Interplay between EGR1 and SP1 is critical for 13-cis retinoic acid-mediated transcriptional repression of angiotensin type 1A receptor

Abstract

Recently, we have demonstrated that 13-cis retinoic acid (13cRA) downregulates rat angiotensin type 1A receptor (Agtr1a) gene transcription through a MAP kinase (ERK1/2)-dependent mechanism in rat liver epithelial and aortic smooth muscle cells. However, the exact mechanism remained unknown. In this study, we determined the signaling intermediates activated by ERK1/2 involved in 13cRA-mediated Agtr1a downregulation. Rat Agtr1a chloramphenicol acetyltransferase (CAT) promoter construct containing a sequence -2541 and -1836 bp upstream of the start site demonstrated reduced CAT activity; this region possesses a specificity protein 1 (SP1) consensus sequence (5'-TGGGGCGGGGCGGGG-3'). Mobility shift analysis using untreated nuclear extracts in the presence of mithramycin A suggests that the trans-acting factor binding to this cis-acting element is SP1. 13cRA significantly reduced specific binding without any change in SP1 protein expression. Studies showed that 13cRA treatment maximally phosphorylates ERK1/2 within 5-10 min, which translocates to the nucleus, activating early growth response protein 1 (Egr1) mRNA expression at 20 min followed by de novo protein synthesis, leading to an EGR1/SP1 interaction. siRNA silencing of Egr1 restored Agtr1a mRNA and protein expression in 13cRA-treated cells, and Sp1 silencing results in complete loss of Agtr1a expression. Our study suggests that 13cRA-mediated activation of ERK1/2, through EGR1, is capable of disrupting SP1, the requisite trans-activator for Agtr1a expression, providing a novel paradigm in Agtr1a gene transcription.