Elsevier

Gene

Volume 421, Issues 1–2, 15 September 2008, Pages 81-88
Gene

Characterization of the bovine angiotensin converting enzyme promoter: Essential roles of Egr-1, ATF-2 and Ets-1 in the regulation by phorbol ester

https://doi.org/10.1016/j.gene.2008.06.005Get rights and content

Abstract

The protease angiotensin converting enzyme (ACE) is a key regulator of blood pressure homeostasis, and is responsible for proteolytic activation of angiotensin I to angiotensin II (Ang II), a potent vasoconstrictor, and proteolytic inactivation of bradykinin, a vasodilator. Recent studies have also implicated ACE and Ang II dysregulation in the progression of fibrotic tissue diseases. Although many studies have utilized bovine tissues and cells for investigating the regulation of ACE gene expression, the bovine ACE promoter has not been previously characterized. Here we present the analysis of the bovine ACE promoter. We investigated cis elements regulated by phorbol 12-myristate 13-acetate (PMA). Sequence analysis shows that the bovine ACE promoter contains several putative binding sites for the transcription factors ATF-2, Ets-1, Egr-1 and SP1/SP3. Chromatin immunoprecipitation (ChIP) indicated that the activation of the bovine ACE promoter by PMA involves histone H4 acetylation, and that PMA induced Egr-1 and ATF-2 binding to the ACE promoter, whereas Ets-1 binding was suppressed by PMA. The regulatory roles of these transcription factors in the bovine ACE gene regulation were confirmed by co-expression of either wild type or dominant negative transcription factors with the luciferase reporter constructs. The bovine and human ACE promoters share similarities in binding sites for transcription factors and PMA regulation within the core regions but contain significant differences in the distal promoter regions.

Introduction

Angiotensin converting enzyme (ACE, E.C. 3.4.15.1), is an extracellular Zn2+ metallopeptidase with broad substrate specificity (Dasarathy and Fanburg, 1989). The ACE enzyme occurs as two splice variations. The somatic isoform is expressed primarily, but not exclusively, in endothelial, epithelial, and neuronal cell types; this splice variant has a molecular weight of 150–180 kDa and contains two structurally related active sites. The second isoform, found in testicular germinal cells, is 90–110 kDa and contains a single active site. The primary activities of ACE for blood pressure homeostasis are the proteolytic activation of the vasoconstrictor angiotensin II (Ang II) and the proteolytic inactivation of the vasodilator bradykinin (Campbell et al., 2004). However, ACE also cleaves a wide variety of other substrates, including pro-hormones and neuropeptides (Corvol et al., 1995, Isaac et al., 1997). It has become recognized that ACE is required for male fertility (Fuchs et al., 2005), erythropoiesis (Savary et al., 2005), modulation of bone marrow maturation (Fuchs et al., 2004), and plays a localized role in the uteroplacental unit during the estrous cycle, gestation, and the postpartum period (Schauser et al., 2001). Dysregulation of ACE is also believed to be involved in fibrotic remodeling diseases, including pulmonary fibrosis and cardiac scarring following myocardial infarction (Zhu et al., 1997, Gaertner et al., 2002, Uhal et al., 2007).

Much of the pioneering work on the endogenous expression of ACE and its pharmacological regulation was performed in bovine tissue-derived cell types. Bovine pulmonary artery endothelial cells (BPAEC) were the first cell type used to demonstrate ACE regulation by hyperoxia (Krulewitz and Fanburg, 1984), cyclic nucleotide analogs (Krulewitz and Fanburg, 1986), cationophores (Dasarathy and Fanburg, 1989), steroid hormones (Krulewitz et al., 1984), and hepatocyte growth factor (Day et al., 2004). Much of the pharmacological findings for ACE regulation in bovine cells was reproduced in human cells, suggesting that similar mechanisms existed for ACE regulation in the two species (Fyhrquist et al., 1983, Iwai et al., 1987, Eyries et al., 2002).

Despite the continued utilization of bovine cell systems for the study of ACE regulation for over 20 years, the sequence of the core promoter was not known. Here, we cloned and characterized the bovine ACE promoter. Using ChIP and luciferase reporter gene assays we determined that the transcription factors Egr-1 and ATF-2 positively regulate the bovine ACE promoter by PMA where as the Ets-1 represses the promoter. Our findings indicate that although there are some similarities between the core promoters of bovine and human ACE, there are substantial sequence differences within the extended promoters which could lead to distinct regulation of the gene.

Section snippets

Reagents

Fetal bovine serum (FBS 100–106) was from Gemini Bio-Products (Woodland, CA). DMEM, RPMI medium, fungizone, tissue culture antibiotics, and Dulbecco's phosphate-buffered saline were purchased from Invitrogen (Carlsbad, CA). PMA was purchased from Sigma-Aldrich (St. Louis, MO). Antibodies for Egr-1, Ets-1, and ATF-2 were purchased from Santa Cruz Biotech (Santa Cruz, CA). Expression plasmids for dominant negative (DN) Egr-1 and DN SP3 have been described previously (Day et al., 2004); the

Cloning and sequence analyses of the 5′-flanking region of the bovine ACE gene

We initially searched promoter elements within genomic sequence of bovine ACE, but did not identify a promoter for the bovine ACE gene. Instead we found a nucleotide sequence coding for a somatic isoform of the bovine ACE (GenBank accession no. NW_929522) that appeared to contain a gap upstream of the coding sequence for the bovine ACE gene and the upstream DNA region. We designed specific primers covering the gap and amplified a 1395 bp fragment which was directly cloned into a pCR-TOPO

Discussion

In the present study we cloned and sequenced the bovine ACE promoter, identified the start site for transcription, and characterized cis elements within the core promoter for their role in basal and PMA-induced transcriptional regulation. The bovine ACE promoter contains a TATA box 26 nucleotides upstream of the transcriptional start site, and translation starts with the ATG codon 28 bp downstream of the transcriptional start site (Fig. 1). Homology studies revealed that the core bovine ACE

Acknowledgements

The opinions expressed in this document are those of the authors and do not reflect the views of the Uniformed Services University of the Health Sciences, the Department of Defense, or the U.S. Federal Government. This work was supported in part by the NIH R01HL073929 and by USUHS starter grant to R.M.D, and by the Deutsche Forschungsgemeinschaft, grant # SFB/C14, to G.T.

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