Faculty Profiles - OKAMURA Hirohiko

Papers - OKAMURA Hirohiko

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PKR-mediated degradation of STAT1 regulates osteoblast differentiation Reviewed

Kaya Yoshida, Hirohiko Okamura, Bruna Rabelo Amorim, Daisuke Hinode, Hideo Yoshida, Tatsuji Haneji

EXPERIMENTAL CELL RESEARCH 315 ( 12 ) 2105 - 2114 2009.7

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

The double-stranded RNA-dependent protein kinase (PI(R) plays a critical role in various biological responses including antiviral defense, cell differentiation, apoptosis, and tumorigenesis. In this study, we investigated whether PKR could affect the post-translational modifications of STAT1 protein and whether these modifications regulate osteoblast differentiation. We demonstrated that PKR was necessary for the ubiquitination of STAT1 protein. The expressions of bone-related genes such as type I Collagen, integrin binding sialoprotein, osteopontin, and osterix were suppressed in osteoblasts lacking PKR activity. In contrast, the expressions of interleukin-6 and matrix metalloproteinases 8 and 13 increased in PKR-mutated osteoblasts. The expression and degradation of STAT1 protein were regulated by PKR in a SLIM-dependent pathway. Inhibition of SLIM by RNA interference resulted in the decreased activity of Runx2 in osteoblasts. Stimulation of interleukin-6 expression and suppression of alkaline phosphatase activity were regulated through by SLIM-dependent pathway. However, expressions of bone-related genes and MMPs were regulated by SLIM-independent pathway. Our present results suggest that the aberrant accumulation of STAT1 protein induced by loss of PKR regulate osteoblast differentiation through both SLIM/STAT1-dependent and -independent pathways. (C) 2009 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.yexcr.2009.02.003

PKR-mediated degradation of STAT1 regulates osteoblast differentiation Reviewed

Kaya Yoshida, Hirohiko Okamura, Bruna Rabelo Amorim, Daisuke Hinode, Hideo Yoshida, Tatsuji Haneji

Experimental Cell Research Vol.315 ( No.12 ) 2105 - 2114 2009.7

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

The double-stranded RNA-dependent protein kinase (PKR) plays a critical role in various biological responses including antiviral defense, cell differentiation, apoptosis, and tumorigenesis. In this study, we investigated whether PKR could affect the post-translational modifications of STAT1 protein and whether these modifications regulate osteoblast differentiation. We demonstrated that PKR was necessary for the ubiquitination of STAT1 protein. The expressions of bone-related genes such as type I collagen, integrin binding sialoprotein, osteopontin, and osterix were suppressed in osteoblasts lacking PKR activity. In contrast, the expressions of interleukin-6 and matrix metalloproteinases 8 and 13 increased in PKR-mutated osteoblasts. The expression and degradation of STAT1 protein were regulated by PKR in a SLIM-dependent pathway. Inhibition of SLIM by RNA interference resulted in the decreased activity of Runx2 in osteoblasts. Stimulation of interleukin-6 expression and suppression of alkaline phosphatase activity were regulated through by SLIM-dependent pathway. However, expressions of bone-related genes and MMPs were regulated by SLIM-independent pathway. Our present results suggest that the aberrant accumulation of STAT1 protein induced by loss of PKR regulate osteoblast differentiation through both SLIM/STAT1-dependent and -independent pathways.

DOI： 10.1016/j.yexcr.2009.02.003

Calcineurin regulates phosphorylation status of transcription factor osterix Reviewed

Hirohiko Okamura, Bruna Rabelo Amorim, Jie Wang, Kaya Yoshida, Tatsuji Haneji

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 379 ( 2 ) 440 - 444 2009.2

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

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. (C) 2008 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2008.12.094

Calcineurin regulates phosphorylation satus of transcriptionfactor osterix Reviewed

Hirohiko Okamura, Bruna Rabelo Amorim, Jie Wang, Kaya Yoshida, Tatsuji Haneji

Biochemical and Biophysical Research Communications Vol.379 ( No.2 ) 440 - 444 2009

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

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.

