Curcumin inhibits osteoclastogenesis by decreasing receptor activator of nuclear factor-kappaB ligand (RANKL) in bone marrow stromal cells

Curcumin (diferuloylmethane), a pigment derived from turmeric, has anti-oxidant and anti-inflammatory activities. Accumulating evidence points to a biochemical link between increased oxidative stress and reduced bone density. Osteoclast formation was evaluated in co-cultures of bone marrow stromal cells (BMSC) and whole bone marrow cells (BMC). Expression of receptor activator of nuclear factor-kappaB ligand (RANKL) was analyzed at the mRNA and protein levels. Exposure to curcumin led to dose-dependent suppression of osteoclastogenesis in the coculture system, and to reduced expression of RANKL in IL-1alpha-stimulated BMSCs. Addition of RANKL abolished the inhibition of osteoclastogenesis by curcumin, whereas the addition of prostaglandin E2(PGE2) did not. The decreased osteoclastogenesis induced by curcumin may reduce bone loss and be of potential benefit in preventing and/or attenuating osteoporosis.     

Defective osteoclastogenesis by IKKbeta-null precursors is a result of receptor activator of NF-kappaB ligand (RANKL)-induced JNK-dependent apoptosis and impaired differentiation

It has been reported previously that inhibitory kappaB kinase (IKK) supports osteoclastogenesis through NF-kappaB-mediated prevention of apoptosis. This finding suggests that the ligand for receptor activator of NF-kappaB (RANKL), the master osteoclastogenic cytokine, induces apoptosis of osteoclast precursors (OCPs) in the absence of IKKbeta/NF-kappaB competency. To validate this hypothesis, we sought to determine the pro-apoptotic signaling factors induced by RANKL in IKKbeta-null osteoclast OCPs and to rescue osteoclast differentiation in the absence of IKKbeta through their inhibition. To accomplish this, we generated mice that lack IKKbeta in multiple hematopoietic lineages, including OCPs. We found that these mice possess both in vitro and in vivo defects in osteoclast generation, in concurrence with previous reports, and that this defect is a result of susceptibility to RANKL-mediated apoptosis as a result of gain-of-function of JNK activation. We demonstrate that differentiation of OCPs depends on IKKbeta because reduced IKKbeta mRNA expression correlates with impaired induction of osteoclast differentiation markers in response to RANKL stimulation. We further show that fine-tuned inhibition of JNK activation in these cells inhibits RANKL-induced apoptosis and restores the ability of IKKbeta-null OCPs to become mature osteoclasts. Our data highlight the pro-osteoclastogenic and anti-apoptotic roles of IKKbeta in OCPs and identify a pro-apoptotic mechanism activated within the RANK signalosome.     

Multimerization of the receptor activator of nuclear factor-kappaB ligand (RANKL) isoforms and regulation of osteoclastogenesis

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.     

Negative regulation of osteoclastogenesis by ectodomain shedding of receptor activator of NF-kappaB ligand

Receptor activator of NF-kappaB ligand (RANKL) is a transmembrane glycoprotein that has an essential role in the development of osteoclasts. The extracellular portion of RANKL is cleaved proteolytically to produce soluble RANKL, but definite RANKL sheddase(s) and the physiologic function of RANKL shedding have not yet been determined. In the present study, we found that matrix metalloproteinase (MMP) 14 and a disintegrin and metalloproteinase (ADAM) 10 have strong RANKL shedding activity. In Western blot analysis, soluble RANKL was detected as two different molecular weight products, and RNA interference of MMP14 and ADAM10 resulted in a reduction of both the lower and higher molecular weight products. Suppression of MMP14 in primary osteoblasts increased membrane-bound RANKL and promoted osteoclastogenesis in cocultures with macrophages. Soluble RANKL produced by osteoblasts from MMP14-deficient mice was markedly reduced, and their osteoclastogenic activity was promoted, consistent with the findings of increased osteoclastogenesis in vivo. RANKL shedding is an important process that down-regulates local osteoclastogenesis.     

