RANKL Signaling Pathways Research Articles

JNK/c-Jun Signaling Mediates an Anti-Apoptotic Effect of RANKL in Osteoclasts

RANKL is known to be important not only for differentiation and activation of osteoclasts but also for their survival. Experimentally, apoptosis of osteoclasts is rapidly induced by the deprivation of RANKL. RANKL activates Elk-related tyrosine kinase (ERK), p38, c-Jun N-terminal kinase (JNK), and NF-kappaB pathways through TRAF6 in osteoclasts and the precursor cells. It has been shown that ERK is critical for regulation of osteoclast survival. However, an involvement of other RANKL signaling pathways such as JNK signaling in survival of osteoclasts has not been fully understood yet. Osteoclasts derived from primary mouse bone marrow cells by soluble RANKL (sRANKL) were treated with a JNK inhibitor, SP600125, or infected with adenovirus carrying dominant-negative (DN)-c-jun, DN-c-fos, mitogen-activated protein kinase kinase 1 (MEKK1), I-kappaBalpha mutant, or NF-kappaB components, p50 and p65. Osteoclasts were cultured with or without sRANKL, and apoptotic phenotype was determined by TUNEL assay, DAPI staining, and expression of cleaved caspase 3 followed by TRACP staining. Overexpression of TRAF6 activated JNK and NF-kappaB signaling pathways and clearly prevented osteoclasts from apoptosis caused by abrogation of sRANKL. An anti-apoptotic effect of RANKL/RANK/TRAF6 signaling on osteoclast was inhibited by JNK-specific inhibitor SP600125 and by overexpression of dominant-negative JNK1, c-jun, and c-fos. Also, overexpression of MEKK1 inhibited apoptosis of osteoclasts even in the absence of sRANKL along with activation of JNK/c-jun signaling. On the other hand, blockade of NF-kappaB signaling by I-kappaBalpha mutant or overexpression of NF-kappaB components showed a marginal effect on apoptosis of osteoclasts. An important role of RANKL-induced activation of MEKK1/JNK/c-jun signaling in the regulation of apoptosis in osteoclasts was shown. Our study suggests that c-fos plays a role as a partner of activator protein-1 factor, c-jun, during the regulation of apoptosis in osteoclasts.

Benzo[ a]pyrene inhibits osteoclastogenesis by affecting RANKL-induced activation of NF-κB

Exposure to polycyclic aryl hydrocarbons is linked to cancer, immunosuppression and other numerous health problems. We previously demonstrated that exposure to benzo[ a]pyrene (BaP), an environmental pollutant present in high concentrations in urban smog and cigarette smoke, inhibits osteoclast differentiation and bone resorption. We hypothesized that this inhibition could be due to crosstalk between the receptor activator of NF-κB ligand (RANKL) and AhR signaling cascades competing for NF-κB, a common transcription factor for both pathways. RAW264.7 cells (a mouse macrophage cell line capable of differentiating into osteoclasts in the presence of RANKL) were exposed to different concentrations of RANKL and BaP and the effect on NF-κB activation, nuclear translocation, as well as the effect of NF-κB inhibitors on BaP-mediated CYP1B1 gene expression was measured. The results demonstrated that BaP inhibited both RANKL-induced NF-κB activation and nuclear translocation. At the same time, BaP-induced CYP1B1 gene expression was inhibited by two NF-κB inhibitors in a dose-dependent manner, demonstrating that NF-κB is involved in a BaP-mediated signaling pathway. A reporter gene assay showed that both BaP and RANKL-induced luciferase reporter gene transcription under the control of NF-κB response elements. Co-immunoprecipitation results demonstrated that AhR interacted with NF-κB p65 in RAW cells and BaP appeared to enhance this interaction. However, in the presence of RANKL, we did not observe any interaction between AhR and p65. These results support our hypothesis that BaP-mediated inhibition of osteoclastogenesis is a consequence of crosstalk between AhR and RANKL signaling pathways competing for the common transcription factor NF-κB.

A Triterpene Glycoside from Black Cohosh that Inhibits Osteoclastogenesis by Modulating RANKL and TNFα Signaling Pathways

Osteoporosis is a major age-related source of morbidity and mortality. Increased bone resorption mediated by osteoclasts is central to its pathogenesis. Cytokines, particularly RANKL and TNFalpha, are often increased under pathologic conditions, leading to enhanced osteoclastogenesis. Black cohosh (Actaea/Cimicifuga racemosa L), a popular herbal supplement for the treatment of menopausal symptoms, was recently shown to have the beneficial effect of preventing bone loss. Here, we demonstrate that 25-acetylcimigenol xylopyranoside (ACCX), a triterpenoid glycoside isolated from black cohosh, potently blocks in vitro osteoclastogenesis induced by either RANKL or TNFalpha. This blockage of osteoclastogenesis elicited by ACCX results from abrogation of the NF-kappaB and ERK pathways induced by either RANKL or TNFalpha, respectively. Importantly, this compound attenuates TNFalpha-induced bone loss in vivo. Therefore, ACCX represents a potential lead for the development of a new class of antiosteoporosis agents.

