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Aquaporins (water channels) selectively enhance water permeability of membranes. Since osteoclast differentiation includes a dramatic increase in cell volume, we hypothesize that aquaporin(s) is/are critical for the formation of the multinucleated osteoclast from its mononuclear precursor. Our studies employ two cell models, bone marrow macrophages (BMMs) and the murine macrophage-like cell line, RAW264.7, as osteoclast precursors. Receptor activator of nuclear factor kappaB (NF-kappaB) ligand (RANKL) and macrophage-colony-stimulating factor or RANKL alone were used to induce osteoclast differentiation in BMMs or RAW264.7 cells, respectively. We first used qualitative reverse transcription (RT)-PCR to examine which of the aquaporins are expressed in osteoclasts and in their precursor cells. Out of the 10 aquaporins examined, only aquaporin 9 (AQP9) was expressed in osteoclast-lineage cells. AQP9 has unique aqueous pore properties mediating the passage of a wide variety of non-charged solutes in addition to water. Western analyses using specific antibodies revealed a higher AQP9 level in RANKL-treated than in untreated cells. Quantitative real-time RT-PCR analyses also demonstrated higher AQP9 mRNA levels in RANKL-treated cells. Finally, we examined the effect of phloretin, an AQP9 inhibitor, on RANKL-induced osteoclast differentiation. Cells were incubated with RANKL for 5 days, and phloretin was added for the last 2 days, when most fusion occurs. A dramatic reduction in osteoclast size and in the number of nuclei per osteoclast was observed in cultures containing phloretin. The inhibitor did not have a significant effect on the number and size of mononuclear phagocytes in cultures not treated with RANKL. Our results suggest a role for AQP9 in osteoclast differentiation, specifically in the fusion process.

Nitric oxide (NO) is a multifunctional signaling molecule and a key vasculoprotective and potential osteoprotective factor. NO regulates normal bone remodeling and pathological bone loss in part through affecting the recruitment, formation, and activity of bone-resorbing osteoclasts. Using murine RAW 264.7 and primary bone marrow cells or osteoclasts formed from them by receptor activator of NF-kappaB ligand (RANKL) differentiation, we found that inducible nitric-oxide synthase (iNOS) expression and NO generation were stimulated by interferon (IFN)-gamma or lipopolysaccharide, but not by interleukin-1 or tumor necrosis factor-alpha. Surprisingly, iNOS expression and NO release were also triggered by RANKL. This response was time- and dose-dependent, required NF-kappaB activation and new protein synthesis, and was specifically blocked by the RANKL decoy receptor osteoprotegerin. Preventing RANKL-induced NO (via iNOS-selective inhibition or use of marrow cells from iNOS-/- mice) increased osteoclast formation and bone pit resorption, indicating that such NO normally restrains RANKL-mediated osteoclastogenesis. Additional studies suggested that RANKL-induced NO inhibition of osteoclast formation does not occur via NO activation of a cGMP pathway. Because IFN-beta is also a RANKL-induced autocrine negative feedback inhibitor that limits osteoclastogenesis, we investigated whether IFN-beta is involved in this novel RANKL/iNOS/NO autoregulatory pathway. IFN-beta was induced by RANKL and stimulated iNOS expression and NO release, and a neutralizing antibody to IFN-beta inhibited iNOS/NO elevation in response to RANKL, thereby enhancing osteoclast formation. Thus, RANKL-induced IFN-beta triggers iNOS/NO as an important negative feedback signal during osteoclastogenesis. Specifically targeting this novel autoregulatory pathway may provide new therapeutic approaches to combat various osteolytic bone diseases.

Dendritic cells (DCs) display the unique ability to activate naive T cells and to initiate primary T cell responses revealed in DC-T cell alloreactions. DCs frequently operate under stress conditions. Oxidative stress enhances the production of inflammatory cytokines by DCs. We performed a proteomic analysis to see which major changes occur, at the protein expression level, during DC differentiation and maturation. Comparative two-dimensional gel analysis of the monocyte, immature DC, and mature DC stages was performed. Manganese superoxide dismutase (Mn-SOD) reached 0.7% of the gel-displayed proteins at the mature DC stage. This important amount of Mn-SOD is a primary antioxidant defense system against superoxide radicals, but its product, H(2)O(2), is also deleterious for cells. Peroxiredoxin (Prx) enzymes play an important role in eliminating such peroxide. Prx1 expression level continuously increased during DC differentiation and maturation, whereas Prx6 continuously decreased, and Prx2 peaked at the immature DC stage. As a consequence, DCs were more resistant than monocytes to apoptosis induced by high amounts of oxidized low density lipoproteins containing toxic organic peroxides and hydrogen peroxide. Furthermore DC-stimulated T cells produced high levels of receptor activator of nuclear factor kappaB ligand, a chemotactic and survival factor for monocytes and DCs. This study provides insights into the original ability of DCs to express very high levels of antioxidant enzymes such as Mn-SOD and Prx1, to detoxify oxidized low density lipoproteins, and to induce high levels of receptor activator of nuclear factor kappaB ligand by the T cells they activate and further emphasizes the role that DCs might play in atherosclerosis, a pathology recognized as a chronic inflammatory disorder.

Because of its ability to suppress tumor cell proliferation, angiogenesis, and inflammation, the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) is currently in clinical trials. How SAHA mediates its effects is poorly understood. We found that in several human cancer cell lines, SAHA potentiated the apoptosis induced by tumor necrosis factor (TNF) and chemotherapeutic agents and inhibited TNF-induced invasion and receptor activator of NF-kappaB ligand-induced osteoclastogenesis, all of which are known to require NF-kappaB activation. These observations corresponded with the down-regulation of the expression of anti-apoptotic (IAP1, IAP2, X chromosome-linked IAP, Bcl-2, Bcl-x(L), TRAF1, FLIP, and survivin), proliferative (cyclin D1, cyclooxygenase 2, and c-Myc), and angiogenic (ICAM-1, matrix metalloproteinase-9, and vascular endothelial growth factor) gene products. Because several of these genes are regulated by NF-kappaB, we postulated that SAHA mediates its effects by modulating NF-kappaB and found that SAHA suppressed NF-kappaB activation induced by TNF, IL-1beta, okadaic acid, doxorubicin, lipopolysaccharide, H(2)O(2), phorbol myristate acetate, and cigarette smoke; the suppression was not cell type-specific because both inducible and constitutive NF-kappaB activation was inhibited. We also found that SAHA had no effect on direct binding of NF-kappaB to the DNA but inhibited sequentially the TNF-induced activation of IkappaBalpha kinase, IkappaBalpha phosphorylation, IkappaBalpha ubiquitination, IkappaBalpha degradation, p65 phosphorylation, and p65 nuclear translocation. Furthermore, SAHA inhibited the NF-kappaB-dependent reporter gene expression activated by TNF, TNFR1, TRADD, TRAF2, NF-kappaB-inducing kinase, IkappaBalpha kinase, and the p65 subunit of NF-kappaB. Overall, our results indicated that NF-kappaB and NF-kappaB-regulated gene expression inhibited by SAHA can enhance apoptosis and inhibit invasion and osteoclastogenesis.

Receptor activator of NF-kappaB (RANK) ligand (RANKL) and its receptor RANK play an essential role in osteoclastogenesis and osteoclast function by activating several signaling pathways. However, several lines of evidence suggest that RANK also transmits an unidentified signaling pathway(s) essential for osteoclastogenesis. To identify the novel pathway(s), we carried out a detailed structure/function study of the RANK cytoplasmic domain. A series of studies using numerous deletion/point mutants elucidated a specific 4-amino acid motif (535IVVY538) essential for osteoclastogenesis. This novel motif plays a crucial role in committing macrophages to the osteoclast lineage but is not implicated in osteoclast function or survival. Moreover, this motif does not activate the known RANK signaling pathways, indicating that it initiates a novel pathway(s). The identification of the novel motif not only provides critical insight into RANK signaling in osteoclastogenesis, but more importantly, the RANK motif and its downstream signaling pathways may represent specific therapeutic targets for various bone diseases, including postmenopausal osteoporosis.

Tumor necrosis factor (TNF) plays an important role in the pathogenesis of inflammatory bone loss through stimulation of osteoclastic bone resorption and inhibition of osteoblastic bone formation. Compared with the well established role of TNF in osteoclastogenesis, mechanisms by which TNF inhibits osteoblast function have not been fully determined. Runx2 is an osteoblast-specific transcription factor whose steady-state protein levels are regulated by proteasomal degradation, mediated by the E3 ubiquitin ligases, Smurf1 and Smurf2. We hypothesized that TNF inhibits osteoblast function through Smurf-mediated Runx2 degradation. We treated C2C12 and 2T3 osteoblast precursor cell lines and primary osteoblasts with TNF and found that TNF, but not interleukin-1, significantly increased Smurf1 and Smurf2 expression. TNF increased the degradation of endogenous or transfected Runx2 protein, which was blocked by treating cells with a proteasomal inhibitor or by infecting cells with small interfering (si)RNA against Smurf1 or Smurf2. TNF inhibited the expression of bone morphogenetic protein and transforming growth factor-beta signaling reporter constructs, and the inhibition of each was blocked by Smurf1 siRNA and Smurf2 siRNA, respectively. Overexpression of Smurf1 and/or Smurf2 siRNAs prevented the inhibitory effect of TNF on Runx2 reporter. Consistent with these in vitro findings, bones from TNF transgenic mice or TNF-injected wild type mice had increased Smurf1 and decreased Runx2 protein levels. We propose that one of the mechanisms by which TNF inhibits bone formation in inflammatory bone disorders is by promoting Runx2 proteasomal degradation through up-regulation of Smurf1 and Smurf2 expression.