The inhibitory effect of melatonin on osteoclastogenesis of RAW 264.7 cells in low concentrations of RANKL and MCSF

RAW 264.7 cells are one of the most recommended cell lines for investigating the activity and differentiation of osteoclasts. These cells differentiate into osteoclasts in the presence of two critical components: receptor activator of nuclear factor kappa B ligand (RANKL) and macrophage colony stimulating factor (MCSF). Melatonin (MEL) hormone has recently become one of the small molecules used in the field of bone regeneration and bone disease treatment, as it has the ability to inhibit the differentiation of osteoclasts directly by suppression of the NF-κB signaling pathway. The main aim of the current study is to determine sufficient RANKL/MCSF concentrations for differentiation of the cells to osteoclasts and to describe the repressive effect of MEL on the osteoclastogenesis of these cells. In this regard, it was found that 10 ng/mL of RANKL- and MCSF-containing medium is suitable for inducing osteoclastogenesis of the cells. In addition, melatonin at doses in the range of 100–1000 µM does not have a cytotoxic effect. Subsequently, results of tartrate resistant acid phosphatase (TRAP) activity, TRAP staining, and relative expressions of cathepsin K, nuclear factor of activated T cells one (NFATC1), and TRAP genes showed a suppressive effect of MEL —especially 800 µM— on RANKL-induced osteoclastogenesis of these cells.     

Receptor activator of NF-kappaB ligand regulates the proliferation of mammary epithelial cells via Id2

Receptor activator of NF-kappaB ligand (RANKL) is a key regulator for mammary gland development during pregnancy. RANKL-deficient mice display impaired development of lobulo-alveolar mammary structures. Similar mammary gland defects have been reported in mice lacking Id2. Here we report that RANKL induces the proliferation of mammary epithelial cells via Id2. RANKL triggers marked nuclear translocation of Id2 in mammary epithelial cells. In vivo studies further demonstrated the defective nuclear translocation of Id2, but the normal expression of cyclin D1, in the mammary epithelial cells of rankl-/- mice. In vitro studies with nuclear localization sequence-tagged Id2 revealed that the nuclear localization of Id2 itself is critical for the downregulation of p21 promoter activity. Moreover, RANKL stimulation failed to induce cell growth and to downregulate p21 expression in Id2-/- mammary epithelial cells. Our results indicate that the inhibitor of helix-loop-helix protein, Id2, is critical to control the proliferation of mammary epithelial cells in response to RANKL stimulation.     

Expression, purification, and characterization of the human receptor activator of NF-kappaB ligand (RANKL) extracellular domain

Receptor activator of NF-kappaB ligand (RANKL) is a type II transmembrane protein found on osteoblasts which functions as a major determinant of osteoclast differentiation and activation. RANKL mediates bone homeostasis through binding to the cognate ligand on osteoclasts, RANK, and a soluble decoy receptor, osteoprotegerin (OPG). We designed a construct encoding the extracellular domain of human RANKL that conformed to reports of native processing. To encourage folding and posttranslational modification of a normally membrane-inserted moiety, we expressed the RANKL truncate as a secreted protein using the signal sequence from OPG in a Trichoplusia ni cell line using a baculovirus expression vector. RANKL was purified by a three-step process including an OPG-Fc affinity column. SDS-PAGE and mass spectral analysis indicated that the protein was >99% pure and glycosylated. Circular dichroism spectra revealed that the protein exhibited structural elements similar to tumor necrosis factor-alpha. By BIAcore analysis, RANKL bound to OPG with an affinity of 6.7 nM. Sedimentation equilibrium analytical ultracentrifugation analyses established that our protein existed as a trimer. We conclude that our expressed human RANKL truncate is folded, is functional, and exhibits self-association consistent with other family members.     

