Transcriptional regulation of Npr1 gene promoter activity by TGF-beta1; role of deltaEF1
Description
Atrial and brain natriuretic peptides (ANP and BNP) intiate their signaling by binding to guanylyl cyclase-A/natriuretic peptide receptor-A (GC-A/NPR-A) which results in production of second messenger cGMP. This signaling pathway plays a critical role in cellular networks regulating hypertension, cardiovascular events, and carcinogenesis. Previous studies have shown that the cis-response elements in the Npr1 gene (coding for GC-A/NPRA) promoter play a key role in regulating the expression and function of the receptor protein. deltaEF1, an E-2 box repressor has been predicted to have binding sites in Npr1 gene promoter. The objective of this study was to determine the repressive effect of TGF-beta1 (Transforming growth factor-beta1) on transcription of murine Npr1 gene promoter and characterize the role of deltaEF1 in this process. Luciferase activities of Npr1 gene promoter constructs was reduced by 70-75% with deltaEF1 co-transfection. Also, Npr1 gene promoter-luciferase constructs embodying deltaEF1 sites showed reduction in luciferase activity after treatment with TGF-beta1 in a time- and dose- dependent manner in mouse mesangial cells (MMCs). Furthermore, site-directed mutagenesis of deltaEF1 binding sites relieved the inhibition on Npr1 gene transcription. Deletion of deltaEF1 sites by deletion mutation eliminated the repression of luciferase activities of Npr1 promoter constructs induced by TGF-beta1,which suggests that deltaEF1 mediates this inhibitory pathway. Electrophoretic mobility shift assay as well as supershift assay confirmed the binding of deltaEF1 to its specific binding sites in Npr1 gene promoter. In vivo binding of deltaEF1 to Npr1 gene promoter region was established by chromatin immunoprecipitation assay. The findings of this study are vital in understanding TGF-beta1 signaling and its effect on Npr1 gene transcription and expression,which plays a critical role in cardiovascular regulation