Receptor activator of NF-kappaB (RANK) cytoplasmic motif, 369PFQEP373, plays a predominant role in osteoclast survival in part by activating Akt/PKB and its downstream effector AFX/FOXO4

Receptor activator of NF-kappaB ligand (RANKL) plays a crucial role in osteoclast differentiation, function, and survival. RANKL exerts its effect by activating its receptor RANK (receptor activator of NF-kappaB), which recruits various intracellular signaling molecules via specific motifs in its cytoplasmic tail. Previously, we identified three RANK cytoplasmic motifs (Motif 1, 369PFQEP373; Motif 2, 559PVQEET564; and Motif 3, 604PVQEQG609) mediating osteoclast formation and function. Here, we investigated RANK cytoplasmic motifs involved in osteoclast survival. Motif 1, in contrast to its minimal role in osteoclast formation and function, plays a predominant role in promoting osteoclast survival. Moreover, whereas Motif 2 and Motif 3 are highly potent in osteoclast formation and function, they exert a moderate effect on osteoclast survival. We also investigated the role of these motifs in activating Akt/protein kinase B (PKB), which has been implicated in RANKL-induced osteoclast survival. Motif 1, but not Motif 2 or Motif 3, is able to stimulate Akt/PKB activation. Because Akt/PKB has been shown to utilize distinct downstream effectors (glycogen synthase kinase-3beta, FKHR/FOXO1a, BAD, and AFX/FOXO4) to regulate cell survival, we next determined which downstream effector(s) is activated by Akt/PKB to promote osteoclast survival. Our data revealed that RANKL only stimulates AFX/FOXO4 phosphorylation, indicating that AFX/FOXO4 is a key downstream target activated by Akt/PKB to modulate osteoclast survival. Taken together, we conclude that Motif 1 plays a predominant role in mediating osteoclast survival in part by activating Akt/PKB and its downstream effector AFX/FOXO4.     

mu-Calpain regulates receptor activator of NF-kappaB ligand (RANKL)-supported osteoclastogenesis via NF-kappaB activation in RAW 264.7 cells

To clarify the role of calpain in the receptor activator of NF-kappaB ligand (RANKL)-supported osteoclastogenesis, RANKL-induced calpain activation was examined by using murine RAW 264.7 cells and bone marrow-derived monocyte/macrophage progenitors. We found that calpain activity increased in response to RANKL in both cell types based on alpha-spectrinolysis and that mu-calpain, rather than m-calpain, was activated during RANKL-supported osteoclastogenesis in RAW 264.7 cells. Overexpression of mu-calpain clearly augmented RANKL-supported osteoclastogenesis in RAW 264.7 cells, thereby implicating its pivotal role in this process. Cell-permeable calpain inhibitors, including calpastatin and calpeptin, were sufficient to suppress RANKL-supported osteoclastogenesis based on decreased expression of the osteoclastogenic marker, matrix metalloproteinase 9, and the generation of tartrate-resistant acid phosphatase-positive multinucleated cells in both cell types. Calpain inhibitors suppressed NF-kappaB activation via inhibition of the cleavage of inhibitor of NF-kappaB(IkappaBalpha)in RAW 264.7 cells. Taken together, our findings suggest that mu-calpain is essential to the regulation of RANKL-supported osteoclastogenesis via NF-kappaB activation.     

Disabling of receptor activator of nuclear factor-kappaB (RANK) receptor complex by novel osteoprotegerin-like peptidomimetics restores bone loss in vivo

The tumor necrosis factor family ligand, tumor necrosis factor-related activation-induced cytokine (TRANCE), and its receptors, receptor activator of nuclear factor-kappaB (RANK) and osteoprotegerin (OPG), are known to be regulators of development and activation of osteoclasts in bone remodeling. Sustained osteoclast activation that occurs through TRANCE-RANK causes osteopenic disorders such as osteoporosis and contributes to osteolytic metastases. Here, we report a rationally designed small molecule mimic of osteoprotegerin to inhibit osteoclast formation in vitro and limit bone loss in an animal model of osteoporosis. One of the mimetics, OP3-4, significantly inhibited osteoclast formation in vitro (IC(50) = 10 microm) and effectively inhibited total bone loss in ovariectomized mice at a dosage of 2 mg/kg/day. Unlike soluble OPG receptors, which preclude TRANCE binding to RANK, OP3-4 shows the ability to modulate RANK-TRANCE signaling pathways and alters the biological functions of the RANK-TRANCE receptor complex by facilitating a defective receptor complex. These features suggest that OPG-derived small molecules can be used as a probe to understand complex biological functions of RANK-TRANCE-OPG receptors and also can be used as a platform to develop more useful therapeutic agents for inflammation and bone disease.     

