Protein Kinase C-Mediated Down-Regulation of b1-Adrenergic Receptor Gene Expression in Rat C6 Glioma Cells

In the current study, we investigated the mechanism by which protein kinase C (PKC) regulates the expression of b1-adrenergic receptor (b1AR) mRNA in rat C6 glioma cells. Exposure of the cells to 4b-phorbol-12-myristate-13-acetate (PMA), an activator PKC, resulted in a down-regulation of both b1AR binding sites and mRNA levels in a timeand 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 b1AR mRNA but significantly decreased the activity of the b1AR 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 2343 to 2336, relative to the translational start site. Mutation of this putative regulatory element, referred to as a b1AR-PKC response element, completely blocked the PKC-mediated down-regulation of b1AR promoter activity. Gel mobility shift analysis detected two specific bands when C6 cell extracts were incubated with a labeled DNA probe containing the b1AR-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 b1AR gene transcription in C6 cells is mediated in part by a cAMP response element binding protein-dependent mechanism acting on a novel response element. Cross-regulation between two of the major signal transduction pathways, the receptor-coupled adenylyl cyclase and phospholipase C systems, is a well recognized phenomenon (Houslay, 1991). The second messengers generated by each of these systems activate protein kinase A and PKC, respectively. The effects of PKC on bAR-stimulated adenylyl cyclase have been investigated extensively and found to be complex. In some cells, desensitization has been observed (Garte and Belman, 1980; Kelleher et al., 1984; Sibley et al., 1984; Kassis et al., 1985). In other cells, potentiation of agonist-stimulated activity has been found (Bell et al., 1985; Sugden et al., 1985; Yoshimasa et al., 1987). The activation of PKC in a third class of cells leads to both potentiation and desensitization (Johnson et al., 1990; Bouvier et al., 1991; Zhou et al., 1994). The potentiation seems to be mediated by phosphorylation of the inhibitory G protein (Houslay, 1991) or the adenylyl cyclase catalyst (Yoshimasa et al., 1987; Simmoteit et al., 1991). The desensitization seems to be caused by PKC-catalyzed phosphorylation of bAR (Kelleher et al., 1984; Sibley et al., 1984; Bouvier et al., 1987, 1991). In this regard, a mutated b2AR that lacks the consensus sites for PKC is no longer susceptible to phorbol ester-mediated phosphorylation and desensitization (Johnson et al., 1990; Bouvier et al., 1991). Less is known about the effects of PKC activation on b1AR expression. In rat C6 glioma cells, exposure of cells to phorbol esters that activate PKC leads to a loss of receptor binding activity (Kassis et al., 1985; Fishman et al., 1987). In addition to its role in the regulation of bAR, PKC may be involved in glial cell proliferation and differentiation (Kronfeld et al., 1995). C6 cells express both b1AR and b2AR subtypes (Fishman et al., 1994; Hosoda et al., 1994). We recently showed 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, 4b-phorbol-12-myristate-13-acetate; CYP, cyanopindolol; SS, supershifted; DMSO, dimethylsulfoxide; EGTA, ethylene glycol bis(b-aminoethyl ether)-N,N,N9,N9-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. 0026-895X/98/010014-08$3.00/0 Copyright © by The American Society for Pharmacology and Experimental Therapeutics All rights of reproduction in any form reserved. MOLECULAR PHARMACOLOGY, 54:14–21 (1998). 14 at A PE T Jornals on Sptem er 0, 2017 m oharm .aspeurnals.org D ow nladed from that on exposure to agonist or other agents that activate protein kinase A, C6 cells coordinately down-regulate both receptor subtypes (Fishman et al., 1994; Hosoda et al., 1994). Receptor down-regulation seems to be mediated in part by down-regulation of receptor mRNA and to involve induction of a repressor protein that blocks gene transcription (Hosoda et al., 1994, 1995; Rydelek-Fitzgerald et al., 1996). We undertook the current study to determine whether similar mechanisms were involved in PKC-mediated down-regulation of bAR in C6 cells. Because b1AR is the major subtype in C6 cells and less is known about its regulation, we concentrated on the effects of PMA on b1AR expression. Experimental Procedures Materials. (2)-I-CYP (2200 Ci/mmol) and a-P-labeled nucleotides were obtained from Dupont-New England Nuclear (Boston, MA). Phorbol esters, bisindolylmaleimide I, HCl, and actinomycin D were purchased from Calbiochem (San Diego, CA). 8-(4-Chlorophenylthio)-cAMP was from Sigma Chemical (St. Louis, MO). Pseudomonas exotoxin A was from List Biological Laboratories (Campbell, CA). CGP 20712A was a generous gift from CIBA-GEIGY (Summit,