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Advances in osteoclast biology: old findings and new insights from mouse models
James R. Edwards and Gregory R. Mundy
Abstract | The maintenance of adequate bone mass is dependent upon the controlled and timely removal of old, damaged bone. This complex process is performed by the highly specialized, multinucleated osteoclast. Over the past 15 years, a detailed picture has emerged describing the origins, differentiation pathways and activation stages that contribute to normal osteoclast function. This information has primarily been obtained by the development and skeletal analysis of genetically modified mouse models. Mice harboring mutations in specific genetic loci exhibit bone defects as a direct result of aberrations in normal osteoclast recruitment, formation or function. These findings include the identification of the RANK–RANKL–OPG system as a primary mediator of osteoclastogenesis, the characterization of ion transport and cellular attachment mechanisms and the recognition that matrix-degrading enzymes are essential components of resorptive activity. This Review focuses on the principal observations in osteoclast biology derived from genetic mouse models, and highlights emerging concepts that describe how the osteoclast is thought to contribute to the maintenance of adequate bone mass and integrity throughout life.
Edwards, J. R. & Mundy, G. R. Nat. Rev. Rheumatol. 7, 235–243 (2011); published online 8 March 2011; doi:10.1038/nrrheum.2011.23

The osteoclast remains one of the most complex and fascinating cells of the body. These cells are destructive yet delicate, short-lived but highly active, modest in numbers but recognized as the only cell of the body capable of degrading and removing large quantities of bone. Although this osteolytic activity in itself is a remarkable achievement considering the complex mixture of organic and inorganic components that constitute the skeleton, other functions of this dynamic cell are beginning to emerge that have broadened our view of the osteoclast to more than a bone-degrading machine. Osteoclasts originate from the hematopoietic stem cell population and develop through the fusion of mono­ nuclear myeloid precursors (Figure 1). As such, mature osteoclasts are large, multinucleated cells located on trabecular and endosteal cortical bone surfaces, often in a resorption pit of their own making. A variety of factors, including tumor necrosis factor (TNF) superfamily ligands and inflammatory proteins from different cell sources, contribute to the formation and function of osteoclasts (Figure 2). Overexpression of such factors ultimately leads to increased bone loss through enhanced osteoclastogenesis or increased resorptive activity. The function of the osteoclast, to resorb bone, involves a complex process that is dependent on a variety of events, which culminates in the degradation of both the inorganic mineral and organic matrix (Figure 1). Once an area of bone becomes targeted for degradation and removal, the osteoclast will migrate and bind tightly to the surrounding
Competing interests The authors declare no competing interests.

matrix. Interactions between bone matrix proteins and integrins, podosomes and polarized actin filaments within the osteoclast create a sealed zone between the osteoclast and the bone surface, separate from the bone marrow cavity. Bone degradation occurs within this isolated area. Mature, bone-resorbing osteoclasts produce a cocktail of matrix-degrading enzymes, hydrogen ions and chloride ions, which are expelled into resorption lacunae through a highly permeable region of the osteoclast membrane known as the ruffled border (Figure 3). The low pH of this environment serves to dissolve the mineral component of bone while simultaneously inducing and enhancing the activity of enzymes that break down the organic matrix. Modifications in the genes encoding critical factors involved in osteoclast activity (such as the cellular compo­nents involved in ion generation and distribution) or random mutations leading to altered osteoclasto­genesis and differentiation, have dramatic effects on normal skeletal modeling and remodeling in a variety of mouse models and human conditions. Our understanding of osteoclast biology in normal and pathological conditions has benefitted immensely from the use of genetically modified animal models. The various skeletal phenotypes generated from lossof-function or gain-of-function mutations in specific genes have highlighted the important independent roles of crucial factors involved in osteoclast differentiation, fusion, function and apoptosis. This Review summarizes the foremost findings in osteoclast biology gained from these animal models, highlights some of the most important studies that have furthered our understanding of this dynamic cell and outlines the principal therapeutic

Institute of Musculoskeletal Sciences, University of Oxford, Nuffield Orthopedic Center, Windmill Road, Oxford OX3 7LD, UK (J. R. Edwards). Vanderbilt Center for Bone Biology, 1235 Medical Research Building IV, Vanderbilt University Medical Center, Nashville, TN 37232‑0575, USA (G. R. Mundy). Correspondence to: J. R. Edwards james.edwards@

© 2011 Macmillan Publishers Limited. All rights reserved

VOLUME 7  |  APRIL 2011  |  235

