Tetsuro Yasui

Epigenetic regulation of osteoclast differentiation: possible involvement of Jmjd3 in the histone demethylation of Nfatc1.

Gene expression is controlled by epigenetic mechanisms such as histone acetylation and methylation, and recent studies have revealed that key developmental steps are regulated by the trimethylation of histone H3 lysine 4 (H3K4me3) and lysine 27 (H3K27me3). Using ChIP sequencing technology combined with real‐time PCR, we here demonstrate that the H3K27me3 observed in the Nfatc1 gene in bone marrow–derived macrophages (BMMs) was markedly reduced in mature osteoclasts. Jumonji domain‐containing 3 (Jmjd3), a H3K27 demethylase, was induced in bone marrow–derived macrophages and in the vicinity of the transcription start site (TSS) of nuclear factor–activated T cells (NFAT) c1 in response to receptor activator of nuclear factor‐κB ligand (RANKL) stimulation. Gene silencing of the Jmjd3 gene by short hairpin RNA reduced demethylation of H3K27me3 at the TSS of Nfatc1 and suppressed RANKL‐induced osteoclastogenesis. These results suggest that the demethylation of H3K27me3 in the Nfatc1 gene locus by Jmjd3 plays a critical role in RANKL‐induced osteoclast differentiation.

Regulation of RANKL-induced osteoclastogenesis by TGF-β through molecular interaction between Smad3 and Traf6.

Previous studies have shown that transforming growth factor β (TGF‐β) promotes receptor activator of nuclear factor‐κB ligand (RANKL)–induced osteoclastogenesis. However, the underlying molecular mechanisms have not been elucidated. When TGF‐β signals were blocked either by a specific inhibitor of TGF‐β type 1 receptor kinase activity, SB431542, or by introducing a dominant‐negative mutant of TGF‐β type 2 receptor, RANKL‐induced osteoclastogenesis was almost completely suppressed. Blockade of Smad signaling by overexpression of Smad7 or c‐Ski markedly suppressed RANKL‐induced osteoclastogenesis, and retroviral induction of an activated mutant of Smad2 or Smad3 reversed the inhibitory effect of SB431542. Immunoprecipitation analysis revealed that Smad2/3 directly associates with the TRAF6‐TAB1‐TAK1 molecular complex, which is generated in response to RANKL stimulation and plays an essential role in osteoclast differentiation. TRAF6‐TAB1‐TAK1 complex formation was not observed when TGF‐β signaling was blocked. Analysis using deletion mutants revealed that the MH2 domain of Smad3 is necessary for TRAF6‐TAB1‐TAK1 complex formation, downstream signal transduction, and osteoclast formation. In addition, gene silencing of Smad3 in osteoclast precursors markedly suppressed RANKL‐induced osteoclast differentiation. In summary, TGF‐β is indispensable in RANKL‐induced osteoclastogenesis, and the binding of Smad3 to the TRAF6‐TAB1‐TAK1 complex is crucial for RANKL‐induced osteoclastogenic signaling.

Anti-apoptotic Molecule Bcl-2 Regulates the Differentiation, Activation, and Survival of Both Osteoblasts and Osteoclasts

The anti-apoptotic molecule Bcl-2 inhibits apoptosis by preventing cytochrome c release from mitochondria. Although several studies have indicated the importance of Bcl-2 in maintaining skeletal integrity, the detailed cellular and molecular mechanisms remain elusive. Bcl-2−/− mice are growth-retarded and exhibit increased bone volume of the primary spongiosa, mainly due to the decreased number and dysfunction of osteoclasts. Osteoblast function is also impaired in Bcl-2−/− mice. Ex vivo studies on osteoblasts and osteoclasts showed that Bcl-2 promoted the differentiation, activation, and survival of both cell types. Because Bcl-2−/− mice die before 6 weeks of age due to renal failure and cannot be compared with adult wild type mice, we generated Bcl-2−/−Bim+/− mice, in which a single Bim allele was inactivated, and compared them with their Bcl-2+/−Bim+/− littermates. Loss of a single Bim allele restored normal osteoclast function in Bcl-2−/− mice but did not restore the impaired function of osteoblasts, and the mice exhibited osteopenia. These data demonstrate that Bcl-2 promotes the differentiation, activity, and survival of both osteoblasts and osteoclasts. The balance between Bcl-2 and Bim regulates osteoclast apoptosis and function, whereas other pro-apoptotic members are important for osteoblasts.