Interleukin (IL)-4 and IL-13 are cytokines that inhibit bone resorption. Data showing an inhibitory effect of IL-4 and IL-13 on RANK mRNA in mouse calvariae were first reported at the 22nd American Society for Bone and Mineral Research Meeting (Lerner, U.H., and Conaway, H. H. 2000) J. Bone Min. Res. 15, Suppl. 1, Abstr. SU 230). In the present study, release of 45Ca from cultured mouse calvarial bones stimulated by different cytokines, peptides, and steroid hormones was inhibited by IL-4 and IL-13. IL-4 and IL-13 decreased receptor activator of nuclear factor-kappaB ligand (RANKL) and RANK mRNA and increased osteoprotegerin (OPG) mRNA in calvariae. Additionally, the cytokines decreased RANKL protein and increased OPG protein in calvarial bones. In osteoblasts isolated from calvariae, both an increase in RANKL mRNA and a decrease in OPG mRNA and protein elicited by vitamin D3 were reversed by IL-4 and IL-13. IL-4 and IL-13 decreased the number of tartrate-resistant acid phosphatase positive multinucleated cells and the mRNA expression of calcitonin receptor, tartrate-resistant acid phosphatase, and cathepsin K in mouse spleen cells and bone marrow macrophages (BMM) treated with macrophage colony-stimulating factor and RANKL. Inhibition of mRNA for RANK and the transcription factor NFAT2 was also noted in spleen cell and BMM cultures treated with IL-4 and IL-13. In addition, RANK mRNA and RANK protein were decreased by IL-4 and IL-13 in RAW 264.7 cells. Osteoblasts, spleen cells, and BMM expressed mRNA for the four proteins making up the IL-4 and IL-13 receptors. No effects by IL-4 on bone resorption and osteoclast formation or on RANKL and RANK mRNA expression were seen in Stat6-/- mice. The data indicate that IL-4 and IL-13, via a STAT6-dependent pathway, inhibit osteoclast differentiation and bone resorption by activating receptors on osteoblasts and osteoclasts that affect the RANKL/RANK/OPG system.

Mechanical input is known to regulate bone remodeling, yet the molecular events involved in mechanical signal transduction are poorly understood. We here investigate proximal events leading to the ERK1/2 activation that is required for mechanical repression of RANKL (receptor activator of NF-kappaB ligand) expression, the factor that controls local recruitment of osteoclasts. Using primary murine bone stromal cells we show that dynamic mechanical strain via substrate deformation activates Ras-GTPase, in particular the H-Ras isoform. Pharmacological inhibition of H-Ras function prevents strain activation of H-Ras as well as the downstream mechanical repression of RANKL. Furthermore, small interfering RNA silencing of H-Ras, but not K-Ras, abrogates mechanical strain repression of RANKL. H-Ras-specific inhibition of mechanorepression of RANKL was also demonstrated in a murine pre-osteoblast cell line (CIMC-4). The requirement of cholesterol for H-Ras activation was probed; cholesterol depletion of rafts using methyl-betacyclodextrin prevented mechanical H-Ras activation. That the mechanical repression of RANKL requires activation of H-Ras, a specific isoform of Ras-GTP that is known to reside in the lipid raft microdomain, suggests that spatial arrangements are critical for generation of specific downstream events in response to mechanical signals. By partitioning signals this way, cells may be able to generate different downstream responses through seemingly similar signaling cascades.

MCP-1 (monocyte chemotactic protein-1) is a CC chemokine that is induced by receptor activator of NFkappaB ligand (RANKL) in human osteoclasts. In the absence of RANKL, treatment of human peripheral blood mononuclear cells with macrophage colony-stimulating factor and MCP-1 resulted in tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cells that are positive for calcitonin receptor (CTR) and a number of other osteoclast markers, including nuclear factor of activated t cells, cytoplasmic, calcineurin-dependent 1 (NFATc1). Although NFATc1 was strongly induced by MCP-1 and was observed in the nucleus, MCP-1 did not permit the formation of bone-resorbing osteoclasts, although these cells had the typical TRAP(+)/CTR(+) multinuclear phenotype of osteoclasts. Despite a similar appearance to osteoclasts, RANKL treatment was required in order for TRAP(+)/CTR(+) multinuclear cells to develop bone resorption activity. The lack of bone resorption was correlated with a deficiency in expression of certain genes related to bone resorption, such as cathepsin K and MMP9. Furthermore, calcitonin blocked the MCP-1-induced formation of TRAP(+)/CTR(+) multinuclear cells as well as blocking osteoclast bone resorption activity, indicating that calcitonin acts at two stages of osteoclast differentiation. Ablation of NFATc1 in mature osteoclasts did not prevent bone resorption activity, suggesting NFATc1 is involved in cell fusion events and not bone resorption. We propose that the MCP-1-induced TRAP(+)/CTR(+) multinuclear cells represent an arrested stage in osteoclast differentiation, after NFATc1 induction and cellular fusion but prior to the development of bone resorption activity.

Osteoclasts, the bone-digesting cells, are polarized cells that secrete acid hydrolases into a resorption lacuna where bone degradation takes place. The molecular mechanisms underlying this process are poorly understood. To analyze the nature of acid hydrolases secreted by osteoclasts, we used the mouse myeloid Raw 264.7 cell line that differentiates in vitro into mature osteoclasts in the presence of the receptor activator of NF-kappaB ligand. Upon differentiation, we observed a strong increase in the secretion of mannose 6-phosphate-containing acid hydrolases. A proteomic analysis of the secreted proteins captured on a mannose 6-phosphate receptor affinity column revealed 58 different proteins belonging to several families of acid hydrolases of which 16 are clearly involved in bone homeostasis. Moreover these acid hydrolases were secreted as proproteins. The expression of most of the identified acid hydrolases is unchanged during osteoclastogenesis. Thus, our data strongly support the notion that the polarized secretion of acid hydrolases by osteoclasts results from a reorganization of key steps of membrane traffic along the lysosomal pathway rather than from a fusion of lysosomes with the membrane facing the resorption lacuna.

The receptor activator of NF-kappaB ligand (RANKL), a critical regulator of osteoclastogenesis, is synthesized as a membrane-anchored protein and cleaved into a soluble form by ectodomain shedding. We developed an assay system to identify molecules regulating the RANKL shedding. Using this system, we found that a splice variant of Ca2+-promoted Ras inactivator (CAPRI), deltaCAPRI, which is expressed in primary osteoblasts, promoted the RANKL shedding. The wild type CAPRI is a member of the Ras GTPase-activating protein (GAP) family and suppresses Ca2+-dependent Ras activation, whereas deltaCAPRI, which lacks one exon in the GAP-related domain, activated the Ras pathway. Overexpression of deltaCAPRI or a constitutive active form of Ras up-regulated the expression level of matrix-metalloproteinase 14 (MMP14), which directly cleaves the ectodomain of RANKL, whereas Erk activation by expressing the constitutive active Mek1 did not affect the MMP14 expression or RANKL shedding. These results suggest that deltaCAPRI is a possible regulator of RANKL shedding by modulating MMP14 expression through Ras signaling cascades other than the Erk pathway.

The receptor activator of NF-kappaB (RANK) belongs to the neuregulin/tumor necrosis factor (TNF) receptor superfamily and is activated by RANK ligand (RANK-L), a homotrimeric, TNF-like cytokine. RANK is present on the surface of osteoclast cell precursors, where its interaction with RANK-L induces their terminal differentiation into osteoclasts, thus increasing bone breakdown. The secreted, soluble receptor osteoprotegerin (OPG) interrupts this activation by binding directly to RANK-L. Therefore, osteoclast maturation (and bone homeostasis) is regulated in vivo by OPG levels of expression. We have studied the assembly state and affinity of OPG for RANK-L by sedimentation analyses and surface plasmon resonance (Biacore). Full-length, homodimeric OPG binds to RANK-L with a KD of 10 nM. OPG is also a member of the TNF receptor superfamily and contains four disulfide-rich ligand-binding domains, yet lacks a transmembrane region separating the ligand-binding region from the two death domains, as observed for other receptor family members. We showed that dimerization of OPG results from noncovalent interactions mediated by the death domains and to a lesser extent by a C-terminal heparin-binding region. In contrast, a C-terminal intermolecular disulfide bond does not contribute to the formation or stability of OPG dimers. A truncate of osteoprotegerin, containing the ligand-binding domains but lacking the dimerization domains, bound RANK-L with a KD of approximately 3 microM, indicating that monomer oligomerization for the OPG provides an increase of 3 orders of magnitude in the affinity for RANK-L. Therefore, OPG dimer formation is required for the mechanism of inhibition of the RANK-L/RANK receptor interaction.

