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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 15years, 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 RANKRANKLOPG 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, 235243 (2011); published online 8 March 2011; doi:10.1038/nrrheum.2011.23

Introduction
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 (Figure1). 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 (Figure2). 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 (Figure1). 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 (Figure3). 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, TN372320575, USA (G.R. Mundy). Correspondence to: J.R. Edwards james.edwards@ ndorms.ox.ac.uk

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Key points
RANKRANKLOPG signaling is an important mediator of osteoclast differentiation Transcriptional regulation of osteoclast formation involves NFB 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 NFB c-Fos NFATc1

Committed myeloid precursor PU.1 MITF

Precursor fusion

Hematopoietic stem cell

RANK NFB 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, demonstrating the capacity of normal hematopoietic cells to rescue this disorder. Subsequently, a mouse strain was identified that harbored a mutation in the Csf1 gene, which encodes macrophage colony-stimulating factor (M-CSF).8 This mutation results in an excessive accumulation of bone throughout the marrow cavities.9 Compared with their wild-type counterparts, osteopetrotic Csf1op/op mice possessed reduced numbers of circulating monocytes in the peripheral blood, few macrophages in the peritoneal cavity and osteoclasts that were both morphologically smaller and fewer in number.9,10 This effect seemed to be related to a failure of monocytemacrophage cells to differentiate fully and suggested that the bone defect in Csf1op/op mice resulted from the presence of insufficient growth factor signals, which are essential for the normal differentiation of cells of the monocytemacrophage lineage. MCSF is a crucial molecule in the control of the maturation and commitment of osteoclast precursor cells, and the upregulation of what has now become accepted as a principal mediator of osteoclast formation, receptor activator of nuclear factorB (RANK; also known as TNFRSF11A)11 (Figure2).

Osteoclast activity (bone resorption)

Figure 1 | Osteoclast differentiation and function. Mature resorbing osteoclasts derive from the hematopoietic stem cell lineage. The activation of early factors, such as PU.1 and MITF, commits undifferentiated cells into myeloid progenitors. These progenitor cells are directed toward a monocytemacrophage lineage following the expression and stimulation of CSF-1R and the activation of intracellular proteins including cFos. The consequent upregulation of the RANK receptor identifies this population of cells as typical osteoclast precursors. Exposure to RANKL represents the primary stimuli for normal osteoclast formation and continued resorptive activity through the activation of the principal transcriptional mediators NFB and NFATc1. The ability of the osteoclast to degrade bone is dependent upon the formation of a tight attachment to the bone surface, after which a cocktail of ions and matrixdegrading enzymes expelled into the sealed lacunae break down the inorganic and organic components of bone, thereby releasing bone-derived factors (including Ca2+ ions and matrix proteins, such as transforming growth factor and osteocalcin) into the marrow cavity. Abbreviations: MITF, micro-ophthalmia-associated transcription factor; NFATc1, nuclear factor of activated Tcells, cytoplasmic 1; NFB, nuclear factor B; RANK, receptor activator of nuclear factorB; RANKL, RANK ligand.

options that have evolved from these studies. The role of matrix metalloproteinases (MMPs) in bone formation and development has been comprehensively reviewed elsewhere1 and is only briefly outlined here.

