Regulation of Osteoclast Function

Osteoclastic bone resorption consists of several complicated processes: osteoclast development, attachment of osteoclasts to calcified tissues, development of a ruffled border and clear zone, followed by the secretion of acids and lysosomal enzymes into the space beneath the ruffled border. During the past decade, several new approaches have been taken to investigate osteoclast function. Highly purified functionally active osteoclasts can be isolated, which allows osteoclast function to be studied biochemically. The mechanisms of reduced bone resorption have been elucidated in several types of congenital osteopetrotic mutant mice. For example, the important role of macrophage-colony stimulating factor (M-CSF, colony-stimulating factor 1) in osteoclast development has been established in the osteopetrotic (op/op) mutant mice. Another osteopetrotic mutant mouse model has been generated by targeted disruption of the proto-oncogene c-src. Studies using this model showed that tyrosine kinase–mediated signals are essentially involved in osteoclast function. Using a purified osteoclast preparation, we showed that osteoclast function was activated by osteoblasts through a mechanism involving cell-to-cell contact. On the basis of these new approaches and experimental findings, we examined several fundamental issues regarding osteoclastic bone resorption. We considered how osteoclasts form ruffled borders and clear zones, which signals are necessary for osteoclast polarization, which mechanisms regulate the life span of osteoclasts, how calcitonin and bisphosphonates inactivate osteoclast function, and how osteoblasts regulate osteoclast function. In this article, we review the regulatory mechanisms of osteoclast function mainly on the basis of the experimental results obtained in our murine culture system. Multinucleated osteoclasts responsible for bone resorption are derived from hemopoietic cells of the monocyte-macrophage lineage.1-4 The progenitor cells are recruited from the hemopoietic tissues such as bone marrow and splenic tissues to bone via the circulating blood stream. They proliferate and differentiate into osteoclasts through a mechanism involving cell-to-cell interaction with osteoblastic stromal cells.4 We have developed a mouse marrow culture system and a coculture system of mouse osteoblastic cells and hemopoietic cells in which osteoclast-like multinucleated cells are formed in response to bone-resorbing factors such as 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3), parathyroid hormone (PTH), prostaglandin E2 (PGE2), and interleukin-6 (IL-6) plus soluble IL-6 receptors.4 The multinucleated cells formed in the mouse culture systems satisfied major criteria of osteoclasts such as tartrate-resistant acid phosphatase (TRAP, a marker enzyme of osteoclasts) activity, calcitonin receptors, p60c-src, vitronectin receptors (αvβ3), and bone-resorbing activity. Recent evidence has suggested that those bone-resorbing hormones and cytokines commonly and directly act on osteoblastic cells for osteoclast development in cocultures of mouse osteoblastic and spleen cells.4, 5 Osteoclasts have several unique characteristics for resorbing bone. The most characteristic morphological features of osteoclasts are the presence of ruffled borders and clear zones.1-4 The clear zone serves in the attachment of osteoclasts to the bone surface. The resorbing area under the ruffled border of osteoclasts is acidic, which favors dissolution of bone mineral. Protons are provided by carbonic anhydrase II in bone-resorbing osteoclasts.6 The vacuolar type proton ATPase exists in the ruffled border membranes of osteoclasts.7, 8 The transport of protons into resorption lacunae is mediated by this vacuolar type proton ATPase. Lysosomal enzymes of osteoclasts are also secreted into a resorbing area to degrade the organic matrix of bone. Cysteine proteinases such as cathepsin K (OC-2)9, 10 and metalloproteinases (MMPs) such as MMP-911 have been reported to be present in osteoclasts, which degrade type I collagen of demineralized bone in an acidic condition. To organize highly polarized cytoplasmic structures such as ruffled borders and clear zones, osteoclasts must adhere to the bone surface. Therefore, bone matrix components were considered essential for inducing osteoclast polarization. However, Jones et al.12, 13 reported that osteoclasts could resorb not only bone and dentine, but also enamel, avian egg shells, and oyster shells. Chambers et al.14 also reported that rabbit osteoclasts could form resorption pits on inorganic bone in the presence of serum. Moreover, Silver et al.15 demonstrated that the space between firmly attached osteoclasts and glass cover slips is maintained at a lower pH than that of culture medium. These findings suggest that bone matrix components are not necessarily required for osteoclasts to obtain polarity and exhibit bone-resorptive activity. Bearing these