Osteoclast differentiation from hematopoietic precursors is controlled by the tumor necrosis factor family member tumor necrosis factor-related activation-induced cytokine (TRANCE) via induction of various transcription factors, including nuclear factor of activated T cells (NFAT) c1. During osteoclast differentiation, NFATc1 is further activated via calcium signaling when costimulatory receptors expressed on osteoclast precursors, such as osteoclast-associated receptor (OSCAR), are stimulated. Here we show that NFATc1 expression precedes that of OSCAR during TRANCE-mediated osteoclastogenesis and that inhibition of NFATc1 by cyclosporin A abolishes TRANCE-induced OSCAR expression and subsequent osteoclast differentiation. Moreover, we show that the 1.0-kb promoter region of the OSCAR gene contains three potential NFATc1-binding sites. Induction of an OSCAR promoter-luciferase reporter is significantly increased when transiently transfected into 293T cells in combination with NFATc1 expression plasmid. Deletion and site-directed mutant constructs confirmed that NFATc1-binding sites are both functional and NFATc1-specific. Furthermore, NFATc1 synergistically activates an OSCAR reporter construct together with microphthalmia transcription factor and PU.1, transcription factors previously shown to be critical for osteoclast differentiation. In addition, a plasmid expressing constitutively active MAP kinase kinase 6 enhances the transactivation activity of NFATc1/microphthalmia transcription factor/PU.1 on the OSCAR promoter. Taken together, our results indicate that NFATc1 is an important transcription factor in the induction of OSCAR during osteoclastogenesis. Elucidation of NFATc1 as a transcription factor for OSCAR expression implies the presence of a positive feedback circuit of TRANCE-induced activation of NFATc1, involving NFATc1-mediated OSCAR expression and its subsequent activation of NFATc1, necessary for efficient differentiation of osteoclasts.

Bone homeostasis depends on the coordination of osteoclastic bone resorption and osteoblastic bone formation. Receptor activator of NF-kappaB ligand (RANKL) induces osteoclast differentiation through activating a transcriptional program mediated by the key transcription factor nuclear factor of activated T cells (NFAT) c1. Immunoreceptors, including osteoclast-associated receptor (OSCAR) and triggering receptor expressed by myeloid cells (TREM)-2, constitute the co-stimulatory signals required for RANKL-mediated activation of calcium signaling, which leads to the activation of NFATc1. However, it remains unknown whether the expression of immunoreceptors are under the control of NFATc1. Here we demonstrate that the expression of OSCAR, but not that of TREM-2, is up-regulated during osteoclastogenesis and markedly suppressed by the calcineurin inhibitor FK506, suggesting that OSCAR is transcriptionally regulated by NFATc1. NFATc1 expression results in the activation of the OSCAR promoter, which was found to be further enhanced by co-expression of PU.1 and microphthalmia-associated transcription factor (MITF). We further provide evidence that NFATc1 specifically regulates OSCAR by chromatin immunoprecipitation assay and quantification of OSCAR and TREM-2 mRNA in NFATc1-/- cells. Thus, OSCAR but not TREM-2 is involved in the positive feedback loop of the immunoreceptor-NFATc1 pathway during osteoclastogenesis. Although several immunoreceptors have been identified as co-stimulatory molecules for RANKL, the expression and function are differentially regulated. These mechanisms, possibly together with the delicate regulation of their ligands on osteoblasts, may provide the exquisite machinery for the modulation of osteoclastogenesis in the maintenance of bone homeostasis.

To clarify the role of calpain in the receptor activator of NF-kappaB ligand (RANKL)-supported osteoclastogenesis, RANKL-induced calpain activation was examined by using murine RAW 264.7 cells and bone marrow-derived monocyte/macrophage progenitors. We found that calpain activity increased in response to RANKL in both cell types based on alpha-spectrinolysis and that mu-calpain, rather than m-calpain, was activated during RANKL-supported osteoclastogenesis in RAW 264.7 cells. Overexpression of mu-calpain clearly augmented RANKL-supported osteoclastogenesis in RAW 264.7 cells, thereby implicating its pivotal role in this process. Cell-permeable calpain inhibitors, including calpastatin and calpeptin, were sufficient to suppress RANKL-supported osteoclastogenesis based on decreased expression of the osteoclastogenic marker, matrix metalloproteinase 9, and the generation of tartrate-resistant acid phosphatase-positive multinucleated cells in both cell types. Calpain inhibitors suppressed NF-kappaB activation via inhibition of the cleavage of inhibitor of NF-kappaB(IkappaBalpha)in RAW 264.7 cells. Taken together, our findings suggest that mu-calpain is essential to the regulation of RANKL-supported osteoclastogenesis via NF-kappaB activation.

The G-protein alpha-subunit G(s)alpha is required for the intracellular cAMP responses to hormones and other agonists. G(s)alpha is known to mediate the cAMP response to parathyroid hormone and other hormones and cytokines in bone and cartilage. To analyze the in vivo role of G(s)alpha signaling in osteoblasts, we developed mice with osteoblast/osteocyte-specific G(s)alpha deficiency (BGsKO) by mating G(s)alpha-floxed mice with collagen Ialpha1 promoter-Cre recombinase transgenic mice. Early skeletal development was normal in BGsKO mice, because formation of the initial cartilage template and bone collar was unaffected. The chondrocytic zones of the growth plates also appeared normal in BGsKO mice. BGsKO mice had a defect in the formation of the primary spongiosa with reduced immature osteoid (new bone formation) and overall length, which led to reduced trabecular bone volume. In contrast, cortical bone was thickened with narrowing of the bone marrow cavity. This was probably due to decreased cortical bone resorption, because osteoclasts were markedly reduced on the endosteal surface of cortical bone. In addition, the expression of alkaline phosphatase, an early osteoblastic differentiation marker, was normal, whereas the expression of the late osteoblast differentiation markers osteopontin and osteocalcin was reduced, suggesting that the number of mature osteoblasts in bone is reduced. Expression of the osteoclast-stimulating factor receptor activator of NF-kappaB ligand was also reduced. Overall, our findings have similarities to parathyroid hormone null mice and confirm that the differential effects of parathyroid hormone on trabecular and cortical bone are primarily mediated via G(s)alpha in osteoblasts. Osteoblast-specific G(s)alpha deficiency leads to reduced bone turnover.

Mutations in the Wnt co-receptor LRP5 alter bone mass in humans, but the mechanisms responsible for Wnts actions in bone are unclear. To investigate the role of the classical Wnt signaling pathway in osteogenesis, we generated mice lacking the beta-catenin or adenomatous polyposis coli (Apc) genes in osteoblasts. Loss of beta-catenin produced severe osteopenia with striking increases in osteoclasts, whereas constitutive activation of beta-catenin in the conditional Apc mutants resulted in dramatically increased bone deposition and a disappearance of osteoclasts. In vitro, osteoblasts lacking the beta-catenin gene exhibited impaired maturation and mineralization with elevated expression of the osteoclast differentiation factor, receptor activated by nuclear factor-kappaB ligand (RANKL), and diminished expression of the RANKL decoy receptor, osteoprotegerin. By contrast, Apc-deficient osteoblasts matured normally but demonstrated decreased expression of RANKL and increased osteoprotegerin. These findings suggest that Wnt/beta-catenin signaling in osteoblasts coordinates postnatal bone acquisition by controlling the differentiation and activity of both osteoblasts and osteoclasts.

Hyaluronic acid (HA) is a component of the extracellular matrix that has been shown to play an important role in bone formation, resorption, and mineralization both in vivo and in vitro. We examined the effects of HA at several molecular weights on osteoclast formation and function induced by RANKL (receptor activator of NF-kappa B ligand) in a mouse monocyte cell line (RAW 264.7). HA at M(r) < 8,000 (low molecular weight HA (LMW-HA)) enhanced tartrate-resistant acid phosphatase-positive multinucleated cell formation and tartrate-resistant acid phosphatase activity induced by RANKL in a dose-dependent manner, whereas HA at M(r) > 900,000 (high molecular weight HA (HMW-HA)) showed no effect on osteoclast differentiation. LMW-HA enhanced pit formation induced by RAW 264.7 cells, whereas HMW-HA did not, and LMW-HA stimulated the expression of RANK (receptor activator of NF-kappa B) protein in RAW 264.7 cells. In addition, we found that LMW-HA enhanced the levels of c-Src protein and phosphorylation of ERKs and p38 MAPK in RAW 264.7 cells stimulated with RANKL, whereas the p38 MAPK inhibitor SB203580 inhibited RANKL-induced osteoclast differentiation. This enhancement of c-Src and RANK proteins induced by LMW-HA was inhibited by CD44 function-blocking monoclonal antibody. These results indicate that LMW-HA plays an important role in osteoclast differentiation and function through the interaction of RANKL and RANK.

It has been established that reactive oxygen species (ROS) such as H2O2 or superoxide anion is involved in bone loss-related diseases by stimulating osteoclast differentiation and bone resorption and that receptor activator of NF-kappaB ligand (RANKL) is a critical osteoclastogenic factor expressed on stromal/osteoblastic cells. However, the roles of ROS in RANKL expression and signaling mechanisms through which ROS regulates RANKL genes are not known. Here we report that increased intracellular ROS levels by H2O2 or xanthine/xanthine oxidase-generated superoxide anion stimulated RANKL mRNA and protein expression in human osteoblast-like MG63 cell line and primary mouse bone marrow stromal cells and calvarial osteoblasts. Further analysis revealed that ROS promoted phosphorylation of cAMP response element-binding protein (CREB)/ATF2 and its binding to CRE-domain in the murine RANKL promoter region. Moreover, the results of protein kinase A (PKA) inhibitor KT5720 and CREB1 RNA interference transfection clearly showed that PKA-CREB signaling pathway was necessary for ROS stimulation of RANKL in mouse osteoblasts. In human MG63 cells, however, we found that ROS promoted heat shock factor 2 (HSF2) binding to heat shock element in human RANKL promoter region and that HSF2, but not PKA, was required for ROS up-regulation of RANKL as revealed by KT5720 and HSF2 RNA interference transfection. We also found that ROS stimulated phosphorylation of extracellular signal-regulated kinases (ERKs) and that PD98059, the inhibitor for ERKs suppressed ROS-induced RANKL expression either in mouse osteoblasts or in MG63 cells. These results demonstrate that ROS stimulates RANKL expression via ERKs and PKA-CREB pathway in mouse osteoblasts and via ERKs and HSF2 in human MG63 cells.