Recent trends in orthopedic surgery performed in Japan for rheumatoid arthritis

The aim of this report was to review the use of orthopedic surgeries performed to manage rheumatoid arthritis (RA). Our review of studies published in English indicates that there has been a decrease in RA-associated surgeries in Western countries. Improvements in medical treatment may partly explain the reduction in numbers of orthopedic joint surgeries, which also suggests a worldwide trend toward improved long-term outcomes. However, the results of our multicenter study in Japan indicate that the number of RA-associated operations has not decreased, and that the numbers of operations performed annually have been relatively stable from 1998 to 2008. Although there definitely has been a decline in the numbers of synovectomy surgeries, the numbers of operations on the upper limbs and foot arthroplasties have increased. With the trend toward milder disease because of improved medical treatment, we speculate that RA patients may want and need better function for the activities of daily living. The combination of medical treatment and surgical intervention is thought to improve outcomes in RA patients who will develop joint destruction. Additional studies, including analyses of RA databases containing long-term data on a variety of surgical interventions, are needed.

Epigenetic regulation of osteoclast differentiation.

Recent studies have uncovered that epigenetic regulation, such as histone methylation and acetylation, plays a critical role in determining cell fate. In particular, the expression of key developmental genes tends to be regulated by trimethylation of histone H3 lysine 4 (H3K4me3) and lysine 27 (H3K27me3). Osteoclasts are primary cells for bone resorption, and their differentiation is tightly regulated by the receptor activator of nuclear factor κB ligand (RANKL) and a transcription factor nuclear factor–activated T cell (NFAT) c1. We found that RANKL‐induced NFATc1 expression is associated with the demethylation of H3K27me3. Jumonji domain containing‐3, a H3K27 demethylase, is induced in bone marrow–derived macrophages in response to RANKL stimulation and may play a critical role in the demethylation of H3K27me3 in the Nfatc1 gene.

Genomewide Comprehensive Analysis Reveals Critical Cooperation Between Smad and c‐Fos in RANKL‐Induced Osteoclastogenesis

We have previously reported that transforming growth factor β (TGF‐β) plays an essential role in receptor activator of nuclear factor‐κB ligand (RANKL)‐induced osteoclastogenesis. However, the detailed underlying molecular mechanisms still remain unclear. Formaldehyde‐assisted isolation of regulatory elements (FAIRE) and chromatin immunoprecipitation (ChIP) followed by sequencing (FAIRE‐seq and ChIP‐seq) analyses indicated the cooperation of Smad2/3 with c‐Fos during osteoclastogenesis. Biochemical analysis and immunocytochemical analysis revealed that physical interaction between Smad2/3 and c‐Fos is required for their nuclear translocation. The gene expression of nuclear factor of activated T‐cells, cytoplasmic 1 (Nfatc1), a key regulator of osteoclastogenesis, was regulated by RANKL and TGF‐β, and c‐Fos binding to open chromatin sites was suppressed by inhibition of TGF‐β signaling by SB431542. Conversely, Smad2/3 binding to Nfatc1 was impaired by c‐Fos deficiency. These results suggest that TGF‐β regulates RANKL‐induced osteoclastogenesis through reciprocal cooperation between Smad2/3 and c‐Fos.

Distinguishing the proapoptotic and antiresorptive functions of risedronate in murine osteoclasts: Role of the Akt pathway and the ERK/Bim axis

OBJECTIVE Nitrogen-containing bisphosphonates are one of the most successful therapeutics for osteoporosis. The aim of this study was to elucidate the functional mechanism of one of the typical nitrogen-containing bisphosphonates, risedronate. METHODS Osteoclasts generated from murine bone marrow macrophages were treated with risedronate in vitro, and its effects on apoptosis and bone-resorbing activity were examined. The mechanism of action of risedronate was examined by gene induction of constitutively active Akt-1 and constitutively active MEK-1, and by gene deletion of Bim. Bim(-/-) mice, in which osteoclasts were resistant to apoptosis, were treated with risedronate and analyzed radiographically, biochemically, and histologically. RESULTS Risedronate induced osteoclast apoptosis through the mitochondria-dependent pathway with an increased expression of Bim, and the proapoptotic effect of risedronate was suppressed by Bim deletion and constitutively active MEK-1 introduction. In contrast, the risedronate-induced suppression of bone resorption was completely reversed by inducing constitutively active Akt-1, but not by Bim deletion or constitutively active MEK-1 introduction. These results suggested that apoptosis and bone-resorbing activity of osteoclasts were regulated through the ERK/Bim axis and the Akt pathway, respectively, both of which were suppressed by risedronate. Although osteoclast apoptosis in response to risedronate administration was suppressed in the Bim(-/-) mice, risedronate treatment increased bone mineral density in Bim(-/-) mice at a level equivalent to that in wild-type mice. CONCLUSION Our findings indicate that the antiresorptive effect of risedronate in vivo is mainly mediated by the suppression of the bone-resorbing activity of osteoclasts and not by the induction of osteoclast apoptosis.

