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.
ASJC Scopus subject areas
- Molecular Medicine