Faculty Profiles - OKAMURA Hirohiko

Papers - OKAMURA Hirohiko

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

Hirohiko Okamura, Di Yang, Kaya Yoshida, Jumpei Teramachi, Tatsuji Haneji

Biochimica et biophysica acta 1843 ( 11 ) 2376 - 84 2014.11

Language：English

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.

DOI： 10.1016/j.bbamcr.2014.06.008

Reduction of PP2A C alpha stimulates adipogenesis by regulating the Wnt/GSK-3 beta/beta-catenin pathway and PPAR gamma expression Reviewed

Hirohiko Okamura, Di Yang, Kaya Yoshida, Jumpei Teramachi, Tatsuji Haneji

BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH 1843 ( 11 ) 2376 - 2384 2014.11

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE BV

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 C alpha in adipocyte differentiation. PP2A C alpha expression and PP2A activity decreased during adipocyte differentiation in C3H10T1/2 and 3T3-L1 cells and the expression of adipocyte marker genes such as PPAR gamma and adiponectin increased. To further clarify the role of PP2A C alpha in adipocyte differentiation, we constructed PP2A knockdown cells by infecting C3H10T1/2 cells with a lentivirus expressing a shRNA specific for the PP2A C alpha (shPP2A cells). Silencing of PP2A C alpha in C3H10T1/2 cells dramatically stimulated adipocyte differentiation and lipid accumulation, which were accompanied by expression of adipocyte marker genes. Silencing of PP2A C alpha suppressed Wnt10b expression and reduced the levels of the inactivated form of GSK-3 beta (phospho-GSK-3 beta), leading to the reduction of beta-catenin levels in the nucleus and its transcriptional activity. Treatment with LiCl, a GSK-3 beta inhibitor, and inhibition of PPAR gamma expression suppressed the accelerated adipogenesis of shPP2A cells. Our data indicate that PP2A C alpha plays an important role in the regulation of adipocyte differentiation by regulating the Wnt/GSK-3 beta/beta-catenin pathway and PPAR gamma expression. (C) 2014 Published by Elsevier B.V.

DOI： 10.1016/j.bbamcr.2014.06.008

Tumor necrosis factor-alpha induces interleukin-34 expression through nuclear factor-kappa B activation in MC3T3-E1 osteoblastic cells Reviewed

Yaqiong Yu, Di Yang, Lihong Qiu, Hirohiko Okamura, Jiajie Guo, Tatsuji Haneji

MOLECULAR MEDICINE REPORTS 10 ( 3 ) 1371 - 1376 2014.9

Language：English Publishing type：Research paper (scientific journal) Publisher：SPANDIDOS PUBL LTD

Osteoblasts produce various types of cytokines under pathological conditions and control osteoclast differentiation. Tumor necrosis factor-alpha (TNF-alpha) 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-alpha through nuclear factor-kappa B (NF-kappa B) activation in MC3T3-E1 osteoblastic cells. TNF-alpha induced IL-34 expression in a dose- and time-dependent manner. Immunocytochemistry with an NF-kappa B antibody demonstrated that NF-kappa B was mainly localized in the cytoplasm of the untreated MC3T3-E1 cells. Rapid translocation of NF-kappa B from the cytoplasm to the nucleus was observed in the cells treated with TNF-alpha for 15 min. Translocation and transcriptional activity of NF-kappa B were also determined by western blotting and a luciferase reporter assay, respectively. Pretreatment with 100 mu M CAPE, an inhibitor of NF-kappa B, significantly inhibited TNF-alpha-induced IL-34 expression. These results indicate that TNF-alpha induces IL-34 expression via NF-kappa B in osteoblasts.

DOI： 10.3892/mmr.2014.2353

Calcium Hydroxide Suppresses Porphyromonas endodontalis Lipopolysaccharide-induced Bone Destruction Reviewed

J. Guo, D. Yang, H. Okamura, J. Teramachi, K. Ochiai, L. Qiu, T. Haneji

JOURNAL OF DENTAL RESEARCH 93 ( 5 ) 508 - 513 2014.5

Language：English Publishing type：Research paper (scientific journal) Publisher：SAGE PUBLICATIONS INC

