Functional analysis of the human somatic angiotensin I-converting enzyme gene promoter

Abstract

Angiotensin I-converting enzyme (ACE) is a key enzyme in the regulation of systemic blood pressure and plays a major role in the renin-angiotensin and bradykinin-kinin systems, at the luminal surface of the vascular endothelia. To identify the promoter region, the transcription regulatory elements and the cell specificity of the ACE gene, five successive DNA deletions of the 5' upstream region (-1214, -754, -472, -343, -132 bp relative to the start site of transcription) were isolated and fused in sense and antisense orientations to the bacterial chloramphenicol acetyltransferase (CAT) reporter gene in the promoterless plasmid pBLCAT3. Promoter activities were measured in transient transfection assays using three different cell lines from rabbit endothelium (RE), human embryocarcinoma (Tera-1) and hepatocarcinoma cells (HepG2). All five fragments of the ACE promoter region directed expression of the CAT gene when transfected into the endothelial and the embryocarcinoma cells, which contain endogenous ACE mRNA and express ACE activity. In contrast only minimal levels of promoter activity were obtained on transfection into hepatocarcinoma cells in which endogenous ACE mRNA and ACE activity were not detected. Transfection of RE and Tera-1 cells demonstrated that promoter activity was defined by the length of the ACE promoter sequence inserted into the construct. The 132 bases located upstream from the transcription start site were sufficient to confer ACE promoter activity, whereas the sequences upstream from -472 bp and between -343 bp and -132 bp were responsible for a decrease of promoter activity. Furthermore, the minimal 132 bp of the ACE promoter contains elements which direct cell-specific CAT expression. In addition, the DNA transfection study in the presence of dexamethasone suggested that the potential glucocorticoid regulatory elements, located in the sequence of the ACE promoter, are not functional.

Phorbol Ester Induction of Angiotensin-Converting Enzyme Transcription Is Mediated by Egr-1 and AP-1 in Human Endothelial Cells via ERK1/2 Pathway

“…AF229986), into the firefly luciferase reporter gene vector pGL 3 -basic (Promega). This construct was designated pACE-4335 and was digested by convenient restriction endonucleases for construction of deletion mutants as previously described by Testut et al 12 Restricted fragments were subcloned into pGL 3 -basic. The plasmids thus obtained were designated pACE-132, pACE-754, pACE-2033, pACE-2795, and pACE-1214.…”

Rat Angiotensin-Converting Enzyme Promoter Regulation by β-Adrenergics and cAMP in Endothelium

“…However, heterologous transfections of the rat endothelin-1 promoter into bovine aortic endothelial cells and of human ACE promoter into rabbit endothelial cells have been used successfully to model conserved mechanisms of gene regulation. 29,30 As shown by studies in bovine 4,12,13 and human cells, 31 cAMP-dependent regulation of somatic ACE is conserved between mammals. Therefore, we used REC 18 to establish cAMP regulation of the rat ACE promoter.…”

“…29 Therefore, endothelial cells of heterologous origin have been used to study regulation of endotheliumspecific promoters. 29,30 This approach can be criticized because promoter regulation can be specific in each species.…”

“…29 Therefore, endothelial cells of heterologous origin have been used to study regulation of endotheliumspecific promoters. 29,30 This approach can be criticized because promoter regulation can be specific in each species. However, heterologous transfections of the rat endothelin-1 promoter into bovine aortic endothelial cells and of human ACE promoter into rabbit endothelial cells have been used successfully to model conserved mechanisms of gene regulation.…”

Haplotype variation in the ACE gene in global populations, with special reference to India, and an alternative model of evolution of haplotypes

“…It is derived from conversion of Ang I by the angiotensin-I-converting enzyme encoded by the ACE gene (MIM 106180). The gene coding for ACE has two promoter sites that code for two major isoforms, somatic ACE (sACE) and testis ACE (tACE) (Testut et al 1993). Variations in the ACE gene, many of which influence levels of the protein, are associated with diseases such as hypertension, myocardial infarction, Alzheimer's disease, major depression, type II diabetes, and various quantitative precursors of these disorders (Rigat et al 1990;Tiret et al 1992;Villard et al 1996;Daniel et al 2006;Kehoe et al 2003;Hadjadj et al 2007).…”