Crimean Journal of Experimental and Clinical Medicine 2013 Volume 3 N 1-2
UDC 616-001.5:616.71-003.93
BONE REMODELING: CELLULAR-MOLECULAR
BIOLOGY AND CYTOKINE RANK-RANKLOSTEOPROTEGERIN (OPG) SYSTEM AND GROWTH
FACTORS
S. Sagalovsky
Abstract
Physiological bone remodeling is a highly coordinated process
responsible for bone resorption and formation and is necessary to
repair damaged bone and maintain mineral homeostasis. In addition
to the traditional bone cells, osteoblasts, osteoclasts and osteocytes,
that are necessary for bone remodeling several biological active
factors have also been implicated in bone disorders. This review
discussed physiologic bone remodeling outlining the traditional bone
biology dogma in light of emerging role growth factors data. Specifically
discussed in detail are the cellular and molecular mechanisms of
bone remodeling, including events that orchestrate the role cytokine
RANK-RANKL-OPG system and growth factors of bone remodeling.
Department of Orthopaedic Clinic
Median, Bad Lausick, Germany.
Correspondence:
Parkstraße 4, 04651 Bad Lausick
Deutschland
e-mail:
stanislav.sagalovsky@mediankliniken.de
Key words: osteoblasts, osteoclasts, bone remodeling, RANK-RANKL-OPG cytokine system,
growth factors.
Bone is a dynamic tissue that undergoes continual
adaption during life to attain and preserve skeletal size,
shape and structural integrity and regulate mineral
homeostasis. Two processes, remodeling and modeling,
underpin development and maintenance of the skeletal
system. Bone modeling is responsible for growth and
mechanically induced adaption of bone and requires that
the process of bone formation and bone resorption, while
globally coordinated, occur independently at distinct
anatomical location. This tightly coordinated event
requires the synchronized activities of multiple cellular
participants to ensure bone resorption and formation
occur sequentially at the same anatomical location to
preserve bone mass. This article reviews the cellular
participants and molecular mechanisms that coordinate
bone remodeling and includes an assessment of cytokine
RANK-RANKL-OPG system and growth factors and
their key role in regulating normal bone physiology.
derive from pluripotent mesenchymal stem cells (MCS)
that prior to osteoblast commitment can also differentiate
into other mesenchymal cells lineages such as fibroblasts,
chondrocytes, myoblasts and bone marrow stromal cells
including adipocytes, depending on the activated signaling
transcription pathways. Understanding the mechanisms
that control the differentiation of osteoblastic cells from
MCS is thus one of the fundamental areas of research of
bone biology. Several specific transcription factors are
responsible for the commitment of pluripotent MSC into the
osteoblast cell lineage [2]. Lineage-specific gene expression
is ultimately under the control of transcription factors that
act to regulate specific gene expression. They act as the
key switching mechanisms to induce gene transcription.
Considerable progress has been made in identifying those
transcription factors which act as “master switches” during
commitment of multipotent cells to specific lineages. A
major breakthrough in understanding genetic regulation of
osteoblast differentiation was made with the identification
of the role of the transcription factor core binding factor 1
(Cbfa-1/RUNX-2) [2, 26, 27]. Cbfa-1/RUNX-2 expression
is an absolute requirement for osteoblast differentiation.
In Cbfa-1 knockout mice there is a normal cartilaginous
skeleton seen but a complete absence of bone formation
[62, 66]. Cbfa-1/RUNX-2 known to interact directly
with the osteocalcin promoter to induce its expression
[10]. However an additional transcription factor, Osterix,
which is a downstream target for Cbfa-1/RUNX-2, has
also been shown to be an absolute requirement for normal
osteoblast differentiation in knockout mice experiments
[27]. More recent studies have shown the existence of
CELLS INVOLVED IN BONE REMODELING:
OSTEOBLASTS AND BONE FORMATION
Bone remodeling is a physiological process that
maintains the integrity of the skeleton by removing old bone
and replacing it with a young matrix. Two principle cell
types are found in bone, the osteoclast, and the osteoblast,
which are the major effectors in the turnover of bone
matrix [51, 55]. Osteoblasts and osteoclasts dictate skeletal
mass, structure, and strength via their respective roles in
resorbing and forming bone. Osteoblasts are specialized
mesenchymal-derived cells whose function is the
deposition and maintenance of skeletal tissue. Osteoblasts
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Review article
distinct isoforms of Cbfa-1, which may have subtly
different roles during normal tissue formation, including
regulation of cartilage expression in addition to bone.