Porphyromonas endodontalis and its main virulence factor, lipopolysaccharide (LPS), are associated with the development of periapical diseases and alveolar bone loss. Calcium hydroxide is commonly used for endodontic therapy. However, the effects of calcium hydroxide on the virulence of P. endodontalis LPS and the mechanism of P. endodontalis LPS-induced bone destruction are not clear. Calcium hydroxide rescued the P. endodontalis LPS-suppressed viability of MC3T3-E1 cells and activity of nuclear factor-kappa B (NF-kappa B) in these cells, resulting in the reduced expression of interleukin-6 and tumor necrosis factor-alpha. In addition, calcium hydroxide inhibited P. endodontalis LPS-induced osteoclastogenesis by decreasing the activities of NF-kappa B, p38, and ERK1/2 and the expression of nuclear factor of activated T-cell cytoplasmic 1 in RAW264.7 cells. Calcium hydroxide also rescued the P. endodontalis LPS-induced osteoclastogenesis and bone destruction in mouse calvaria. Taken together, our present results indicate that calcium hydroxide suppressed bone destruction by attenuating the virulence of P. endodontalis LPS on bone cells.

DOI： 10.1177/0022034514526886

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

Makoto Ishikawa, Kaya Yoshida, Hirohiko Okamura, Kazuhiko Ochiai, Haruna Takamura, Natsumi Fujiwara, Kazumi Ozaki

Biochimica et biophysica acta 1832 ( 12 ) 2035 - 43 2013.12

Language：English Publishing type：Research paper (scientific journal)

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.

DOI： 10.1016/j.bbadis.2013.07.012

Oral porphyromonas gingivalis translocates to the liver and regulates hepatic glycogen synthesis through the Akt/GSK-3b signaling pathway Reviewed

Ishikawa Makoto, Kaya Yoshida, Hirohiko Okamura, Kazuhiko Ochiai, Takamura Haruna, Natsumi Fujiwara, Kazumi Ozaki

Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease Vol.1832 ( No.12 ) 2035 - 2043 2013.12

Language：English Publishing type：Research paper (scientific journal)

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.

DOI： 10.1016/j.bbadis.2013.07.012

Histone Demethylase Jmjd3 Regulates Osteoblast Differentiation via Transcription Factors Runx2 and Osterix Reviewed

Di Yang, Hirohiko Okamura, Yoshiki Nakashima, Tatsuji Haneji

JOURNAL OF BIOLOGICAL CHEMISTRY 288 ( 47 ) 33530 - 33541 2013.11

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

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.

DOI： 10.1074/jbc.M113.497040

Protein phosphatase 2A C alpha regulates osteoblast differentiation and the expressions of bone sialoprotein and osteocalcin via osterix transcription factor Reviewed

Hirohiko Okamura, Kaya Yoshida, Di Yang, Tatsuji Haneji

JOURNAL OF CELLULAR PHYSIOLOGY 228 ( 5 ) 1031 - 1037 2013.5

Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY-BLACKWELL

Serine/threonine protein phosphatase 2A (PP2A) participates in regulating many important physiological processes such as cell cycle, growth, apoptosis, and signal transduction. Osterix is a zinc-finger-containing transcription factor that is essential for osteoblast differentiation and regulation of many bone-related genes. We have recently reported that decrease in a-isoform of PP2A catalytic subunit (PP2A Ca) accelerates osteoblast differentiation through the expression of bone-related genes. In this study, we further examined the role of PP2A Ca in osteoblast differentiation by establishing the stable cell lines that overexpress PP2A Ca. Overexpression of PP2A Ca reduced alkaline phosphatase (ALP) activity. Osteoblast differentiation and mineralization were also decreased in PP2A Ca-overexpressing cells, with reduction of bone-related genes including osterix, bone sialoprotein (Bsp), and osteocalcin (OCN). Luciferase assay showed that the transcriptional activity of the Osterix promoter region was decreased in PP2A Ca-overexpressing cells. Introduction of ectopic Osterix rescued the expression of Bsp and OCN in PP2A Ca-overexpressing cells. These results indicate that PP2A Ca and its activity play a negative role in osteoblast differentiation and Osterix is a key factor responsible for regulating the expressions of Bsp and OCN during PP2A Ca-mediated osteoblast differentiation. J. Cell. Physiol. (C) 2012 Wiley Periodicals, Inc.

