Vol. 54, Issue 1, 14-21, July 1998

Protein Kinase C-Mediated Down-Regulation of ₁-Adrenergic Receptor Gene Expression in Rat C6 Glioma Cells

Zhongwei Li, Vidita A. Vaidya, John D. Alvaro, Philip A. Iredale, Richard Hsu, Ginger Hoffman, Laura Fitzgerald, Patricia K. Curran, Curtis A. Machida, Peter H. Fishman, and Ronald S. Duman

Division of Molecular Psychiatry, Departments of Psychiatry and Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06508 (Z.L., V.A.V., J.D.A., P.A.I., R.H., G.H., L.F., R.S.D.), Membrane Biochemistry Section, Laboratory of Molecular and Cellular Neurobiology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892 (P.K.C., P.H.F.), Division of Neuroscience, Oregon Regional Primate Research Center, Beaverton, Oregon 97006 (C.A.M.), and Graduate Programs in Neuroscience and Molecular and Cell Biology (C.A.M.), Oregon Health Sciences University, Portland, Oregon 97201

In the current study, we investigated the mechanism by which protein kinase C (PKC) regulates the expression of ₁-adrenergic receptor (₁AR) mRNA in rat C6 glioma cells. Exposure of the cells to 4-phorbol-12-myristate-13-acetate (PMA), an activator PKC, resulted in a down-regulation of both ₁AR binding sites and mRNA levels in a time- and concentration-dependent manner. This effect was not observed with phorbol esters that do not activate PKC and was blocked by bisindolylmaleimide, a specific PKC inhibitor. Activation of PKC did not reduce the half-life of ₁AR mRNA but significantly decreased the activity of the ₁AR promoter, as determined by reporter analysis. A putative response element, with partial homology to a consensus cAMP response element, was identified by mutation analysis of the promoter at positions 343 to 336, relative to the translational start site. Mutation of this putative regulatory element, referred to as a ₁AR-PKC response element, completely blocked the PKC-mediated down-regulation of ₁AR promoter activity. Gel mobility shift analysis detected two specific bands when C6 cell extracts were incubated with a labeled DNA probe containing the ₁AR-PKC response element sequence. Formation of one of these bands was inhibited by an oligonucleotide probe containing a consensus CRE and disrupted by an antibody for cAMP response element binding protein. Based on these studies, we propose that the PKC-induced down-regulation of ₁AR gene transcription in C6 cells is mediated in part by a cAMP response element binding protein-dependent mechanism acting on a novel response element.