The angiotensin II type 1 receptor (AT1R)-associated protein (ATRAP/Agtrap) promotes constitutive internalization of the AT1R so as to specifically inhibit the pathological activation of its downstream signaling yet preserve the base-line physiological signaling activity of the AT1R. Thus, tissue-specific regulation of Agtrap expression is relevant to the pathophysiology of cardiovascular and renal disease. However, the regulatory mechanism of Agtrap gene expression has not yet been fully elucidated. In this study, we show that the proximal promoter region from -150 to +72 of the mouse Agtrap promoter, which contains the X-box, E-box, and GC-box consensus motifs, is able to elicit substantial transcription of the Agtrap gene. Among these binding motifs, we showed that the E-box specifically binds upstream stimulatory factor (Usf) 1 and Usf2, which are known E-box-binding transcription factors. It is indicated that the E-box-Usf1/Usf2 binding regulates Agtrap expression because of the following: 1) mutation of the E-box to prevent Usf1/Usf2 binding reduces Agtrap promoter activity; 2) knockdown of Usf1 or Usf2 affects both endogenous Agtrap mRNA and Agtrap protein expression, and 3) the decrease in Agtrap mRNA expression in the afflicted kidney by unilateral ureteral obstruction is accompanied by changes in Usf1 and Usf2 mRNA. Furthermore, the results of siRNA transfection in mouse distal convoluted tubule cells and those of unilateral ureteral obstruction in the afflicted mouse kidney suggest that Usf1 decreases but Usf2 increases the Agtrap gene expression by binding to the E-box. The results also demonstrate a functional E-box-USF1/USF2 interaction in the human AGTRAP promoter, thereby suggesting that a strategy of modulating the E-box-USF1/USF2 binding has novel therapeutic potential.
Keywords: Angiotensin; Distal Tubule; Gene Transcription; Receptors; Renin Angiotensin System; Transcription Regulation.