Targeting MEK1/2 blocks osteoclast differentiation, function and cytokine secretion in multiple myeloma

Osteolytic bone disease in multiple myeloma (MM) is associated with upregulation of osteoclast (OCL) activity and constitutive inhibition of osteoblast function. The extracellular signal-regulated kinase 1/2 (ERK1/2) pathway mediates OCL differentiation and maturation. We hypothesized that inhibition of ERK1/2 could prevent OCL differentiation and downregulate OCL function. It was found that AZD6244, a mitogen-activated or extracellular signal-regulated protein kinase (MEK) inhibitor, blocked OCL differentiation and formation in a dose-dependent manner, evidenced by decreased alphaVbeta3-integrin expression and tartrate-resistant acid phosphatase positive (TRAP+) cells. Functional dentine disc cultures showed inhibition of OCL-induced bone resorption by AZD6244. Major MM growth and survival factors produced by OCLs including B-cell activation factor (BAFF) and a proliferation-inducing ligand (APRIL), as well as macrophage inflammatory protein (MIP-1alpha), which mediates OCL differentiation and MM, were also significantly inhibited by AZD6244. In addition to ERK inhibition, NFATc1 (nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1) and c-fos were both downregulated, suggesting that AZD6244 targets a later stage of OCL differentiation. These results indicate that AZD6244 inhibits OCL differentiation, formation and bone resorption, thereby abrogating paracrine MM cell survival in the bone marrow microenvironment. The present study therefore provides a preclinical rationale for the evaluation of AZD6244 as a potential new therapy for patients with MM.     

Interleukin-7 is a direct inhibitor of in vitro osteoclastogenesis

We examined the direct effects of IL-7 on osteoclastogenesis in murine bone marrow cultures, using cells from wild-type and IL-7- and IL-7 receptor (IL-7R)-deficient mice. IL-7 inhibited osteoclast-like cells (OCL) formation in macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappaB ligand (RANKL)-stimulated (both at 30 ng/ml) murine bone marrow cultures. Significant inhibitory effects were seen at 1 ng/ml (57%) and 10 ng/ml (86%). IL-7 also inhibited (P < 0.05) OCL formation in bone marrow cultures that were stimulated with vitamin D(3) (10(-8) M, 60%), bovine PTH (bPTH) (100 ng/ml, 54%), or RANKL alone (30 ng/ml, 50%). IL-7 (10 ng/ml) increased expression of the B lymphocyte marker B220 from 40-86% of total nonadherent cells in cultures treated with M-CSF and RANKL. Bone marrow cells from IL-7-deficient [IL-7 knockout (KO)] mice showed a significant (P < 0.05) increase in tartrate-resistant acid phosphatase(+) OCL numbers in cultures that were stimulated with vitamin D(3) (136 +/- 13.3%), bPTH (196 +/- 18.8%), or M-CSF and RANKL (160 +/- 7.2%). In contrast, in vitro osteoclast formation in bone marrow from IL-7R-deficient (IL-7R KO) mice showed a significant decrease in tartrate-resistant acid phosphatase(+) OCL numbers in cultures that were stimulated with vitamin D(3), PTH, RANKL, or M-CSF and RANKL. These results demonstrate that there are differences in the mechanisms regulating OCL formation between IL-7 KO and IL-7R KO cells. It seems that IL-7 is a direct inhibitor of OCL formation in vitro, based on results of adding IL-7 to wild-type cultures and the responses of IL-7 KO cells. It is unknown why IL-7R KO cells behave differently from IL-7 KO cells in vitro. However, it is possible that additional cytokines interact with IL-7R and that loss of these signals contributes to the responses of IL-7R KO cells. Alternatively, IL-7 may interact with multiple receptors.     

