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The modeling and remodeling of bone requires activation and polarization of osteoclasts, achieved by reorganization of the cytoskeleton. Members of the Rho subfamily of small GTPases, including Cdc42, are known regulators of cytoskeletal components, but the role of these proteins in bone physiology and pathophysiology remains unclear. Here, we examined loss-of-function mice in which Cdc42 was selectively ablated in differentiated osteoclasts and gain-of-function animals wherein Cdc42Gap, a protein that inactivates the small GTPase, was deleted globally. Cdc42 loss-of-function mice were osteopetrotic and resistant to ovariectomy-induced bone loss, while gain-of-function animals were osteoporotic. Isolated Cdc42-deficient osteoclasts displayed suppressed bone resorption, while osteoclasts with increased Cdc42 activity had enhanced resorptive capacity. We further demonstrated that Cdc42 modulated M-CSF-stimulated cyclin D expression and phosphorylation of Rb and induced caspase 3 and Bim, thus contributing to osteoclast proliferation and apoptosis rates. Furthermore, Cdc42 was required for multiple M-CSF- and RANKL-induced osteoclastogenic signals including activation and expression of the differentiation factors MITF and NFATc1 and was a component of the Par3/Par6/atypical PKC polarization complex in osteoclasts. These data suggest that Cdc42 regulates osteoclast formation and function and may represent a promising therapeutic target for prevention of pathological bone loss.

The pathogenesis of glucocorticoid-induced (GC-induced) bone loss is unclear. For example, osteoblast apoptosis is enhanced by GCs in vivo, but they stimulate bone formation in vitro. This conundrum suggests that an intermediary cell transmits a component of the bone-suppressive effects of GCs to osteoblasts in the intact animal. Bone remodeling is characterized by tethering of the activities of osteoclasts and osteoblasts. Hence, the osteoclast is a potential modulator of the effect of GCs on osteoblasts. To define the direct impact of GCs on bone-resorptive cells, we compared the effects of dexamethasone (DEX) on WT osteoclasts with those derived from mice with disruption of the GC receptor in osteoclast lineage cells (GRoc-/- mice). While the steroid prolonged longevity of osteoclasts, their bone-degrading capacity was suppressed. The inhibitory effect of DEX on bone resorption reflects failure of osteoclasts to organize their cytoskeleton in response to M-CSF. DEX specifically arrested M-CSF activation of RhoA, Rac, and Vav3, each of which regulate the osteoclast cytoskeleton. In all circumstances GRoc-/- mice were spared the impact of DEX on osteoclasts and their precursors. Consistent with osteoclasts modulating the osteoblast-suppressive effect of DEX, GRoc-/- mice are protected from the steroid's inhibition of bone formation.

Skin and bone - what comes to mind at hearing this phrase? While certainly a metaphor for disease, it also defines two very different tissues, one a flexible and contiguous outer covering, the other a morphologically diverse hard tissue distributed at over 200 sites in the body. As the accompanying series of Reviews highlights, these tissues are indeed diverse, but there are also surprising similarities. Skin is the interface between the internal organs and the environment, and as such plays a crucial role in the body's defense mechanism. The skin and its many appendages are responsible for functions as diverse as epidermal barrier and defense, immune surveillance, UV protection, thermoregulation, sweating, lubrication, pigmentation, the sensations of pain and touch, and, importantly, the protection of various stem cell niches in the skin. Bone serves a number of purposes: it provides protection for vital organs, a lever for locomotion, a reservoir for calcium, and the site of adult hematopoiesis. The tissue is composed of osteoblasts, osteoclasts, and their individual precursors plus a complex mixture of mesenchymal, myeloid, and lymphoid cells in the marrow space. Finally, the endothelial microenvironment provides nutrition and is a conduit for the influx and emigration of cells that impact bone biology in several important ways. This Review series guides the reader through these various facets of 2 diverse, yet interdependent, tissues.