Inhibition of osteoclast differentiation by polycyclic aryl hydrocarbons is dependent on cell density and RANKL concentration

We investigated the effect of representative polycyclic aryl hydrocarbons (PAHs), benzo[ a]pyrene (BaP), and 7,12-dimethylbenz[ a]anthracene (DMBA) on osteoclast differentiation and function by using dispersed cancellous bone derived rabbit osteoclasts and the RAW264.7 cells. These cells differentiate into osteoclasts when exposed to receptor activator of NF-κB ligand (RANKL). The rabbit osteoclasts were exposed to 10 −6 to 10 −9 M BaP or DMBA and the tartrate-resistant acid phosphatase (TRAP)-positive cells were counted. The effect of PAHs on osteoclast differentiation in dispersed rabbit osteoclast-containing stromal cell populations was cell density dependent, suggesting that the cell density of stromal cells, osteoclast precursors, and/or mature osteoclasts are factors regulating the effect of PAHs. To investigate the direct effect of BaP on osteoclast differentiation, RAW264.7 cells were exposed to 10 −5 to 10 −6 M BaP. Treatment of RAW264.7 cells cultured with 25 ng/ml soluble RANKL and 10 −5 M BaP for 5 days decreased osteoclast differentiation, TRAP activity levels, and resorption of bone-like substrata. The inhibition was prevented by 10 −6 to 10 −7 M resveratrol, an aryl hydrocarbon receptor (AhR) antagonist, and by higher concentrations of RANKL. To investigate the ability of RANKL to reverse BaP-mediated inhibition, gene expression was determined by RT-PCR. Cytochrome P450 1B1 (CYP1B1) mRNA, one of the genes activated by BaP, was present only in the groups exposed to BaP; the levels of CYP1B1 mRNA decreased in the presence of increasing concentrations of RANKL. These results suggest that the inhibitory effects of PAHs on osteoclastogenesis are direct and likely involve interaction of the RANKL and PAH signaling pathways.

Evidence of reciprocal regulation between the high extracellular calcium and RANKL signal transduction pathways in RAW cell derived osteoclasts

During bone resorption, osteoclasts are exposed to high Ca2+ concentrations (up to 40 mM). The role of high extracellular Ca2+ in receptor activator of NF-kappaB ligand (RANKL)-mediated osteoclast survival and their functional interrelationship is unclear. In this study, we show that RANKL enhances osteoclast tolerance to high extracellular Ca2+ by protecting the cell from cell death in a dose dependent manner. We have provided evidence that RANKL does this by attenuating high extracellular Ca2+-induced Ca2+ elevations. Moreover, we have found that high extracellular Ca2+-induced cell death was partially inhibited by a caspase-3 inhibitor, suggesting caspase-3-mediated apoptosis is involved. Conversely, using reporter gene assays and Western blot analysis, we have demonstrated that high extracellular Ca2+ desensitizes the RANKL-induced activation of NF-kappaB and c-Jun N-terminal kinase (JNK), and inhibits constitutive and RANKL-stimulated ERK phosphorylation, indicating a negative feed-back mechanism via specific RANKL signaling pathways. Taken together, this study provides evidence for a reciprocal regulation between high extracellular Ca2+ and RANKL signaling in RAW cell derived osteoclasts. Our data imply a cross talk mechanism of extracellular Ca2+ on osteoclast survival through the regulation of RANKL.

12-O-tetradecanoylphorbol-13-acetate (TPA) inhibits osteoclastogenesis by suppressing RANKL-induced NF-kappaB activation.

