indeed have a direct effect on bone resorption (343, 344). Yet some studies show the opposite (340). Overall, the results appear to be very context-dependent, illustrating the complex nature of the biological effects of glucocorticoids. Measurements of biochemical markers of bone turnover in human subjects on glucocorticoid therapy provided diverse results, which appeared to be dependent on the dose of glucocorticoid used (331, 345, 346). However, biochemical marker data indicate that bone resorption Endocrine Reviews, February 2011, 32(1):31– 63 increases short term, whereas bone formation is attenuated long term (345) in response to glucocorticoid therapy, which corresponds well to mouse studies (335–339). The short-term increase in bone resorption and long-term suppression of bone formation are also observed with histomorphometry in mice (347, 348). In summary, glucocorticoids exert detrimental effects on bone, and whereas the effects on osteoclasts are not completely clear yet, further investigation of the effect on the coupling between osteoclasts and osteoblasts could explain the overall beneficial effect of antiresorptives on a syndrome mediated primarily by suppressed bone formation (227, 349). These findings further highlight the importance of understanding the interplay between bone cells to provide the optimal treatment. B. Future treatments A series of interesting targets for osteoporosis treatment are currently under investigation. The targets of these treatments to some extent employ novel modes of action on osteoclasts. These novel modes of action are of importance when investigating whether they may have secondary effects on bone formation, and subsequently on bone quality. 1. Denosumab Denosumab is a fully humanized monoclonal antibody to RANKL; it has gone through a phase III fracture efficacy trial in which it was shown to reduce fracture rates by 68% in vertebrae and 40% in hip (351); and it was recently accepted for treatment of severe osteoporosis in both the United States and Europe. In line with in vitro studies of inhibition of RANKL (210, 352), denosumab prevents osteoclastogenesis, blocks bone resorption, and increases osteoclast apoptosis. It induces a massive reduction of osteoclasts in vivo and, thereby, almost complete suppression of bone resorption in both humans and mice (352–354) (Table 1). Denosumab treatment also leads to a marked suppression of bone formation markers in humans (353, 354), as well as a marked suppression in bone formation rates measured by histomorphometry in animal models (352, 355). Thus, denosumab treatment is consistent with the classical perception of coupling. A key point with respect to denosumab is whether the suppression is too severe and could lead to detrimental effects on bone quality long term (6). However, as is the case with bisphosphonate treatment, this is not clear at present. 2. Cathepsin K inhibitors Cathepsin K is a critical protease for degradation of the type I collagen matrix in the resorption pits during bone edrv.endojournals.org 47 resorption by osteoclasts (36, 65, 66). Studies conducted in pycnodysostosis patients before the final identification of cathepsin K showed massive accumulation of nondigested bone collagen fibers in the resorption pit below the osteoclasts (175). These findings were matched by those from investigations in cathepsin K-deficient mice (66), demonstrating a critical role for cathepsin K in degradation of the organic matrix. Further studies in cathepsin K-deficient systems have indicated that cells of the osteoblast lineage, namely bone-lining cells (68); cells of hematopoietic origin (69); and a general up-regulation of the osteoclastic stimuli, osteoclast numbers, and proteases, especially RANKL and MMPs (103), are involved in compensating for the lack of cathepsin K. Interestingly, a hallmark of the absence of cathepsin K function is the presence of the MMP-derived collagen fragment ICTP, which is seen in pycnodysostosis patients, cathepsin K-deficient mice, and cell cultures (67, 69, 103, 356), strongly indicating a compensation by MMPs in the absence of cathepsin K (Table 1). An interesting study by Fuller et al. (357) showed that inhibition of cathepsin K in cultured osteoclasts led to augmented secretion of IGF-I. Furthermore, increased numbers of osteoclasts, containing granules of matrix proteins, have been observed in monkey studies of cathepsin K inhibitors (175, 181), thus indicating the potential of this protease for anabolic stimulation of the osteoblasts. Cathepsin K-deficient mice have been studied extensively, and recent experiments indicate that bone formation in trabecular bone is increased after cathepsin K administration and thus that bone resorption and bone formation are not coupled (100, 176). However, clinical studies of cathepsin K inhibitors, such as odanacatib, have shown that whereas a robust reduction in CTX and N-terminal crosslinked peptide of type I collagen occurred and no changes in TRACP 5b were observed, a significant decrease in the bone formation marker pro-peptide of collagen type I and nonsignificant reductions in bone formation rates by histomorphometry were seen (178). Furthermore, a study of osteoclast morphology as a function of cathepsin K inhibition in humans indicated increased size of the osteoclasts and the presence of large TRACP-positive vacuoles, yet no increase in osteoclast numbers (358). Studies in monkeys clearly demonstrated that bone formation in the trabecular compartments was dose-dependent and significantly reduced by cathepsin K inhibitors, whereas an induction of bone formation was observed at cortical sites (179, 180). Further studies are needed to clarify whether the osteoclasts in cathepsin Kdeficient situations indeed signal to the osteoblasts. An indication came from a pycnodysostosis case study that showed no bone formation response to PTH (174), and 48 Henriksen et al. Osteoclast Subtypes thus indicated that secretion of the coupling signals may be attenuated at least in human systems. A possible explanation for the lack of secondary anabolic effects induced by inhibition of cathepsin K is the presence of demineralized collagen fibers in the resorption pit, which are removed by bone-lining cells (68). Although it is not well understood how the presence of fibers and their subsequent removal affect osteoblasts, a study indicated that RGD sequences, which are numerous in collagen, antagonize osteoblast function (359). These findings again illustrate the importance of carefully investigating the osteoclast subtype as a function of cathepsin K inhibition to more accurately predict potential secondary effects on bone formation. 3. Glucagon-like peptide-2 GLP-2 is a 33-amino acid peptide. GLP-2 is created by specific posttranslational proteolytic cleavage of proglucagon in a process that also liberates the related GLP-1 (124). GLP-2 is produced by the intestinal endocrine L cell and by various neurons in the central nervous system (124). Intestinal GLP-2 is cosecreted along with GLP-1 upon nutrient ingestion. GLP-2 has in clinical settings been demonstrated to inhibit bone resorption (124 –126) (Table 1). Reductions in bone resorption by exogenous GLP-2 require an intact gastrointestinal tract (125, 361, 362). The decreased mealinduced inhibition of bone resorption in jejunostomy patients, who lack a GLP-2 response, supports the view that GLP-2 plays a role in postprandial reduction in bone resorption (361, 362). GLP-2 has in addition been suggested to inhibit bone resorption without affecting bone formation (125), highlighting this mode of inhibition of resorption for further investigation with respect to osteoclast subtypes. 4. Acid secretion inhibitors Acid secretion by osteoclasts has been an interesting therapeutic target since the discovery that this process is controlled by the a3 subunit of the V-ATPase and ClC-7, both of which are quite specific to osteoclasts (37, 39, 56). Furthermore, in vitro studies of osteoclasts treated with inhibitors of these ion transporters have shown that the osteoclasts are unable to resorb bone and that they therefore survive longer (8, 159, 363), thereby mimicking the elevated numbers of osteoclasts observed in patients with mutations in the genes for a3 and ClC-7 (37, 97) (Table 1). In aged ovariectomized rats, early low-potency chloride channel inhibitors were able to prevent bone resorption by approximately 50%, as monitored by both BMD and the biochemical markers of bone resorption CTX-I or deoxypyridinoline, while augmenting the number of osteoclasts Endocrine Reviews, February 2011, 32(1):31– 63 and showing no inhibition of bone formation markers (8, 364). Similar findings were published for an inhibitor of the V-ATPase (365). In a study of prosthetic implants coated with bafilomycin, osteoclast numbers were elevated, and indications of increased bone formation were observed (366). These studies were the first to provide proof of concept that inhibition of acidification is a really promising target for osteoporosis treatment. Most interestingly, bone formation levels, as measured by osteocalcin and by evaluation of the dynamic histomorphometry parameters mineral apposition rate and the mineralizing surface vs. bone surface, were not affected. These data therefore suggest that inhibition of acidification of the osteoclastic resorption lacunae results in an uncoupling of bone formation and bone resorption, thereby possibly improving the potential efficacy of the treatment. This is in contrast to other antiresorptive treatments where a secondary decrease in bone formation is observed (3, 367). These data also indicate that the subtype of osteoclasts obtained—nonresorbing yet alive—when targeting acid secretion is active with respect to bone formation, and thus might possibly be combined with PTH treatment in the future. Finally, other compounds that appear to modulate the activity of osteoclasts are in development for osteoporosis. These include calcilytics, PTHrP, and sclerostin, but their effects on osteoclasts, which most likely are indirect, are not clear yet (368 –370), and thus these will not be described further. VII. The Bone Anabolic Effects of the Osteoclasts Since the early discovery that osteoclast activities were involved in regulation of bone formation during targeted remodeling (17, 18, 371, 372), a series of studies have investigated the nature of this process. The early studies focused mainly on the release of molecules from the bone matrix during bone resorption and identified molecules such as IGF-I and TGF-␤ (357, 373, 374). However, with the recent discovery that mature osteoclasts, not osteoclast precursors and not necessarily bone resorption, are needed for stimulation of bone formation (8, 9), a series of studies have investigated this phenomenon. Zhao et al. (12) demonstrated that osteoclast-mediated expression of EphrinB2 and osteoblast-mediated expression of EphB4 were involved in a bidirectional communication between these cell types. EphrinB2 on osteoclasts stimulated bone formation by the osteoblasts via binding to EphB4, while EphB4 expression on osteoblasts in turn inhibited osteoclastogenesis via binding to EphrinB2 (12). Endocrine Reviews, February 2011, 32(1):31– 63 However, ephrin signaling requires close contact between the osteoclasts and their target cells. This has led to the speculation that ephrin signaling could be involved in the interplay between osteoclasts and bone-lining cells, which are found in close contact and appear to regulate the activity of each other (68, 375). Stimulatory signals from osteoclasts directly to mature bone-forming osteoblasts are, on the other hand, likely to be paracrine because these cell types are not found in close contact (264). Both TGF-␤ and IGF-I are produced by the osteoclasts and are known to stimulate bone formation under various circumstances (376 –380). In relation to these findings, it is interesting that the anabolic effect of PTH in mice was shown to be mediated through IGF-I (350), an effect that is absent in the absence of osteoclasts (166). This confirms that IGF-I is a coupling factor. A recent study demonstrated the mature human osteoclasts, independent of their resorptive activity, secrete factors that activate nodule formation by the osteoblasts (11). This study was followed by a study showing that osteoclasts produce the anabolic factors bone morphogenetic protein 6, Wnt10b, and sphingosine-1-phosphate, again independent of bone resorption (13). Furthermore, inhibition of bone morphogenetic protein 6, Wnt10b, and sphingosine-1-phosphate led to inhibition of the osteoclast-mediated stimulation of bone formation in vitro. Finally, osteoclasts have also been shown to produce cardiotrophin-1 (CT-1), which activates bone formation by osteoblasts, although the role of CT-1 was clearly shown to be dependent on age because loss of CT-1 in newborn mice caused osteopenia, whereas in larger mice it caused mild osteopetrosis due to defective bone resorption (360). In summary, the presence of mature osteoclasts is associated with the secretion of stimulation of bone anabolic signals, and whereas several candidate factors have been identified, a clear demonstration that removal of one of the molecules specifically in the osteoclasts in vivo leads to loss of bone formation is still missing. VIII. Conclusions and Future Perspectives Osteoclasts have traditionally been viewed as bone resorption “machines”; however, studies of osteoclasts have highlighted that these cells are highly context-specific, and the context of the individual osteoclasts is important for the continued regulation of bone remodeling. As described in detail in this review, the osteoclasts possess at least two highly important functions: 1) bone resorption, a process that is highly dependent on a series of external stimuli, such as matrix type, remodeling status, hormones involved in calcium homeostasis, genotype, inflammation, and importantly also on intervention strate- edrv.endojournals.org 49 gies; and 2) stimulation of bone formation by the osteoblasts, a process that as illustrated by studies conducted in osteopetrotic patients is, to a large extent, independent of bone resorption. It is presently not completely clear when the osteoclasts are anabolically active, yet it appears to be related to the presence of large multinuclear osteoclasts because bone anabolic responses are seen under these circumstances (3, 15). Understanding osteoclast functioning may be useful for developing drugs that not only inhibit bone resorption but also enable bone resorption levels that ensure targeted remodeling and, importantly, support continued anabolic signaling from osteoclasts to osteoblasts in the bone remodeling compartment. This has the triple effect of: 1) maintaining a sufficient resorption level and thereby avoiding excessive aging of the bones; 2) sustaining a local stimulation of bone formation at the resorption site only; and 3) not initiating induction of bone formation in otherwise quiescent sites. Theoretically, this type of inhibition of bone resorption would allow a continuous, ongoing increase in BMD, which is in contrast to the effects of the presently approved antiresorptives where a plateau effect on BMD is observed within the first 12–18 months. This means that even with less powerful suppression of bone resorption, such as that seen with the oral formulation of salmon calcitonin, the long-term effects would surpass those of the bisphosphonates. A deeper understanding of both the differences in the resorption process depending on circumstances and the knowledge relating to when the osteoclasts are anabolically active will aid in the identification of novel treatment opportunities for bone diseases. Finally, the use of biochemical markers of bone turnover is becoming increasingly relevant for the continued understanding of osteoclasts. Markers provide systemic information on the outcome of a given treatment and can help answer questions such as whether glucocorticoids exert detrimental effects on bone formation, and whether antiresorptives antagonize bone formation secondary to bone resorption because of suppression of osteoclast numbers or activity. Acknowledgments The Danish Research Foundation “Den Danske Forskningsfond” is acknowledged for financial support. Address all correspondence and requests for reprints to: K. Henriksen, Nordic Bioscience A/S, Herlev Hovedgade 207, DK-2730 Herlev, Denmark. E-mail: [email protected] Disclosure Summary: M.A.K. owns stocks in Nordic Bioscience A/S. All other authors have no conflicts of interest. 