Another runt-related gene that plays an important role in
the commitment of multipotent MSC to the osteoblastic
lineage and for osteoblast differentiation at an early stage is
RUNX-2. Cbfa-1/RUNX-2 are involved in the production
of bone matrix proteins [69], as it is able to up-regulate the
expression of major bone matrix protein genes, such as type
I collagen, osteopontin, bone sialoprotein and osteocalcin
leading to an increase of immature osteoblasts from MCS;
the immature osteoblasts from immature bone [26,27].
Osteoblast commitment, differentiation and growth are
controlled by several local and systemic factors that can
also act in a paracrine and/or autocrine way and that can
regulate the activity of specific transcription factor [10].
Huge advances have been made in the understanding of
cellular and molecular control of bone formation in the past
decade. The establishment of in vitro models of osteoblast
differentiation and formation have been essential for
determining the effects of specific growth factors and
growth factor-induced transcription factors on osteogenesis.
Growth factors are soluble proteins that act as signaling
agents for cells and influence critical functions, such as
cell division, matrix synthesis and tissue differentiation,
by receptor-ligand binding. Growth factors involved in
the local regulation of bone formation and their functions
summarized in Table. Results of experimental studies have
established that growth factors play many important roles
in bone formation and bone repair [38]. Growth factors
that are known to affect osteogenic cells include fibroblast
growth factor (FGF), transforming growth factor-ß (TGF-ß),
insulin-like growth factor (IGF), platelet-derived growth
factor (PDGF) and vascular endothelial growth factor
(VEGF), and additional cytokine modulators [1,44,53],
which potently modulate the functions of osteoblasts in
an autocrine manner [1,53]. Growth factors signaling first
transmits signal across the plasma membrane through the
formation of heteromeric complexes of specific type I and
type II serine/treonine kinase receptors. The type I receptor
is phosphorylated following the activation of specific type
II receptor. Activated type I receptors initiate intracellular
signaling cascade through phosphorilation of specific
proteins (selective MAPKs pathway). The physiological
function of this complex cascade are summarized in
Figure 1. Recent results demonstrate [8] that following
growth factors induction MAPK and other pathways
converge at the RUNX-2 gene to control osteoblasts
cells differentiation. Growth factors-induced activation
of ERK-MAPK is an important signaling component that
stimulates cell proliferation to enrich osteoprogenitor cells,
thereby promoting their differentiation into osteoblasts.
Table The role in bone remodeling of the growth factors
Molecule
TGF-ß
FGF
IGF
VEGF
PDGF
Role in bone remodeling
Mesenchymal stromal cell proliferation; osteoblast precursor recruitment; osteoblast and chonrocyte
differentiation; bone matrix production; recruitment of osteoclast precursor but inhibition of terminal differentiation and induction of apoptosis.
Osteoblast recruitment and proliferation.
Osteoblast proliferation; bone matrix synthesis; bone resorption.
Osteoblast proliferation and differentiation; conversion of cartilage into bone
Osteoprogenitor migration, proliferation and differentiation; osteoclastogenesis.
None: TGF-ß - transforming growth factor – ß; FGF – fibroblast growth factor; IGF – insulin-like growth factor; VEGF – vascular
endothelial growth factor; PDGF – platelet-derived growth factor.
providing a pool of early osteoblasts. In contrast, during
later phases of osteoblast differentiation, transforming
growth factor- ß1 blocks differentiation and mineralization.
These effects appear to be highly dependent on bone cell
source, dose applied and the local environment, which
may be a result of the inhibition of DNA synthesis at high
TGF-ß1 concentrations. Additionally, TGF-ß1 inhibits the
expression of the RUNX-2 and osteocalcin genes, whose
expression is controlled by Cbfa-1/RUNX-2 in osteoblastlike cell lines [14]. Likewise, TGF-ß1 inhibits resorption on
reducing the formation and differentiation of the osteoclasts,
as well as mature osteoclast activity and stimulating their
apoptosis [6]. Transforming growth factor-ß1 interacts with
a range of other growth factors in bone with a resulting
complex response. Further work [1] is needed to clarify the
role of transforming factor-ß1 during bone regeneration
and wound healing and to determine the inter-relationship
between TGF-ß1 and other growth factors that have
effects during different stages of osteoblast differentiation.
Fibroblast growth factor (FGF) is a family of
structurally related polypeptides that are known to play
a critical role in angiogenesis and mesenchymal cell
Transforming
growth
factor-ß1
(TGF-ß1).
