Evidence that receptor activator of nuclear factor (NF)-kappaB ligand can suppress cell proliferation and induce apoptosis through activation of a NF-kappaB-independent and TRAF6-dependent mechanism

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.     

Upregulation of receptor activator of nuclear factor-κB ligand expression in the thymic subcapsular, paraseptal, perivascular, and medullary epithelial cells during thymus regeneration

The receptor activator of nuclear factor (NF)-B ligand (RANKL; also termed TRANCE/OPGL/ODF/TNFSF11), a new member of the tumor-necrosis factor (TNF) superfamily, was identified as a key cytokine involved in the differentiation of the immune system and the regulation of immunity as well as in bone metabolism. In particular, RANKL-deficient mice showed defects in the early differentiation of T lymphocytes, suggesting that RANKL is a novel regulator of early thymocyte development. Here, we describe the expression of RANKL during regeneration following acute involution induced by cyclophosphamide in the rat thymus. The present study demonstrates the presence and upregulated expression of the RANKL in thymic subcapsular, paraseptal, perivascular, and medullary epithelial cells during thymus regeneration. Our results suggest that the RANKL expressed in these thymic epithelial cells plays a role in the development of T cells during thymic regeneration.     

TWEAK/Fn14 interaction regulates RANTES production, BMP-2-induced differentiation, and RANKL expression in mouse osteoblastic MC3T3-E1 cells

Tumour necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK), a member of the TNF family, is a multifunctional cytokine that regulates cell growth, migration, and survival principally through a TWEAK receptor, fibroblast growth factor-inducible 14 (Fn14). However, its physiological roles in bone are largely unknown. We herein report various effects of TWEAK on mouse osteoblastic MC3T3-E1 cells. MC3T3-E1 cells expressed Fn14 and produced RANTES (regulated upon activation, healthy T cell expressed and secreted) upon TWEAK stimulation through PI3K-Akt, but not nuclear factor-kappaB (NF-kappaB), pathway. In addition, TWEAK inhibited bone morphogenetic protein (BMP)-2-induced expression of osteoblast differentiation markers such as alkaline phosphatase through mitogen-activated protein kinase (MAPK) Erk pathway. Furthermore, TWEAK upregulated RANKL (receptor activation of NF-kappaB ligand) expression through MAPK Erk pathway in MC3T3-E1 cells. All these effects of TWEAK on MC3T3-E1 cells were abolished by mouse Fn14-Fc chimera. We also found significant TWEAK mRNA or protein expression in osteoblast- and osteoclast-lineage cell lines or the mouse bone tissue, respectively. Finally, we showed that human osteoblasts expressed Fn14 and induced RANTES and RANKL upon TWEAK stimulation. Collectively, TWEAK/Fn14 interaction regulates RANTES production, BMP-2-induced differentiation, and RANKL expression in MC3T3-E1 cells. TWEAK may thus be a novel cytokine that regulates several aspects of osteoblast function.     

Epidermal growth factor receptor regulates osteoclast differentiation and survival through cross-talking with RANK signaling

The epidermal growth factor receptor (EGFR) functions in various cellular physiological processes such as proliferation, differentiation, and motility. Although recent studies have reported that EGFR signaling is involved in osteoclast recruitment and formation, the molecular mechanism of EGFR signaling for the regulation of osteoclastogenesis remains unclear. We investigated the role of the EGFR in osteoclast differentiation and survival and show that the expression of the EGFR was highly up-regulated by receptor activator of nuclear factor-kappaB ligand (RANKL) during osteoclast differentiation. EGFR-specific tyrosine kinase inhibitors and EGFR knockdown blocked RANKL-dependent osteoclast formation, suggesting that EGFR signaling plays an important role in osteoclastogenesis. EGFR inhibition impaired the RANKL-mediated activation of osteoclastogenic signaling pathways, including c-Jun N-terminal kinase (JNK), NF-kappaB, and Akt/protein kinase B (PKB). In addition, EGFR inhibition in differentiated osteoclasts caused apoptosis through caspase activation. Inhibition of the phosphoinositide-3 kinase (PI3K)-Akt/PKB pathway and subsequent activation of BAD and caspases-9 and -3 may be responsible for the EGFR inhibition-induced apoptosis. The EGFR physically associated with receptor activator of nuclear factor-kappaB (RANK) and Grb2-associated binder 2 (Gab2). Moreover, RANKL transactivated EGFR. These data indicate that EGFR regulates RANKL-activated signaling pathways by cross-talking with RANK, suggesting that the EGFR may play a crucial role as a RANK downstream signal and/or a novel type of RANK co-receptor in osteoclast differentiation and survival.     