DOI： 10.1016/j.bbrc.2008.12.094

Histone H1.2 is translocated to mitochondria and associates with Bak in bleomycin-induced apoptotic cells Reviewed

Hirohiko Okamura, Kaya Yoshida, Bruna Rabelo Amorim, Tatsuji Haneji

Journal of Cellular Biochemistry Vol.103 ( No.5 ) 1488 - 1496 2008.4

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

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 (gamma-H2AX). Incubation of SCCTF cells for 48 h with 10 microM 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.

DOI： 10.1002/jcb.21537

Histone H1.2 is translocated to mitochondria and associates with Bak in bleomycin-induced apoptotic cells Reviewed

Hirohiko Okamura, Kaya Yoshida, Bruna Rabelo Amorim, Tatsuji Haneji

JOURNAL OF CELLULAR BIOCHEMISTRY 103 ( 5 ) 1488 - 1496 2008.4

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

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 (gamma-H2AX). Incubation of SCCTF cells for 48 h with 10 mu M 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.

DOI： 10.1002/jcb.21537

The transcriptional factor Osterix directly interacts with RNA helicase A. Invited Reviewed

Amorim BR, Okamura H, Yoshida K, Wang J, Qiu LH, Haneji T

Ocean Papers & Printers, New Delhi 216 - 222 2008

Cobalt chloride induces apoptosis and zinc chloride suppresses cobalt-induced apoptosis by Bcl-2 expression in human submandibular gland HSG cells Reviewed

Kazumi Akita, Hirohiko Okamura, Kaya Yoshida, Hiroyuki Morimoto, Hiroaki Ogawa-Iyehara, Tatsuji Haneji

INTERNATIONAL JOURNAL OF ONCOLOGY 31 ( 4 ) 923 - 929 2007.10

Language：English Publishing type：Research paper (scientific journal) Publisher：PROFESSOR D A SPANDIDOS

To determine the effects of cobalt chloride on human submandibular gland cells, HSG cells were exposed to various concentrations of cobalt chloride. Cobalt chloride induced cytotoxicity and cell death in HSG cells as determined by phase-contrast microscopy and WST-1 cell viability assay. By using the Hoechst 33342 staining, marked nuclear condensation and fragmentation of chromatin were observed in cobalt chloride-treated cells. Cobalt chloride induced DNA ladder formation in HSG cells in both dose- and time-dependent manner with maximal effect at a concentration of 0.5 mM and 48 h, respectively. Cobalt chloride inhibited the expression of both Bcl-2 protein and mRNA in dose- and time-dependent manner. Zinc chloride recovered the cobalt-suppressed Bcl-2 expression and protected against cobalt-induced apoptosis in HSG cells. Our results show that the pathway of the apoptosis in HSG cells is regulated by cobalt chloride and zinc chloride. Our results also indicate that cobalt-induced apoptotic steps in HSG cells are related to the production of Bcl-2 protein.

Calyculin A induces apoptosis and stimulates phosphorylation of p65NF-kappa B in human osteoblastic osteosarcoma MG63 cells Reviewed

Hiroaki Tanaka, Kaya Yoshida, Hirohiko Okamura, Hiroyuki Morimoto, Toshihiko Nagata, Tatsuji Haneji

INTERNATIONAL JOURNAL OF ONCOLOGY 31 ( 2 ) 389 - 396 2007.8

Language：English Publishing type：Research paper (scientific journal) Publisher：PROFESSOR D A SPANDIDOS