50 Henriksen et al. Osteoclast Subtypes Endocrine Reviews, February 2011, 32(1):31– 63 References 1. Segovia-Silvestre T, Neutzsky-Wulff AV, Sorensen MG, Christiansen C, Bollerslev J, Karsdal MA, Henriksen K 2009 Advances in osteoclast biology resulting from the study of osteopetrotic mutations. Hum Genet 124: 561–577 2. Everts V, de Vries TJ, Helfrich MH 2009 Osteoclast heterogeneity: lessons from osteopetrosis and inflammatory conditions. Biochim Biophys Acta 1792:757–765 3. Karsdal MA, Martin TJ, Bollerslev J, Christiansen C, Henriksen K 2007 Are nonresorbing osteoclasts sources of bone anabolic activity? J Bone Miner Res 22:487– 494 4. Everts V, Korper W, Jansen DC, Steinfort J, Lammerse I, Heera S, Docherty AJ, Beertsen W 1999 Functional heterogeneity of osteoclasts: matrix metalloproteinases participate in osteoclastic resorption of calvarial bone but not in resorption of long bone. FASEB J 13:1219 –1230 5. Marks Jr SC 1983 The origin of osteoclasts: evidence, clinical implications and investigative challenges of an extraskeletal source. J Oral Pathol 12:226 –256 6. Leeming DJ, Henriksen K, Byrjalsen I, Qvist P, Madsen SH, Garnero P, Karsdal MA 2009 Is bone quality associated with collagen age? Osteoporos Int 20:1461–1470 7. Henriksen K, Leeming DJ, Byrjalsen I, Nielsen RH, Sorensen MG, Dziegiel MH, Martin TJ, Christiansen C, Qvist P, Karsdal MA 2007 Osteoclasts prefer aged bone. Osteoporos Int 18:751–759 8. Karsdal MA, Henriksen K, Sørensen MG, Gram J, Schaller S, Dziegiel MH, Heegaard AM, Christophersen P, Martin TJ, Christiansen C, Bollerslev J 2005 Acidification of the osteoclastic resorption compartment provides insight into the coupling of bone formation to bone resorption. Am J Pathol 166:467– 476 9. Martin TJ, Sims NA 2005 Osteoclast-derived activity in the coupling of bone formation to resorption. Trends Mol Med 11:76 – 81 10. Martin TJ, Quinn JM, Gillespie MT, Ng KW, Karsdal MA, Sims NA 2006 Mechanisms involved in skeletal anabolic therapies. Ann NY Acad Sci 1068:458 – 470 11. Karsdal MA, Neutzsky-Wulff AV, Dziegiel MH, Christiansen C, Henriksen K 2008 Osteoclasts secrete non-bone derived signals that induce bone formation. Biochem Biophys Res Commun 366:483– 488 12. Zhao C, Irie N, Takada Y, Shimoda K, Miyamoto T, Nishiwaki T, Suda T, Matsuo K 2006 Bidirectional ephrinB2EphB4 signaling controls bone homeostasis. Cell Metab 4:111–121 13. Pederson L, Ruan M, Westendorf JJ, Khosla S, Oursler MJ 2008 Regulation of bone formation by osteoclasts involves Wnt/BMP signaling and the chemokine sphingosine-1phosphate. Proc Natl Acad Sci USA 105:20764 –20769 14. Seeman E, Delmas PD 2006 Bone quality—the material and structural basis of bone strength and fragility. N Engl J Med 354:2250 –2261 15. Henriksen K, Neutzsky-Wulff AV, Bonewald LF, Karsdal MA 2009 Local communication on and within bone controls bone remodeling. Bone 44:1026 –1033 16. Chavassieux P, Seeman E, Delmas PD 2007 Insights into material and structural basis of bone fragility from diseases associated with fractures: how determinants of the biome- 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. chanical properties of bone are compromised by disease. Endocr Rev 28:151–164 Takahashi H, Epker B, Frost HM 1964 Resorption precedes formative activity. Surg Forum 15:437– 438 Hattner R, Epker BN, Frost HM 1965 Suggested sequential mode of control of changes in cell behaviour in adult bone remodelling. Nature 206:489 – 490 Parfitt AM 1982 The coupling of bone formation to bone resorption: a critical analysis of the concept and of its relevance to the pathogenesis of osteoporosis. Metab Bone Dis Relat Res 4:1– 6 Martin TJ 1993 Hormones in the coupling of bone resorption and formation. Osteoporos Int 3(Suppl 1):121–125 Nakamura M, Udagawa N, Matsuura S, Mogi M, Nakamura H, Horiuchi H, Saito N, Hiraoka BY, Kobayashi Y, Takaoka K, Ozawa H, Miyazawa H, Takahashi N 2003 Osteoprotegerin regulates bone formation through a coupling mechanism with bone resorption. Endocrinology 144:5441–5449 Teitelbaum SL, Ross FP 2003 Genetic regulation of osteoclast development and function. Nat Rev Genet 4:638 – 649 Goltzman D 2002 Discoveries, drugs and skeletal disorders. Nat Rev Drug Discov 1:784 –796 Martin TJ, Seeman E 2007 New mechanisms and targets in the treatment of bone fragility. Clin Sci (Lond) 112:77–91 Harvey N, Earl S, Cooper C 2006 Epidemiology of osteoporotic fractures. In: Favus MJ, ed. Primer on the metabolic bone diseases and disorders of mineral metabolism. Chap 42. 6th ed. Philadelphia: Lippincott Williams & Wilkins; 244 –248 Albers-Schönberg HE 1904 Röntgenbilder einer seltenen Knockenerkrankung. 5th ed. Munch. Med. Wochenschr, Münich: Germany; 365–368 Tolar J, Teitelbaum SL, Orchard PJ 2004 Osteopetrosis. N Engl J Med 351:2839 –2849 Sobacchi C, Frattini A, Guerrini MM, Abinun M, Pangrazio A, Susani L, Bredius R, Mancini G, Cant A, Bishop N, Grabowski P, Del Fattore A, Messina C, Errigo G, Coxon FP, Scott DI, Teti A, Rogers MJ, Vezzoni P, Villa A, Helfrich MH 2007 Osteoclast-poor human osteopetrosis due to mutations in the gene encoding RANKL. Nat Genet 39:960 –962 Guerrini MM, Sobacchi C, Cassani B, Abinun M, Kilic SS, Pangrazio A, Moratto D, Mazzolari E, Clayton-Smith J, Orchard P, Coxon FP, Helfrich MH, Crockett JC, Mellis D, Vellodi A, Tezcan I, Notarangelo LD, Rogers MJ, Vezzoni P, Villa A, Frattini A 2008 Human osteoclast-poor osteopetrosis with hypogammaglobulinemia due to TNFRSF11A (RANK) mutations. Am J Hum Genet 83: 64 –76 Henriksen K, Gram J, Høegh-Andersen P, Jemtland R, Ueland T, Dziegiel MH, Schaller S, Bollerslev J, Karsdal MA 2005 Osteoclasts from patients with autosomal dominant osteopetrosis type I (ADOI) caused by a T253I mutation in LRP5 are normal in vitro, but have decreased resorption capacity in vivo. Am J Pathol 167:1341–1348 Glass 2nd DA, Karsenty G 2006 Canonical Wnt signaling in osteoblasts is required for osteoclast differentiation. Ann NY Acad Sci 1068:117–130 Henriksen K, Tanko LB, Qvist P, Delmas PD, Christiansen C, Karsdal MA 2007 Assessment of osteoclast number and Endocrine Reviews, February 2011, 32(1):31– 63 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. function: application in the development of new and improved treatment modalities for bone diseases. Osteoporos Int 18:681– 685 Teitelbaum SL 2007 Osteoclasts: what do they do and how do they do it? Am J Pathol 170:427– 435 Hayman AR, Jones SJ, Boyde A, Foster D, Colledge WH, Carlton MB, Evans MJ, Cox TM 1996 Mice lacking tartrate-resistant acid phosphatase (Acp 5) have disrupted endochondral ossification and mild osteopetrosis. Development 122:3151–3162 Sly WS, Hewett-Emmett D, Whyte MP, Yu YS, Tashian RE 1983 Carbonic anhydrase II deficiency identified as the primary defect in the autosomal recessive syndrome of osteopetrosis with renal tubular acidosis and cerebral calcification. Proc Natl Acad Sci USA 80:2752–2756 Gelb BD, Shi GP, Chapman HA, Desnick RJ 1996 Pycnodysostosis, a lysosomal disease caused by cathepsin K deficiency. Science 273:1236 –1238 Frattini A, Orchard PJ, Sobacchi C, Giliani S, Abinun M, Mattsson JP, Keeling DJ, Andersson AK, Wallbrandt P, Zecca L, Notarangelo LD, Vezzoni P, Villa A 2000 Defects in TCIRG1 subunit of the vacuolar proton pump are responsible for a subset of human autosomal recessive osteopetrosis. Nat Genet 25:343–346 Vu TH, Shipley JM, Bergers G, Berger JE, Helms JA, Hanahan D, Shapiro SD, Senior RM, Werb Z 1998 MMP-9/gelatinase B is a key regulator of growth plate angiogenesis and apoptosis of hypertrophic chondrocytes. Cell 93:411– 422 Kornak U, Kasper D, Bösl MR, Kaiser E, Schweizer M, Schulz A, Friedrich W, Delling G, Jentsch TJ 2001 Loss of the ClC-7 chloride channel leads to osteopetrosis in mice and man. Cell 104:205–215 Chalhoub N, Benachenhou N, Rajapurohitam V, Pata M, Ferron M, Frattini A, Villa A, Vacher J 2003 Grey-lethal mutation induces severe malignant autosomal recessive osteopetrosis in mouse and human. Nat Med 9:399 – 406 Findlay DM, Martin TJ 1997 Receptors of calciotropic hormones. Horm Metab Res 29:128 –134 Del Fattore A, Cappariello A, Teti A 2008 Genetics, pathogenesis and complications of osteopetrosis. Bone 42:19 –29 Väänänen HK, Horton M 1995 The osteoclast clear zone is a specialized cell-extracellular matrix adhesion structure. J Cell Sci 108:2729 –2732 Roodman GD 1999 Cell biology of the osteoclast. Exp Hematol 27:1229 –1241 Boyle WJ, Simonet WS, Lacey DL 2003 Osteoclast differentiation and activation. Nature 423:337–342 Zou W, Kitaura H, Reeve J, Long F, Tybulewicz VL, Shattil SJ, Ginsberg MH, Ross FP, Teitelbaum SL 2007 Syk, c-Src, the ␣v␤3 integrin, and ITAM immunoreceptors, in concert, regulate osteoclastic bone resorption. J Cell Biol 176: 877– 888 Meadows NA, Sharma SM, Faulkner GJ, Ostrowski MC, Hume DA, Cassady AI 2007 The expression of Clcn7 and Ostm1 in osteoclasts is coregulated by microphthalmia transcription factor. J Biol Chem 282:1891–1904 Motyckova G, Weilbaecher KN, Horstmann M, Rieman DJ, Fisher DZ, Fisher DE 2001 Linking osteopetrosis and pycnodysostosis: regulation of cathepsin K expression by edrv.endojournals.org 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 51 the microphthalmia transcription factor family. Proc Natl Acad Sci USA 98:5798 –5803 Luchin A, Purdom G, Murphy K, Clark MY, Angel N, Cassady AI, Hume DA, Ostrowski MC 2000 The microphthalmia transcription factor regulates expression of the tartrate-resistant acid phosphatase gene during terminal differentiation of osteoclasts. J Bone Miner Res 15:451– 460 Zhao H, Laitala-Leinonen T, Parikka V, Väänänen HK 2001 Downregulation of small GTPase Rab7 impairs osteoclast polarization and bone resorption. J Biol Chem 276:39295–39302 Rodan GA, Martin TJ 2000 Therapeutic approaches to bone diseases. Science 289:1508 –1514 Blair HC, Teitelbaum SL, Ghiselli R, Gluck S 1989 Osteoclastic bone resorption by a polarized vacuolar proton pump. Science 245:855– 857 Blair HC, Teitelbaum SL, Tan HL, Koziol CM, Schlesinger PH 1991 Passive chloride permeability charge coupled to H(⫹)-ATPase of avian osteoclast ruffled membrane. Am J Physiol 260:C1315–C1324 Baron R, Neff L, Louvard D, Courtoy PJ 1985 Cell-mediated extracellular acidification and bone resorption: evidence for a low pH in resorbing lacunae and localization of a 100-kD lysosomal membrane protein at the osteoclast ruffled border. J Cell Biol 101:2210 –2222 Scimeca JC, Franchi A, Trojani C, Parrinello H, Grosgeorge J, Robert C, Jaillon O, Poirier C, Gaudray P, Carle GF 2000 The gene encoding the mouse homologue of the human osteoclast-specific 116-kDa V-ATPase subunit bears a deletion in osteosclerotic (oc/oc) mutants. Bone 26:207–213 Li YP, Chen W, Liang Y, Li E, Stashenko P 1999 Atp6ideficient mice exhibit severe osteopetrosis due to loss of osteoclast-mediated extracellular acidification. Nat Genet 23:447– 451 Kornak U, Schulz A, Friedrich W, Uhlhaas S, Kremens B, Voit T, Hasan C, Bode U, Jentsch TJ, Kubisch C 2000 Mutations in the a3 subunit of the vacuolar H(⫹)-ATPase cause infantile malignant osteopetrosis. Hum Mol Genet 9:2059 –2063 Henriksen K, Gram J, Schaller S, Dahl BH, Dziegiel MH, Bollerslev J, Karsdal MA 2004 Characterization of osteoclasts from patients harboring a G215R mutation in ClC-7 causing autosomal dominant osteopetrosis type II. Am J Pathol 164:1537–1545 Graves AR, Curran PK, Smith CL, Mindell JA 2008 The Cl(⫺)/H(⫹) antiporter ClC-7 is the primary chloride permeation pathway in lysosomes. Nature 453:788 –792 Weinert S, Jabs S, Supanchart C, Schweizer M, Gimber N, Richter M, Rademann J, Stauber T, Kornak U, Jentsch TJ 2010 Lysosomal pathology and osteopetrosis upon loss of H⫹-driven lysosomal Cl⫺ accumulation. Science 328:1401–1403 Josephsen K, Praetorius J, Frische S, Gawenis LR, Kwon TH, Agre P, Nielsen S, Fejerskov O 2009 Targeted disruption of the Cl⫺/HCO3⫺ exchanger Ae2 results in osteopetrosis in mice. Proc Natl Acad Sci USA 106:1638 –1641 Jansen ID, Mardones P, Lecanda F, de Vries TJ, Recalde S, Hoeben KA, Schoenmaker T, Ravesloot JH, van Borren MM, van Eijden TM, Bronckers AL, Kellokumpu S, Medina JF, Everts V, Oude Elferink RP 2009 Ae2a,b-Deficient 52 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 74. 75. 76. Henriksen et al. Osteoclast Subtypes mice exhibit osteopetrosis of long bones but not of calvaria. FASEB J 23:3470 –3481 Wu J, Glimcher LH, Aliprantis AO 2008 HCO3⫺/Cl⫺ anion exchanger SLC4A2 is required for proper osteoclast differentiation and function. Proc Natl Acad Sci USA 105: 16934 –16939 Bossard MJ, Tomaszek TA, Thompson SK, Amegadzie BY, Hanning CR, Jones C, Kurdyla JT, McNulty DE, Drake FH, Gowen M, Levy MA 1996 Proteolytic activity of human osteoclast cathepsin K. Expression, purification, activation, and substrate identification. J Biol Chem 271: 12517–12524 Saftig P, Hunziker E, Wehmeyer O, Jones S, Boyde A, Rommerskirch W, Moritz JD, Schu P, von Figura K 1998 Impaired osteoclastic bone resorption leads to osteopetrosis in cathepsin-K-deficient mice. Proc Natl Acad Sci USA 95: 13453–13458 Gowen M, Lazner F, Dodds R, Kapadia R, Feild J, Tavaria M, Bertoncello I, Drake F, Zavarselk S, Tellis I, Hertzog P, Debouck C, Kola I 1999 Cathepsin K knockout mice develop osteopetrosis due to a deficit in matrix degradation but not demineralization. J Bone Miner Res 14:1654 –1663 Nishi Y, Atley L, Eyre DE, Edelson JG, Superti-Furga A, Yasuda T, Desnick RJ, Gelb BD 1999 Determination of bone markers in pycnodysostosis: effects of cathepsin K deficiency on bone matrix degradation. J Bone Miner Res 14:1902–1908 Everts V, Delaissé JM, Korper W, Jansen DC, TigchelaarGutter W, Saftig P, Beertsen W 2002 The bone lining cell: its role in cleaning Howship’s lacunae and initiating bone formation. J Bone Miner Res 17:77–90 Henriksen K, Sørensen MG, Nielsen RH, Gram J, Schaller S, Dziegiel MH, Everts V, Bollerslev J, Karsdal MA 2006 Degradation of the organic phase of bone by osteoclasts: a secondary role for lysosomal acidification. J Bone Miner Res 21:58 – 66 Everts V, Korper W, Hoeben KA, Jansen ID, Bromme D, Cleutjens KB, Heeneman S, Peters C, Reinheckel T, Saftig P, Beertsen W 2006 Osteoclastic bone degradation and the role of different cysteine proteinases and matrix metalloproteinases: differences between calvaria and long bone. J Bone Miner Res 21:1399 –1408 Shorey S, Heersche JN, Manolson MF 2004 The relative contribution of cysteine proteinases and matrix metalloproteinases to the resorption process in osteoclasts derived from long bone and scapula. Bone 35:909 –917 Salo J, Lehenkari P, Mulari M, Metsikkö K, Väänänen HK 1997 Removal of osteoclast bone resorption products by transcytosis. Science 276:270 –273 Nesbitt SA, Horton MA 1997 Trafficking of matrix collagens through bone-resorbing osteoclasts. Science 276: 266 –269 Baron R 2005 General principles of bone biology. In: Rosen CJ, Compston JE, Lian JB, eds. Primer on the metabolic bone diseases and disorders of mineral metabolism. Chap 1. 5th ed. Hoboken, NJ: Wiley; 1– 8 Bollerslev J 1989 Autosomal dominant osteopetrosis: bone metabolism and epidemiological, clinical, and hormonal aspects. Endocr Rev 10:45– 67 Perez-Amodio S, Jansen DC, Schoenmaker T, Vogels IM, Reinheckel T, Hayman AR, Cox TM, Saftig P, Beertsen W, Everts V 2006 Calvarial osteoclasts express a higher level Endocrine Reviews, February 2011, 32(1):31– 63 77. 78. 79. 80. 81. 82. 83. 84. 85. 86. 87. 88. 89. of tartrate-resistant acid phosphatase than long bone osteoclasts and activation does not depend on cathepsin K or L activity. Calcif Tissue Int 79:245–254 Hollberg K, Hultenby K, Hayman A, Cox T, Andersson G 2002 Osteoclasts from mice deficient in tartrate-resistant acid phosphatase have altered ruffled borders and disturbed intracellular vesicular transport. Exp Cell Res 279: 227–238 Roberts HC, Knott L, Avery NC, Cox TM, Evans MJ, Hayman AR 2007 Altered collagen in tartrate-resistant acid phosphatase (TRAP)-deficient mice: a role for TRAP in bone collagen metabolism. Calcif Tissue Int 80:400 – 410 van den Bos T, Speijer D, Bank RA, Brömme D, Everts V 2008 Differences in matrix composition between calvaria and long bone in mice suggest differences in biomechanical properties and resorption: special emphasis on collagen. Bone 43:459 – 468 Engsig MT, Chen QJ, Vu TH, Pedersen AC, Therkidsen B, Lund LR, Henriksen K, Lenhard T, Foged NT, Werb Z, Delaissé JM 2000 Matrix metalloproteinase 9 and vascular endothelial growth factor are essential for osteoclast recruitment into developing long bones. J Cell Biol 151: 879 – 889 Delaissé JM, Andersen TL, Engsig MT, Henriksen K, Troen T, Blavier L 2003 Matrix metalloproteinases (MMP) and cathepsin K contribute differently to osteoclastic activities. Microsc Res Tech 61:504 –513 Shibata S, Yamashita Y 2001 An ultrastructural study of osteoclasts and chondroclasts in poorly calcified mandible induced by high doses of strontium diet to fetal mice. Ann Anat 183:357–361 Bromley M, Woolley DE 1984 Chondroclasts and osteoclasts at subchondral sites of erosion in the rheumatoid joint. Arthritis Rheum 27:968 –975 Karsdal MA, Leeming DJ, Dam EB, Henriksen K, Alexandersen P, Pastoureau P, Altman RD, Christiansen C 2008 Should subchondral bone turnover be targeted when treating osteoarthritis? Osteoarthritis Cartilage 16:638 – 646 Mansell JP, Collins C, Bailey AJ 2007 Bone, not cartilage, should be the major focus in osteoarthritis. Nat Clin Pract Rheumatol 3:306 –307 Ortega N, Behonick DJ, Werb Z 2004 Matrix remodeling during endochondral ossification. Trends Cell Biol 14:86 –93 Kong YY, Yoshida H, Sarosi I, Tan HL, Timms E, Capparelli C, Morony S, Oliveira-dos-Santos AJ, Van G, Itie A, Khoo W, Wakeham A, Dunstan CR, Lacey DL, Mak TW, Boyle WJ, Penninger JM 1999 OPGL is a key regulator of osteoclastogenesis, lymphocyte development and lymphnode organogenesis. Nature 397:315–323 Wiktor-Jedrzejczak W, Bartocci A, Ferrante Jr AW, Ahmed-Ansari A, Sell KW, Pollard JW, Stanley ER 1990 Total absence of colony-stimulating factor 1 in the macrophage-deficient osteopetrotic (op/op) mouse. Proc Natl Acad Sci USA 87:4828 – 4832 Wiktor-Jedrzejczak W, Urbanowska E, Aukerman SL, Pollard JW, Stanley ER, Ralph P, Ansari AA, Sell KW, Szperl M 1991 Correction by CSF-1 of defects in the osteopetrotic op/op mouse suggests local, developmental, and humoral Endocrine Reviews, February 2011, 32(1):31– 63 90. 91. 92. 93. 94. 95. 96. 97. 98. 99. 100. 101. 102. 