Gαq-TRPC6-mediated Ca2+ entry induces RhoA activation and resultant endothelial cell shape change in response to thrombin

RhoA activation and increased intracellular Ca2+ concentration mediated by the activation of transient receptor potential channels (TRPC) both contribute to the thrombin-induced increase in endothelial cell contraction, cell shape change, and consequently to the mechanism of increased endothelial permeability. Herein, we addressed the possibility that TRPC signals RhoA activation and thereby contributes in actinomyosin-mediated endothelial cell contraction and increased endothelial permeability. Transduction of a constitutively active G alpha(q) mutant in human pulmonary arterial endothelial cells induced RhoA activity. Preventing the increase in intracellular Ca2+ concentration by the inhibitor of Gaq or phospholipase C and the Ca2+ chelator, BAPTA-AM, abrogated thrombin-induced RhoA activation. Depletion of extracellular Ca2+ also inhibited RhoA activation, indicating the requirement of Ca2+ entry in the response. RhoA activation could not be ascribed to store-operated Ca2+ (SOC) entry because SOC entry induced with thapsigargin or small interfering RNA-mediated inhibition of TRPC1 expression, the predominant SOC channel in these endothelial cells, failed to alter RhoA activity. However, activation of receptor-operated Ca2+ entry by oleoyl-2-acetyl-sn-glycerol, the membrane permeable analogue of the Ga-q-phospholipase C product diacylglycerol, induced RhoA activity. Receptor-operated Ca2+ activation was mediated by TRPC6 because small interfering RNA-induced TRPC6 knockdown significantly reduced Ca2+ entry. TRPC6 knockdown also prevented RhoA activation, myosin light chain phosphorylation, and actin stress fiber formation as well as inter-endothelial-junctional gap formation in response to either oleoyl-2-acetyl-snglycerol or thrombin. TRPC6-mediated RhoA activity was shown to be dependent on PKC alpha activation. Our results demonstrate that Gaq activation of TRPC6 signals the activation of PKC alpha, and thereby induces RhoA activity and endothelial cell contraction.