Osteoclast differentiation
Sporadic mutations associated with bone defects in mice revealed initial clues to the origins and functions of the osteoclast. Early studies identified specific mouse strains with skeletal features (such as increased bone mass) that strongly resembled those of human osteopetrosis. These characteristics resulted from mutations in the Mitf,2 Ostm13 and Tcirg14 genes. Several important studies provided a key contribution to our understanding of the cellular nature of this condition.57 In these seminal experiments, the osteopetrotic phenotype of Mitf mi/mi and
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PU.1, MITF, CSF-1R and cFos The earliest molecule known to influence the differentiation and commitment of precursor myeloid cells to an osteoclast lineage is the PU.1 transcription factor. In humans, PU.1 has been implicated in the transcriptional regulation of CSF1R (which encodes the MCSF receptor),12 and ITGAM (which encodes integrin M; also known as CD11b),13 both of which are intrinsic to normal osteoclast formation. Mice deficient in PU.1 die within 48h of birth. However, they show classic features of osteopetrosis, such as cartilaginous remnants within trabecular bone and a failure of tooth eruption.14 The develop ment of both osteoclasts and macrophages is arrested in PU.1deficient mice; however, transplantation of cells from normal animals into the bone marrow of these mice restored osteoclast and macro phage differen tiation.14 Together, these studies indicate that PU.1 is central to the differentiation of hematopoietic pre cursors. In addition, the observation that PU.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. Interactions between PU.1 and the micro-ophthalmiaassociated transcription factor (MITF) are also central to normal osteoclast biology. 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-helixleucine-zipper protein family of transcription factors. 15 As a consequence, MITF target genes that encode essential molecules in osteoclast formation and function, such as tartrate-resistant acid phosphatase (TRAP) and carbonic anhydrase 2 (CA-II),16,17 remain inactivated. However, unlike PU.1-deficient animals, Mitf mi/mi mice contain abundant macrophages,18
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suggesting that the control of osteoclast formation by MITF is downstream of regulation by PU.1. One of the first mouse models of an osteoclast-related bone defect was generated by deletion of Fos (also known as c-fos). Mice deficient in c-Fos developed osteopetrosis through a decrease in osteoclast numbers, which could be rescued by bone marrow transplantation or ectopic overexpression of Fos . 19 Interestingly, macrophage numbers were increased in these mice, suggesting that, in the absence of c-Fos, inhibition of osteoclast commitment favors the differentiation of monocytes towards a macrophage lineage. Deletion of other components of the activator protein 1 (AP1) and mitogen-activated protein kinase (MAPK) signaling pathways resulted in embryonic lethality, although bone marrow cultures from Mapk14 +/ heterozygous mice showed reduced osteoclast formation.20

Ca2+

RANKL

OPG M-CSF

RANK TRAF2 Ca2+calmodulin complex TRAF5 CSF-1R PU.1

TRAF6

c-Fos MAPK JNKERKMAPK14 IB P AP-1 NFB

Src PYK2

Calcineurin c-Fos NFATc1

NFB

IB

NFB

RANKRANKLOPG signaling axis In the late 1990s a family of biologically related TNF-like proteins was identified, which considerably advanced our understanding of osteoclast formation and allowed for the widespread development and use of invitro osteoclast assays. Osteoprotegerin (OPG; also known as TNFRSF11B) was initially identified as a soluble protein capable of blocking osteoclast formation invitro and bone resorption invivo.21,22 Expression cloning with OPG as a probe led to the identification of RANK ligand (RANKL, also known as TNFSF11) as a target of OPG,2225 followed by the identification of the transmembrane signaling receptor RANK.25 A role for the RANKRANKLOPG signaling axis in skeletal development was suggested by the detection of high levels of RANK messenger RNA in osteoclast progenitors and mature osteoclasts.26 However, 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. Radiographs of genetically modified mice with elevated circulating levels of OPG indicated a generalized osteopetrosis with increased radiodensity in long bones, vertebrae and pelvis.21 Histological analysis showed a dramatic increase in the volume of trabecular bone, which occupied large areas within the marrow cavity and contained frequent cartilaginous remnants.21 These effects correlated with a profound decrease in osteoclast numbers, suggesting that impaired resorption of the cartilage template leads to dysregulated skeletal modeling and remodeling. Also, in marked contrast to Csf1op/op mice, macrophage numbers were normal in this model, suggesting that OPG blocks mature osteoclast formation only, rather than precursor commitment. Mice deficient in the OPG gene (Tnfrsf11b) were viable and appeared histologically normal at birth, but exhibited a marked increase in mortality during the first 2weeks of life. This phenomenon was attributed to an increased incidence of fracture in axial and appendicular skeletal regions, thought to result from either a severe loss of bone volume or heightened porosity of trabecular and cortical bone. These bone changes were mediated by elevated osteoclast number
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NFATc1