DOI： 10.1002/jcp.24250

Protein phosphatase 2A Cα regulates osteoblast differentiation and the expressions of bone sialoprotein and osteocalcin via osterix transcription factor. Reviewed

Hirohiko Okamura, Kaya Yoshida, Di Yang, Tatsuji Haneji

Journal of Cellular Physiology Vol.228 ( No.5 ) 1031 - 1037 2013.5

Language：English Publishing type：Research paper (scientific journal)

Serine/threonine protein phosphatase 2A (PP2A) participates in regulating many important physiological processes such as cell cycle, growth, apoptosis, and signal transduction. Osterix is a zinc-finger-containing transcription factor that is essential for osteoblast differentiation and regulation of many bone-related genes. We have recently reported that decrease in α-isoform of PP2A catalytic subunit (PP2A Cα) accelerates osteoblast differentiation through the expression of bone-related genes. In this study, we further examined the role of PP2A Cα in osteoblast differentiation by establishing the stable cell lines that overexpress PP2A Cα. Overexpression of PP2A Cα reduced alkaline phosphatase (ALP) activity. Osteoblast differentiation and mineralization were also decreased in PP2A Cα-overexpressing cells, with reduction of bone-related genes including osterix, bone sialoprotein (Bsp), and osteocalcin (OCN). Luciferase assay showed that the transcriptional activity of the Osterix promoter region was decreased in PP2A Cα-overexpressing cells. Introduction of ectopic Osterix rescued the expression of Bsp and OCN in PP2A Cα-overexpressing cells. These results indicate that PP2A Cα and its activity play a negative role in osteoblast differentiation and Osterix is a key factor responsible for regulating the expressions of Bsp and OCN during PP2A Cα-mediated osteoblast differentiation.

DOI： 10.1002/jcp.24250

Protein phosphatase 2A C is involved in osteoclastogenesis by regulating RANKL and OPG expression in osteoblasts. Reviewed

Hirohiko Okamura, Di Yang, Kaya Yoshida, Tatsuji Haneji

FEBS Letters Vol.587 ( No.1 ) 48 - 53 2013.1

Language：English Publishing type：Research paper (scientific journal)

We examined whether alteration of PP2A Cα expression in osteoblasts is involved in osteoclast differentiation. Reduction of PP2A Cα in MC3T3-E1 cells (shPP2A) decreased receptor activator of nuclear factor κB ligand (RANKL) expression and increased osteoprotegerin (OPG) expression. The conditioned medium from shPP2A cells failed to induce NFATc1 as well as the expression of osteoclast marker genes cathepsin K and osteoclast-associated receptor (OSCAR) in bone marrow macrophage cells. Treatment of bone marrow macrophage cells with the conditioned medium from shPP2A cells impaired osteoclastogenesis. These results suggest that alteration of PP2A Cα expression in osteoblasts modulates the expressions of RANKL and OPG, which are involved in osteoclastogenesis via the NFATc1 transcription factor.

DOI： 10.1016/j.febslet.2012.10.041

Protein phosphatase 2A C alpha is involved in osteoclastogenesis by regulating RANKL and OPG expression in osteoblasts Reviewed

Hirohiko Okamura, Di Yang, Kaya Yoshida, Tatsuji Haneji

FEBS LETTERS 587 ( 1 ) 48 - 53 2013.1

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE BV

We examined whether alteration of PP2A C alpha expression in osteoblasts is involved in osteoclast differentiation. Reduction of PP2A C alpha in MC3T3-E1 cells (shPP2A) decreased receptor activator of nuclear factor kappa B ligand (RANKL) expression and increased osteoprotegerin (OPG) expression. The conditioned medium from shPP2A cells failed to induce NFATc1 as well as the expression of osteoclast marker genes cathepsin K and osteoclast-associated receptor (OSCAR) in bone marrow macrophage cells. Treatment of bone marrow macrophage cells with the conditioned medium from shPP2A cells impaired osteoclastogenesis. These results suggest that alteration of PP2A C alpha expression in osteoblasts modulates the expressions of RANKL and OPG, which are involved in osteoclastogenesis via the NFATc1 transcription factor. (C) 2012 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.

DOI： 10.1016/j.febslet.2012.10.041

PKR plays a positive role in osteoblast differentiation by regulating GSK-3β activity through a β-catenin-independent pathway Reviewed

Kaya Yoshida, Hirohiko Okamura, Kazuhiko Ochiai, Yumi Hoshino, Tatsuji Haneji, Masami Yoshioka, Daisuke Hinode, Hideo Yoshida

Molecular and Cellular Endocrinology 361 ( 1-2 ) 99 - 105 2012.9

Publishing type：Research paper (scientific journal)