Synovial macrophage-osteoclast differentiation in inflammatory arthritis

BACKGROUND: Pathological bone resorption (marginal erosions and juxta-articular osteoporosis) by osteoclasts commonly occurs in rheumatoid arthritis (RA). OBJECTIVES: To define the nature of the mononuclear precursor cells from which osteoclasts are formed in inflamed synovial tissues and to determine the cellular and humoral factors which influence osteoclast differentiation. METHOD: Macrophage (CD14+), non-macrophage (CD14-), and unsorted (CD14+/CD14-) synovial cell populations from RA and inflammatory/non-inflammatory osteoarthritis (OA) synovium were cultured in the presence of receptor activator for nuclear factor kappaB ligand (RANKL) and monocyte-colony stimulating factor (M-CSF; in the presence/absence of prostaglandin E(2) (PGE(2)), interleukin 1beta (IL1beta), tumour necrosis factor alpha (TNFalpha), and IL6). Osteoclast differentiation was assessed by expression of tartrate resistant acid phosphatase (TRAP), vitronectin receptor (VNR), and lacunar resorption. RESULTS: TRAP+ and VNR+ multinucleated cells capable of lacunar resorption were only formed in cultures of CD14+-containing synovial cell populations (that is, CD14+ and CD14+/CD14- cells). No difference in the extent of osteoclast formation was noted in cultures of CD14+ cells isolated from RA, inflammatory OA, and non-inflammatory OA synovium. However, more TRAP+/VNR+ cells and more lacunar resorption was noted in CD14+/CD14- cells from RA and inflammatory OA synovial tissues. The addition of PGE(2), IL1beta, TNFalpha, and IL6 did not increase RANKL/M-CSF-induced osteoclast formation and lacunar resorption of both CD14+/CD14- and CD14+ synovial cell populations. CONCLUSIONS: Osteoclast precursors in synovial tissues are CD14+ monocyte/macrophages. The increase in osteoclast formation in cultures of CD14+/CD14- compared with CD14+ synovial cells in RA and inflammatory OA points to a role for CD14- cells in promoting osteoclast differentiation and bone resorption in inflamed synovial tissues by a mechanism which does not involve a direct effect of proinflammatory cytokines/prostaglandins on RANKL-induced macrophage-osteoclast differentiation.     

The synthetic triterpenoid TP-222 inhibits RANKL stimulation of osteoclastogenesis and matrix metalloproteinase-9 expression

OBJECTIVE: Receptor activator of nuclear factor-kappaB ligand (RANKL) promotes osteoclast differentiation from monocyte precursors by inducing a cohort of genes, including tartrate-resistant acid phosphatase (TRAP) and matrix metalloproteinase-9 (MMP-9). A family of synthetic triterpenoids with antiinflammatory and pro-apoptotic properties was described to modulate differentiation in monocytic cell lineages. We therefore investigated the ability of the potent and bioavailable synthetic triterpenoid TP-222 to inhibit RANKL-induced osteoclast formation and MMP-9 expression from monocytic precursor cells. METHODS: Osteoclast formation was assayed by staining for TRAP-positive multinucleated cells. MMP-9 expression was measured by quantitative RT-PCR, Western blot, immunohistochemistry, and gel zymography. In vivo effects of TP-222 were assessed by daily intraperitoneal injection of 4-week-old mice for 7 days followed by measurement of osteoclast number and MMP-9 expression at the cartilage/bone junction of the epiphyseal growth plate. RESULTS: RANKL promoted and TP-222 (300 nM) inhibited osteoclast formation in cultures of RAW264.7 cells or bone marrow-derived monocytes. RANKL also induced MMP-9 expression in RAW264.7 cells and this was reduced by concurrent or subsequent addition of TP-222. TP-222 treatment significantly reduced the mean number of osteoclasts present at the cartilage/bone interface compared to vehicle-injected control mice. Morphometric analyses of tissue sections showed that TP-222 treatment reduced the amount of immunoreactive MMP-9 present in both mononucleated pre-osteoclasts and osteoclasts. CONCLUSION: Our data demonstrate that TP-222 inhibits osteoclast formation and MMP-9 expression in vitro and in vivo, and suggest that triterpenoids may be useful compounds for modulating bone resorption diseases.     

Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL

Osteoclasts, the multinucleated cells that resorb bone, develop from hematopoietic cells of monocyte/macrophage lineage. Osteoclast-like cells (OCLs) are formed by coculturing spleen cells with osteoblasts or bone marrow stromal cells in the presence of bone-resorbing factors. The cell-to-cell interaction between osteoblasts/stromal cells and osteoclast progenitors is essential for OCL formation. Recently, we purified and molecularly cloned osteoclastogenesis-inhibitory factor (OCIF), which was identical to osteoprotegerin (OPG). OPG/OCIF is a secreted member of the tumor necrosis factor receptor family and inhibits osteoclastogenesis by interrupting the cell-to-cell interaction. Here we report the expression cloning of a ligand for OPG/OCIF from a complementary DNA library of mouse stromal cells. The protein was found to be a member of the membrane-associated tumor necrosis factor ligand family and induced OCL formation from osteoclast progenitors. A genetically engineered soluble form containing the extracellular domain of the protein induced OCL formation from spleen cells in the absence of osteoblasts/stromal cells. OPG/OCIF abolished the OCL formation induced by the protein. Expression of its gene in osteoblasts/stromal cells was up-regulated by bone-resorbing factors. We conclude that the membrane-bound protein is osteoclast differentiation factor (ODF), a long-sought ligand mediating an essential signal to osteoclast progenitors for their differentiation into osteoclasts. ODF was found to be identical to TRANCE/RANKL, which enhances T-cell growth and dendritic-cell function. ODF seems to be an important regulator in not only osteoclastogenesis but also immune system.     