Curcumin (diferuloylmethane) inhibits receptor activator of NF-kappa B ligand-induced NF-kappa B activation in osteoclast precursors and suppresses osteoclastogenesis

Numerous studies have indicated that inflammatory cytokines play a major role in osteoclastogenesis, leading to the bone resorption that is frequently associated with cancers and other diseases. Gene deletion studies have shown that receptor activator of NF-kappaB ligand (RANKL) is one of the critical mediators of osteoclastogenesis. How RANKL mediates osteoclastogenesis is not fully understood, but an agent that suppresses RANKL signaling has potential to inhibit osteoclastogenesis. In this report, we examine the ability of curcumin (diferuloylmethane), a pigment derived from turmeric, to suppress RANKL signaling and osteoclastogenesis in RAW 264.7 cells, a murine monocytic cell line. Treatment of these cells with RANKL activated NF-kappaB, and preexposure of the cells to curcumin completely suppressed RANKL-induced NF-kappaB activation. Curcumin inhibited the pathway leading from activation of IkappaBalpha kinase and IkappaBalpha phosphorylation to IkappaBalpha degradation. RANKL induced osteoclastogenesis in these monocytic cells, and curcumin inhibited both RANKL- and TNF-induced osteoclastogenesis and pit formation. Curcumin suppressed osteoclastogenesis maximally when added together with RANKL and minimally when it was added 2 days after RANKL. Whether curcumin inhibits RANKL-induced osteoclastogenesis through suppression of NF-kappaB was also confirmed independently, as RANKL failed to activate NF-kappaB in cells stably transfected with a dominant-negative form of IkappaBalpha and concurrently failed to induce osteoclastogenesis. Thus overall these results indicate that RANKL induces osteoclastogenesis through the activation of NF-kappaB, and treatment with curcumin inhibits both the NF-kappaB activation and osteoclastogenesis induced by RANKL.     

Cadherin2 (N-cadherin) plays an essential role in zebrafish cardiovascular development

Background  

Cadherins are cell surface adhesion molecules that play important roles in development of vertebrate tissues and organs. We studied cadherin2 expression in developing zebrafish heart using in situ hybridization and immunocytochemical methods, and we found that cadherin2 was strongly expressed by the myocardium of the embryonic zebrafish. To gain insight into cadherin2 role in the formation and function of the heart, we analyzed cardiac differentiation and performance in a cadherin2 mutant, glass onion (glo).     

Warm temperature-sensitive transient receptor potential vanilloid 4 (TRPV4) plays an essential role in thermal hyperalgesia

Animals sense various ranges of temperatures by cutaneous thermal stimuli. Transient receptor potential vanilloid 4 (TRPV4) is a cation channel activated at a warm temperature (over 30 degrees C) in exogenously expressed cells. We found in the present study that TRPV4 is essential in thermal hyperalgesia at a warm temperature in vivo. TRPV4-/- and TRPV4+/+ mice exhibited the same latency of escape from 35-50 degrees C hotplates. Neuronal activity in the femoral nerve, however, revealed that the number and activity level of neurons decreased in response to a warm temperature in TRPV4-/- mice. TRPV4-/- mice displayed a significantly longer latency to escape from the plates at 35- 45 degrees C when hyperalgesia was induced by carrageenan without changes in foot volumes. TRPV4 therefore determines the sensitivity rather than the threshold of painful heat detection and plays an essential role in thermal hyperalgesia.     

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.     