Transforming growth factor-ß1 belongs to a large
superfamily of related proteins that also includes BMPs,
growth and differentiation factors, activins, inhibitins and
anti-Mullerian hormone. All members play important
roles in regulating cell proliferation and differentiation
and the production of extracellular matrix. There are
five isoforms of TGF-ß1 (transforming growth factor-ß1
to transforming factor-ß5). Most cells synthesize and
respond to TGF-ß, but high levels are found in bone,
platelets and cartilage. TGF-ß1 is the most abundant
isoform at the protein level (for a recent comprehensive
review see Arvidson et al. [1]). The activation of
TGF-ß1 is highly regulated, and once activated it
interacts with transmembrane serine/threonine kinase
receptors. Binding TGF-ß1 with its receptor tyrosine
kinase (RTK) on the surface of osteoblast/osteoblast
precursor recruits the adapter proteins and shown to
signal through the Ras/mitogen-activated protein kinase
(MAPK) pathway [44]. During the early stages of bone
formation, the action of transforming growth factor is to
recruit and stimulate osteoprogenitor cells to proliferate,
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Crimean Journal of Experimental and Clinical Medicine 2013 Volume 3 N 1-2
ubiquitous. Although fibroblast growth factor signaling
has been implicated in bone development, studies on null
mutant mice have not yet fully shown the role of this family
in skeletal development [44]. Fibroblast growth factor-1
and fibroblast growth factor-2 in vitro stimulate osteoblast
proliferation but do not increase collagen production
or alkaline phosphatase in differentiated osteoblasts,
although these effects may be differentiation stagespecific as constitutive FGF signaling inhibits osteoblastic
differentiation and dramatically increases apoptosis when
cells are exposed to differentiating conditions. FGFs are
strongly mitogenic to bone marrow stromal cells and are able
to maintain the self-renewal of these cells in culture [57].
Insulin-like growth factors (IGFs). Growth hormone
and insulin-like growth factors play critical roles in
skeletal development. Growth hormone participates in
the regulation of skeletal growth and triggers the release
of insulin-like growth factor in target cells. The insulinlike growth factors are bound to binding proteins, adding
another crucial tier to modulate the activity of insulinlike growth factor. Two insulin-like growth factors have
been identified – insulin-like growth factor-1 and insulinlike growth factor-2 – both of which are found in high
concentration in serum. In bone, whilst insulin-like growth
factor-2 is more abundant, insulin-like growth factor-1
may be more potent, although this might be different both
between and within species [1]. The regulation of insulinlike growth factor is complex, and the growth hormone
mode of action in skeletal cells is largely unknown. Of
the major hormones that regulate the skeleton, all have
significant effects on skeletal insulin-like growth factor,
as do many growth factors, such as transforming growth
factor-ß1 and fibroblast growth factor. Insulin-like growth
factors increase proliferation and play a major role in
stimulating mature osteoblast function. IGF-1 secreted
from osteoblasts in the bone tissue has been demonstrated
to be a potent chemotactic factor that might play a major
role in the recruitment of osteoblast during bone formation
[41]. As with other growth factors detailed in this section,
the way that osteoblasts respond to insulin-like growth
factor signal may well depend on both the differentiation
status of the cell and cell type. At the molecular level,
insulin-like growth factor-1 upregulates the osteoblastassociated transcription factor, Osterix, but not Cbfa-1/
RUNX-2. Although it is widely accepted that insulin-like
growth factors have a defining role in bone remodeling,
their actual role is still unclear and need to be understood
within the complex inter-relationship of the components
of the insulin-like growth factor system that evidently
occur in vivo. Overall, the evidence suggests that the
major effects of insulin-like growth factors are to promote
the late-stage differentiation and activity of osteoblasts.
Platelet-derived growth factor (PDGF) is secreted by
platelets during the early phases of fracture healing, but its
presence has been found in various tissues, including bone.
Owing to its expression by a range of tissues, it is thought
to have both systemic and local actions. Platelet-derived
growth factor is composed of two polypeptide chains and
can exist in three different isoforms of two gene products
(AA;BB;AB) and these bind to two separate a and b receptors.