Previously, we reported that okadaic acid, a specific inhibitor of serine/threonine protein phosphatases, induced apoptosis in human osteoblastic cells. However, it is not clear whether calyculin A, another inhibitor of protein phosphatases, would induce apoptosis in human osteoblastic cells and if so, which mechanisms are involved and whether the phosphorylation status of NF-kappa B could be affected by the treatment with calyculin A. In this report, we demonstrate that calyculin A induced apoptosis in MG63 cells, as judged by WST-8 assay, nuclear fragmentation, and DNA ladder formation. Expression of PTEN, FasL, and FasR mRNA was stimulated by calyculin A treatment in MG63 cells. Calyculin A also enhanced the phosphorylation level of NF-kappa B, as judged from the results of Western blot analysis and an in vitro dephosphorylation assay. Western blot analysis with antiphospho-p65NF-kappa B antibody disclosed that the NF-kappa B was phosphorylated on serine 536 in cytosol and translocated into nucleus with calyculin A-treatment. The phosphorylation status of p65NF-kappa B was further confirmed by using the phosphorylation site-mutated p65NF-kappa B gene transfected into HEK293 cells. Unlike TNF-alpha, calyculin A treatment did not degraded I kappa B alpha within 10 min, while it degraded I kappa B alpha at 2-h treatment. Our findings indicate that calyculin A elicit phosphorylation of NF-kappa B on serine 536 in MG63 cells, resulting in the translocation of phospho-NF-kappa B to the nucleus, thereby promoting transcriptional activity of NF-kappa B-related genes.

Differential expression of protein phosphatase type 1 isotypes and nucleolin during cell cycle arrest Reviewed

Hiroyuki Morimoto, Akiko Ozaki, Hirohiko Okamura, Kaya Yoshida, Bruna Rabelo Amorim, Hiroaki Tanaka, Seiichiro Kitamura, Tatsuji Haneji

CELL BIOCHEMISTRY AND FUNCTION 25 ( 4 ) 369 - 375 2007.7

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

In the present study, we examined the expression and cytolocalization of protein phosphatase type 1 (PPI) isoforms and nucleolin in human osteoblastic cell line MG63 cells at two boundaries in the cell cycle. We treated MG63 cells with hydroxyurea and nocodazole to arrest the cells at the G(1)/S and G(2)/M boundaries, respectively. As judged from the results of Western blot analysis, PPI isoforms were expressed differently at each boundary of the cell cycle. Nucleolin was also shown to have a different expression pattern at each boundary. In the hydroxyurea-treated cells, nucleolus-like bodies were bigger in size and decreased in number compared with those in asynchronized cells. However, the subcellular localization of PPIs and nucleolin was not changed. Anti-nucleolin antibody interacted with 1 10-kDa and 95-kDa proteins present in asynchronized cells and in the cells treated with hydroxyurea. Treatment of the cells with nocodazole decreased the level of the 95-kDa form of nucleolin. In the nocodazole-treated cells, it was impossible to distinguish the distribution of each protein. The phosphorylation status of nucleolin in the cell cycle arrested samples was examined by 2D-IEF-PAGE followed by Western blot analysis. In the case of asynchronized cells or hydroxyurea-treated ones, nucleolin was located at a basic isoelectric point (dephosphorylated status); whereas in the G2/M arrest cells, the isoelectric point of nucleolin shifted to an acidic status, indicating that nucleolin was phosphorylated. The present results indicate that PPI and nucleolin were differently expressed at G(1)/S and G2/M boundaries of the cell cycle and acted in a different fashion during cell-cycle progression. Copyright (C) 2005 John Wiley & Sons, Ltd.

DOI： 10.1027/cbf.1300

Calyculin A stimulates the expression of TNF-alpha mRNA via phosphorylation of Akt in mouse osteoblastic MC3T3-E1 cells Reviewed