findings in mind, we examined the factor(s) that would be essential for inducing osteoclast polarization, in particular those required for the formation of the ringed structure consisting of F-actin dots (actin ring) in osteoclasts. Actin rings are thought to correspond to clear zones in bone-resorbing osteoclasts in vivo. Many investigators have observed that osteoclasts exhibit actin rings during resorption.16-19 Moreover, the disruption of actin rings by calcitonin, dibutyryl cyclic adenosine monophosphate (cAMP), and various other inhibitors suppresses the pit-forming activity of osteoclasts.20-23 In this sense, the actin ring formation in osteoclasts is closely related to the polarity and bone-resorbing activity of osteoclasts. When osteoclasts formed in cocultures of mouse osteoblastic cells and bone marrow cells are placed on a plastic dish, calcified dentine, or calcium phosphate in the presence of fetal bovine serum (FBS), osteoclasts similarly form actin rings.24 However, osteoclasts placed on demineralized dentine or type I collagen gel matrix do not form actin rings even in the presence of FBS. This suggests that the polarization of osteoclasts is substrate-dependent and that bone matrix components are not necessarily required for osteoclasts to obtain polarity. Actin ring formation is also induced when osteoclasts are placed on plastic dishes coated with vitronectin, fibronectin, or type I collagen in the absence of FBS, but not on those coated with laminin, poly-L-lysine, or bovine serum albumin. The GRGDS, but not the GRGES, peptide inhibits actin ring formation in a dose-dependent manner.24 Osteoclasts express abundant αvβ3 vitronectin receptors.25, 26 Recognition of proteins containing the RGD sequence by osteoclasts through vitronectin receptors appears essential for inducing osteoclast polarization. An immunoelectron microscopic examination revealed that vacuolar proton ATPase, a marker enzyme of ruffled borders, is localized along the apical membrane in a much higher density than the basolateral membrane in osteoclasts placed on plastic dishes.24 In osteoclasts placed on a collagen gel, however, vacuolar proton ATPase distributes throughout the cytoplasm without polarity, even in the presence of FBS. These findings again suggest that some proteins containing the RGD sequence are essential but not sufficient for osteoclast polarization, and that some physical properties of the matrix components, such as rigidity, hardness, or roughness, may be necessary. Chambers et al.14 reported that osteoclasts form resorption pits on bone but not on demineralized bone or on osteoids. These results suggest that the attachment of osteoclasts to some proteins containing the RGD sequence present in bone through αvβ3 integrins is the initial step to forming a bone-resorbing apparatus, such as clear zones and ruffled borders. In addition to the RGD sequence, some physical properties of bone, such as rigidity and hardness, appear necessary to induce the polarization of osteoclasts (Fig. 1). The mineral components may also be important to maintain the polarized state of osteoclasts. Further studies are necessary to elucidate how integrins, physical properties, and mineral components relate to each other to induce and maintain osteoclast polarization. In 1991, Soriano et al.27 reported that the targeted disruption of c-src in mice induces osteopetrosis. Soon after that, it was revealed that osteoclasts express high levels of p60c-src28, 29 and that osteoclasts from p60c-src-deficient mice have defects in ruffled border formation.30, 31 These results indicated that the signaling mediated by p60c-src tyrosine kinase is involved in the polarization of osteoclasts. Actually, herbimycin A, an inhibitor of tyrosine kinases, inhibits osteoclastic bone resorption in vivo and in vitro.22, 32, 33 Since then, many investigators including our group have explored the downstream signal transduction of p60c-src for osteoclast polarization. Focal adhesion kinase (p125FAK), a tyrosine kinase found at focal adhesions of normal cells, was originally identified as one of the tyrosine-phosphorylated proteins in chick embryo fibroblasts transformed by v-src or activated variants of c-src.34 Berry et al.35 and Tanaka et al.22 independently showed that p125FAK is expressed in osteoclasts and possibly involved in osteoclastic bone resorption. However, no osteopetrotic changes in bone have been identified in p125FAK gene knockout mice. Therefore, proteins other than p125FAK appear to be the target of p60c-src tyrosine kinase in osteoclasts. The product of the proto-oncogene c-cbl is a 120 kD protein (c-Cbl) that is tyrosine phosphorylated in response to the activation of various signaling pathways and in v-src–transformed cells.34 Tanaka et al.38 reported that the level of tyrosine phosphorylation of c-Cbl immunoprecipitated from c-src–deficient osteoclasts is strikingly reduced, compared with that found in wild-type osteoclasts. c-Cbl