TNF-alpha is the dominant cytokine in inflammatory osteolysis. Using mice whose BM stromal cells and osteoclast precursors are chimeric for the presence of TNF receptors, we found that both cell types mediated the cytokine's osteoclastogenic properties. The greater contribution was made, however, by stromal cells that express the osteoclastogenic cytokine M-CSF. TNF-alpha stimulated M-CSF gene expression, in vivo, only in the presence of TNF-responsive stromal cells. M-CSF, in turn, induced the key osteoclastogenic cytokine receptor, receptor activator of NF-kappaB (RANK), in osteoclast precursors. In keeping with the proproliferative and survival properties of M-CSF, TNF-alpha enhanced osteoclast precursor number only in the presence of stromal cells bearing TNF receptors. To determine the clinical relevance of these observations, we induced inflammatory arthritis in wild-type mice and treated them with a mAb directed against the M-CSF receptor, c-Fms. Anti-c-Fms mAb selectively and completely arrested the profound pathological osteoclastogenesis attending this condition, the significance of which is reflected by similar blunting of the in vivo bone resorption marker tartrate-resistant acid phosphatase 5b (TRACP 5b). Confirming that inhibition of the M-CSF signaling pathway targets TNF-alpha, anti-c-Fms also completely arrested osteolysis in TNF-injected mice with nominal effect on macrophage number. M-CSF and its receptor, c-Fms, therefore present as candidate therapeutic targets in states of inflammatory bone erosion.

TNF receptor-associated factor 6 (TRAF6) associates with the cytoplasmic domain of receptor activator of NF-kappaB (RANK). This event is central to normal osteoclastogenesis. We discovered that TRAF6 also interacts with FHL2 (four and a half LIM domain 2), a LIM domain--only protein that functions as a transcriptional coactivator or corepressor in a cell-type--specific manner. FHL2 mRNA and protein are undetectable in marrow macrophages and increase pari passu with osteoclast differentiation in vitro. FHL2 inhibits TRAF6-induced NF-kappaB activity in wild-type osteoclast precursors and, in keeping with its role as a suppressor of TRAF6-mediated RANK signaling, TRAF6/RANK association is enhanced in FHL2-/- osteoclasts. FHL2 overexpression delays RANK ligand-induced (RANKL-induced) osteoclast formation and cytoskeletal organization. Interestingly, osteoclast-residing FHL2 is not detectable in naive wild-type mice, in vivo, but is abundant in those treated with RANKL and following induction of inflammatory arthritis. Reflecting increased RANKL sensitivity, osteoclasts generated from FHL2-/- mice reach maturation and optimally organize their cytoskeleton earlier than their wild-type counterparts. As a consequence, FHL2-/- osteoclasts are hyperresorptive, and mice lacking the protein undergo enhanced RANKL and inflammatory arthritis-stimulated bone loss. FHL2 is, therefore, an antiosteoclastogenic molecule exerting its effect by attenuating TRAF6-mediated RANK signaling.

TNF-induced receptor activator NF-kappaB ligand (RANKL) synthesis by bone marrow stromal cells is a fundamental component of inflammatory osteolysis. We found that this process was abolished by IL-1 receptor antagonist (IL-1Ra) or in stromal cells derived from type I IL-1 receptor-deficient (IL-1RI-deficient) mice. Reflecting sequential signaling of the cytokines TNF and IL-1, TNF induces stromal cell expression of IL-1 and IL-1RI. These data suggest that TNF regulates RANKL expression via IL-1, and, therefore, IL-1 plays a role in TNF-induced periarticular osteolysis. Consistent with this posture, TNF-stimulated osteoclastogenesis in cultures consisting of WT marrow macrophages and stromal cells exposed to IL-1Ra or in cocultures established with IL-1RI-deficient stromal cells was reduced approximately 50%. The same magnitude of osteoclast inhibition occurred in IL-1RI-deficient mice following TNF administration in vivo. Like TNF, IL-1 directly targeted osteoclast precursors and promoted the osteoclast phenotype in a TNF-independent manner in the presence of permissive levels of RANKL. IL-1 is able to induce RANKL expression by stromal cells and directly stimulate osteoclast precursor differentiation under the aegis of p38 MAPK. Thus, IL-1 mediates the osteoclastogenic effect of TNF by enhancing stromal cell expression of RANKL and directly stimulating differentiation of osteoclast precursors.

