Tumor necrosis factor-α induces nuclear factor-κB-dependent TRPC1 expression in endothelial cells

Abstract

We investigated the role of tumor necrosis factor-α (TNF-α) in activating the store-operated Ca2+ channels in endothelial cells via the expression of transient receptor potential channel (TRPC) isoforms. We observed that TNF-α exposure of human umbilical vein endothelial cells resulted in TRPC1 mRNA and protein expression, whereas it had no effect on TRPC3, TRPC4, or TRPC5 expression. The TRPC1 expression was associated with increased Ca2+ influx after intracellular Ca2+ store depletion with either thrombin or thapsigargin. We cloned the 5′-regulatory region of the human TRPC1 (hTRPC1) gene which contained a TATA box and CCAAT sequence close to the transcription initiation site. We also identified four nuclear factor-κB (NF-κB)-binding sites in the 5′-regulatory region. To address the contribution of NF-κB in the mechanism of TRPC1 expression, we determined the effects of TNF-α on expression of the reporter luciferase after transfection of hTRPC1 promoter-luciferase (hTRPC1-Pro-Luc) construct in the human dermal microvascular endothelial cell line. Reporter activity increased >4-fold at 4 h after TNF-α challenge. TNF-α-induced increase in reporter activity was markedly reduced by co-expression of either kinase-defective IKKβ kinase mutant or non-phosphorylatable IκB mutant. Treatment with NEMO-binding domain peptide, which prevents NF-κB activation by selectively inhibiting IKKγ interaction with IKK complex, also blocked the TNF-α-induced TRPC1 expression. Thus, TNF-α induces TRPC1 expression through an NF-κB-dependent pathway in endothelial cells, which can trigger augmented Ca2+ entry following Ca2+ store depletion. The augmented Ca2+ entry secondary to TRPC1 expression may be an important mechanism of endothelial injury induced by TNF-α.