Regulatory roles and molecular signaling of TNF family members in osteoclasts

The tumor necrosis factor (TNF) family has been one of the most intensively studied families of proteins in the past two decades. The TNF family constitutes 19 members that mediate diverse biological functions in a variety of cellular systems. The TNF family members regulate cellular functions through binding to membrane-bound receptors belonging to the TNF receptor (TNFR) family. Members of the TNFR family lack intrinsic kinase activity and thus they initiate signaling by interacting intracellular signaling molecules such as TNFR associated factor (TRAF), TNFR associated death domain (TRADD) and Fas-associated death domain (FADD). In bone metabolism, it has been shown that numerous TNF family members including receptor activator of nuclear factor kappaB ligand (RANKL), TNF-alpha, Fas ligand (FasL) and TNF-related apoptosis-inducing ligand (TRAIL) play pivotal roles in the differentiation, function, survival and/or apoptosis of osteoclasts, the principal bone-resorbing cells. These TNF family members not only regulate physiological bone remodeling but they are also implicated in the pathogenesis of various bone diseases such as osteoporosis and bone loss in inflammatory conditions. This review will focus on our current understanding of the regulatory roles and molecular signaling of these TNF family members in osteoclasts.     

Crystal structure of the extracellular domain of mouse RANK ligand at 2.2-A resolution.

Bone remodeling involves the resorption of bone by osteoclasts and the synthesis of bone matrix by osteoblasts. Receptor activator of NF-kappa B ligand (RANKL, also known as ODF and OPGL), a member of the tumor necrosis factor (TNF) family, triggers osteoclastogenesis by forming a complex with its receptor, RANK. We have determined the crystal structure of the extracellular domain of mouse RANKL at 2.2-A resolution. The structure reveals that the RANKL extracellular domain is trimeric, which was also shown by analytical ultracentrifugation, and each subunit has a beta-strand jellyroll topology like the other members of the TNF family. A comparison of RANKL with TNF beta and TNF-related apoptosis-inducing ligand (TRAIL), whose structures were determined to be in the complex form with their respective receptor, reveals conserved and specific features of RANKL in the TNF superfamily and suggests the presence of key residues of RANKL for receptor binding.     

Bone metabolism compensates for the delayed growth in small for gestational age neonates

The goal of the present study is to investigate the relationship between anthropometric and bone metabolism markers in a sample of neonates and their mothers. A sample of 20 SGA (small for the gestational age), AGA (appropriate for the gestational age) and LGA (large for the gestational age) term neonates and their 20 mothers was analyzed at birth and at exit. Elisa method was used to measure the OPG (Osteoprotegerin), RANK (Receptor activator of nuclear factor-kappaB), RANKL (Receptor activator of nuclear factor-kappaB Ligand), IGF-1 (Insulin-like growth factor 1), IGFBP3 (Insulin-like Growth Factor Binding Protein 3) and Leptin levels. Birth weight and length were positively correlated with RANKL, IGF-1 and IGFBP3 and negatively with the ratio OPG/RANKL. SGA neonates presented lower RANKL values and higher OPG/RANKL ratio while LGA neonates had higher RANK levels than AGA neonates. Positive association was shown between neonatal IGFBP3 and maternal IGF-1 values and between neonatal and maternal RANK values at birth and at exit. These results reveal a remarkable upregulation of OPG/RANKL ratio in SGA neonates, pointing out the role of bone turnover in compensating for the delayed neonatal growth.     