AP-1

NFB

Integrins

Figure 2 | Principal signaling pathways governing osteoclast formation and activation. The differentiation of a hematopoietic stem cell into a mature resorbing osteoclast is controlled by the sequential expression of specific regulatory factors, which are activated by a converging cascade of intracellular signaling molecules. Principally, the upregulation of CSF-1R by PU.1, and the consequent activation of CSF-1R by the MCSF protein, leads to the recruitment of a SrcPYK2 complex, which is capable of recruiting transcriptional regulators and also mediating cytoplasmic signaling via integrin proteins. Activation of RANK by RANKL represents a wellaccepted signaling mechanism that initiates osteoclast formation and activity. Recruitment of TRAF2 and TRAF5 to the RANKRANKL complex and the subsequent involvement of the essential TRAF6 protein leads to the phosphorylation of the inhibitory IB protein by IB kinase. IB is degraded by the proteasome, thereby liberating active NFB to induce gene transcription and contribute in part to the activation of NFATc1, the principal downstream mediator of calcium signaling. Abbreviations: NFATc1, nuclear factor of activated Tcells, cytoplasmic 1; NFB, nuclear factorB; OPG, osteoprotegerin; RANK, receptor activator of nuclear factorB; RANKL, RANK ligand; TRAF, TNF receptor-associated factor.

and activity, although increased vascular calcification and consequent aortic aneurysms (which are noted in twothirds of OPG-deficient animals) might also be involved.27 Together, these models indicate that endogenous OPG is not required for embryonic skeletal development, but is essential for postnatal bone modeling through its suppression of normal osteoclast formation. Consistent with these findings, mice deficient in either RANK or RANKL develop severe osteopetrosis owing to a complete absence of osteoclasts. 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).26,28,29 Downstream of the RANK receptor, TNF receptorassociated factor (TRAF) proteins, such as TRAF2, TRAF5 and TRAF6, initiate a signaling cascade that culminates in nuclear factorB (NFB) activation.3033 Mice deficient in TRAF5 or TRAF2 have no obvious skeletal malformations, although TRAF2-deficient animals have an increased sensitivity of cells of the hematopoietic lineage
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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 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. 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 degradation of inorganic mineral by the low pH, in concert with the activation of matrix-degrading enzymes (for example, cathepsinK) that are released across the ruffled border membrane by fused vesicles, results in the typical resorption of an area of bone.

to TNF-induced stimuli,32,34 suggesting that TRAF2 and TRAF5 have a complementary role in NFB activation and osteoclasto genesis. On the other hand, TRAF6knockout animals exhibit a distinctive impairment in bone model ing, charac terized by reduced femur length, widened metaphyses and medullary cavities filled with bony trabecu lae and cartilage.35,36 However, controversy persists with regard to whether osteoclasts are actually present in TRAF6-knockout animals. One strain demonstrated a complete absence of multi nucleated TRAP-positive cellsa defining feature of mature osteoclasts35and another had a normal number of poorly functioning osteoclasts that appeared detached from the bone surface; only a few of these cells had formed structures reminiscent of attachment zones. 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. The disparate results might be explained in part by the different approaches used to generate each strain. TRAF6 function can also be modulated by accessory proteins such as FHL2, which is only detectable in osteoclasts following RANKL stimulation or in inflammatory arthritis; FHL2 binds TRAF6 to inhibit its association with RANK.37,38 Mice lacking FHL2 have hyper-resorptive osteoclasts as a result of an aggressive cytoskeletal organization, indicating that RANKL stimulation and downstream mediators regulate the mature resorptive cell in addition to promoting osteoclast differentiation.37,38

