title: Ca2+ influx induced by protease-activated receptor-1 activates a feed-forward mechanism of TRPC1 expression via nuclear factor-kappaB activation in endothelial cells.
abstract: Thrombin activation of protease-activated receptor-1 induces Ca(2+) influx through store-operated cation channel TRPC1 in endothelial cells. We examined the role of Ca(2+) influx induced by the depletion of Ca(2+) 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 Ca(2+) 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-kappaB (NF-kappaB) binding sites. Co-expression of dominant negative IkappaBalpha mutant prevented the thrombin-induced increase in TRPC1 expression, indicating the key role of NF-kappaB activation in mediating the response. Using TRPC1 promoter-deletion mutant constructs, we showed that NF-kappaB 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-kappaB to NF-kappaB consensus sites were located in this domain. Supershift assays using NF-kappaB protein-specific antibodies demonstrated the binding of p65 homodimer to the TRPC1 promoter. Inhibition of store Ca(2+) depletion, buffering of intracellular Ca(2+), or down-regulation of protein kinase Calpha downstream of Ca(2+) influx all blocked thrombin-induced NF-kappaB activation and the resultant TRPC1 expression in endothelial cells. Thus, Ca(2+) influx via TRPC1 is a critical feed-forward pathway responsible for TRPC1 expression. The NF-kappaB-regulated TRPC1 expression may be an essential mechanism of vascular inflammation and, hence, a novel therapeutic target.
authors.affiliation: Department of Pharmacology and Center for Lung and Vascular Biology, University of Illinois College of Medicine, Chicago, Illinois 60612.
authors.forename: Biman C
authors.initials: BC
authors.lastname: Paria
authors.affiliation: Department of Pharmacology and Center for Lung and Vascular Biology, University of Illinois College of Medicine, Chicago, Illinois 60612.
authors.forename: Angela M
authors.initials: AM
authors.lastname: Bair
authors.affiliation: Department of Pharmacology and Center for Lung and Vascular Biology, University of Illinois College of Medicine, Chicago, Illinois 60612.
authors.forename: Jiaping
authors.initials: J
authors.lastname: Xue
authors.affiliation: Department of Pharmacology and Center for Lung and Vascular Biology, University of Illinois College of Medicine, Chicago, Illinois 60612.
authors.forename: Yanni
authors.initials: Y
authors.lastname: Yu
authors.affiliation: Department of Pharmacology and Center for Lung and Vascular Biology, University of Illinois College of Medicine, Chicago, Illinois 60612.
authors.forename: Asrar B
authors.initials: AB
authors.lastname: Malik
authors.affiliation: Department of Pharmacology and Center for Lung and Vascular Biology, University of Illinois College of Medicine, Chicago, Illinois 60612. Electronic address: tiruc@uic.edu.
authors.forename: Chinnaswamy
authors.initials: C
authors.lastname: Tiruppathi
mesh_terms: D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D015536:Down-Regulation; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D005799:Genes, Dominant; D006801:Humans; D009154:Mutation; D016328:NF-kappa B / Q000378:metabolism*; D009841:Oligonucleotides / Q000737:chemistry; D011401:Promoter Regions, Genetic; D044463:Receptor, PAR-1 / Q000378:metabolism*; D050052:TRPC Cation Channels / Q000378:metabolism; D013917:Thrombin / Q000737:chemistry / Q000378:metabolism
chemical_list: D016328:NF-kappa B; D009841:Oligonucleotides; D044463:Receptor, PAR-1; D050052:TRPC Cation Channels; C096351:transient receptor potential cation channel, subfamily C, member 1; D013917:Thrombin; D002118:Calcium