Lihong Qiu, Kaya Yoshida, Bruna Rabelo Amorim, Hirohiko Okamura, Tatsuji Haneji

MOLECULAR AND CELLULAR ENDOCRINOLOGY 271 ( 1-2 ) 38 - 44 2007.6

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER IRELAND LTD

Intracellular phosphatase activity has been recognized to play a central role in signal transduction. In the present study, we investigated the effects of calyculin A, an inhibitor of protein phosphatases, on the expression of TNF-alpha mRNA and the possible signaling pathways in mouse osteoblastic MC3T3-E1 cells. The result of semiquantitative RT-PCR showed that calyculin A increased the expression of TNF-alpha mRNA in MC3T3-E1 cells. Pre-treatment of LY294002 and Wortmannin, inhibitors of PI3K, inhibited the calyculin A-stimulated TNF-alpha mRNA expression. Western blot result disclosed that calyculin A increased the phosphorylation status of Akt at Ser473. However, U0126 and S8203580, specific inhibitor of MEK1/2 and p38MAPK, respectively, had no effect on calyculin A-stimulated expression of TNF-alpha mRNA. BAY11-7085 and CAPE, inhibitors of NF-kappa B activity, did not alter the calyculin A-stimulated TNF-alpha mRNA expression. Indirect immunofluorescent study confirmed that NF-kappa B was not translocated to the nucleus by calyculin A treatment. Our present results suggest that inhibition of phosphatase activity by calyculin A stimulate the phosphorylation of Akt at Ser473 by PI3K/Akt signaling pathway, resulting in the expression TNF-alpha mRNA. (c) 2007 Elsevier Ireland Ltd. All rights reserved.

DOI： 10.1016/j.mce.2007.03.005

The transcriptional factor Osterix directly interacts with RNA helicase A Reviewed

Bruna Rabelo Amorim, Hirohiko Okamura, Kaya Yoshida, Lihong Qiu, Hiroyuki Morimoto, Tatsuji Haneji

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 355 ( 2 ) 347 - 351 2007.4

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

Osterix is an osteoblast-specific transcriptional factor, required for bone formation and osteoblast differentiation. Here, we identified new Osterix interacting factors by using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Among the candidates, RNA helicase A was identified to interact with Osterix. To determine the interaction of Osterix with RNA helicase A, immunoprecipitation assay was performed. Western analysis confirmed the association between Osterix and RNA helicase A. Immunocytochemical analysis also showed that Osterix and RNA helicase A were co-localized in HEK 293 cells. Our data suggest that RNA helicase A might be a component of Osterix regulation. (c) 2007 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2007.01.150

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

Hirohiko Okamura, Kaya Yoshida, Eiko Sasaki, Lihong Qiu, Bruna Rabelo Amorim, Hiroyuki Morimoto, Tatsuji Haneji

CELL BIOLOGY INTERNATIONAL 31 ( 2 ) 119 - 125 2007.2

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

PTEN is a tumor suppressor gene encoding a phosphatase, and it negatively regulates cell survival mediated by the phosphomositol 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 mu g/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. (c) 2006 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.cellbi.2006.09.014

口腔扁平上皮癌細胞の薬剤耐性とアポトーシス ~ 転写調節因子の役割 ~ Reviewed

岡村裕彦

四国歯学会雑誌 19 171 - 17 2007

Role of transcription factors in multidrug resistance and apoptosis. Invited Reviewed

Okamura H, Haneji T

Dentistry in Japan 43 ( 1 ) 1 - 6 2007

Okadaic acid induces phosphorylation of p65NF-kappa B on serine 536 and activates NF-kappa B transcriptional activity in human osteoblastic MG63 tells Reviewed

Akiko Ozaki, Hiroyuki Morimoto, Hiroaki Tanaka, Hirohiko Okamura, Kaya Yoshida, Bruna Rabelo Amorim, Tatsuji Haneji

JOURNAL OF CELLULAR BIOCHEMISTRY 99 ( 5 ) 1275 - 1284 2006.12

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

Nuclear factor-kappa B (NF-kappa B) is an essential. transcription factor in the control of expression of genes involved in coil growth, differentiation, inflammation, and neoplastic transformation. Previously, we reported that okadaic acid (OA), which is a specific inhibitor of serine/threonine protein phosphatases, induced apoptosis in cells of human osteosarcoma cell line MG63. However, to date, it is hot clear whether the phosphorylation status of NF-kappa B could be affected by the treatment with OA. in this report, we demonstrate that treatment of MG63 cells with OA enhanced the phosphorylation level of NF-kappa B, as judged from the results of Western blot analysis and a protein phosphatase dephosphorylation assay. The phosphorylation level of NF-kappa B was enhanced in both time- and dose-dependent manners. In the cells treated with 100nM OA for 3 h, consequential translocation of NF-kappa B from the cytosol to the nucleus occurred. Western blotting experiments with an anti-phospho-p65NF-kappa B antibody disclosed that the NF-kappa B was phosphorylated on serine 536. Furthermore, OA stimulated the transcriptional activity of NF-kappa B in MG63 cells, as judged from the results of a luciferase assay. Our findings indicate that OA elicit phosphorylation of NF-kappa B on serine 536 in MG63 cells, resulting in the translocation of phospho-NF-kappa B to the nucleus, thereby promoting transcriptional activity of genes.

