Enhanced Sp1/YY1 Expression Directs CBS Transcription to Mediate VEGF-Stimulated Pregnancy-Dependent H2S Production in Human Uterine Artery Endothelial Cells

Abstract

Pregnancy and VEGF (vascular endothelial growth factor) stimulate uterine artery endothelial cell (UAEC) hydrogen sulfide production via selectively upregulating CBS (cystathionine β-synthase) but not CSE (cystathionine γ-lyase) expression. This study was conducted to determine the mechanisms by which VEGF utilizes to stimulate pregnancy-dependent upregulation of CBS and hydrogen sulfide production in human UAEC. The proximal human CBS promoter contains 4 Sp1 (specificity protein 1; a/b/c/d) sites and 1 YY1 (Yin Yang 1) site; luciferase assays using reporter genes driven by human CBS promoter with a series of 5′-deletions identified a promoter sequence (-574 to -394) containing Sp1d and the YY1 sites critical for basal and VEGF-stimulated CBS promoter activation. VEGF stimulated pregnancy-dependent recruitment of Sp1 to Sp1d and YY1 to YY1 and also recruited YY1 to Sp1c and increased Sp1/YY1 association in pregnant human UAEC, suggesting formation of a Sp1/YY1 complex at the Sp1c site. Endothelial Sp1 and YY1 proteins were significantly greater in pregnant than nonpregnant human uterine artery. VEGF stimulated pregnancy-dependent Sp1 and YY1 protein expression in vitro. Treatment with Sp1 and YY1 siRNAs completely blocked Sp1/YY1-mediated pregnancy-dependent CBS protein upregulation and hydrogen sulfide production by VEGF in human UAEC. VEGF did not trans-activate CSE promoter or increase CSE expression, and Sp1/YY1 knockdown did not affect CSE expression in human UAEC. Thus, pregnancy augments EC Sp1 and YY1 expression and promotes the recruitment of Sp1/YY1 to their DNA-binding sequences in proximal human CBS promoter to upregulate CBS transcription, underlying a novel mechanism to mediate VEGF-stimulated pregnancy-dependent endothelial hydrogen sulfide production in the human uterine artery.