Hirohiko Okamura

Histone demethylase Jmjd3 regulates osteoblast differentiation via transcription factors Runx2 and osterix.

Background: The roles of histone demethylase Jmjd3 in osteoblasts are not fully understood. Results: Jmjd3 expression increased during osteoblast differentiation. Silencing of Jmjd3 impaired osteoblast differentiation. Introduction of the exogenous Runx2 and osterix partly rescued osteoblast differentiation in shJmjd3 cells. Conclusion: Jmjd3 regulates osteoblast differentiation via transcription factors Runx2 and osterix. Significance: This study provides new insights about the roles of Jmjd3 in osteoblast differentiation. Post-translational modifications of histones including methylation play important roles in cell differentiation. Jumonji domain-containing 3 (Jmjd3) is a histone demethylase, which specifically catalyzes the removal of trimethylation of histone H3 at lysine 27 (H3K27me3). In this study, we examined the expression of Jmjd3 in osteoblasts and its roles in osteoblast differentiation. Jmjd3 expression in the nucleus was induced in response to the stimulation of osteoblast differentiation as well as treatment of bone morphogenetic protein-2 (BMP-2). Either treatment with Noggin, an inhibitor of BMP-2, or silencing of Smad1/5 suppressed Jmjd3 expression during osteoblast differentiation. Silencing of Jmjd3 expression suppressed osteoblast differentiation through the expression of bone-related genes including Runx2, osterix, osteopontin, bone sialoprotein (BSP), and osteocalcin (OCN). Silencing of Jmjd3 decreased the promoter activities of Runx2 and osterix and increased the level of H3K27me3 on the promoter regions of Runx2 and osterix. Introduction of the exogenous Runx2 and osterix partly rescued osteoblast differentiation in the shJmjd3 cells. The present results indicate that Jmjd3 plays important roles in osteoblast differentiation and regulates the expressions of BSP and OCN via transcription factors Runx2 and osterix.

Oral Porphyromonas gingivalis translocates to the liver and regulates hepatic glycogen synthesis through the Akt/GSK-3β signaling pathway

Periodontal diseases are common chronic inflammatory disorders that result in the destruction of tissues around teeth. Many clinical studies suggest that periodontal diseases are risk factors for insulin resistance and diabetic mellitus development. However, the molecular mechanisms by which periodontal diseases regulate the progress of diabetes mellitus remain unknown. In this study, we investigated whether Porphyromonas gingivalis (P.g.), a major pathogen of periodontal diseases, present in the oral cavity, moves to the liver and affects hepatic glycogen synthesis. SNAP26b-tagged P.g. (SNAP-P.g.) was introduced into the oral cavity to induce periodontal disease in 4-week old female Balb/c mice. SNAP-P.g. was detected in the liver extracted from SNAP-P.g.-treated mice using nested PCR analysis. High blood glucose levels tended to promote SNAP-P.g. translocation from the oral cavity to the liver in mice. Periodic acid-Schiff staining suggested that hepatic glycogen synthesis decreased in SNAP-P.g.-treated mice. SNAP-P.g. was also internalized into the human hepatoma cell line HepG2, and this attenuated the phosphorylation of insulin receptor substrate (IRS)-1, Akt and glycogen synthase kinase-3β induced by insulin. Insulin-induced glycogen synthesis was suppressed by SNAP-P.g. in HepG2 cells. Our results suggest that P.g. translocation from the oral cavity to the liver may contribute to the progress of diabetes mellitus by influencing hepatic glycogenesis.

Histone H1.2 is translocated to mitochondria and associates with bak in bleomycin‐induced apoptotic cells

Bleomycin induces single‐ and double‐stranded breaks in DNA, with consequent mitochondrial membrane aberrations that lead to the apoptotic cell death. It is poorly understood how DNA damage‐inducing apoptotic signals are transmitted to mitochondria, from which apoptotic factors are released into the cytoplasm. Here, we investigated the localization of histone H1.2 in the bleomycin‐treated human squamous carcinoma SCCTF cells. The presence of DNA double‐strand breaks in the bleomycin‐treated cells was examined by Western analysis using antibody against phosphorylated histone H2AX (γ‐H2AX). Incubation of SCCTF cells for 48 h with 10 µM bleomycin induced apoptosis, as determined by cleavage of lamin B1 to 28 kDa fragment and DNA ladder formation. The mitochondrial permeabilization causing apoptotic feature was also detected with MitoCapture in the bleomycin‐treated cells. Histone H1.2 was translocated from the nucleus to the mitochondria after treatment with bleomycin and co‐localized with Bak in mitochondria. Our present results suggest that histone H1.2 plays an important role in transmitting apoptotic signals from the nucleus to the mitochondria following double‐stranded breaks of DNA by bleomycin. J. Cell. Biochem. 103: 1488–1496, 2008.