103. requirements for this growth factor. Exp Hematol 19:1049 –1054 Li J, Sarosi I, Yan XQ, Morony S, Capparelli C, Tan HL, McCabe S, Elliott R, Scully S, Van G, Kaufman S, Juan SC, Sun Y, Tarpley J, Martin L, Christensen K, McCabe J, Kostenuik P, Hsu H, Fletcher F, Dunstan CR, Lacey DL, Boyle WJ 2000 RANK is the intrinsic hematopoietic cell surface receptor that controls osteoclastogenesis and regulation of bone mass and calcium metabolism. Proc Natl Acad Sci USA 97:1566 –1571 Gerber HP, Vu TH, Ryan AM, Kowalski J, Werb Z, Ferrara N 1999 VEGF couples hypertrophic cartilage remodeling, ossification and angiogenesis during endochondral bone formation. Nat Med 5:623– 628 Blavier L, Delaissé JM 1995 Matrix metalloproteinases are obligatory for the migration of preosteoclasts to the developing marrow cavity of primitive long bones. J Cell Sci 108:3649 –3659 Dieudonné SC, Foo P, van Zoelen EJ, Burger EH 1991 Inhibiting and stimulating effects of TGF-␤ 1 on osteoclastic bone resorption in fetal mouse bone organ cultures. J Bone Miner Res 6:479 – 487 Lowe C, Yoneda T, Boyce BF, Chen H, Mundy GR, Soriano P 1993 Osteopetrosis in Src-deficient mice is due to an autonomous defect of osteoclasts. Proc Natl Acad Sci USA 90:4485– 4489 Neutzsky-Wulff AV, Karsdal MA, Henriksen K 2008 Characterization of the bone phenotype in ClC-7-deficient mice. Calcif Tissue Int 83:425– 437 Bollerslev J, Steiniche T, Melsen F, Mosekilde L 1989 Structural and histomorphometric studies of iliac crest trabecular and cortical bone in autosomal dominant osteopetrosis: a study of two radiological types. Bone 10:19 –24 Bollerslev J, Marks Jr SC, Pockwinse S, Kassem M, Brixen K, Steiniche T, Mosekilde L 1993 Ultrastructural investigations of bone resorptive cells in two types of autosomal dominant osteopetrosis. Bone 14:865– 869 Nordahl J, Andersson G, Reinholt FP 1998 Chondroclasts and osteoclasts in bones of young rats: comparison of ultrastructural and functional features. Calcif Tissue Int 63: 401– 408 Sawae Y, Sahara T, Sasaki T 2003 Osteoclast differentiation at growth plate cartilage-trabecular bone junction in newborn rat femur. J Electron Microsc (Tokyo) 52:493–502 Pennypacker B, Shea M, Liu Q, Masarachia P, Saftig P, Rodan S, Rodan G, Kimmel D 2009 Bone density, strength, and formation in adult cathepsin K (⫺/⫺) mice. Bone 44: 199 –207 Garnero P, Ferreras M, Karsdal MA, Nicamhlaoibh R, Risteli J, Borel O, Qvist P, Delmas PD, Foged NT, Delaissé JM 2003 The type I collagen fragments ICTP and CTX reveal distinct enzymatic pathways of bone collagen degradation. J Bone Miner Res 18:859 – 867 Sørensen MG, Henriksen K, Schaller S, Henriksen DB, Nielsen FC, Dziegiel MH, Karsdal MA 2007 Characterization of osteoclasts derived from CD14⫹ monocytes isolated from peripheral blood. J Bone Miner Metab 25:36 – 45 Kiviranta R, Morko J, Alatalo SL, NicAmhlaoibh R, Risteli J, Laitala-Leinonen T, Vuorio E 2005 Impaired bone resorption in cathepsin K-deficient mice is partially compen- edrv.endojournals.org 104. 105. 106. 107. 108. 109. 110. 111. 112. 113. 114. 115. 116. 117. 118. 119. 53 sated for by enhanced osteoclastogenesis and increased expression of other proteases via an increased RANKL/OPG ratio. Bone 36:159 –172 Zenger S, Hollberg K, Ljusberg J, Norgård M, EkRylander B, Kiviranta R, Andersson G 2007 Proteolytic processing and polarized secretion of tartrate-resistant acid phosphatase is altered in a subpopulation of metaphyseal osteoclasts in cathepsin K-deficient mice. Bone 41:820 – 832 Burr DB 2002 Targeted and nontargeted remodeling. Bone 30:2– 4 Parfitt AM 2002 Targeted and nontargeted bone remodeling: relationship to basic multicellular unit origination and progression. Bone 30:5–7 Noble B 2003 Bone microdamage and cell apoptosis. Eur Cell Mater 6:46 –55; discussion 55 Verborgt O, Gibson GJ, Schaffler MB 2000 Loss of osteocyte integrity in association with microdamage and bone remodeling after fatigue in vivo. J Bone Miner Res 15: 60 – 67 Tatsumi S, Ishii K, Amizuka N, Li M, Kobayashi T, Kohno K, Ito M, Takeshita S, Ikeda K 2007 Targeted ablation of osteocytes induces osteoporosis with defective mechanotransduction. Cell Metab 5:464 – 475 Talmage RV, Talmage DW 2006 Calcium homeostasis: solving the solubility problem. J Musculoskelet Neuronal Interact 6:402– 407 Parfitt AM 2003 Misconceptions (3): calcium leaves bone only by resorption and enters only by formation. Bone 33:259 –263 Marenzana M, Shipley AM, Squitiero P, Kunkel JG, Rubinacci A 2005 Bone as an ion exchange organ: evidence for instantaneous cell-dependent calcium efflux from bone not due to resorption. Bone 37:545–554 Dent CE, Smellie JM, Watson L 1965 Studies in osteopetrosis. Arch Dis Child 40:7–15 Manolagas SC 2000 Birth and death of bone cells: basic regulatory mechanisms and implications for the pathogenesis and treatment of osteoporosis. Endocr Rev 21: 115–137 Karsdal MA, Hjorth P, Henriksen K, Kirkegaard T, Nielsen KL, Lou H, Delaissé JM, Foged NT 2003 Transforming growth factor-␤ controls human osteoclastogenesis through the p38 MAPK and regulation of RANK expression. J Biol Chem 278:44975– 44987 Van Wesenbeeck L, Odgren PR, Coxon FP, Frattini A, Moens P, Perdu B, MacKay CA, Van Hul E, Timmermans JP, Vanhoenacker F, Jacobs R, Peruzzi B, Teti A, Helfrich MH, Rogers MJ, Villa A, Van Hul W 2007 Involvement of PLEKHM1 in osteoclastic vesicular transport and osteopetrosis in incisors absent rats and humans. J Clin Invest 117:919 –930 Valcourt U, Merle B, Gineyts E, Viguet-Carrin S, Delmas PD, Garnero P 2007 Non-enzymatic glycation of bone collagen modifies osteoclastic activity and differentiation. J Biol Chem 282:5691–5703 Schlemmer A, Hassager C, Jensen SB, Christiansen C 1992 Marked diurnal variation in urinary excretion of pyridinium cross-links in premenopausal women. J Clin Endocrinol Metab 74:476 – 480 Gertz BJ, Clemens JD, Holland SD, Yuan W, Greenspan S 1998 Application of a new serum assay for type I collagen 54 120. 121. 122. 123. 124. 125. 126. 127. 128. 129. 130. 131. 132. Henriksen et al. Osteoclast Subtypes cross-linked N-telopeptides: assessment of diurnal changes in bone turnover with and without alendronate treatment. Calcif Tissue Int 63:102–106 Qvist P, Christgau S, Pedersen BJ, Schlemmer A, Christiansen C 2002 Circadian variation in the serum concentration of C-terminal telopeptide of type I collagen (serum CTx): effects of gender, age, menopausal status, posture, daylight, serum cortisol, and fasting. Bone 31:57– 61 Bjarnason NH, Henriksen EE, Alexandersen P, Christgau S, Henriksen DB, Christiansen C 2002 Mechanism of circadian variation in bone resorption. Bone 30:307–313 Karsdal MA, Byrjalsen I, Riis BJ, Christiansen C 2008 Optimizing bioavailability of oral administration of small peptides through pharmacokinetic and pharmacodynamic parameters: the effect of water and timing of meal intake on oral delivery of salmon calcitonin. BMC Clin Pharmacol 8:5 Karsdal MA, Byrjalsen I, Azria M, Arnold M, Choi L, Riis BJ, Christiansen C 2009 Influence of food intake on the bioavailability and efficacy of oral calcitonin. Br J Clin Pharmacol 67:413– 420 Henriksen DB, Alexandersen P, Byrjalsen I, Hartmann B, Bone HG, Christiansen C, Holst JJ 2004 Reduction of nocturnal rise in bone resorption by subcutaneous GLP-2. Bone 34:140 –147 Henriksen DB, Alexandersen P, Hartmann B, Adrian CL, Byrjalsen I, Bone HG, Holst JJ, Christiansen C 2007 Disassociation of bone resorption and formation by GLP-2: a 14-day study in healthy postmenopausal women. Bone 40: 723–729 Henriksen DB, Alexandersen P, Hartmann B, Adrian CL, Byrjalsen I, Bone HG, Holst JJ, Christiansen C 2009 Fourmonth treatment with GLP-2 significantly increases hip BMD: a randomized, placebo-controlled, dose-ranging study in postmenopausal women with low BMD. Bone 45:833– 842 Tankó LB, Bagger YZ, Alexandersen P, Devogelaer JP, Reginster JY, Chick R, Olson M, Benmammar H, Mindeholm L, Azria M, Christiansen C 2004 Safety and efficacy of a novel salmon calcitonin (sCT) technology-based oral formulation in healthy postmenopausal women: acute and 3-month effects on biomarkers of bone turnover. J Bone Miner Res 19:1531–1538 Eghbali-Fatourechi G, Khosla S, Sanyal A, Boyle WJ, Lacey DL, Riggs BL 2003 Role of RANK ligand in mediating increased bone resorption in early postmenopausal women. J Clin Invest 111:1221–1230 Clowes JA, Eghbali-Fatourechi GZ, McCready L, Oursler MJ, Khosla S, Riggs BL 2009 Estrogen action on bone marrow osteoclast lineage cells of postmenopausal women in vivo. Osteoporos Int 20:761–769 Taxel P, Kaneko H, Lee SK, Aguila HL, Raisz LG, Lorenzo JA 2008 Estradiol rapidly inhibits osteoclastogenesis and RANKL expression in bone marrow cultures in postmenopausal women: a pilot study. Osteoporos Int 19:193–199 Cao JJ, Wronski TJ, Iwaniec U, Phleger L, Kurimoto P, Boudignon B, Halloran BP 2005 Aging increases stromal/ osteoblastic cell-induced osteoclastogenesis and alters the osteoclast precursor pool in the mouse. J Bone Miner Res 20:1659 –1668 D’Amelio P, Grimaldi A, Pescarmona GP, Tamone C, Roato I, Isaia G 2005 Spontaneous osteoclast formation Endocrine Reviews, February 2011, 32(1):31– 63 133. 134. 135. 136. 137. 138. 139. 140. 141. 142. 143. 144. 145. from peripheral blood mononuclear cells in postmenopausal osteoporosis. FASEB J 19:410 – 412 Jevon M, Hirayama T, Brown MA, Wass JA, Sabokbar A, Ostelere S, Athenasou NA 2003 Osteoclast formation from circulating precursors in osteoporosis. Scand J Rheumatol 32:95–100 D’Amelio P, Grimaldi A, Di Bella S, Brianza SZ, Cristofaro MA, Tamone C, Giribaldi G, Ulliers D, Pescarmona GP, Isaia G 2008 Estrogen deficiency increases osteoclastogenesis up-regulating T cells activity: a key mechanism in osteoporosis. Bone 43:92–100 Shevde NK, Bendixen AC, Dienger KM, Pike JW 2000 Estrogens suppress RANK ligand-induced osteoclast differentiation via a stromal cell independent mechanism involving c-Jun repression. Proc Natl Acad Sci USA 97:7829 –7834 Srivastava S, Toraldo G, Weitzmann MN, Cenci S, Ross FP, Pacifici R 2001 Estrogen decreases osteoclast formation by down-regulating receptor activator of NF-B ligand (RANKL)-induced JNK activation. J Biol Chem 276: 8836 – 8840 Sørensen MG, Henriksen K, Dziegiel MH, Tankó LB, Karsdal MA 2006 Estrogen directly attenuates human osteoclastogenesis, but has no effect on resorption by mature osteoclasts. DNA Cell Biol 25:475– 483 Huber DM, Bendixen AC, Pathrose P, Srivastava S, Dienger KM, Shevde NK, Pike JW 2001 Androgens suppress osteoclast formation induced by RANKL and macrophage-colony stimulating factor. Endocrinology 142:3800 – 3808 Nakamura T, Imai Y, Matsumoto T, Sato S, Takeuchi K, Igarashi K, Harada Y, Azuma Y, Krust A, Yamamoto Y, Nishina H, Takeda S, Takayanagi H, Metzger D, Kanno J, Takaoka K, Martin TJ, Chambon P, Kato S 2007 Estrogen prevents bone loss via estrogen receptor ␣ and induction of Fas ligand in osteoclasts. Cell 130:811– 823 Bord S, Horner A, Beavan S, Compston J 2001 Estrogen receptors ␣ and ␤ are differentially expressed in developing human bone. J Clin Endocrinol Metab 86:2309 –2314 Martin-Millan M, Almeida M, Ambrogini E, Han L, Zhao H, Weinstein RS, Jilka RL, O’Brien CA, Manolagas SC 2010 The estrogen receptor-␣ in osteoclasts mediates the protective effects of estrogens on cancellous but not cortical bone. Mol Endocrinol 24:323–334 Riggs BL, Melton Iii 3rd LJ, Robb RA, Camp JJ, Atkinson EJ, Peterson JM, Rouleau PA, McCollough CH, Bouxsein ML, Khosla S 2004 Population-based study of age and sex differences in bone volumetric density, size, geometry, and structure at different skeletal sites. J Bone Miner Res 19: 1945–1954 Recker R, Lappe J, Davies KM, Heaney R 2004 Bone remodeling increases substantially in the years after menopause and remains increased in older osteoporosis patients. J Bone Miner Res 19:1628 –1633 Garnero P, Sornay-Rendu E, Chapuy MC, Delmas PD 1996 Increased bone turnover in late postmenopausal women is a major determinant of osteoporosis. J Bone Miner Res 11:337–349 Arlot ME, Delmas PD, Chappard D, Meunier PJ 1990 Trabecular and endocortical bone remodeling in postmenopausal osteoporosis: comparison with normal postmenopausal women. Osteoporos Int 1:41– 49 Endocrine Reviews, February 2011, 32(1):31– 63 146. Viguet-Carrin S, Garnero P, Delmas PD 2006 The role of collagen in bone strength. Osteoporos Int 17:319 –336 147. Herrmann M, Widmann T, Colaianni G, Colucci S, Zallone A, Herrmann W 2005 Increased osteoclast activity in the presence of increased homocysteine concentrations. Clin Chem 51:2348 –2353 148. Saito M, Marumo K 2010 Collagen cross-links as a determinant of bone quality: a possible explanation for bone fragility in aging, osteoporosis, and diabetes mellitus. Osteoporos Int 21:195–214 149. Miyata T, Notoya K, Yoshida K, Horie K, Maeda K, Kurokawa K, Taketomi S 1997 Advanced glycation end products enhance osteoclast-induced bone resorption in cultured mouse unfractionated bone cells and in rats implanted subcutaneously with devitalized bone particles. J Am Soc Nephrol 8:260 –270 150. Schwartz AV, Garnero P, Hillier TA, Sellmeyer DE, Strotmeyer ES, Feingold KR, Resnick HE, Tylavsky FA, Black DM, Cummings SR, Harris TB, Bauer DC 2009 Pentosidine and increased fracture risk in older adults with type 2 diabetes. J Clin Endocrinol Metab 94:2380 –2386 151. Raska Jr I, Broulík P 2005 The impact of diabetes mellitus on skeletal health: an established phenomenon with inestablished causes? Prague Med Rep 106:137–148 152. Sanguineti R, Storace D, Monacelli F, Federici A, Odetti P 2008 Pentosidine effects on human osteoblasts in vitro. Ann NY Acad Sci 1126:166 –172 153. Henriksen K, Gram J, Neutzsky-Wulff AV, Jensen VK, Dziegiel MH, Bollerslev J, Karsdal MA 2009 Characterization of acid flux in osteoclasts from patients harboring a G215R mutation in ClC-7. Biochem Biophys Res Commun 378:804 – 809 154. Taranta A, Migliaccio S, Recchia I, Caniglia M, Luciani M, De Rossi G, Dionisi-Vici C, Pinto RM, Francalanci P, Boldrini R, Lanino E, Dini G, Morreale G, Ralston SH, Villa A, Vezzoni P, Del Principe D, Cassiani F, Palumbo G, Teti A 2003 Genotype-phenotype relationship in human ATP6idependent autosomal recessive osteopetrosis. Am J Pathol 162:57– 68 155. Maranda B, Chabot G, Décarie JC, Pata M, Azeddine B, Moreau A, Vacher J 2008 Clinical and cellular manifestations of OSTM1-related infantile osteopetrosis. J Bone Miner Res 23:296 –300 156. Rajapurohitam V, Chalhoub N, Benachenhou N, Neff L, Baron R, Vacher J 2001 The mouse osteopetrotic greylethal mutation induces a defect in osteoclast maturation/ function. Bone 28:513–523 157. Semba I, Ishigami T, Sugihara K, Kitano M 2000 Higher osteoclastic demineralization and highly mineralized cement lines with osteocalcin deposition in a mandibular cortical bone of autosomal dominant osteopetrosis type II: ultrastructural and undecalcified histological investigations. Bone 27:389 –395 158. Garnero P, Thompson E, Woodworth T, Smolen JS 2010 Rapid and sustained improvement in bone and cartilage turnover markers with the anti-interleukin-6 receptor inhibitor tocilizumab plus methotrexate in rheumatoid arthritis patients with an inadequate response to methotrexate: results from a substudy of the multicenter double-blind, placebo-controlled trial of tocilizumab in inadequate responders to methotrexate alone. Arthritis Rheum. 62:33– 43 edrv.endojournals.org 55 159. Nielsen RH, Karsdal MA, Sørensen MG, Dziegiel MH, Henriksen K 2007 Dissolution of the inorganic phase of bone leading to release of calcium regulates osteoclast survival. Biochem Biophys Res Commun 360:834 – 839 160. Alatalo SL, Ivaska KK, Waguespack SG, Econs MJ, Väänänen HK, Halleen JM 2004 Osteoclast-derived serum tartrate-resistant acid phosphatase 5b in Albers-Schonberg disease (type II autosomal dominant osteopetrosis). Clin Chem 50:883– 890 161. Del Fattore A, Peruzzi B, Rucci N, Recchia I, Cappariello A, Longo M, Fortunati D, Ballanti P, Iacobini M, Luciani M, Devito R, Pinto R, Caniglia M, Lanino E, Messina C, Cesaro S, Letizia C, Bianchini G, Fryssira H, Grabowski P, Shaw N, Bishop N, Hughes D, Kapur RP, Datta HK, Taranta A, Fornari R, Migliaccio S, Teti A 2006 Clinical, genetic, and cellular analysis of 49 osteopetrotic patients: implications for diagnosis and treatment. J Med Genet 43: 315–325 162. Soriano P, Montgomery C, Geske R, Bradley A 1991 Targeted disruption of the c-src proto-oncogene leads to osteopetrosis in mice. Cell 64:693–702 163. Grigoriadis AE, Wang ZQ, Cecchini MG, Hofstetter W, Felix R, Fleisch HA, Wagner EF 1994 c-Fos: a key regulator of osteoclast-macrophage lineage determination and bone remodeling. Science 266:443– 448 164. Marzia M, Sims NA, Voit S, Migliaccio S, Taranta A, Bernardini S, Faraggiana T, Yoneda T, Mundy GR, Boyce BF, Baron R, Teti A 2000 Decreased c-Src expression enhances osteoblast differentiation and bone formation. J Cell Biol 151:311–320 165. Demiralp B, Chen HL, Koh AJ, Keller ET, McCauley LK 2002 Anabolic actions of parathyroid hormone during bone growth are dependent on c-fos. Endocrinology 143: 4038 – 4047 166. Koh AJ, Demiralp B, Neiva KG, Hooten J, Nohutcu RM, Shim H, Datta NS, Taichman RS, McCauley LK 2005 Cells of the osteoclast lineage as mediators of the anabolic actions of parathyroid hormone in bone. Endocrinology 146:4584 – 4596 167. Holmen SL, Zylstra CR, Mukherjee A, Sigler RE, Faugere MC, Bouxsein ML, Deng L, Clemens TL, Williams BO 2005 Essential role of ␤-catenin in postnatal bone acquisition. J Biol Chem 280:21162–21168 168. Gong Y, Slee RB, Fukai N, Rawadi G, Roman-Roman S, Reginato AM, Wang H, Cundy T, Glorieux FH, Lev D, Zacharin M, Oexle K, Marcelino J, Suwairi W, Heeger S, Sabatakos G, Apte S, Adkins WN, Allgrove J, ArslanKirchner M, Batch JA, Beighton P, Black GC, Boles RG, Boon LM, Borrone C, Brunner HG, Carle GF, Dallapiccola B, De Paepe A, Floege B, Halfhide ML, Hall B, Hennekam RC, Hirose T, Jans A, Jüppner H, Kim CA, KepplerNoreuil K, Kohlschuetter A, LaCombe D, Lambert M, Lemyre E, Letteboer T, Peltonen L, Ramesar RS, Romanengo M, Somer H, Steichen-Gersdorf E, Steinmann B, Sullivan B, Superti-Furga A, Swoboda W, van den Boogaard MJ, Van Hul W, Vikkula M, Votruba M, Zabel B, Garcia T, Baron R, Olsen BR, Warman ML 2001 LDL receptor-related protein 5 (LRP5) affects bone accrual and eye development. Cell 107:513–523 169. Van Wesenbeeck L, Cleiren E, Gram J, Beals RK, Bénichou O, Scopelliti D, Key L, Renton T, Bartels C, Gong Y, Warman ML, De Vernejoul MC, Bollerslev J, Van Hul W 2003 56 170. 