skeletal malformations as a result of defects in osteoclast formation and function. Under nonstimulated conditions, the NFB protein complex remains in the cytoplasm bound to the inhibitor of B (IB). Upstream activation signals trigger IB kinase (IKK) to phosphorylate IB, which targets this inhibitory protein for proteasomal degradation and allows active NFB to translocate to the nucleus where it stimulates osteoclastogenic genes. In mouse studies, loss of the individual p50 or p52 subunits of NFB (encoded by the Nfkb1 and Nfkb2 genes, respectively) does not lead to obvious bone malformations;39 however, deletion of both Nfkb1 and Nfkb2 results in postnatal growth retardation and craniofacial abnormalities.40,41 These defects were attributed to a reduction in osteoclast numbers, which resulted in decreased resorption and osteopetrosis. Further studies demonstrated that the p50 and p52 subunits are not essential for osteoclast precursor formation, but are required for the formation of mature, bone-resorbing cells.42 Skeletal abnormalities are also reported in mice deficient in the catalytic subunits of IKK (IKK or IKK). The Chuk/ mouse (which lacks IKK) has defective limb-bud outgrowth and tooth eruption, but otherwise normal skeletal development; however, this mutation is perinatally lethal.43,44 The effects of IKK on bone might be mediated by epidermal-tissue-derived molecules, such as fibroblast growth factor, rather than through a defect in osteoclasts.45 Also, specific deletion of Ikbkb (which encodes IKK) in cells of the myeloid lineage indicated that IKK rather than IKK is critical for osteoclast formation and survival. 46 Complementing this finding, deletion of one allele of the gene coding for the subunit of IB also resulted in considerable bone loss through enhanced osteoclastogeneis and osteoclast activity.47 Together, these animal models firmly establish NFB as a crucial regulator of normal osteoclast formati on and activity.