Platelet-derived growth factor is a powerful mitogen for
Figure 1. Growth factors, RTK receptor, and transducer
proteins are expressed in the osteoblast-precursor cell
and growth-factor signaling pathways. Activated of the
growth factor receptor tyrosine kinase (RTK) activate class I
phosphatidylinositol 3-kinase (PI3K) or guanosine-nucleotidebinding protein (Ras) and through direct binding orthrough
tyrosine phosphorylattion of scaffolding adaptors, such
as Act/PKB/PKC and IKK/IkB or Raf/MEK, which then bind
and activate NF-kB or MAPK. Activated NF-kB and MAPK
are through direct binding phosphorylation of ERK/JnKcJun, which then leading activate gene regulation proteins /
HCO3Cbfa1/RUNX2. Abbreviations: Act/PKB – protein kinase
B; Cbfa1/RUNX2 – transcription factors; ERK – extracellular
signal-regulated kinase; Fos – transcription factor; IKK/
IKB – enzyme complex NF-kB signal transduction cascade;
Jnk/Jun – N-terminal kinase; MAPK/MEK/MEKK – mitogen
activated protein kinase; NF-kB – nuclear factor kappa B;
PI3K – phosphatidylinositol 3-kinase; Ras/Raf – guanosinnucleotide-binding protein and serin/threonin-protein kinases;
RTK – receptor tyrosin kinase.
mitogenes. To mediate their range of effects, FDF proteins
signal via membrane-spanning tyrosine kinases receptor
and there are a wide variety of mechanisms for receptor
regulation and availability. Mutations in these receptors are
associated with abnormalities in ossification and activating
mutations FGF receptor II cause several craniosynostosis
syndromes by affecting the proliferation and differentiation
of osteoblasts [44], highlighting a key role for these
molecules in the control of bone formation. In normal adult
tissues, the most abundant proteins are fibroblast growth
factor-1 and fibroblast growth factor-2. FGF-2 is expressed
by osteoblasts and is generally more potent then FGF-1,
although the expression of other FGFs are not nearly as
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Review article
and sufficient stimuli that control the bechavior of the
osteoclast, an event that occurs via cell-cell interaction
The bone resorption cascade involves a series of steps
directed towards the removal of both the mineral and
organic constituents of bone matrix by osteoclasts, aided by
osteoblasts. The role of the osteoclast as a major resorbing
cell, and its structure and biochemical properties have
been well characterized [6, 54]. The first stage involves
the recruitment and dissemination of osteoclast progenitors
to bone. The progenitor cells are recruited from the
haemopoietic tissue such as bone marrow and slenic tissue
to bone via the circulating blood stream. They proliferate
and differentiate into osteoclasts through a mechanism
involving cell-to-cell interaction with osteoblast stromal
cells. Osteoclast formation from osteoclast precursor
is regulated predominantly by osteoblastic cells during
normal bone remodeling (Figure 2). Osteoblastic cells in
the bone marrow express two cytokines that are required
for osteoclast-progenitor differentiation into osteoclasts:
receptor activator of NF-kB ligand (RANKL) and
osteoprotegerin (OPG) [31]. The discoveries of the receptor
activator of NF-kB ligand and osteoprotegerin have
revolutionized our understanding of the process underlying
osteoclast formation and activation [ 23,61]. RANKL and
OPG potently stimulate and inhibit, respectively, osteoclast
differentiation. RANKL is a membrane bound factor that
is produced by osteoblasts and stromal cells in response to
a variety of signals such as parathyroid hormone (PTH),
tumor necrosis factor-α (TNF-α) and interleukin-1 (Il1). RANKL bind to the cytoplasmic membrane receptor
RANK (receptor activator of NF-kB), which is a member
of the tumor necrosis factor (TNF) receptor super family
and subsequently induces both osteoclast differentiation
and activation. OPG is a soluble decoy receptor for
RANKL and can inhibit its effects, thereby preventing
osteoclast development and subsequent bone resorption
[4]. Over expression of OPG in transgenic mice results in
osteopetrosis [49], and, conversely, OPG deficient mice
exhibit severe osteoporosis [15, 36]. Many of the same
agent that stimulate RANKL expression (including PTH,
IL-1, PGE) also inhibit OPG expression [40,46], which
enhances osteoclastogenesis even further. While FGF-2
induces RANKL expression by osteoblasts, it also inhibits
osteoclast differentiation directly by interfering with the
action of macrophage colony stimulating factor (M-CSF)
[43, 57]. In contrast, to the stimulatory effects of the
agents described above, estrogen inhibits the production of
RANKL by osteoblasts [25]. Transforming growth factor-ß
(TGF-ß) also strongly suppresses RANKL expression by
osteoblasts, whereas it stimulates OPG expression [8].