Double-stranded RNA-dependent protein kinase (PKR) is involved in various cellular functions. We previously reported that PKR regulates osteoblast differentiation, but the specific mechanisms by which this occurs remain unclear. In this study, we investigated the role of PKR in Glycogen synthase kinase 3β (GSK-3β) regulation of osteoblast differentiation. Lithium chloride (LiCl), a GSK-3β inhibitor, increased GSK-3β phosphorylation in MC3T3-E1 and MG-63 cells. LiCl also inhibited Runx2 and expression of its regulated genes, causing inhibition of Alkaline phosphatase activity and mineralization. LiCl injection to the calvaria in mice suppressed bone formation. Further, GSK-3β phosphorylation was increased in osteoblasts, by Akt-independent mechanisms, in which PKR was constitutively inactivated. A PKR inhibitor, 2-aminopurine, also induced GSK-3β phosphorylation in MC3T3-E1 and MG-63 cells. Further, Runx2 and its regulated genes were inhibited in PKR-inactivated osteoblasts, and differentiation was suppressed through a β-catenin-independent pathway. PKR positively regulates the differentiation of osteoblasts by mediating GSK-3β activity through a β-catenin-independent pathway. © 2012 Elsevier Ireland Ltd.

DOI： 10.1016/j.mce.2012.03.019

PKR plays a positive role in osteoblast differentiation by regulating GSK-3b activity through a b-catenin-independent pathway Reviewed

Kaya Yoshida, Hirohiko Okamura, Kazuhiko Ochiai, Yumi Hoshimo, Tatsuji Haneji, Masami Yoshioka, Daisuke Hinode, Hideo Yoshida

Molecular and Cellular Endocrinology Vol.361 ( No.1-2 ) 99 - 105 2012.9

Language：English Publishing type：Research paper (scientific journal)

Double-stranded RNA-dependent protein kinase (PKR) is involved in various cellular functions. We previously reported that PKR regulates osteoblast differentiation, but the specific mechanisms by which this occurs remain unclear. In this study, we investigated the role of PKR in Glycogen synthase kinase 3 (GSK-3) regulation of osteoblast differentiation. Lithium chloride (LiCl), a GSK-3 inhibitor, increased GSK-3 phosphorylation in MC3T3-E1 and MG-63 cells. LiCl also inhibited Runx2 and expression of its regulated genes, causing inhibition of Alkaline phosphatase activity and mineralization. LiCl injection to the calvaria in mice suppressed bone formation. Further, GSK-3 phosphorylation was increased in osteoblasts, by Akt-independent mechanisms, in which PKR was constitutively inactivated. A PKR inhibitor, 2-aminopurine, also induced GSK-3 phosphorylation in MC3T3-E1 and MG-63 cells. Further, Runx2 and its regulated genes were inhibited in PKR-inactivated osteoblasts, and differentiation was suppressed through a -catenin-independent pathway. PKR positively regulates the differentiation of osteoblasts by mediating GSK-3 activity through a -catenin-independent pathway.

DOI： 10.1016/j.mce.2012.03.019

Interaction between PKR and PACT mediated by LPS-inducible NF-?B in human gingival cells Reviewed

Kaya Yoshida, Hirohiko Okamura, Yumi Hoshino, Masayuki Shono, Masami Yoshioka, Daisuke Hinode, Hideo Yoshida

JOURNAL OF CELLULAR BIOCHEMISTRY 113 ( 1 ) 165 - 173 2012.1

Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY-BLACKWELL

The double-stranded RNA-dependent protein kinase (PKR) is a serine/threonine kinase expressed constitutively in mammalian cells. PKR is activated upon virus infection by double-stranded RNA (dsRNA), and plays a critical role in host antiviral defense mechanisms. PKR is also known to regulate various biological responses, including cell differentiation and apoptosis. However, whether PKR is involved in the progress of periodontitis is not clear. The present study explained the phosphorylation of PKR by LPS in the human gingival cell line, Sa3. Expression of genes encoding LPS receptors was detected in Sa3 cells and treatment of cells with 1 mu g/mL LPS for 6 h caused PKR phosphorylation. LPS elevated the expression of the protein activator of PKR (PACT) mRNA and protein, followed by the enhanced association between PACT and PKR within 3 h. In addition, LPS treatment induced the translocation of NF-?B to the nucleus after 30 min, and inhibition of NF-B decreased the PACTPKR interaction induced by LPS. The level of pro-inflammatory cytokine mRNA, including interleukin-6 (IL-6) and tumor necrosis factor alpha (TNFa), appeared within 45 min and reached at the maximal levels by 90 min after the addition of LPS. This induction of pro-inflammatory cytokines was not affected by RNAi-mediated silencing of PKR and a pharmacological inhibitor of PKR, whereas the inhibition of NF-?B decreased it. These results indicated that LPS induces PKR phosphorylation and the PACTPKR association in Sa3 cells. Our results also suggest that NF-?B is involved in the PACTPKR interaction and the production of pro-inflammatory cytokines in periodontitis. J. Cell. Biochem. 113: 165173, 2012. (C) 2011 Wiley Periodicals, Inc.