and p60c-src associate with each other and colocalize in osteoclasts in vivo. Antisense oligonucleotides for c-Cbl as well as p60c-src inhibit osteoclastic bone resorption.38 These results indicate that c-Cbl may act downstream of p60c-src in a signaling pathway required for osteoclast function. The postulated polarization process of osteoclasts. Attachment of osteoclasts to some proteins containing the RGD sequence in the extracellular matrix through vitronectin receptors induces the formation of clear zones and ruffled borders in osteoclasts. Osteoclasts placed on demineralized dentine or type I collagen gels do not polarize even in the presence of FBS, in which abundant RGD sequence–containing proteins are present.24 In addition to proteins containing RGD, some physical properties of the substrate such as rigidity or hardness are required to induce osteoclast polarity.24 Several signaling molecules such as p60c-src, p125FAK, c-Cbl, p130Cas, PI-3 kinase, and rho p21 are involved in the intracellular processes of osteoclast polarization. The possible roles of each molecule are described in the text. The novel signaling molecule, p130Cas (Cas, Crk associated substrate), is highly phosphorylated on tyrosine residues and is stably associated with the oncogene products p47v-crk and p60v-src.39 We found that a 130 kD protein is tyrosine phosphorylated in association with actin ring formation in osteoclasts, and we immunologically identified it as p130Cas.40 This protein localizes mainly in the peripheral region of osteoclasts placed on plastic dishes and colocalizes with F-actin. Incubating osteoclasts with cytochalasin D, an inhibitory agent of actin polymerization, induced the disruption of actin rings, which resulted in a marked decrease in tyrosine phosphorylation of p130Cas in osteoclasts.40 These results suggest that p130Cas is a possible signaling molecule downstream of the tyrosine kinases associated with polarization of osteoclasts. Schwartzberg et al.41 generated transgenic mice that express wild-type and mutated versions of c-src under the control of the promoter of TRAP and crossed them into the p60c-src–deficient background. They found that complementation of the defect does not require kinase activity but does require intact SH2 and SH3 domains of p60c-src. These results suggest that the tyrosine kinase activity of p60c-src is not important for osteoclast function. Alternatively, other c-src family tyrosine kinases could be substituted for the portion of kinase activity, but not for the SH2 and SH3 domains of p60c-src in osteoclastic bone resorption. Phosphatidylinositol (PI)-3 kinase is a key enzyme activated in the signaling pathways of growth factors, and it catalyzes phosphorylation of PI to PI-3-P, PI-4-P to PI-3,4-P2, and PI-4,5-P2 to PI-3,4,5-P3, respectively.42 The role of PI-3 kinase has been studied extensively over the past few years. Accumulating evidence suggests that PI-3 kinase is involved in several aspects of intracellular membrane trafficking.43, 44 We examined the role of PI-3 kinase in osteoclastic bone resorption using wortmannin, a selective inhibitor of PI-3 kinase.23 PI-3 kinase is present preferentially along the ruffled border membrane and the limiting membrane of associated intracellular vacuoles in rat authentic osteoclasts. Wortmannin dose-dependently inhibits the pit-forming activity of osteoclasts and disrupts actin rings accompanied by a decrease in the PI-3 kinase activity.23 Electron microscopic analysis revealed that osteoclasts incubated with wortmannin do not form ruffled borders, and numerous electron lucent vacuoles with differing sizes appear throughout the cytoplasm.23, 45 Hall et al.46 also showed that wortmannin inhibits the pit-forming activity of isolated rat osteoclasts. Hruska et al.47 reported that the binding of avian osteoclast precursors to osteopontin stimulates PI-3 kinase activity in immunoprecipitates formed by antibodies against integrin αvβ3. Taken together, these results indicate that PI-3 kinase may play an important role in the process of ruffled border formation in osteoclasts, probably in the trafficking of intracellular vacuoles.45 Rho protein (rho p21), a p21ras-related small guanine nucleotide binding protein, regulates cytoskeletal organization in several types of cells.48, 49 Clostridium botulinum-derived ADP-ribosyltransferase (C3 exoenzyme) specifically ADP-ribosylates rho p21 at Asn 41 and renders it functionally inactive.50 We examined the involvement of rho p21 in osteoclastic bone resorption using the C3 exoenzyme.20 The C3 exoenzyme added to the culture of osteoclasts placed on dentine slices disrupted actin rings and inhibited pit formation by osteoclasts in a dose-dependent manner. The amount of unribosylated rho p21, the activated form of rho p21, in the osteoclasts was concomitantly decreased. Microinjecting C3 exoenzyme into osteoclasts placed on culture dishes completely disrupts actin rings within 20 