The differential expression of osteoprotegerin (OPG) and receptor activator of nuclear factor kappaB ligand (RANKL) in human osteoarthritic subchondral bone osteoblasts is an indicator of the metabolic state of these disease cells

OBJECTIVE: We previously reported that human OA subchondral bone osteoblasts could be discriminated into two subpopulations identified by their levels of endogenous production (low [L] or high [H]) of PGE(2). Here, we investigated the OPG and RANKL expression levels, the histologic analysis of the subchondral bone as well as the osteoclast differentiation effect of osteoblasts on normal and both OA subpopulations (L and H), and further examined on the L OA osteoblasts the modulation of bone remodelling factors on the OPG and RANKL levels, as well as on the resorption activity. METHODS: Gene expression was determined using real-time PCR, PGE2 and OPG levels by specific ELISA, and membranous RANKL by flow cytometry. Histological observation of the subchondral bone was performed on human knee specimens. Osteoclast differentiation and formation was assayed by using the pre-osteoclastic cell line RAW 264.7. OPG and RANKL modulation on L OA osteoblasts was monitored following treatment with osteotropic factors, and the resorption activity was studied by the co-culture of differentiated PBMC/osteoblasts. RESULTS: Human OA subchondral bone osteoblasts expressed less OPG than normal. Compared to normal, RANKL gene expression levels were increased in L OA and decreased in H OA cells. The OPG/RANKL mRNA ratio was significantly diminished in L OA compared to normal or H OA (p<0.02, p<0.03), and markedly increased in H OA compared to normal. Inhibition of endogenous PGE(2) levels by indomethacin markedly decreased the ratio of OPG/RANKL on the H OA. In contrast to H OA osteoblasts, L OA cells induced a significantly higher level of osteoclast differentiation and formation (p<0.05).Histological analysis showed a reduced subchondral bone on the L OA and an increased bone mass on the H OA compared to normal. Treatment of L OA osteoblasts with osteotropic factors revealed that the OPG/RANKL mRNA expression ratio was significantly reduced by vitamin D(3) and significantly increased by TNF-alpha, PTH and PGE(2), while IL-1Beta demonstrated no effect. OPG protein levels showed similar profiles. No true effect was noted on membranous RANKL upon treatment with IL-1Beta, PGE(2) and PTH, but a significant increase was observed with vitamin D3 and TNF-alpha. The resorption activity of the L OA cells was significantly inhibited by all treatments except IL-1Beta, with maximum effect observed with vitamin D(3) and PGE(2). CONCLUSION: OPG and RANKL levels, and consequently the OPG/RANKL ratio, differed according to human OA subchondral bone osteoblast classification; it is decreased in L and increased in H OA. These findings, in addition to those showing that L OA osteoblasts have a reduced subchondral bone mass and induce a higher level of osteoclast differentiation, strongly suggest that the metabolic state of the L OA osteoblasts favours bone resorption.     

Osteoblasts and bone formation

Bone is constantly being remodelled in a dynamic process where osteoblasts are responsible for bone formation and osteoclasts for its resorption. Osteoblasts are specialized mesenchymal cells that undergo a process of maturation where genes like core-binding factor alpha1 (Cbfa1) and osterix (Osx) play a very important role. Moreover, it was found recently that Wnt/ beta-catenin pathway plays a part on osteoblast differentiation and proliferation. In fact, mutations on some of the proteins involved in this pathway, like the low-density lipoprotein receptor related protein 5/6 (LRP5/6) lead to bone diseases. Osteoblast have also a role in regulation of bone resorption through receptor activator of nuclear factor-kappaB (RANK) ligand (RANKL), that links to its receptor, RANK, on the surface of pre-osteoblast cells, inducing their differentiation and fusion. On the other hand, osteoblasts secrete a soluble decoy receptor (osteoprotegerin, OPG) that blocks RANK/RANKL interaction by binding to RANKL and, thus, prevents osteoclast differentiation and activation. Therefore, the balance between RANKL and OPG determines the formation and activity of osteoclasts. Another factor that influences bone mass is leptin, a hormone produced by adipocytes that have a dual effect. It can act through the central nervous system and diminish osteoblasts activity, or can have an osteogenic effect by binding directly to its receptors on the surface of osteoblast cells.     