Highly active antiretroviral therapy (HAART), which includes HIV protease inhibitors (PIs), has been associated with bone demineralization. To determine if this complication reflects accelerated resorptive activity, we studied the impact of two common HIV PIs, ritonavir and indinavir, on osteoclast formation and function. Surprisingly, we find that ritonavir, but not indinavir, inhibits osteoclast differentiation in a reversible manner and also abrogates bone resorption by disrupting the osteoclast cytoskeleton, without affecting cell number. Ritonavir given in vivo completely blunts parathyroid hormone-induced osteoclastogenesis in mice, which confirms that the drug is bone sparing. In keeping with its antiresorptive properties, ritonavir impairs receptor activator of nuclear factor kappaB ligand-induced (RANKL-induced) activation of NF-kappaB and Akt signaling pathways, both critical to osteoclast formation and function. In particular, ritonavir is found to inhibit RANKL-induced Akt signaling by disrupting the recruitment of TNF receptor-associated factor 6/c-Src complex to lipid rafts. Thus, ritonavir may represent a bone-sparing PI capable of preventing development of osteopenia in patients currently on HAART.

beta(3) integrin-null osteoclasts are dysfunctional, but their numbers are increased in vivo. In vitro, however, the number of beta(3)(-/-) osteoclasts is reduced because of arrested differentiation. This paradox suggests cytokine regulation of beta(3)(-/-) osteoclastogenesis differs in vitro and in vivo. In vitro, additional MCSF, but not receptor activator of NF-kappaB ligand (RANKL), completely rescues beta(3)(-/-) osteoclastogenesis. Similarly, activation of extracellular signal-regulated kinases (ERKs) and expression of c-Fos, both essential for osteoclastogenesis, are attenuated in beta(3)(-/-) preosteoclasts, but completely restored by additional MCSF. In fact, circulating and bone marrow cell membrane-bound MCSFs are enhanced in beta(3)(-/-) mice, correlating with the increase in the osteoclast number. To identify components of the MCSF receptor that is critical for osteoclastogenesis in beta(3)(-/-) cells, we retrovirally transduced authentic osteoclast precursors with chimeric c-Fms constructs containing various cytoplasmic domain mutations. Normalization of osteoclastogenesis and ERK activation, in beta(3)(-/-) cells, uniquely requires c-Fms tyrosine 697. Finally, like high-dose MCSF, overexpression of c-Fos normalizes the number of beta(3)(-/-) osteoclasts in vitro, but not their ability to resorb dentin. Thus, while c-Fms and alpha(v)beta(3) collaborate in the osteoclastogenic process via shared activation of the ERK/c-Fos signaling pathway, the integrin is essential for matrix degradation.

RANK, the receptor activator of NF-kappaB, and its ligand RANKL (initially termed TRANCE, also termed ODF and OPGL), are a TNF superfamily receptor-ligand pair that govern the development and function of osteoclasts, lymphoid tissue, and mammary epithelium. While TNF family cytokines share a common structural scaffold, individual receptor-ligand pairs associate with high specificity. Given the low level of amino acid conservation among members of the TNF superfamily, the means by which these molecules achieve specificity cannot be completely understood without knowledge of their three-dimensional structures. To determine the elements of RANKL that mediate RANK activation, we have crystallized the ectodomain of murine RANKL and solved its structure to a resolution of 2.6 A. RANKL self-associates as a homotrimer with four unique surface loops that distinguish it from other TNF family cytokines. Mutagenesis of selected residues in these loops significantly modulates RANK activation, as evidenced by in vitro osteoclastogenesis, thereby establishing their necessity in mediating the biological activities of RANKL. Such structural determinants of RANKL-RANK specificity may be of relevance in the pharmacologic design of compounds to ameliorate osteopenic disorders of bone.

Osteoclastic bone resorption requires cell-matrix contact, an event mediated by the alpha v beta 3 integrin. The structural components of the integrin that mediate osteoclast function are, however, not in hand. To address this issue, we generated mice lacking the beta 3 integrin gene, which have dysfunctional osteoclasts. Here, we show the full rescue of beta 3(-/-) osteoclast function following expression of a full-length beta 3 integrin. In contrast, truncated beta 3, lacking a cytoplasmic domain (h beta 3c), is completely ineffective in restoring function to beta 3(-/-) osteoclasts. To identify the components of the beta 3 cytoplasmic domain regulating osteoclast function, we generated six point mutants known, in other circumstances, to mediate beta integrin signaling. Of the six, only the S(752)P substitution, which also characterizes a form of the human bleeding disorder Glanzmann's thrombasthenia, fails to rescue beta 3(-/-) osteoclasts or restore ligand-activated signaling in the form of c-src activation. Interestingly, the double mutation Y(747)F/Y(759)F, which disrupts platelet function, does not affect the osteoclast. Thus similarities and distinctions exist in the mechanisms by which the beta 3 integrin regulates platelets and osteoclasts.