Human osteoclast formation from monocyte precursors under the action of receptor activator of nuclear factor-kappaB ligand (RANKL) was suppressed by granulocyte macrophage colony-stimulating factor (GM-CSF), with down-regulation of critical osteoclast-related nuclear factors. GM-CSF in the presence of RANKL and macrophage colony-stimulating factor resulted in mononuclear cells that were negative for tartrate-resistant acid phosphatase (TRAP) and negative for bone resorption. CD1a, a dendritic cell marker, was expressed in GM-CSF, RANKL, and macrophage colony-stimulating factor-treated cells and absent in osteoclasts. Microarray showed that the CC chemokine, monocyte chemotactic protein 1 (MCP-1), was profoundly repressed by GM-CSF. Addition of MCP-1 reversed GM-CSF suppression of osteoclast formation, recovering the bone resorption phenotype. MCP-1 and chemokine RANTES (regulated on activation normal T cell expressed and secreted) permitted formation of TRAP-positive multinuclear cells in the absence of RANKL. However, these cells were negative for bone resorption. In the presence of RANKL, MCP-1 significantly increased the number of TRAP-positive multinuclear bone-resorbing osteoclasts (p = 0.008). When RANKL signaling through NFATc1 was blocked with cyclosporin A, both MCP-1 and RANTES expression was down-regulated. Furthermore, addition of MCP-1 and RANTES reversed the effects of cyclosporin A and recovered the TRAP-positive multinuclear cell phenotype. Our model suggests that RANKL-induced chemokines are involved in osteoclast differentiation at the stage of multinucleation of osteoclast precursors and provides a rationale for increased osteoclast activity in inflammatory conditions where chemokines are abundant.

We studied estrogen effects on osteoclastic differentiation using RAW264.7, a murine monocytic cell line. Differentiation, in response to RANKL and colony-stimulating factor 1, was evaluated while varying estrogen receptor (ER) stimulation by estradiol or nonsteroidal ER agonists was performed. The RAW264.7 cells were found to express ERalpha but not ERbeta. In contrast to RANKL, which decreased ERalpha expression and induced osteoclast differentiation, 10 nm estradiol, 3 microm genistein, or 3 microm daidzein all increased ERalpha expression, stimulated cell proliferation, and decreased multinucleation, with the effects of estrogen > or = daidzein > genistein. However, no estrogen agonist reduced RANKL stimulation of osteoclast differentiation markers or its down-regulation of ERalpha expression by more than approximately 50%. Genistein is also an Src kinase antagonist in vitro, but it did not decrease Src phosphorylation in RAW264.7 cells relative to other estrogen agonists. However, both phytoestrogens and estrogen inhibited RANKL-induced IkappaB degradation and NF-kappaB nuclear localization with the same relative potency as seen in proliferation and differentiation assays. This study demonstrates, for the first time, the direct effects of estrogen on osteoclast precursor differentiation and shows that, in addition to effecting osteoblasts, estrogen may protect bone by reducing osteoclast production. Genistein, which activates ERs selectively, inhibited osteoclastogenesis less effectively than the nonselective phytoestrogen daidzein, which effectively reproduced effects of estrogen.

Prostaglandin E2 (PGE2) synergistically enhances the receptor activator for NF-kappa B ligand (RANKL)-induced osteoclastic differentiation of the precursor cells. Here we investigated the mechanisms of the stimulatory effect of PGE2 on osteoclast differentiation. PGE2 enhanced osteoclastic differentiation of RAW264.7 cells in the presence of RANKL through EP2 and EP4 prostanoid receptors. RANKL-induced degradation of I kappa B alpha and phosphorylation of p38 MAPK and c-Jun N-terminal kinase in RAW264.7 cells were up-regulated by PGE2 in a cAMP-dependent protein kinase A (PKA)-dependent manner, suggesting that EP2 and EP4 signals cross-talk with RANK signals. Transforming growth factor beta-activated kinase 1 (TAK1), an important MAPK kinase kinase in several cytokine signals, possesses a PKA recognition site at amino acids 409-412. PKA directly phosphorylated TAK1 in RAW264.7 cells transfected with wild-type TAK1 but not with the Ser412 --> Ala mutant TAK1. Ser412 --> Ala TAK1 served as a dominant-negative mutant in PKA-enhanced degradation of I kappa B alpha, phosphorylation of p38 MAPK, and PGE2-enhanced osteoclastic differentiation in RAW264.7 cells. Furthermore, forskolin enhanced tumor necrosis factor alpha-induced I kappa B alpha degradation, p38 MAPK phosphorylation, and osteoclastic differentiation in RAW264.7 cells. Ser412 --> Ala TAK1 abolished the stimulatory effects of forskolin on those cellular events induced by tumor necrosis factor alpha. Ser412 --> Ala TAK1 also inhibited the forskolin-induced up-regulation of interleukin 6 production in RAW264.7 cells treated with lipopolysaccharide. These results suggest that the phosphorylation of the Ser412 residue in TAK1 by PKA is essential for cAMP/PKA-induced up-regulation of osteoclastic differentiation and cytokine production in the precursor cells.

Receptor activator of NF-kappa B ligand (RANKL) and its receptor activator of NF-kappa B (RANK) play pivotal roles in osteoclast differentiation and function. However, the structural determinants of the RANK that mediate osteoclast formation and function have not been definitively identified. To address this issue, we developed a chimeric receptor approach that permits a structure/function study of the RANK cytoplasmic domain in osteoclasts. Using this approach, we examined the role of six RANK putative tumor necrosis factor receptor-associated factor-binding motifs (PTM) (PTM1, ILLMT-REE(286-293); PTM2, PSQPS(349-353); PTM3, PFQEP(369-373); PTM4, VYVSQTSQE(537-545); PTM5, PVQEET(559-564); and PTM6, PVQEQG(604-609)) in osteoclast formation and function. Our data revealed that the RANK cytoplasmic domain possesses three functional motifs (PFQEP(369-373), PVQEET(559-564), and PVQEQG(604-609)) capable of mediating osteoclast formation and function. Moreover, we demonstrated that these motifs play distinct roles in activating intracellular signaling. PFQEP(369-373) initiates NF-kappa B, c-Jun N-terminal kinase, extracellular signal-regulated kinase, and p38 signaling pathways and PVQEET(559-564) activates NF-kappa B and p38 pathways in osteoclasts, whereas PVQEQG(604-609) is only capable of activating NF-kappa B pathway. Significantly, the revelation of these functional RANK cytoplasmic motifs has not only laid a foundation for further delineating RANK signaling pathways in osteoclasts, but, more importantly, these RANK motifs themselves represent potential therapeutic targets for bone disorders such as osteoporosis.

CpG oligodeoxynucleotides (CpG-ODNs), mimicking bacterial DNA, stimulate osteoclastogenesis via Toll-like receptor 9 (TLR9) in receptor activator of NF-kappa B ligand (RANKL)-primed osteoclast precursors. This activity is mediated via tumor necrosis factor (TNF)-alpha induction by CpG-ODN. To further reveal the role of the cytokine in TLR9-mediated osteoclastogenesis, we compared the ability of CpG-ODN to induce osteoclastogenesis in two murine strains, BALB/c and C57BL/6, expressing different TNF-alpha alleles. The induction of osteoclastogenesis and TNF-alpha release by CpG-ODN was by far more noticeable in BALB/c-derived than in C57BL/6-derived osteoclast precursors. Unexpectedly, as revealed by Northern analysis, CpG-ODN induction of TNF-alpha mRNA increase was more efficient in C57BL/6-derived cells. The cytokine transcript abundance was increased due to both increased message stability and rate of transcription. The difference between the two cell types was the result of a higher transcription rate in CpG-ODN-induced C57BL/6-derived cells caused by a single nucleotide polymorphism in kappa B2a site within the TNF-alpha promoter sequence. CpG-ODN enhanced the rate of the cytokine translation in BALB/c-derived cells. Thus, CpG-ODN modulated both transcription and translation of TNF-alpha. The induction of transcription was more evident in C57BL/6-derived cells, while the induction of translation took place only in BALB/c-derived osteoclast precursors. Altogether the cytokine was induced to a larger extent in BALB/c-derived osteoclast precursors, consistent with the increased CpG-ODN osteoclastogenic effect in these cells.

