James H. Steer

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

The mechanism by which TPA‐induced PKC activity modulates osteoclastogenesis is not clear. Using a RAW264.7 cell culture system and assays for NF‐κB nuclear translocation, NF‐κB reporter gene activity, and MAPK assays, we demonstrated that TPA inhibits osteoclastogenesis through the suppression of RANKL‐induced NF‐κβ activation.

A Novel Mutation (K378X) in the Sequestosome 1 Gene Associated With Increased NF-κB Signaling and Paget's Disease of Bone With a Severe Phenotype†

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 Pagets disease of bone. p62 mutants increased NF‐κB activation and significantly potentiated osteoclast formation and bone resorption in human primary cell cultures.

Sesquiterpene Lactone Parthenolide Blocks Lipopolysaccharide‐Induced Osteolysis Through the Suppression of NF‐κB Activity

Effective treatment for bacteria‐induced bone lytic diseases is not yet available. In this study, we showed that PAR, an NF‐κB 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‐κB activity.

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 Ca2+ 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 γ 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‐κB) 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. J. Cell. Physiol. 212:787–795, 2007.

Myocyte Enhancer Factor 2 and Microphthalmia-associated Transcription Factor Cooperate with NFATc1 to Transactivate the V-ATPase d2 Promoter during RANKL-induced Osteoclastogenesis

The V-ATPase d2 protein constitutes an important subunit of the V-ATPase proton pump, which regulates bone homeostasis; however, currently little is known about its transcriptional regulation. Here, in an attempt to understand regulation of the V-ATPase d2 promoter, we identified the presence of NFATc1, microphthalmia-associated transcription factor (MITF)- and myocyte enhancer factor 2 (MEF2)-binding sites within the V-ATPase d2 promoter using complementary bioinformatic analyses, chromatin immunoprecipitation, and electromobility shift assay. Intriguingly, activation of the V-ATPase d2 promoter by NFATc1 was enhanced by either MEF2 or MITF overexpression. By comparison, coexpression of MITF and MEF2 did not further enhance V-ATPase d2 promoter activity above that of expression of MITF alone. Consistent with a role in transcriptional regulation, both NFATc1 and MITF proteins translocated from the cytosol to the nucleus during RANKL-induced osteoclastogenesis, whereas MEF2 persisted in the nucleus of both osteoclasts and their mononuclear precursors. Targeted mutation of the putative NFATc1-, MITF-, or MEF2-binding sites in the V-ATPase d2 promoter impaired its transcriptional activation. Additionally retroviral overexpression of MITF or MEF2 in RAW264.7 cells potentiated RANKL-induced osteoclastogenesis and V-ATPase d2 gene expression. Based on these data, we propose that MEF2 and MITF function cooperatively with NFATc1 to transactivate the V-ATPase d2 promoter during RANKL-induced osteoclastogenesis.

Caffeic acid phenethyl ester, an active component of honeybee propolis attenuates osteoclastogenesis and bone resorption via the suppression of RANKL-induced NF-κB and NFAT activity.

Receptor activator NF‐κB ligand (RANKL)‐activated signaling is essential for osteoclast differentiation, activation and survival. Caffeic acid phenethyl ester (CAPE), a natural NF‐κB inhibitor from honeybee propolis has been shown to have anti‐tumor and anti‐inflammatory properties. In this study, we investigated the effect of CAPE on the regulation of RANKL‐induced osteoclastogenesis, bone resorption and signaling pathways. Low concentrations of CAPE (<1 µM) dose dependently inhibited RANKL‐induced osteoclastogenesis in RAW264.7 cell and bone marrow macrophage (BMM) cultures, as well as decreasing the capacity of human osteoclasts to resorb bone. CAPE inhibited both constitutive and RANKL‐induced NF‐κB and NFAT activation, concomitant with delayed IκBα degradation and inhibition of p65 nuclear translocation. At higher concentrations, CAPE induced apoptosis and caspase 3 activities of RAW264.7 and disrupts the microtubule network in osteoclast like (OCL) cells. Taken together, our findings demonstrate that inhibition of NF‐κB and NFAT activation by CAPE results in the attenuation of osteoclastogenesis and bone resorption, implying that CAPE is a potential treatment for osteolytic bone diseases. J. Cell. Physiol. 221: 642–649, 2009.

