Open AccessCa2+influx via TRPC channels induces NF-κB-dependent A20 expression to prevent thrombin-induced apoptosis in endothelial cells
Author(s) -
Prabhakar B. Thippegowda,
Vandana Singh,
Premanand Sundivakkam,
Jiaping Xue,
Asrar B. Malik,
Chinnaswamy Tiruppathì
Publication year - 2010
Publication title -
american journal of physiology. cell physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.432
H-Index - 181
eISSN - 1522-1563
pISSN - 0363-6143
DOI - 10.1152/ajpcell.00456.2009
Subject(s) - gene knockdown , trpc1 , thrombin , small interfering rna , trpc , microbiology and biotechnology , small hairpin rna , apoptosis , chemistry , rna interference , transfection , biology , receptor , transient receptor potential channel , gene , immunology , biochemistry , rna , platelet
NF-kappaB signaling is known to induce the expression of antiapoptotic and proinflammatory genes in endothelial cells (ECs). We have shown recently that Ca(2+) influx through canonical transient receptor potential (TRPC) channels activates NF-kappaB in ECs. Here we show that Ca(2+) influx signal prevents thrombin-induced apoptosis by inducing NF-kappaB-dependent A20 expression in ECs. Knockdown of TRPC1 expressed in human umbilical vein ECs with small interfering RNA (siRNA) suppressed thrombin-induced Ca(2+) influx and NF-kappaB activation in ECs. Interestingly, we observed that thrombin induced >25% of cell death (apoptosis) in TRPC1-knockdown ECs whereas thrombin had no effect on control or control siRNA-transfected ECs. To understand the basis of EC survival, we performed gene microarray analysis using ECs. Thrombin stimulation increased only a set of NF-kappaB-regulated genes 3- to 14-fold over basal levels in ECs. Expression of the antiapoptotic gene A20 was the highest among these upregulated genes. Like TRPC1 knockdown, thrombin induced apoptosis in A20-knockdown ECs. To address the importance of Ca(2+) influx signal, we measured thrombin-induced A20 expression in control and TRPC1-knockdown ECs. Thrombin-induced p65/RelA binding to A20 promoter-specific NF-kappaB sequence and A20 protein expression were suppressed in TRPC1-knockdown ECs compared with control ECs. Furthermore, in TRPC1-knockdown ECs, thrombin induced the expression of proapoptotic proteins caspase-3 and BAX. Importantly, thrombin-induced apoptosis in TRPC1-knockdown ECs was prevented by adenovirus-mediated expression of A20. These results suggest that Ca(2+) influx via TRPC channels plays a critical role in the mechanism of cell survival signaling through A20 expression in ECs.