Pharmacogenomic analysis of mechanisms mediating ethanol regulation of dopamine beta-hydroxylase.
We previously showed that ethanol regulates dopamine beta-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.
Hassan
Sajida
S
Departments of Pharmacology Toxicology and Neurology and the Center for Study of Biological Complexity, Virginia Commonwealth University, Richmond, Virginia 23298, USA.
Duong
Bao
B
Kim
Kwang-Soo
KS
Miles
Michael F
MF
D047390:Casein Kinase II; D002492:Central Nervous System Depressants / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D004299:Dopamine beta-Hydroxylase / Q000235:genetics* / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D000431:Ethanol / Q000494:pharmacology*; D017353:Gene Deletion; D005799:Genes, Dominant; D006801:Humans; D008957:Models, Genetic; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D020411:Oligonucleotide Array Sequence Analysis; D010597:Pharmacogenetics; D010716:Phosphatidylinositols / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors; D018075:RNA, Complementary / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured
D002492:Central Nervous System Depressants; D004791:Enzyme Inhibitors; D010716:Phosphatidylinositols; D018075:RNA, Complementary; D012333:RNA, Messenger; D000431:Ethanol; D004299:Dopamine beta-Hydroxylase; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases