Angiotensin II-induced transcriptional activation of the cyclin D1 gene is mediated by Egr-1 in CHO-AT1A cells

Cyclin D1 protein expression is regulated by mitogenic stimuli and is a critical component in the regulation of G(1) to S phase progression of the cell cycle. Angiotensin II (Ang II) binds to specific G protein-coupled receptors and is mitogenic in Chinese hamster ovary cells stably expressing the rat vascular Ang II type IA receptor (CHO-AT(1A)). We recently reported that in these cells, Ang II induced cyclin Dl promoter activation and protein expression in a phosphatidylinositol 3-kinase (PI3K)-, SHP-2-, and mitogen-activated protein kinase/ extracellular signal-regulated kinase (MAPK/ERK)-dependent manner (Guillemot, L., Levy, A., Zhao, Z. J., Bereziat, G., and Rothhut, B. (2000) J. Biol. Chem. 275, 26349-26358). In this report, transfection studies using a series of deleted cyclin D1 promoters revealed that two regions between base pairs (bp) -136 and -96 and between by -29 and +139 of the human cyclin Dl promoter contained regulatory elements required for Ang II-mediated induction. Mutational analysis in the -136 to -96 by region provided evidence that a Sp1/early growth response protein (Egr) motif was responsible for cyclin Dl promoter activation by Ang II. Gel shift and supershift studies showed that Ang II-induced Egr-1 binding involved de novo protein synthesis and correlated well with Egr-1 promoter activation. Both U0126 (an inhibitor of the MAPK/ERK kinase MEK) and wortmannin (an inhibitor of PI3K) abrogated Egr-1 endogenous expression and Egr-1 promoter activity induced by Ang II. Moreover, using a co-transfection approach, we found that Ang II induction of Egr-1 promoter activity was blocked by dominant-negative p21(ras), Raf-1, and tyrosine phosphatase SHP-2 mutants. Identical effects were obtained when inhibitors and dominant negative mutants were tested on the -29 to +139 by region of the cyclin DI promoter. Taken together, these findings demonstrate that Ang II-induced cyclin Dl up-regulation is mediated by the activation and specific interaction of Egr-1 with the -136 to -96 by region of the cyclin Dl promoter and by activation of the -29 to +139 by region, both in a p21(ras)/Raf-1/MEK/ERK-dependent manner, and also involves PI3K and SHP-2.