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: manolagasstavros@exchange.uams.edu *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 TGF1 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 ␣v3 and ␣21, 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 17estradiol 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 (17E2-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. 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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: stady2000@unitours.co.il or naorzvi@post.tau.ac.il 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. 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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 ␣v3 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 ␣v3 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: kh@nordicbioscience.com. 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. 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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: e.f.eriksen@medisin.uio.no (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 [1] 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 [2]. 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 [5]. 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 [6]. In normal bone the duration of the remodeling cycle in cortical is shorter than in cancellous bone with a median of 120 days [6]. 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 [7], 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 [8]. 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 [9]. During this sequence of cellular proliferation Runx2 regulates expression of genes encoding osteocalcin, VEGF, RANKL, sclerostin, and dentin matrix protein 1 [DMP1] [10]. Osterix is another transcription factor essential for osteoblast differentiation [11]. 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 [12]. The action of PTH and BMPs is closely associated with activation of Wnt signalling pathways [13]. 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 [14]. 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 [15]. 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 [13]. 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 [16]. 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 [20]. Canonical Wnt signaling in osteoblast differentiation is modulated by Runx2 and osterix [21]. 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 [22]. Sclerostin is the product of the SOST gene, which is mutated and downregulated in patients with sclerosteosis and van Buchem disease and sclerosteosis [23], which are diseases characterized by high bone density. Expression levels of sclerostin are repressed in response to mechanical loading and intermittent PTH treatment [24]. Preliminary studies with a humanized monoclonal antibody against sclerostin have shown bone anabolism in animals as well as humans [25]. 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 [26]. The promotion of osteoclast differentiation by RANKL is inhibited by the decoy receptor osteoprotegerin (OPG), which is also produced by osteoblasts [26] (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 [26]. 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 [27] than bisphosphonates and has demonstrated excellent reduction of fracture risk in postmenopausal osteoporosis [28]. 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 [29]. 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 [30] 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 [31]. 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 [32]. Also the osteoclastic factor sphingosine 1-phosphate (S1P) [33] 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 [34]. Resorption lacunae are 3 times more frequent in association with microcracks, indicating that remodeling is associated with repair of such microdamage [35]. Damaged osteocytes promote differentiation of osteoclast precursors driven by secretion of M-CSF and RANKL [36]. 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 [37]. 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 [38]. The relation between microdamage and initiation of bone remodeling is further corroborated by the fact that osteoclastic resorption is augmented in old bone [39]. 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. [40] 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 [41]. In addition blood vessels serve as a way of transporting circulating osteoblast [42] and osteoclast precursors [43] 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 [44] Endothelin and VEGF are also involved in signaling between vasculature and bone [45], 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 [46]. Expression of VEGF is closely associated with the early phases of bone modeling and remodeling events [47] and it induces osteoblast chemotaxis and differentiation [48] 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 [51]. 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 [51]. The dominating isoform of nitrogen oxide synthase (eNOS) is expressed in osteocytes and lining cells, but not in cuboidal osteoblasts [52]. Acidosis and hypoxia generally increase bone resorption [53–56] and inhibit bone formation [57]. As hypoxia may cause acidosis through increased anaerobic metabolism, the two factors may act synergistically at the tissue level [56]. 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 [58] (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 [59]. 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 [60] and in humans [61], 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 [62]. 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 [62], 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 [63]. 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. 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