Reduction of protein phosphatase 2A Cα enhances bone formation and osteoblast differentiation through the expression of bone-specific transcription factor Osterix

The serine/threonine protein phosphatase 2A (PP2A) participates in regulating many important physiological processes such as control of cell cycle, growth, and division. On the other hand, Osterix is a zinc-finger-containing transcription factor that is essential for the differentiation of osteoblasts and regulation of many bone-related genes. Here we examined the effect of okadaic acid (OA), a specific inhibitor of PP2A, on bone formation in vivo and the molecular mechanism regulated by PP2A Cα in osteoblast differentiation. Administration of 1nM OA to the calvarial region in mice increased bone mineral density, as shown by μCT, while histomorphological analysis showed an increase in mineral apposition and bone thickness in the same region. In addition, treatment with 1nM OA stimulated osteoblast differentiation and the expression of Osterix, bone sialoprotein (Bsp), and osteocalcin (OCN) in mouse osteoblastic MC3T3-E1 cells. Moreover, the expression and phosphatase activity of PP2A Cα was decreased in the initial step of osteoblast differentiation, which was in parallel with an increase in Osterix expression. To further clarify the role of PP2A Cα in osteoblast differentiation, we constructed PP2A knock-down cells by infecting MC3T3-E1 cells with a lentivirus expressing shRNA specific for the PP2A Cα. Accordingly, the silencing of PP2A Cα in MC3T3-E1 cells dramatically increased osteoblast differentiation and mineralization, which were accompanied with expressions of Osterix, Bsp, and OCN. Our data indicate that PP2A Cα plays an important role in the regulation of bone formation and osteoblast differentiation through the bone-related genes.

Tumor necrosis factor-α induces interleukin-34 expression through nuclear factor-κB activation in MC3T3-E1 osteoblastic cells

Osteoblasts produce various types of cytokines under pathological conditions and control osteoclast differentiation. Tumor necrosis factor-α (TNF-α) has been demonstrated to exert complex effects in osteoblasts under local inflammatory conditions, including in periodontal and periapical diseases. Interleukin-34 (IL-34) has been recently identified as a novel regulatory factor for the differentiation and function of osteoclasts. The present study provides the first evidence, to the best of our knowledge, that the expression of IL-34 is induced by TNF-α through nuclear factor-κB (NF-κB) activation in MC3T3-E1 osteoblastic cells. TNF-α induced IL-34 expression in a dose- and time-dependent manner. Immunocytochemistry with an NF-κB antibody demonstrated that NF-κB was mainly localized in the cytoplasm of the untreated MC3T3-E1 cells. Rapid translocation of NF-κB from the cytoplasm to the nucleus was observed in the cells treated with TNF-α for 15 min. Translocation and transcriptional activity of NF-κB were also determined by western blotting and a luciferase reporter assay, respectively. Pretreatment with 100 μM CAPE, an inhibitor of NF-κB, significantly inhibited TNF-α-induced IL-34 expression. These results indicate that TNF-α induces IL-34 expression via NF-κB in osteoblasts.

Calcineurin regulates phosphorylation status of transcription factor osterix

Osterix is an osteoblast-specific transcriptional factor that is essential for osteoblast differentiation and bone formation. Calcineurin regulates bone formation through modulating osteoblast differentiation. However, post-translational modification of osterix such as phosphorylation and interactions between osterix and calcineurin remains unclear. In the present study, we demonstrated that calcineurin interacted with osterix determined by immunoprecipitation assay and Western analysis. Immunocytochemical study also revealed that osterix and calcineurin were co-localized in nucleus. Deletion of calcineurin binding motif on osterix molecule disrupted osterix-calcineurin interaction. Phosphorylation status of osterix was augmented by treatment with phosphatase inhibitors, FK506 and calyculin A. In contrast, treatment of recombinant calcineurin reduced phosphorylation status of osterix. Our present study suggests that calcineurin has an important role in the function of osterix through its modification of phosphorylation.

Expression of PTEN and Akt phosphorylation in lipopolysaccharide-treated NIH3T3 cells.

PTEN is a tumor suppressor gene encoding a phosphatase, and it negatively regulates cell survival mediated by the phosphoinositol 3‐kinase (PI3‐Kinase)‐Akt pathway. To elucidate PTEN expression and its effect on the PI3‐kinase‐Akt pathway in fibroblasts and macrophages, we investigated the expression of PTEN and the phosphorylation status of Akt in NIH3T3 and RAW264.7 cells treated with LPS. Phosphorylation of Akt was induced by LPS treatment in a dose‐dependent manner in RAW264.7 cells, but not in NIH3T3 cells. LPS induced the expression of PTEN in a dose and time‐dependent manner in NIH3T3 cells (0–1 μg/ml, 0–6 h). However, LPS did not stimulate PTEN expression in RAW264.7 cells. These data indicate the existence of diverse mechanisms for PTEN expression and Akt activation in fibroblasts and macrophages. RNA interference using double‐stranded RNA specific for the PTEN gene reduced both mRNA and protein levels of PTEN in NIH3T3 cells treated or not with LPS. The phosphorylation status of Akt in NIH3T3 cells stimulated with LPS did not change when the PTEN expression had been inhibited by RNA interference. The present results suggest that the up‐regulation of PTEN expression by LPS is not involved in the activation of Akt in NIH3T3 cells. PTEN expression might be involved in the diverse inflammatory responses to LPS in fibroblasts and macrophages.