Mechanisms of bone resorption in periodontal disease

Periodontal diseases are infectious, chronic inflammatory diseases that result both in loss of alveolar bone and destruction of connective tissue in periodontal regions. The bone resorption is triggered through immune responses, and results from inflammatory reactions directed against periodontopathic bacteria. Osteoclasts, bone resorbing cells, differentiate from macrophage/monocyte lineage cells, and are activated by various cytokines including osteoclast differentiation factor (ODF) / receptor activator of NF-kappaB ligand (RANKL). Activated antigen-specific CD4 positive T-cells directed against periodontopathic bacteria produce ODF/RANKL, which has been shown to play a critical role in bone resorption in periodontal diseases. This review describes recent progress towards elucidating mechanisms of bone resorption in periodontal disease.     

Osteoclasts formed by measles virus-infected osteoclast precursors from hCD46 transgenic mice express characteristics of pagetic osteoclasts.

Pagetic osteoclasts (OCLs) are abnormal in size and contain paramyxoviral-like nuclear inclusions that cross-react with antibodies to measles virus (MV). However, the role that MV infection plays in Paget's disease is unknown, because no animal model of Paget's disease is available. Therefore, we targeted a cellular MV receptor, human CD46 (hCD46), to cells in the OCL lineage in transgenic mice using the mouse tartrate-resistant acid phosphatase (TRAP) gene promoter. In vitro infection of OCL precursors from hCD46 transgenic mice with MV significantly increased OCL formation in bone marrow cultures. The numbers of TRAP-positive mononuclear cells and CFU-GM, the earliest identifiable OCL precursor, were also significantly increased. MV-infected OCLs formed from hCD46 marrow were increased in size, contained markedly increased numbers of nuclei, and had increased bone-resorbing capacity per OCL compared with OCLs formed from marrow of nontransgenic littermates. Furthermore, IL-6 and 24-hydroxylase messenger RNA expression levels were increased in MV-infected hCD46 transgenic mouse bone marrow cultures. Treatment of MV-infected hCD46 marrow cultures with a neutralizing antibody to IL-6 blocked the increased OCL formation seen in these cultures. These data demonstrate that MV infection of OCL precursors results in OCLs that have many features of pagetic OCLs, that the enhanced OCL formation is in part mediated by increased IL-6 expression induced by MV infection, and suggest that the hCD46 transgenic mouse may be a useful model for examining the effects of MV infection on OCL formation in vivo.     

Osteoblasts provide a suitable microenvironment for the action of receptor activator of nuclear factor-kappaB ligand

Deficiency of osteoprotegerin (OPG), a soluble decoy receptor for receptor activator of nuclear factor-kappaB ligand (RANKL), in mice induces osteoporosis caused by enhanced bone resorption. Serum concentrations of RANKL are extremely high in OPG-deficient (OPG(-/-)) mice, suggesting that circulating RANKL is involved in osteoclastogenesis. RANKL(-/-) mice exhibit osteopetrosis, with the absence of osteoclasts. We examined the requirements for osteoclastogenesis using OPG(-/-) mice, RANKL(-/-) mice, and a system involving bone morphogenetic protein 2 (BMP-2)-induced ectopic bone formation. When collagen disks containing BMP-2 (BMP-2-disks) or vehicle were implanted into OPG(-/-) mice, osteoclast-like cells (OCLs) and alkaline phosphatase-positive OCLs appeared in BMP-2-disks but not the control disks. F4/80-positive osteoclast precursors were similarly distributed in both BMP-2- and control disks. Cells expressing RANKL were detected in the BMP-2-disks, and the addition of OPG to the disk inhibited OCL formation. Muscle cells in culture differentiated into alkaline phosphatase-positive cells in the presence of BMP-2 and accordingly expressed RANKL mRNA in response to PTH. This suggests that RANKL expressed by osteoblasts is a requirement for osteoclastogenesis. We then examined how osteoblasts are involved in osteoclastogenesis other than RANKL expression, using RANKL(-/-) mice. BMP-2- and control disks were implanted into RANKL(-/-) mice, which were injected with RANKL for 7 d. Many OCLs were observed in the BMP-2-disks and bone tissues but not the control disks. These results suggest that osteoblasts also play important roles in osteoclastogenesis through offering the critical microenvironment for the action of RANKL.     

