Tumor necrosis factor-α-induced TRPC1 expression amplifies store-operated Ca2+influx and endothelial permeability

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Abstract
We determined the effects of TNF-α on the expression of transient receptor potential channel (TRPC) homologues in human vascular endothelial cells and the consequences of TRPC expression on the endothelial permeability response. We observed that TNF-α exposure increased TRPC1 expression without significantly altering expression of other TRPC isoforms in human pulmonary artery endothelial cells (HPAEC). Because TRPC1 belongs to the store-operated cation channel family, we measured the Ca2+ store depletion-mediated Ca2+ influx in response to thrombin exposure. We observed that thrombin-induced Ca2+ influx in TNF-α-stimulated HPAEC was twofold greater than in control cells. To address the relationship between store-operated Ca2+ influx and TRPC1 expression, we overexpressed TRPC1 by three- to fourfold in the human dermal microvascular endothelial cell line (HMEC) using the TRPC1 cDNA. Thrombin-induced store Ca2+ depletion in these cells caused approximately twofold greater increase in Ca2+ influx than in control cells. Furthermore, the inositol 1,4,5-trisphosphate-sensitive store-operated cationic current was increased greater than twofold in TRPC1-transfected cells compared with control. To address the role of Ca2+ influx via TRPC1 in signaling endothelial permeability, we measured actin-stress fiber formation and transendothelial monolayer electrical resistance (TER) in the TRPC1 cDNA-transfected HMEC and TNF-α-challenged HPAEC. Both thrombin-induced actin-stress fiber formation and a decrease in TER were augmented in TRPC1-overexpressing HMEC compared with control cells. TNF-α-induced increased TRPC1 expression in HPAEC also resulted in marked endothelial barrier dysfunction in response to thrombin. These findings indicate the expression level of TRPC1 in endothelial cells is a critical determinant of Ca2+ influx and signaling of the increase in endothelial permeability.