few macrophages in the peritoneal cavity and osteoclasts that were both morphologically smaller and fewer in number.1 has been implicated in the transcriptional regulation of CSF1R (which encodes the M‑CSF receptor). these studies indicate that PU. a mouse strain was identified that harbored a mutation in the Csf1 gene. such as cartilaginous remnants within trabecular bone and a failure of tooth eruption. such as transforming growth factor β and osteocalcin) into the marrow cavity.1deficient mice.14 Together.REVIEWS Key points ■■ RANK–RANKL–OPG signaling is an important mediator of osteoclast differentiation ■■ Transcriptional regulation of osteoclast formation involves NFκB and NFATc1 ■■ Osteoclast attachment to the bone surface is necessary for resorption to proceed ■■ Osteoclast activity is dependent upon effective ion transport across the cell membrane ■■ Matrix-degrading enzymes are essential for effective breakdown of organic bone ■■ Osteoclast-targeted therapies are the first-line treatment for excessive bone loss Osteoclast precursor Osteoclast differentiation and fusion CSF-1R RANK NFκB c-Fos NFATc1 Committed myeloid precursor PU. options that have evolved from these studies.8 This mutation results in an excessive accumulation of bone throughout the marrow cavities. which are essential for the normal differentiation of cells of the monocyte–macrophage lineage.9 Compared with their wild-type counterparts. micro-ophthalmia-associated transcription factor. Exposure to RANKL represents the primary stimuli for normal osteoclast formation and continued resorptive activity through the activation of the principal transcriptional mediators NFκB and NFATc1.1-deficient myeloid progenitors are capable of commitment to the monocytic lineage suggests that other unidentified molecules controlling later (or earlier) commitment are also involved. such as PU. All rights reserved . RANK. after which a cocktail of ions and matrixdegrading enzymes expelled into the sealed lacunae break down the inorganic and organic components of bone.nature.1 is central to the differentiation of hematopoietic pre­ cursors. NFκB.1-deficient animals. However. Osteoclast activity (bone resorption) Figure 1 | Osteoclast differentiation and function.12 and ITGAM (which encodes integrin αM. Mice deficient in PU. osteopetrotic Csf1op/op mice possessed reduced numbers of circulating monocytes in the peripheral blood.17 remain inactivated.9. thereby releasing bone-derived factors (including Ca2+ ions and matrix proteins. However. The consequent upregulation of the RANK receptor identifies this population of cells as typical osteoclast precursors. nuclear factor of activated T  © 2011 Macmillan Publishers Limited. however. M‑CSF is a crucial molecule in the control of the maturation and commitment of osteoclast pre­cursor cells. nuclear factor κB. Osteoclast differentiation Sporadic mutations associated with bone defects in mice revealed initial clues to the origins and functions of the osteoclast.10 This effect seemed to be related to a failure of monocyte–macrophage cells to differentiate fully and suggested that the bone defect in Csf1op/op mice resulted from the presence of insufficient growth factor signals.18 www. MITF target genes that encode essential molecules in osteoclast formation and function. commits undifferentiated cells into myeloid progenitors. receptor activator of nuclear factor κB (RANK. NFATc1.14 The develop­ ment of both osteoclasts and macrophages is arrested in PU. Interactions between PU. also known as CD11b). Several important studies provided a key contribution to our understanding of the cellular nature of this condition.13 both of which are intrinsic to normal osteoclast formation. In addition. Subsequently. unlike PU.16. RANKL. Mutations in the Mitf locus account for the osteopetrosis in the Mitf mi/mi mouse model and are thought to be the result of a defect in the gene encoding an essential member of the basic helixloop-helix–leucine-zipper protein family of transcription factors.1 MITF Precursor fusion Hematopoietic stem cell RANK NFκB NFATc1 Osteoclast attachment to bone Release of bone-derived factors Mature resorbing osteoclast Bone Ostm1gl/gl mice was reversed following transplantation of bone marrow from wild-type mice. RANK ligand. These characteristics resulted from mutations in the Mitf.5–7 In these seminal experiments. Mature resorbing osteoclasts derive from the hematopoietic stem cell lineage. PU. The ability of the osteoclast to degrade bone is dependent upon the formation of a tight attachment to the bone surface. Mitf mi/mi mice contain abundant macrophages. receptor activator of nuclear factor κB. cytoplasmic 1. MITF.1 die within 48 h of birth. which encodes macrophage colony-stimulating factor (M-CSF). The role of matrix metalloproteinases (MMPs) in bone formation and development has been comprehensively reviewed elsewhere1 and is only briefly outlined here. Early studies identified specific mouse strains with skeletal features (such as increased bone mass) that strongly resembled those of human osteopetrosis.1 transcription factor. In humans. also known as TNFRSF11A)11 (Figure 2). such as tartrate-resistant acid phosphatase (TRAP) and carbonic anhydrase 2 (CA-II). 15 As a consequence. Abbreviations: MITF. the osteopetrotic phenotype of Mitf mi/mi and 236  |  APRIL 2011  |  VOLUME 7 PU. transplantation of cells from normal animals into the bone marrow of these mice restored osteoclast and macro­ phage differen­ tiation.1 and MITF. CSF-1R and c‑Fos The earliest molecule known to influence the differentiation and commitment of precursor myeloid cells to an osteoclast lineage is the PU. and the upregulation of what has now become accepted as a principal mediator of osteoclast formation. The activation of early factors. These progenitor cells are directed toward a monocyte–macrophage lineage following the expression and stimulation of CSF-1R and the activation of intracellular proteins including c‑Fos. they show classic features of osteopetrosis.1 and the micro-­ophthalmiaassociated transcription factor (MITF) are also central to normal osteoclast biology.1. the observation that PU.2 Ostm13 and Tcirg14 genes. demonstrating the capacity of normal hematopoietic cells to rescue this disorder.

