Objective. To clarify the function of osteoclast-like multinuclear cells differentiated from bone marrowderived macrophages (BMMs) by a combination of tumor necrosis factor ␣ (TNF␣) and interleukin-6 (IL-6), and to investigate the molecular mechanisms underlying the differentiation.Methods. BMMs were stimulated by TNF␣ and/or IL-6. The cells were then compared with conventional osteoclasts differentiated in vitro by RANKL. An in vitro pit formation assay on dentine slices and an in vivo resorption assay of calvarial bones were performed. We also evaluated the activities and expression levels of NF-B, c-Fos, and NF-ATc1, which are essential to the differentiation of conventional osteoclasts. Small interfering RNA was used to knock down c-Fos. The effects of genetic ablation of STAT-3 and pharmacologic inhibitors of NF-AT, JAK, and ERK were also studied.Results. Osteoclast-like cell differentiation depended on TNF␣ and IL-6 and was not inhibited by osteoprotegerin. These differentiated cells were associated with both in vitro and in vivo bone resorption activity. TNF␣ and IL-6 had a synergistic effect on the activity and expression of c-Fos. Knockdown of c-Fos inhibited the expression of NF-ATc1 and the differentiation of osteoclast-like cells. All of these inhibitors blocked differentiation of the cells in vitro, but surprisingly, the conditional knockout of STAT-3 did not. Tofacitinib also inhibited the bone destruction caused by TNF␣ and IL-6 in vivo.Conclusion. Our results demonstrate that a combination of the inflammatory cytokines TNF␣ and IL-6 can induce osteoclast-like cells that have in vitro and in vivo bone-resorptive activity.

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Objective. Synovial fibroblasts (SFs) produce matrix-degrading enzymes that cause joint destruction in rheumatoid arthritis (RA). Epigenetic mechanisms play a pivotal role in autoimmune diseases. This study was undertaken to elucidate the epigenetic mechanism that regulates the transcription of matrix metalloproteinases (MMPs) in RASFs.Methods. MMP gene expression and histone methylation profiles in the MMP promoters were examined in RASFs. The effect of WD repeat domain 5 (WDR5) silencing on histone methylation and MMP gene expression in RASFs was analyzed. MMP gene expression, surface expression of the interleukin-6 (IL-6) receptor, phosphorylation of STAT-3, and binding of STAT-3 in the MMP promoters were investigated in RASFs stimulated with IL-6.Results. The MMP-1, MMP-3, MMP-9, and MMP-13 genes were actively transcribed in RASFs. Correspondingly, the level of histone H3 trimethylated at lysine 4 (H3K4me3) was elevated, whereas that of H3K27me3 was suppressed in the MMP promoters in RASFs. The decrease in H3K4me3 via WDR5 small interfering RNA reduced the levels of messenger RNA for MMP-1, MMP-3, MMP-9, and MMP-13 in RASFs. Interestingly, IL-6 signaling significantly increased the expression of MMP-1, MMP-3, and MMP-13, but not MMP-9, in RASFs. Although the IL-6 signaling pathway was similarly active in RASFs and osteoarthritis SFs, STAT-3 bound to the MMP-1, MMP-3, and MMP-13 promoters, but not the MMP-9 promoter, after IL-6 stimulation in RASFs.Conclusion. Our findings indicate that histone methylation and STAT-3 regulate spontaneous and IL-6-induced MMP gene activation in RASFs. The combination of chromatin structure and transcription factors may regulate distinct arthritogenic properties of RASFs.Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease that results in progressive joint destruction and is difficult to treat effectively (1). Several lines of evidence suggest that synovial fibroblasts (SFs) are characterized by an activated and aggressive phenotype with a tumor-like appearance and play a major role in the pathogenesis of RA (2). RASFs produce a variety of proteolytic enzymes, such as matrix metalloproteinases (MMPs) and cathepsins. These enzymes degrade articular cartilage, which is mainly composed of an extracellular matrix (ECM) that consists of proteoglycans and type II collagen (3).The MMPs are a family of zinc-dependent endopeptidases that have the catalytic extracellular activity of degrading the ECM components in a neutral pH environment (4). Twenty-three human MMPs have been identified and grouped into different subfamilies, including the collagenases, gelatinases, stromelysins, matrilysins, and membrane-type MMPs, according to

