Quercetin induced tissue-type plasminogen activator expression is mediated through Sp1 and p38 mitogen-activated protein kinase in human endothelial cells.

Abstract

Wine polyphenol quercetin upregulates tissue-type plasminogen activator (t-PA) transcription in cultured human umbilical cord vein endothelial cells (HUVECs). However, the regulatory elements and signaling pathways involved in this regulation are unknown. We aimed to localize quercetin-responsive t-PA promoter elements, identify the proteins that bind these elements, and decipher signaling pathways involved in the regulation of t-PA. To localize quercetin-responsive elements, HUVECs were transiently transfected with various t-PA promoter-reporter constructs. Element functionality was evaluated by mutational analysis. Nuclear protein-t-PA element interactions were evaluated by electrophoretic mobility shift assays (EMSAs) and chromatin immunoprecipitation (ChIP) analysis. Mitogen-activated protein kinase (MAPK) inhibitors were used to determine the signaling pathways involved in t-PA regulation. MAPK inhibition effects were evaluated by real-time PCR, immunoblotting analysis, and transfections. Coimmunoprecipitation was used to evaluate MAPK and transcription factor interaction. Deletion of the t-PA promoter region - 288 to - 250 resulted in loss of quercetin responsiveness. This region contains putative Sp1-binding elements, which we termed Sp1a and Sp1b. Sp1b mutation abolished the quercetin-inducible response, whereas Sp1a mutation had no effect. EMSA and ChIP analysis demonstrated quercetin-enhanced Sp1 binding to Sp1b. Inhibition of p38 MAPK abrogated basal and quercetin-induced t-PA expression and promoter activity, as well as quercetin-induced Sp1 binding to Sp1b. Quercetin enhanced p38 MAPK and Sp1 physical association, which was similarly diminished by p38 MAPK inhibition. We showed, for the first time, the presence of a functional Sp1-binding element in the t-PA promoter controlling quercetin induction via the p38 MAPK pathway. Understanding these mechanisms may provide new insights into polyphenol cardioprotective effects.