Transgenic mice that overexpress a soluble RANK fusion protein have a severely osteo­ petrotic pheno­ type similar to that of OPG-transgenic mice (these animals overexpress OPG). which considerably advanced our understanding of osteoclast formation and allowed for the widespread development and use of in vitro osteoclast assays.30–33 Mice deficient in TRAF5 or TRAF2 have no obvious skeletal malformations.21. which is capable of recruiting transcriptional regulators and also mediating cytoplasmic signaling via integrin proteins. Mice deficient in the OPG gene (Tnfrsf11b) were viable and appeared histologically normal at birth.22–25 followed by the identification of the transmembrane signaling receptor RANK. and activity. although increased vascular calcification and consequent aortic aneurysms (which are noted in twothirds of OPG-deficient animals) might also be involved. which occupied large areas within the marrow cavity and contained frequent cartilaginous remnants. TNF receptor-associated factor. The differentiation of a hematopoietic stem cell into a mature resorbing osteoclast is controlled by the sequential expression of specific regulatory factors. in marked contrast to Csf1op/op mice. TRAF5 and TRAF6. All rights reserved . which are activated by a converging cascade of intracellular signaling molecules.1 TRAF6 c-Fos MAPK JNK–ERK–MAPK14 IκB P AP-1 NFκB Src PYK2 Calcineurin c-Fos NFATc1 NFκB IκB NFκB RANK–RANKL–OPG signaling axis In the late 1990s a family of biologically related TNF-like proteins was identified. osteoprotegerin. the principal downstream mediator of calcium signaling. although TRAF2-deficient animals have an increased sensitivity of cells of the hematopoietic lineage VOLUME 7  |  APRIL 2011  |  237 © 2011 Macmillan Publishers Limited. rather than precursor commitment. nuclear factor κB. Osteoprotegerin (OPG. initiate a signaling cascade that culminates in nuclear factor κB (NFκB) activation. macrophage numbers were increased in these mice. This phenomenon was attributed to an increased incidence of fracture in axial and appendicular skeletal regions. also known as TNFSF11) as a target of OPG. leads to the recruitment of a Src–PYK2 complex. Radiographs of genetically modified mice with elevated circulating levels of OPG indicated a generalized osteopetrosis with increased radiodensity in long bones.20 Ca2+ RANKL OPG M-CSF RANK TRAF2 Ca2+–calmodulin complex TRAF5 CSF-1R PU. thought to result from either a severe loss of bone volume or heightened porosity of trabecular and cortical bone. suggesting that impaired resorption of the cartilage template leads to dysregulated skeletal modeling and remodeling. 19 Interestingly.28.21 These effects correlated with a profound decrease in osteoclast numbers. but is essential for postnatal bone modeling through its suppression of normal osteoclast formation.1. inhibition of osteoclast commitment favors the differentiation of monocytes towards a macrophage lineage.21 Histological analysis showed a dra­matic increase in the volume of trabecular bone.29 Downstream of the RANK receptor. suggesting that OPG blocks mature osteoclast formation only. in the absence of c-Fos. mice deficient in either RANK or RANKL develop severe osteopetrosis owing to a complete absence of osteoclasts. TNF receptorassociated factor (TRAF) proteins.22 Expression cloning with OPG as a probe led to the identification of RANK ligand (RANKL. Principally.1. Recruitment of TRAF2 and TRAF5 to the RANK–RANKL complex and the subsequent involvement of the essential TRAF6 protein leads to the phosphorylation of the inhibitory IκB protein by IκB kinase. which could be rescued by bone marrow transplantation or ectopic overexpression of Fos . cytoplasmic 1.27 Together.25 A role for the RANK–RANKL–OPG signaling axis in skeletal development was suggested by the detection of high levels of RANK messenger RNA in osteoclast progenitors and mature osteoclasts. One of the first mouse models of an osteoclast-related bone defect was generated by deletion of Fos (also known as c-fos). macrophage numbers were normal in this model. Consistent with these findings. IκB is degraded by the proteasome. RANK ligand. the true physiological importance of this signaling system in osteoclast formation and function was not realized until the effects of mutations in their respective genes were studied independently in mouse models. these models indicate that endogenous OPG is not required for embryonic skeletal development. Abbreviations: NFATc1.REVIEWS suggesting that the control