While TNF-alpha is pivotal to the pathogenesis of inflammatory osteolysis, the means by which it recruits osteoclasts and promotes bone destruction are unknown. We find that a pure population of murine osteoclast precursors fails to undergo osteoclastogenesis when treated with TNF-alpha alone. In contrast, the cytokine dramatically stimulates differentiation in macrophages primed by less than one percent of the amount of RANKL (ligand for the receptor activator of NF-kappaB) required to induce osteoclast formation. Mirroring their synergistic effects on osteoclast differentiation, TNF-alpha and RANKL markedly potentiate NF-kappaB and stress-activated protein kinase/c-Jun NH(2)-terminal kinase activity, two signaling pathways essential for osteoclastogenesis. In vivo administration of TNF-alpha prompts robust osteoclast formation in chimeric animals in which ss-galactosidase positive, TNF-responsive macrophages develop within a TNF-nonresponsive stromal environment. Thus, while TNF-alpha alone does not induce osteoclastogenesis, it does so both in vitro and in vivo by directly targeting macrophages within a stromal environment that expresses permissive levels of RANKL. Given the minuscule amount of RANKL sufficient to synergize with TNF-alpha to promote osteoclastogenesis, TNF-alpha appears to be a more convenient target in arresting inflammatory osteolysis.

Osteoclasts express the alphavbeta3 integrin, an adhesion receptor that has been implicated in bone resorption and that is therefore a potential therapeutic target. To assess the role of this heterodimer in skeletal development in vivo, we engineered mice in which the gene for the beta3 integrin subunit was deleted. Bone marrow macrophages derived from these mutants differentiate in vitro into numerous osteoclasts, thus establishing that alphavbeta3 is not necessary for osteoclast recruitment. Furthermore, the closely related integrin, alphavbeta5, does not substitute for alphavbeta3 during cytokine stimulation or authentic osteoclastogenesis. beta3 knockout mice, but not their heterozygous littermates, develop histologically and radiographically evident osteosclerosis with age. Despite their increased bone mass, beta3-null mice contain 3.5-fold more osteoclasts than do heterozygotes. These mutant osteoclasts are, however, dysfunctional, as evidenced by their reduced ability to resorb whale dentin in vitro and the significant hypocalcemia seen in the knockout mice. The resorptive defect in beta3-deficient osteoclasts may reflect absence of matrix-derived intracellular signals, since their cytoskeleton is distinctly abnormal and they fail to spread in vitro, to form actin rings ex vivo, or to form normal ruffled membranes in vivo. Thus, although it is not required for osteoclastogenesis, the integrin alphavbeta3 is essential for normal osteoclast function.

Central to the bone-sparing effect of estrogen (E(2)) is its ability to block the monocytic production of the osteoclastogenic cytokine TNF-alpha (TNF). However, the mechanism by which E(2) downregulates TNF production is presently unknown. Transient transfection studies in HeLa cells, an E(2) receptor-negative line, suggest that E(2) inhibits TNF gene expression through an effect mediated by estrogen receptor beta (ERbeta). We also report that in RAW 264.7 cells, an E(2) receptor-positive murine monocytic line, E(2) downregulates cytokine-induced TNF gene expression by decreasing the activity of the Jun NH(2)-terminal kinase (JNK). The resulting diminished phosphorylation of c-Jun and JunD at their NH(2)-termini decreases the ability of these nuclear proteins to autostimulate the expression of the c-Jun and JunD genes, thus leading to lower production of c-Jun and JunD. The consequent decrease in the nuclear levels of c-Jun and JunD leads to diminished binding of c-Jun/c-Fos and JunD/c-Fos heterodimers to the AP-1 consensus sequence in the TNF promoter and, thus, to decreased transactivation of the TNF gene.

beta3 integrins have been implicated in a wide variety of functions, including platelet aggregation and thrombosis (alphaIIbbeta3) and implantation, placentation, angiogenesis, bone remodeling, and tumor progression (alphavbeta3). The human bleeding disorder Glanzmann thrombasthenia (GT) can result from defects in the genes for either the alphaIIb or the beta3 subunit. In order to develop a mouse model of this disease and to further studies of hemostasis, thrombosis, and other suggested roles of beta3 integrins, we have generated a strain of beta3-null mice. The mice are viable and fertile, and show all the cardinal features of GT (defects in platelet aggregation and clot retraction, prolonged bleeding times, and cutaneous and gastrointestinal bleeding). Implantation appears to be unaffected, but placental defects do occur and lead to fetal mortality. Postnatal hemorrhage leads to anemia and reduced survival. These mice will allow analyses of the other suggested functions of beta3 integrins and we report that postnatal neovascularization of the retina appears to be beta3-integrin-independent, contrary to expectations from inhibition experiments.