Biology of RANK,RANKL, and osteoprotegerin

The discovery of the receptor activator of nuclear factor-kappaB ligand (RANKL)/RANK/osteoprotegerin (OPG) system and its role in the regulation of bone resorption exemplifies how both serendipity and a logic-based approach can identify factors that regulate cell function. Before this discovery in the mid to late 1990s, it had long been recognized that osteoclast formation was regulated by factors expressed by osteoblast/stromal cells, but it had not been anticipated that members of the tumor necrosis factor superfamily of ligands and receptors would be involved or that the factors involved would have extensive functions beyond bone remodeling. RANKL/RANK signaling regulates the formation of multinucleated osteoclasts from their precursors as well as their activation and survival in normal bone remodeling and in a variety of pathologic conditions. OPG protects the skeleton from excessive bone resorption by binding to RANKL and preventing it from binding to its receptor, RANK. Thus, RANKL/OPG ratio is an important determinant of bone mass and skeletal integrity. Genetic studies in mice indicate that RANKL/RANK signaling is also required for lymph node formation and mammary gland lactational hyperplasia, and that OPG also protects arteries from medial calcification. Thus, these tumor necrosis factor superfamily members have important functions outside bone. Although our understanding of the mechanisms whereby they regulate osteoclast formation has advanced rapidly during the past 10 years, many questions remain about their roles in health and disease. Here we review our current understanding of the role of the RANKL/RANK/OPG system in bone and other tissues.     

Adiponectin inhibits induction of TNF-alpha/RANKL-stimulated NFATc1 via the AMPK signaling

We investigated here whether adiponectin can exhibit an inhibitory effect on tumor necrosis factor-alpha (TNF-alpha)- and receptor activator of nuclear factor-kappaB ligand (RANKL)-induced osteoclastogenesis by using RAW264 cell D clone with a high efficiency to form osteoclasts. Globular adiponectin (gAd) strongly inhibited TNF-alpha/RANKL-induced differentiation of osteoclasts by interfering with TNF receptor-associated factor 6 production and calcium signaling; consequently, the induction of nuclear factor of activated T cells c1 (NFATc1) was strongly inhibited. Moreover, we observed that inhibition of AMP-activated protein kinase abrogated gAd inhibition for TNF-alpha/RANKL-induced NFATc1 expression. Our data suggest that adiponectin acts as a potent regulator of bone resorption observed in diseases associated with cytokine activation.     

Regulation of osteoprotegerin pro- or anti-tumoral activity by bone tumor microenvironment

Tumor development in bone is often associated with fractures, bone loss and bone pain, and improvement is still needed in therapeutic approaches. Bone tumors are related to the existence of a vicious cycle between bone resorption and tumor proliferation in which the molecular triad osteoprotegerin (OPG)/receptor activator of NF-kappaB (RANK)/RANK ligand (RANKL) plays a pivotal role. RANKL, a member of the TNF superfamily, is one of the main inducers of bone resorption. Its soluble receptor OPG represents a promising therapeutic candidate as it prevents bone lesions and inhibits associated tumor growth. However, its therapeutic use in bone tumors remains controversial due to its ability to bind and inhibit another member of the TNF superfamily, TNF related apoptosis inducing ligand (TRAIL), which is a potent inducer of tumor cell apoptosis. Through its heparin binding domain, OPG is also able to bind proteoglycans present in the bone matrix. This paper is an overview of the involvement of the micro-environment, as represented by the balance of RANKL/TRAIL and the presence of proteoglycans in the regulation of OPG biological activity in bone tumors.     

RIP kinase is involved in arsenic-induced apoptosis in multiple myeloma cells

These studies explore the molecular effect of arsenicals on MM cells. Freshly isolated cells derived from patients with advanced, chemo-refractory myeloma as well as human myeloma cell lines, ARP-1, RPMI-8226 and H929 were exposed to the organic arsenical melarsoprol and to the inorganic compound AT. Both agents potently induced apoptosis in myeloma cells. Exposure to 1-5 microM AT or melarsoprol for 6 hours suppressed NF-kappa B DNA binding and enhanced of c-Jun kinase (JNK) activity. Arsenic also activated caspase-3 resulting in the cleavage of poly (ADP-ribose) polymerase (PARP) and Fas/TNF alpha related receptor interacting protein (RIP). In contrast to reported observations in acute promyelocytic leukemia, myeloma cell apoptosis was not associated with either the downregulation of Bcl-2 protein or with alterations in the expression of other Bcl-2 family members, Bax, Bak, Bag, and Bcl-xl. This study first shows that arsenic induces apoptotic signaling in MM through the cleavage of TNF alpha related receptor interacting protein (RIP). RIP is a key downstream protein in FasL/ TNF alpha /TRAIL induced apoptosis and a major antiapoptotic adaptor of pathways through NF-kappa B and JNK. RIP has not been previously characterized in myeloma. This study supports the hypothesis that arsenicals share common mediators (RIP, NF-kappa B, PARP, caspase-3) with death receptor induced apoptosis. These studies provide an important insight into the molecular mechanism of AT induced apoptosis and can be used in the development of adjuvant therapy for MM, presently an incurable disease.     