of osteoclast formation by MITF is downstream of regulation by PU. RANK. OPG. receptor activator of nuclear factor κB. suggesting that.26 However. and the consequent activation of CSF-1R by the M‑CSF protein. Deletion of other components of the activator protein 1 (AP‑1) and mitogen-activated protein kinase (MAPK) signaling pathways resulted in embryonic lethality. also known as TNFRSF11B) was initially identified as a soluble protein capable of blocking osteoclast formation in vitro and bone resorption in vivo. Also. but exhibited a marked increase in mortality during the first 2 weeks of life. such as TRAF2. vertebrae and pelvis. Activation of RANK by RANKL represents a wellaccepted signaling mechanism that initiates osteoclast formation and activity. although bone marrow cultures from Mapk14 +/– heterozygous mice showed reduced osteoclast formation. the upregulation of CSF-1R by PU. RANKL. These bone changes were mediated by elevated osteoclast number NATURE REVIEWS | RHEUMATOLOGY NFATc1 AP-1 NFκB Integrins Figure 2 | Principal signaling pathways governing osteoclast formation and activation. nuclear factor of activated T cells. NFκB. thereby liberating active NFκB to induce gene transcription and contribute in part to the activation of NFATc1. TRAF. Mice deficient in c-Fos developed osteopetrosis through a decrease in osteoclast numbers.26.

37. such as fibroblast growth factor. widened metaphyses and medullary cavities filled with bony trabecu­ lae and cartilage. indicating that RANKL stimulation and downstream mediators regulate the mature resorptive cell in addition to promoting osteoclast differentiation.53 In addition. but are required for the formation of mature. 36 These findings suggest that deficiency of TRAF6 results in a defect in either osteoclast differentiation or osteoclast function and implicate this protein as a possible mediator of osteoclast formation and activity. On the other hand. The disparate results might be explained in part by the different approaches used to generate each strain. charac­ terized by reduced femur length. cathepsin K) that are released across the ruffled border membrane by fused vesicles. bone-resorbing cells. TRAF6knockout animals exhibit a distinctive impairment in bone model­ ing. 46 Complementing this finding. leading to severe osteopetrosis and absence of tooth eruption. FHL‑2 binds TRAF6 to inhibit its association with RANK. All rights reserved . the NFκB protein complex remains in the cytoplasm bound to the inhibitor of κB (IκB). One strain demonstrated a complete absence of multi­ nucleated TRAP-positive cells—a defining feature of mature ­osteoclasts35—and another had a normal number of poorly functioning osteoclasts that appeared detached from the bone surface. which is only detectable in osteoclasts following RANKL stimulation or in inflammatory arthritis. these animal models firmly establish NFκB as a crucial regulator of normal osteoclast formati­ on and activity.43. controversy persists with regard to whether osteoclasts are actually present in TRAF6-knockout animals. a genome-wide search led to the identification of nuclear factor of activated T cells. In mouse studies. The degradation of inorganic mineral by the low pH. however. NFκB activation Unsurprisingly.45 Also.38 Mice lacking FHL‑2 have hyper-­resorptive osteoclasts as a result of an aggressive cytoskeletal organization. to TNF-induced stimuli. in concert with the activation of matrix-degrading enzymes (for example.48 Mice deficient in NFATc1 die in utero owing to a defect in cardiac valve formation.40. cytoplasmic 1 (NFATc1) as the transcription factor most strongly induced by RANKL.37.50 however.REVIEWS CO2 CO2 CO2 + H2O Carbonic anhydrase 2 HCO3 CI– CI– – CI– CI – HCO3 – H2CO3 H+ H+ H+ Cathepsin K TRAP H+ Figure 3 | Osteoclastic resorption. the receptor for TYRO protein tyrosine kinase-binding protein) together with RANK on the surface of precursor cells activates members of www. loss of the individual p50 or p52 subunits of NFκB (encoded by the Nfkb1 and Nfkb2 genes.47 Together.35. Further studies demonstrated that the p50 and p52 subunits are not essential for osteoclast precursor formation. respectively) does not lead to obvious bone malformations. TRAF6 function can also be modulated by accessory proteins such as FHL‑2. animals engineered to express NFATc1 only in the heart are viable and show a notable reduction in osteoclast number and size. specific deletion of Ikbkb (which encodes IKK‑β) in cells of the myeloid lineage indicated that IKK‑β