RANKL-treated Cells Research Articles

RANKL interferes with osteoclastogenesis in PEG-fused U937 cells through LGR4

ABSTRACT Introduction RANKL plays an important role in the differentiation and maturation process of preosteoclast cells. The osteoclast is a multinucleated cell that can have various sizes and a variable number of nuclei. However, there are no models that allow us to understand how successive cell fusions have a limit, or how cell fusion is regulated. Methodology The present investigation was aimed to determine whether fusing U937 cells with PEG to generate osteoclast-like cells expresses LGR4 and whether applying RANKL to these cells modifies osteoclastic activity compared to non-PEG-fused and RANKL-treated cells. Results By fusing U937 cells with PEG, it was found that the LGR4 receptor expression was promoted as early as 24 hours of culture. Applying RANKL before or after fusion inhibits osteoclastic activity. Interfering RANKL interaction with LGR4 in PEG-treated cells recovers and increases cell fusion and osteoclastic activity. PEG-fused U937 cells show osteoclast markers similar to those observed in the classical RANKL-stimulated cell model. Conclusion Our model allows us to understand that RANKL has fusogenic activity during the first days of culture and in fused cells modulates fusion, contributing to differentiate the role of RANKL before and after fusion through LGR4.

Myc and Bet Proteins Orchestrate the Early Regulatory Genome Changes Required for Osteoclast Lineage Commitment

BACKGROUND: Bone disease, a common source of morbidity in multiple myeloma (MM), is caused by RANKL-induced aberrant activation of osteoclasts (OC). RANKL-induced OC lineage commitment requires repression of an Irf-8 dependent macrophage inflammatory transcriptional programme commensurate with activation of an OC lineage-specific programme. Functional data have shown the requirement for the histone acetylation readers Brd2-4 BET proteins and of cMyc for OC lineage development. However, how Brd2-4 and Myc co-operate genome-wide to regulate transcriptome changes that underpin the very early stages of RANKL-induced OC lineage commitment has not been defined. METHODS: The OC progenitor-like murine RAW264.7 cell line was used for osteoclastogenesis. OC were assayed by TRAP staining. We performed RNA-seq for transcriptome analysis and ChIP-seq against Brd2-4, cMyc, and H3K27Ac mark for epigenomic profiling. The pan-Bet inhibitor IBET151 was used alone or in combination with RANKL. ChIP-seq/RNA-seq data were processed using standard bioinformatics pipelines; downstream analyses (pathway and motif enrichment, factor differential binding) were performed by various tools including EnrichR, R packages ChIPpeakAnno/DiffBind, Rose. RESULTS: Transcriptomic profiling of OC progenitors at 0, 4, 14 and 24h post-RANKL treatment identified 12 distinct clusters of expression trends. The 4h activated cluster includes OC master transcription factors (TFs; cMyc, Nfatc1, Fosl), and is enriched in OC-defining pathways. Notably, by 14h the majority of the genes required for mature OC formation and activation are already highly expressed (e.g. Ctsk, Mmp9). The downregulated clusters include monocyte defining TFs (e.g. Irf8, Mafb and Bcl6). These RANKL-dependent transcriptome changes are completely abrogated by iBET151, highlighting the critical role of Brd2-4 in osteoclastogenesis. Differential chromatin binding analysis upon RANKL induction revealed an overall enhanced Brd2-4 binding at already existing or de novo gained sites. This was more pronounced for Brd2&4 and much less for Brd3, with differentially binding sites (DBS) comprising 50% and 20% respectively of all binding sites in RANKL-treated cells. For Brd2&3, DBS were primarily distributed at promoters and for Brd4 at intergenic, candidate enhancers regions. Notably, nearly all gained DBS were sensitive to and abrogated by iBET151. Combinatorial profiling of Brd2 and Brd4 showed that almost half of Brd2 DBS peaks overlap with Brd4 (47%; 897/1896), while only 24% (766/3234) of Brd4 DBS peaks are co-occupied by Brd2. Transcriptome and Brd2&4 DBS integration in combination with motif enrichment analysis, identified genes that are predicted to be regulated by Brd2 and/or Brd4. EnrichR analysis suggests that enhanced binding of Brd2&4, singly or in combination, is required for activation of the critical OC lineage-specific and repression of the macrophage-defining transcriptional programs highlighting the non-redundant roles of Brd2&4 in OC development. Cell lineage commitment often requires 'commissioning' of cell-specific super-enhancers (SE). Combined analysis of genome-wide Brd4/H3K27ac profiles identified 678 RANKL-induced SE and their respective target genes. Further, 110 of these SE showed enhanced Brd4 binding in 2 peaks: 20/110 were linked to significantly up- and 90/100 to down-regulated genes. The repressed genes were significantly enriched to previously described Irf8, MafB and RunX1 targets, suggesting a critical role of SE in the repression of the monocyte/macrophage inflammatory programme during OC lineage commitment. Strikingly, among top hits, we detected a SE linked to the regulation of cMyc. To further investigate its role in OC development, we obtained the cistrome of cMyc after RANKL induction. We identified 560 binding sites which were highly enriched in cMyc, Max, Fli1, Fosl2 and Irf8 motifs. Cistrome-transcriptome integration suggested direct activation of 141 and repression of 52 genes by cMyc in response to RANKL; these are enriched in ribosome biogenesis pathways and Irf8-dependent targets respectively. CONCLUSIONS: Myc and Brd4 mark SE that repress an Irf8-dependent transcriptional programme, a requirement for OC lineage commitment. The non-redundant roles of Brd2&4 suggest that selective targeting of either could inhibit aberrant OC activation associated with MM. Disclosures Caputo: GSK: Research Funding. Auner:Amgen: Other: Consultancy and Research Funding; Takeda: Consultancy; Karyopharm: Consultancy. Karadimitris:GSK: Research Funding.

