Transcriptional Induction of Heme Oxygenase-1 Gene Expression by Okadaic Acid in Primary Rat Hepatocyte Cultures
- 1Institut für Klinische Chemie und Pathobiochemie der Justus-Liebig-Universität Gieβen, Gieβen (S.I., V.H., N.K.); and 2Institut für Biochemie und Molekulare Zellbiologie der Georg-August-Universität Göttingen, Göttingen, Germany (T.K.)
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.
Footnotes
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Send reprint requests to: Dr. Stephan Immenschuh, Zentrum Innere Medizin, Abteilung Gastroenterologie und Endokrinologie, Georg-August-Universität Göttingen, Robert-Koch-Str. 40, 37075 Göttingen, Germany. E-mail:simmens{at}gwdg.de
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↵1 Present address: Zentrum Innere Medizin, Abteilung Gastroenterologie und Endokrinologie, Georg-August-Universität Göttingen, Robert Koch Str. 40, 37075 Göttingen; Germany.
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This work was supported by grants from the Deutsche Forschungsgemeinschaft Im 2–1 (S.I.) and SFB 402 A1 (T.K.).
- Abbreviations:
- HO
- heme oxygenase
- ActD
- actinomycin D
- AP-1
- activator protein-1
- Bt2cAMP
- dibutyryl cAMP
- CAT
- chloramphenicol acetytransferase
- CHX
- cycloheximide
- CRE
- cAMP response element
- CREB
- CRE-binding protein
- CYP
- cytochrome P450
- ERK
- extracellular signal-regulated kinase
- GAPDH
- glyceraldehyde-3-phosphate dehydrogenase
- iNOS
- inducible nitric-oxide synthase
- OA
- okadaic acid
- PCK
- phosphoenolpyruvate carboxykinase
- PK
- protein kinase
- PCR
- polymerase chain reaction
- PKA
- cAMP-dependent protein kinase
- PKG
- cGMP-dependent protein kinase
- PP
- protein phosphatase
- rRNA
- ribosomal RNA
- TF
- transcription factor
- PMSF
- phenylmethylsulfonyl fluoride
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- Received May 13, 1999.
- Accepted November 19, 1999.
- The American Society for Pharmacology and Experimental Therapeutics
