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 degr adation and regulates the cellular heme content. The gene expression of the inducible isoform of HO, HO-1, is up-regulated in response to various agen ts causing oxidative stress. To investigate the regulatory role of protein phosphatases in the hepatic regulation of HO-1 gene expression, primary cul tures of rat hepatocytes were treated with okadaic acid (OA), which specifi cally inhibits the serine threonine protein phosphatases 1 and 2A. Both pro tein synthesis and mRNA expression of HO-1 were induced by OA in cultured h epatocytes, but not in cultured tissue macrophages of rat liver. The HO-1 m RNA 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 k inase A inhibitor KT5720 markedly reduced the OA-dependent HO-1 mRNA induct ion. In contrast, the dibutyryl cAMP-dependent induction of the phosphoenol pyruvate carboxykinase mRNA expression and enzyme activity was inhibited by simultaneous treatment with OA in hepatocytes. The induction of the HO-1 g ene expression by OA was transcriptional as determined by studies with acti nomycin 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 trans fected 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.