minutes.20 These results suggest that rho p21 also plays an important role in bone resorption by regulating the cytoskeletal organization in osteoclasts. Thus, several signal transducing molecules appear to be involved in osteoclast polarization (Fig. 1). Genetic and pharmacological experiments indicate that p60c-src and other tyrosine kinases, rho p21, PI-3 kinase, and possibly c-Cbl, may be involved in osteoclast polarization. Other molecules such as p125FAK and p130Cas may also be implicated in osteoclast activation, but their roles remain to be determined. At present, the hierarchy of these signaling molecules involved in osteoclast polarization is unknown (Fig. 1). Further studies will elucidate the role of each signaling molecule in the polarization process of osteoclasts. Osteoclasts are terminally differentiated cells with a limited life span. Isolated murine osteoclasts placed on culture dishes survive for no longer than a week. Time course studies on osteoclast formation in murine bone marrow cultures or cocultures showed that most osteoclasts formed in these cultures disappear within 5 days.4 Two cytokines, IL-151 and M-CSF,51, 52 act on mature osteoclasts directly and elongate their life span in culture. In contrast, estrogen stimulates apoptosis of osteoclasts, possibly mediated by transforming growth factor-β (TGF-β).53 IL-1 is a potent osteoclast-activating factor that promotes bone resorption both in vitro and in vivo.54-56 Thomson et al.57 first reported that IL-1 stimulates pit-forming activity of isolated rat osteoclasts through a soluble factor(s) secreted by osteoblasts. However, recent evidence indicates that osteoclasts are also the direct target cells of IL-1. Yu and Ferrier58 reported that IL-1 acts directly on isolated rabbit osteoclasts and increases intracellular [Ca2+] levels. We also showed that IL-1 prolongs the viability of purified osteoclasts.51 When ST2 cells were removed from the cocultures by dispase to purify osteoclasts, more than 80% of the purified osteoclasts detached from culture dishes within 24 h. Of various soluble factors examined, IL-1 and M-CSF stimulated osteoclast survival (Fig. 2).51 Moreover, IL-1 activated NF-κB–like factor, an important mediator of IL-1–induced gene transcription, in purified osteoclasts within 5 minutes.59 By means of in situ hybridization, Xu et al.60 identified mRNA expression of type I (IL-1RI) and type II receptors (IL-1RII) for IL-1 in murine and rat osteoclasts in normal bone and in rat osteoclasts in inflammatory bone tissues. These results suggest that the direct effect of IL-1 on osteoclasts is an important mechanism by which IL-1 mediates physiological and pathological bone resorption. M-CSF is a crucial factor involved in osteoclast development, demonstrated by the discovery of a point mutation of the gene encoding M-CSF in osteopetrotic (op/op) mice.61-63 Immunocytochemical and in situ hybridization studies revealed that mature osteoclasts as well as their mononuclear precursors have abundant M-CSF receptors (c-Fms, a receptor-type tyrosine kinase).64 This suggests that M-CSF plays important roles not only in osteoclast development but also in osteoclast function. Effects of osteotropic hormones and cytokines on the survival of osteoclasts. ST2 cells and bone marrow cells were cocultured in the presence of 1α,25(OH)2D3 (10−8 M) and dexamethasone (10−7 M) for 10 days.51 Cocultures were then with dispase to ST2 cells from the cocultures The purified osteoclasts were incubated for 24 with various factors including 1α,25(OH)2D3 (10−8 calcitonin (10−7 IL-6 M-CSF or IL-1 and for TRAP were and after the culture for 24 and the osteoclasts were osteoclasts were also in the cocultures without dispase The results are expressed as means of et reported that M-CSF inhibits pit formation by isolated rat osteoclasts. This M-CSF was as an essential factor for osteoclastic bone resorption. et reported that M-CSF strikingly stimulates osteoclastic and and that it osteoclast survival by They also demonstrated that the of M-CSF on bone resorption is not to the of resorption by osteoclasts but to the of osteoclasts that are resorbing with these findings, we showed that M-CSF added to purified osteoclasts prolongs their life span (Fig. 2).51 Therefore, tyrosine kinase–mediated signals appear to be involved in the and the survival of mature osteoclasts. is considered to be an important hormone that regulates bone not only in but also in of this by or of the results in a marked bone by osteoclastic bone Recent studies have suggested that cytokines such as IL-1 and IL-6 are involved in the bone resorption induced by the estrogen et also reported that estrogen promotes the apoptosis of murine osteoclasts in vivo and in increases the of by which also stimulates the apoptosis of osteoclasts in and osteoclast This indicates that estrogen may bone and after by the life span of osteoclasts through Taken together, these results indicate that the and