Transcriptional Induction of Heme Oxygenase-1 Gene Expression by Okadaic Acid in Primary Rat Hepatocyte Cultures

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Abstract
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.