rather than IKK‑α is critical for osteoclast formation and survival.44 The effects of IKK‑α on bone might be mediated by epidermal-tissue-derived molecules.41 These defects were attributed to a reduction in osteoclast numbers.36 However. but otherwise normal skeletal development. rather than through a defect in osteoclasts. this mutation is perinatally lethal. Upstream activation signals trigger IκB kinase (IKK) to phosphorylate IκB.34 suggesting that TRAF2 and TRAF5 have a complementary role in NFκB activation and osteoclasto­ genesis. deletion of both Nfkb1 and Nfkb2 results in postnatal growth retardation and craniofacial abnormalities. which resulted in decreased resorption and osteopetrosis. only a few of these cells had formed structures reminiscent of attachment zones. ligand binding to ­immunoglobulin-like receptors (such as TREM2. results in the typical resorption of an area of bone.39 however.nature.52  © 2011 Macmillan Publishers Limited. mutations in genes coding for components of the NFκB signaling pathway also give rise to 238  |  APRIL 2011  |  VOLUME 7 NFATc1 and immunomodulators of bone Following the confirmation of RANKL as a principal mediator of osteoclast formation. deletion of one allele of the gene coding for the α subunit of IκB also resulted in considerable bone loss through enhanced osteoclastogeneis and osteoclast activity. mutations in each of these molecules lead to impaired osteoclast differentiation and result in severe osteo­petrosis. which targets this inhibitory protein for proteasomal degradation and allows active NFκB to translocate to the nucleus where it stimulates osteoclastogenic genes. Adequate ion transport is then necessary to ensure that the components needed to acidify the resorption lacunae (H+ and Cl–) along with the active intracellular enzymes (carbonic anhydrase 2) to process these products are available. The first and essential stage of osteoclastic activity is the efficient identification of and adherence to a region of bone and the formation of a resorption space isolated from the bone marrow cavity.51 NFATc1 is regulated by calcineurin and thus is involved in both calcium signaling and activation of immune cells via adaptors harboring an immuno­ receptor tyrosine-based activation motif (such as Fcγ receptor and TYRO protein tyrosine kinase-binding protein).42 Skeletal abnormalities are also reported in mice deficient in the catalytic subunits of IKK (IKK‑α or IKK‑β).38 skeletal malformations as a result of defects in osteoclast formation and function. Under nonstimulated conditions.32.49. The Chuk–/– mouse (which lacks IKK‑α) has defective limb-bud outgrowth and tooth eruption.

54 Although the true effect of oscillations in intra­cellular levels of calcium ions on osteoclast differentiation remains to be fully determined.REVIEWS the Tec family of tyrosine kinases (Tec and Btk).6 kb deletion in this gene in the osteopetrotic Tcirg1oc/oc mouse model. rather than a reduction in H+ release and osteoclast activity. This interaction occurs primarily through the activity of integrin complexes that have two distinct α and β chains.70 Mice deficient in the Ca2 gene. they failed to attach to the bone surface. CA‑II-deficient mice also have extensive renal tubular acidosis. effective resorption is dependent on the acidification of the lacunar space below the ruffled border membrane.57 of bone. known as the podosome. 65. Integrin αVβ3 forms a complex with filamentous actin. integrins might also influence the rearrangement of the cyto­ skeleton during osteoclast polarization.55 Activated T cells are also closely linked to excessive osteoclastogenesis and pathological bone destruction through mechanisms including the manufacture and solubilization of RANKL56 and TNF production.61. which encodes CA‑II. To achieve the low pH needed to activate matrix-degrading enzymes and the dissolution of the mineral component NATURE REVIEWS | RHEUMATOLOGY . osteoclast numbers were unchanged. as Src–/– mice have impaired bone remodeling as a result of defective osteoclast spreading rather than a reduction in osteoclast numbers. owing to a marked increase in osteoclast numbers that correlated with a substantial decrease in OPG levels in the bone marrow. and was attributed to the same mechanism (in which impaired T‑cell promotion of OPG production by B cells led to enhanced osteoclast formation). Itgb3–/– animals had over threefold more osteoclasts per mm2 of trabecular bone surface area compared with wild-type mice. H+ and Cl– ions are expelled through