of apoptosis of osteoclasts may also be important in regulating osteoclastic bone resorption. are with a high for and the to inhibit osteoclastic bone The are as in various associated with stimulated bone However, the mechanism by which bisphosphonates inhibit bone resorption has not been We examined the of on osteoclastic bone resorption using our culture not inhibit osteoclast formation induced by 1α,25(OH)2D3 in the and the osteoclasts from cocultures incubated with on collagen dishes formed as many resorption pits as those obtained from the control The of to inhibit osteoclast formation was in osteoclasts formed in the presence of other bisphosphonates such as and These results suggest that bisphosphonates do not osteoclast formation and that osteoclasts formed in a containing bisphosphonates can resorb bone. However, pit formation by osteoclasts was inhibited by directly added to the pit formation Osteoclasts placed on plastic dishes or dentine slices similarly formed actin rings osteoclasts) of the presence or absence of However, disrupted actin rings after were Osteoclasts placed on a collagen gel to form actin rings osteoclasts) and not to Osteoclasts formed from spleen cells developed actin rings but not ruffled borders on dentine Actin rings formed by spleen osteoclasts were not disrupted by added to the but when those of osteoclasts were These results indicate that bisphosphonates could polarized osteoclasts with the ringed structure of and ruffled borders (Fig. et reported that inhibits the activity of protein tyrosine phosphatase that is highly expressed in osteoclasts. The of other such as and phosphatase are not by We also found that osteoclasts with increases the tyrosine phosphorylation levels of several proteins in The levels to be to the of protein tyrosine by tyrosine kinase activity of the p60c-src of osteoclasts was not at by directly added to the tyrosine kinase stimulated of tyrosine-phosphorylated such as and growth factor inhibited of those induced by the osteoclast These findings indicate that bisphosphonates specifically into polarized osteoclasts may bone-resorbing activity by protein tyrosine other than of tyrosine have also been for the inhibitory of bisphosphonates on osteoclastic bone resorption. et reported that inhibited proton transport in membrane derived from osteoclasts, and the of to inhibit proton transport was higher than that of other bisphosphonates such as and is possible to that bisphosphonates may inhibit osteoclast function through et reported that the effect of bisphosphonates on bone resorption is mediated by their on osteoblasts. estrogen and bisphosphonates induce apoptosis of osteoclasts in vivo and in is a peptide hormone consisting of acids that inhibits osteoclastic bone resorption through receptors, which are expressed on the basolateral membrane of signal transduction pathways kinase intracellular and protein kinase may be involved in the of osteoclastic bone resorption by To how calcitonin inhibits osteoclast function, we examined morphological changes of the of disrupted actin rings and inhibited pit formation by osteoclasts placed on dentine also disrupted actin rings formed in spleen osteoclasts and induced the of F-actin even in osteoclasts placed on collagen This suggests that calcitonin osteoclasts of the presence or absence of their polarity (Fig. and dibutyryl which the of and also inhibit the pit-forming activity of osteoclasts, but the actin rings of osteoclasts. The and dose-dependent of calcitonin for the activation of in osteoclasts were to those for the disruption of actin Moreover, activated into osteoclasts disrupted actin rings within 10 These results suggest that than activation, is involved in the of calcitonin through the disruption of actin et reported that calcitonin promotes survival of rat osteoclasts in vitro on glass or bone by the of The survival effect of calcitonin is also by dibutyryl of the inhibitory of bisphosphonates and calcitonin on osteoclastic bone resorption. When osteoclasts formed in cocultures of normal murine osteoblastic cells and bone marrow cells were placed on plastic dishes or dentine formed actin rings osteoclasts placed on collagen gels to form actin rings Osteoclasts formed from spleen cells of mice developed actin rings but not ruffled borders on dentine This indicates that osteoclasts added to the culture disrupted actin rings in normal but not in osteoclasts placed on dentine disrupted actin rings of both normal and osteoclasts placed on dentine but not induced the of F-actin in osteoclasts placed on collagen These results indicate that bisphosphonates polarized osteoclasts, calcitonin osteoclasts of their polarity. The pathway may also regulate osteoclast function. increases calcium in the stably with the calcitonin An in the intracellular calcium in osteoclasts with disrupts actin rings within 10 These results suggest that signaling pathways other than are also involved in the of bone