171. 172. 173. 174. 175. 176. 177. 178. 179. 180. 181. 182. 183. 184. Henriksen et al. Osteoclast Subtypes Six novel missense mutations in the LDL receptor-related protein 5 (LRP5) gene in different conditions with an increased bone density. Am J Hum Genet 72:763–771 Bollerslev J, Ueland T, Odgren PR 2003 Serum levels of TGF-␤ and fibronectin in autosomal dominant osteopetrosis in relation to underlying mutations and well-described murine counterparts. Crit Rev Eukaryot Gene Expr 13:163–171 Sarnsethsiri P, Hitt OK, Eyring EJ, Frost HM 1971 Tetracycline-based study of bone dynamics in pycnodysostosis. Clin Orthop Relat Res 74:301–312 Ho N, Punturieri A, Wilkin D, Szabo J, Johnson M, Whaley J, Davis J, Clark A, Weiss S, Francomano C 1999 Mutations of CTSK result in pycnodysostosis via a reduction in cathepsin K protein. J Bone Miner Res 14:1649 –1653 Hou WS, Brömme D, Zhao Y, Mehler E, Dushey C, Weinstein H, Miranda CS, Fraga C, Greig F, Carey J, Rimoin DL, Desnick RJ, Gelb BD 1999 Characterization of novel cathepsin K mutations in the pro and mature polypeptide regions causing pycnodysostosis. J Clin Invest 103: 731–738 Chavassieux P, Asser Karsdal M, Segovia-Silvestre T, Neutzsky-Wulff AV, Chapurlat R, Boivin G, Delmas PD 2008 Mechanisms of the anabolic effects of teriparatide on bone: insight from the treatment of a patient with pycnodysostosis. J Bone Miner Res 23:1076 –1083 Everts V, Aronson DC, Beertsen W 1985 Phagocytosis of bone collagen by osteoclasts in two cases of pycnodysostosis. Calcif Tissue Int 37:25–31 Li CY, Jepsen KJ, Majeska RJ, Zhang J, Ni R, Gelb BD, Schaffler MB 2006 Mice lacking cathepsin K maintain bone remodeling but develop bone fragility despite high bone mass. J Bone Miner Res 21:865– 875 Fratzl-Zelman N, Valenta A, Roschger P, Nader A, Gelb BD, Fratzl P, Klaushofer K 2004 Decreased bone turnover and deterioration of bone structure in two cases of pycnodysostosis. J Clin Endocrinol Metab 89:1538 –1547 Bone HG, McClung MR, Roux C, Recker RR, Eisman JA, Verbruggen N, Hustad CM, DaSilva C, Santora AC, Ince BA 2010 Odanacatib, a cathepsin-K inhibitor for osteoporosis: a two-year study in postmenopausal women with low bone density. J Bone Miner Res 25:937–947 Pennypacker B, Wesolowski G, Heo J, Duong LT 2009 Effects of odanacatib on central femur cortical bone in estrogen-deficient adult rhesus monkeys. J Bone Miner Res 24(Suppl 1):1171 (Abstract) Cusick T, Pennypacker B, Scott K, Duong LT, Kimmel D 2009 Effects of odanacatib on bone mass, turnover and strength in the femoral neck of estrogen deficient adult rhesus monkeys. J Bone Miner Res 24(Suppl 1):FR0416 (Abstract) Scott K, Cusick T, Duong LT, Pennypacker B, Kimmel D 2009 Effects of odanacatib on bone turnover and osteoclast morphology in the lumbar vertebra of ovariectomized adult rhesus monkeys. J Bone Miner Res 24(Suppl 1): SU0227 (Abstract) Helfrich M, Crockett JC, Hocking LJ, Coxon FP 2007 The pathogenesis of osteoclast diseases: some knowns, but still many unknowns. BoneKEy-Osteovision 4:61–77 Roodman GD 1996 Paget’s disease and osteoclast biology. Bone 19:209 –212 Goode A, Layfield R 2010 Recent advances in understand- Endocrine Reviews, February 2011, 32(1):31– 63 185. 186. 187. 188. 189. 190. 191. 192. 193. 194. 195. 196. 197. 198. 199. 200. 201. ing the molecular basis of Paget’s disease of bone. J Clin Pathol 63:199 –203 Roodman GD, Windle JJ 2005 Paget disease of bone. J Clin Invest 115:200 –208 Ralston SH, Langston AL, Reid IR 2008 Pathogenesis and management of Paget’s disease of bone. Lancet 372: 155–163 Neale SD, Smith R, Wass JA, Athanasou NA 2000 Osteoclast differentiation from circulating mononuclear precursors in Paget’s disease is hypersensitive to 1,25-dihydroxyvitamin D(3) and RANKL. Bone 27:409 – 416 Singer FR, Mills BG, Gruber HE, Windle JJ, Roodman GD 2006 Ultrastructure of bone cells in Paget’s disease of bone. J Bone Miner Res 21(Suppl 2):P51–P54 Kurihara N, Hiruma Y, Zhou H, Subler MA, Dempster DW, Singer FR, Reddy SV, Gruber HE, Windle JJ, Roodman GD 2007 Mutation of the sequestosome 1 (p62) gene increases osteoclastogenesis but does not induce Paget disease. J Clin Invest 117:133–142 Hiruma Y, Kurihara N, Subler MA, Zhou H, Boykin CS, Zhang H, Ishizuka S, Dempster DW, Roodman GD, Windle JJ 2008 A SQSTM1/p62 mutation linked to Paget’s disease increases the osteoclastogenic potential of the bone microenvironment. Hum Mol Genet 17:3708 –3719 Gennari L, Merlotti D, Mossetti G, Rendina D, De Paola V, Martini G, Nuti R 2009 The use of intravenous aminobisphosphonates for the treatment of Paget’s disease of bone. Mini Rev Med Chem 9:1052–1063 Clines GA, Guise TA 2008 Molecular mechanisms and treatment of bone metastasis. Expert Rev Mol Med 10:e7 Guise TA, Mohammad KS, Clines G, Stebbins EG, Wong DH, Higgins LS, Vessella R, Corey E, Padalecki S, Suva L, Chirgwin JM 2006 Basic mechanisms responsible for osteolytic and osteoblastic bone metastases. Clin Cancer Res 12:6213s– 6216s Hu MI, Lu H, Gagel RF 2010 Cancer therapies and bone health. Curr Rheumatol Rep 12:177–185 Akhtari M, Mansuri J, Newman KA, Guise TM, Seth P 2008 Biology of breast cancer bone metastasis. Cancer Biol Ther 7:3–9 Lipton A 2006 Future treatment of bone metastases. Clin Cancer Res 12:6305s– 6308s Clezardin P, Teti A 2007 Bone metastasis: pathogenesis and therapeutic implications. Clin Exp Metastasis 24: 599 – 608 Cicek M, Oursler MJ 2006 Breast cancer bone metastasis and current small therapeutics. Cancer Metastasis Rev 25: 635– 644 Leeming DJ, Koizumi M, Byrjalsen I, Li B, Qvist P, Tankó LB 2006 The relative use of eight collagenous and noncollagenous markers for diagnosis of skeletal metastases in breast, prostate, or lung cancer patients. Cancer Epidemiol Biomarkers Prev 15:32–38 Leeming DJ, Delling G, Koizumi M, Henriksen K, Karsdal MA, Li B, Qvist P, Tankó LB, Byrjalsen I 2006 ␣ CTX as a biomarker of skeletal invasion of breast cancer: immunolocalization and the load dependency of urinary excretion. Cancer Epidemiol Biomarkers Prev 15:1392–1395 Koopmans N, de Jong IJ, Breeuwsma AJ, van der Veer E 2007 Serum bone turnover markers (PINP and ICTP) for the early detection of bone metastases in patients with Endocrine Reviews, February 2011, 32(1):31– 63 202. 203. 204. 205. 206. 207. 208. 209. 210. 211. 212. 213. 214. 215. 216. 217. prostate cancer: a longitudinal approach. J Urol 178:849 – 853 Winding B, NicAmhlaoibh R, Misander H, HøeghAndersen P, Andersen TL, Holst-Hansen C, Heegaard AM, Foged NT, Brünner N, Delaissé JM 2002 Synthetic matrix metalloproteinase inhibitors inhibit growth of established breast cancer osteolytic lesions and prolong survival in mice. Clin Cancer Res 8:1932–1939 Le Gall C, Bonnelye E, Clézardin P 2008 Cathepsin K inhibitors as treatment of bone metastasis. Curr Opin Support Palliat Care 2:218 –222 Le Gall C, Bellahcène A, Bonnelye E, Gasser JA, Castronovo V, Green J, Zimmermann J, Clézardin P 2007 A cathepsin K inhibitor reduces breast cancer induced osteolysis and skeletal tumor burden. Cancer Res 67:9894 – 9902 Coussens LM, Fingleton B, Matrisian LM 2002 Matrix metalloproteinase inhibitors and cancer: trials and tribulations. Science 295:2387–2392 Pavlaki M, Zucker S 2003 Matrix metalloproteinase inhibitors (MMPIs): the beginning of phase I or the termination of phase III clinical trials. Cancer Metastasis Rev 22:177–203 Coleman R, Gnant M 2009 New results from the use of bisphosphonates in cancer patients. Curr Opin Support Palliat Care 3:213–218 Machado M, Cruz LS, Tannus G, Fonseca M 2009 Efficacy of clodronate, pamidronate, and zoledronate in reducing morbidity and mortality in cancer patients with bone metastasis: a meta-analysis of randomized clinical trials. Clin Ther 31:962–979 Fizazi K, Lipton A, Mariette X, Body JJ, Rahim Y, Gralow JR, Gao G, Wu L, Sohn W, Jun S 2009 Randomized phase II trial of denosumab in patients with bone metastases from prostate cancer, breast cancer, or other neoplasms after intravenous bisphosphonates. J Clin Oncol 27:1564 –1571 Kearns AE, Khosla S, Kostenuik PJ 2008 Receptor activator of nuclear factor B ligand and osteoprotegerin regulation of bone remodeling in health and disease. Endocr Rev 29:155–192 Rachner TD, Singh SK, Schoppet M, Benad P, Bornhäuser M, Ellenrieder V, Ebert R, Jakob F, Hofbauer LC 2010 Zoledronic acid induces apoptosis and changes the TRAIL/ OPG ratio in breast cancer cells. Cancer Lett 287:109 –116 Schett G, Teitelbaum SL 2009 Osteoclasts and arthritis. J Bone Miner Res 24:1142–1146 Schett G 2007 Erosive arthritis. Arthritis Res Ther 9(Suppl 1):S2 Gravallese EM, Harada Y, Wang JT, Gorn AH, Thornhill TS, Goldring SR 1998 Identification of cell types responsible for bone resorption in rheumatoid arthritis and juvenile rheumatoid arthritis. Am J Pathol 152:943–951 Feldmann M, Brennan FM, Maini RN 1996 Role of cytokines in rheumatoid arthritis. Annu Rev Immunol 14: 397– 440 Keffer J, Probert L, Cazlaris H, Georgopoulos S, Kaslaris E, Kioussis D, Kollias G 1991 Transgenic mice expressing human tumour necrosis factor: a predictive genetic model of arthritis. EMBO J 10:4025– 4031 Singh JA, Christensen R, Wells GA, Suarez-Almazor ME, Buchbinder R, Lopez-Olivo MA, Ghogomu ET, Tugwell P 2009 A network meta-analysis of randomized controlled edrv.endojournals.org 218. 219. 220. 221. 222. 223. 224. 225. 226. 227. 228. 229. 57 trials of biologics for rheumatoid arthritis: a Cochrane overview. CMAJ 181:787–796 Licastro F, Chiappelli M, Ianni M, Porcellini E 2009 Tumor necrosis factor-␣ antagonists: differential clinical effects by different biotechnological molecules. Int J Immunopathol Pharmacol 22:567–572 Pettit AR, Ji H, von Stechow D, Müller R, Goldring SR, Choi Y, Benoist C, Gravallese EM 2001 TRANCE/ RANKL knockout mice are protected from bone erosion in a serum transfer model of arthritis. Am J Pathol 159:1689 – 1699 Romas E, Sims NA, Hards DK, Lindsay M, Quinn JW, Ryan PF, Dunstan CR, Martin TJ, Gillespie MT 2002 Osteoprotegerin reduces osteoclast numbers and prevents bone erosion in collagen-induced arthritis. Am J Pathol 161:1419 –1427 Redlich K, Hayer S, Ricci R, David JP, Tohidast-Akrad M, Kollias G, Steiner G, Smolen JS, Wagner EF, Schett G 2002 Osteoclasts are essential for TNF-␣-mediated joint destruction. J Clin Invest 110:1419 –1427 Cohen SB, Dore RK, Lane NE, Ory PA, Peterfy CG, Sharp JT, van der Heijde D, Zhou L, Tsuji W, Newmark R 2008 Denosumab treatment effects on structural damage, bone mineral density, and bone turnover in rheumatoid arthritis: a twelve-month, multicenter, randomized, doubleblind, placebo-controlled, phase II clinical trial. Arthritis Rheum 58:1299 –1309 Zwerina J, Redlich K, Polzer K, Joosten L, Krönke G, Distler J, Hess A, Pundt N, Pap T, Hoffmann O, Gasser J, Scheinecker C, Smolen JS, van den Berg W, Schett G 2007 TNF-induced structural joint damage is mediated by IL-1. Proc Natl Acad Sci USA 104:11742–11747 Jarrett SJ, Conaghan PG, Sloan VS, Papanastasiou P, Ortmann CE, O’Connor PJ, Grainger AJ, Emery P 2006 Preliminary evidence for a structural benefit of the new bisphosphonate zoledronic acid in early rheumatoid arthritis. Arthritis Rheum 54:1410 –1414 Breuil V, Euller-Ziegler L 2006 Bisphosphonate therapy in rheumatoid arthritis. Joint Bone Spine 73:349 –354 Lems WF, Lodder MC, Lips P, Bijlsma JW, Geusens P, Schrameijer N, van de Ven CM, Dijkmans BA 2006 Positive effect of alendronate on bone mineral density and markers of bone turnover in patients with rheumatoid arthritis on chronic treatment with low-dose prednisone: a randomized, double-blind, placebo-controlled trial. Osteoporos Int 17:716 –723 Reid DM, Devogelaer JP, Saag K, Roux C, Lau CS, Reginster JY, Papanastasiou P, Ferreira A, Hartl F, Fashola T, Mesenbrink P, Sambrook PN 2009 Zoledronic acid and risedronate in the prevention and treatment of glucocorticoid-induced osteoporosis (HORIZON): a multicentre, double-blind, double-dummy, randomised controlled trial. Lancet 373:1253–1263 Sims NA, Green JR, Glatt M, Schlict S, Martin TJ, Gillespie MT, Romas E 2004 Targeting osteoclasts with zoledronic acid prevents bone destruction in collagen-induced arthritis. Arthritis Rheum 50:2338 –2346 Herrak P, Görtz B, Hayer S, Redlich K, Reiter E, Gasser J, Bergmeister H, Kollias G, Smolen JS, Schett G 2004 Zoledronic acid protects against local and systemic bone loss in tumor necrosis factor-mediated arthritis. Arthritis Rheum 50:2327–2337 58 Henriksen et al. Osteoclast Subtypes 230. Morko JP, Söderström M, Säämänen AM, Salminen HJ, Vuorio EI 2004 Up regulation of cathepsin K expression in articular chondrocytes in a transgenic mouse model for osteoarthritis. Ann Rheum Dis 63:649 – 655 231. Morko J, Kiviranta R, Joronen K, Säämänen AM, Vuorio E, Salminen-Mankonen H 2005 Spontaneous development of synovitis and cartilage degeneration in transgenic mice overexpressing cathepsin K. Arthritis Rheum 52: 3713–3717 232. Schurigt U, Hummel KM, Petrow PK, Gajda M, Stöckigt R, Middel P, Zwerina J, Janik T, Bernhardt R, Schüler S, Scharnweber D, Beckmann F, Saftig P, Kollias G, Schett G, Wiederanders B, Bräuer R 2008 Cathepsin K deficiency partially inhibits, but does not prevent, bone destruction in human tumor necrosis factor-transgenic mice. Arthritis Rheum 58:422– 434 233. Hou WS, Li Z, Gordon RE, Chan K, Klein MJ, Levy R, Keysser M, Keyszer G, Brömme D 2001 Cathepsin K is a critical protease in synovial fibroblast-mediated collagen degradation. Am J Pathol 159:2167–2177 234. Yasuda Y, Kaleta J, Brömme D 2005 The role of cathepsins in osteoporosis and arthritis: rationale for the design of new therapeutics. Adv Drug Deliv Rev 57:973–993 235. Ainola M, Valleala H, Nykänen P, Risteli J, Hanemaaijer R, Konttinen YT 2008 Erosive arthritis in a patient with pycnodysostosis: an experiment of nature. Arthritis Rheum 58:3394 –3401 236. Svelander L, Erlandsson-Harris H, Astner L, Grabowska U, Klareskog L, Lindstrom E, Hewitt E 2009 Inhibition of cathepsin K reduces bone erosion, cartilage degradation and inflammation evoked by collagen-induced arthritis in mice. Eur J Pharmacol 613:155–162 237. Salminen-Mankonen HJ, Morko J, Vuorio E 2007 Role of cathepsin K in normal joints and in the development of arthritis. Curr Drug Targets 8:315–323 238. Hakala M, Risteli J, Aman S, Kautiainen H, Korpela M, Hannonen P, Leirisalo-Repo M, Laasonen L, Paimela L, Möttönen T 2008 Combination drug strategy in recentonset rheumatoid arthritis suppresses collagen I degradation and is associated with retardation of radiological progression. Scand J Rheumatol 37:90 –93 239. Sassi ML, Aman S, Hakala M, Luukkainen R, Risteli J 2003 Assay for cross-linked carboxyterminal telopeptide of type I collagen (ICTP) unlike CrossLaps assay reflects increased pathological degradation of type I collagen in rheumatoid arthritis. Clin Chem Lab Med 41:1038 –1044 240. Chopin F, Garnero P, le Henanff A, Debiais F, Daragon A, Roux C, Sany J, Wendling D, Zarnitsky C, Ravaud P, Thomas T 2008 Long-term effects of infliximab on bone and cartilage turnover markers in patients with rheumatoid arthritis. Ann Rheum Dis 67:353–357 241. Kadono Y, Tanaka S, Nishino J, Nishimura K, Nakamura I, Miyazaki T, Takayanagi H, Nakamura K 2009 Rheumatoid arthritis associated with osteopetrosis. Mod Rheumatol 19:687– 690 242. Russell RG, Watts NB, Ebetino FH, Rogers MJ 2008 Mechanisms of action of bisphosphonates: similarities and differences and their potential influence on clinical efficacy. Osteoporos Int 19:733–759 243. Chavassieux PM, Arlot ME, Reda C, Wei L, Yates AJ, Meunier PJ 1997 Histomorphometric assessment of the long-term effects of alendronate on bone quality and re- Endocrine Reviews, February 2011, 32(1):31– 63 244. 245. 246. 247. 248. 249. 250. 251. 252. 253. 254. 255. 256. modeling in patients with osteoporosis. J Clin Invest 100: 1475–1480 Recker RR, Weinstein RS, Chesnut 3rd CH, Schimmer RC, Mahoney P, Hughes C, Bonvoisin B, Meunier PJ 2004 Histomorphometric evaluation of daily and intermittent oral ibandronate in women with postmenopausal osteoporosis: results from the BONE study. Osteoporos Int 15: 231–237 Recker RR, Delmas PD, Halse J, Reid IR, Boonen S, García-Hernandez PA, Supronik J, Lewiecki EM, Ochoa L, Miller P, Hu H, Mesenbrink P, Hartl F, Gasser J, Eriksen EF 2008 Effects of intravenous zoledronic acid once yearly on bone remodeling and bone structure. J Bone Miner Res 23:6 –16 Nenonen A, Cheng S, Ivaska KK, Alatalo SL, Lehtimaki T, Schmidt-Gayk H, Uusi-Rasi K, Heinonen A, Kannus P, Sievanen H, Vuori I, Vaananen HK, Halleen JM 2005 Serum TRACP 5b is a useful marker for monitoring alendronate treatment: comparison with other markers of bone turnover. J Bone Miner Res 20:1804 –1812 Hannon RA, Clowes JA, Eagleton AC, Al Hadari A, Eastell R, Blumsohn A 2004 Clinical performance of immunoreactive tartrate-resistant acid phosphatase isoform 5b as a marker of bone resorption. Bone 34:187–194 Muñoz-Torres M, Reyes-García R, Mezquita-Raya P, Fernández-García D, Alonso G, Luna Jde D, Ruiz-Requena ME, Escobar-Jiménez F 2009 Serum cathepsin K as a marker of bone metabolism in postmenopausal women treated with alendronate. Maturitas 64:188 –192 D’Amelio P, Grimaldi A, Di Bella S, Tamone C, Brianza SZ, Ravazzoli MG, Bernabei P, Cristofaro MA, Pescarmona GP, Isaia G 2008 Risedronate reduces osteoclast precursors and cytokine production in postmenopausal osteoporotic women. J Bone Miner Res 23:373–379 D’Amelio P, Grimaldi A, Cristofaro MA, Ravazzoli M, Molinatti PA, Pescarmona GP, Isaia GC 1 December 2009 Alendronate reduces osteoclast precursors in osteoporosis. Osteoporos Int 10.1007/s00198-009-1129-1 Weinstein RS, Roberson PK, Manolagas SC 2009 Giant osteoclast formation and long-term oral bisphosphonate therapy. N Engl J Med 360:53– 62 Mori S, Harruff R, Ambrosius W, Burr DB 1997 Trabecular bone volume and microdamage accumulation in the femoral heads of women with and without femoral neck fractures. Bone 21:521–526 Chapurlat RD, Arlot M, Burt-Pichat B, Chavassieux P, Roux JP, Portero-Muzy N, Delmas PD 2007 Microcrack frequency and bone remodeling in postmenopausal osteoporotic women on long-term bisphosphonates: a bone biopsy study. J Bone Miner Res 22:1502–1509 Burr DB, Allen MR 2008 Low bone turnover and microdamage? How and where to assess it? J Bone Miner Res 23:1150 –1151; author reply 1152–1153 Ravn P, Hosking D, Thompson D, Cizza G, Wasnich RD, McClung M, Yates AJ, Bjarnason NH, Christiansen C 1999 Monitoring of alendronate treatment and prediction of effect on bone mass by biochemical markers in the early postmenopausal intervention cohort study. J Clin Endocrinol Metab 84:2363–2368 Ravn P, Clemmesen B, Christiansen C 1999 Biochemical markers can predict the response in bone mass during alendronate treatment in early postmenopausal women. Alen- Endocrine Reviews, February 2011, 32(1):31– 63 257. 