Regulation of receptor activator of NF-kappa B ligand-induced osteoclastogenesis by endogenous interferon-beta (INF-beta ) and suppressors of cytokine signaling (SOCS). The possible counteracting role of SOCSs- in IFN-beta-inhibited osteoclast formation

Bone resorption and the immune system are correlated with each other, and both are controlled by a variety of common cytokines produced in the bone microenvironments. Among these immune mediators, the involvement of type I interferons (IFNs) in osteoclastic bone resorption remains unknown. In this study, we investigated the participation of IFN-beta and suppressors of cytokine signaling (SOCS)-1 and -3 in osteoclastogenesis. Addition of exogenous IFN-beta to osteoclast progenitors (bone-derived monocytes/macrophages) inhibited their differentiation toward osteoclasts induced by the receptor activator of NF-kappaB ligand (RANKL) and macrophage colony-stimulating factor with/without transforming growth factor-beta, where inhibition was associated with down-regulation of the gene expressions of molecules related to osteoclast differentiation. In addition, RANKL induced the expression of IFN-beta; furthermore, neutralizing antibody against type I IFNs accelerated the osteoclast formation, indicating type I IFNs as potential intrinsic inhibitors. On the other hand, RANKL also induced the expression of SOCS-1 and -3, suppressors of the IFN signaling. Pretreatment with RANKL for a sufficient time for the induction of SOCSs attenuated phosphorylation of STAT-1 in response to IFN-beta in osteoclast progenitors, causing a decrease in the binding activity of nuclear extracts toward the interferon-stimulated response element. mRNA levels of STAT-1, STAT-2, and IFN-stimulated gene factor-3gamma, comprising IFN-stimulated gene factor-3, were not altered by RANKL. Thus, although the inhibitory cytokine such as IFN-beta is produced in response to RANKL, the inhibition of osteoclastogenesis may be rescued by the induction of signaling suppressors such as SOCSs.     

gamma-tubulin plays an essential role in the coordination of mitotic events

Recent data from multiple organisms indicate that gamma-tubulin has essential, but incompletely defined, functions in addition to nucleating microtubule assembly. To investigate these functions, we examined the phenotype of mipAD159, a cold-sensitive allele of the gamma-tubulin gene of Aspergillus nidulans. Immunofluorescence microscopy of synchronized material revealed that at a restrictive temperature mipAD159 does not inhibit mitotic spindle formation. Anaphase A was inhibited in many nuclei, however, and after a slight delay in mitosis (approximately 6% of the cell cycle period), most nuclei reentered interphase without dividing. In vivo observations of chromosomes at a restrictive temperature revealed that mipAD159 caused a failure of the coordination of late mitotic events (anaphase A, anaphase B, and chromosomal disjunction) and nuclei reentered interphase quickly even though mitosis was not completed successfully. Time-lapse microscopy also revealed that transient mitotic spindle abnormalities, in particular bent spindles, were more prevalent in mipAD159 strains than in controls. In experiments in which microtubules were depolymerized with benomyl, mipAD159 nuclei exited mitosis significantly more quickly (as judged by chromosomal condensation) than nuclei in a control strain. These data reveal that gamma-tubulin has an essential role in the coordination of late mitotic events, and a microtubule-independent function in mitotic checkpoint control.     

Activation of NF-kappaB by RANK requires tumor necrosis factor receptor-associated factor (TRAF) 6 and NF-kappaB-inducing kinase. Identification of a novel TRAF6 interaction motif

Various members of the tumor necrosis factor (TNF) receptor superfamily activate nuclear factor kappaB (NF-kappaB) and the c-Jun N-terminal kinase (JNK) pathways through their interaction with TNF receptor-associated factors (TRAFs) and NF-kappaB-inducing kinase (NIK). We have previously shown that the cytoplasmic domain of receptor activator of NF-kappaB (RANK) interacts with TRAF2, TRAF5, and TRAF6 and that its overexpression activates NF-kappaB and JNK pathways. Through a detailed mutational analysis of the cytoplasmic domain of RANK, we demonstrate that TRAF2 and TRAF5 bind to consensus TRAF binding motifs located in the C terminus at positions 565-568 and 606-611, respectively. In contrast, TRAF6 interacts with a novel motif located between residues 340 and 358 of RANK. Furthermore, transfection experiments with RANK and its deletion mutants in human embryonic 293 cells revealed that the TRAF6-binding region (340-358), but not the TRAF2 or TRAF5-binding region, is necessary and sufficient for RANK-induced NF-kappaB activation. Moreover, a kinase mutant of NIK (NIK-KM) inhibited RANK-induced NF-kappaB activation. However, RANK-mediated JNK activation required a distal portion (427-603) of RANK containing the TRAF2-binding domain. Thus, our results indicate that RANK interacts with various TRAFs through distinct motifs and activates NF-kappaB via a novel TRAF6 interaction motif, which then activates NIK, thus leading to NF-kappaB activation, whereas RANK most likely activates JNK through a TRAF2-interacting region in RANK.     