Signaling through the receptor activator of nuclear factor kappa B (RANK) is required for both osteoclast differentiation and mammary gland development, yet the extent to which RANK utilizes similar signaling pathways in these tissues remains unclear. Mice expressing a kinase-inactive form of the inhibitor of kappa B kinase alpha (IKK alpha) have mammary gland defects similar to those of RANK-null mice yet have apparently normal osteoclast function. Because mice that completely lack IKK alpha have severe skin and skeletal defects that are not associated with IKK alpha-kinase activity, we wished to directly examine osteoclastogenesis in IKK alpha(-/-) mice. We found that unlike RANK-null mice, which completely lack osteoclasts, IKK alpha(-/-) mice did possess normal numbers of TRAP(+) osteoclasts. However, only 32% of these cells were multinucleated compared with 57% in wild-type littermates. A more profound defect in osteoclastogenesis was observed in vitro using IKK alpha(-/-) hematopoietic cells treated with colony-stimulating factor 1 and RANK ligand (RANKL), as the cells failed to form large, multinucleated osteoclasts. Additionally, overall RANKL-induced global gene expression was significantly blunted in IKK alpha(-/-) cells, including osteoclast-specific genes such as TRAP, MMP-9, and c-Src. IKK alpha was not required for RANKL-mediated I kappa B alpha degradation or phosphorylation of mitogen-activated protein kinases but was required for RANKL-induced p100 processing. Treatment of IKK alpha(-/-) cells with tumor necrosis factor alpha (TNF alpha) in combination with RANKL led to partial rescue of osteoclastogenesis despite a lack of p100 processing. However, the ability of TNF alpha alone or in combination with transforming growth factor beta to induce osteoclast differentiation was dependent on IKK alpha, suggesting that synergy between RANKL and TNFalpha can overcome p100 processing defects in IKK alpha(-/-) cells.

The receptor activator of NF-kappaB ligand (RANKL) induces various osteoclast-specific marker genes during osteoclast differentiation mediated by mitogen-activated protein (MAP) kinase cascades. However, the results of transcriptional programming of an osteoclast-specific cathepsin K gene are inconclusive. Here we report the regulatory mechanisms of RANKL-induced cathepsin K gene expression during osteoclastogenesis in a p38 MAP kinase-dependent manner. The reporter gene analysis with sequential 5'-deletion constructs of the cathepsin K gene promoter indicates that limited sets of the transcription factors such as NFATc1, PU.1, and microphthalmia transcription factor indeed enhance synergistically the gene expression when overexpressed in RAW264 cells. In addition, the activation of p38 MAP kinase is required for the maximum enhancement of the gene expression. RANKL-induced NFATc1 forms a complex with PU.1 in nuclei of osteoclasts following the nuclear accumulation of NFATc1 phosphorylated by the activated p38 MAP kinase. These results suggest that the RANKL-induced cathepsin K gene expression is cooperatively regulated by the combination of the transcription factors and p38 MAP kinase in a gradual manner.

Osteoprotegerin Ligand (OPGL) is a member of the tumor necrosis factor ligand superfamily and has been shown to be involved in interactions between T cells and dendritic cells. Its role in monocyte effector function, however, has not been defined. In the present study a role for OPGL in activating monocytes/macrophages has been characterized. OPGL was found to up-regulate receptor activator of NF-kappaB (RANK) receptor expression on monocytes, regulate their effector function by inducing cytokine and chemokine secretion, activate antigen presentation through up-regulation of co-stimulatory molecule expression, and promote survival. This activation is mediated through the MAPK pathway as evidenced by activation of p38 and p42/44 MAPK and up-regulation of BCL-XL protein levels. A physiological role for OPGL in monocyte activation and effector function was tested in a model of lipopolysaccharide-induced endotoxic shock. Administration of receptor activator of NF-kappaB (RANK)-Fc to block OPGL activity in vivo was able to protect mice from death induced by sepsis, indicating a hitherto undescribed role for OPGL in monocyte function and in mediating inflammatory response. This was further tested in an animal model of inflammation-mediated arthritis. Treatment with RANK-Fc significantly ameliorated disease development and attenuated bone destruction. Thus, our study strongly suggests that administration of receptor fusion proteins to specifically block OPGL activity in vivo may result in blocking development of monocyte/macrophage-mediated diseases.

Osteoclasts are specialized macrophages that resorb bone. Mice lacking the AP-1 component c-Fos are osteopetrotic because of a lack of osteoclast differentiation and show an increased number of macrophages. The nature of the critical function of c-Fos in osteoclast differentiation is not known. Microarray analysis revealed that Nfatc1, another key regulator of osteoclastogenesis, was down-regulated in Fos(-/-) osteoclast precursors. Chromatin immunoprecipitation assay showed that c-Fos bound to the Nfatc1 and Acp5 promoters in osteoclasts. In vitro promoter analyses identified nuclear factor of activated T-cells (NFAT)/AP-1 sites in the osteoclast-specific Acp5 and Calcr promoters. Moreover, in Fos(-/-) precursors gene transfer of an active form of NFAT restored transcription of osteoclast-specific genes in the presence of receptor activator of the NF-kappaB ligand (RANKL), rescuing bone resorption. In the absence of RANKL, however, Fos(-/-) precursors were insensitive to NFAT-induced osteoclastogenesis unlike wild-type precursors. These data indicate that lack of Nfatc1 expression is the cause of the differentiation block in Fos(-/-) osteoclast precursors and that transcriptional induction of Nfatc1 is a major function of c-Fos in osteoclast differentiation.

Bacterial infection sometimes impairs bone metabolism. In this study, we infected the osteoblastic cell line MC3T3-E1 with Mycobacterium bovis bacillus Calmette-Guérin (BCG) and identified genes that were up-regulated in the BCG-infected cells by the suppression subtractive hybridization method. A gene encoding 4-1BB (CD137), a member of the tumor necrosis factor-alpha receptor family, was found to be one of the up-regulated genes. Up-regulation of 4-1BB was also observed by infection with Escherichia coli, Salmonella typhimurium, and Staphylococcus aureus, and by treatment with lipopolysaccharides and heat-killed BCG. Bone marrow cells and the macrophage-like cell lines J774 and RAW264.7 were found to express 4-1BB ligand (4-1BBL). Recombinant 4-1BB (r4-1BB) that was immobilized on culture plates strongly inhibited macrophage colony stimulating factor (M-CSF)/receptor activator of nuclear factor-kappaB ligand (RANKL)-induced in vitro osteoclast formation from bone marrow cells. Anti-4-1BBL antibody also inhibited osteoclast formation to a lesser extent, indicating involvement of reverse signaling through 4-1BBL during inhibition of osteoclast formation. A casein kinase I (CKI) inhibitor markedly suppressed the inhibitory effect of r4-1BB on M-CSF/RANKL-induced osteoclast formation, suggesting that CKI might be involved in 4-1BB/4-1BBL reverse signaling. r4-1BB showed no effects on M-CSF- or RANKL-induced phosphorylation of I-kappaB, ERK1/2, p38, or JNK, whereas RANKL-induced phosphorylation of Akt, a downstream target of phosphatidylinositol 3-kinase (PI3K), was completely abolished by r4-1BB, suggesting that 4-1BB/4-1BBL reverse signaling may interfere with PI3K/Akt pathway. r4-1BB also abolished RANKL-mediated induction of nuclear factor of activated T cells-2. This study may elucidate a novel role of 4-1BB in cell metabolism, especially osteoclastogenesis.

RANK Ligand (RANKL) is a critical osteoclastogenic factor that is expressed on stromal cells and osteoblasts. Most resorption stimuli induce osteoclast formation by modulating RANKL gene expression in marrow stromal/osteoblast cells. However, it is unclear how these stimuli modulate RANKL gene expression in the bone microenvironment. To characterize the transcriptional control of human RANKL gene expression in stromal/osteoblast cells, we PCR-amplified and cloned a 2-kb 5'-flanking sequence of the RANKL gene, using normal human osteoblast derived genomic DNA as a template. Sequence analysis identified the presence of several potential Heat Shock Factor (HSF) responsive elements (HSE) in the human RANKL gene promoter region. Co-expression of HSF-1 or HSF-2 with the RANKL gene promoter-luciferase reporter plasmid in human osteoblastic cells (NOBC) demonstrated a 2-fold and 4.5-fold increase in promoter activity, respectively. RT-PCR analysis for HSF-1 and 2 mRNA expression in human bone marrow-derived stromal cells (SAKA-T) and osteoblast cells detected only HSF-2 expression. As evident from EMSA analysis, in contrast to 1,25(OH)(2)D(3) SAKA-T cells treated with b-FGF demonstrated increased levels of HSF-2 binding to the HSE present in the RANKL gene promoter region. Immunocytochemical staining further confirmed nuclear localization of HSF-2 in both SAKA-T transformed stromal cells and human bone marrow derived primary stromal/preosteoblastic cells in response to b-FGF treatment. Furthermore, b-FGF treatment of SAKA-T cells transfected with the luciferase reporter plasmid containing the hRANKL HSE region (-2 kb to -1275 bp) upstream to a heterologous promoter showed increased levels of transactivation. Western blot analysis further demonstrated enhanced levels of RANKL expression and HSP-27 phosphorylation in SAKA-T cells treated with b-FGF. In addition, overexpression of HSF-2 in SAKA-T cells resulted in a 5-fold increase in the levels of RANKL expression in these cells. These data further suggest that HSF-2 is a downstream target of b-FGF to induce RANKL expression in stromal/osteoblast cells, and that HSF may play an important role in modulating RANKL gene expression in the bone microenvironment.

