Abstract
RhoA activation and increased intracellular Ca(2+) 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 Galphaq mutant in human pulmonary arterial endothelial cells induced RhoA activity. Preventing the increase in intracellular Ca2+ concentration by the inhibitor of Galphaq 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 storeoperated 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 Galphaq-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-sn-glycerol or thrombin. TRPC6-mediated RhoA activity was shown to be dependent on PKCalpha activation. Our results demonstrate that Galphaq activation of TRPC6 signals the activation of PKCalpha, and thereby induces RhoA activity and endothelial cell contraction.
Highlights
TRPC1 expression, the predominant store-operated Ca2ϩ (SOC) channel in these endothelial cells, failed to alter RhoA activity
Thrombin by binding to the endothelial cell surface protease-activated receptor-1 (PAR-1)2 induces a signaling cascade resulting in the development of minute inter-endothelial junctional gaps that lead to increased endothelial permeability, the hallmark of tissue inflammation [1]
Because at the mRNA level TRPC1 and TRPC6 are more abundantly expressed in human endothelial cells than other cell types [11, 18, 23, 26], in the present study we addressed the roles of TRPC1 and TRPC6 in regulating RhoA activation
Summary
TRPC1 expression, the predominant SOC channel in these endothelial cells, failed to alter RhoA activity. We determined the role of Ca2ϩ entry mediated by SOC and ROC channels in inducing RhoA activation in response to thrombin. Inhibition of TRPC6 expression prevented OAG-induced Ca2ϩ entry as compared with cells transfected with control siRNA
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