258. 259. 260. 261. 262. 263. 264. 265. 266. 267. 268. 269. 270. dronate Osteoporosis Prevention Study Group. Bone 24: 237–244 Ravn P, Thompson DE, Ross PD, Christiansen C 2003 Biochemical markers for prediction of 4-year response in bone mass during bisphosphonate treatment for prevention of postmenopausal osteoporosis. Bone 33:150 –158 Eriksen EF, Melsen F, Sod E, Barton I, Chines A 2002 Effects of long-term risedronate on bone quality and bone turnover in women with postmenopausal osteoporosis. Bone 31:620 – 625 Black DM, Schwartz AV, Ensrud KE, Cauley JA, Levis S, Quandt SA, Satterfield S, Wallace RB, Bauer DC, Palermo L, Wehren LE, Lombardi A, Santora AC, Cummings SR 2006 Effects of continuing or stopping alendronate after 5 years of treatment: the Fracture Intervention Trial Longterm Extension (FLEX): a randomized trial. JAMA 296: 2927–2938 Durie BG, Katz M, Crowley J 2005 Osteonecrosis of the jaw and bisphosphonates. N Engl J Med 353:99 –102 Watts NB, Diab DL 2010 Long-term use of bisphosphonates in osteoporosis. J Clin Endocrinol Metab 95:1555– 1565 Khosla S, Burr D, Cauley J, Dempster DW, Ebeling PR, Felsenberg D, Gagel RF, Gilsanz V, Guise T, Koka S, McCauley LK, McGowan J, McKee MD, Mohla S, Pendrys DG, Raisz LG, Ruggiero SL, Shafer DM, Shum L, Silverman SL, Van Poznak CH, Watts N, Woo SB, Shane E 2007 Bisphosphonate-associated osteonecrosis of the jaw: report of a task force of the American Society for Bone and Mineral Research. J Bone Miner Res 22:1479 –1491 Cheng A, Daly CG, Logan RM, Stein B, Goss AN 2009 Alveolar bone and the bisphosphonates. Aust Dent J 54(Suppl 1):S51–S61 Tran Van PT, Vignery A, Baron R 1982 Cellular kinetics of the bone remodeling sequence in the rat. Anat Rec 202: 445– 451 Tran Van P, Vignery A, Baron R 1982 An electron-microscopic study of the bone-remodeling sequence in the rat. Cell Tissue Res 225:283–292 Baron R, Neff L, Tran Van P, Nefussi JR, Vignery A 1986 Kinetic and cytochemical identification of osteoclast precursors and their differentiation into multinucleated osteoclasts. Am J Pathol 122:363–378 Favia G, Pilolli GP, Maiorano E 2009 Histologic and histomorphometric features of bisphosphonate-related osteonecrosis of the jaws: an analysis of 31 cases with confocal laser scanning microscopy. Bone 45:406 – 413 Bedogni A, Blandamura S, Lokmic Z, Palumbo C, Ragazzo M, Ferrari F, Tregnaghi A, Pietrogrande F, Procopio O, Saia G, Ferretti M, Bedogni G, Chiarini L, Ferronato G, Ninfo V, Lo Russo L, Lo Muzio L, Nocini PF 2008 Bisphosphonate-associated jawbone osteonecrosis: a correlation between imaging techniques and histopathology. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 105: 358 –364 Hansen T, Kunkel M, Weber A, James Kirkpatrick C 2006 Osteonecrosis of the jaws in patients treated with bisphosphonates— histomorphologic analysis in comparison with infected osteoradionecrosis. J Oral Pathol Med 35:155–160 Ramalho AC, Couttet P, Baudoin C, Morieux C, Graulet AM, de Vernejoul MC, Cohen-Solal ME 2002 Estradiol edrv.endojournals.org 271. 272. 273. 274. 275. 276. 277. 278. 279. 280. 281. 282. 283. 284. 285. 59 and raloxifene decrease the formation of multinucleate cells in human bone marrow cultures. Eur Cytokine Netw 13:39 – 45 Saintier D, Burde MA, Rey JM, Maudelonde T, de Vernejoul MC, Cohen-Solal ME 2004 17␤-Estradiol downregulates ␤3-integrin expression in differentiating and mature human osteoclasts. J Cell Physiol 198:269 –276 Michael H, Härkönen PL, Väänänen HK, Hentunen TA 2005 Estrogen and testosterone use different cellular pathways to inhibit osteoclastogenesis and bone resorption. J Bone Miner Res 20:2224 –2232 Michael H, Härkönen PL, Kangas L, Väänänen HK, Hentunen TA 2007 Differential effects of selective oestrogen receptor modulators (SERMs) tamoxifen, ospemifene and raloxifene on human osteoclasts in vitro. Br J Pharmacol 151:384 –395 Taranta A, Brama M, Teti A, De luca V, Scandurra R, Spera G, Agnusdei D, Termine JD, Migliaccio S 2002 The selective estrogen receptor modulator raloxifene regulates osteoclast and osteoblast activity in vitro. Bone 30:368 – 376 Oursler MJ, Osdoby P, Pyfferoen J, Riggs BL, Spelsberg TC 1991 Avian osteoclasts as estrogen target cells. Proc Natl Acad Sci USA 88:6613– 6617 Oursler MJ, Pederson L, Fitzpatrick L, Riggs BL, Spelsberg T 1994 Human giant cell tumors of the bone (osteoclastomas) are estrogen target cells. Proc Natl Acad Sci USA 91:5227–5231 Oursler MJ, Pederson L, Pyfferoen J, Osdoby P, Fitzpatrick L, Spelsberg TC 1993 Estrogen modulation of avian osteoclast lysosomal gene expression. Endocrinology 132: 1373–1380 Kremer M, Judd J, Rifkin B, Auszmann J, Oursler MJ 1995 Estrogen modulation of osteoclast lysosomal enzyme secretion. J Cell Biochem 57:271–279 Parikka V, Lehenkari P, Sassi ML, Halleen J, Risteli J, Härkönen P, Väänänen HK 2001 Estrogen reduces the depth of resorption pits by disturbing the organic bone matrix degradation activity of mature osteoclasts. Endocrinology 142:5371–5378 Ott SM, Oleksik A, Lu Y, Harper K, Lips P 2002 Bone histomorphometric and biochemical marker results of a 2-year placebo-controlled trial of raloxifene in postmenopausal women. J Bone Miner Res 17:341–348 Steiniche T, Hasling C, Charles P, Eriksen EF, Mosekilde L, Melsen F 1989 A randomized study on the effects of estrogen/gestagen or high dose oral calcium on trabecular bone remodeling in postmenopausal osteoporosis. Bone 10:313–320 Patel S, Pazianas M, Tobias J, Chambers TJ, Fox S, Chow J 1999 Early effects of hormone replacement therapy on bone. Bone 24:245–248 Eriksen EF, Langdahl B, Vesterby A, Rungby J, Kassem M 1999 Hormone replacement therapy prevents osteoclastic hyperactivity: a histomorphometric study in early postmenopausal women. J Bone Miner Res 14:1217–1221 Vedi S, Purdie DW, Ballard P, Bord S, Cooper AC, Compston JE 1999 Bone remodeling and structure in postmenopausal women treated with long-term, high-dose estrogen therapy. Osteoporos Int 10:52–58 Ettinger B, Black DM, Mitlak BH, Knickerbocker RK, Nickelsen T, Genant HK, Christiansen C, Delmas PD, 60 286. 287. 288. 289. 290. 291. 292. 293. 294. 295. 296. 297. Henriksen et al. Osteoclast Subtypes Zanchetta JR, Stakkestad J, Glüer CC, Krueger K, Cohen FJ, Eckert S, Ensrud KE, Avioli LV, Lips P, Cummings SR 1999 Reduction of vertebral fracture risk in postmenopausal women with osteoporosis treated with raloxifene: results from a 3-year randomized clinical trial. Multiple Outcomes of Raloxifene Evaluation (MORE) Investigators. JAMA 282:637– 645 Meunier PJ, Vignot E, Garnero P, Confavreux E, Paris E, Liu-Leage S, Sarkar S, Liu T, Wong M, Draper MW 1999 Treatment of postmenopausal women with osteoporosis or low bone density with raloxifene. Raloxifene Study Group. Osteoporos Int 10:330 –336 Lufkin EG, Whitaker MD, Nickelsen T, Argueta R, Caplan RH, Knickerbocker RK, Riggs BL 1998 Treatment of established postmenopausal osteoporosis with raloxifene: a randomized trial. J Bone Miner Res 13:1747–1754 Chambers TJ, Moore A 1983 The sensitivity of isolated osteoclasts to morphological transformation by calcitonin. J Clin Endocrinol Metab 57:819 – 824 Suzuki H, Nakamura I, Takahashi N, Ikuhara T, Matsuzaki K, Isogai Y, Hori M, Suda T 1996 Calcitonin-induced changes in the cytoskeleton are mediated by a signal pathway associated with protein kinase A in osteoclasts. Endocrinology 137:4685– 4690 Shyu JF, Shih C, Tseng CY, Lin CH, Sun DT, Liu HT, Tsung HC, Chen TH, Lu RB 2007 Calcitonin induces podosome disassembly and detachment of osteoclasts by modulating Pyk2 and Src activities. Bone 40:1329 –1342 Karsdal MA, Henriksen K, Bay-Jensen AC, Molloy B, Arnold M, John MR, Byrjalsen I, Azria M, Riis BJ, Qvist P, Christiansen C 2010 Lessons learned from the development of oral calcitonin: the first tablet formulation of a peptide in phase III clinical trials. J Clin Pharmacol 10.1177/0091270010372625 Karsdal MA, Byrjalsen I, Riis BJ, Christiansen C 2008 Investigation of the diurnal variation in bone resorption for optimal drug delivery and efficacy in osteoporosis with oral calcitonin. BMC Clin Pharmacol 8:12 Kung AW, Pasion EG, Sofiyan M, Lau EM, Tay BK, Lam KS, Wilawan K, Ongphiphadhanakul B, Thiebaud D 2006 A comparison of teriparatide and calcitonin therapy in postmenopausal Asian women with osteoporosis: a 6-month study. Curr Med Res Opin 22:929 –937 Hwang JS, Tu ST, Yang TS, Chen JF, Wang CJ, Tsai KS 2006 Teriparatide vs. calcitonin in the treatment of Asian postmenopausal women with established osteoporosis. Osteoporos Int 17:373–378 Trovas GP, Lyritis GP, Galanos A, Raptou P, Constantelou E 2002 A randomized trial of nasal spray salmon calcitonin in men with idiopathic osteoporosis: effects on bone mineral density and bone markers. J Bone Miner Res 17:521–527 Chesnut 3rd CH, Majumdar S, Newitt DC, Shields A, Van Pelt J, Laschansky E, Azria M, Kriegman A, Olson M, Eriksen EF, Mindeholm L 2005 Effects of salmon calcitonin on trabecular microarchitecture as determined by magnetic resonance imaging: results from the QUEST study. J Bone Miner Res 20:1548 –1561 Ikegame M, Ejiri S, Ozawa H 2004 Calcitonin-induced change in serum calcium levels and its relationship to osteoclast morphology and number of calcitonin receptors. Bone 35:27–33 Endocrine Reviews, February 2011, 32(1):31– 63 298. Jiang Y, Zhao J, Geusens P, Liao EY, Adriaensens P, Gelan J, Azria M, Boonen S, Caulin F, Lynch JA, Ouyang X, Genant HK 2005 Femoral neck trabecular microstructure in ovariectomized ewes treated with calcitonin: MRI microscopic evaluation. J Bone Miner Res 20:125–130 299. Hoff AO, Catala-Lehnen P, Thomas PM, Priemel M, Rueger JM, Nasonkin I, Bradley A, Hughes MR, Ordonez N, Cote GJ, Amling M, Gagel RF 2002 Increased bone mass is an unexpected phenotype associated with deletion of the calcitonin gene. J Clin Invest 110:1849 –1857 300. Dacquin R, Davey RA, Laplace C, Levasseur R, Morris HA, Goldring SR, Gebre-Medhin S, Galson DL, Zajac JD, Karsenty G 2004 Amylin inhibits bone resorption while the calcitonin receptor controls bone formation in vivo. J Cell Biol 164:509 –514 301. Davey RA, Turner AG, McManus JF, Chiu WS, Tjahyono F, Moore AJ, Atkins GJ, Anderson PH, Ma C, Glatt V, MacLean HE, Vincent C, Bouxsein M, Morris HA, Findlay DM, Zajac JD 2008 Calcitonin receptor plays a physiological role to protect against hypercalcemia in mice. J Bone Miner Res 23:1182–1193 302. Turner C, Tjahyono A, Moore A, Findlay D, Morris H, Zajac J, Davey R 2009 The calcitonin receptor expressed by osteoclasts plays a biological role to protect against induced hypercalcemia in mice. J Bone Miner Res 24(Suppl 1):1049 (Abstract) 303. Karsdal MA, Byrjalsen I, Leeming DJ, Delmas PD, Christiansen C 2008 The effects of oral calcitonin on bone collagen maturation: implications for bone turnover and quality. Osteoporos Int 19:1355–1361 304. Holtrop ME, King GJ, Cox KA, Reit B 1979 Time-related changes in the ultrastructure of osteoclasts after injection of parathyroid hormone in young rats. Calcif Tissue Int 27:129 –135 305. Ma YL, Cain RL, Halladay DL, Yang X, Zeng Q, Miles RR, Chandrasekhar S, Martin TJ, Onyia JE 2001 Catabolic effects of continuous human PTH (1–38) in vivo is associated with sustained stimulation of RANKL and inhibition of osteoprotegerin and gene-associated bone formation. Endocrinology 142:4047– 4054 306. Hodsman AB, Kisiel M, Adachi JD, Fraher LJ, Watson PH 2000 Histomorphometric evidence for increased bone turnover without change in cortical thickness or porosity after 2 years of cyclical hPTH(1–34) therapy in women with severe osteoporosis. Bone 27:311–318 307. Arlot M, Meunier PJ, Boivin G, Haddock L, Tamayo J, Correa-Rotter R, Jasqui S, Donley DW, Dalsky GP, Martin JS, Eriksen EF 2005 Differential effects of teriparatide and alendronate on bone remodeling in postmenopausal women assessed by histomorphometric parameters. J Bone Miner Res 20:1244 –1253 308. Recker RR, Marin F, Ish-Shalom S, Möricke R, Hawkins F, Kapetanos G, de la Peña MP, Kekow J, Farrerons J, Sanz B, Oertel H, Stepan J 2009 Comparative effects of teriparatide and strontium ranelate on bone biopsies and biochemical markers of bone turnover in postmenopausal women with osteoporosis. J Bone Miner Res 24:1358 – 1368 309. Dobnig H, Turner RT 1995 Evidence that intermittent treatment with parathyroid hormone increases bone formation in adult rats by activation of bone lining cells. Endocrinology 136:3632–3638 Endocrine Reviews, February 2011, 32(1):31– 63 310. Jilka RL, Weinstein RS, Bellido T, Roberson P, Parfitt AM, Manolagas SC 1999 Increased bone formation by prevention of osteoblast apoptosis with parathyroid hormone. J Clin Invest 104:439 – 446 311. Luiz de Freitas PH, Li M, Ninomiya T, Nakamura M, Ubaidus S, Oda K, Udagawa N, Maeda T, Takagi R, Amizuka N 2009 Intermittent PTH administration stimulates pre-osteoblastic proliferation without leading to enhanced bone formation in osteoclast-less c-fos(⫺/⫺) mice. J Bone Miner Res 24:1586 –1597 312. Black DM, Greenspan SL, Ensrud KE, Palermo L, McGowan JA, Lang TF, Garnero P, Bouxsein ML, Bilezikian JP, Rosen CJ 2003 The effects of parathyroid hormone and alendronate alone or in combination in postmenopausal osteoporosis. N Engl J Med 349:1207–1215 313. Finkelstein JS, Hayes A, Hunzelman JL, Wyland JJ, Lee H, Neer RM 2003 The effects of parathyroid hormone, alendronate, or both in men with osteoporosis. N Engl J Med 349:1216 –1226 314. Ettinger B, San Martin J, Crans G, Pavo I 2004 Differential effects of teriparatide on BMD after treatment with raloxifene or alendronate. J Bone Miner Res 19:745–751 315. Johnston S, Andrews S, Shen V, Cosman F, Lindsay R, Dempster DW, Iida-Klein A 2007 The effects of combination of alendronate and human parathyroid hormone(134) on bone strength are synergistic in the lumbar vertebra and additive in the femur of C57BL/6J mice. Endocrinology 148:4466 – 4474 316. Samadfam R, Xia Q, Goltzman D 2007 Co-treatment of PTH with osteoprotegerin or alendronate increases its anabolic effect on the skeleton of oophorectomized mice. J Bone Miner Res 22:55– 63 317. Meunier PJ, Roux C, Seeman E, Ortolani S, Badurski JE, Spector TD, Cannata J, Balogh A, Lemmel EM, PorsNielsen S, Rizzoli R, Genant HK, Reginster JY 2004 The effects of strontium ranelate on the risk of vertebral fracture in women with postmenopausal osteoporosis. N Engl J Med 350:459 – 468 318. Seeman E, Vellas B, Benhamou C, Aquino JP, Semler J, Kaufman JM, Hoszowski K, Varela AR, Fiore C, Brixen K, Reginster JY, Boonen S 2006 Strontium ranelate reduces the risk of vertebral and nonvertebral fractures in women eighty years of age and older. J Bone Miner Res 21:1113– 1120 319. Reginster JY, Seeman E, De Vernejoul MC, Adami S, Compston J, Phenekos C, Devogelaer JP, Curiel MD, Sawicki A, Goemaere S, Sorensen OH, Felsenberg D, Meunier PJ 2005 Strontium ranelate reduces the risk of nonvertebral fractures in postmenopausal women with osteoporosis: Treatment of Peripheral Osteoporosis (TROPOS) study. J Clin Endocrinol Metab 90:2816 –2822 320. Seeman E, Boonen S, Borgström F, Vellas B, Aquino JP, Semler J, Benhamou CL, Kaufman JM, Reginster JY 2010 Five years treatment with strontium ranelate reduces vertebral and nonvertebral fractures and increases the number and quality of remaining life-years in women over 80 years of age. Bone 46:1038 –1042 321. Ferrari S 2009 Continuous broad protection against osteoporotic fractures with strontium ranelate. Rheumatology (Oxford) 48(Suppl 4):iv20 –iv24 322. Arlot ME, Jiang Y, Genant HK, Zhao J, Burt-Pichat B, Roux JP, Delmas PD, Meunier PJ 2008 Histomorphomet- edrv.endojournals.org 323. 324. 325. 326. 327. 328. 329. 330. 331. 332. 333. 334. 335. 336. 337. 