the ruffled border into the sealed zone between the osteoclast and the bone surface (Figure 3). however. The V1 domain hydrolyzes ATP whereas the V0 domain anchors this large protein complex to the membrane and employs a rotary mechanism to export protons through the cell membrane. which then dissociates into H+ and HCO3– ions. This phenomenon is clearly illustrated by the bone abnormalities present in genetically modified mice lacking either T cells or B cells. Interestingly. However. an increase in overall bone mass can be detected with radio­graphy at age 4 months.59 In addition to their mechanical role in cell–bone attachment. it is necessary for the formation of the ruffled border and resorptive activity of the mature cell.68 Another mechanism dictating the osteoclastic resorptive rate is the generation of H + ions. have a bone phenotype with long bones of reduced size and a widened metaphysis.66 By contrast. Mice lacking the proto-oncogene Src also demonstrate defective osteoclast function as a result of impaired H+-ATPase generation and vesicular trafficking to the ruffled border membrane. 60. as B cells are thought to be the source of the majority of bone-marrow-derived OPG.68 A polarization signal (probably generated by integrin–bone surface inter­ actions69) stimulates the transport of H +-ATPase via Src-containing vesicles towards the ruffled border membrane. Mice lacking Btk and Tec show severe osteopetrosis caused by a defect during late-stage osteoclast differentiation. mice deficient in the d2 domain of the V0 subunit exhibit only a mild increase in bone mass67 and defective fusion of preosteoclasts to mature multinucleated osteoclasts. 71 Although cortical bone volume seemed relatively normal. The essential enzyme CA‑II catalyzes the conversion of H2O and CO2 into H2CO3. These phenotypes are thought to be the result of an altered RANKL:OPG ratio in these animals. trabecular bone volume in these mice was increased by almost 50% compared with wild-type animals.5 is created within the resorption lacunae by a vacuolar ATPase electrogenic proton pump consisting of at least 14 different subunits. A similar effect was observed in mice lacking T cells. Findings from these animals are also supported by the identification of human mutations in loci encoding the electrogenic proton pump of the osteoclast. Consequently.60 Src-deficient osteoclasts fail to form a ruffled border and are consequently unable to resorb bone efficiently. by transmitting matrix-derived signals to the interior of the cell. Although bone resorption was minimal to absent in these animals. This mechanism probably involves the activation of Src family members. which VOLUME 7  |  APRIL 2011  |  239 © 2011 Macmillan Publishers Limited.61 A pH of approximately 4. Mice harboring a deletion in Src die shortly after birth with an osteopetrotic phenotype that includes thickened growth plates and a shortened diaphy­sis containing extensions of trabecular bone. these models highlight common factors that link immunoregulatory pathways with osteoclast differentiation and offer insight into the aberrant bone homeostasis associated with immuno­ deficiencies.62 The importance of this cellular machinery to normal osteoclast function was highlighted by a mouse model harboring a deletion in the α3 domain of the V0 subunit gene Tcirg163 and the subsequent identification of a 1.55 Mice lacking B cells were unsurprisingly osteoporotic. resulting in a lack of enzyme activity within the ruffled border of the osteoclast and decreased resorptive activity in these models.64 These critical gene defects inactivate the ATPase subunit.58 which recognizes proteins in the organic matrix of bone that include ArgGlyAsp (RGD) amino-acid motifs. These characteristics suggest that the vacuolar H+-ATPase complex might function as more than just a proton pump. many of which exist as different isoforms.60 Ion exchange Following osteoclast attachment to the bone surface. Insertion of vesicles containing H+-ATPase into the plasma­ lemma leads to the complex and osteoclastdefining structure of the ruffled border. Although mice lacking the gene for the β3 integrin (Itgb3–/–) seem normal. these cells showed marked disruption of the actin cytoskeleton and lacked a defined ruffled border membrane. indicating that although Src is not required for osteoclast formation. All rights reserved Osteoclast function Integrin binding and signaling Efficient osteoclast activity requires the cell to interact with and bind tightly to the surface of the bone.