RANK (receptor activator of nuclear factor-kappaB) and RANKL expression in multiple myeloma

The new members of the tumour necrosis factor (TNF) receptor-ligand family, receptor activator of nuclear factor-kappaB ligand (RANKL) and its receptor RANK, play a crucial role in osteoclast differentiation and activation. An increased expression of RANKL and/or RANK may be involved in the excessive bone resorption observed in multiple myeloma (MM). We used immunohistochemistry to study RANK and RANKL expression in bone marrow (BM) biopsies obtained at diagnosis in 15 MM patients, six patients with monoclonal gammopathy of undetermined significance (MGUS) and 10 normal BM biopsies. Plasma cells were not labelled with anti-RANKL or anti-RANK antibodies. In all biopsies, RANKL was expressed in endosteal bone surface, around vessels and in cells characterized by cytoplasmic expansions. These last cells did not express CD45 and were vimentin positive, corresponding to bone marrow stromal cells. Numerous stromal cells expressed RANKL in MM and MGUS specimens, with a greater expression in MM than in MGUS. Very few cells were stained with anti-RANKL in normal BM specimens. With the anti-RANK antibody, small mononuclear cells in the bone microenvironment were positive and were identified as erythroblast cells. In conclusion, we showed that RANKL was expressed in reticular stromal cells, with a greater intensity in myeloma specimens. These results suggest that RANKL overexpressed by bone marrow stromal cells may contribute to the high rate of bone resorption observed in MM.     

Alpha-lipoic acid suppresses osteoclastogenesis despite increasing the receptor activator of nuclear factor kappaB ligand/osteoprotegerin ratio in human bone marrow stromal cells

Growing evidence has shown a biochemical link between increased oxidative stress and reduced bone density. Although alpha-lipoic acid (alpha-LA) has been shown to act as a thiol antioxidant, its effect on bone cells has not been determined. Using proteomic analysis, we identified six differentially expressed proteins in the conditioned media of alpha-LA-treated human bone marrow stromal cell line (HS-5). One of these proteins, receptor activator of nuclear factor kappaB ligand (RANKL), was significantly up-regulated, as confirmed by immunoblotting with anti-RANKL antibody. ELISA showed that alpha-LA stimulated RANKL production in cellular extracts (membranous RANKL) about 5-fold and in conditioned medium (soluble RANKL) about 23-fold, but had no effect on osteoprotegerin (OPG) secretion. Despite increasing the RANKL/OPG ratio, alpha-LA showed a dose-dependent suppression of osteoclastogenesis, both in a coculture system of mouse bone marrow cells and osteoblasts and in a mouse bone marrow cell culture system, and reduced bone resorption in a dose-dependent manner. In addition, alpha-LA-induced soluble RANKL was not inhibited by matrix metalloprotease inhibitors, indicating that soluble RANKL is produced by alpha-LA without any posttranslational processing. In contrast, alpha-LA had no significant effect on the proliferation and differentiation of HS-5 cells. These results suggest that alpha-LA suppresses osteoclastogenesis by directly inhibiting RANKL-RANK mediated signals, not by mediating cellular RANKL production. In addition, our findings indicate that alpha-LA-induced soluble RANKL is not produced by shedding of membranous RANKL.     

Increase in expression of receptor activator of nuclear factor kappaB at sites of bone erosion correlates with progression of inflammation in evolving collagen-induced arthritis