Lactosylceramide is essential for the osteoclastogenesis mediated by macrophage-colony-stimulating factor and receptor activator of nuclear factor-kappa B ligand.

Glycosphingolipids and their metabolites play important roles in a variety of biological processes. Several signal molecules are localized in a glycolipid-enriched microdomain on the cell surface, and their signals are regulated by the glycolipid composition. However, the function of glycolipids in osteoclastogenesis has not been clearly understood. We found that D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), a glucosylceramide synthase inhibitor, completely inhibits the osteoclast formation induced by macrophage-colony-stimulating factor and receptor activator of nuclear factor-kappa B ligand (RANKL) in a dose-dependent manner. Expression of RANK, the receptor of RANKL, induced by macrophage colony-stimulating factor, was reduced markedly in D-PDMP-treated cells. d-PDMP also inhibited the phosphorylation of the inhibitor of nuclear factor-kappa B and extracellular signal-regulated kinase 1/2 induced by RANKL. In several experiments with the addition of glycolipids to D-PDMP-treated purified bone marrow cells, lactosylceramide (LacCer) strongly affected the differentiation into tartrate-resistant acid phosphatase mononucleated cells, but not positive multinucleated cells. GM3 and GM1 also recovered, but less effectively compared with LacCer. Moreover, exogenous LacCer recovered the reduced expression of RANK and the phosphorylation of inhibitor of NF-kappa B and extracellular signal-regulated kinase 1/2 after stimulation by RANKL at the same level of cells without D-PDMP treatment. Our data suggest that glycosphingolipids, especially LacCer, are necessary for the initiation step of RANKL-induced osteoclastogenesis.     

The suppressor domain of inositol 1,4,5-trisphosphate receptor plays an essential role in the protection against apoptosis

The N-terminal 1-225 amino acids (aa) of the type 1 inositol 1,4,5-trisphosphate receptor (IP(3)R1) function as a suppressor/coupling domain. In this study we used IP(3)R-deficient B-lymphocytes to investigate the effects of modifications in this domain on IP(3) binding and Ca(2+)-release activity. Although the N-terminal 1-225 aa of IP(3)R3 had the same role as in IP(3)R1, the suppression of IP(3) binding for IP(3)R1 was lost when the suppressor/coupling domains were exchanged between the two isoforms. Resulting chimeric receptors showed a higher sensitivity to IP(3)-induced activation (IICR). Deletion of 11 aa in IP(3)R1 ([Delta76-86]-IP(3)R1) or replacing aa 76-86 of the IP(3)R1 in the suppressor/coupling domain by 13 aa of IP(3)R3 ([75-87 T3]-IP(3)R1) also resulted in increased IP(3) binding and sensitivity of IICR. These residues constitute the only part of the suppressor/coupling domain that is strikingly different between the two isoforms. Expression of [Delta76-86]-IP(3)R1 and of [75-87 T3]-IP(3)R1 increased the propensity of cells to undergo staurosporine-induced apoptosis, but had no effect on the Ca(2+) content in the endoplasmic reticulum. In the cell model used, our observations suggest that the sensitivity of the Ca(2+)-release activity of IP(3)R1 to IP(3) influences the sensitivity of the cells to apoptotic stimuli and that the suppressor/coupling domain may have an anti-apoptotic function by attenuating the sensitivity of IICR.     

Characterization of osteoprotegerin binding to glycosaminoglycans by surface plasmon resonance: role in the interactions with receptor activator of nuclear factor kappaB ligand (RANKL) and RANK