Double Stranded RNA‐Dependent Protein Kinase is Necessary for TNF‐α‐Induced Osteoclast Formation In Vitro and In Vivo

Double‐stranded RNA‐dependent protein kinase (PKR) is involved in cell cycle progression, cell proliferation, cell differentiation, tumorgenesis, and apoptosis. We previously reported that PKR is required for differentiation and calcification in osteoblasts. TNF‐α plays a key role in osteoclast differentiation. However, it is unknown about the roles of PKR in the TNF‐α‐induced osteoclast differentiation. The expression of PKR in osteoclast precursor RAW264.7 cells increased during TNF‐α‐induced osteoclastogenesis. The TNF‐α‐induced osteoclast differentiation in bone marrow‐derived macrophages and RAW264.7 cells was markedly suppressed by the pretreatment of PKR inhibitor, 2‐aminopurine (2AP), as well as gene silencing of PKR. The expression of gene markers in the differentiated osteoclasts including TRAP, Calcitonin receptor, cathepsin K, and ATP6V0d2 was also suppressed by the 2AP treatment. Bone resorption activity of TNF‐α‐induced osteoclasts was also supressed by 2AP treatment. Inhibition of PKR supressed the TNF‐α‐induced activation of NF‐κB and MAPK in RAW264.7 cells. 2AP inhibited both the nuclear translocation of NF‐κB and its transcriptional activity in RAW264.7 cells. 2AP inhibited the TNF‐α‐induced expression of NFATc1 and c‐fos, master transcription factors in osteoclastogenesis. TNF‐α‐induced nuclear translocation of NFATc1 in mature osteoclasts was clearly inhibited by the 2AP treatment. The PKR inhibitor C16 decreased the TNF‐α‐induced osteoclast formation and bone resorption in mouse calvaria. The present study indicates that PKR is necessary for the TNF‐α‐induced osteoclast differentiation in vitro and in vivo. J. Cell. Biochem. 116: 1957–1967, 2015.

Histone Demethylase Utx Regulates Differentiation and Mineralization in Osteoblasts

Alteration of methylation status of lysine 27 on histone H3 (H3K27) associates with dramatic changes in gene expression in response to various differentiation signals. Demethylation of H3K27 is controlled by specific histone demethylases including ubiquitously transcribed tetratricopeptide repeat X chromosome (Utx). However, the role of Utx in osteoblast differentiation remains unknown. In this study, we examined whether Utx should be involved in osteoblast differentiation. Expression of Utx increased during osteoblast differentiation in MC3T3‐E1 cells and primary osteoblasts. GSK‐J1, a potent inhibitor of H3K27 demethylase, increased the levels of trimethylated H3K27 (H3K27me3) and decreased the expressions of Runx2 and Osterix and ALP activity in MC3T3‐E1 cells. Stable knockdown of Utx by shRNA attenuated osteoblast differentiation and decreased ALP activity, calcium content, and bone‐related gene expressions. Silencing of Utx increased the level of H3K27me3 on the promoter regions of Runx2 and Osterix and decreased the promoter activities of Runx2 and Osterix. Taken together, our present results propose that Utx plays important roles in osteoblast differentiation by controlling the expressions of Runx2 and Osterix. J. Cell. Biochem. 116: 2628–2636, 2015.

Reduction of PP2A Cα stimulates adipogenesis by regulating the Wnt/GSK-3β/β-catenin pathway and PPARγ expression.

Serine/threonine protein phosphatase 2A (PP2A) regulates several physiological processes such as the cell cycle, cell growth, apoptosis, and signal transduction. In this study, we examined the expression and role of PP2A Cα in adipocyte differentiation. PP2A Cα expression and PP2A activity decreased during adipocyte differentiation in C3H10T1/2 and 3T3-L1 cells and the expression of adipocyte marker genes such as PPARγ and adiponectin increased. To further clarify the role of PP2A Cα in adipocyte differentiation, we constructed PP2A knockdown cells by infecting C3H10T1/2 cells with a lentivirus expressing a shRNA specific for the PP2A Cα (shPP2A cells). Silencing of PP2A Cα in C3H10T1/2 cells dramatically stimulated adipocyte differentiation and lipid accumulation, which were accompanied by expression of adipocyte marker genes. Silencing of PP2A Cα suppressed Wnt10b expression and reduced the levels of the inactivated form of GSK-3β (phospho-GSK-3β), leading to the reduction of β-catenin levels in the nucleus and its transcriptional activity. Treatment with LiCl, a GSK-3β inhibitor, and inhibition of PPARγ expression suppressed the accelerated adipogenesis of shPP2A cells. Our data indicate that PP2A Cα plays an important role in the regulation of adipocyte differentiation by regulating the Wnt/GSK-3β/β-catenin pathway and PPARγ expression.