1'-Acetoxychavicol acetate inhibits RANKL-induced osteoclastic differentiation of RAW 264.7 monocytic cells by suppressing nuclear factor-kappaB activation

Osteoclastogenesis is commonly associated with various age-related diseases, including cancer. A member of the tumor necrosis factor superfamily, receptor activator of nuclear factor-kappaB (NF-kappaB) ligand (RANKL), has been shown to play a critical role in osteoclast formation and bone resorption. Thus, agents that suppress RANKL signaling have a potential to suppress bone loss. In this report, we investigated the effect of 1'-acetoxychavicol acetate (ACA), a component of Alpina galanga, on RANKL signaling and consequent osteoclastogenesis in RAW 264.7 cells, a murine monocytic cell line. Treatment of these cells with RANKL activated NF-kappaB, and coexposure of the cells to ACA completely suppressed RANKL-induced NF-kappaB activation in a time- and concentration-dependent manner. The suppression of NF-kappaB by ACA was mediated through suppression of RANKL-induced activation of IkappaBalpha kinase, IkappaBalpha phosphorylation, and IkappaBalpha degradation. Furthermore, incubation of monocytic cells with RANKL induced osteoclastogenesis, and ACA suppressed it. Inhibition of osteoclastogenesis was maximal when cells were simultaneously exposed to ACA and RANKL and minimum when ACA was added 2 days after RANKL. ACA also inhibited the osteoclastogenesis induced by human breast cancer MCF-7 cells, multiple myeloma MM1 cells, and head and neck squamous cell carcinoma LICR-LON-HN5 cells. These results indicate that ACA is an effective blocker of RANKL-induced NF-kappaB activation and of osteoclastogenesis induced by RANKL and tumor cells, suggesting its potential as a therapeutic agent for osteoporosis and cancer-associated bone loss.     

Differential regulation of the expression of CD95 ligand, receptor activator of nuclear factor-kappa B ligand (RANKL), TNF-related apoptosis-inducing ligand (TRAIL), and TNF-alpha during T cell activation

Members of TNF superfamily are characterized by their ability to inflict apoptosis upon binding to their cognate receptors in a homotrimeric manner. These proteins are expressed on different cell types under various conditions. However, the mechanisms governing the expression of these molecules remain elusive. We have found that the TCR signal can elicit the expression of receptor activator of NF-kappaB ligand (RANKL), TNF-alpha, CD95L, and TNF-related apoptosis inducing ligand (TRAIL) in T cell hybridoma A1.1 cells, thus allowing us to examine the expression pattern of these molecules under precisely the same conditions. We have previously reported that CD95L expression requires both protein kinase C (PKC) translocation and Ca2+ mobilization and is inhibited by cyclosporin A, and dexamethasone. We demonstrate now that activation-induced expression of RANKL is mediated by Ca2+ mobilization. PKC activation does not induce RANKL expression nor does it synergize with the Ca2+ signal. Activation-induced RANKL expression is blocked by cyclosporin A, but not by dexamethasone. The expression of TNF, in contrast, is mediated by PKC, but not by Ca2+. TNF-alpha expression is not inhibited by cyclosporin A, but is sensitive to dexamethasone. A1.1 cells constitutively express TRAIL at low levels. Stimulation with anti-CD3 leads to an initial reduction and subsequent increase in TRAIL expression. TRAIL induction is not inhibited by cyclosporin A, but highly sensitive to dexamethasone. Therefore, expression of the TNF superfamily genes is regulated by distinct signals. Detailed understanding of the regulatory mechanisms could provide crucial information concerning the role of these molecules in the modulation of the immune system.     

