NF-kappaB modulators in osteolytic bone diseases.

Osteoclasts are responsible for bone resorption and play a pivotal role in the pathogenesis of osteolytic disorders. NF-kappaB is a set of nuclear factors that bind to consensus DNA sequences called kappaB sites, and is essential for osteoclast formation and survival. NF-kappaB signalling pathways are strictly regulated to maintain bone homeostasis by cytokines such as RANKL, TNF-alpha and IL-1, which differentially regulate classical and/or alternative NF-kappaB pathways in osteoclastic cells. These pathways are also modulated by NF-kappaB mediators, including TRAF6, aPKC, p62/SQSTM1 and deubiquitinating enzyme CYLD that are involved in the ubiquitin-proteasome system during RANK-mediated osteoclastogenesis. Abnormal activation of NF-kappaB signalling in osteoclasts has been associated with excessive osteoclastic activity, and frequently observed in osteolytic conditions, including periprosthetic osteolysis, arthritis, Paget's disease of bone, and periodontitis. NF-kappaB modulators such as parthenolide and NEMO-binding domain peptide demonstrate therapeutic effects on inflammation-induced bone destruction in mouse models. Unravelling the structure and function of NF-kappaB pathways in osteoclasts and other cell types will be important in developing new strategies for treatments of bone diseases.

Cytoplasmic terminus of vacuolar type proton pump accessory subunit Ac45 is required for proper interaction with V(0) domain subunits and efficient osteoclastic bone resorption.

Solubilization of mineralized bone by osteoclasts is largely dependent on the acidification of the extracellular resorption lacuna driven by the vacuolar (H+)-ATPases (V-ATPases) polarized within the ruffled border membranes. V-ATPases consist of two functionally and structurally distinct domains, V(1) and V(0). The peripheral cytoplasmically oriented V(1) domain drives ATP hydrolysis, which necessitates the translocation of protons across the integral membrane bound V(0) domain. Here, we demonstrate that an accessory subunit, Ac45, interacts with the V(0) domain and contributes to the vacuolar type proton pump-mediated function in osteoclasts. Consistent with its role in intracellular acidification, Ac45 was found to be localized to the ruffled border region of polarized resorbing osteoclasts and enriched in pH-dependent endosomal compartments that polarized to the ruffled border region of actively resorbing osteoclasts. Interestingly, truncation of the 26-amino acid residue cytoplasmic tail of Ac45, which encodes an autonomous internalization signal, was found to impair bone resorption in vitro. Furthermore, biochemical analysis revealed that although both wild type Ac45 and mutant were capable of associating with subunits a3, c, c'', and d, deletion of the cytoplasmic tail altered its binding proximity with a3, c'', and d. In all, our data suggest that the cytoplasmic terminus of Ac45 contains elements necessary for its proper interaction with V(0) domain and efficient osteoclastic bone resorption.

Calcium/calmodulin-dependent kinase activity is required for efficient induction of osteoclast differentiation and bone resorption by receptor activator of nuclear factor kappa B ligand (RANKL).

Calcium/calmodulin-dependent protein kinase (CaMK) is a major down stream mediator of Ca(2+) signaling in a wide range of cellular functions, including ion channel and cell cycle regulation and neurotransmitter synthesis and release. Here we have investigated the role of the CaMK signaling pathway in osteoclast differentiation and bone resorption. We observed that the CaMKI, CaMKII gamma isoforms were present in both bone-marrow derived macrophages and RAW264.7 murine macrophage cell line, and that expression persisted during osteoclast differentiation in the presence of receptor activator of nuclear factor kappa B (NF-kappaB) ligand (RANKL). RANKL-induced differentiation was accompanied by increased cyclic AMP response element transcriptional activity, and ERK phosphorylation, which are both downstream targets of CaMK. Two selective inhibitors of CaMKs, KN-93 and KN-62, inhibited osteoclastogenesis in a time and concentration-dependent manner. This was accompanied by suppression of cathepsin K expression and osteoclastic bone resorption, which are markers for differentiated osteoclast function. KN-93 and KN-62 both inhibited RANKL-induced ERK phosphorylation and CREB transcriptional activity. These findings imply a role for CaMK in osteoclast differentiation and bone resorption.

p62 ubiquitin binding-associated domain mediated the receptor activator of nuclear factor-kappaB ligand-induced osteoclast formation: a new insight into the pathogenesis of Paget's disease of bone.

