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22UDC 616-001.5:616.71-003.93
BONE REMODELING: CELLULAR-MOLECULAR BIOLOGY AND CYTOKINE RANK-RANKL-OSTEOPROTEGERIN (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.
Key words: osteoblasts, osteoclasts, bone remodeling, RANK-RANKL-OPG cytokine system, growth factors.
Department of Orthopaedic Clinic Median, Bad Lausick, Germany. Correspondence:
Parkstraße 4, 04651 Bad Lausick Deutschland e-mail:
stanislav. [email protected]
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.
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
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
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-B (TGF-B), 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 Role in bone remodeling
TGF-ß 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. FGF Osteoblast recruitment and proliferation.
IGF Osteoblast proliferation; bone matrix synthesis; bone resorption.
VEGF Osteoblast proliferation and differentiation; conversion of cartilage into bone
PDGF Osteoprogenitor migration, proliferation and differentiation; osteoclastogenesis.
None: TGF-B - transforming growth factor - B; FGF - fibroblast growth factor; IGF - insulin-like growth factor; VEGF - vascular
endothelial growth factor; PDGF - platelet-derived growth factor.
Transforming growth factor-fi1 (TGF-fi1). Transforming growth factor-B1 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-B1 (transforming growth factor-B1 to transforming factor-B5). Most cells synthesize and respond to TGF-B, but high levels are found in bone, platelets and cartilage. TGF-B1 is the most abundant isoform at the protein level (for a recent comprehensive review see Arvidson et al. [1]). The activation of TGF-B1 is highly regulated, and once activated it interacts with transmembrane serine/threonine kinase receptors. Binding TGF-B1 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,
providing a pool of early osteoblasts. In contrast, during later phases of osteoblast differentiation, transforming growth factor- B1 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-B1 concentrations. Additionally, TGF-B1 inhibits the expression of the RUNX-2 and osteocalcin genes, whose expression is controlled by Cbfa-1/RUNX-2 in osteoblast-like cell lines [14]. Likewise, TGF-B1 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-B1 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-B1 during bone regeneration and wound healing and to determine the inter-relationship between TGF-B1 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
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-nucleotide-binding 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/JnK-cJun, 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/ 1KB - 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 - guanosin-nucleotide-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
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 stage-specific 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-M 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 osteoblast-associated 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
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-B1 [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-K-dependent 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-RANK-
OPG-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
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-a (TNF-a) and interleukin-1 (Il-1). 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-B (TGF-B) 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
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-a 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].
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 non-resorbing 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.
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
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. NF-kB increases c-Fos expression and c-Fos interacts with NFATcl to trigger the transcription of osteoclastogenic genes. OPG inhibits the initiation of the process by binding to RANKL .Abbreviations: NFATcl, 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.
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: avB3, avB5, a2p1 and avM [70]. The role of avfi3 has received much attention, because antibodies against avB3, 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]. AvB3 is highly expressed in osteoclasts and is found bot hat the plasma membrane and in various intracellular vacuoles. However, the precise function of avB3 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 avB3 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 avB3 [70]. Some authors have suggested that avB3 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
Figure 3. The mechanisms of osteoclastic bone resorption: several transport systems including the H+-ATPase proton pump, CT/HCO3-exchanger 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 CL-channel 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 avB3-integrin in osteoclast formation, adhesion, polarization and migration. The natural ligand for avB3-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.
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 cell-soecific 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 [3 9]. In order to generate protons, the presence of carbonic anhydrase II (CA II) is essential. It
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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