Identifying the regulatory element for human angiotensin-converting enzyme 2 (ACE2) expression in human cardiofibroblasts

Autor: Mu-Yuan Chen, Tang-Ching Kuan, I-Liang Lee, Cheng-Hao Wen, Chih-Sheng Lin, Tzu-Hui Yang
Rok vydání: 2011
Předmět:
Transcriptional Activation
Physiology
Blotting
Western
Electrophoretic Mobility Shift Assay
Plasma protein binding
Peptidyl-Dipeptidase A
Biology
Transfection
Biochemistry
Gene Expression Regulation
Enzymologic
Receptor
Angiotensin
Type 1
Transforming Growth Factor beta1
Cellular and Molecular Neuroscience
Endocrinology
Humans
Electrophoretic mobility shift assay
Luciferase
Regulatory Elements
Transcriptional
Cloning
Molecular
Binding site
Luciferases
Promoter Regions
Genetic
Cells
Cultured
Sequence Deletion
Regulation of gene expression
Binding Sites
Base Sequence
Genome
Human
Tumor Necrosis Factor-alpha
Angiotensin II
Promoter
Fibroblasts
Molecular biology
Up-Regulation
Mutagenesis
Site-Directed
Angiotensin-Converting Enzyme 2
hormones
hormone substitutes
and hormone antagonists
Protein Binding
Signal Transduction
Binding domain
Zdroj: Peptides. 32:1832-1839
ISSN: 0196-9781
DOI: 10.1016/j.peptides.2011.08.009
Popis: 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.
Databáze: OpenAIRE
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