61 ric and microCT analysis of bone biopsies from postmenopausal osteoporotic women treated with strontium ranelate. J Bone Miner Res 23:215–222 Meunier PJ, Slosman DO, Delmas PD, Sebert JL, Brandi ML, Albanese C, Lorenc R, Pors-Nielsen S, De Vernejoul MC, Roces A, Reginster JY 2002 Strontium ranelate: dosedependent effects in established postmenopausal vertebral osteoporosis—a 2-year randomized placebo controlled trial. J Clin Endocrinol Metab 87:2060 –2066 Axmann R, Böhm C, Krönke G, Zwerina J, Smolen J, Schett G 2009 Inhibition of interleukin-6 receptor directly blocks osteoclast formation in vitro and in vivo. Arthritis Rheum 60:2747–2756 Bruyère O, Collette J, Rizzoli R, Decock C, Ortolani S, Cormier C, Detilleux J, Reginster JY 2010 Relationship between 3-month changes in biochemical markers of bone remodelling and changes in bone mineral density and fracture incidence in patients treated with strontium ranelate for 3 years. Osteoporos Int 21:1031–1036 Bonnelye E, Chabadel A, Saltel F, Jurdic P 2008 Dual effect of strontium ranelate: stimulation of osteoblast differentiation and inhibition of osteoclast formation and resorption in vitro. Bone 42:129 –138 Canalis E, Hott M, Deloffre P, Tsouderos Y, Marie PJ 1996 The divalent strontium salt S12911 enhances bone cell replication and bone formation in vitro. Bone 18:517–523 Takahashi N, Sasaki T, Tsouderos Y, Suda T 2003 S 12911–2 inhibits osteoclastic bone resorption in vitro. J Bone Miner Res 18:1082–1087 Hurtel-Lemaire AS, Mentaverri R, Caudrillier A, Cournarie F, Wattel A, Kamel S, Terwilliger EF, Brown EM, Brazier M 2009 The calcium-sensing receptor is involved in strontium ranelate-induced osteoclast apoptosis. New insights into the associated signaling pathways. J Biol Chem 284:575–584 Brennan TC, Rybchyn MS, Green W, Atwa S, Conigrave AD, Mason RS 2009 Osteoblasts play key roles in the mechanisms of action of strontium ranelate. Br J Pharmacol 157:1291–1300 Engvall IL, Svensson B, Tengstrand B, Brismar K, Hafström I 2008 Impact of low-dose prednisolone on bone synthesis and resorption in early rheumatoid arthritis: experiences from a two-year randomized study. Arthritis Res Ther 10:R128 Caplan L, Saag KG 2009 Glucocorticoids and the risk of osteoporosis. Expert Opin Drug Saf 8:33– 47 Canalis E, Mazziotti G, Giustina A, Bilezikian JP 2007 Glucocorticoid-induced osteoporosis: pathophysiology and therapy. Osteoporos Int 18:1319 –1328 van Brussel MS, Bultink IE, Lems WF 2009 Prevention of glucocorticoid-induced osteoporosis. Expert Opin Pharmacother 10:997–1005 Weinstein RS, Jilka RL, Parfitt AM, Manolagas SC 1998 Inhibition of osteoblastogenesis and promotion of apoptosis of osteoblasts and osteocytes by glucocorticoids. Potential mechanisms of their deleterious effects on bone. J Clin Invest 102:274 –282 Hofbauer LC, Zeitz U, Schoppet M, Skalicky M, Schüler C, Stolina M, Kostenuik PJ, Erben RG 2009 Prevention of glucocorticoid-induced bone loss in mice by inhibition of RANKL. Arthritis Rheum 60:1427–1437 Jia D, O’Brien CA, Stewart SA, Manolagas SC, Weinstein 62 338. 339. 340. 341. 342. 343. 344. 345. 346. 347. 348. 349. 350. Henriksen et al. Osteoclast Subtypes RS 2006 Glucocorticoids act directly on osteoclasts to increase their life span and reduce bone density. Endocrinology 147:5592–5599 Yao W, Cheng Z, Busse C, Pham A, Nakamura MC, Lane NE 2008 Glucocorticoid excess in mice results in early activation of osteoclastogenesis and adipogenesis and prolonged suppression of osteogenesis: a longitudinal study of gene expression in bone tissue from glucocorticoid-treated mice. Arthritis Rheum 58:1674 –1686 Weinstein RS, Chen JR, Powers CC, Stewart SA, Landes RD, Bellido T, Jilka RL, Parfitt AM, Manolagas SC 2002 Promotion of osteoclast survival and antagonism of bisphosphonate-induced osteoclast apoptosis by glucocorticoids. J Clin Invest 109:1041–1048 Kim HJ, Zhao H, Kitaura H, Bhattacharyya S, Brewer JA, Muglia LJ, Ross FP, Teitelbaum SL 2006 Glucocorticoids suppress bone formation via the osteoclast. J Clin Invest 116:2152–2160 Sivagurunathan S, Muir MM, Brennan TC, Seale JP, Mason RS 2005 Influence of glucocorticoids on human osteoclast generation and activity. J Bone Miner Res 20:390 –398 Hofbauer LC, Gori F, Riggs BL, Lacey DL, Dunstan CR, Spelsberg TC, Khosla S 1999 Stimulation of osteoprotegerin ligand and inhibition of osteoprotegerin production by glucocorticoids in human osteoblastic lineage cells: potential paracrine mechanisms of glucocorticoid-induced osteoporosis. Endocrinology 140:4382– 4389 Soares-Schanoski A, Gómez-Piña V, del Fresno C, Rodríguez-Rojas A, García F, Glaría A, Sánchez M, VallejoCremades MT, Baos R, Fuentes-Prior P, Arnalich F, López-Collazo E 2007 6-Methylprednisolone down-regulates IRAK-M in human and murine osteoclasts and boosts bone-resorbing activity: a putative mechanism for corticoid-induced osteoporosis. J Leukoc Biol 82:700 –709 Søe K, Delaissé JM 30 April 2010 Glucocorticoids maintain human osteoclasts in the active mode of their resorption cycle. J Bone Miner Res 10.1002/jbmr.113 Dovio A, Perazzolo L, Osella G, Ventura M, Termine A, Milano E, Bertolotto A, Angeli A 2004 Immediate fall of bone formation and transient increase of bone resorption in the course of high-dose, short-term glucocorticoid therapy in young patients with multiple sclerosis. J Clin Endocrinol Metab 89:4923– 4928 Minisola S, Del Fiacco R, Piemonte S, Iorio M, Mascia ML, Fidanza F, Cipriani C, Raso I, Porfiri ML, Francucci CM, D’Erasmo E, Romagnoli E 2008 Biochemical markers in glucocorticoid-induced osteoporosis. J Endocrinol Invest 31:28 –32 Dalle Carbonare L, Bertoldo F, Valenti MT, Zenari S, Zanatta M, Sella S, Giannini S, Cascio VL 2005 Histomorphometric analysis of glucocorticoid-induced osteoporosis. Micron 36:645– 652 Stellon AJ, Webb A, Compston JE 1988 Bone histomorphometry and structure in corticosteroid treated chronic active hepatitis. Gut 29:378 –384 Stoch SA, Saag KG, Greenwald M, Sebba AI, Cohen S, Verbruggen N, Giezek H, West J, Schnitzer TJ 2009 Onceweekly oral alendronate 70 mg in patients with glucocorticoid-induced bone loss: a 12-month randomized, placebo-controlled clinical trial. J Rheumatol 36:1705–1714 Bikle DD, Sakata T, Leary C, Elalieh H, Ginzinger D, Endocrine Reviews, February 2011, 32(1):31– 63 351. 352. 353. 354. 355. 356. 357. 358. 359. 360. 361. 362. Rosen CJ, Beamer W, Majumdar S, Halloran BP 2002 Insulin-like growth factor I is required for the anabolic actions of parathyroid hormone on mouse bone. J Bone Miner Res 17:1570 –1578 Cummings SR, San Martin J, McClung MR, Siris ES, Eastell R, Reid IR, Delmas P, Zoog HB, Austin M, Wang A, Kutilek S, Adami S, Zanchetta J, Libanati C, Siddhanti S, Christiansen C 2009 Denosumab for prevention of fractures in postmenopausal women with osteoporosis. N Engl J Med 361:756 –765 Kostenuik PJ, Nguyen HQ, McCabe J, Warmington KS, Kurahara C, Sun N, Chen C, Li L, Cattley RC, Van G, Scully S, Elliott R, Grisanti M, Morony S, Tan HL, Asuncion F, Li X, Ominsky MS, Stolina M, Dwyer D, Dougall WC, Hawkins N, Boyle WJ, Simonet WS, Sullivan JK 2009 Denosumab, a fully human monoclonal antibody to RANKL, inhibits bone resorption and increases BMD in knock-in mice that express chimeric (murine/human) RANKL. J Bone Miner Res 24:182–195 Bone HG, Bolognese MA, Yuen CK, Kendler DL, Wang H, Liu Y, San Martin J 2008 Effects of denosumab on bone mineral density and bone turnover in postmenopausal women. J Clin Endocrinol Metab 93:2149 –2157 McClung MR, Lewiecki EM, Cohen SB, Bolognese MA, Woodson GC, Moffett AH, Peacock M, Miller PD, Lederman SN, Chesnut CH, Lain D, Kivitz AJ, Holloway DL, Zhang C, Peterson MC, Bekker PJ 2006 Denosumab in postmenopausal women with low bone mineral density. N Engl J Med 354:821– 831 Ominsky MS, Schroeder J, Jolette J, Smith SY, Farrell DJ, Atkinson JE, Kostenuik PJ 2007 Decreased bone turnover and porosity are associated with improved bone strength in ovariectomized (OVX) cynomolgus monkeys treated with denosumab, a fully human RANKL antibody. J Bone Miner Res 22:S126 (Abstract) Sassi ML, Eriksen H, Risteli L, Niemi S, Mansell J, Gowen M, Risteli J 2000 Immunochemical characterization of assay for carboxyterminal telopeptide of human type I collagen: loss of antigenicity by treatment with cathepsin K. Bone 26:367–373 Fuller K, Lawrence KM, Ross JL, Grabowska UB, Shiroo M, Samuelsson B, Chambers TJ 2008 Cathepsin K inhibitors prevent matrix-derived growth factor degradation by human osteoclasts. Bone 42:200 –211 Chappard D, Libouban H, Mindeholm L, Baslé MF, Legrand E, Audran M 2010 The cathepsin K inhibitor AAE581 induces morphological changes in osteoclasts of treated patients. Microsc Res Tech 73:726 –732 Adams CS, Shapiro IM 2003 Mechanisms by which extracellular matrix components induce osteoblast apoptosis. Connect Tissue Res 44(Suppl 1):230 –239 Walker EC, McGregor NE, Poulton IJ, Pompolo S, Allan EH, Quinn JM, Gillespie MT, Martin TJ, Sims NA 2008 Cardiotrophin-1 is an osteoclast-derived stimulus of bone formation required for normal bone remodeling. J Bone Miner Res 23:2025–2032 Gottschalck IB, Jeppesen PB, Hartmann B, Holst JJ, Henriksen DB 2008 Effects of treatment with glucagon-like peptide-2 on bone resorption in colectomized patients with distal ileostomy or jejunostomy and short-bowel syndrome. Scand J Gastroenterol 43:1304 –1310 Gottschalck IB, Jeppesen PB, Holst JJ, Henriksen DB 2008 Endocrine Reviews, February 2011, 32(1):31– 63 363. 364. 365. 366. 367. 368. 369. 370. 371. Reduction in bone resorption by exogenous glucagon-like peptide-2 administration requires an intact gastrointestinal tract. Scand J Gastroenterol 43:929 –937 Sørensen MG, Henriksen K, Neutzsky-Wulff AV, Dziegiel MH, Karsdal MA 2007 Diphyllin, a novel and naturally potent V-ATPase inhibitor, abrogates acidification of the osteoclastic resorption lacunae and bone resorption. J Bone Miner Res 22:1640 –1648 Schaller S, Henriksen K, Sveigaard C, Heegaard AM, Hélix N, Stahlhut M, Ovejero MC, Johansen JV, Solberg H, Andersen TL, Hougaard D, Berryman M, Shiødt CB, Sørensen BH, Lichtenberg J, Christophersen P, Foged NT, Delaissé JM, Engsig MT, Karsdal MA 2004 The chloride channel inhibitor n53736 prevents bone resorption in ovariectomized rats without changing bone formation. J Bone Miner Res 19:1144 –1153 Visentin L, Dodds RA, Valente M, Misiano P, Bradbeer JN, Oneta S, Liang X, Gowen M, Farina C 2000 A selective inhibitor of the osteoclastic V-H(⫹)-ATPase prevents bone loss in both thyroparathyroidectomized and ovariectomized rats. J Clin Invest 106:309 –318 Rzeszutek K, Sarraf F, Davies JE 2003 Proton pump inhibitors control osteoclastic resorption of calcium phosphate implants and stimulate increased local reparative bone growth. J Craniofac Surg 14:301–307 Schaller S, Henriksen K, Sørensen MG, Karsdal MA 2005 The role of chloride channels in osteoclasts: ClC-7 as a target for osteoporosis treatment. Drug News Perspect 18: 489 – 495 Brown EM 2007 The calcium-sensing receptor: physiology, pathophysiology and CaR-based therapeutics. Subcell Biochem 45:139 –167 Martin TJ 2005 Osteoblast-derived PTHrP is a physiological regulator of bone formation. J Clin Invest 115:2322– 2324 Kramer I, Keller H, Leupin O, Kneissel M 2010 Does osteocytic SOST suppression mediate PTH bone anabolism? Trends Endocrinol Metab 21:237–244 Thompson ER, Baylink DJ, Wergedal JE 1975 Increases edrv.endojournals.org 372. 373. 374. 375. 376. 377. 378. 379. 380. 63 in number and size of osteoclasts in response to calcium or phosphorus deficiency in the rat. Endocrinology 97: 283–289 Howard GA, Bottemiller BL, Turner RT, Rader JI, Baylink DJ 1981 Parathyroid hormone stimulates bone formation and resorption in organ culture: evidence for a coupling mechanism. Proc Natl Acad Sci USA 78:3204 –3208 Lazowski DA, Fraher LJ, Hodsman A, Steer B, Modrowski D, Han VK 1994 Regional variation of insulin-like growth factor-I gene expression in mature rat bone and cartilage. Bone 15:563–576 Robinson JA, Riggs BL, Spelsberg TC, Oursler MJ 1996 Osteoclasts and transforming growth factor-␤: estrogenmediated isoform-specific regulation of production. Endocrinology 137:615– 621 Karsdal MA, Fjording MS, Foged NT, Delaissé JM, Lochter A 2001 Transforming growth factor-␤-induced osteoblast elongation regulates osteoclastic bone resorption through a p38 mitogen-activated protein kinase- and matrix metalloproteinase-dependent pathway. J Biol Chem 276:39350 –39358 Mundy GR, Bonewald LF 1990 Role of TGF ␤ in bone remodeling. Ann NY Acad Sci 593:91–97 Baylink DJ, Finkelman RD, Mohan S 1993 Growth factors to stimulate bone formation. J Bone Miner Res 8(Suppl 2):S565–S572 Hayden JM, Mohan S, Baylink DJ 1995 The insulin-like growth factor system and the coupling of formation to resorption. Bone 17:93S–98S Janssens K, ten Dijke P, Janssens S, Van Hul W 2005 Transforming growth factor-␤1 to the bone. Endocr Rev 26: 743–774 Zhang M, Xuan S, Bouxsein ML, von Stechow D, Akeno N, Faugere MC, Malluche H, Zhao G, Rosen CJ, Efstratiadis A, Clemens TL 2002 Osteoblast-specific knockout of the insulin-like growth factor (IGF) receptor gene reveals an essential role of IGF signaling in bone matrix mineralization. J Biol Chem 277:44005– 44012 Rev Endocr Metab Disord (2010) 11:219–227 DOI 10.1007/s11154-010-9153-1 Cellular mechanisms of bone remodeling Erik Fink Eriksen Published online: 29 December 2010 # The Author(s) 2010. This article is published with open access at Springerlink.com Abstract Bone remodeling is a tightly regulated process securing repair of microdamage (targeted remodeling) and replacement of old bone with new bone through sequential osteoclastic resorption and osteoblastic bone formation. The rate of remodeling is regulated by a wide variety of calcitropic hormones (PTH, thyroid hormone, sex steroids etc.). In recent years we have come to appreciate that bone remodeling proceeds in a specialized vascular structure,— the Bone Remodeling Compartment (BRC). The outer lining of this compartment is made up of flattened cells, displaying all the characteristics of lining cells in bone including expression of OPG and RANKL. Reduced bone turnover leads to a decrease in the number of BRCs, while increased turnover causes an increase in the number of BRCs. The secretion of regulatory factors inside a confined space separated from the bone marrow would facilitate local regulation of the remodeling process without interference from growth factors secreted by blood cells in the marrow space. The BRC also creates an environment where cells inside the structure are exposed to denuded bone, which may enable direct cellular interactions with integrins and other matrix factors known to regulate osteoclast/ osteoblast activity. However, the denuded bone surface inside the BRC also constitutes an ideal environment for the seeding of bone metastases, known to have high affinity for bone matrix. Circulating osteoclast- and osteoblast precursor cells have been demonstrated in peripheral blood. The dominant pathway regulating osteoclast recruitment is the RANKL/OPG system, while many different factors E. F. Eriksen (*) Department Of Clinical Endocrinology, Oslo University Hospital, Aker, Trondheimsveien 235, 0514 Oslo, Norway e-mail: [email protected] (RUNX, Osterix) are involved in osteoblast differentiation. Both pathways are modulated by calcitropic hormones. Keywords Osteoblasts . Osteoclasts . Lining cells . Growth factors . Cytokines . Bone remodeling . Osteoporosis . Bone remodeling compartment 1 Introduction Bone histomorphometry has given us great insights into bone physiology and bone remodeling in particular. Histomorphometric indices obtained using tetracycline double labeling techniques are unique, because they contrary to DXA and bone markers reflect cellular activity of osteoclasts and osteoblasts using the incorporation of a time marker, namely spaced administration of an agent (tetracycline) reflecting ongoing active bone formation. The study of bone remodeling originated with the classical works of Harold Frost 40 years ago  and our ever expanding understanding of this process is the basis for the development of highly effective treatments for osteoporosis, that we have seen over the last 20 years. 2 The bone remodeling cycle Although macroscopically the skeleton seems to be a static organ, it is an extremely dynamic tissue at the microscopic level. The ability of bone to sustain the tremendous loads placed on it in everyday life depends on, constant repair of mechanical microdamage that develops both in cancellous bone—the “spongy” bone present in the vertebrae, pelvis, and ends (metaphyses) of long bones—and in cortical bone—the compact bone present in the shafts (diaphyses) of the long bones and surrounding cancellous bone as a thin layer in the 220 Rev Endocr Metab Disord (2010) 11:219–227 vertebrae and pelvis. Bone remodeling is based on the concerted action of resorptive and formative cell populations in order to replace old bone with new bone and thus secure the integrity of the skeleton. This sequence has to be tightly regulated by both local and systemic factors, because significant deviations from a neutral balance between resorption and formation would mean severe accelerated bone loss or bone gains with possible disastrous consequences in terms of increased fracture risk or compression syndromes. Bone remodeling takes place in what Frost termed the Basic Multicellular Unit (BMU), which comprises the osteoclasts, osteoblasts, and osteocytes within the boneremodeling cavity (Fig. 1). In cancellous bone remodeling occurs on the surface of trabeculae and lasts about 200 days in normal bone. The remodeling cycle can be as short as 100 days in thyrotoxicosis and primary hyperparathyroidism and exceed 1,000 days in low turnover states like Myxedema and after bisphosphonate treatment . Remodeling is initiated by osteoclastic resorption, which erodes a resorption lacuna, the depth of which varies between 60 in young individuals and 40 μm in older individuals. The resorption period has a median duration of 30–40 days and is followed by bone formation over a period of 150 days (Fig. 1) [3, 4]. In normal bone the result of the remodeling cycle is complete refilling of the resorption lacuna with new bone. In disease states like osteoporosis, the main defect is that the osteoblast is unable to refill the resorption lacuna leading to a net loss of bone with each remodeling event Resorption Formation Marrow capillary Cancellous bone 50 µm 80 µm Cortical bone Haversian system Central vessel 0 100 200 Time -days Fig. 1 Schematic representation of Bone Multicellular Units (BMUs) in cancellous and cortical bone. Broken lines denote the outer limit of Bone Remodeling Compartment associated with the resorptive and formative sites of the BMU. The mean thickness of the structure in cancellous bone is 50 μm and 80 μm in cortical bone equivalent to a mean Haversian system diameter of 160 μm. The Blood supply for the BRCs is provided by capillaries either coming from the marrow space as is the case for cancellous BMUs or from the central vessel of Haversian systems in cortical bone. The duration of the remodeling sequence is somewhat longer in cancellous than in cortical bone. The position of marrow cappillaries is hypothetical, as the exact distribution is poorly elucidated . In cortical bone remodeling proceeds in tunnels with osteoclasts forming “cutting cones” removing damaged bone followed by refilling by osteoblasts in the “closing cone” occurring behind the osteoclasts . In normal bone the duration of the remodeling cycle in cortical is shorter than in cancellous bone with a median of 120 days . The total surface of cancellous bone is completely remodeled over a period of 2 years. Contrary to remodeling sites in cancellous bone, which are close to red marrow, known to contain osteoprogenitor cells , remodeling sites in cortical bone are distant from red marrow. Therefore, it was assumed that the mechanisms of bone remodeling were different in cancellous versus cortical bone, i.e. that the cells needed for bone remodeling in cancellous bone traveled directly from the red marrow to bone surfaces in cancellous bone, while cells reached cortical remodeling sites bone via the vasculature . 