Substrate recognition by osteoclast precursors induces C-src/microtubule association.

The osteoclast is distinguished from other macrophage polykaryons by its polarization, a feature induced by substrate recognition. The most striking component of the polarized osteoclast is its ruffled membrane, probably reflecting insertion of intracellular vesicles into the bone apposed plasmalemma. The failure of osteoclasts in c-src-/- osteopetrotic mice to form ruffled membranes indicates pp60(c-src) (c-src) is essential to osteoclast polarization. Interestingly, c-src itself is a vesicular protein that targets the ruffled membrane. This being the case, we hypothesized that matrix recognition by osteoclasts, and their precursors, induces c-src to associate with microtubules that traffic proteins to the cell surface. We find abundant c-src associates with tubulin immunoprecipitated from avian marrow macrophages (osteoclast precursors) maintained in the adherent, but not nonadherent, state. Since the two proteins colocalize only within adherent avian osteoclast-like cells examined by double antibody immunoconfocal microscopy, c-src/tubulin association reflects an authentic intracellular event. C-src/tubulin association is evident within 90 min of cell-substrate recognition, and the event does not reflect increased expression of either protein. In vitro kinase assay demonstrates tubulin-associated c-src is enzymatically active, phosphorylating itself as well as exogenous substrate. The increase in microtubule-associated kinase activity attending adhesion mirrors tubulin-bound c-src and does not reflect enhanced specific activity. The fact that microtubule-dissociating drugs, as well as cold, prevent adherence-induced c-src/tubulin association indicates the protooncogene complexes primarily, if not exclusively, with polymerized tubulin. Association of the two proteins does not depend upon protein tyrosine phosphorylation and is substrate specific, as it is induced by vitronectin and fibronectin but not type 1 collagen. Finally, consistent with cotransport of c-src and the osteoclast vacuolar proton pump to the polarized plasmalemma, the H+-ATPase decorates microtubules in a manner similar to the protooncogene, specifically coimmunoprecipitates with c-src from the osteoclast light Golgi membrane fraction, and is present, with c-src, in preparations enriched with acidifying vesicles reconstituted from the osteoclast ruffled membrane.

A role for the alphavbeta3 integrin in the transmigration of monocytes.

The beta2 integrins and intercellular adhesion molecule-1 (ICAM-1) are important for monocyte migration through inflammatory endothelium. Here we demonstrate that the integrin alphavbeta3 is also a key player in this process. In an in vitro transendothelial migration assay, monocytes lacking beta3 integrins revealed weak migratory ability, whereas monocytes expressing beta3 integrins engaged in stronger migration. This migration could be partially blocked by antibodies against the integrin chains alphaL, beta2, alphav, or IAP, a protein functionally associated with alphavbeta3 integrin. Transfection of beta3 integrin chain cDNA into monocytes lacking beta3 integrins resulted in expression of the alphavbeta3 integrin and conferred on these cells an enhanced ability to transmigrate through cell monolayers expressing ICAM-1. These monocytes also engaged in alphaLbeta2-dependent locomotion on recombinant ICAM-1 which was enhanced by alphavbeta3 integrin occupancy. Antibodies against IAP were able to revert this alphavbeta3 integrin-dependent cell locomotion to control levels. Finally, adhesion assays revealed that occupancy of alphavbeta3 integrin could decrease monocyte binding to ICAM-1. In conclusion, we show that alphavbeta3 integrin modulates alphaLbeta2 integrin-dependent monocyte adhesion to and migration on ICAM-1. This could represent a novel mechanism to promote monocyte motility on vascular ICAM-1 and initiate subsequent transendothelial migration.

Lipopolysaccharide-stimulated osteoclastogenesis is mediated by tumor necrosis factor via its P55 receptor.

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.

TNF-alpha induces osteoclastogenesis by direct stimulation of macrophages exposed to permissive levels of RANK ligand.

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.

Crystal structure of the TRANCE/RANKL cytokine reveals determinants of receptor-ligand specificity.

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.

Estrogen decreases TNF gene expression by blocking JNK activity and the resulting production of c-Jun and JunD.

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.

Estrogen blocks M-CSF gene expression and osteoclast formation by regulating phosphorylation of Egr-1 and its interaction with Sp-1.

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.

A Glanzmann's mutation in beta 3 integrin specifically impairs osteoclast function.

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.