is antiresorptive therapy. show skeletal features attributed pri­ marily to other bone cells. growth factors and. the release of MMPs through the ruffled border membrane is thought to contribute to the breakdown of the organic matrix during active bone resorption. Histological examination of bone from these mice revealed a normal distribution of fully differen­tiated osteoclasts.71 The HCO3– ions generated by CA‑II enzyme activity are exchanged for Cl– ions via other membranous regions of the osteoclast not involved in resorption. atypical subtrochanteric fractures and bone fragility. MMP-deficient animals can. with abnormal joint morphology and a reduced bone marrow cavity with large areas of abnormal. but cathepsin K is now accepted as the principal protease mediating in vivo matrix degradation within the low-pH environment of resorption lacunae. These enzymes include cathepsin K. new compounds capable of effectively reducing the resorptive activity of osteoclasts are highly sought. such as osteoporosis or cancer-induced bone disease. resulting in elevated bone density in early adulthood and a delay in fracture repair.85. however. Convincing support for this role comes from observations made in both mice and humans harboring mutations in the cathepsin K gene. poorly arranged bone matrix.nature. CLCN7 mutations underlie infantile malignant osteopetrosis. such as osteoblasts. II and X. which results in poor acidification of the resorption lacunae. but has been linked to adverse effects. Cathepsin K Several cathepsin enzymes have been linked to normal osteoclast function in vitro (for example. namely osteo­necrosis of the jaw.73 The dense. highlighted the vast potential for targeting this factor with an exo­genous soluble inhibitor. The generally mild pheno­ types that result from deletions of individual MMP genes suggest that a substantial degree of compensation occurs. All rights reserved . gained from mutant mouse models. brittle bones and severe osteopetrosis in this model are caused by an inefficient proton-neutralizing current of Cl– ions through the ClC‑7 channel. but the capacity of these cells to degrade bone matrix was severely compromised. illustrated the potential of target­ ing cathepsin K www. pycnodysostosis.82 Tartrate-resistant acid phosphatase Expression of TRAP is also implicated in normal osteoclast function on the basis of observations from TRAPdeficient mice.79. resulting in the short stature and dense bones characteristic of pycnodysostosis.86 The FDA approved this agent in June 2010 for women at high risk of osteoporotic fracture. First-line treatment for diseases that involve excessive bone loss.84 As such. was developed follow­ing the identification of RANKL as a principal osteo­ clastogenic factor in vivo.78 Of interest.74 Confirmation of these genetic origins was achieved through the generation of ­cathepsin‑K-deficient mice.73 cleavage of cytokines. for example. a chloride-channel gene. The severe skeletal effects associated with mutations in the human cathepsin K gene (pycnodysostosis). leading to defective tissue organization and brittle bones despite a high overall bone mass. These mice have similar numbers of osteoclasts as wildtype mice. has contributed sub­ stantially to the development of therapeutics aimed at preserving bone mass. Denosumab is a mono­clonal antibody directed against RANKL that can rapidly reduce osteoclastic resorption and increase bone mineral density (BMD) in post­menopausal women. The detailed characterization of the bone phenotypes described above has undoubtedly contributed to the development of novel therapeutics aimed specifically at osteoclasts.75–77 Matrix metalloproteinases MMPs are expressed at high levels within bone and cartilage. extracellular matrix components such as collagen types I. but the function of these cells is impaired such that they cannot acidify the resorption lacunae and thus fail to resorb bone. was mapped to the region encoding cathepsin K. which are abundant within the ruffled border membrane. denosumab. typi­ cally bisphosphonates. by revealing new targets that regulate osteoclast differentiation and function. together with the effects of controlled deletion of this gene in mouse models.72 Mice that lack Clcn7. which were (unsurprisingly) osteopetrotic.81. cathepsins B–E and G).1 In osteoclasts.83 Matrix-degrading proteases In addition to the low pH of the resorption lacunae. osteocytes and chondro­ cytes. and are suggested to mediate normal skeletal development and postnatal bone remodeling through the 240  |  APRIL 2011  |  VOLUME 7 Osteoclast-targeted therapies A clear understanding of the central mechanisms underlying mammalian osteoclast formation and function.80 and that osteoclast function overall is reduced in Mmp13–/– mice. efficient degradation of bone is dependent on the production and activation of various matrix-degrading enzymes (Figure 3). as demonstrated in these genetic mouse models. several MMPs and TRAP. is supported by mutations identified in human families. are smaller than wild-type littermates and have shortened limbs with bones that lack a well-defined marrow cavity. and confirmed by the OPG-transgenic mouse. more importantly. are findings in mice indicating that deletion of Mmp9 or Mmp13 impairs osteoclast recruitment and invasion during skeletal development. The use of bisphosphonate drugs to effectively block osteoclast activity is well documented.73 The importance of the ClC‑7 channel in normal osteoclast function.REVIEWS probably clouds the exact interpretation of the effects of this enzyme in skeletal biology. Intracellular pH is maintained by export of these Cl– ions into the resorption lacunae via chloride channels. The striking elevation in bone mass demon­ strated by the RANKL knockout model. however.74 This mutation completely prevents the production of detectable cathepsin K protein. The best-described of the nonbisphosphonate anti­ resorptive  © 2011 Macmillan Publishers Limited. The genetic defect underlying the osteochondro­ dysplastic syndrome. These animals display an early-onset osteopetrotic bone phenotype with normal osteoclast formation but reduced osteoclast activity.