2.1 Osteoblast differentiation Osteoblasts are mesenchymal cells derived from mesodermal and neural crest progenitor cells and their formation entails differentiation from progenitors into proliferating preosteoblasts, bone matrix-producing osteoblasts, and eventually into osteocytes or a bone-lining cells. The earliest osteoblastic marker, Runt-related transcription factor 2 (Runx2) is necessary for progenitor cell differentiation along the osteoblast lineage . During this sequence of cellular proliferation Runx2 regulates expression of genes encoding osteocalcin, VEGF, RANKL, sclerostin, and dentin matrix protein 1 [DMP1] . Osterix is another transcription factor essential for osteoblast differentiation . A large number of paracrine, autocrine, and endocrine factors affect osteoblast development and maturation like: bone morphogenetic proteins (BMPs), growth factors like FGF and IGF, angiogenic factors like endothelin-1, hormones like PTH and prostaglandin agonists, all modulate osteoblast differentiation . The action of PTH and BMPs is closely associated with activation of Wnt signalling pathways . The fully differentiated osteoblast is characterized by coexpression of alkaline phosphatase and type I collagen, both important for synthesis of bone matrix and subsequent mineralization thereof . Mature osteoblasts also produce regulators of matrix mineralization like osteocalcin, osteopontin and ostenectin, RANKL which is necessary for osteoclast differentiation as well as the receptor for PTH (PTHR1). At the end of their lifespan osteoblasts transform into either osteocytes which become embedded in the mineralized matrix or lining cells, which cover all surfaces of bone. Specific molecules expressed by osteocytes include DMP1, FGF 23 and Rev Endocr Metab Disord (2010) 11:219–227 sclerostin, which control bone formation and phosphate metabolism . 2.1.1 Wnts and osteoblast differentiation Wnts are secreted glycoproteins crucial for the development and renewal of many tissues, including bone. Wnt signalling dominate osteoblast differentiation pathways and act via binding to a receptor complex consisting of LDL receptor-related protein 5 (LRP5) orLRP6 and one of ten Frizzled molecules . The so called canonical Wnt signaling pathway is active in all cells of the osteoblastic lineage, and involves the stabilization of β-catenin and regulation of multiple transcription factors . Wnt/βcatenin signaling is also important for mechanotransduction, fracture healing and osteoclast maturation [17–19]. The activation of canonical Wnt-signaling promotes osteoblast differentiation from mesenchymal progenitors at the expense of adipogenesis, which leads to improved bone strength, while suppression causes bone loss . Canonical Wnt signaling in osteoblast differentiation is modulated by Runx2 and osterix . Wnt signaling is a prime target for bone active drugs and the approaches include inhibition of Wnt antagonist like Dkk1, sclerostin, and Sfrp1 with neutralizing antibodies and inhibition of glycogen synthase kinase 3 β (GSK3 β), which promotes phosphorylation and degradation of βcatenin. One of the most promising approaches so far has been inhibition of the osteocyte protein sclerostin, which exerts tonic inhibition of osteoblast activity . Sclerostin is the product of the SOST gene, which is mutated and downregulated in patients with sclerosteosis and van Buchem disease and sclerosteosis , which are diseases characterized by high bone density. Expression levels of sclerostin are repressed in response to mechanical loading and intermittent PTH treatment . Preliminary studies with a humanized monoclonal antibody against sclerostin have shown bone anabolism in animals as well as humans . 2.2 Osteoclast differentiation The dominating pathway regulating osteoclast differentiation is the RANKL/RANK/OPG pathway. This pathway is based on osteoblasts promoting osteoclast differentiation through membrane presentation of RANKL and binding of this factor to the membrane receptor RANK on mononuclear osteoclast precursors. Osteoclast differentiation is also modulated by M-CSF . The promotion of osteoclast differentiation by RANKL is inhibited by the decoy receptor osteoprotegerin (OPG), which is also produced by osteoblasts  (74). Estrogens increase OPG and decrease RANKL expression in osteblasts, thus favoring bone formation. Postmenopausal bone loss is linked to 221 reduced estrogen levels favoring increased resorption. In many instances . PTH given as daily injections favors bone anabolism, reduces RANKL and increases OPG levels. In cases with chronic elevation of circulating PTH levels as seen in primary hyperparathyroidism the opposite pattern is seen with elevated RANKL and reduced OPG levels. A humanized monoclonal antibody against RANKL has been shown to elicit even more pronounced reduction in osteoclast numbers  than bisphosphonates and has demonstrated excellent reduction of fracture risk in postmenopausal osteoporosis . 2.3 Coupling between resorption and formation During normal bone remodeling, the amount of resorbed bone is completely replaced in location and amount by new bone. This is secured through tight coupling of bone resorption to bone formation. The mechanisms underlying the coupling process still remains largely elusive, although the last 15 years has increased our knowledge significantly. The dominating hypothesis years ago was that liberation of growth factors like IGF 1 and 2 and cytokines embedded in bone matrix during bone resorption secured the balance between resorption and formation during bone remodeling . Later work showing that osteoblastic bone formation proceeds unperturbed despite lack of bone resorption in the presence of defective osteoclasts lacking for example chloride channels or important factors for ruffled border formation in osteoclasts like c-Src  has supplemented this hypothesis. The important role that osteoclasts play in the regulation of bone formation is also corroborated by studies on mice lacking c-fos or M-CSF, which display absence of osteoclasts and defective bone formation . Other system involved in coupling of bone resorption to bone formation are the transmembrane proteins, ephrinB2, which are expressed on osteoblasts and EPH receptor B4 (EphB4), which are expressed on osteoclasts . Also the osteoclastic factor sphingosine 1-phosphate (S1P)  seems to play a significant role. The interaction of Ephrin and EPH by cell to cell contact promotes osteoblast differentiation and represses osteoclast differentiation. Secretion of S1P by osteoclast seems to recruit osteoblast progenitor cells to sites of bone resorption and stimulate differentiation of these progenitor cells by stimulating EphB4 signaling, This causes a shut down of bone resorption and initiate the formative phase of bone remodeling in the so called transition phase. 3 Targeted and non-targeted remodeling Through its constant removal and renewal of damaged bone, bone remodeling secures skeletal integrity throughout 222 life. It has become customary to distinguish between targeted and non-targeted (stochastic) remodeling. Nontargeted remodeling denotes regulation remodeling by hormones like PTH, thyroxine, growth hormone and estrogen, but also antiresorptive drugs like bisphosphonates may affect non-targeted remodeling. It seems that the main pathway is via modulation of osteoclasts, which then via the coupling between resorption and formation subsequently affects osteoblast activity. Targeted remodeling secures removal of damaged bone through targeted resorption. Osteocytes are the most abundant cells in bone, and their death by microdamage has been suggested to be the major event leading in the initiation of osteoclastic bone resorption. In normal bone . Resorption lacunae are 3 times more frequent in association with microcracks, indicating that remodeling is associated with repair of such microdamage . Damaged osteocytes promote differentiation of osteoclast precursors driven by secretion of M-CSF and RANKL . In cortical bone there is evidence to suggest, that microdamage not only activates new BMUs, but may also may direct the movement of existing BMUs as they tunnel through the cortex. It also seems that the degree of damage to the osteocyte network determines osteocyte metabolic responses to loading and influences targeted remodeling . Analysis of the relationship of between mean microcrack length and BMU resorption space density in cortical bone indicates that BMUs have an effective area about 40 times greater than their actual cross-section, which suggests that osteoclasts in the cutting cone of cortical BMUs are able to sense and steer toward microdamage . The relation between microdamage and initiation of bone remodeling is further corroborated by the fact that osteoclastic resorption is augmented in old bone . Rev Endocr Metab Disord (2010) 11:219–227 Table 1 Osteoblastic and endothelial markers detected on cells lining the Bone remodeling Compartment (BRC) vs. vascular endothelial cells as assessed by immuno- and enzyme histochemical staining Antigen BRC Vascular endothelium VEGF Von Willebrand Factor CD 34 Alkaline Phosphatase* Osteocalcin Osteonectin IGF 1,2 TGF β 1,2,3 bFGF OPG RANKL – – – + + + + + + + + + + + – – – – – – – – EG EG Ob CV OC OC 4 The bone remodeling compartment The work by Hauge et al.  demonstrated that the cells in the BMU, even in cancellous bone, were not directly contiguous to the bone marrow, but rather they were covered by a “canopy” of cells forming the outer lining of a specialized vascular structure with the denuded bone surface as the other delineation. The cells of this canopy display all classicial markers of the osteoblastic phenotype (Table 1), and are therefore most probably bone-lining cells, which seem to be connected to bone-lining cells on the quiescent bone surface. The structure has been demonstrated in cortical as well as cortical bone (Fig. 2). In turn, these bone-lining cells on the quiescent bone surface are in communication with osteocytes embedded within the bone matrix. Penetrating the canopy of bone-lining cells, and presumably serving as a conduit for the cells needed in the BMU, are capillaries. Fig. 2 Different representations of BRC structures in cortical (upper panel) and trabecular bone (lower panel). In cortical bone the BRC (outer demarcation by the broken line) is filled with erythrocyte ghosts (EG) and is located at the closing cone of the Haversian system situated over osteoblasts (OB). A few osteoclasts (OC) are also seen. CV denotes the central vessel of the Haversian system. In trabecular bone (lower panel) the outer lining of the BRC is clearly discernible, demarcating a vascular structure on top of osteoblasts (OB). Picture in upper panel courtesy of Pierre Delmas, Lyon, France Rev Endocr Metab Disord (2010) 11:219–227 Angiogenesis is closely associated with bone resorption and bone and angiogenic factors like VEGF and endothelin regulate osteoclast and osteoblast activity . In addition blood vessels serve as a way of transporting circulating osteoblast  and osteoclast precursors  to sites undergoing active remodeling. The involvement of vascular cells during the initiation of bone resorption is still unresolved. Is the very first step adhesion of a blood vessel to bone lining cells at a site where targeted repair is needed? Conceivably, osteocyte apoptosis and possible release of osteotropic growth factors and cytokines could be attractants for blood vessels, which would then subsequently initiate the formation of a resorptive BRC. But, as outlined above, the framework for signaling within the osteocyte-lining cell-BRC network could also be a way by which remodeling events on bony surfaces are triggered from damage accumulation or changes in mechanical strain within bone. There is increasing evidence for a common lineage and close interaction between vascular endothelial cells and bone cells. Endothelial cells drive differentiation of marrow stromal cell towards the osteoblastic phenotype  Endothelin and VEGF are also involved in signaling between vasculature and bone , and VEGF as well as other angiogenic factors are expressed during intramembranous osteogenesis. Osteoblastic cells, as well as osteoclasts, possess receptors for VEGF and also produce VEGF . Expression of VEGF is closely associated with the early phases of bone modeling and remodeling events  and it induces osteoblast chemotaxis and differentiation  and differentiation. Cells may enter the remodeling space either via diapedesis through the lining cell dome covering the BRC or via the circulation. It is still debatable whether all cells involved in remodeling arrive via the circulation, but while circulating osteoclast precursors were demonstrated more than a decade ago, there is now increasing evidence that osteoblast lineage cells are also present in the circulation strengthening the involvement of circulating precursor cells in the process [42, 49, 50]. While the systemic hormonal regulation of the remodeling process has to occur via factors arriving at individual remodeling sites via the bloodstream, the way by which local regulatory factors exert their action on individual cell populations involved is still obscure. Over the last decades, however, we have increased our knowledge about the different growth factors and cytokines involved in local regulation of bone remodeling tremendously (Fig. 3). Apart from growth factors and cytokines, simple molecules like nitrogen oxide (NO), as well as hypoxia and acidosis have been shown to exert pronounced effects on bone remodeling balance and activity. NO exerts biphasic effects on osteoclast activity with low concentrations potentiating and 223 high concentrations inhibiting bone resorption . Similarly, osteoblastic growth and differentiation are inhibited by high concentrations of NO, while lower concentrations may play a role in regulating normal osteoblast growth and in mediating the effects of estrogens on bone formation, mechanotransduction and bone anabolic responses . The dominating isoform of nitrogen oxide synthase (eNOS) is expressed in osteocytes and lining cells, but not in cuboidal osteoblasts . Acidosis and hypoxia generally increase bone resorption [53–56] and inhibit bone formation . As hypoxia may cause acidosis through increased anaerobic metabolism, the two factors may act synergistically at the tissue level . Hypoxia and acidosis also affect secretion of pro-angiogenic factors like VEGF as outlined below. 5 Key functions of the bone remodeling compartment 1. The BRC provides a closed microenvironment permitting tight regulation of bone remodeling. Current concepts regarding local regulation of bone remodeling generally assumes that the local growth factors, cytokines and even NO come either from cells in the marrow space or vascular cells having free access to the remodeling site without barriers, or are produced by osteoclasts and osteoblasts at the remodeling site. The BRC concept implies that the all factors liberated from the cells or vessels in the marrow space exert their regulatory role either through diffusion through the outer layer of the BRC, transport via the bloodstream to the interior of the BRC or indirectly via modulation of cell activity in the outer wall of the BRC. The presence of a specific compartment in which remodeling can proceed without interference from local factors liberated in the marrow space seems to be logical. If the access to the marrow space was open, the very high levels of growth factors in the marrow microenvironment might offset eventual localized regulatory effects by local growth factors, crucial to osteoclast and osteoblast differentiation and the remodeling process. 