A peptidomimetic antagonist of the alpha(v)beta3 integrin inhibits bone resorption in vitro and prevents osteoporosis in vivo.

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.

Mice lacking beta3 integrins are osteosclerotic because of dysfunctional osteoclasts.

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.

Beta3-integrin-deficient mice are a model for Glanzmann thrombasthenia showing placental defects and reduced survival.

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.

Estrogen enhances alpha v beta 3 integrin expression by avian osteoclast precursors via stabilization of beta 3 integrin mRNA.

Although bone resorption is accelerated with menopause, the means by which diminished circulating 17 beta-estradiol (E2) promotes osteoclastic activity are unknown. We hypothesized that since the integrin alpha v beta 3 is essential to the resorptive process, reduced E2 levels may increase the integrin's expression by osteoclast precursors. Thus, avian osteoclast precursors (known to contain E2 receptors) were exposed +/- E2, surface iodinated, and lysed. The lysate was immunoprecipitated with an antibody recognizing the intact alpha v beta 3 heterodimer. We find E2 alone fails to impact on alpha v beta 3 expression. Most importantly, however, picomolar (i.e. post-menopausal), but not nanomolar (i.e. premenopausal) concentrations of E2, when added in conjunction with 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], enhance alpha v beta 3 expression on the plasma membrane of avian osteoclast precursors relative to 1,25(OH)2D3 alone. Induction of alpha v beta 3 by picomolar levels of E2 is dose-dependent, maximizing at 10(-11)-10(-12) M, wherein the sex steroid enhances 1,25-(OH)2-D3-stimulated integrin expression approximately 2.5-fold. Northern analysis reveals that beta 3 mRNA levels parallel those of alpha v beta 3. E2 (10(-12) M) increases expression of beta 3 mRNA induced by a range of 1,25-(OH)2D3 concentrations extending from 10(-10) m-10(-8) M. The E2 + 1,25-(OH)2D3 additive effect on beta 3 mRNA appears as early as 1 day of treatment and progresses for at least 3 days. Consistent with evidence that the beta 3 subunit regulates heterodimer expression, the sex steroid does not impact alpha v mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Granulocyte macrophage-colony stimulating factor reciprocally regulates alphav-associated integrins on murine osteoclast precursors.

The integrins alphavbeta3 and alphavbeta3 are expressed reciprocally during murine osteoclastogenesis in vitro. Specifically, immature osteoclast precursors, in the form of bone marrow macrophages, contain exclusively alphavbeta5, surface expression of which declines with commitment to the osteoclast phenotype, while levels of alphavbeta3 increase concomitantly. The distinct functional significance of alphavbeta5 is underscored by the integrin's capacity, unlike alphavbeta3, to mediate both attachment and spreading on ligand, of marrow macrophages, suggesting alphavbeta3 negotiates initial recognition, by osteoclast precursors, of bone matrix. Northern analysis demonstrates changes in the two beta-subunits, and not alphav, are responsible for these alterations. Treatment of early precursors with granulocyte-macrophage colony stimulating factor (GM-CSF) leads to alterations in beta3 and beta5 mRNA and alphavbeta5 and alphavbeta3, paralleling those occurring during osteoclastogenesis. Nuclear run-on and message stability studies demonstrate that while GM-CSF treatment of precursors alters beta5 transcriptionally, the changes in beta3 arise from prolonged mRNA t1/2. Similar to GM-CSF treatment, the rate of beta5 transcription falls during authentic osteoclastogenesis. In contrast to cytokine-induced alphavbeta3, however, that attending osteoclastogenesis reflects accelerated transcription of the beta3-subunit. Thus, while GM-CSF may participate in modulation of alphavbeta5 during osteoclast differentiation, signals other than those derived from the cytokine must regulate expression of alphavbeta3.

Osteoclasts, macrophages, and the molecular mechanisms of bone resorption.

The osteoclast is a physiological polykaryon and the major if not exclusive resorptive cell of bone. It participates in bone remodeling, repair, and growth and mobilization of mineral to meet homeostatic demands. Most importantly, osteoporosis, a disease endemic in Western society and Asia, is always a reflection of enhanced osteoclastic activity relative to bone formation by osteoblasts. In fact, all forms of anti-osteoporosis therapy proven successful involve inhibition of osteoclastic bone resorption. Bone resorption is regulated either by altering recruitment of osteoclast precursors into fully differentiated resorptive polykaryons or modulating the rate at which mature osteoclasts degrade bone. With this in mind, our laboratory has focused on the molecular mechanisms of osteoclast differentiation and the means by which the cell degrades bone matrix.