Thapsigargin Modulates Osteoclastogenesis Through the Regulation of RANKL‐Induced Signaling Pathways and Reactive Oxygen Species Production

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‐κB and AP‐1 activities.

Altered leucocyte trafficking and suppressed tumour necrosis factor alpha release from peripheral blood monocytes after intra-articular glucocorticoid treatment

OBJECTIVES A generalised transient improvement may follow intra-articular administration of glucocorticoids to patients with inflammatory arthropathy. This may represent a systemic anti-inflammatory effect of glucocorticoid released from the joint, mediated through processes such as altered leucocyte trafficking or suppressed release of pro-inflammatory cytokines. Patients, who had received intra-articular injections of glucocorticoids were therefore studied for evidence of these two systemic effects. METHODS Patients with rheumatoid arthritis were studied. Peripheral blood leucocyte counts, tumour necrosis factor α (TNFα) release by peripheral blood monocytes, blood cortisol concentrations, and blood methylprednisolone concentration were measured for 96 hours after intra-articular injection of methylprednisolone acetate. RESULTS Measurable concentrations of methylprednisolone were present in blood for up to 96 hours after injection. Significant suppression of the hypothalamic-pituitary-adrenal axis persisted throughout this time. Altered monocyte and lymphocyte trafficking, as evidenced by peripheral blood monocytopenia and lymphopenia, was apparent by four hours after injection and resolved in concordance with the elimination of methylprednisolone. Granulocytosis was observed at 24 and 48 hours. Release of TNFα by endotoxin stimulated peripheral blood monocytes was suppressed at four hours and thereafter. Suppression was maximal at eight hours and was largely reversed by the glucocorticoid antagonist, mifepristone. CONCLUSIONS After intra-articular injection of methylprednisolone, blood concentrations of glucocorticoid are sufficient to suppress monocyte TNFα release for at least four days and to transiently alter leucocyte trafficking. These effects help to explain the transient systemic response to intra-articular glucocorticoids. Suppression of TNFα is principally a direct glucocorticoid effect, rather than a consequence of other methylprednisolone induced changes to blood composition.

Dexamethasone suppresses release of soluble TNF receptors by human monocytes concurrently with TNF-α suppression

Glucocorticoids suppress many monocyte functions, including endotoxin‐stimulated release of TNF‐α. Monocytes also release soluble receptors for TNF (sTNF‐R), which can modulate TNF bioactivity. We therefore examined the effects of the glucocorticoid, dexamethasone, on the release of soluble forms of the 55 kDa and 75 kDa receptors for TNF (sTNF‐R55 and sTNF‐R75) by human monocytes and the human monocytic Mono Mac 6 cell line. Peripheral blood mononuclear cells (PBMC) spontaneously released 406 ± 181 pg/106 cells of STNF‐R75 over 18 h in culture and Mono Mac 6 cells released 554 ± 29pg/106 cells. Lipopolysaccharide (LPS) exposure increased release of sTNF‐R75 by 54 and 217%, respectively. Dexamethasone suppressed both spontaneous and LPS‐stimulated release. The effect of dexamethasone was concentration dependent. At 1 μmol/L, dexamethasone suppressed the LPS‐stimulated release of sTNF‐R75 by 86% in PBMC and by 40% in Mono Mac 6 cells. Neither PBMC nor Mono Mac 6 cells released measurable amounts of sTNF‐R55, but spontaneous release of sTNF‐R55 from purified human monocytes (55 ± 2 pg/106 cells over 18 h) was reduced by 45% in the presence of dexamethasone. Dexamethasone reduced bioactive TNF in PBMC cultures, as well as immunoassayable TNF‐a, which indicates that suppression of TNF‐α release was biologically more important than suppressed release of soluble inhibitors. Similar concurrent suppression of IL‐1β and IL‐Ira release occurred in PBMC and Mono Mac 6 cultures exposed to dexamethasone.

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‐κB 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‐κB 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.