Hydrogen gas protects against ovariectomy-induced osteoporosis by inhibiting NF-κB activation.

Osteoporosis is a prevalent condition among postmenopausal women, and lacks satisfactory therapeutic options. Hydrogen (H2) has been shown to be effective in alleviating many diseases. This study aimed to investigate the effects of H2 on inhibiting osteoclastogenesis and bone loss in ovariectomized mice. Osteoclast differentiation from Raw264.7 cells was induced with receptor activator NF-κB ligand (RANKL) with or without 60% H2. The number and resorption activity of osteocalsts were assessed by tartrate-resistant acid phosphatase staining and pit formation assay, respectively. The expression of osteoclast markers and NF-κB phosphorylation were detected by western blot. NF-κB nuclear translocation was assessed by immunofluorescence. NF-κB transcriptional activity was analyzed by luciferase assay. Bone loss in mice was induced by ovariectomy (OVX). OVX mice were given either regular air or 60% H2. Bone structure was analyzed by micro-computed tomography and hematoxylin and eosin staining. Cytokine levels were measured by enzyme-linked immunosorbent assay. The data were analyzed with one-way or two-way ANOVA followed by Bonferroni post hoc tests. H2 did not have any measurable effect on the proliferation of Raw264.7 cells. The number of osteoclasts and size of resorption pits of RANKL+H2-treated cells were 3 to 4 times less than RANKL treated cells. The expression of osteoclast marker genes of RANKL+H2-treated cells was 30% to 60% lower than RANKL-treated cells (P < 0.05). H2 markedly inhibited RANKL-induced activation, nuclear translocation, and transcriptional activity of NF-κB (P < 0.05, RANKL+H2 vs RANKL). The amount and density of trabecular bone and bone mineral density of ovariectomized mice were significantly less than sham-operated mice (P < 0.05 OVX vs sham). The amount of trabecular bone and bone mineral density of OVX mice that inhaled H2 were more than 40% higher, whereas the levels of serum proinflammatory cytokine interleukin 1β, IL-6, and tumor necrosis factor-α were more than 50% lower than those of OVX mice (P < 0.05). These results demonstrated that H2 could be an effective therapeutic agent of postmenopausal osteoporosis.

Gene expression profiling in osteoclast precursors by insulin using microarray analysis.