DOI： 10.1002/jcb.23340

Interaction between PKR and PACT mediated by LPS-inducible NF-kB in human gingival cells Reviewed

Kaya Yoshida, Hirohiko Okamura, Yumi Hoshimo, Masayuki Shono, Masami Yoshioka, Daisuke Hinode, Hideo Yoshida

Journal of Cellular Biochemistry Vol.113 165 - 173 2012.1

Language：English Publishing type：Research paper (scientific journal)

The double-stranded RNA-dependent protein kinase (PKR) is a serine/threonine kinase expressed constitutively in mammalian cells. PKR is activated upon virus infection by double-stranded RNA (dsRNA), and plays a critical role in host antiviral defense mechanisms. PKR is also known to regulate various biological responses, including cell differentiation and apoptosis. However, whether PKR is involved in the progress of periodontitis is not clear. The present study explained the phosphorylation of PKR by LPS in the human gingival cell line, Sa3. Expression of genes encoding LPS receptors was detected in Sa3 cells and treatment of cells with 1 μg/mL LPS for 6 h caused PKR phosphorylation. LPS elevated the expression of the protein activator of PKR (PACT) mRNA and protein, followed by the enhanced association between PACT and PKR within 3 h. In addition, LPS treatment induced the translocation of NF-κB to the nucleus after 30 min, and inhibition of NF-κB decreased the PACT-PKR interaction induced by LPS. The level of pro-inflammatory cytokine mRNA, including interleukin-6 (IL-6) and tumor necrosis factor alpha (TNFα), appeared within 45 min and reached at the maximal levels by 90 min after the addition of LPS. This induction of pro-inflammatory cytokines was not affected by RNAi-mediated silencing of PKR and a pharmacological inhibitor of PKR, whereas the inhibition of NF-κB decreased it. These results indicated that LPS induces PKR phosphorylation and the PACT-PKR association in Sa3 cells. Our results also suggest that NF-κB is involved in the PACT-PKR interaction and the production of pro-inflammatory cytokines in periodontitis.

DOI： 10.1002/jcb.23340

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

Hirohiko Okamura, Kaya Yoshida, Kazuhiko Ochiai, Tatsuji Haneji

BONE 49 ( 3 ) 368 - 375 2011.9

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE INC

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 C alpha in osteoblast differentiation. Administration of 1 nM OA to the calvarial region in mice increased bone mineral density, as shown by mu CT, while histomorphological analysis showed an increase in mineral apposition and bone thickness in the same region. In addition, treatment with 1 nM 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 C alpha 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 C alpha in osteoblast differentiation, we constructed PP2A knock-down cells by infecting MC3T3-E1 cells with a lentivirus expressing shRNA specific for the PP2A C alpha. Accordingly, the silencing of PP2A Cot in MC3T3-E1 cells dramatically increased osteoblast differentiation and mineralization, which were accompanied with expressions of Osterix, lisp, and OCN. Our data indicate that PP2A C alpha plays an important role in the regulation of bone formation and osteoblast differentiation through the bone-related genes. (C) 2011 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bone.2011.06.004

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

Hirohiko Okamura, Kaya Yoshida, Kazuhiko Ochiai, Tatsuji Haneji

Bone Vol.49 ( No.3 ) 368 - 375 2011.6

Language：English Publishing type：Research paper (scientific journal)

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.

DOI： 10.1016/j.bone.2011.06.004

Intermedilysin induces EGR-1 expression through calcineurin/NFAT pathway in human cholangiocellular carcinoma cells Reviewed

Heni Susilowati, Hirohiko Okamura, Katsuhiko Hirota, Masayuki Shono, Kaya Yoshida, Keiji Murakami, Atsushi Tabata, Hideaki Nagamune, Tatsuji Haneji, Yoichiro Miyake

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 404 ( 1 ) 57 - 61 2011.1

Language：English Publishing type：Research paper (scientific journal) Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE

