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J. Biol. Chem., Vol. 278, Issue 40, 38860-38869, October 3, 2003

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Pharmacogenomic Analysis of Mechanisms Mediating Ethanol Regulation of Dopamine -Hydroxylase^*

Sajida Hassan , Bao Duong  **, Kwang-Soo Kim ¶ and Michael F. Miles  ||

From the Departments of Pharmacology Toxicology and Neurology and the Center for Study of Biological Complexity, Virginia Commonwealth University, Richmond, Virginia 23298, the ^**Ernest Gallo Clinic and Research Center, University of California at San Francisco, Emeryville, CA 94608, and the ^¶Molecular Neurobiology Laboratory, McLean Hospital, Harvard Medical School, Belmont, Massachusetts 02178

We previously showed that ethanol regulates dopamine -hydroxylase (DBH) mRNA and protein levels in human neuroblastoma cells (Thibault, C., Lai, C., Wilke, N., Duong, B., Olive, M. F., Rahman, S., Dong, H., Hodge, C. W., Lockhart, D. J., and Miles, M. F. (2000) Mol. Pharmacol. 58, 1593-1600). DBH catalyzes norepinephrine synthesis, and several studies have suggested a role for norepinephrine in ethanol-mediated behaviors. Here, we performed a detailed analysis of mechanism(s) underlying ethanol regulation of DBH expression in SH-SY5Y cells. Transient transfection analysis showed that ethanol (25-200 mM) caused concentration- and time-dependent increases in DBH gene transcription. Progressive deletions identified ethanol-responsive sequences in the -262 to -142 bp region of the DBH gene promoter. Mutagenesis of cAMP-response element (CRE) sequences in this region abolished ethanol responsiveness while maintaining responsiveness to phorbol esters. Coexpression of dominant-negative CRE-binding protein greatly reduced ethanol induction of DBH. Inhibitors of protein kinase A, casein kinase II, and MAPK reduced ethanol induction of DBH promoter activity. Pharmacogenomic studies with microarrays showed that protein kinase A, MEK, and casein kinase II inhibitors blocked induction of DBH and a large subset of ethanol-responsive genes. These genes had diverse functional groupings, including multiple members of the MAPK and phosphatidylinositol signaling cascades. Real-time PCR analysis validated select microarray results. Taken together, these results suggest that ethanol regulation of DBH requires a functional CRE and its binding protein and may require interaction of multiple kinase pathways. This mechanism may also mediate ethanol responsiveness of a complex subset of genes in neural cells. These studies may have implications for behavioral responses to ethanol or mechanisms underlying ethanol-related neurological disease.

Received for publication, May 13, 2003 , and in revised form, June 18, 2003.

^* This work was supported by Grant AA13200 from the National Institute on Alcohol Abuse and Alcoholism (to M. F. M.) and by the State of California for medical research on alcohol and substance abuse through the University of California, San Francisco. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: Elan Pharmaceuticals, South San Francisco, CA 94080.

^|| To whom correspondence should be addressed: Dept. of Pharmacology/Toxicology, Virginia Commonwealth University, P. O. Box 980599, Richmond, VA 23298. Tel.: 804-225-4054; Fax: 804-828-6432; E-mail: mfmiles{at}vcu.edu.

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