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

Background: 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. Objectives: 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. Methods: 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. Results: 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. Conclusions: 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.