title: Identifying the regulatory element for human angiotensin-converting enzyme 2 (ACE2) expression in human cardiofibroblasts.
abstract: Angiotensin-converting enzyme 2 (ACE2) has been proposed as a potential target for cardioprotection in regulating cardiovascular functions, owing to its key role in the formation of the vasoprotective peptides angiotensin-(1-7) from angiotensin II (Ang II). The regulatory mechanism of ace2 expression, however, remains to be explored. In this study, we investigated the regulatory element within the upstream of ace2. The human ace2 promoter region, from position -2069 to +20, was cloned and a series of upstream deletion mutants were constructed and cloned into a luciferase reporter vector. The reporter luciferase activity was analyzed by transient transfection of the constructs into human cardiofibroblasts (HCFs) and an activating domain was identified in the -516/-481 region. Deletion or reversal of this domain within ace2 resulted in a significant decrease in promoter activity. The nuclear proteins isolated from the HCFs formed a DNA-protein complex with double stranded oligonucleotides of the -516/-481 domain, as detected by electrophoretic mobility shift assay. Site-directed mutagenesis of this region identified a putative protein binding domain and a potential binding site, ATTTGGA, homologous to that of an Ikaros binding domain. This regulatory element was responsible for Ang II stimulation via the Ang II-Ang II type-1 receptor (AT1R) signaling pathway, but was not responsible for pro-inflammatory cytokines TGF-β1 and TNF-α. Our results suggest that the nucleotide sequences -516/-481 of human ace2 may be a binding domain for an as yet unidentified regulatory factor(s) that regulates ace2 expression and is associated with Ang II stimulation.
authors.affiliation: Department of Biological Science and Technology, National Chiao Tung University, No. 75 Po-Ai Street, Hsinchu 30068, Taiwan.
authors.forename: Tang-Ching
authors.initials: TC
authors.lastname: Kuan
authors.forename: Tzu-Hui
authors.initials: TH
authors.lastname: Yang
authors.forename: Cheng-Hao
authors.initials: CH
authors.lastname: Wen
authors.forename: Mu-Yuan
authors.initials: MY
authors.lastname: Chen
authors.forename: I-Liang
authors.initials: IL
authors.lastname: Lee
authors.forename: Chih-Sheng
authors.initials: CS
authors.lastname: Lin
mesh_terms: D000804:Angiotensin II / Q000378:metabolism / Q000494:pharmacology; D000085962:Angiotensin-Converting Enzyme 2; D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D002478:Cells, Cultured; D003001:Cloning, Molecular; D024202:Electrophoretic Mobility Shift Assay; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D015971:Gene Expression Regulation, Enzymologic; D015894:Genome, Human; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D007703:Peptidyl-Dipeptidase A / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D044140:Receptor, Angiotensin, Type 1 / Q000235:genetics / Q000378:metabolism; D050436:Regulatory Elements, Transcriptional; D017384:Sequence Deletion; D015398:Signal Transduction; D015533:Transcriptional Activation; D014162:Transfection; D053773:Transforming Growth Factor beta1 / Q000235:genetics / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism; D015854:Up-Regulation
chemical_list: D044140:Receptor, Angiotensin, Type 1; C508960:TGFB1 protein, human; D053773:Transforming Growth Factor beta1; D014409:Tumor Necrosis Factor-alpha; D000804:Angiotensin II; D008156:Luciferases; D007703:Peptidyl-Dipeptidase A; C000705307:ACE2 protein, human; D000085962:Angiotensin-Converting Enzyme 2