Abstract
The binding of atrial and brain natriuretic peptides (ANP and BNP) to guanylyl cyclase-A/natriuretic peptide receptor-A (GC-A/NPR-A) produces second messenger cGMP, which lowers blood pressure and prevents cardiovascular events. The objective of the present study was to examine the repressive effect of transforming growth factor (TGF-β1) in the regulation of Npr1 (coding for GC-A/NPRA) gene expression and function. The rat thoracic aortic vascular smooth muscle cells (RTASMC) were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum and treated with TGF-β1. The luciferase assay results showed that TGF-β1 significantly repressed Npr1 promoter activity in a dose- and time-dependent manner by 82% and 85% (2.5 ng/ml, 25.51 ± 2.2 and 24 h, 7.0 ± 0.6 vs. untreated control 70.06 ± 4.6, p < 0.001, respectively). Treatment with TGF-β1 decreased NPRA mRNA and protein levels by 62% (treated, 0.42 ± 0.05 vs. control, 0.9 ± 0.02, p < 0.01) and 55% (treated, 9603 ± 860 vs. untreated, 22211 ± 1449, p < 0.01), respectively. TGF-β1 attenuated ANP-dependent intracellular accumulation of cGMP by 59% (TGF-β1 + ANP-treated, 8.66 ± 0.9 pmol/1x 106 cells vs. ANP-treated cells 23.51 ± 2.2; p < 0.001). Chromatin immunoprecipitation and electrophoretic mobility shift assay showed that TGF-β1 enhanced the recruitment of transcription factor delta EF1 (δEF1) to form a transcriptional repressor complex with their binding sites in Npr1 promoter. Western blot analysis showed significant increase in δEF1 protein expression by 2.4-fold (treated, 907.9 ± 36.5. vs. untreated, 378.5 ± 10.3; p < 0.001) and phosphorylation of mothers against decapentaplegic homolog 2/3 (SMAD2/3) proteins by 2.3-fold (treated, 620.9 ± 10.6 vs. untreated, 269 ± 9.7; p < 0.01) in TGF-β1-treated cells. Collectively, the present results demonstrate that TGF-β1 represses Npr1 gene transcription and expression by interactive actions of δEF1 and phosphorylated SMADs. Together, the present results suggest that TGF-β1 and δEF1 inhibit Npr1 gene expression and function, which may be critical in regulating the blood pressure and cardiovascular homeostasis.
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