OBJECTIVE: The receptor activator of nuclear factor kappaB (RANK)/RANK ligand (RANKL) pathway is critical in osteoclastogenesis and bone resorption and has been implicated in the process of focal bone erosion in arthritis. This study was undertaken to identify in vivo the hitherto-unknown origin and localization of RANK-expressing osteoclast precursor cells at sites of bone erosion in arthritis. METHODS: DBA-1 mice were immunized with bovine type II collagen/Freund's complete adjuvant and were given an intraperitoneal booster injection of type II collagen on day 21. Arthritis was monitored visually, and joint pathology was examined histologically. RANK and RANKL expression were analyzed using specific immunohistochemistry, and tartrate-resistant acid phosphatase (TRAP) staining was performed. In addition, TRAP and cathepsin K messenger RNA expression were analyzed by in situ hybridization. RESULTS: A marked increase in the number of cells expressing RANK correlated with the progression of synovial inflammation and clinical disease severity in evolving collagen-induced arthritis (CIA). Interestingly, RANK expression demonstrated a gradient pattern with increased numbers of RANK-positive cells within the synovial infiltrate in areas closer to periosteum and cortical bone. Cells expressing RANK included cells in synovial tissue, bone lining cells on the surface of trabecular bone at sites of erosion, and cells in periosteal areas adjacent to synovial inflammation. In areas where RANK-positive cells were abundant, TRAP-positive, multinucleated osteoclast-like cells were also present at sites of focal bone erosion, suggesting differentiation of synovially derived RANK-positive osteoclast precursor cells into osteoclasts. In addition, TRAP- and cathepsin K-double-positive osteoclast-like cells were detected on the synovial side of cortical bone at sites of early and advanced cortical bone erosion. Sites of RANK expression also correlated well with sites of RANKL expression, and there was a close correlation of the temporal expression of the receptor-ligand pair. CONCLUSION: Cells expressing RANK increased in abundance with the progression of arthritis in evolving CIA, and sites of RANK-expressing cells correlated with sites of TRAP-positive, multinucleated osteoclast-like cells as well as with sites of RANKL expression. These data support the hypothesis that the RANK/RANKL pathway plays an important role in the process of bone erosion in CIA.     

Bone marrow microenvironment controls the in vivo differentiation of murine dendritic cells into osteoclasts

Finding that activated T cells control osteoclast (OCL) differentiation has revealed the importance of the interactions between immune and bone cells. Dendritic cells (DCs) are responsible for T-cell activation and share common precursors with OCLs. Here we show that DCs participate in bone resorption more directly than simply through T-cell activation. We show that, among the splenic DC subsets, the conventional DCs have the higher osteoclastogenic potential in vitro. We demonstrate that conventional DCs differentiate into functional OCLs in vivo when injected into osteopetrotic oc/oc mice defective in OCL resorptive function. Moreover, this differentiation involves the presence of activated CD4(+) T cells controlling a high RANK-L expression by bone marrow stromal cells. Our results open new insights in the differentiation of OCLs and DCs and offer new basis for analyzing the relations between bone and immune systems.     

Bradykinin potentiates cytokine-induced prostaglandin biosynthesis in osteoblasts by enhanced expression of cyclooxygenase 2, resulting in increased RANKL expression

OBJECTIVE: Bradykinin (BK) stimulates bone resorption in vitro and synergistically potentiates interleukin-1 (IL-1)-induced bone resorption and prostaglandin (PG) formation, suggesting that kinins are important in inflammation-induced bone loss. The present study was undertaken to study 1) the role of the kinin B1 and B2 receptors in the synergistic interaction with IL-1 and tumor necrosis factor alpha (TNFalpha), 2) the molecular mechanisms involved in synergistic enhancement of PG formation, and 3) the effects of kinins on cytokine-induced expression of RANKL, RANK, and osteoprotegerin (OPG) (the latter being crucial molecules in osteoclast differentiation). METHODS: Formation of PGs, expression of enzymes involved in arachidonic acid metabolism, and expression of RANKL, RANK, and OPG were assessed in the human osteoblastic cell line MG-63 and in mouse calvarial bones. The role of NF-kappaB and MAP kinases was studied using pharmacologic inhibitors. RESULTS: PGE(2) formation and cyclooxygenase 2 (COX-2) protein expression were induced by IL-1beta and potentiated by kinins with affinity for the B1 or B2 receptors, resulting in PGE(2)-dependent enhancement of RANKL. The enhancements of PGE(2) formation and COX-2 were markedly decreased by inhibition of p38 and JNK MAP kinases, whereas inhibition of NF-kappaB resulted in abolishment of the PGE(2) response with only slight inhibition of COX-2. CONCLUSION: Kinin B1 and B2 receptors synergistically potentiate IL-1- and TNFalpha-induced PG biosynthesis in osteoblasts by a mechanism involving increased levels of COX-2, resulting in increased RANKL. The synergistic stimulation is dependent on NF-kappaB and MAP kinases. These mechanisms might help to explain the enhanced bone resorption associated with inflammatory disorders, including that in rheumatoid arthritis.