DOI： 10.1002/jcb.20873

Double-stranded RNA-dependent protein kinase is required for bone calcification in MC3T3-E1 cells in vitro Reviewed

K Yoshida, H Okamura, BR Amorim, A Ozaki, H Tanaka, H Morimoto, T Haneji

EXPERIMENTAL CELL RESEARCH 311 ( 1 ) 117 - 125 2005.11

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

In this study, we demonstrated that double-stranded RNA-dependent protein kinase (PKR) is required for the calcification of osteoblasts via the signal transducers and activators of transcription 1 alpha(STAT1 alpha) signaling in vitro. A dominant-negative mutant PKR cDNA, in which the amino acid lysine at 296 was replaced with arginine and which does not have catalytic activity, was transfected into mouse ostcoblastic MC3T3-E1 cells, thereby, we established cells that stably expressed the PKR mutant gene (PKR-K/R). Phosphorylation of PKR was not stimulated by polyinosic-polycytidylic acid in the mutant cells. The PKR-K/R mutant cells exhibited up-regulated cell growth and had low alkaline phosphatase (ALP) activity. The PKR-K/R mutant cells were not able to form bone nodules in vitro. In the PKR-K/R mutant cells, runt-related gene 2 (Runx2)-mediated transcription decreased compared with the levels in the control cells. The expression of STAT1 alpha protein increased and the protein was translocated to the nucleus in the PKR-K/R mutant cells. When the expression of STAT1 alpha a protein in PKR Mutant cells was suppressed using RNAi, the activity of Runx2-mediated transcription recovered to the control level. Our results indicate that PKR is a stimulator of Runx2 transcription and is a negative modulator of STAT1 alpha expression. Our findings also suggest that PKR plays important roles in the differentiation and calcification of osteoblasts by modulating STAT1 alpha and/or Runx2 expression. (c) 2005 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.yexcr.2005.09.006

Okadaic acid induces tyrosine phosphorylation of I kappa B alpha that mediated by PKR pathway in human osteoblastic MG63 cells Reviewed

H Morimoto, A Ozaki, H Okamura, K Yoshida, S Kitamura, T Haneji

MOLECULAR AND CELLULAR BIOCHEMISTRY 276 ( 1-2 ) 211 - 217 2005.8

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

Treatment of human osteosarcoma cell line MG63 cells with okadaic acid stimulated phosphorylation of I kappa B alpha, as judged from the results of Western blot analysis and a lambda protein phosphatase dephosphorylation assay. The stimulated phosphorylation of I kappa B alpha was both time- and dose-dependent. The phosphorylation sites of I kappa B alpha were taken to be tyrosine residues because the anti-phospho-tyrosine antibody bound to the samples immunoprecipitated with the anti-I kappa B alpha antibody. In the cells treated with 100 nM okadaic acid consequential translocation of NF-kappa B p65 from the cytosol to the nucleus occurred. Double-stranded RNA-dependent protein kinase (PKR) is a player in the cellular antiviral response and is involved in transcriptional stimulation through activation of NF-kappa B. We investigated the functional relationship between PKR and I kappa B alpha phosphorylation by constructing MG63 PKR K/R cells that produced a catalytically inactive mutant PKR. NF-kappa B p65 was detected in the nucleus of these cells, even in the unstimulated cells. Although I kappa B alpha was degraded phosphorylation of eIF-2 alpha, a substrate of PKR, did not occur in the mutant cells treated with okadaic acid. Our results suggest that okadaic acid-induced tyrosine phosphorylation of I kappa B alpha was mediated by PKR kinase activity, thus indicating the involvement of this kinase in the control mechanism governing the activation of NF-kappa B.