TANK Potentiates Tumor Necrosis Factor Receptor-Associated Factor-Mediated c-Jun N-Terminal Kinase/Stress-Activated Protein Kinase Activation through the Germinal Center Kinase Pathway

Tumor necrosis factor (TNF) receptor-associated factors (TRAFs) are mediators of many members of the TNF receptor superfamily and can activate both the nuclear factor kappaB (NF-kappaB) and stress-activated protein kinase (SAPK; also known as c-Jun N-terminal kinase) signal transduction pathways. We previously described the involvement of a TRAF-interacting molecule, TRAF-associated NF-kappaB activator (TANK), in TRAF2-mediated NF-kappaB activation. Here we show that TANK synergized with TRAF2, TRAF5, and TRAF6 but not with TRAF3 in SAPK activation. TRAF2 and TANK individually formed weak interactions with germinal center kinase (GCK)-related kinase (GCKR). However, when coexpressed, they formed a strong complex with GCKR, thereby providing a potential mechanism for TRAF and TANK synergy in GCKR-mediated SAPK activation, which is important in TNF family receptor signaling. Our results also suggest that TANK can form potential intermolecular as well as intramolecular interactions between its amino terminus and carboxyl terminus. This study suggests that TANK is a regulatory molecule controlling the threshold of NF-kappaB and SAPK activities in response to activation of TNF receptors. In addition, CD40 activated endogenous GCKR in primary B cells, implicating GCK family proteins in CD40-mediated B-cell functions.     

Activin A stimulates IkappaB-alpha/NFkappaB and RANK expression for osteoclast differentiation,but not AKT survival pathway in osteoclast precursors

Recent studies have reported that activin A enhances osteoclastogenesis in cultures of mouse bone marrow cells stimulated with receptor activator of nuclear factor-kappaB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). However, the exact mechanisms by which activin A functions during osteoclastogenesis are not clear. RANKL stimulation of RANK/TRAF6 signaling increases nuclear factor-kappaB (NFkappaB) nuclear translocation and activates the Akt/PKB cell survival pathway. Here we report that activin A alone activates IkappaB-alpha, and stimulates nuclear translocation of NFkappaB and receptor activator of nuclear factor-kappaB (RANK) expression for osteoclastogenesis, but not Akt/PKB survival signal transduction including BAD and mammalian target of rapamycin (mTOR) for survival in osteoclast precursors in vitro. Activin A alone failed to activate Akt, BAD, and mTOR by immunoblotting, and it also failed to prevent apoptosis in osteoclast precursors. While activin A activated IkappaB-alpha and induced nuclear translocation of phosphorylated-NFkappaB, and it also enhanced RANK expression in osteoclast precursors. Moreover, activin A enhanced RANKL- and M-CSF-stimulated nuclear translocation of NFkappaB. Our data suggest that activin A enhances osteoclastogenesis treated with RANKL and M-CSF via stimulation of RANK, thereby increasing the RANKL stimulation. Activin A alone activated the NFkappaB pathway, but not survival in osteoclast precursors in vitro, but it is, thus, insufficient as a sole stimulus to osteoclastogenesis.     

TRAF-6 dependent signaling pathway is essential for TNF-related apoptosis-inducing ligand (TRAIL) induces osteoclast differentiation

Human osteoclast formation from mononuclear phagocyte precursors involves interactions between tumor necrosis factor (TNF) ligand superfamily members and their receptors. Recent evidence indicates that in addition to triggering apoptosis, the TNF-related apoptosis-inducing ligand (TRAIL) induces osteoclast differentiation. To understand TRAIL-mediated signal transduction mechanism in osteoclastogenesis, we demonstrated that TRAIL induces osteoclast differentiation via a Tumor necrosis factor receptor-associated factor 6 (TRAF-6)-dependent signaling pathway. TRAIL-induced osteoclast differentiation was significantly inhibited by treatment with TRAF-6 siRNA and TRAF6 decoy peptides in both human monocytes and murine RAW264.7 macrophage cell lines, as evaluated in terms of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells and bone resorption activity. Moreover, TRAIL-induced osteoclast differentiation was also abolished in TRAF6 knockout bone marrow macrophages. In addition to induction of NFATc1, treatment of TRAIL also induced ubiquitination of TRAF6 in osteoclast differentiation. Thus, our data demonstrate that TRAIL induces osteoclastic differentiation via a TRAF-6 dependent signaling pathway. This study suggests TRAF6-dependent signaling may be a central pathway in osteoclast differentiation, and that TNF superfamily molecules other than RANKL may modify RANK signaling by interaction with TRAF6-associated signaling.