Regulation of bone resorption and sealing zone formation in osteoclasts occurs through protein kinase b–mediated microtubule stabilization

We investigated the role of protein kinase B (Akt), a downstream effector of phosphatidylinositol 3‐kinase, in bone‐resorbing activity of mature osteoclasts. Treatment with a specific Akt inhibitor disrupted sealing zone formation and decreased the bone‐resorbing activity of osteoclasts. The normal microtubule structures were lost and the Akt inhibitor reduced the amount of acetylated tubulin, which reflects stabilized microtubules, whereas forced Akt activation by adenovirus vectors resulted in the opposite effect. Forced Akt activation increased the binding of the microtubule‐associated protein adenomatous polyposis coli (APC), the APC‐binding protein end‐binding protein 1 (EB1) and dynactin, a dynein activator complex, with microtubules. Depletion of Akt1 and Akt2 resulted in a disconnection of APC/EB1 and a decrease in bone‐resorbing activity along with reduced sealing zone formation, both of which were recovered upon the addition of LiCl, a glycogen synthase kinase‐3β (GSK‐3β) inhibitor. The Akt1 and Akt2 double‐knockout mice exhibited osteosclerosis due to reduced bone resorption. These findings indicate that Akt controls the bone‐resorbing activity of osteoclasts by stabilizing microtubules via a regulation of the binding of microtubule associated proteins.

Impact of biologics on the prevalence of orthopedic surgery in the National Database of Rheumatic Diseases in Japan

The aim of this study was to investigate changes in the treatment strategy of rheumatoid arthritis (RA) and the prevalence of RA-related surgeries after approval of biologics in Japan and to analyze the impact of biologics on the incidence of orthopedic surgeries using a nationwide observational cohort database of rheumatic diseases [National Database of Rheumatic Diseases by iR-net in Japan (NinJa)]. The proportion of patients using biologics linearly increased from 2004 (1.8%) to 2007 (10.0%), but neither the number nor type of RA-related surgery significantly changed during this period. Patients treated with biologics exhibited relatively more severe disease activity and lower physical function. Among patients using biologics, those who underwent RA-related surgeries exhibited background characteristics of longer disease duration and worse physical function, while disease activity was not different from patients without surgery. These results suggest that the potential value of biologics in avoiding surgical procedure is limited in patients with severe functional disorders caused by long disease duration. Further investigation with a longer observation period is required to obtain more definite conclusions as to the impact of biologics usage on orthopedic surgeries.

Quantitative and qualitative characterization of expanded CD4 + T cell clones in rheumatoid arthritis patients

Rheumatoid arthritis (RA) is an autoimmune destructive arthritis associated with CD4+ T cell-mediated immunity. Although expanded CD4+ T cell clones (ECs) has already been confirmed, the detailed characteristics of ECs have not been elucidated in RA. Using combination of a single-cell analysis and next-generation sequencing (NGS) in TCR repertoire analysis, we here revealed the detailed nature of ECs by examining peripheral blood (PB) from 5 RA patients and synovium from 1 RA patient. When we intensively investigated the single-cell transcriptome of the most expanded clones in memory CD4+ T cells (memory-mECs) in RA-PB, senescence-related transcripts were up-regulated, indicating circulating ECs were constantly stimulated. Tracking of the transcriptome shift within the same memory-mECs between PB and the synovium revealed the augmentations in senescence-related gene expression and the up-regulation of synovium-homing chemokine receptors in the synovium. Our in-depth characterization of ECs in RA successfully demonstrated the presence of the specific immunological selection pressure, which determines the phenotype of ECs. Moreover, transcriptome tracking added novel aspects to the underlying sequential immune processes. Our approach may provide new insights into the pathophysiology of RA.