Paget's disease of bone (PDB) is a debilitating bone disorder characterized by giant osteoclasts, enhanced bone destruction, and irregular bone formation. Recently, mutations in SQSTM1 (also known as p62) have been detected in PDB sufferers, with all mutations resulting in either loss of function or truncation/deletion of the ubiquitin binding-associated (UBA) domain. We hypothesized that mutation in the p62 gene resulting in either deletion or premature termination of the UBA domain accounts for the elevated osteoclastic formation and bone resorption associated with PDB. Remarkably, overexpression of the p62 UBA domain deletion mutant (p62DeltaUBA) significantly enhanced osteoclastogenesis in vitro compared to cells expressing either wild-type p62 (p62WT) or a control vector in a RAW264.7 osteoclastogenic system. Overexpression of p62DeltaUBA potentiated the formation of abnormally large multinucleated osteoclasts and resorption of bone, reminiscent of PDB. Consistent with the enhancement of osteoclastogenesis, overexpression of p62DeltaUBA potentiated receptor activator of nuclear factor-kappaB ligand-induced activation of nuclear factor-kappaB, NFAT, and ERK phosphorylation. Furthermore, as determined by confocal microscopy, deletion of the p62 UBA domain impaired the association of p62 with TRAF6 in the proteasomal compartment. These results suggest that the UBA domain encodes essential regulatory elements required for receptor activator of nuclear factor-kappaB ligand-induced osteoclast formation and bone resorption that may be directly associated with the progression of PDB.

A novel mutation (K378X) in the sequestosome 1 gene associated with increased NF-kappaB signaling and Paget's disease of bone with a severe phenotype.

UNLABELLED: Sequestosome 1/p62 (p62) mutations are associated with PDB; however, there are limited data regarding functional consequences. We report a novel mutation in exon 7 (K378X) in a patient with polyostotic Paget's disease of bone. p62 mutants increased NF-kappaB activation and significantly potentiated osteoclast formation and bone resorption in human primary cell cultures. INTRODUCTION: Sequestosome 1/p62 (p62) mutations are associated with Paget's disease of bone (PDB); however, there are limited data regarding functional consequences. One report has linked the common P392L mutation in the p62 ubiquitin binding associated (UBA) domain with increases in NF-kappaB activity, a transcription factor essential for osteoclastogenesis. To further clarify the functional impact of p62 mutations associated with PDB, we assessed the effect of p62 mutation (a novel mutation: K378X, and previously reported mutations: P392L and E396X) on RANK-induced NF-kappaB activation and compared this with the effect of wildtype p62. In addition, we studied the effect of p62 mutation on osteoclast formation and bone resorption. MATERIALS AND METHODS: We performed co-transfection experiments with expression plasmids for p62 (wildtype or mutated) and RANK and an NF-kappaB luciferase reporter gene. Luciferase activities were recorded after addition of luciferin to cellular lysates. RAW(264.7) cells stably expressing enhanced green fluorescent protein (EGFP)-tagged p62 (wildtype, K378X, or P392L) or EGFP alone were assessed for changes in cell proliferation. Additionally, these cells were stimulated with RANKL to produce osteoclast-like cells (OLCs). Primary human monocytes collected from the K378X-affected patient and a control subject were stimulated to form OLCs and bone resorption data were obtained. RESULTS: The novel mutation introduces a premature stop codon in place of Lys-378 and thereby eliminates the entire p62 UBA domain; this and two additional natural mutations (P392L, E396X) increased NF-kappaB activation compared with wildtype p62. Wildtype p62 consistently inhibited NF-kappaB activation compared with empty vector. UBA mutations (K378X and P392L) significantly increased the number of OLCs formed in response to RANKL and also the number of nuclei of the OLCs. K378X-affected human monocytes formed more OLCs with more nuclei and increased bone resorption compared with control monocytes. CONCLUSIONS: Our data show that mutation of the p62 UBA domain results in increased activation of NF-kappaB and osteoclast formation and function compared with wildtype p62. These results may partially explain the mechanism by which p62 mutation contributes to the pathogenesis of PDB.

Thapsigargin modulates osteoclastogenesis through the regulation of RANKL-induced signaling pathways and reactive oxygen species production.

UNLABELLED: The mechanism by which TG modulates osteoclast formation and apoptosis is not clear. In this study, we showed a biphasic effect of TG on osteoclast formation and apoptosis through the regulation of ROS production, caspase-3 activity, cytosolic Ca2+, and RANKL-induced activation of NF-kappaB and AP-1 activities. INTRODUCTION: Apoptosis and differentiation are among the consequences of changes in intracellular Ca2+ levels. In this study, we investigated the effects of the endoplasmic reticular Ca2+-ATPase inhibitor, thapsigargin (TG), on osteoclast apoptosis and differentiation. MATERIALS AND METHODS: Both RAW264.7 cells and primary spleen cells were used to examine the effect of TG on RANKL-induced osteoclastogenesis. To determine the action of TG on signaling pathways, we used reporter gene assays for NF-kappaB and activator protein-1 (AP-1) activity, Western blotting for phospho-extracellular signal-related kinase (ERK), and fluorescent probes to measure changes in levels of intracellular calcium and reactive oxygen species (ROS). To assess rates of apoptosis, we measured changes in annexin staining, caspase-3 activity, and chromatin and F-actin microfilament structure. RESULTS: At concentrations that caused a rapid rise in intracellular Ca2+, TG increased caspase-3 activity and promoted apoptosis in osteoclast-like cells (OLCs). Low concentrations of TG, which were insufficient to measurably alter intracellular Ca2+, unexpectedly suppressed caspase-3 activity and enhanced RANKL-induced osteoclastogenesis. At these lower concentrations, TG potentiated ROS production and RANKL-induced NF-kappaB activity, but suppressed RANKL-induced AP-1 activity and had little effect on ERK phosphorylation. CONCLUSION: Our novel findings of a biphasic effect of TG are incompletely explained by our current understanding of TG action, but raise the possibility that low intensity or local changes in subcellular Ca2+ levels may regulate intracellular differentiation signaling. The extent of cross-talk between Ca2+ and RANKL-mediated intracellular signaling pathways might be important in determining whether cells undergo apoptosis or differentiate into OLCs.

