Glucose mediates transcriptional repression of the human angiotensin type-1 receptor gene: role for a novel cis-acting element.

Abstract

Human angiotensin type 1 receptor (hAT1R) gene is regulated by hormones, second messengers, and both pathophysiological and developmental states. The focus of the present study was to determine the role of glucose in the trans-repression of hAT1R gene transcription and to identify the functional cis-acting response element(s). Serial deletions of the hAT1R promoter region indicated that an area between -1717 and -1543 base pairs upstream of the 5' end of the cDNA sequence has a glucose responsive regulatory element (GluRE) to down-regulate the gene expression. Further analysis revealed a putative 29-bp (5'-AACTGATTTTTGTATATTGATCTTGTATT-3') repressor element located between -1582 and -1610 bp was necessary for transcriptional repression. Removal of this region from promoter construct abolished repression of the hAT1R gene transcription in human proximal tubule epithelial cells (hPTECs). Using mobility shift assays, we demonstrated DNA binding activity to the labeled repressor element in hPTEC nuclear extracts. Additional studies demonstrated increased DNA binding activity to the labeled repressor element in nuclear extracts treated with high glucose (25 mM). Southwestern analysis identified two GluRE binding proteins of 34 and 36 kDa in glucose-treated extracts. Glucose-induced activity of the repressor trans-acting factor(s) reached a maximum at 4 h, which correlated with decreased transcriptional activity of the hAT1R gene, suggesting that glucose can down-regulate the transcription of the hAT1R gene through the repressor element. Furthermore, insertion of the glucose response element into heterologous SV40 promoter (SV40) chloramphenicol acetyl transferase (CAT) vector showed orientation/distance-independent repression of SV40 promoter-mediated CAT activity in hPTECs. Our results show that the glucose response factor(s) acts as trans-acting factor(s) binding to the cis-acting repressor element in the hAT1R promoter, which may participate in the control of basal transcription as well as glucose-mediated transcriptional inhibition of the hAT1R gene.