NFB activation Unsurprisingly, mutations in genes coding for components of the NFB signaling pathway also give rise to
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NFATc1 and immunomodulators of bone Following the confirmation of RANKL as a principal mediator of osteoclast formation, a genome-wide search led to the identification of nuclear factor of activated Tcells, cytoplasmic 1 (NFATc1) as the transcription factor most strongly induced by RANKL.48 Mice deficient in NFATc1 die inutero owing to a defect in cardiac valve formation;49,50 however, animals engineered to express NFATc1 only in the heart are viable and show a notable reduction in osteoclast number and size, leading to severe osteopetrosis and absence of tooth eruption.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 Fc receptor and TYRO protein tyrosine kinase-binding protein).52 Interestingly, mutations in each of these molecules lead to impaired osteoclast differentiation and result in severe osteopetrosis.53 In addition, ligand binding to immunoglobulin-like receptors (such as TREM2, the receptor for TYRO protein tyrosine kinase-binding protein) together with RANK on the surface of precursor cells activates members of
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the Tec family of tyrosine kinases (Tec and Btk). Mice lacking Btk and Tec show severe osteopetrosis caused by a defect during late-stage osteoclast differentiation.54 Although the true effect of oscillations in intracellular levels of calcium ions on osteoclast differentiation remains to be fully determined, these models highlight common factors that link immunoregulatory pathways with osteoclast differentiation and offer insight into the aberrant bone homeostasis associated with immuno deficiencies. This phenomenon is clearly illustrated by the bone abnormalities present in genetically modified mice lacking either Tcells or Bcells. These phenotypes are thought to be the result of an altered RANKL:OPG ratio in these animals, as Bcells are thought to be the source of the majority of bone-marrow-derived OPG.55 Mice lacking Bcells were unsurprisingly osteoporotic, owing to a marked increase in osteoclast numbers that correlated with a substantial decrease in OPG levels in the bone marrow. A similar effect was observed in mice lacking Tcells, and was attributed to the same mechanism (in which impaired Tcell promotion of OPG production by Bcells led to enhanced osteoclast formation).55 Activated Tcells are also closely linked to excessive osteoclastogenesis and pathological bone destruction through mechanisms including the manufacture and solubilization of RANKL56 and TNF production.57 of bone, H+ and Cl ions are expelled through the ruffled border into the sealed zone between the osteoclast and the bone surface (Figure3).61 A pH of approximately 4.5is created within the resorption lacunae by a vacuolar ATPase electrogenic proton pump consisting of at least 14 different subunits, many of which exist as different isoforms. 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.61,62 The importance of this cellular machinery to normal osteoclast function was highlighted by a mouse model harboring a deletion in the 3 domain of the V0 subunit gene Tcirg163 and the subsequent identification of a 1.6kb deletion in this gene in the osteopetrotic Tcirg1oc/oc mouse model.64 These critical gene defects inactivate the ATPase subunit, resulting in a lack of enzyme activity within the ruffled border of the osteoclast and decreased resorptive activity in these models. Findings from these animals are also supported by the identification of human mutations in loci encoding the electrogenic proton pump of the osteoclast. 65,66 By contrast, 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, rather than a reduction in H+ release and osteoclast activity. These characteristics suggest that the vacuolar H+-ATPase complex might function as more than just a proton pump. 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. 60,68 A polarization signal (probably generated by integrinbone surface inter actions69) stimulates the transport of H +-ATPase via Src-containing vesicles towards the ruffled border membrane. Insertion of vesicles containing H+-ATPase into the plasma lemma leads to the complex and osteoclastdefining structure of the ruffled border. 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. Although bone resorption was minimal to absent in these animals, osteoclast numbers were unchanged.60 Src-deficient osteoclasts fail to form a ruffled border and are consequently unable to resorb bone efficiently, indicating that although Src is not required for osteoclast formation, 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. The essential enzyme CAII catalyzes the conversion of H2O and CO2 into H2CO3, which then dissociates into H+ and HCO3 ions.70 Mice deficient in the Ca2 gene, which encodes CAII, have a bone phenotype with long bones of reduced size and a widened metaphysis. 71 Although cortical bone volume seemed relatively normal, trabecular bone volume in these mice was increased by almost 50% compared with wild-type animals. However, CAII-deficient mice also have extensive renal tubular acidosis, which
