Angiotensin II-induced Transcriptional Activation of the Cyclin D1 Gene Is Mediated by Egr-1 in CHO-AT1A Cells

Abstract

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., Bé ré ziat, 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 control of mammalian cell proliferation by extracellular signals in G 1 to S phase progression of the cell cycle is largely mediated by serine/threonine cyclin-dependent kinases CDK4 1 and CDK6, which interact with specific D-type cyclins. The CDK-D-type cyclin complexes induce phosphorylation of the retinoblastoma protein (pRb), thereby releasing the transcription factor E2F, which is required for the transcription of S phase-specific genes (1-4). Activation by mitogenic stimuli of D-type cyclins during the G 1 phase appears to be an essential and rate-limiting step in G 1 to S phase progression of the cell cycle (5-7). The cyclin D1 gene expression seems to be essentially regulated at the transcription level. The promoter region of the cyclin D1 gene contains multiple potential cis-regulatory elements including binding sites for AP1, E2F, Oct, Egr-1...

Upregulation of rat Ccnd1 gene by exendin-4 in pancreatic beta cell line INS-1: interaction of early growth response-1 with cis-regulatory element

“…Meanwhile, the oligo 3′ probe containing the binding site of the consensus SP1 was supershifted by anti-SP1 antibody but not by anti-EGR1 or anti-AP2 antibodies (data not shown). These results are consistent with previous studies showing binding of EGR1 to an overlapping EGR1/ SP1 site without displacement of SP1 in the human Ccnd1 promoter [26,27].…”

“…Results of the promoter analysis revealed that the region between −174 and −73 was largely involved in Ccnd1 promoter activity, implicating this region as a major exendin-4-responsive element. The importance of this region has also been emphasised in human Ccnd1 promoters of oesophageal squamous carcinoma [26] and of Chinese hamster ovary cells stably overexpressing angiotensin II type 1A receptor [27]. Meanwhile, other cis-regulatory elements located between −73 and +130 of the promoter also appear to be involved in promoter activities in response to exendin-4.…”

“…However, the oligo 3′ probe that included another putative EGR1 binding site (−108 to −100) failed to form a complex with EGR1. In humans, this EGR1 binding site, overlapping two SP1 binding sites, corresponds to the consensus EGR1 site and is essential for the binding of EGR1 in order to activate Ccnd1 transcription in response to growth factors [26,27]. Comparing the sequences of the rat (5′-CGCCTCGGC-3′) and human (5′-CGCCCCCGC-3′), the thymine and guanine at −104 and −102 in rats are replaced with cytosine in humans.…”

Berberine suppresses MEK/ERK-dependent Egr-1 signaling pathway and inhibits vascular smooth muscle cell regrowth after in vitro mechanical injury

“…Once activated, Egr-1 regulates the expression of several genes, including monocyte chemotactic protein-1 (MCP-1), platelet-derived growth factor (PDGF) A and B [7,9,10], and Cyclin D1 [11,12]. Previous studies showed that mechanical injury-induced up-regulation of Egr-1 is through activation of extracellular signal-regulated kinase (ERK), a key transducer of extracellular signals that promote cell growth and movement, which are critical for the initiation and progression of vascular lesions [13].…”

“…ERK transmits mitogenic signals by translocation to the nucleus where many of its substrates, including transcription factors, Egr-1, c-Fos and Elk-1 are activated [16][17][18]. Since Egr-1 induces the expression of multiple genes implicated in pathogenesis of atherosclerosis and restenosis after angioplasty [11,12,19], indicating that blocking of ERK/Egr-1 signaling pathway might be an important therapeutic strategy in reducing the incidence of restenosis after angioplasty.…”