Angiotensin II-induced transcriptional activation of the cyclin D1 gene is mediated by Egr-1 in CHO-AT(1A) 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 1A receptor (CHO-AT(1A)). We recently reported that in these cells, Ang II induced cyclin D1 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., Béréziat, 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 bp -29 and +139 of the human cyclin D1 promoter contained regulatory elements required for Ang II-mediated induction. Mutational analysis in the -136 to -96 bp region provided evidence that a Sp1/early growth response protein (Egr) motif was responsible for cyclin D1 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 bp region of the cyclin D1 promoter. Taken together, these findings demonstrate that Ang II-induced cyclin D1 up-regulation is mediated by the activation and specific interaction of Egr-1 with the -136 to -96 bp region of the cyclin D1 promoter and by activation of the -29 to +139 bp region, both in a p21(ras)/Raf-1/MEK/ERK-dependent manner, and also involves PI3K and SHP-2.     

The nfkb1 promoter is controlled by proteins of the Ets family.

The gene encoding NFKB1 is autoregulated, responding to NF-kappa B/Rel activation through NF-kappa B binding sites in its promoter, which also contains putative sites for Ets proteins. One of the Ets sites, which we refer to as EBS4, is located next to an NF-kappa B/Rel binding site, kB3, which is absolutely required for activity of the promoter in Jurkat T cells in response to activation by phorbol 12-myristate 13-acetate (PMA), PMA/ionomycin, or the Tax protein from human T cell leukemia virus type I. We show that EBS4 is, required for the full response of the nfkb1 promoter to PMA or PMA/ionomycin in Jurkat cells. EBS4 is bound by Ets-1, Elf-1, and other species. Overexpression of Ets-1 augments the response to PMA/ionomycin and this is reduced by mutation of EBS4. Elf-1 has less effect in conjunction with PMA/ionomycin, but by itself activates the promoter 12-fold. This activation is only partly affected by mutation of EBS4, and a mutant promoter that binds Ets-1, but not Elf-1, at the EBS4 site responds to PMA/ionomycin as efficiently as the wild-type. Ets proteins may be responsible for fine-tuning the activity of the nfkb1 gene in a cell-type-specific manner.