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Osteoclast function
Integrin binding and signaling Efficient osteoclast activity requires the cell to interact with and bind tightly to the surface of the bone. This interaction occurs primarily through the activity of integrin complexes that have two distinct and chains. IntegrinV3 forms a complex with filamentous actin, known as the podosome,58 which recognizes proteins in the organic matrix of bone that include ArgGlyAsp (RGD) amino-acid motifs. Although mice lacking the gene for the 3integrin (Itgb3/) seem normal, an increase in overall bone mass can be detected with radiography at age 4months. Interestingly, Itgb3/ animals had over threefold more osteoclasts per mm2 of trabecular bone surface area compared with wild-type mice; however, these cells showed marked disruption of the actin cytoskeleton and lacked a defined ruffled border membrane. Consequently, they failed to attach to the bone surface.59 In addition to their mechanical role in cellbone attachment, integrins might also influence the rearrangement of the cyto skeleton during osteoclast polarization, by transmitting matrix-derived signals to the interior of the cell. This mechanism probably involves the activation of Src family members, as Src/ mice have impaired bone remodeling as a result of defective osteoclast spreading rather than a reduction in osteoclast numbers.60 Ion exchange Following osteoclast attachment to the bone surface, effective resorption is dependent on the acidification of the lacunar space below the ruffled border membrane. To achieve the low pH needed to activate matrix-degrading enzymes and the dissolution of the mineral component
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probably clouds the exact interpretation of the effects of this enzyme in skeletal biology.71 The HCO3 ions generated by CAII enzyme activity are exchanged for Cl ions via other membranous regions of the osteoclast not involved in resorption. Intracellular pH is maintained by export of these Cl ions into the resorption lacunae via chloride channels, which are abundant within the ruffled border membrane.72 Mice that lack Clcn7, a chloride-channel gene, are smaller than wild-type littermates and have shortened limbs with bones that lack a well-defined marrow cavity.73 The dense, brittle bones and severe osteopetrosis in this model are caused by an inefficient proton-neutralizing current of Cl ions through the ClC7 channel, which results in poor acidification of the resorption lacunae. These mice have similar numbers of osteoclasts as wildtype mice, but the function of these cells is impaired such that they cannot acidify the resorption lacunae and thus fail to resorb bone.73 The importance of the ClC7 channel in normal osteoclast function, as demonstrated in these genetic mouse models, is supported by mutations identified in human families; for example, CLCN7 mutations underlie infantile malignant osteopetrosis.73 cleavage of cytokines, growth factors and, more importantly, extracellular matrix components such as collagen types I, II and X.1 In osteoclasts, the release of MMPs through the ruffled border membrane is thought to contribute to the breakdown of the organic matrix during active bone resorption. The generally mild pheno types that result from deletions of individual MMP genes suggest that a substantial degree of compensation occurs. MMP-deficient animals can, however, show skeletal features attributed pri marily to other bone cells, such as osteoblasts, osteocytes and chondro cytes.78 Of interest, however, are findings in mice indicating that deletion of Mmp9 or Mmp13 impairs osteoclast recruitment and invasion during skeletal development,79,80 and that osteoclast function overall is reduced in Mmp13/ mice, resulting in elevated bone density in early adulthood and a delay in fracture repair.81,82
Tartrate-resistant acid phosphatase Expression of TRAP is also implicated in normal osteoclast function on the basis of observations from TRAPdeficient mice. These animals display an early-onset osteopetrotic bone phenotype with normal osteoclast formation but reduced osteoclast activity.83