The tumor necrosis factor family ligand, tumor necrosis factor-related activation-induced cytokine (TRANCE), and its receptors, receptor activator of nuclear factor-kappaB (RANK) and osteoprotegerin (OPG), are known to be regulators of development and activation of osteoclasts in bone remodeling. Sustained osteoclast activation that occurs through TRANCE-RANK causes osteopenic disorders such as osteoporosis and contributes to osteolytic metastases. Here, we report a rationally designed small molecule mimic of osteoprotegerin to inhibit osteoclast formation in vitro and limit bone loss in an animal model of osteoporosis. One of the mimetics, OP3-4, significantly inhibited osteoclast formation in vitro (IC(50) = 10 microm) and effectively inhibited total bone loss in ovariectomized mice at a dosage of 2 mg/kg/day. Unlike soluble OPG receptors, which preclude TRANCE binding to RANK, OP3-4 shows the ability to modulate RANK-TRANCE signaling pathways and alters the biological functions of the RANK-TRANCE receptor complex by facilitating a defective receptor complex. These features suggest that OPG-derived small molecules can be used as a probe to understand complex biological functions of RANK-TRANCE-OPG receptors and also can be used as a platform to develop more useful therapeutic agents for inflammation and bone disease.

A novel bone-resorbing factor was cloned using an expression cloning technique, which involved a Xenopus oocyte expression system and an assay for osteoclast formation. A candidate clone was isolated from a BW5147 mouse T-lymphoma cell cDNA library. Sequencing analysis identified the factor as gamma-glutamyltranspeptidase (GGT), which is an enzyme involved in glutathione metabolism. The addition of purified GGT protein to mouse bone marrow culture effectively induced formation of osteoclasts. An antibody against GGT inhibited osteoclast formation but not the enzymatic activity. We also demonstrated that an inactive form of GGT, the enzymatic activity of which had been blocked by chemical modification with a specific inhibitor, acivicin, supported osteoclast formation. These results indicate that GGT acts on osteoclast formation independent of its own enzymatic activity. Furthermore, both native GGT and inactive GGT stimulated the expression of the receptor activator of nuclear factor-kappaB ligand (RANKL) mRNA and protein from bone marrow stromal cells. This report is the first demonstration of a novel biological activity of GGT protein in a manner independent of its enzymatic activity.

The receptor activator of NF-kappaB ligand (RANKL), a recently identified member of the tumor necrosis factor (TNF) superfamily, has been shown to induce osteoclastogenesis and dendritic cell survival. Most members of the TNF superfamily suppress cell proliferation and induce apoptosis, but whether RANKL does so is not known. We demonstrate that treatment of monocyte RAW 264.7 cells with RANKL induces dose-dependent growth inhibition (IC50 = 10 ng/ml) as determined by dye uptake and [3H]thymidine incorporation methods. Suppression of RANKL-induced NF-kappaB activation by dominant-negative IkappaBalpha or by the NEMO-peptide had no effect on RANKL-induced cell growth inhibition. Inhibition of RANKL-induced JNK activation, however, abolished the RANKL-induced apoptosis. Suppression of interaction of RANK with TRAF6 by TRAF6-binding peptide abrogated the anti-proliferative effects of RANKL, suggesting the critical role of TRAF6. Flow cytometric analysis of cells treated with RANKL showed accumulation of cells in G0/G1 phase of the cell cycle, and this accumulation correlated with a decline in the levels of cyclin D1, cyclin D3, and cyclin E and an increase in cyclin-dependent kinase inhibitor p27 (Kip). Flow cytometric analysis showed the presence of annexin V-positive cells in cultures treated with RANKL. RANKL-induced apoptosis was further confirmed using calcein AM/ethidium homodimer-1 dye and cleavage of poly(ADP-ribose) polymerase (PARP), procaspase 3, and procaspase 9; benzyloxycarbonyl-VAD, the pancaspase inhibitor, suppressed the PARP cleavage. Thus, overall, our studies indicate that RANKL can inhibit cell proliferation and induce apoptosis through a TRAF-6-dependent but NF-kappaB-independent mechanism.

It is unknown why sustained elevation of parathyroid hormone (PTH) stimulates bone resorption, whereas intermittent administration stimulates bone formation. We show in mice that daily injections of PTH attenuate osteoblast apoptosis, thereby increasing osteoblast number, bone formation rate, and bone mass, but do not affect osteoclast number. In contrast, sustained elevation of PTH, achieved either by infusion or by raising endogenous hormone secretion with a calcium-deficient diet, does not affect osteoblast apoptosis but increases osteoclast number. Attenuation of apoptosis by PTH in cultured osteoblastic cells requires protein kinase A-mediated phosphorylation and inactivation of the pro-apoptotic protein Bad as well as transcription of survival genes, like Bcl-2, mediated by CREB (cAMP response element-binding protein) and Runx2. But, PTH also increases proteasomal proteolysis of Runx2. Moreover, the anti-apoptotic effect of PTH is prolonged by inhibition of proteasomal activity, by overexpressing a dominant negative form of the E3 ligase (ubiquitin-protein isopeptide ligase) that targets Runx2 for degradation (Smurf1), or by overexpressing Runx2 itself. The duration of the anti-apoptotic effect of PTH, thus, depends on the level of Runx2, which in turn is decreased by PTH via Smurf1-mediated proteasomal proteolysis. The self-limiting nature of PTH-induced survival signaling might explain why intermittent administration of the hormone is required for bone anabolism.

Development of bone depends on a continuous supply of bone-degrading osteoclasts. Although several factors such as the matrix metalloproteinases and the integrins have been shown to be important for osteoclast recruitment, the mechanism of action remains poorly understood. In this study we investigated the molecular mechanisms homing osteoclasts to their future site of resorption during bone development. We show that RANKL and VEGF, two cytokines known to be present in bone, possess chemotactic properties toward osteoclasts cultured in modified Boyden chambers. Furthermore, in ex vivo cultures of embryonic murine metatarsals, a well established model of osteoclast recruitment, antagonists of RANKL and VEGF reduced calcium release, showing that both cytokines play roles during bone development. In cultures of purified osteoclasts both RANKL and VEGF induced phosphorylation of ERK1/2 MAP kinase. M-CSF, a well-known chemoattractant of osteoclast, also induced activation of ERK1/2, although this activation followed a kinetic pattern differing from that of RANKL and VEGF. RANKL and VEGF-induced, but not M-CSF-induced, osteoclast invasion was completely blocked by the specific inhibitor of ERK1/2 phosphorylation, PD98059. In addition, PD98059 was able to inhibit calcium release in cultures of embryonic metatarsals. In contrast, PD98059 was unable to abrogate the RANKL-induced calcium release in the tibia model, demonstrating that only some of the RANKL functions on osteoclast physiology are regulated through the ERK1/2 pathway. Taken together, these results show that RANKL and VEGF, in addition to their role in osteoclast differentiation and activation of resorption, are important components of the processes regulating osteoclast chemotaxis.

Accelerated bone resorption leading to osteopenia and osteoporosis has been noted in human immunodeficiency virus (HIV) seropositive, treatment-naive patients, but it may be greatly increased in incidence in those receiving highly active anti-retroviral therapies that incorporate certain protease inhibitors (PI). The pathophysiology of these processes is unclear. We have documented the induction of the primary cytokine responsible for osteoclast differentiation and bone resorption, the receptor activator of nuclear factor kappa B ligand (RANKL), in T cells exposed to soluble HIV-1 envelope glycoprotein gp120. Using a murine osteoclast precursor cell line as well as primary human osteoclast precursors, we demonstrate that pharmacologic levels of two PIs that are linked clinically to osteopenia, ritonavir and saquinavir, abrogate a physiological block to RANKL activity, interferon-gamma-mediated degradation of the RANKL signaling adapter protein, TRAF6 (tumor necrosis factor receptor-associated protein 6) in proteasomes. In contrast, indinavir and nelfinavir, PIs that may promote or stabilize bone formation in vivo, had no impact on this system. These findings offer a molecular basis for the acceleration of bone resorption by certain PIs and provide the first example of clinically useful drugs that can interfere with the cross-talk between RANKL and interferon-gamma via the proteasome. They also suggest a novel therapeutic approach to HIV osteopenia through modulation of these two molecules.

The receptor activator of nuclear factor-kappaB ligand (RANKL), a member of the tumor necrosis factor family, is a transmembrane protein, which is known as an essential initiation factor of osteoclastogenesis. Previously, we identified three RANKL isoforms. RANKL1 was identical to the originally reported RANKL. RANKL2 had a shorter intracellular domain. RANKL3 did not have the intracellular or transmembrane domains and was suggested to act as a soluble form protein. Here, we show that RANKL forms homo- or heteromultimers. NIH3T3 cells transfected with RANKL1 or RANKL2 form mononuclear tartrate-resistant acid phosphatase-positive preosteoclasts in an in vitro osteoclastogenesis assay system. Coexpression of RANKL1 and RANKL2 induces multinucleated osteoclasts. RANKL3 has no effect on the formation of preosteoclasts or osteoclasts but significantly inhibits fusion of preosteoclasts when coexpressed with RANKL1 and RANKL2. These findings imply the presence of multiple multimeric structures of RANKL, which may regulate bone metabolism.