Central to the pathogenesis of osteoporosis is the ability of estrogen deficiency to increase osteoclast formation by enhancing stromal cell production of the osteoclastogenic cytokine macrophage colony-stimulating factor (M-CSF). We report that stromal cells from ovariectomized mice exhibit increased casein kinase II-dependent phosphorylation of the nuclear protein Egr-1. Phosphorylated Egr-1 binds less avidly to the transcriptional activator Sp-1 and the resulting higher levels of free Sp-1 stimulate transactivation of the M-CSF gene. Estrogen replacement fails to block M-CSF mRNA expression and osteoclast formation in ovariectomized mice lacking Egr-1, confirming the critical role played by this transcription factor in mediating the antiosteoclastogenic effects of estrogen. Thus, by downregulating formation of a novel Egr-1/Sp-1 complex in stromal cells, estrogen deficiency results in enhanced levels of free Sp-1 and increased M-CSF gene expression and osteoclast formation.

Chronic bone infection, as attends periodontitis, is often complicated by severe osteolysis. While LPS is believed to be central to the pathogenesis of the osteolytic lesion, the mechanisms by which this bacteria-derived molecule promotes bone resorption are unknown. We find that LPS induces bone marrow macrophages (BMMs) to express c-src, a protooncogene product that we demonstrate is a specific marker of commitment to the osteoclast phenotype. We next turned to possible soluble mediators of LPS-induced c-src. Of a number of osteoclastogenic cytokines tested, only TNF-alpha mirrors the c-src-enhancing effect of LPS. Suggesting that LPS augmentation of c-src is TNF-mediated, endotoxin sequentially induces BMM expression of TNF, followed by c-src. TNF and c-src expression, by cultured BMMs derived from LPS-injected mice, reflects duration of exposure to circulating endotoxin, intimating that endotoxin's effect in vivo is also mediated by TNF. Consistent with these findings, thalidomide (which antagonizes TNF action) attenuates c-src induction by LPS. An anti-TNF antibody blocks LPS enhancement of c-src mRNA, validating the cytokine's modulating role in vitro. Using BMMs of TNF receptor-deleted mice, we demonstrate that TNF induction of c-src is transmitted through the cytokine's p55, but not p75, receptor. Most importantly, LPS administered to wild-type mice prompts osteoclast precursor differentiation, manifest by profound osteoclastogenesis in marrow cultured ex vivo, and by a profusion of marrow-residing cells expressing the osteoclast marker tartrate resistant acid phosphatase, in vivo. In contrast, LPS does not substantially enhance osteoclast proliferation in mice lacking the p55TNF receptor, confirming that LPS-induced osteoclastogenesis is mediated by TNF in vivo via this receptor. Thus, therapy targeting TNF and/or its p55 receptor presents itself as a means of preventing the osteolysis of chronic bacterial infection.

Osteoclastic bone degradation requires intimacy between the matrix and the resorptive cell. While the precise role the integrin alpha(v)beta3 plays in the process is not yet understood, occupancy of the heterodimer by soluble ligand or by blocking antibody effectively inhibits bone resorption in vitro and in vivo, suggesting that alpha(v)beta3 blockade may prevent postmenopausal osteoporosis. Thus, we identified a synthetic chemical peptide mimetic, beta-[2-[[5-[(aminoiminomethyl)amino]-1-oxopentyl]amino]-1-+ ++oxoethyl]amino-3-pyridinepropanoic acid, bistrifluoroacetate (SC56631) based upon the alpha(v)beta3 ligand, Arg-Gly-Asp (RGD), which recognizes the isolated integrin, and its relative, alpha(v)beta5, as effectively as does the natural peptide. The mimetic dampens osteoclastic bone resorption in vitro and in vivo. Most importantly, intravenous administration of the mimetic prevents the 55% loss of trabecular bone sustained by rats within 6 wk of oophorectomy. Histological examination of bones taken from SC56631-treated, oophorectomized animals also demonstrates the compound's bone sparing properties and its capacity to decrease osteoclast number. Thus, an RGD mimetic prevents the rapid bone loss that accompanies estrogen withdrawal.