Identifying the regulatory element for human angiotensin-converting enzyme 2 (ACE2) expression in human cardiofibroblasts |
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Authors: | Kuan Tang-Ching Yang Tzu-Hui Wen Cheng-Hao Chen Mu-Yuan Lee I-Liang Lin Chih-Sheng |
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Institution: | Department of Biological Science and Technology, National Chiao Tung University, No. 75 Po-Ai Street, Hsinchu 30068, Taiwan |
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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. |
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Keywords: | Ang I angiotensin I Ang II angiotensin II AT1R angiotensin II Type 1 Receptor Ang-(1-7) angiotensin-(1-7) Ang-(1-9) angiotensin-(1-9) ACE angiotensin-converting enzyme ACE2 angiotensin-converting enzyme 2 EMSA electrophoretic mobility shift assay HCFs human cardiofibroblasts RAS renin-angiotensin system TGF-β1 transforming growth factor-beta1 TNF-α tumor necrosis factor-alpha |
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