We have assessed characteristics of primary human osteoblasts, shedding light on signaling mediated by beta1 integrin. beta1 integrins are major receptors for these matrix glycoproteins. 1) Integrins beta1, alpha2, alpha3, alpha4, alpha5, alpha6, and alphav were highly expressed on primary osteoblasts. 2) Engagement of beta1 integrins on osteoblasts by cross-linking with specific antibody or ligand matrices, such as fibronectin or collagen, augmented expression of intercellular adhesion molecule 1 (ICAM-1) and receptor activator of nuclear factor kappaB ligand (RANKL) on the surface. 3) Up-regulation of ICAM-1 and RANKL on osteoblasts by beta1 stimulation was completely abrogated by pretreatment with herbimycin A and genistein, tyrosine kinase inhibitors, or transfection of dominant negative truncations of focal adhesion kinase (FAK). 4) Engagement of beta1 integrins on osteoblasts induced tartrate-resistant acid phosphatase-positive multinuclear cell formation in the coculture system of osteoblasts and peripheral monocytes. 5) Up-regulation of tartrate-resistant acid phosphatase-positive multinuclear cell formation by beta1 stimulation was completely abrogated by transfection of dominant negative truncations of FAK. Our results indicate that beta1 integrin-dependent adhesion of osteoblasts to bone matrices induces ICAM-1 and RANKL expression and osteoclast formation via tyrosine kinase, especially FAK. We here propose that beta1 integrin/FAK-mediated signaling on osteoblasts could be involved in ICAM-1- and RANKL-dependent osteoclast maturation.

Prolactin (PRL) is the primary hormone that, in conjunction with local factors, leads to lobuloalveolar development during pregnancy. Recently, receptor activator of NF-kappaB ligand (RANKL) has been identified as one of the effector molecules essential for lobuloalveolar development. The molecular mechanisms by which PRL may induce RANKL expression have not been carefully examined. Here we report that RANKL expression in the mammary gland is developmentally regulated and dependent on PRL and progesterone, whereas its receptor RANK (receptor activator of NF-kappaB) and decoy receptor osteoprotegerin (OPG) are constitutively expressed at all stages in both normal (PRL+/-) and prolactin knockout (PRL-/-) mice. In vitro, PRL markedly increased RANKL expression in primary mammary epithelial cells and RANKL-luciferase reporter activity in CHOD6 cells, which constitutively express the PRL receptor. We identified a gamma-interferon activation sequence (GAS) in the region between residues -965 to -725 of the RANKL promoter, which conferred a PRL response. Using dominant negative mutants of recombinant Jak2 and Stat5 in CHOD6 cells, and by reconstituting the Jak2/Stat5 pathway in COS7 cells, we determined that Jak2 and Stat5a are essential for the PRL-induced RANKL expression in mammary gland.

Although RANK-L is essential for osteoclast formation, factors such as transforming growth factor-beta (TGF-beta) are potent modulators of osteoclastogenic stimuli. To systematically investigate the role of TGF-beta in human osteoclastogenesis, monocytes were isolated from peripheral blood by three distinct approaches, resulting in either a lymphocyte-rich, a lymphocyte-poor, or a pure osteoclast precursor (CD14-positive) cell population. In each of these osteoclast precursor populations, the effect of TGF-beta on proliferation, TRAP activity, and bone resorption was investigated with respect to time and length of exposure. When using the highly pure CD14 osteoclast precursor cell population, the effect of TGF-beta was strongly dependent on the stage of osteoclast maturation. When monocytes were exposed to TGF-beta during the initial culture period (days 1-7), TRAP activity and bone resorption were increased by 40%, whereas the cell number was reduced by 25%. A similar decrease in cell number was observed when TGF-beta was present during the entire culture period (days 1-21), but in direct contrast, TRAP activity, cell fusion, cathepsin K, and matrix metalloproteinase (MMP)-9 expression as well as bone resorption were almost completely abrogated. Moreover, we found that latent TGF-beta was strongly activated by incubation with MMP-9 and suggest this to be a highly relevant mechanism for regulating osteoclast activity. To further investigate the molecular mechanism responsible for the divergent effects of continuous versus discontinuous exposure to TGF-beta, we examined RANK expression and p38 MAPK activation. We found the TGF-beta strongly induced p38 MAPK in monocytes, but not in mature osteoclasts, and that continuous exposure of TGF-beta to monocytes down-regulated RANK expression. The current results suggest that TGF-beta promotes human osteoclastogenesis in monocytes through stimulation of the p38 MAPK, whereas continuous exposure to TGF-beta abrogates osteoclastogenesis through down-regulation of RANK expression and therefore attenuation of RANK-RANK-L signaling.

Long-term administration of glucocorticoids (GCs) causes osteoporosis with a rapid and severe bone loss and with a slow and prolonged bone disruption. Although the involvement of GCs in osteoblastic proliferation and differentiation has been studied extensively, their direct action on osteoclasts is still controversial and not conclusive. In this study, we investigated the direct participation of GCs in osteoclastogenesis. Dexamethasone (Dex) at <10(-8) M stimulated, but at >10(-7) M depressed, receptor activator of NF-kappaB ligand (RANKL)-induced osteoclast formation synergistically with transforming growth factor-beta. The stimulatory action of Dex was restricted to the early phase of osteoclast differentiation and enhanced the priming of osteoclast progenitors (bone marrow-derived monocytes/macrophages) toward differentiation into cells of the osteoclast lineage. The osteoclast differentiation depending on RANKL requires the activation of NF-kappaB and AP-1, and the DNA binding of these transcription factors to their respective consensus cis-elements was enhanced by Dex, consistent with the stimulation of osteoclastogenesis. However, Dex did not affect the RANKL-induced signaling pathways such as the activation of IkappaB kinase followed by NF-kappaB nuclear translocation or the activation of JNK. On the other hand, Dex significantly decreased the endogenous production of interferon-beta, and this cytokine depressed the RANKL-elicited DNA binding of NF-kappaB and AP-1, as well as osteoclast formation. Thus, the down-regulation of inhibitory cytokines such as interferon-beta by Dex may allow the osteoclast progenitors to be freed from the suppression of osteoclastogenesis, resulting in an increased number of osteoclasts, as is observed in the early phase of GC-induced osteoporosis.

Vascular endothelial growth factor (VEGF) is known as a key regulator of angiogenesis during endochondral bone formation. Recently, we demonstrated that TNF-related activation-induced cytokine (TRANCE or RANKL), which is essential for bone remodeling, also had an angiogenic activity. Here we report that VEGF up-regulates expression of receptor activator of NF-kappa B (RANK) and increases angiogenic responses of endothelial cells to TRANCE. Treatment of human umbilical vein endothelial cells (HUVECs) with VEGF increased both RANK mRNA and surface protein expression. Although placenta growth factor specific to VEGF receptor-1 had no significant effect on RANK expression, inhibition of downstream signaling molecules of the VEGF receptor-2 (Flk-1/KDR) such as Src, phospholipase C, protein kinase C, and phosphatidylinositol 3'-kinase suppressed VEGF-stimulated RANK expression in HUVECs. Moreover, the MEK inhibitor PD98059 or expression of dominant negative MEK1 inhibited induction of RANK by VEGF but not the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA-AM). VEGF potentiated TRANCE-induced ERK activation and tube formation via RANK up-regulation in HUVECs. Together, these results show that VEGF enhances RANK expression in endothelial cells through Flk-1/KDR-protein kinase C-ERK signaling pathway, suggesting that VEGF plays an important role in modulating the angiogenic action of TRANCE under physiological or pathological conditions.

Amelogenins, major components of developing enamel, are predominantly involved in the formation of tooth enamel. Although amelogenins are also implicated in cementogenesis, their precise spatial expression pattern and molecular role are not clearly understood. Here, we report for the first time the expression of two alternate splice forms of amelogenins, M180 and the leucine-rich amelogenin peptide (LRAP), in the periodontal region of mouse tooth roots. Lack of M180 and LRAP mRNA expression correlated with cementum defects observed in the amelogenin-null mice. The cementum defects were characterized by an increased presence of multinucleated cells, osteoclasts, and cementicles. These defects were associated with an increased expression of the receptor activator of the nuclear factor-kappa B ligand (RANKL), a critical regulator of osteoclastogenesis. These findings indicate that the amelogenin splice variants, M180 and LRAP, are critical in preventing abnormal resorption of cementum.

Exercise promotes positive bone remodeling through controlling cellular processes in bone. Nitric oxide (NO), generated from endothelial nitric-oxide synthase (eNOS), prevents resorption, whereas receptor activator of nuclear kappa B ligand (RANKL) promotes resorption through regulating osteoclast activity. Here we show that mechanical strain differentially regulates eNOS and RANKL expression from osteoprogenitor stromal cells in a magnitude-dependent fashion. Strain (0.25-2%) induction of eNOS expression was magnitude-dependent, reaching a plateau at 218 +/- 36% of control eNOS. This was accompanied by increases in eNOS protein and a doubling of NO production. Concurrently, 0.25% strain inhibited RANKL expression with increasing response up to 1% strain (44 +/- 3% of control RANKL). These differential responses to mechanical input were blocked when an ERK1/2 inhibitor was present during strain application. Inhibition of NO generation did not prevent strain-activated ERK1/2. To confirm the role of ERK1/2, cells were treated with an adenovirus encoding a constitutively activated MEK; Ad.caMEK significantly increased eNOS expression and NO production by more than 4-fold and decreased RANKL expression by half. In contrast, inhibition of strain-activated c-Jun kinase failed to prevent strain effects on either eNOS or RANKL. Our data suggest that physiologic levels of mechanical strain utilize ERK1/2 kinase to coordinately regulate eNOS and RANKL in a manner leading to positive bone remodeling.