DOI： 10.1007/s11010-005-4440-y

PTEN expression elicited by EGR-1 transcription factor in calyculin a-induced apoptotic cells Reviewed

H Okamura, K Yoshida, H Morimoto, T Haneji

JOURNAL OF CELLULAR BIOCHEMISTRY 94 ( 1 ) 117 - 125 2005.1

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

PTEN is a tumor suppressor gene encoding a phosphatase that negatively regulates cell survival mediated by the PI3-kinase-Akt pathway. The gene for transcription factor EGR-1 is an early response gene essential for cellular growth, proliferation, and differentiation. Protein phosphatase inhibitors including calyculin A and okadaic acid are potent inducers of apoptosis in several cell lines; however, the molecular mechanisms underlying their action are unknown. The purpose of this study was to examine the expression of PTEN and EGR-1 and the phosphorylation status of EGR-1 and Akt in calyculin A-treated human squamous carcinoma cells (SCCTF). Phosphorylation of EGR-1 and upregulation of PTEN expression were observed to occur in SCCTF cells treated with calyculin A in time- and dose-dependent fashions. The level of phosphorylated Akt decreased as the expression of PTEN protein increased in the calyculin A-treated SCCTF cells. Calyculin A-stimulated expression of FGR-1 and PTEN might be p53 independent, because the expression of them was also detected in p53-null Saos-2 cells. RNA interference using double-stranded RNA specific for the EGR-1 gene inhibited not only EGR-1 expression but also PTEN expression in SCCTF cells treated or not with calyculin A. Calyculin A induced nuclear fragmentation and chromatin condensation in SCCTF cells. The present results suggest that the level of PTEN expression and the phosphorylation status of Akt were associated with apoptosis induced by calyculin A. These observations also support the view that EGR-1 regulates PTEN expression in the initial steps of the apoptotic pathway. (C) 2004 Wiley-Liss, Inc.

DOI： 10.1002/jcb.20283

Okadaic acid induces apoptosis through double-stranded RNA-dependent protein kinase/eukaryotic initiation factor-2 alpha pathway in human osteoblastic MG63 cells Reviewed

H Morimoto, H Okamura, K Yoshida, S Kitamura, T Haneji

JOURNAL OF BIOCHEMISTRY 136 ( 4 ) 433 - 438 2004.10

Language：English Publishing type：Research paper (scientific journal) Publisher：JAPANESE BIOCHEMICAL SOC

Double-stranded RNA-dependent protein kinase (PKR) is a participant in the cellular antiviral response and phosphorylates the a-subunit of eukaryotic translation initiation factor 2alpha (eIF-2alpha) to block protein synthesis. Treatment of human osteosarcoma cell line MG63 cells with a serine and threonine protein phosphatase inhibitor, okadaic acid, at the concentration of 100 nM, but not at 20 nM, induced apoptosis. To investigate the functional relationship between phosphatases and apoptosis, we examined the phosphorylation levels of PKR and eIF-2a by Western blot analysis. During treatment of cells with it at the higher concentration (100 nM), okadaic acid increased the level of phosphorylated PKR in MG63 cells, this kinase phosphorylating eIF-2alpha. However, at the lower concentration (20 nM), okadaic acid did not affect the level of phosphorylated PKR. In the cells treated with 100 nM okadaic acid, activation of NF-kappaB also occurred. Even though inhibition of translation occurred simultaneously in MG63 cells, the expression of pro-apoptotic proteins Fas and Bax was not affected by 100 nM okadaic acid in these cells. We concluded that the inhibition of translation decreased anti-apoptotic protein expression, thus resulting in apoptosis. Our results also suggest that the inhibition of the protein phosphatase activity by okadaic acid induced apoptosis in MG63 cells through PKR and eIF-2alpha.

DOI： 10.1093/jb/mvh144

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