The balance between bone formation by osteoblasts and destruction of mineralized bone matrix by osteoclasts is important for bone homeostasis. The increase of osteoclast differentiation by RANKL induces bone diseases such as osteoporosis. Recent studies have shown that insulin is one of main factors mediating the cross-talk between bone remodeling and energy metabolism. However, the systemic examination of insulin-induced differential gene expression profiles in osteoclasts has not been extensively studied. Here, we investigated the global effects of insulin on osteoclast precursors at the level of gene transcription by microarray analysis. The number of genes that were up-regulated by ≥ 1.5 fold after insulin treatment for 6 h, 12 h, or 24 h was 76, 73, and 39; and 96, 83, and 54 genes were down-regulated, respectively. The genes were classified by 20 biological processes or 24 molecular functions and the number of genes involved in ‘development processes’ and ‘cell proliferation and differentiation’ was 25 and 18, respectively, including Inhba, Socs, Plk3, Tnfsf4, and Plk1. The microarray results of these genes were verified by real-time RT-PCR analysis. We also compared the effects of insulin and RANKL on the expression of these genes. Most genes had a very similar pattern of expressions in insulin- and RANKL-treated cells. Interestingly, Tnfsf4 and Inhba genes were affected by insulin but not by RANKL. Taken together, these results suggest a potential role for insulin in osteoclast biology, thus contributing to the understanding of the pathogenesis and development of therapeutics for numerous bone and metabolic diseases.

Expression profiling of microRNAs in RAW264.7 cells treated with a combination of tumor necrosis factor alpha and RANKL during osteoclast differentiation

Tumor necrosis factor alpha (TNF-α), a cytokine involved in the pathogenesis of periodontal disease, induces osteoclast differentiation and indirectly promotes alveolar bone resorption. We investigated TNF-α-regulated osteoclast differentiation, focusing on microRNAs. MicroRNAs are small, noncoding RNAs that are involved in various biological processes, including cellular differentiation, proliferation and apoptosis. Aside from miR-21, miR-155 and miR-223, the identities of the microRNAs that play roles in osteoclast differentiation are unknown. Notably, no previous studies have reported the expression profiling of microRNAs during TNF-α-regulated osteoclast differentiation. We used microarrays to screen the levels of expression of mature microRNAs in RAW264.7 cells treated with a combination of TNF-α and RANKL, or RANKL alone for 0, 24 or 82 h during osteoclast formation. We validated the results of the microarray analyses through quantitative RT-PCR analyses of representative microRNAs in RAW264.7 cells and murine bone marrow macrophages. During osteoclast formation, the expression of 44 mature microRNAs differed by more than twofold between untreated cells and cells treated with a combination of TNF-α and RANKL, and the expression of 52 mature microRNAs differed upon RANKL treatment. According to quantitative RT-PCR analyses, miR-378 was upregulated and miR-223 was downregulated during osteoclast formation. Furthermore, miR-21, miR-29b, miR-146a, miR-155 and miR-210 were highly expressed during osteoclast differentiation in TNF-α/RANKL-treated cells compared with RANKL-treated cells. These results suggest that miR-223 and miR-378 may play important roles in osteoclastogenesis, and that miR-21, miR-29b, miR-146a, miR-155 and miR-210 are involved in TNF-α-regulated osteoclast differentiation.

Inositol Hexakisphosphate Inhibits Osteoclastogenesis on RAW 264.7 Cells and Human Primary Osteoclasts

BackgroundInoxitol hexakisphosphate (IP6) has been found to have an important role in biomineralization and a direct effect inhibiting mineralization of osteoblasts in vitro without impairing extracellular matrix production and expression of alkaline phosphatase. IP6 has been proposed to exhibit similar effects to those of bisphosphonates on bone resorption, however, its direct effect on osteoclasts (OCL) is presently unknown.Methodology/Principal FindingsThe aim of the present study was to investigate the effect of IP6 on the RAW 264.7 monocyte/macrophage mouse cell line and on human primary osteoclasts. On one hand, we show that IP6 decreases the osteoclastogenesis in RAW 264.7 cells induced by RANKL, without affecting cell proliferation or cell viability. The number of TRAP positive cells and mRNA levels of osteoclast markers such as TRAP, calcitonin receptor, cathepsin K and MMP-9 was decreased by IP6 on RANKL-treated cells. On the contrary, when giving IP6 to mature osteoclasts after RANKL treatment, a significant increase of bone resorption activity and TRAP mRNA levels was found. On the other hand, we show that 1 µM of IP6 inhibits osteoclastogenesis of human peripheral blood mononuclear cells (PBMNC) and their resorption activity both, when given to undifferentiated and to mature osteoclasts.Conclusions/SignificanceOur results demonstrate that IP6 inhibits osteoclastogenesis on human PBMNC and on the RAW264.7 cell line. Thus, IP6 may represent a novel type of selective inhibitor of osteoclasts and prove useful for the treatment of osteoporosis.