Selective culture of primate marrow-derived macrophages in medium devoid of protein additives.

Viable cultures of bone marrow-derived macrophages (BMM phi) from a primate source, the baboon, were maintained for up to 4 weeks in culture in the absence of any exogenous protein in the medium. Baboon peripheral blood monocytes, spleen, lung, and liver M phi s or human BMM phi failed to survive for greater than 4 days. The protein-free BMM phi cultures were morphologically distinctive by virtue of the extremely dendritic appearance of the M phi s. In contrast baboon marrow cultured in the presence of fetal calf serum led to the overgrowth of fibroblastoid cells and in the presence of horse serum produced numbers of giant cells or polykaryocytes in addition to M phi s. The BMM phi were capable of nonimmune phagocytosis of yeast particles, expressed Ia antigen on their surfaces (59%), and were positive cytochemically for nonspecific (alpha-naphthyl acetate) esterase, oil red O, and tartrate resistant acid phosphatase. The addition of sera to established protein-free BMM phi cultures induced a rapid change of shape, viz., retraction of the dendritic processes and rounding up of the M phi s apparent within 10 min. This shape change was not induced by the addition of hemopoietic growth factors granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte CSF (G-CSF), macrophage CSF (M-CSF), or interleukin 3 (IL-3), nor could it be inhibited by the calcium channel blocking agent Nifedipine. Low levels of M-CSF activity, assayed by the murine bone marrow proliferation assay, were detected in the supernatant.

Activity of ornithine decarboxylase and creatine kinase in soft and hard tissue of vitamin D-deficient chicks following parenteral application of 1,25-dihydroxyvitamin D3 or 24R,25-dihydroxyvitamin D3.

We investigated the stimulation of creatine kinase (CK) and ornithine decarboxylase (ODC) by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and 24R,25-dihydroxyvitamin D3 [24R,25(OH)2D3] in doses ranging from 1.625 to 6500 pmol in 4-week-old vitamin D-deficient chicks. Enzyme activities were monitored for 72 h. 1,25(OH)2D3 but not 24R,25(OH)2D3 enhanced the activity of ODC in duodenum and bone. The time course of ODC activity in bone was biphasic, with an increase after 1 h and a higher peak after 24 h. Diaphyses and epiphyses responded equally well after a dose of 6500 pmol. The kidney, liver, and lung showed 1.5-3.8-fold increase in CK activity following 1,25(OH)2D3, reaching a maximum between 3-5 h. However, sustained stimulation of CK activity could still be demonstrated after 72 h, and the 48-h levels in the lung even exceeded the 5-h values. No change of activity of either enzyme was noted in heart and brain after application of 1,25(OH)2D3. There was no coincidence of stimulation of ODC and CK by 1,25(OH)2D3 in the same tissue, and the dose-responsiveness of both enzymes differed considerably. Near maximum activities of ODC were achieved with 19.5 pmol 1,25(OH)2D3 in duodenum and pancreas, while maximum responses of CK occurred in the liver at 195 pmol and in lung and kidney at 6500 pmol. 24R,25(OH)2D3 failed to produce any consistent effects of either enzyme in all tissues examined. These results, particularly the lack of response to 24R,25(OH)2D3, are different from those reported in rats.

Expression and regulation of RAB3 proteins in osteoclasts and their precursors.

The ruffled membrane, the resorptive organelle of the osteoclast, is generated by fusion of intracytoplasmic acidifying vesicles with the plasma membrane, an event analogous to regulated exocytosis. While the ruffled membrane is essential to the bone resorptive process, the mechanisms governing its generation are unknown. However, regulated exocytosis is mediated, in part, by isoforms of the Rab3 subset of Rab GTPases. Because of similarities between exocytosis and ruffled membrane formation, we asked if Rab3 proteins are expressed by osteoclasts or their precursors, and if so, are these molecules regulated by agents known to prompt the osteoclast phenotype? We find murine osteoclast precursors, in the form of bone marrow macrophages (BMMs), express at least two Rab3 isoforms, namely A and B/C, which are individually enhanced by a variety of hematopoietic cytokines. Consistent with the osteoclastogenic properties of a number of these cytokines, differentiation of BMMs into osteoclasts, in vitro, is associated with increased expression of both isoforms, particularly Rab3B/C. Finally, Rab3B/C localizes with the avian osteoclast H+ATPase (vacuolar proton pump) and pp60c-src, both intracellularly and within acidifying vesicles derived largely from the ruffled membrane. Thus, expression of specific rab3 proteins, an event which may control formation of the osteoclast ruffled membrane, is modulated by cytokines during osteoclastogenesis.

