Transcriptional induction of heme oxygenase-1 gene expression by okadaic acid in primary rat hepatocyte cultures.
Heme oxygenase (HO) catalyzes the rate-limiting enzymatic step of heme degradation and regulates the cellular heme content. The gene expression of the inducible isoform of HO, HO-1, is up-regulated in response to various agents causing oxidative stress. To investigate the regulatory role of protein phosphatases in the hepatic regulation of HO-1 gene expression, primary cultures of rat hepatocytes were treated with okadaic acid (OA), which specifically inhibits the serine threonine protein phosphatases 1 and 2A. Both protein synthesis and mRNA expression of HO-1 were induced by OA in cultured hepatocytes, but not in cultured tissue macrophages of rat liver. The HO-1 mRNA induction by OA occurred in a time- and concentration-dependent manner. Simultaneous treatment with OA plus dibutyryl cAMP caused a synergistic up-regulation of steady-state levels of HO-1 mRNA, and the specific protein kinase A inhibitor KT5720 markedly reduced the OA-dependent HO-1 mRNA induction. In contrast, the dibutyryl cAMP-dependent induction of the phosphoenolpyruvate carboxykinase mRNA expression and enzyme activity was inhibited by simultaneous treatment with OA in hepatocytes. The induction of the HO-1 gene expression by OA was transcriptional as determined by studies with actinomycin D, nuclear run-off assay, and measurement of the half-life of HO-1 mRNA. Luciferase reporter constructs containing DNA sequences of the rat HO-1 promoter 5'-flanking region were up-regulated by OA in transiently transfected hepatocytes. Mutation of the cAMP response element/activator protein-1 (-665/-654) site obliterated the OA-dependent induction, suggesting that this element is involved in the transcriptional induction of the rat HO-1 gene by OA.
Immenschuh
S
S
Institut für Klinische Chemie und Pathobiochemie der Justus-Liebig-Universität Giebetaen, Giebetaen, Germany. simmens@gwdg.de
Hinke
V
V
Katz
N
N
Kietzmann
T
T
D000818:Animals; D000242:Cyclic AMP / Q000378:metabolism; D004790:Enzyme Induction; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D006419:Heme Oxygenase (Decyclizing) / Q000096:biosynthesis / Q000235:genetics*; D051547:Heme Oxygenase-1; D066298:In Vitro Techniques; D008099:Liver / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D008297:Male; D019319:Okadaic Acid / Q000494:pharmacology*; D011401:Promoter Regions, Genetic; D011493:Protein Kinase C / Q000096:biosynthesis; D012333:RNA, Messenger / Q000187:drug effects / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D015533:Transcriptional Activation; D014162:Transfection
D004791:Enzyme Inhibitors; D012333:RNA, Messenger; D019319:Okadaic Acid; D000242:Cyclic AMP; D006419:Heme Oxygenase (Decyclizing); D051547:Heme Oxygenase-1; D011493:Protein Kinase C