Protein Kinase C-Mediated Down-Regulation of β1-Adrenergic Receptor Gene Expression in Rat C6 Glioma Cells
- Zhongwei Li1,
- Vidita A. Vaidya1,
- John D. Alvaro1,
- Philip A. Iredale1,
- Richard Hsu1,
- Ginger Hoffman1,
- Laura Fitzgerald1,
- Patricia K. Curran2,
- Curtis A. Machida3,4,
- Peter H. Fishman2 and
- Ronald S. Duman1
- 1Division 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.), 2Membrane 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.), 3Division of Neuroscience, Oregon Regional Primate Research Center, Beaverton, Oregon 97006 (C.A.M.), and Graduate Programs in 4Neuroscience and Molecular and Cell Biology (C.A.M.), Oregon Health Sciences University, Portland, Oregon 97201
Abstract
In the current study, we investigated the mechanism by which protein kinase C (PKC) regulates the expression of β1-adrenergic receptor (β1AR) mRNA in rat C6 glioma cells. Exposure of the cells to 4β-phorbol-12-myristate-13-acetate (PMA), an activator PKC, resulted in a down-regulation of both β1AR 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 β1AR mRNA but significantly decreased the activity of the β1AR 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 β1AR-PKC response element, completely blocked the PKC-mediated down-regulation of β1AR promoter activity. Gel mobility shift analysis detected two specific bands when C6 cell extracts were incubated with a labeled DNA probe containing the β1AR-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 β1AR gene transcription in C6 cells is mediated in part by a cAMP response element binding protein-dependent mechanism acting on a novel response element.
Footnotes
-
Send reprint requests to: Dr. Ronald S. Duman, Department of Psychiatry, 34 Park Street, New Haven, CT 06508. E-mail:ronald.duman{at}yale.edu
-
This work is supported by United States Public Health Service Grants MH45481, MH53199, and 2-PO1-MH25642 and by a Veterans Administration National Center Grant for Post-traumatic Stress Disorder, VA Medical Center.
- Abbreviations:
- PKC
- protein kinase C
- AR
- adrenergic receptor
- CRE
- cAMP response element
- CREB
- cAMP response element binding protein
- PMA
- 4β-phorbol-12-myristate-13-acetate
- CYP
- cyanopindolol
- SS
- supershifted
- DMSO
- dimethylsulfoxide
- EGTA
- ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid
- HEPES
- 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
- AP-1
- activator protein-1
- PRE
- protein kinase C response element
- CREM
- cAMP response modulator transcription factor
-
- Received January 21, 1998.
- Accepted March 19, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