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) is a member of the tumor necrosis factor family that is implicated in apoptosis, proliferation, migration, and inflammation. We describe our findings showing that TWEAK mediated the differentiation of RAW264.7 (RAW) monocyte/macrophage cells into multinuclear, functional osteoclasts. The effect of TWEAK was direct and not mediated by the receptor activator of nuclear factor-kappa B (NF-kappa B) ligand (RANKL) as shown by the use of TWEAK- or RANKL-neutralizing antibodies and by osteoprotegerin, a decoy receptor for RANKL. Recently, fibroblast growth factor-inducible 14 (Fn14) was suggested to be a receptor for TWEAK. We show that the Fn14/TWEAK receptor (TweakR) was not responsible for the osteoclastic effect of TWEAK on RAW cells. Flow cytometry analysis did not reveal the expression of Fn14/TweakR on RAW cells. Moreover, Fn14/TweakR-neutralizing antibodies did not block TWEAK-induced RAW cell differentiation into osteoclasts. This indicated that a second TweakR, TweakR2, exists on RAW cells and is responsible for mediating TWEAK-induced differentiation. We next compared the signaling pathways that are activated by the two receptors. TWEAK binding to TweakR2 activated the NF-kappa B, mitogen-activated protein kinase and c-Jun N-terminal kinase signaling cascades in RAW cells. In contrast, TWEAK binding to Fn14/TweakR activated the NF-kappa B and c-Jun N-terminal kinase pathways but induced only a weak activation of MAPK in HT-29 human colon adenocarcinoma cells expressing endogenous Fn14/TweakR. We propose that the biological effects of TWEAK are mediated by binding to one of at least two distinct receptors that induce differential activation of downstream signaling pathways.

We have recently reported the identification of a novel member of the leukocyte receptor family, osteoclast-associated receptor (OSCAR), which has two Ig-like domains and functions as a bone-specific regulator of osteoclast differentiation. Here, we have cloned the OSCAR promoter region to examine its regulation by transcription factors. The 1.7-kb promoter region of the mouse OSCAR gene contains two potential E-box elements for microphthalmia transcription factor (MITF) and three putative PU.1 sites. MITF or PU.1 alone activates the OSCAR reporter construct 5-6-fold, and the combination of MITF and PU.1 synergistically activates the OSCAR reporter activity up to 110-fold. The mRNA expression patterns of MITF, PU.1, and OSCAR in TRANCE-treated (RAW 264.7) or TRANCE/M-CSF-treated cells (primary osteoclasts) reveal that MITF mRNA expression is induced at a much earlier time point than OSCAR gene expression. In contrast to MITF, PU.1 mRNA levels remain relatively constant at all time points, suggesting that TRANCE-induced MITF, not PU.1 expression, is one of the critical regulatory mechanisms for optimal OSCAR expression during osteoclastogenesis. In addition, we have shown that the combination of MITF and constitutively active MKK6-expressing plasmids synergistically activates OSCAR reporter activity. Taken together, our results strongly suggest that PU.1 and MITF transcription factors synergistically activate OSCAR gene expression. Moreover, the activation of OSCAR gene expression by PU.1/MITF is further enhanced by the TRANCE-induced MKK6/p38 signaling cascade.

Receptor activator of nuclear factor-kappaB ligand (RANKL), osteoprotegerin (OPG), and macrophage-colony stimulating factor play essential roles in the regulation of osteoclastogenesis. Runx2-deficient (Runx2-/-) mice showed a complete lack of bone formation because of maturational arrest of osteoblasts and disturbed chondrocyte maturation. Further, osteoclasts were absent in these mice, in which OPG and macrophage-colony stimulating factor were normally expressed, but RANKL expression was severely diminished. We investigated the function of Runx2 in osteoclast differentiation. A Runx2-/- calvaria-derived cell line (CA120-4), which expressed OPG strongly but RANKL barely, severely suppressed osteoclast differentiation from normal bone marrow cells in co-cultures. Adenoviral introduction of Runx2 into CA120-4 cells induced RANKL expression, suppressed OPG expression, and restored osteoclast differentiation from normal bone marrow cells, whereas the addition of OPG abolished the osteoclast differentiation induced by Runx2. Addition of soluble RANKL (sRANKL) also restored osteoclast differentiation in co-cultures. Forced expression of sRANKL in Runx2-/- livers increased the number and size of osteoclast-like cells around calcified cartilage, although vascular invasion into the cartilage was superficial because of incomplete osteoclast differentiation. These findings indicate that Runx2 promotes osteoclast differentiation by inducing RANKL and inhibiting OPG. As the introduction of sRANKL was insufficient for osteoclast differentiation in Runx2-/- mice, however, our findings also suggest that additional factor(s) or matrix protein(s), which are induced in terminally differentiated chondrocytes or osteoblasts by Runx2, are required for osteoclastogenesis in early skeletal development.

Osteoclasts generate a massive acid flux to mobilize bone calcium. Local extracellular acidification is carried out by vacuolar type H+-ATPase (V-ATPase) localized in the plasma membrane. We have shown that a3, one of the four subunit a isoforms (a1, a2, a3, and a4), is a component of the plasma membrane V-ATPase (Toyomura, T., Oka, T., Yamaguchi, C., Wada, Y., and Futai, M. (2000) J. Biol. Chem. 275, 8760-8765). To establish the unique localization of V-ATPase, we have used a murine macrophage cell line, RAW 264.7, that can differentiate into multinuclear osteoclast-like cells on stimulation with RANKL (receptor activator of nuclear factor kappaB ligand). The V-ATPase with the a3 isoform was localized to late endosomes and lysosomes, whereas those with the a1 and a2 isoforms were localized to organelles other than lysosomes. After stimulation, the V-ATPase with the a3 isoform was immunochemically colocalized with lysosome marker lamp2 and was detected in acidic organelles. These organelles were also colocalized with microtubules, and the signals of lamp2 and a3 were dispersed by nocodazole, a microtubule depolymerizer. In RAW-derived osteoclasts cultured on mouse skull pieces, the a3 isoform was transported to the plasma membrane facing the bone and accumulated inside podosome rings. These findings indicate that V-ATPases with the a3 isoform localized in late endosomes/lysosomes are transported to the cell periphery during differentiation and finally assembled into the plasma membrane of mature osteoclasts.

Tartrate-resistant acid phosphatase (TRAP) plays an important role in bone resorption. TRAP expression in osteoclasts is regulated by receptor activator of NF-kappaB (RANKL), a potent activator of osteoclast differentiation. However, the molecular mechanism underlying the RANKL-induced TRAP expression remains unknown. Here we show that two regions in the mouse TRAP promoter (one at -1858 to -1239 and the other at -1239 to -1039, relative to the translation start site) are implicated in RANKL-induced TRAP transcription in RAW264.7 cells. A detailed characterization of the region at -1239 to -1039 identifies a 12-bp sequence, AGCCACGTGGTG, that specifically binds nuclear proteins from RAW264.7 cells and primary bone marrow macrophages (BMMs) in an electrophoretic mobility shift assay (EMSA). Moreover, the binding is significantly enhanced in EMSA with nuclear extracts from RANKL-treated RAW264.7 cells and BMMs, suggesting that the 12-bp sequence may be involved in RANKL-induced TRAP transcription. Various assays reveal that nuclear proteins binding to the 12-bp sequence are upstream stimulatory factors (USF) 1 and 2. Importantly, mutation of the USF-binding site partially blocks RANKL-induced TRAP transcription in RAW264.7 cells, confirming that USF1 and USF2 are functionally involved in RANKL-induced TRAP transcription. In summary, our data show that USF1 and USF2 play a functional role in RANKL-dependent TRAP expression during osteoclast differentiation.

Fibroblast growth factor (FGF)-2 and parathyroid hormone (PTH) are potent inducers of osteoclast (OCL) formation, and PTH increases FGF-2 mRNA and protein expression in osteoblasts. To elucidate the role of endogenous FGF-2 in PTH responses, we examined PTH-induced OCL formation in bone marrow cultures from wild type and mice with a disruption of the Fgf2 gene. FGF-2-induced OCL formation was similar in marrow culture from both genotypes. In contrast, PTH-stimulated OCL formation in bone marrow cultures or co-cultures of osteoblast-spleen cells from Fgf2-/mice was significantly impaired. PTH increased RANKL mRNA expression in osteoblasts cultures from both genotypes. After 6 days of treatment, osteoprotegerin protein in cell supernatants was 40-fold higher in vehicle-treated and 30-fold higher in PTH-treated co-cultures of osteoblast and spleen cells from Fgf2-/mice compared with Fgf2+/+ mice. However, a neutralizing antibody to osteoprotegerin did not rescue reduced OCL formation in response to PTH. Injection of PTH caused hypercalcemia in Fgf2+/+ but not Fgf2-/mice. We conclude that PTH stimulates OCL formation and bone resorption in mice in part by endogenous FGF-2 synthesis by osteoblasts. Because RANKL- and interleukin-11-induced OCL formation was also reduced in bone marrow cultures from Fgf2-/mice, we further conclude that endogenous FGF-2 is necessary for maximal OCL formation by multiple bone resorbing factors.