The integrin alphavbeta5 is expressed on avian osteoclast precursors and regulated by retinoic acid.

Osteoclasts arise by proliferation, differentiation, and subsequent fusion of marrow-derived precursors, all processes requiring attachment to matrix. Integrins are important mediators of cell-matrix recognition and bone is rich in proteins containing the Arg-Gly-Asp motif, recognized primarily by alphav integrins. Thus, we determined if avian osteoclast precursors express integrins capable of mediating initial attachment to matrix proteins. Early, marrow-derived osteoclast precursors, when first isolated, contain no detectable alphavbeta3, but express an alphav integrin with an 80 kDa associated beta subunit. Immunoprecipitation with an antibody raised against the conserved beta5 cytoplasmic tail sequence indicates the the alphav associated the integrin is alphavbeta5. Retinoic acid is a resorptive steroid, and its exposure to early osteoclast precursors prompts a time- and dose-dependent decrease in alphavbeta5 expression, while simultaneously stimulating alphavbeta3 expression. Northern analysis reveals that retinoic acid decreases beta5 steady-state mRNA, nontranscriptionally, without altering that of alphav. The finding alphavbeta5 expression decreases under the influence of retinoic acid, an osteoclastogenic steroid, while those of alphavbeta3 rise, suggests that these closely related integrins play separate and complementary roles during osteoclast differentiation.

Receptor activator of nuclear factor-kappa b ligand activates nuclear factor-kappa b in osteoclast precursors.

Receptor activator of nuclear factor-kappa B ligand [RANK ligand (RANK-L)] stimulates mature osteoclasts to resorb bone, a process associated with NF-kappa B activation. RANK-L also prompts macrophages to develop the osteoclast phenotype. Although NF-kappa B is essential for osteoclast differentiation, it is not known whether RANK-L activates this transcription complex in osteoclast precursors. We report that RANK-L rapidly induces NF-kappa B activation in both authentic osteoclast precursors, namely bone marrow macrophages, and RAW 264.7 cells, a murine macrophage line also capable of RANK-L-mediated osteoclastogenesis. Supershift studies reveal the RANK-L-induced DNA binding moiety contains p50/p65, the most common NF-kappa B complex. Subcellular translocation of p50 and p65 subunits is confirmed by Western blots and immunofluorescence analysis. RANK-L activates NF-kappa B in both bone marrow macrophages and RAW 264.7 cells by serine phosphorylation of I kappa B alpha within 5 min, resulting in rapid I kappa B alpha degradation and resynthesis. Attesting to function, RANK-L treatment of RAW 264.7 cells transiently transfected with a plasmid containing NF-kappa B consensus elements linked to luciferase greatly enhances reporter activity. Our data suggest that activation of the NF-kappa B pathway is an integral component of RANK-L-induced osteoclast differentiation.

1,25-Dihydroxyvitamin D3 transcriptionally activates the beta 3-integrin subunit gene in avian osteoclast precursors.

Osteoclasts are polykaryons and the principal, if not exclusive, resorptive cell of bone. They are members of the monocyte/macrophage family whose precursors differentiate under the influence of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. Bone resorption is dependent on osteoclast-bone attachment, and we have shown that the integrin alpha v beta 3 is critical to the resorptive process. Thus, we asked whether 1,25-(OH)2D3 enhances the expression of alpha v beta 3 on the surface of osteoclast precursors and if the steroid modulates expression of the beta 3-integrin subunit. We found that 1,25-(OH)2D3 promotes the plasma membrane appearance of alpha v beta 3 on avian bone marrow-derived osteoclast precursors and does so at physiological concentrations (10(-11) M) of the steroid. The effect is time dependent, appearing within 1 day of treatment. A full-length avian cDNA was cloned to explore the molecular mechanisms of beta 3 expression. The deduced amino acid sequence of the cDNA is 81% identical and 89% similar to that of human beta 3. Northern analysis demonstrates that beta 3 mRNA levels in vitamin D-treated osteoclast precursors mirror protein expression. Nuclear run-on experiments document the transcriptional nature of the event.