Administration of RANKL to mice causes osteoporosis
[32], whereas disruption of the RANKL gene in mice
leads to severe osteopetrosis, impaired tooth eruption,
and the absence of osteoclasts [33, 64]. Membrane bound
macrophage colony stimulating factor (M-CSF) is also a
critical early modulator in the differentiation of osteoclasts
[29]. M-CSF binds to c- Fms on the surface of osteoclast
precursors, and this event enhances their proliferation and
survival. M-CSF enhances the survival of monocyte stem
cells thereby permitting them to respond to direct inducers
of differentiation such as RANKL. A combination of
connective tissue cells, and although it can stimulate, and
is synthesized by, mesenchymal cells and osteoblast like
cells, it does not have powerful bone-induction properties.
PDGF isoforms have a strong chemotactic effect on
osteoblasts and other connective tissue cells, and may act
to recruit mesenchymal cells during bone development and
remodeling [1, 44]. In addition to PDGF autoregulation in
osteoblasts, there is paracrine regulation by other growth
factors, such as transforming growth factor-ß1 [44].
Osteoblasts play a crucial role in the process of bone
formation, in the induction and regulation of extracellular
matrix mineralization and in the control of bone remodeling
[52]. During bone formation, mature osteoblasts synthesize
and secrete type I collagen (which represents the greated
part of the organic extracellular bone matrix) and various
non-collagen proteins such as osteocalcin, osteopontin
and bone sialoprotein (which exert various essential
functions, including the regulation of bone turnover,
the control of bone mineral deposition and regulation of
bone cell activity). Osteocalcin (Gla) is a vitamin-Kdependent osteoblast-specific protein and whose synthesis
is enhanced by 1,25 OH vitamin D3 and reflects metabolic
cellular activity. Of the de novo synthesized osteocalcin,
60-90% is incorporated into the bone matrix where it
binds to hydroxyapatite during matrix mineralization.
Osteopontin (OPN) is a phosphorylated acidic glycoprotein
that is present in large amounts in immature bone. OPN is
synthesized by osteoblast but is expressed by other cellular
types, such as chondrocytes; it is involved in various
physiological and pathological events. Bone sialoproteins
is a glycosylated, phosphorylated and sulfated protein
that promotes hydroxyapatite crystal nucleation and
osteoblast differentiation [19]. This has been confirmed
by the observation that bone-sialoprotein-knockout mice
present hypo-mineralized bone, a reduction in the size of
their long bones and aberrant levels of osteoblast markers
[34]. Osteoblasts also synthesize cytokine interleukin-1
(IL-1) and interleukin-6 (IL-6), which control bone cells
in an autocrine and/or paracrine manner. Various in vitro
studies of human and murine osteoblastic cell lines suggest
that IL-1 can affect proliferation, collagen and osteocalcin
synthesis and alkaline phosphatase production [30, 40].
Osteoblasts express receptors for various hormones
including parathyroid hormone (PTH) [51], 1,25 (OH)2D3
[43], estrogenes [25], wchich are involved in the regulation
of osteoblast differentiation and activity. Vitamin D3 is
able to modulate the methabolic activity of osteoblasts
through the activation of a series of Vitamin- D-responsive
genes that reflect a more mature osteoblast phenotype.
CONTROL OF BONE REMODELING BY
OSTEOBLASTS: THE ROLE RANKL-RANKOPG-SYSTEM AND OF THE OSTEOCLAST
DEVELOPMENT
In recent years it has become evident that osteoblasts
have a global role in orchestrating the bone remodeling
process. Their function is not restricted solely to bone
formation, but it is now firmly established that they are
responsible for initiating bone resorption In cellular terms,
apart from forming the mineral and organic extracellular
compartment of bone, the osteoblast provides the essential
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Crimean Journal of Experimental and Clinical Medicine 2013 Volume 3 N 1-2
M-CSF and RANKL is sufficient for human, mouse, and
rat multinucleated osteoclast formation in vitro [22, 65].
Although RANKL is critical for osteoclast formation and
activation, a series of complementary studies has revealed
a number of additional gene products that are necessary for
osteoclastogenesis and a variety of hormones and cytokines
that modulate osteoclast formation [24, 40]. Deletion of the
genes for M-CSF, c-fos, RANK and NF-kB results in absent
osteoclast formation [31] confirming their requirement for
osteoclastogenesis. Osteoclasts are formed in mice whom
the genes for TRAF6 (TNF receptor activating factor 6)
and the c-fos have been deleted; however, these osteoclasts
exhibit defects in bone resorption resulting in osteopetrosis
[9]. Interestingly, another TRAF6 knockout mice exhibits
defective osteoclastogenesis [20]. TRAF6 activates the
MAP kinase cascade, and eventually activates JNK, JKK
and N-kB have been directly implicated in the response
to RANKL (Figure 2) [9]. Different domains of TRAF6
modulate both the initial differentiation and subsequent
maturation of osteoclasts bz activating various kinase
cascades. RANKL also activates NF-kB in osteoclasts (see
Figure 2), in large part via TRAF stimulation of Ik kinase
(IKK) to phosphorylate IkB, which then dissociates from
NF-kB, and permits NF-kB translocation into the nucleus
and subsequent binding to NF-kB responsive genes. TNF-α
also acts to induce osteoclast formation and activation in
concert with RANKL via the TNF receptor and TRAF2/6
and subsequently to activate NF-kB signaling [20].
