Ser9 phosphorylation of mitochondrial GSK-3beta is a primary mechanism of cardiomyocyte protection by erythropoietin against oxidant-induced apoptosis.
The aim of this study was to determine the role of GSK-3beta in cardiomyocyte protection afforded by erythropoietin (EPO) against oxidant stress-induced apoptosis. Treatment with EPO (10 units/ml) induced Ser473 phosphorylation of Akt and Ser9 phosphorylation of GSK-3beta and significantly reduced the proportion of apoptotic H9c2 cardiomyocytes after exposure to H2O2 from 38.3 +/- 2.7% to 26.0 +/- 2.9%. This protection was not detected in cells transfected with constitutively active GSK-3beta (S9A), which lacks Ser9 for inhibitory phosphorylation. The antiapoptotic effect of EPO was mimicked completely by GSK-3beta knockdown using small interfering RNA and partly by the transfection with kinase-deficient GSK-3beta (K85R). The level of colocalization of intracellular GSK-3beta with mitochondria assessed by enhanced green fluorescent protein-tagged GSK-3beta or immunocytochemistry was not altered by EPO treatment. However, EPO increased the level of Ser9-phospho-GSK-3beta colocalized with mitochondria by 50% in a phosphatidylinositol 3-kinase-dependent manner. Mitochondrial translocation of Bcl-2-associated X protein (BAX) after exposure to H2O2 was inhibited by EPO pretreatment and by GSK-3beta knockdown. These results suggest that the suppression of GSK-3beta activity by Akt-mediated Ser9 phosphorylation in the mitochondria affords cardiomyocytes tolerance against oxidant-induced apoptosis, possibly by inhibiting the access of BAX to the mitochondria.
Ohori
Katsuhiko
K
Second Department of Internal Medicine, Sapporo Medical University School of Medicine, South-1 West-16, Sapporo 060-8543, Japan.
Miura
Tetsuji
T
Tanno
Masaya
M
Miki
Takayuki
T
Sato
Takahiro
T
Ishikawa
Satoko
S
Horio
Yoshiyuki
Y
Shimamoto
Kazuaki
K
D000818:Animals; D000975:Antioxidants / Q000378:metabolism*; D017209:Apoptosis; D002460:Cell Line; D019610:Cytoprotection; D004921:Erythropoietin / Q000378:metabolism*; D038362:Glycogen Synthase Kinase 3 / Q000235:genetics / Q000378:metabolism*; D000071679:Glycogen Synthase Kinase 3 beta; D006861:Hydrogen Peroxide / Q000633:toxicity; D008929:Mitochondria, Heart / Q000201:enzymology* / Q000473:pathology; D032383:Myocytes, Cardiac / Q000201:enzymology* / Q000473:pathology; D018384:Oxidative Stress; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation; D021381:Protein Transport; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D051381:Rats; D017467:Receptors, Erythropoietin / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012694:Serine; D014162:Transfection; D051028:bcl-2-Associated X Protein / Q000378:metabolism
D000975:Antioxidants; C494828:Bax protein, rat; D019253:Proto-Oncogene Proteins c-bcl-2; D034741:RNA, Small Interfering; D017467:Receptors, Erythropoietin; D011993:Recombinant Fusion Proteins; D051028:bcl-2-Associated X Protein; D004921:Erythropoietin; D012694:Serine; D006861:Hydrogen Peroxide; D019869:Phosphatidylinositol 3-Kinases; D000071679:Glycogen Synthase Kinase 3 beta; C000605508:Gsk3b protein, rat; D051057:Proto-Oncogene Proteins c-akt; D038362:Glycogen Synthase Kinase 3