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Objective We have previously reported that stimulation of mouse bone marrow–derived macrophages with tumor necrosis factor (TNF) and interleukin‐6 (IL‐6) induces differentiation of osteoclast‐like cells. We undertook this study to clarify the characterization and function of human TNF and IL‐6–induced osteoclasts using peripheral blood collected from patients with rheumatoid arthritis (RA) and healthy donors. Methods Peripheral blood monocytes were cultured with a combination of TNF and IL‐6, TNF alone, IL‐6 alone, or with RANKL, and their bone resorption ability was evaluated. Expression levels of NFATc1, proinflammatory cytokines, and matrix metalloproteinase 3 were analyzed. The effects of NFAT inhibitor and JAK inhibitor were examined. Furthermore, the relationship between the number of TNF and IL‐6–induced osteoclasts or RANKL‐induced osteoclasts differentiated from peripheral blood mononuclear cells (PBMCs) in patients with RA and the modified total Sharp score (mTSS) or whole‐body bone mineral density (BMD) was examined. Results Peripheral blood monocytes stimulated with a TNF and IL‐6–induced osteoclasts were shown to demonstrate the ability to absorb bone matrix. Cell differentiation was not inhibited by the addition of osteoprotegerin. Stimulation with a combination of TNF and IL‐6 promoted NFATc1 expression, whereas the NFAT and JAK inhibitors prevented TNF and IL‐6–induced osteoclast formation. Expression levels of IL1β, TNF, IL12p40, and MMP3 were significantly increased in TNF and IL‐6–induced osteoclasts, but not in RANKL‐induced osteoclasts. The number of TNF and IL‐6–induced osteoclasts differentiated from PBMCs in patients with RA positively correlated with the mTSS, whereas RANKL‐induced osteoclast numbers negatively correlated with the whole‐body BMD of the same patients. Conclusion Our results demonstrate that TNF and IL‐6–induced osteoclasts may contribute to the pathology of inflammatory arthritis associated with joint destruction, such as RA.

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The oxidation properties of an H-terminated Si(001) surface following the use of a hyperthermal broad atomic oxygen beam have been studied at low temperatures. Effects of sample temperature, flux and energy of the impinging atomic oxygen on the oxidation of silicon were investigated by X-ray photoelectron spectroscopy. It was confirmed that oxide growth at the surface was achieved even at room temperature and the oxide thickness reached a terminal thickness of a few nanometers depending upon the oxidation conditions. The oxidation process was divided into two stages: the fast oxidation stage and the subsequent slow oxidation stage. It was also confirmed that the reaction yield of atomic oxygen with Si increased with increasing translational energy. The characteristic feature of beam oxidation was explained by the effect of energy accommodation from the impinging oxygen atom to the surface silicon atom.

Wear-life of the MoS 2 film was evaluated by in situ tribological testing under 5 eV atomic oxygen exposures which simulated atomic oxygen environment in low Earth orbit. A combination of a laser-detonation atomic oxygen source and a conventional pinon-disk friction tester was used to perform tribological tests. It was confirmed that the friction coefficient was not affected by atomic oxygen exposure when atomic oxygen fluence was low; however, the friction coefficient increased with increasing atomic oxygen fluence and it reached as high as 0.05 at the atomic oxygen fluence of 3.4 · 10 16 atoms/cm 2 /cycle (nine times larger than the normal value). Effect of atomic oxygen on the wear-life of the film has much more drastic. With atomic oxygen fluence of 1.7 · 10 16 atoms/ cm 2 /cycle, wear-life of the film was reduced less than one-tenth of that during ex situ testing result of the same film. It was also observed that the wear-life of the film was inversely proportional to the atomic oxygen fluence between sliding passes.