Figure 2. RANKL-RANK-OPG system and regulation of osteoclast
precursor by osteoblast. Under physiologic condition, RANKL
produced by osteoclasts binds to its receptor RANK on the surface
of osteoclast precursors and recruits the adaptor proteinTRAF6,
leading to NF-kB activation and translocation to the nucleus. NFkB increases c-Fos expression and c-Fos interacts with NFATc1 to
trigger the transcription of osteoclastogenic genes. OPG inhibits
the initiation of the process by binding to RANKL .Abbreviations:
NFATc1, nuclear factor of activated T cells; NF-kB, nuclear factor
– kB; OPG, osteoprotegerin; RANKL, receptor activator of nuclear
factor- kB ligand; TRAF, tumor necrosis factor receptor associated
factor.
OSTEOCLAST AND BONE RESORPTION
The development of an in vitro bone resorption model
using isolated primary osteoclasts and mineralized bone
matrix as a substrate almost twenty years ago provided
an excellent system for detailed cell biological studies of
bone resorption [3, 58]. Although this model has several
limitations in attempts to study the whole physiological
cascade of bone resorption, it provides an excellent tool
for detailed studies of the cellular mechanisms involved
in the destruction of mineralized bone matrix. The
sequence of cellular events needed for bone resorption is
called the resorption cycle. Resorption requires cellular
activates : migration of the osteoclast to the resorption
site, its attachment to bone, polarization and formation of
new membrane domains, dissolution of hydroxyapatite,
degradation of organic matrix, removal of degradation
products from the resorption lacuna, and finally either
apoptosis of the osteoclasts or their return to the nonresorbing stage. The term resorption cycle covers neither
the differentiation pathway nor the cellular activities
needed for the fusion of mononuclear precursorto form the
multinuclear mature osteoclast. It should not be mistaken
for the more wiedely used term remodeling cycle, which
is used to describe the bone remodeling at the tissue level
that involves the activities of several different cell types.
surroundings (Figure 3).The molecular interactions
between the plasma membrane and the bone matrix at the
sealing zone are still unknown. Several lines of evidence
have shown, however, that integrins play an important
role in early phases of the resorption cycle [63, 70]. At last
four different integrins are expressed in osteoclasts: αvß3,
αvß5, α2β1 and αvß1 [70]. The role of αvß3 has received
much attention, because antibodies against αvß3, as well
as argynine-glycine- aspartic acid ( RGD)-containing
peptides such as echistation and kistrin, are defective
inhibitors of bone resorption both in vitro and in vivo [12].
Αvß3 is highly expressed in osteoclasts and is found bot hat
the plasma membrane and in various intracellular vacuoles.
However, the precise function of αvß3 in resorbing
osteoclasts remains unknown; the integrin could play a
role both in adhesion and migration of osteoclasts and in
endocytosis of resorption products. The latter possibility
is supported by the observation that high amount of αvß3
are present at the ruffled border and by recent data from
receptor-binding assays showing that denatured type I
collagen has a high affinity for αvß3 [70]. Some authors
have suggested that αvß3 integrin also mediates the
attachment of the sealing zone to the bone matrix [63, 70].
Previous ultrastructural studies indicated that resorbing
osteoclasts are highly polarized cells [5]. Current data
OSTEOCLAST ATTACH TO BONE MATRIX
THROUGH THE SEALING ZONE
After migration of the osteoclast to a resorption site, a
specific membrane domain, the sealing zone, forms under
the osteoclast. The plasma membrane attached tightly
to the bone matrix and seals the resorption site form its
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Review article
Before proteolytic enzymes can reach and degrade
collagenous bone matrix, tightly packed hydroxyapatite
crystals must be dissolved. It is now generally accepted
that the dissolution of mineral occurs by targeted secretion
of HCl through the ruffled border into the resorption
lacuna [13]. This is an extracellular space between the
ruffled border membrane and the bone matrix, and is
sealed from the extracellular fluid by the sealing zone.