Matrix-degrading proteases In addition to the low pH of the resorption lacunae, efficient degradation of bone is dependent on the production and activation of various matrix-degrading enzymes (Figure3). These enzymes include cathepsinK, several MMPs and TRAP.
Cathepsin K Several cathepsin enzymes have been linked to normal osteoclast function invitro (for example, cathepsins BE and G), but cathepsinK is now accepted as the principal protease mediating invivo matrix degradation within the low-pH environment of resorption lacunae. Convincing support for this role comes from observations made in both mice and humans harboring mutations in the cathepsinK gene. The genetic defect underlying the osteochondro dysplastic syndrome, pycnodysostosis, was mapped to the region encoding cathepsinK.74 This mutation completely prevents the production of detectable cathepsinK protein, resulting in the short stature and dense bones characteristic of pycnodysostosis.74 Confirmation of these genetic origins was achieved through the generation of cathepsinK-deficient mice, which were (unsurprisingly) osteopetrotic, with abnormal joint morphology and a reduced bone marrow cavity with large areas of abnormal, poorly arranged bone matrix. Histological examination of bone from these mice revealed a normal distribution of fully differentiated osteoclasts, but the capacity of these cells to degrade bone matrix was severely compromised, leading to defective tissue organization and brittle bones despite a high overall bone mass.7577 Matrix metalloproteinases MMPs are expressed at high levels within bone and cartilage, and are suggested to mediate normal skeletal development and postnatal bone remodeling through the
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Osteoclast-targeted therapies
A clear understanding of the central mechanisms underlying mammalian osteoclast formation and function, gained from mutant mouse models, has contributed sub stantially to the development of therapeutics aimed at preserving bone mass. First-line treatment for diseases that involve excessive bone loss, such as osteoporosis or cancer-induced bone disease, is antiresorptive therapy, typi cally bisphosphonates. The use of bisphosphonate drugs to effectively block osteoclast activity is well documented, but has been linked to adverse effects, namely osteonecrosis of the jaw, atypical subtrochanteric fractures and bone fragility.84 As such, new compounds capable of effectively reducing the resorptive activity of osteoclasts are highly sought. The detailed characterization of the bone phenotypes described above has undoubtedly contributed to the development of novel therapeutics aimed specifically at osteoclasts, by revealing new targets that regulate osteoclast differentiation and function. The best-described of the nonbisphosphonate anti resorptive drugs, denosumab, was developed following the identification of RANKL as a principal osteo clastogenic factor invivo. The striking elevation in bone mass demon strated by the RANKL knockout model, and confirmed by the OPG-transgenic mouse, highlighted the vast potential for targeting this factor with an exogenous soluble inhibitor. Denosumab is a monoclonal antibody directed against RANKL that can rapidly reduce osteoclastic resorption and increase bone mineral density (BMD) in postmenopausal women.85,86 The FDA approved this agent in June 2010 for women at high risk of osteoporotic fracture. The severe skeletal effects associated with mutations in the human cathepsin K gene (pycnodysostosis), together with the effects of controlled deletion of this gene in mouse models, illustrated the potential of target ing cathepsin K
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invivo and encouraged the development of compounds aimed at blocking the function of this matrix-degrading enzyme. The cathepsin K inhibitor odanacatib has demon strated a clear and potent specificity for cathep sinK. 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.87,88 This compound has a different mechanism of action compared with that of previously employed antiresorptive agents, which trigger osteoclast apoptosis or prevent the formation of these cells. Odanacatib, by contrast, would be expected to lead to the formation of nonfunctional osteoclasts, a feature that could confer benefits through retaining osteoclast communication with other cells of the bone marrow microenvironment, the continued ex amination of which might reveal distinct properties. Other approaches being investigated include pharmaco logical targeting of the MCSF receptor, which reversed ovariectomy-induced bone loss in rodents.89 In addition, integrin-targeted therapy with RGD mimetics prevented cancellous bone loss in ovariectomized rats90 and increased BMD in women with postmenopausal osteoporosis.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. Genetically modified mice have revealed the involvement of novel molecules and highlighted their invivo relevance, confirmed human mutations and collectively expanded our understanding of the mechanisms governing normal and pathological osteoclast formation and function. In 2009, 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). The causative mutation was not associ ated with genetic loci previously linked to osteopetrosis. Despite closely mapping to the region encoding chloride channels on mouse chromosome 17, no changes in ClC7DNA or protein were observed.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 TGF1 in the migration of bone mesenchymal stem cells following bone resorption.93 Ablated osteoclast function and elevated bone mass were also observed in mice lacking Adora1,94 the putative cannabinoid receptor Gpr5595 and the Bcl2 family member Bcl-xL (also known as Bcl2L1),96 respectively. 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, but these animals demonstrated decreased trabecular bone,
1. Krane, S.M. & Inada, M. Matrix metalloproteinases and bone. Bone 43, 718 (2008). Hertwig, P . Sechs neue mutationen bei der hausmaus in ihrer bedeutung fr allgemeine vererbungsfragen. Z. Menschl. Vererb. Konstitutionsl. 26, 121 (1942). 3.

enhanced osteoclastogenesis97 and increased lifespan of mature resorbing cells.98 Altered osteoclast biology can also be observed in genetically modified mice lacking the DNA-modifying proteins ID199 and NAD-dependent deacetylase sirtuin 1 (SIRT1)100 or the microRNA-related endoribonuclease, Dicer.101 The discovery of new molecular mechanisms and physio logical pathways inevitably raises further un answered questions (Box1). Although osteoclast differen tiation and commitment from precursor cells of the monocyte macrophage lineage is well established, the identification and functional characterization of specific subgroups within these populations is only beginning. As we continue to explore the role of osteoclasts in the biology of bone, 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. Instead, we now appreciate that osteoclasts are complex cells that are fully integrated into the bone marrow microenvironment. 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.
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, mouse/murine model, genetic mutation, knockout, transgenic, osteopetrosis and osteoporosis. The authors focused mainly on well-established and validated studies.

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Author contributions J.R. Edwards and G.R. Mundy discussed article content. J.R. Edwards researched data, wrote the article and revised/edited the manuscript during submission.

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