Ca2+ influx induced by protease-activated receptor-1 activates a feed-forward mechanism of TRPC1 expression via nuclear factor-κB activation in endothelial cells

Thrombin activation of protease-activated receptor-1 induces Ca2+ influx through store-operated cation channel TRPC1 in endothelial cells. We examined the role of Ca2+ influx induced by the depletion of Ca2+ stores in signaling TRPC1 expression in endothelial cells. Both thrombin and a protease-activated receptor-1-specific agonist peptide induced TRPC1 expression in human umbilical vein endothelial cells, which was coupled to an augmented store-operated Ca2+ influx and increase in endothelial permeability. To delineate the mechanisms of thrombin-induced TRPC1 expression, we transfected in endothelial cells TRPC1-promoter-luciferase ( TRPC1-Pro-Luc) construct containing multiple nuclear factor-kappa B ( NF-kappa B) binding sites. Co-expression of dominant negative I kappa B alpha mutant prevented the thrombin-induced increase in TRPC1 expression, indicating the key role of NF-kappa B activation in mediating the response. Using TRPC1 promoter-deletion mutant constructs, we showed that NF-kappa B binding sites located between - 1623 and - 871 in the TRPC1 5'-regulatory region were required for thrombin-induced TRPC1 expression. Electrophoretic mobility shift assay utilizing TRPC1 promoter-specific oligonucleotides identified that the DNA binding activities of NF-kappa B to NF-kappa B consensus sites were located in this domain. Supershift assays using NF-kappa B protein-specific antibodies demonstrated the binding of p65 homodimer to the TRPC1 promoter. Inhibition of store Ca2+ depletion, buffering of intracellular Ca2+, or down-regulation of protein kinase C alpha downstream of Ca2+ influx all blocked thrombin-induced NF-kappa B activation and the resultant TRPC1 expression in endothelial cells. Thus, Ca2+ influx via TRPC1 is a critical feed-forward pathway responsible for TRPC1 expression. The NF-kappa B-regulated TRPC1 expression may be an essential mechanism of vascular inflammation and, hence, a novel therapeutic target.