Ca2+ influx via TRPC channels induces NF-κB-dependent A20 expression to prevent thrombin-induced apoptosis in endothelial cells

• Published in: American Journal of Physiology: Cell Physiology 2010-03, Vol.298 (3), p.C656-C664
• Main Authors: Thippegowda, Prabhakar B., Singh, Vandana, Sundivakkam, Premanand C., Xue, Jiaping, Malik, Asrar B., Tiruppathi, Chinnaswamy
• Format: Article
• Language: English
• Subjects: Vascular Biology
• ISSN: 0363-6143; 1522-1563
• DOI: 10.1152/ajpcell.00456.2009

NF-κB 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-κB in ECs. Here we show that Ca 2+ influx signal prevents thrombin-induced apoptosis by inducing NF-κB-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-κB 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-κB-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-κB 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.