RANKL Up-regulates Brain-type Creatine Kinase via Poly(ADP-ribose) Polymerase-1 during Osteoclastogenesis

Receptor activator of nuclear factor κB ligand (RANKL) is the key regulator for osteoclast formation and function. During osteoclastogenesis, RANKL-stimulated signals differentially modulate expression of a large number of proteins. Using proteomics approaches, we identified that brain-type cytoplasmic creatine kinase (Ckb) was greatly induced in mature osteoclasts. Ckb has been shown to contribute to osteoclast function. However, the mechanisms of Ckb regulation and the contribution of other isoforms of creatine kinase during RANKL-induced osteoclastogenesis are unknown. We found that Ckb was the predominant isoform of creatine kinase during osteoclastogenesis. Real-time PCR confirmed that RANKL induced ckb mRNA expression by over 40-fold in primary mouse bone marrow macrophages and Raw 264.7 cells. The RANKL-responsive region was identified within the -0.4- to -0.2-kb 5'-flanking region of the ckb gene. Affinity binding purification followed by mass spectrometry analysis revealed that poly(ADP-ribose) polymerase-1 (PARP-1) bound to the -0.4/-0.2-kb fragment that negatively regulated expression of ckb in response to RANKL stimulation. Electrophoretic mobility shift assays with PARP-1-specific antibody located the binding site of PARP-1 to the TTCCCA consensus sequence. The expression of PARP-1 was reduced during RANKL-induced osteoclastogenesis, concurrently with increased expression of Ckb. Consistently, knockdown of PARP-1 by lentivirus-delivered shRNA enhanced ckb mRNA expression. The activity of PARP-1 was determined to be required for its inhibitory effect on the ckb expression. In summary, we have demonstrated that PARP-1 is a negative regulator of the ckb expression. Down-regulation of PARP-1 is responsible for the up-regulation of ckb during RANKL-induced osteoclastogenesis.

Retinoblastoma protein-interacting zinc finger 1 (RIZ1) participates in RANKL-induced osteoclast formation via regulation of NFATc1 expression

The role of retinoblastoma protein-interacting zinc finger 1 (RIZ1) in receptor activator of NF-κB ligand (RANKL)-induced osteoclast formation was examined in mouse RAW 264.7 macrophage-like cells. The expression of RIZ1 was significantly augmented by RANKL-treated cells. Silencing of RIZ1 with the siRNA significantly reduced the appearance of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells as osteoclasts in RANKL-treated cells. The expression of nuclear factor of activated T cell 1 (NFATc1) as the terminal transcription factor of osteoclast formation was prevented by RIZ1 siRNA. It was suggested that that RIZ1 might participate in RANKL-induced osteoclast formation through the regulation of NFATc1 expression.

Osteoclastic Differentiation of Monocytes Is Inhibited by Non-Transcriptional Effects of Estrogen Mediated by BCAR1.