the continued ex­ amination of which might reveal distinct properties. Review criteria Data for this Review were identified by searching the PubMed database for original articles and reviews on genetically modified mouse models resulting from defects in osteoclasts using the search terms “osteoclast”. which reversed ovariectomy-induced bone loss in rodents. Administration of this agent is associ­ated with a consider­ able reduction in levels of bone resorption bio­ markers and increased bone density at the lumbar spine and hip of postmenopausal women with low BMD. “transgenic”.92 Further insight into the mechanism coupling bone forma­tion to a period of osteoclast activity was revealed by immune-deficient mice lacking Tgfb1: this model demonstrated a mechanistic role for TGF‑β1 in the migration of bone mesenchymal stem cells following bone resorption. Walker.89 In addition. Vererb. Spleen cells transmit osteopetrosis in mice. Control of bone resorption by hematopoietic tissue. 7–18 (2008). Z. 4. by contrast. A new sub-lethal colour mutation in the house mouse. These cells are capable of responding to a vast array of stimuli and influenc­ ing formation and function of other cells of the bone micro­ environment to contribute to the maintenance of adequate bone mass and integrity throughout life. Menschl. Dicer. Lond. which trigger osteoclast apoptosis or prevent the formation of these cells. the identification and functional characterization of specific subgroups within these populations is only beginning.91 Box 1 | Unanswered questions ■■ Can osteoclasts exist in a quiescent state? ■■ What is the role (if any) of nonresorbing osteoclasts? ■■ Do cells outside of the myeloid lineage directly fuse with developing osteoclasts? ■■ How might fusion of precursor cells with non-myeloid cells influence the resorptive capabilities of osteoclasts? ■■ What is the relevance of converging mechanisms in immune cell activation and osteoclast formation? ■■ Are current osteoclast-targeted therapies sufficient or are they now solely responsible for new pathologies of decreased bone turnover? ■■ What can we learn from new models and emerging studies about the effects of long-term disruption of osteoclast formation and activity on the skeleton? Conclusions and future directions The use of mutant mouse models continues to shape the way we study bone biology. integrin-targeted therapy with RGD mimetics prevented cancellous bone loss in ovariectomized rats90 and increased BMD in women with postmenopausal osteoporosis. Konstitutionsl. B 118. Dickie. D. M. Walker. M. D. Hertwig. Odanacatib. The induction and reversal of congenital osteopetrosis in mice through use NATURE REVIEWS | RHEUMATOLOGY © 2011 Macmillan Publishers Limited. G. 39 (1967). a clear inference is that the use of genetically modified mouse models has expanded upon the original concept of the osteoclast as simply a large polykaryon specialized for bone resorption alone. Science 190. 2. “knockout”. enhanced osteoclastogenesis97 and increased lifespan of mature resorbing cells.88 This compound has a different mechanism of action com­pared with that of previously employed antiresorptive agents. 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Sechs neue mutationen bei der hausmaus in ihrer bedeutung für allgemeine vererbungsfragen. The authors focused mainly on well-established and validated studies. G.96 respectively. “osteopetrosis” and “osteoporosis”. 1–21 (1942). Walker. Our understanding of the effects of hormones on bone have been advanced by observations that mice lacking the estrogen receptor in cells of the osteoclast lineage displayed no change in cortical bone. discovery of a spontaneous mutation resulting in an osteopetrotic phenotype associated with defective osteoclasts led to the characterization of a new mouse strain (termed ntl). Matrix metalloproteinases and bone. Instead. The causative mutation was not associ­ ated with genetic loci previously linked to osteopetrosis. R. Other approaches being investigated include pharmaco­ logical targeting of the M‑CSF receptor. Bone 43. Proc.101 The discovery of new molecular mechanisms and physio­ logical pathways inevitably raises further un­ answered questions (Box 1). Despite closely mapping to the region encoding chloride channels on mouse chromosome 17. 5. but these animals demonstrated decreased trabecular bone.93 Ablated osteoclast function and elevated bone mass were also observed in mice lacking Adora1. Although osteoclast differen­ tiation and commitment from precursor cells of the monocyte–­ macrophage lineage is well established. As we continue to explore the role of osteoclasts in the biology of bone. confirmed human mutations and collectively expanded our understanding of the mechanisms governing normal and pathological osteoclast formation and function. Soc. “genetic mutation”.87. & Inada. All rights reserved VOLUME 7  |  APRIL 2011  |  241 . 1.98 Altered osteoclast biology can also be observed in genetically modified mice lacking the DNA-modifying proteins ID‑199 and NAD-dependent deacetylase sirtuin 1 (SIRT1)100 or the microRNA-related endoribonuclease. no changes in ClC‑7 DNA or protein were observed. The cathepsin K inhibitor odanacatib has demon­ strated a clear and potent specificity for cathep­ sin K. Private communication.

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