Osteoprotegerin (OPG) is a decoy receptor for receptor activator of nuclear factor kappaB ligand (RANKL), a key inducer of osteoclastogenesis via its receptor RANK. We previously showed that RANK, RANKL, and OPG are able to form a tertiary complex and that OPG must be also considered as a direct effector of osteoclast functions. As OPG contains a heparin-binding domain, the present study investigated the interactions between OPG and glycosaminoglycans (GAGs) by surface plasmon resonance and their involvement in the OPG functions. Kinetic data demonstrated that OPG binds to heparin with a high-affinity (KD: 0.28 nM) and that the pre-incubation of OPG with heparin inhibits in a dose-dependent manner the OPG binding to the complex RANK-RANKL. GAGs from different structure/origin (heparan sulfate, dermatan sulfate, and chondroitin sulfate) exert similar activity on OPG binding. The contribution of the sulfation pattern and the size of the oligosaccharide were determined in this inhibitory mechanism. The results demonstrated that sulfation is essential in the OPG-blocking function of GAGs since a totally desulfated heparin loses its capacity to bind and to block OPG binding to RANKL. Moreover, a decasaccharide is the minimal structure that totally inhibits the OPG binding to the complex RANK-RANKL. Western blot analysis performed in 293 cells surexpressing RANKL revealed that the pre-incubation of OPG with these GAGs strongly inhibits the OPG-induced decrease of membrane RANKL half-life. These data support an essential function of the related glycosaminoglycans heparin and heparan sulfate in the activity of the triad RANK-RANKL-OPG.     

IL-23 induces receptor activator of NF-kappaB ligand expression on CD4+ T cells and promotes osteoclastogenesis in an autoimmune arthritis model

IL-23, a clinically novel cytokine, targets CD4(+) T cells. Recent IL-1Ra(-/-) mouse studies have demonstrated that IL-23 indirectly stimulates the differentiation of osteoclast precursors by enhancing IL-17 release from CD4(+) T cells. IL-17, in turn, stimulates osteoclastogenesis in osteoclast precursor cells. In this study, we found that IL-23 up-regulates receptor activator of NF-kappaB ligand expression by CD4(+) T cells, and thus contributes to osteoclastogenesis. This indirect pathway is mediated by NF-kappaB and STAT3. We have also demonstrated that IL-23 can influence osteoclastogenesis positively under the special conditions in the IL-1-dominant milieu of IL-1Ra(-/-) mice. We propose that IL-23-enhanced osteoclastogenesis is mediated mainly by CD4(+) T cells. The results of this study show that IL-23 is a promising therapeutic target for the treatment of arthritis-associated bone destruction.     

A novel interaction between DNA ligase III and DNA polymerase gamma plays an essential role in mitochondrial DNA stability

The data in the present study show that DNA polymerase gamma and DNA ligase III interact in mitochondrial protein extracts from cultured HT1080 cells. An interaction was also observed between the two recombinant proteins in vitro. Expression of catalytically inert versions of DNA ligase III that bind DNA polymerase gamma was associated with reduced mitochondrial DNA copy number and integrity. In contrast, overexpression of wild-type DNA ligase III had no effect on mitochondrial DNA copy number or integrity. Experiments revealed that wild-type DNA ligase III facilitates the interaction of DNA polymerase gamma with a nicked DNA substrate in vitro, and that the zinc finger domain of DNA ligase III is required for this activity. Mitochondrial protein extracts prepared from cells overexpressing a DNA ligase III protein that lacked the zinc finger domain had reduced base excision repair activity compared with extracts from cells overexpressing the wild-type protein. These data support the interpretation that the interaction of DNA ligase III and DNA polymerase gamma is required for proper maintenance of the mammalian mitochondrial genome.     

5-Lipoxygenase plays an essential role in 4-HNE-enhanced ROS production in murine macrophages via activation of NADPH oxidase

Abstract

4-Hydroxynonenal (HNE) mediates oxidative stress-linked pathological processes; however, its role in the generation of reactive oxygen species (ROS) in macrophages is still unclear. Thus, this study investigated the sources and mechanisms of ROS generation in macrophages stimulated with HNE. Exposure of J774A.1 cells to HNE showed an increased production of ROS, which was attenuated by NADPH oxidase as well as 5-lipoxygenase (5-LO) inhibitors. Linked to these results, HNE increased membrane translocation of p47phox promoting NADPH oxidase activity, which was attenuated in peritoneal macrophages from 5-LO-deficient mice as well as in J774A.1 cells treated with a 5-LO inhibitor, MK886 or 5-LO siRNA. In contrast, HNE-enhanced 5-LO activity was not affected by inhibition of NADPH oxidase. Furthermore, leukotriene B₄, 5-LO metabolite, was found to enhance NADPH oxidase activity in macrophages. Altogether, these results suggest that 5-LO plays a critical role in HNE-induced ROS generation in murine macrophages through activation of NADPH oxidase.