The low pH in the resorption lacuna is achieved by the
action of ATP- consuming vacuolar proton pumps both at
the ruffled border membrane and in intracellular vacuoles.
A model of osteoclast ion transport activities involved
in the acidification of bone surface is shown in Figure
3. Osteoclasts attach to bone and form a circumferential
sealing zone that isolates the bone resorption compartment
from the extracellular space. Osteoclast plasma membrane
within the sealing zone develops into the ruffled border. The
observation that NH4Cl reversibly inhibits bone resorption
by osteoclasts indicates that the resorption compartment
is acidic and that the sealing zone is impermeant to H +
and NH+4. The osteoclast cytoplasm is rich in carbonic
anhydrase [16], proving a continuous supply of protons and
bicarbonate. Protons are transported across this membrane
into the bone resorption compartment by vacuolar-type H+ATPase (V-type ATPase) [50, 67]. Chloride ions passively
follow the protons through conductive anion channels
[48]. The combined activities of the proton pump and
chloride channel acidify the resorption compartment and
alkalinize the cytoplasm. Bicarbonate exits the cell into the
extracellular space in exchange for chloride via a basolateral
electroneutral anion exchanger, correcting the cytoplasmic
alkalinization and compensating for cytoplasmic chloride
loss [11]. The net result of these coordinated transport
activities is the transcellular movement of HCl into the
bone resorption compartment. This model predicts that both
the ruffled border proton pump and chloride channel play
key roles in bone resorption. The proton pump provides the
proton-motive force necessary to generate a pH gradient.
However, the pump is electrogenic. The chloride channel
shot-circuits the electrogenic pump and allows maximal
proton transport. It follows that limitation of the chloride
conductance could inhibit acid transport independently of
the intrinsic activity of the proton pump. Analogous to a
current model for regulation of the pH of some intracellular
organells, regulation of the anion conductance rather than
proton pump activity could be the key point at which the
rate of osteoclast acid transport, and hence bone resorption,
is governed. Thus, molecular characterization of the
ruffled border chloride channel may provide insight into
regulation of osteoclast bone resorption and could define a
pharmacological target for the treatment of metabolic bone
disease [6, 67]. The osteoclast proton pump is sensitive
to bafilomycin A1, which also effectively inhibits bone
resorption both in vitro and in vivo [42]. The recent finding
that vacuolar ATPase at the ruffled border contains cellsoecific subunits has further encouraged development
of resorption inhibitors that inhibit the osteoclast proton
pump [47]. Protons for the proton pump are produced by
cytoplasmic carbonic anhydrase II, high levels of which are
synthesized in osteoclasts [39]. In order to generate protons,
the presence of carbonic anhydrase II (CA II) is essential. It
Figure 3. The mechanisms of osteoclastic bone resorption: several
transport systems including the H+-ATPase proton pump, Cl-/HCO3exchanger and chloride channel are responsible for the acidification
in the osteoclastic resorption lacune. The osteoclast attaches to bone,
which promts formation of a convoluted ruffled membrane and a
resorptive microenvironment beneath the cell. Hydrocarbonic acid,
the product of a vacuolar-type H+-ATPase and charge-coupled CLchannel concentrated in the ruffled membrane, is secreted, resulting
in mineral dissolution. Vesicles containing acidic collagenolytic
enzymes in the form of cathepsins K, fuse with the bone-apposed
membrane, leading matrix degradation. Intracellular pH balance is
maintained by a passive Cl-/HCO3- exchanger on the contra-resorptive
surface of the cell. In the right corner: this figure summarized current
information and hypotheses regulating the role of avß3-integrin in
osteoclast formation, adhesion, polarization and migration. The
natural ligand for avß3-integrin is not known, however osteopontin
and bone sialoprotein are two RGD (arginine-glycine-aspartate)
containing proteins which could potentially be ligandes. See text for
future details.
suggest that resorbing osteoclasts contain not only the
sealing zone but also at least three other specialized
membrane domains: a ruffled border, a functional secretory
domain and a basolateral membrane [51, 56, 58]. As
the osteoclast prepares to resorb bone, it attaches to the
bone matrix through the sealing zone and forms another
specific membrane domain, the ruffled border. The ruffled
border is a resorbing organelle, and it is formed by fusion
of intracellular acidic vesicles with the region of plasma
membrane facing the bone [31, 58, 59]. During this fusion
process much internal membrane is transferred, and forms
long, finger-like projections that penetrate the bone matrix.
The characteristics of the ruffled border to not match
those of any other plasma membrane domain described.