Involvement of p38 mitogen-activated protein kinase signaling pathway in osteoclastogenesis mediated by receptor activator of NF-kappa B ligand (RANKL)

The receptor activator of NF-kappaB ligand (RANKL) induces osteoclast differentiation from bone marrow cells in the presence of macrophage colony-stimulating factor. We found that treatment of bone marrow cells with SB203580 inhibited osteoclast differentiation via inhibition of the RANKL-mediated signaling pathway. To elucidate the role of p38 mitogen-activated protein (MAP) kinase pathway in osteoclastogenesis, we employed RAW264 cells which could differentiate into osteoclast-like cells following treatment with RANKL. In a dose-dependent manner, SB203580 but not PD98059, inhibited RANKL-induced differentiation. Among three MAP kinase families tested, this inhibition profile coincided only with the activation of p38 MAP kinase. Expression in RAW264 cells of the dominant negative form of either p38alpha MAP kinase or MAP kinase kinase (MKK) 6 significantly inhibited RANKL-induced differentiation of the cells. These results indicate that activation of the p38 MAP kinase pathway plays an important role in RANKL-induced osteoclast differentiation of precursor bone marrow cells.     

Evaluation of efficacy on RANKL induced osteoclast from RAW264.7 cells

Established RAW264.7 cell lines for osteoclastic differentiation has been widely engaged in bone homeostasis research, however, the efficacy of RANKL independently stimulating has rarely been defined, because protocols were usually developed and modified by various laboratories. Otherwise, problematic issues are also lie in the cell's seeding density, RANKL stimulating time point, and distinguishing osteoclastogenesis ability of RANKL-treated RAW264.7 cells. Therefore, in the current study, we examined the efficacy of various concentrations of RANKL-treated RAW264.7 for its osteoclastic differentiation with or without pretreated other costimulators such as: LPS and/or M-CSF. The oteoclastogenesis ability of RANKL-treated RAW264.7 cells was demonstrated by bone resorption pit, F-actin, and osteoclastogenesis specific marker studies. Besides that, through tartrate-resistant acid phosphatase (TRAP) staining, we clarified to start the treatment with 30 ng/ml RANKL at 12 hr after seeded RAW264.7 with the density of 6.25 × 10 ³ cells/cm ² manifested an significantly increased number of multinucleated osteoclastic cells. Overall, our results establishing an optimal method for RANKL independently inducing RAW 264.7 cell osteoclastic differentiation, which could efficiently generate osteoclasts in vitro for significant advances in our understanding of bone biology.     

HIV envelope gp120-mediated regulation of osteoclastogenesis via receptor activator of nuclear factor kappa B ligand (RANKL) secretion and its modulation by certain HIV protease inhibitors through interferon-gamma/RANKL cross-talk

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.     

Epidermal receptor activator of NF-kappaB ligand controls Langerhans cells numbers and proliferation

Langerhans cells (LC) are the dendritic APC population of the epidermis, where they reside for long periods and are self-replicating. The molecular signals underlying these characteristics are unknown. The TNF superfamily member receptor activator of NF-kappaB ligand (RANKL, TNFSF11) has been shown to sustain viability of blood dendritic cells in addition to its role in promoting proliferation and differentiation of several cell types, notably osteoclasts. In this study, we have studied expression of the RANKL system in skin and have defined a key role for this molecule in LC homeostasis. In vitro and in vivo, human KC expressed RANKL and epidermal LC expressed cell surface RANK. In vitro, RANKL sustained CD34(+) progenitor-derived LC viability following 72-h cultures in cytokine-free medium (79.5 +/- 1% vs 55.2 +/- 5.7% live cells, respectively; n = 4; p < 0.05). In vivo, RANKL-deficient mice displayed a marked reduction in epidermal LC density (507.1 +/- 77.2 vs 873.6 +/- 41.6 LC per mm(2); n = 9; p < 0.05) and their proliferation was impaired without a detectable effect on apoptosis. These data indicate a key role for the RANKL system in the regulation of LC survival within the skin and suggest a regulatory role for KC in the maintenance of epidermal LC homeostasis.