2. The BRC is the structure translating microdamage into targeted remodeling by which mechanosensory signals from the osteocyte network are translated into changes in osteoclast and osteoblast activity on trabecular surfaces. Lining cells are connected to the osteocyte network via gap junctions between lining cells on quiescent surfaces and osteocyte cannaliculi  (Fig. 4). Signals from lining cells indicating damage or stress could be transmitted to the outer lining cell layer of the BRC and trigger osteoclast recruitment. By analogy with remodeling in cortical bone, which is clearly associated with growth of a blood vessel into 224 Rev Endocr Metab Disord (2010) 11:219–227 MONOCYTES VEGF, FGF, ENDOTHELIN,N O, H+, HYPOXIA IL1,6,7,11, TNFα Marrow capillary/sinusoids E2, PTH, 1,25D,T3 IGF-2, IGF-1, TGFβ, β - - OB OC IL,M-CSF. C-fos TNF, EphrinB2 Osteoblastic Lining cells Vascular endothelium EphB4 RANKL EphrinB2 Osteoclast precursor EphB4 RANK reseptor IGFs, S1P Osteoblast Osteoclast Monocyte Osteoclast Fig. 3 Depiction of some of the main local regulatory factors operating at remodeling sites with osteoclasts (OC) and osteoblasts (OB). Interleukins (IL), tumor necrosis factors (TNF), transforming growth factors (TGF), colony stimulating factors (CSF), Insulin like growth factors (IGF), fibroblast growth factors (FGF), platelet derived growth factors (PDGF), bone morphogenetic proteins (BMP)) are formed by both monocytic cells in the marrow space or circulation, as well as bone cells in the BMU. NFκB- or RANK- ligand (RANKL) and osteoprotegerin (OPG) are formed specifically by osteoblasts. Factors from the marrow space as well as factors liberated by MARROW CAPILLARY OC OB OSTEOCYTES Fig. 4 Connections between the osteocyte network, lining cells and the BRC. All cells in this network are connected with gap junctions, which may provide a pathway (block arrows), by which signals generated deep within bone may reach the surface and elicit remodeling events by osteoclasts (OC) and osteoblasts (OB) in response to mechanical stimuli. The response may be modulated by factors liberated from the vascular endothelium or marrow capillaries/sinusoids and paracrine factors (broken arrow) liberated from lining cells may also play a role endothelial cells (vascular endothelial growth factor (VEGF), endothelin, nitrogen oxide (NO)) may diffuse to receptors on osteoclasts or osteoblasts. The cellular responses in the BMU are then further modulated by systemic hormones in the circulation (estrogen (E2), parathyroid hormone (PTH), active vitamin D (1,25D), thyroid hormone (T3)). Left lower insert depicts in detail osteoblastosteoclast interactions inside the BRC and right lower insert depict an alternativ, still hypohetical, version of that interaction based on lining cells acting as the osteoblastic component in thata interaction the remodeling site (5), the presumed ingrowth of a capillary into the BRC provides the vascular supply for the cells in the BMU of cancellous bone and might also provide the necessary osteoclasts and, subsequently, the osteoblasts that are needed for bone remodeling in both cancellous and cortical bone The BRC would also be a site where hormonal modulation (e.g. ERT) of the mechanosensory input could take place . 3. The BRC is the most probable structure at which coupling between osteoclasts and osteoblasts occurs. The RANKL/OPG pathway involves presentation of osteoblastic, membrane bound RANKL to the RANK receptor on osteoclast precursors by cell to cell contact. Due to the timing and sequence of bone resorption and bone formation, however, resorption and formation are generally separated in time and space, which makes the needed cell to cell contact between osteoclast precursors and active osteoblasts highly unlikely on a broader basis, and even if soluble RANKL played a major role it had no RANKL on precursor cells within the BRC to Rev Endocr Metab Disord (2010) 11:219–227 bind to. A more likely cell, which could present RANKL to RANK on osteoclast precursors, would be the lining cell. As demonstrated in animals  and in humans , lining cells exhibit positive immunoreactivity for OPG and RANKL, and might therefore be responsible for the cell to cell contact with osteoclast precursors. 4. The BRC also obviates the need for a “postal code” system ensuring that resorptive and formative cells adhere to areas on the bone surface, where they are needed. Bone surfaces are generally covered by lining cells, which would prevent direct contact between bone cells and integrins or other adhesion molecules known to modulate cell activity. The BRC would be the only place where these cells (circulating osteoclasts as well as circulating osteoblast precursors) would be exposed to these matrix constituents, because the formation of the BRC involves detachment of lining cells from the bone surface. 5. The BRC may play a crucial role in the spread of bone metastases. It is well established that apart from entering bone via local ingrowth, tumor cells reach bony surfaces via the circulation. The growth of metastatic cells in bone is enhanced by the so called “vicious cycle”, where PTHrp produced by tumor cells (e.g. breast cancer cells) induces increased local bone resorption and subsequent liberation of TGFβ from the bone matrix . The local effects of TGFβ in the bone microenvironment are two-fold: 1) it enhances the growth of bone metastases and 2) increases PTHrp formation from tumor cells further , thus maintaining the vicious cycle. As shown above, one of the key components of the vicious cycle, TGFβ, is produced by the cells lining the BRC. Other key promoters of bone metastases like IL-1 and IL-6 are also produced by the lining cell layer covering BRCs. It is therefore probable that the microenvironment in the BRC is highly conducive to metastatic seeding and the formation of the vicious cycle, further enabling growth of the bone metastasis. Moreover, the existence of a closed compartment would make vicious cycle formation easier due to absence of interference with cytokine and growth factors from the marrow space. Several large scale studies have established that bisphosphonates reduce the number of skeletal events in breast cancer, prostate cancer and myelomatosis, and iv bisphosphonates are now used routinely in advanced cancer . There is still debate as to how much of the beneficial effects of bisphosphonates in advanced cancer are due to inhibition of angiogenesis or to other, direct antitumor effects. Bisphosphonate, however, could exert their inhibitory effects on bone metastases simply by reducing the number of BRCs and thereby the surface of denuded bone available for metastatic seeding. 225 6 Conclusion Bone remodeling involves tight coupling and regulation of osteoclasts and osteoblasts and is modulated by a wide variety of hormones and osteocyte products secreted in response to mechanical stimulation and microdamage. Bone remodeling proceeds in a specialized vascular entity the “Bone Remodeling Compartment” (BRC), which provides the structural basis for coupling and regulation of cellular activity. Increased knowledge about the interplay between different factors and cells surrounding the BRC will most likely result in even better treatment options for skeletal disease. Open Access This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. References 1. Frost HM. Tetracycline-based histological analysis of bone remodeling. Calcif Tissue Res. 1969;3:211–37. 2. Eriksen EF. Normal and pathological remodeling of human trabecular bone: three dimensional reconstruction of the remodeling sequence in normals and in metabolic bone disease. Endocr Rev. 1986;7:379–408. 3. Eriksen EF, Gundersen HJ, Melsen F, Mosekilde L. Reconstruction of the formative site in iliac trabecular bone in 20 normal individuals employing a kinetic model for matrix and mineral apposition. Metab Bone Dis Relat Res. 1984;5:243–52. 4. Eriksen EF, Melsen F, Mosekilde L. Reconstruction of the resorptive site in iliac trabecular bone: a kinetic model for bone resorption in 20 normal individuals. Metab Bone Dis Relat Res. 1984;5:235–42. 5. Eriksen EF, Hodgson SF, Eastell R, Cedel SL, O’Fallon WM, Riggs BL. Cancellous bone remodeling in type I (postmenopausal) osteoporosis: quantitative assessment of rates of formation, resorption, and bone loss at tissue and cellular levels. J Bone Miner Res. 1990;5:311–9. 6. Agerbaek MO, Eriksen EF, Kragstrup J, Mosekilde L, Melsen F. A reconstruction of the remodelling cycle in normal human cortical iliac bone. Bone Miner. 1991;12:101–12. 7. Parfitt AM. The mechanism of coupling: a role for the vasculature. Bone. 2000;26:319–23. 8. Burkhardt R. In: Arlet J, Ficat RP, Hungerford DS, editors. The structural relationship of bone forming and endothelial cells of the bone marrow. Baltimore: Williams & Wilkins; 1984. p. 2–14. 9. Komori T, Yagi H, Nomura S, Yamaguchi A, Sasaki K, Deguchi K, et al. Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts. Cell. 1997;89:755–64. 10. Lian JB, Stein GS, Javed A, van Wijnen AJ, Stein JL, Montecino M, et al. Networks and hubs for the transcriptional control of osteoblastogenesis. Rev Endocr Metab Disord. 2006;7:1–16. 11. Nakashima K, Zhou X, Kunkel G, Zhang Z, Deng JM, Behringer RR, et al. The novel zinc finger-containing transcription factor osterix is required for osteoblast differentiation and bone formation. Cell. 2002;108:17–29. 226 12. Qin L, Qiu P, Wang L, Li X, Swarthout JT, Soteropoulos P, et al. Gene expression profiles and transcription factors involved in parathyroid hormone signaling in osteoblasts revealed by microarray and bioinformatics. J Biol Chem. 2003;278:19723– 31. 13. Westendorf JJ, Kahler RA, Schroeder TM. Wnt signaling in osteoblasts and bone diseases. Gene. 2004;341:19–39. 14. Murshed M, Harmey D, Millan JL, McKee MD, Karsenty G. Unique coexpression in osteoblasts of broadly expressed genes accounts for the spatial restriction of ECM mineralization to bone. Genes Dev. 2005;19:1093–104. 15. Bonewald L. Osteocytes as multifunctional cells. J Musculoskelet Neuronal Interact. 2006;6:331–3. 16. Hens JR, Wilson KM, Dann P, Chen X, Horowitz MC, Wysolmerski JJ. TOPGAL mice show that the canonical Wnt signaling pathway is active during bone development and growth and is activated by mechanical loading in vitro. J Bone Miner Res. 2005;20:1103–13. 17. Robinson JA, Chatterjee-Kishore M, Yaworsky PJ, Cullen DM, Zhao W, Li C, et al. Wnt/beta-catenin signaling is a normal physiological response to mechanical loading in bone. J Biol Chem. 2006;281:31720–8. 18. Chen Y, Whetstone HC, Lin AC, Nadesan P, Wei Q, Poon R, et al. Beta-catenin signaling plays a disparate role in different phases of fracture repair: implications for therapy to improve bone healing. PLoS Med. 2007;4:e249. 19. Spencer GJ, Utting JC, Etheridge SL, Arnett TR, Genever PG. Wnt signalling in osteoblasts regulates expression of the receptor activator of NFkappaB ligand and inhibits osteoclastogenesis in vitro. J Cell Sci. 2006;119:1283–96. 20. Bodine PV, Komm BS. Wnt signaling and osteoblastogenesis. Rev Endocr Metab Disord. 2006;7:33–9. 21. Hill TP, Spater D, Taketo MM, Birchmeier W, Hartmann C. Canonical Wnt/beta-catenin signaling prevents osteoblasts from differentiating into chondrocytes. Dev Cell. 2005;8:727–38. 22. van Bezooijen RL, Svensson JP, Eefting D, Visser A, van der Horst G, Karperien M, et al. Wnt but not BMP signaling is involved in the inhibitory action of sclerostin on BMP-stimulated bone formation. J Bone Miner Res. 2007;22:19–28. 23. Balemans W, Ebeling M, Patel N, Van HE, Olson P, Dioszegi M, et al. Increased bone density in sclerosteosis is due to the deficiency of a novel secreted protein (SOST). Hum Mol Genet. 2001;10:537–43. 24. Keller H, Kneissel M. SOST is a target gene for PTH in bone. Bone. 2005;37:148–58. 25. Padhi D, Jang G, Stouch B, Fang L, Posvar E. Single-dose, placebo-controlled, randomized study of AMG 785, a sclerostin monoclonal antibody. J Bone Miner Res. 2010. 26. Khosla S. Minireview: the OPG/RANKL/RANK system. Endocrinology. 2001;142:5050–5. 27. Reid IR, Miller PD, Brown JP, Kendler DL, Fahrleitner-Pammer A, Valter I, et al. Effects of denosumab on bone histomorphometry: the FREEDOM and STAND studies. J Bone Miner Res. 2010;25:2256–65. 28. Cummings SR, San MJ, McClung MR, Siris ES, Eastell R, Reid IR, et al. Denosumab for prevention of fractures in postmenopausal women with osteoporosis. N Engl J Med. 2009;361:756– 65. 29. Mohan S, Baylink DJ. Insulin-like growth factor system components and the coupling of bone formation to resorption. Horm Res. 1996;45 Suppl 1:59–62. 30. Karsdal MA, Martin TJ, Bollerslev J, Christiansen C, Henriksen K. Are nonresorbing osteoclasts sources of bone anabolic activity? J Bone Miner Res. 2007;22:487–94. 31. Dai XM, Zong XH, Akhter MP, Stanley ER. Osteoclast deficiency results in disorganized matrix, reduced mineralization, and Rev Endocr Metab Disord (2010) 11:219–227 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. abnormal osteoblast behavior in developing bone. J Bone Miner Res. 2004;19:1441–51. Zhao C, Irie N, Takada Y, Shimoda K, Miyamoto T, Nishiwaki T, et al. Bidirectional ephrinB2-EphB4 signaling controls bone homeostasis. Cell Metab. 2006;4:111–21. Ryu J, Kim HJ, Chang EJ, Huang H, Banno Y, Kim HH. Sphingosine 1-phosphate as a regulator of osteoclast differentiation and osteoclast-osteoblast coupling. EMBO J. 2006;25:5840– 51. Cardoso L, Herman BC, Verborgt O, Laudier D, Majeska RJ, Schaffler MB. Osteocyte apoptosis controls activation of intracortical resorption in response to bone fatigue. J Bone Miner Res. 2009;24:597–605. Li J, Mashiba T, Burr DB. Bisphosphonate treatment suppresses not only stochastic remodeling but also the targeted repair of microdamage. Calcif Tissue Int. 2001;69:281–6. Kurata K, Heino TJ, Higaki H, Vaananen HK. Bone marrow cell differentiation induced by mechanically damaged osteocytes in 3D gel-embedded culture. J Bone Miner Res. 2006;21:616–25. Colopy SA, Benz-Dean J, Barrett JG, Sample SJ, Lu Y, Danova NA, et al. Response of the osteocyte syncytium adjacent to and distant from linear microcracks during adaptation to cyclic fatigue loading. Bone. 2004;35:881–91. Burger EH, Klein-Nulend J, Smit TH. Strain-derived canalicular fluid flow regulates osteoclast activity in a remodelling osteon—a proposal. J Biomech. 2003;36:1453–9. Henriksen K, Leeming DJ, Byrjalsen I, Nielsen RH, Sorensen MG, Dziegiel MH, et al. Osteoclasts prefer aged bone. Osteoporos Int. 2007;18:751–9. Hauge EM, Qvesel D, Eriksen EF, Mosekilde L, Melsen F. Cancellous bone remodeling occurs in specialized compartments lined by cells expressing osteoblastic markers. J Bone Miner Res. 2001;16:1575–82. Brandi ML, Collin-Osdoby P. Vascular biology and the skeleton. J Bone Miner Res. 2006;21:183–92. Eghbali-Fatourechi GZ, Lamsam J, Fraser D, Nagel D, Riggs BL, Khosla S. Circulating osteoblast-lineage cells in humans.[see comment]. N Engl J Med. 2005;352:1959–66. Kassem M, Risteli L, Mosekilde L, Melsen F, Eriksen EF. Formation of osteoblast-like cells from human mononuclear bone marrow cultures. APMIS. 1991;99:269–74. Kaigler D, Krebsbach PH, Polverini PJ, Mooney DJ. Role of vascular endothelial growth factor in bone marrow stromal cell modulation of endothelial cells. Tissue Eng. 2003;9:95–103. Veillette CJ, von Schroeder HP. Endothelin-1 down-regulates the expression of vascular endothelial growth factor-A associated with osteoprogenitor proliferation and differentiation. Bone. 2004;34:288– 96. Tombran-Tink J, Barnstable CJ. Osteoblasts and osteoclasts express PEDF, VEGF-A isoforms, and VEGF receptors: possible mediators of angiogenesis and matrix remodeling in the bone. Biochem Biophys Res Commun. 2004;316:573–9. Xiong H, Rabie AB. Neovascularization and mandibular condylar bone remodeling in adult rats under mechanical strain. Front Biosci. 2005;10:74–82. Li G, Cui Y, McIlmurray L, Allen WE, Wang H. rhBMP-2, rhVEGF(165), rhPTN and thrombin-related peptide, TP508 induce chemotaxis of human osteoblasts and microvascular endothelial cells. J Orthop Res. 2005;23:680–5. Zvaifler NJ, Marinova-Mutafchieva L, Adams G, Edwards CJ, Moss J, Burger JA, et al. Mesenchymal precursor cells in the blood of normal individuals. Arthritis Res. 2000;2:477–88. Kuznetsov SA, Mankani MH, Gronthos S, Satomura K, Bianco P, Robey PG. Circulating skeletal stem cells. J Cell Biol. 2001;153:1133–40. Rev Endocr Metab Disord (2010) 11:219–227 51. Ralston SH, Ho LP, Helfrich MH, Grabowski PS, Johnston PW, Benjamin N. Nitric oxide: a cytokine-induced regulator of bone resorption. J Bone Miner Res. 1995;10:1040–9. 52. Fox SW, Chow JW. Nitric oxide synthase expression in bone cells. Bone. 1998;23:1–6. 53. Bushinsky DA. Acid-base imbalance and the skeleton [Review] [67 refs]. Eur J Nutr. 2001;40:238–44. 54. Goldhaber P, Rabadjija L. H+ stimulation of cell-mediated bone resorption in tissue culture. Am J Physiol. 1987;253:E90–8. 55. Arnett TR, Dempster DW. Effect of pH on bone resorption by rat osteoclasts in vitro. Endocrinology. 1986;119:119–24. 56. Arnett TR, Gibbons DC, Utting JC, Orriss IR, Hoebertz A, Rosendaal M, et al. Hypoxia is a major stimulator of osteoclast formation and bone resorption. J Cell Physiol. 2003;196:2–8. 57. Utting JC, Robins SP, Brandao-Burch A, Oriss IR, Behar J, Arnett TR. Hypoxia inhibits the growth, differentiation and bone forming capacity of rat osteoblasts. Exp Cell Res. 2006;312:1694–702. 227 58. Marotti G, Ferretti M, Muglia MA, Palumbo C, Palazzini S. A quantitative evaluation of osteoblast-osteocyte relationships on growing endosteal surface of rabbit tibiae. Bone. 1992;13:363–8. 59. Bonewald LF. Osteocyte biology: its implications for osteoporosis. J Musculoskelet Neuronal Interact. 2004;4:101–4. 60. Silvestrini G, Ballanti P, Patacchioli F, Leopizzi M, Gualteri N, Monnazzi P, et al. Detection of osteoprotegerin (OPG) and its ligand (RANKL) mRNA and protein in femur and tibia of the rat. J Mol Histol. 2005;36:59–67. 61. Eriksen EF, Qvesel D, Hauge EM, Melsen F. Further evidence that vascular remodeling spaces are lined by cells of osteogenic origin: characterization of a possible coupling structure. J Bone Miner Res. 2005;15:S371. 62. Mundy GR. Mechanisms of bone metastasis [Review] [99 refs]. Cancer. 1997;80:1546–56. 63. Polascik TJ. Bisphosphonates in oncology: evidence for the prevention of skeletal events in patients with bone metastases. Drug Des Devel Ther. 2009;3:27–40.