Sp1-like activity mediates angiotensin-II-induced plasminogen-activator inhibitor type-1 (PAI-1) gene expression in mesangial cells

Abstract

Angiotensin II (Ang II) up-regulates plasminogen-activator inhibitor type-1 (PAI-1) expression in mesangial cells to enhance extracellular matrix formation. The proximal promoter region (bp -87 to -45) of the human PAI-1 gene contains several potent binding sites for transcription factors [two phorbol-ester-response-element (TRE)-like sequences; D-box (-82 to -76) and P-box (-61 to 54), and one Sp1 binding site-like sequence, Sp1-box 1 (-72 to -67)]. We studied this region to determine the transcription factor(s) that mediates Ang-II-induced transcriptional activation of the PAI-1 gene. Various double-stranded decoy oligodeoxynucleotides (ODNs) corresponding to various sequences in the proximal promoter region were transfected to mesangial cells to examine the effects on Ang-II-induced PAI-1 mRNA expression. Transfection with the full-length decoy (bp -87 to -45, D-P-ODN) markedly attenuated Ang-II-induced PAI-1 mRNA expression by up to 70%. Transfection with D-ODN (-87 to -71) and P-ODN (-66 to -45), which correspond to each of the two TRE-like sequences, did not attenuate the expression. Gel-shift assays using nuclear extracts prepared from Ang-II-treated mesangial cells and D-P-ODN showed three specific complexes. The major complex was supershifted by anti-Sp1 antibody. The methylation-interference experiment demonstrated that human recombinant Sp1 bound to the so-called GT box (TGGGTGGGGCT, -78 to -69), which contains the Sp1-box 1. The complex that migrated with anti-Sp1 antibody was enhanced in the cells treated with Ang II. Further, D-Sp1-ODN (-85 to -63) containing the GT box attenuated up-regulation of PAI-1 mRNA expression induced by Ang II to a level (68±9% inhibition) comparable to D-P-ODN, whereas ODN with four mutations in the GT box had no effect. Our findings suggest that binding of Sp1 or an Sp1-like transcription factor to the GT box in the PAI-1 promoter up-regulates PAI-1 gene transcription in mesangial cells stimulated with Ang II. This transcription-factor binding site may be targeted to control Ang-II-dependent extracellular matrix formation by mesangial cells.