Estrogen is a major positive regulator of bone mass with important inhibitory effects on bone resorption. However, knowledge of the mechanisms underlying estrogen effects remains incomplete. We investigated whether estrogen altered osteoclastic differentiation of human monocytes in response to Receptor Activator of NF-kB (RANK) Ligand (RANKL), a critical stimulus for osteoclast formation. For these studies, human monocytic cells were isolated from peripheral blood by positive selection for CD14 expression (“CD14 cells”) and maintained in charcoal stripped medium with monocyte colony-stimulating factor. Compared to RANKL treatment alone, co-treatment with 17-β -estradiol reduced osteoclast formation, as shown by decreased development of tartrate resistant acid phosphatase (TRAP) activity, multinucleation, and resorption pit formation on bone. A possible mechanism for the estrogen effects was interference with RANKL signal transduction. We examined estrogen effects on a key event in the RANKL signaling: nuclear translocation of NF-kB following phosphorylation and degradation of IkB. Estrogen cotreatment of RANKL-stimulated CD14 cells inhibited the nuclear translocation of NF-kB observed with RANKL alone. This effect was evident after only 30 minutes of treatment. Cotreatment with estrogen also inhibited RANKL-stimulated IkB phosphorylation; this was evident within 5 minutes after treatment. The time course of the estrogen effects argued strongly for a non-transcriptional mechanism. We therefore investigated the involvement of proteins previously implicated in rapid estrogen effects, including the breast cancer anti-estrogen resistance protein 1(BCAR1, also called p130Cas), a scaffolding/adaptor protein recently reported to interact with estrogen receptors in breast cancer cells. Co-immunoprecipitation from ligand treated CD14 cells revealed an estrogen-dependent association of estrogen receptor-α (ER) with BCAR1 that was enhanced by cotreatment with RANKL. The coordinate regulation of this complex by both RANKL and estrogen suggested that BCAR1 might represent a link between the two signaling systems. In addition, in cells cotreated with estrogen and RANKL, co-immunoprecipitation revealed a further association of ER with Traf6, a critical proximal signaling intermediate for RANKL. These protein interactions were also rapid, evident 5 minutes after ligand addition. To test the functional significance of BCAR1 in estrogen effects, its expression was inhibited by transduction with BCAR1 siRNA. In cells deficient in BCAR1, estrogen no longer inhibited RANKL-stimulated NF-kB nuclear translocation. Unexpectedly, suppression of BCAR1 itself partially blocked RANKL stimulated NF-kB translocation, suggesting a role for BCAR1 even in the absence of estrogen. Furthermore, when BCAR1 deficient CD14 cells were treated with RANKL, osteoclastic differentiation, as assessed by formation of TRAP-positive multinucleated cells, was reduced compared to RANKL-treated cells transduced with control siRNA. The results suggest that BCAR1 is involved in the RANK-NF-kB pathway that mediates osteoclast differentiation signals in monocytes. In addition, BCAR1 appears necessary for estrogen inhibition of RANKL signaling. The estrogen dependent association of ER with BCAR1 may, like BCAR1 deficiency, block BCAR1 actions in the RANKL-NF-kB pathway, and thus inhibit the osteoclastic differentiation of monocytes.

RANKL Treatment Releases the Negative Regulation of the Poly(ADP-Ribose) Polymerase-1 on Tcirg1 Gene Expression During Osteoclastogenesis

The Tcirg1 gene encodes the osteoclast-specific a3 isoform of the V-ATPase a subunit. Using the mouse osteoclastic model RAW264.7 cells, we studied Tcirg1 gene expression, and we identified PARP-1 as a transcriptional repressor negatively regulated by RANKL during osteoclastogenesis. The TCIRG1 gene encodes the a3 isoform of the V-ATPase a subunit, and mutations at this locus account for approximately 60% of infantile malignant osteopetrosis cases. Using RAW264.7 cells as an osteoclastic differentiation model, we undertook a transcriptional study of the mouse Tcirg1 gene focused on the 4-kb region upstream of the transcription starting point. The promoter activity of serial-deletion fragments of the Tcirg1 gene promoter was monitored throughout the RAW264.7 cell differentiation process. We next performed EMSA, UV cross-linking, affinity purification, mass spectrometry analysis, gel supershift, and siRNA transfection experiments to identify the factor(s) interacting with the promoter. The -3946/+113 region of the mouse Tcirg1 gene displayed a high basal promoter activity, which was enhanced by RANKL treatment of RAW264.7 cells. Constructs deleted up to -1589 retained this response to RANKL. A deletion up to -1402 induced a 3-fold enhancement of the basal activity, whereas RANKL response was not affected. EMSA experiments led us to identify within the -1589/-1402 region, a 10-nucleotide sequence, which bound a nuclear protein present in nondifferentiated RAW264.7 cells. This interaction was lost using nuclear extracts derived from RANKL-treated cells. Affinity purification followed by mass spectrometry analysis and gel supershift assay allowed the identification of poly(ADP-ribose) polymerase-1 (PARP-1) as this transcriptional repressor, whereas Western blot experiments revealed the cleavage of the DNA-binding domain of PARP-1 on RANKL treatment. Finally, both PARP-1 depletion after siRNA transfection and RAW264.7 cell treatment by an inhibitor of PARP-1 activity induced an increase of a3 mRNA expression. We provide evidence that the basal transcription activity of the Tcirg1 gene is negatively regulated by the binding of PARP-1 protein to its promoter region in mouse pre-osteoclast. On RANKL treatment, PARP-1 protein is cleaved and loses its repression effect, allowing an increase of Tcirg1 gene expression that is critical for osteoclast function.