The mechanism by which TPA-induced PKC activity modulates osteoclastogenesis is not clear. Using a RAW(264.7) cell culture system and assays for NF-kappaB nuclear translocation, NF-kappaB reporter gene activity, and MAPK assays, we demonstrated that TPA inhibits osteoclastogenesis through the suppression of RANKL-induced NF-kappaB activation. The protein kinase C (PKC) pathway has been suggested to be an important regulator of osteoclastic bone resorption. The role of PKC in RANKL-induced osteoclastogenesis, however, is not clear. In this study, we examined the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA), a PKC activator, on osteoclastogenesis and studied its role in RANKL-induced signaling. RANKL-induced RAW(264.7) cell differentiation into osteoclast-like cells was used to assess the effect of TPA on osteoclastogenesis. Assays for NF-kappaB nuclear translocation, NF-kappaB reporter gene activity, protein kinase activity, and Western blotting were used to examine the effects of TPA on RANKL-induced NF-kappaB, c-Jun N-terminal kinase (JNK), and MEK/ERK and p38 signal transduction pathways. We found that TPA inhibited RANKL-induced RAW(264.7) cell differentiation into osteoclasts in a dose-dependent manner. Time course analysis showed that the inhibitory effect of TPA on RANKL-induced osteoclastogenesis occurs predominantly at an early stage of osteoclast differentiation. TPA alone had little effect on NF-kappaB activation in RAW(264.7) cells, but it suppresses the RANKL-induced NF-kappaB activation in a dose-dependent fashion. Interestingly, the suppressive effect of TPA on RANKL-induced NF-kappaB activation was prevented by a conventional PKC inhibitor, Go6976. Supershift studies revealed that the RANKL-induced DNA binding of NF-kappaB complexes consisted of C-Rel, NF-kappaB1 (p50), and RelA (p65). In addition, TPA induced the activation of JNK in RAW(264.7) cells but had little effect on RANKL-induced activation of JNK. TPA also inhibited RANKL-induced activation of ERK but had little effect on p38 activation. Given that NF-kappaB activation is obligatory for osteoclast differentiation, our studies imply that inhibition of osteoclastogenesis by TPA is, at least in part, caused by the suppression of RANKL-induced activation of NF-kappaB during an early stage of osteoclastogenesis. Selective modulation of RANKL signaling pathways by PKC activators may have important therapeutic implications for the treatment of bone diseases associated with enhanced bone resorption.

Defective mammary gland morphogenesis in mice lacking the progesterone receptor B isoform.

Progesterone (P) regulates female reproduction via two nuclear receptors, PR-A and PR-B. Although both receptors display overlapping and distinct transcription regulatory properties, their individual physiological roles are unclear. To address the physiological role of PR-A, we generated a mouse model in which expression of PR-B was specifically ablated (PRBKO-/-). We show that selective activation of PR-A in PRBKO-/- mice is sufficient to elicit normal ovarian and uterine responses to P but results in reduced mammary gland morphogenesis. In the absence of PR-B, pregnancy-associated ductal sidebranching and lobuloalveolar development are markedly reduced due to decreased ductal and alveolar epithelial cell proliferation and decreased survival of alveolar epithelium. In an effort to elucidate the molecular genetic signaling pathways that are differentially regulated by PRs in the mammary gland, we have identified receptor activator of nuclear factor kappa B ligand (RANKL) as a paracrine mediator of P-dependent alveologenesis. Further, we demonstrate that the defects in PRBKO-/- mice are associated with an inability of PR-A to activate the RANKL signaling pathway in response to P. Our data indicate that functional interaction between PR-A and PR-B is not required for reproductive activity and that selective modulation of PR-A activity by progestin agonists may have a protective effect against both uterine and mammary gland hyperplasias.

Regulation of RANKL signaling in arthritic bone destruction

Formation of osteoclasts is induced by a tumor necrosis factor family cytokine, RANKL (receptor activator of NF-κB ligand). To maintain the normal bone homeostasis and to prevent the pathological bone resorption, RANKL signaling must be strictly kept in control. During the course of our study on the bone loss in rheumatoid arthritis (RA), we found that RANKL expressed on synovial fibroblasts is responsible for osteoclastogenesis from synoviocytes. However, it has been also reported that RANKL-expressing T cells are involved in osteoclastogenesis in RA. We then focused on the regulation of osteoclast differentiation by T cells. Using mice lacking a receptor component for IFN-γ, we revealed that T-cell production of IFN-γ strongly suppresses osteoclastogenesis by interfering with the RANKL signaling pathway [1]. We have shed light on a new biological function of IFN-γ, which is to protect against calcified tissue destruction upon T-cell activation, demonstrating that activated T cells not only positively regulate, but also negatively affect osteoclastogenesis. To explore the molecular targets for suppressing bone destruction in RA, we performed a genome-wide screening of RANKL-inducible genes. We found that RANKL induces IFN-β, a critical cytokine for antiviral defense. Mice deficient in IFN-β signaling exhibited severe osteopenia accompanied by enhanced osteoclastogenesis, suggesting that IFN-β is essential for normal bone remodeling by suppressing excessive osteoclast differentiation [2]. In addition, we revealed beneficial effects of IFN-β in animal models of pathological bone resorption. We have recently identified that the transcription factor NFATc1 is specifically induced by RANKL [3]. We demonstrate that NFATc1-deficient embryonic stem cells fail to differentiate into osteoclasts in response to RANKL stimulation, and the ectopic expression of NFATc1 causes the precursor cells to undergo efficient differentiation without RANKL signaling. Thus, NFATc1 may be a master switch regulator for the terminal differentiation of osteoclasts, functioning downstream of RANKL signaling. The activation of NFATc1 by RANKL is regulated by calcium-dependent phosphatase, calcineurin, and calcineurin inhibitors such as FK506 and cyclosporin A strongly suppress osteoclastogenesis. The possibility of NFATc1 as a therapeutic target of bone destruction in RA will be discussed.