Sesquiterpene lactone parthenolide blocks lipopolysaccharide-induced osteolysis through the suppression of NF-kappaB activity.

UNLABELLED: Effective treatment for bacteria-induced bone lytic diseases is not yet available. In this study, we showed that PAR, an NF-kappaB inhibitor found in medicinal herbs, can block LPS-induced osteolysis. PAR does this by inhibiting osteoclastogenesis and bone resorption and promoting apoptosis of osteoclasts through the suppression of NF-kappaB activity. INTRODUCTION: Osteolysis induced by chronic gram-negative bacterial infection underlies many bone diseases such as osteomyelitis, septic arthritis, and periodontitis. Drugs that inhibit lipopolysaccharide (LPS)-induced osteolysis are critically needed for the prevention of bone destruction in infective bone diseases. In this study, we investigated the effect of parthenolide (PAR) on LPS-induced osteolysis in vivo and studied its role in osteoclastogenesis, bone resorption, apoptosis, and NF-kappaB activity. MATERIALS AND METHODS: The LPS-induced osteolysis in the mouse calvarium model was used to examine the effect of PAR in vivo. RANKL-induced osteoclast differentiation from RAW264.7 cells and bone resorption assays were used to assess the effect of PAR in vitro. Assays for NF-kappaB activation, p65 translocation, and IkappaB-alpha degradation were used to determine the mechanism of action of PAR in osteoclasts and their precursors. Flow cytometry and confocal microscopic analysis were used to examine cell apoptosis. Semiquantitative RT-PCR was performed to examine the effect of PAR on gene expression of RANK and TRAF6. RESULTS: We found that PAR (0.5 and 1 mg/kg), injected simultaneously with LPS (25 mg/kg) or 3 days later, blocked the LPS-induced osteolysis in the mouse calvarium model. In vitro studies showed that low concentrations of PAR (<1 microM) inhibited in vitro osteoclastogenesis and osteoclastic bone resorption, whereas higher concentrations (>5 microM) triggered apoptotic cell death of osteoclasts and their precursor cells in a dose-dependent manner. Furthermore, PAR inhibited LPS-induced NF-kappaB activation, p65 translocation, and IkappaB-alpha degradation both in mature osteoclasts and their precursors in a time- and dose-dependent manner. In addition, PAR inhibited NF-kappaB activation induced by osteoclastogenic factors RANKL, interleukin (IL)-1beta, or TNF-alpha to varying degrees and reduced the gene expression of RANK and TRAF6. CONCLUSION: The NF-kappaB pathway is known to mediate both osteoclast differentiation and survival. These findings indicate that PAR blocks LPS-induced osteolysis through the suppression of NF-kappaB activity and suggest that it might have therapeutic value in bacteria-induced bone destruction.

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

UNLABELLED: 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. INTRODUCTION: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Effects of Bafilomycin A1: an inhibitor of vacuolar H (+)-ATPases on endocytosis and apoptosis in RAW cells and RAW cell-derived osteoclasts.

Bafilomycin A1, a specific inhibitor of V-ATPases, is a potent inhibitor of bone resorption, but the underlying mechanisms of its action remain unclear. In this study, we investigated the effect of Bafilomycin A1 on endocytosis and apoptosis in RAW cells and RAW cell-derived osteoclasts. Quantitative analysis by flow cytometry showed that Bafilomycin A1 increased total transferrin levels when RAW cells were exposed to labeled transferrin and decreased the total uptake of Dextran-rhodamine B, both in a dose- and time-dependent fashion, indicating that Bafilomycin influences receptor-mediated and fluid phase endocytosis in these cells. Furthermore, Bafilomycin A1 induced apoptosis of RAW cells in a dose dependent manner as evidenced by Annexin V flow cytometry. The action of Bafilomycin A1 on endocytotic events appeared to be more sensitive and occurred earlier than on its apoptosis inducing effects, suggesting that interrupting of endocytosis might be an early sign of Bafilomycin-mediated osteoclast inhibition. Semi-quantitative RT-PCR analysis showed that the gene transcripts of putative Bafilomycin A1 binding subunit, V-ATPase-subunit a3, were expressed in the preosteoclastic RAW cell line, and up-regulated during RANKL-induced osteoclastogenesis. Osteoclasts treated with Bafilomycin A1 exhibited apoptosis as well as altered cellular localization of Transferrin Alexa 647. Given that endocytosis and apoptosis are important processes during osteoclastic bone resorption, the potent effect of Bafilomycin A1 on endocytosis and apoptosis of osteoclasts and their precursor cells may in part account for Bafilomycin A1 inhibited bone resorption.