Involvement of Aquaporin 9 in Osteoclast Differentiation

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.

MCP-1-induced Human Osteoclast-like Cells Are Tartrate-resistant Acid Phosphatase, NFATc1, and Calcitonin Receptor-positive but Require Receptor Activator of NFκB Ligand for Bone Resorption

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.

Effects of the phytoestrogen coumestrol on RANK-ligand-induced differentiation of osteoclasts.

Phytoestrogens, which have structural similarity to 17beta-estradiol, have been reported to act as agonists/antagonists of estrogen in animals and humans. Estrogen is known to have an important role in maintaining bone mass, because the concentration of serum estrogen decreases after menopause and the estrogen deficiency causes bone loss. In this study, we investigated the effects of coumestrol and other phytoestrogens on osteoclast differentiation using estrogen receptor alpha-transfected RAW264.7 (RAW264.7-ERalpha) cells. When the cells were cultured with the receptor activator of nuclear factor kappa B-ligand (RANKL), both formation of tartrate-resistant acid phosphatase (TRAP) positive multinucleated cells and TRAP activity were increased compared with control cells that were cultured in the absence of RANKL. Coumestrol decreased RANKL-induced formation of TRAP-positive multinucleated cells and TRAP activity dose-dependently. RANKL-stimulated RAW264.7-ERalpha cells formed resorption pits on calcium phosphate films and the pit formation was inhibited by coumestrol in a dose-dependent manner. RT-PCR analyses revealed that coumestrol (10 microM) decreased mRNA levels of calcitonin receptor (CTR) and matrix metalloproteinase-9 (MMP9) in RANKL-treated cells. In addition, pretreatment of coumestrol decreased RANKL-induced phosphorylation of extracellular signal-regulated kinases/p44/42 (ERK1/2). These results suggest that coumestrol has an inhibitory effect on the differentiation of osteoclasts, at least partially via ERK1/2 pathway.

Gene array identification of osteoclast genes: differential inhibition of osteoclastogenesis by cyclosporin A and granulocyte macrophage colony stimulating factor.

Treatment of adherent peripheral blood mononuclear cells (PBMCs) with macrophage colony stimulating factor (M-CSF) and receptor activator of NF-kappaB ligand (RANKL) stimulates the formation of multinucleate osteoclast-like cells. Treatment with M-CSF alone results in the formation of macrophage-like cells. Through the use of Atlas human cDNA expression arrays, genes regulated by RANKL were identified. Genes include numerous cytokines and cytokine receptors (RANTES and CSF2R proportional, variant ), transcription factors (nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) and GA binding protein transcription factor alpha (GABPalpha)), and ribosomal proteins (60S L17 and 40S S20). Real-time PCR analysis showed significant correlation (R2 of 0.98 P < 0.01) with array data for all genes tested. Time courses showed differential activation patterns of transcription factors with early induction of FUSE binding protein 1 (FBP) and c-Jun, and later steady upregulation of NFATc1 and GABP by RANKL. Treatment with cyclosporin A, a known NFATc1 inhibitor, resulted in a blockade of osteoclast formation. The mononuclear cells resulting from high cyclosporin treatment (1,000 ng/ml) were cathepsin K (CTSK) and tartrate-resistant acid phosphatase (TRAP) positive but expression of calcitonin receptor (CTR) was downregulated by more than 30-fold. Constant exposure of M-CSF- and RANKL-treated cells to GM-CSF resulted in inhibition of osteoclast formation and the downregulation of CTSK and TRAP implicating the upregulation of CSF2R in a possible feedback inhibition of osteoclastogenesis.