Intermedilysin (ILY) is a cholesterol-dependent cytolysin produced by Streptococcus intermedius, which is associated with human brain and liver abscesses. Although intrahepatic bile duct cells play a valuable role in the pathogenesis of liver abscess, the molecular mechanism of ILY-treated intrahepatic bile duct cells remains unknown. In this study, we report that ILY induced a nuclear accumulation of intracellular calcium ([Ca(2+)]i) in human cholangiocellular cells HuCCT1. We also demonstrate that 10 ng/ml ILY induced NFAT1 dephosphorylation and its nuclear translocation in HuCCT1 cells. In contrast to the result that ILY induced NF-kappa B translocation in human hepatic HepG2 cells, ILY did not affect NF-kappa B localization in HuCCT1 cells. Dephosphorylation and nuclear translocation of NFAT1 caused by ILY were prevented by [Ca(2+)]i calcium chelator, BAPTA/AM, and calcineurin inhibitors, cyclosporine A and tacrolimus. ILY induced early growth response-1 (EGR-1) expression and it was inhibited by the pre-treatment with cyclosporine A, indicating that the calcineurin/NFAT pathway was involved in EGR-1 expression in response to ILY. ILY-induced calcineurin/NFAT1 activation and sequential EGR-1 expression might be related to the pathogenesis of S. intermedius in human bile duct cells. (C) 2010 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2010.11.057

Intermedilysin induces EGR-1 expression through calcineurin/NFAT pathway in human cholangiocellular carcinoma cells. Reviewed

Heni Susilowati, Hirohiko Okamura, Katsuhiko Hirota, Masayuki Shono, Kaya Yoshida, Keiji Murakami, Atsushi Tabata, Hideaki Nagamune, Tatsuji Haneji, Yoichiro Miyake

Biochemical and Biophysical Research Communications Vol.404 ( No.1 ) 57 - 61 2011

Language：English Publishing type：Research paper (scientific journal)

(Intermedilysin (ILY) is a cholesterol-dependent cytolysin produced by Streptococcus intermedius, which is associated with human brain and liver abscesses. Although intrahepatic bile duct cells play a valuable role in the pathogenesis of liver abscess, the molecular mechanism of ILY-treated intrahepatic bile duct cells remains unknown. In this study, we report that ILY induced a nuclear accumulation of intracellular calcium ([Ca(2+)]i) in human cholangiocellular cells HuCCT1. We also demonstrate that 10 ng/ml ILY induced NFAT1 dephosphorylation and its nuclear translocation in HuCCT1 cells. In contrast to the result that ILY induced NF-B translocation in human hepatic HepG2 cells, ILY did not affect NF-B localization in HuCCT1 cells. Dephosphorylation and nuclear translocation of NFAT1 caused by ILY were prevented by [Ca(2+)]i calcium chelator, BAPTA/AM, and calcineurin inhibitors, cyclosporine A and tacrolimus. ILY induced early growth response-1 (EGR-1) expression and it was inhibited by the pre-treatment with cyclosporine A, indicating that the calcineurin/NFAT pathway was involved in EGR-1 expression in response to ILY. ILY-induced calcineurin/NFAT1 activation and sequential EGR-1 expression might be related to the pathogenesis of S. intermedius in human bile duct cells.)

DOI： 10.1016/j.bbrc.2010.11.057

Negative regulation of TIMP1 is mediated by transcription factor TWIST1 Reviewed

Hirohiko Okamura, Kaya Yoshida, Tatsuji Haneji

INTERNATIONAL JOURNAL OF ONCOLOGY 35 ( 1 ) 181 - 186 2009.7

Language：English Publishing type：Research paper (scientific journal) Publisher：SPANDIDOS PUBL LTD

TWIST1 is involved in tumor invasion and metastasis by promoting epithelial-to-mesenchymal transition. However, the molecular target of TWIST1 involving this mechanism is poorly understood. To identify the TWIST-regulated genes, we established the Saos-2 cells stably expressing TWIST1 gene by transfecting TWIST1 cDNA into the cells and performed a differential display approach by using annealing control primers. Here, we report that one of the genes that were down-regulated in TWIST1 expressing cells is predicted to be TIMP1. TIMP1 has been reported as the naturally occurring specific inhibitor of matrix metalloproteinases (MMPs). Overexpression of TWIST1 gene suppressed the expression of TIMP1 mRNA but had no effect on TIMP2 and MMP-2 expression, as determined by semi-quantitative RT-PCR. We showed that TWIST1 was up-regulated in SCCBHY cells, which have a strong capacity of invasion into mandibular bone compared with SCCHN cells. Our present results suggest that TWIST1 is involved in tumor invasion by regulating the expression of TIMP1.

DOI： 10.3892/ijo_00000327

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