resorption. Further studies are necessary to elucidate how and are and to each other in osteoclasts and osteoclast function by osteoblasts may regulate osteoclast function, the mechanism has not been We developed a of highly and functionally active osteoclasts from cocultures of mouse osteoblastic cells and bone marrow Osteoclasts in the were on density Highly osteoclasts for 24 on dentine slices to form resorption When osteoblasts were the area of resorption pits was strikingly with the in the of osteoblasts the osteoblastic and and bone stromal and pit formation by osteoclasts. In contrast, the and and a to osteoclast function. When cell-to-cell between cells and osteoclasts was osteoclasts to form resorption pits (Fig. activity of purified authentic rat osteoclasts was also stimulated by These results suggest that osteoblasts are involved in not only osteoclast formation but also osteoclast activation through the mechanism of cell-to-cell (Fig. of cell-to-cell between cells and osteoclasts for osteoclast function. Highly osteoclasts were placed on dentine slices and incubated for 24 in the absence or presence of cells were directly added to a dentine on which osteoclasts been To osteoclasts from directly cells, the dentine on which osteoclasts been were placed a of cells in a culture cells were with and added with the from cells to the dentine on which osteoclasts been an of 24 dentine slices were with to resorption Many resorption pits were observed only in et isolated highly mononuclear from cocultures of bone marrow cells and osteoblastic cells using a containing The mononuclear showed pit-forming activity on bone slices when both osteoblastic cells and 1α,25(OH)2D3 were et also reported that cells pit-forming activity of osteoclasts through the mechanism of cell-to-cell contact. In et showed that stromal cells calcitonin formation in cocultures with spleen cells, but these calcitonin cells to form resorption pits on bone osteoblastic cells such as and cells to the bone resorption by calcitonin These results may indicate that osteoblasts directly the function of mature osteoclasts. In contrast, et reported that highly rabbit osteoclasts formed resorption pits on dentine without stromal hormones such as 1α,25(OH)2D3 and not pit-forming activity of purified rabbit osteoclasts in the absence of osteoblastic stromal The activity was by 1α,25(OH)2D3 only in the presence of rabbit stromal These results suggest that osteoblastic cells are involved in the activation of osteoclasts induced by osteotropic factors, some characteristics of osteoclasts formed in vitro may be from those of authentic osteoclasts in of the activation of osteoclast function. In our and to the pit forming activity of osteoclasts even in the presence of osteoblasts. cytokines, such as and which their through as a signal not pit-forming activity of osteoclasts. In showed no effect on pit formation by These results indicate that the signals mediated by may be important for osteoclast development but not for osteoclast function. of experiments by et demonstrated that avian and osteoclasts estrogen receptors, and estrogen directly acts on osteoclasts to inhibit their bone-resorbing activity. However, in our mouse estrogen from to showed no effect on the pit-forming activity of may be present between mouse osteoclasts formed in vitro and authentic osteoclasts from other of The osteoclasts formed in cocultures with 1α,25(OH)2D3 may have been activated during Further studies are to elucidate the mechanism by which osteoblasts osteoclast function and how osteotropic hormones and cytokines regulate the process of bone resorption. Osteoclasts are highly polarized cells that form ruffled borders and clear zones the bone surface. Attachment of osteoclasts to some proteins containing through vitronectin receptors is the first step involved in inducing osteoclast polarization. In addition to the RGD sequence, some physical properties of bone, such as rigidity, hardness, or roughness, appear to be required to induce polarity. Since the by Soriano et c-src knockout mice in 1991, the intracellular process for osteoclast polarization has been examined by many During the past 5 it has been that the signal transducing molecules described in this are involved in the of osteoclast function. However, how each signaling molecule with other molecules to induce or osteoclast polarization The targeted disruption of each molecule in mice will more on the of osteoclast function. Several osteotropic factors regulate the survival of osteoclasts. This suggests that the of the life span of osteoclasts is also closely related to their bone-resorbing activity. In addition to the that osteoblasts an osteoclast recent findings have a new that osteoblasts may bone resorption by osteoclasts through a mechanism involving cell-to-cell contact. are to investigate osteoclast function more studies on osteoclast function will to of the regulatory mechanism of bone resorption.