Activator protein-1 mediates shear stress-induced prostaglandin D synthase gene expression endothelial cells

Objective-We attempted to determine the molecular mechanism of fluid shear stress-induced lipocalin-type prostaglandin D synthase (L-PGDS) expression in vascular endothelial cells. Methods and Results-We examined the promoter region of the L-PGDS gene by loading laminar shear stress (20 dyne/cm(2)), using a parallel-plate flow chamber, on endothelial cells transfected with luciferase reporter vectors containing the 5'-flanking regions of the human L-PGDS gene. A deletion mutant analysis revealed that a shear stress-responsive element resided in the region between -2607 and -2523 bp. A mutation introduced into the putative binding site for activator protein-1 (AP-1) within this region eliminated the response to shear stress. In an electrophoretic mobility shift assay, shear stress stimulated nuclear protein binding to the AP-1 binding site, which was supershifted by antibodies to c-Fos and c-Jun. Shear stress elevated the c-Jun phosphorylation level in a time-dependent manner, similar to that of L-PGDS gene expression. SP600125, a c-Jun N-terminal kinase inhibitor, decreased the c-Jun phosphorylation, DNA binding of AP-1, and L-PGDS expression induced by shear stress. Additionally, an mRNA chase experiment using actinomycin D demonstrated that shear stress did not stabilize L-PGDS mRNA. Conclusions-Shear stress induces L-PGDS expression by transcriptional activation through the AP-1 binding site.