Although facing the extracellular matrix, it has several
features that are typical of late endosomal membranes.
Several late endosomal markers, such as CIC-7, V-type H+ATPase, are densely concentrated at the ruffled border [50].
BONE MATRIX IS DEGRADED IN THE
RESORPTION LACUNA
The main physiological function of osteoclast is
degrade mineralized bone matrix [21, 53]. This involves
dissolution of crystalline hydroxyapatite and proteolytic
cleavage of the organic matrix, which is rich in collagen.
41
Crimean Journal of Experimental and Clinical Medicine 2013 Volume 3 N 1-2
catalyzes the conversion of H2O and CO2 into H2CO3, which
then is ionized into H+ and HCO-3 [45]. Mutation in CA II
can cause osteopetrosis due to non-functional osteoclasts
[35]. The HCO-3 ions are exchanged for Cl- through
an anion exchanger, membrane transport protein AE2,
located in the basolateral membrane, leading to continued
of Cl- for acidification of the resorption lacuna [37, 42].
After solubilization of the mineral phase, several
proteolytic enzymes degrade the organic bone matrix,
although the detailed sequence of events at the resorption
lacuna is still obscure. Two major classes of proteolytic
enzymes, lysosomal cysteine proteinases and matrix
metalloproteinases (MMPs) have been studied most
extensively. Osteoclasts produce proteases, of which
cysteine proteinase cathepsin K has provent to be the
most important [17], aiding the degradation of the organic
bone matrix. Eleven different types have been described
(B, C, F, H, K, L and other) with cathepsin K being the
most important with respect to bone remodeling, since it
is a protease with intense collagenase activity, especially
with respect to acid pH, which is essential to dissolve
calcic hydroxyapatite, the main mineral component of
bone. It degrades the two types of collagen, I and II and is
predominantly expressed in osteoclasts [28]. Cathepsin K
gives rise to specific degradation products-like C-terminal
cross-linking telopeptide of type I collagen (CTX-I),
which can be used for measurements of bone resorption
[18]. The role of cathepsin K in bone resorption was
determined using evidence from an autosomal recessive
osteochondrodysplasia named pycnodysostosis, a very
rare disease characterized by high bone mineral density,
acroosteolysis of the distal phalanxes, shot stature, and
cranial deformaties with late closing of the fontanelles [68].
Studies in mice submitted to nonfunctional mutations of
cathepsin have given rise to different models of osteopetrosis
[17, 33]. Matrix in bone resorption [16, 60], during which,
MMP activity is known to give rise to a specific degradation
fragment, C-trminal telopeptide of type I collagen (ICTP)
[7]. After matrix degradation, the degradation products are
removed from the resorption lacuna through a transcytotic
vascular pathway from the ruffled border to the functional
secretory domain, where they are liberated into the
extracellular space. Quantitative data are still missing, but
clear large amounts of degraded extracellular material must
be transported through the resorbing cell, given that the
volume of the resorption pit can easily exceed the volume
of the entre cell. The extent to which the degradation of
collagen and other matrix components is extracellular
and the extent to which this takes place in intracellular
transcytotic compartments are not known.Recent results
have suggested that tartrate-resistant acid phosphatase
(TRAP), a widely used osteoclast marker, is licalised
in the transcytotic vesicles of resorbing osteoclasts, and
that it can generate highly destructive reactive oxygen
species able to destroy collagen. This activity, together
with the co-localisation of TRAP and collagen fragments
in transcytotic vesicles, suggests that TRAP functions in
furtger destruction of matrix-degradation products in the
transcytotic vesicles. The observed mild osteopetrosis
in TRAP-knockout mice support this hypothesis [7].
CONCLUSIONS
Bone remodeling is a complex process involving a
number of cellular function directly towards to co-ordinated
resorption and formation of new bone. Bone is a rise source
of growth factors with important actions in the regulation of
bone formation and bone resorption. Frequently, these local
factors (GFs) are synthesized by skeletal cells, although
some cytokine RANK-RANKL-OPG system are secreted
by stromal cells. This factors regulate the synthesis,
activation and direct action on cellular metabolism,
and they modify the replication and differentiated
function of cells of the osteoclast and osteoblast lineage.
The rapidly accumulating new knowledge about the
multiple possible regulatory mechanisms withing bone
should aid the understanding of physiological bone
remodeling and also offer potential explanations for
the change in bone turnover seen in a variety of disease
states. This knowledge will be important in devising
new therapeutic strategies to control bone formation and
resorption based upon these novel regulatory mechanisms.
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