Ca2+ Entry via TRPC Channels Is Necessary for Thrombin-induced NF-κB Activation in Endothelial Cells through AMP-activated Protein Kinase and Protein Kinase Cδ

The transient receptor potential canonical (TRPC) family channels are proposed to be essential for store-operated Ca2+ entry in endothelial cells. Ca2+ signaling is involved in NF-κB activation, but the role of store-operated Ca2+ entry is unclear. Here we show that thrombin-induced Ca2+ entry and the resultant AMP-activated protein kinase (AMPK) activation targets the Ca2+-independent protein kinase Cδ (PKCδ) to mediate NF-κB activation in endothelial cells. We observed that thrombin-induced p65/RelA, AMPK, and PKCδ activation were markedly reduced by knockdown of the TRPC isoform TRPC1 expressed in human endothelial cells and in endothelial cells obtained from Trpc4 knock-out mice. Inhibition of Ca2+/calmodulin-dependent protein kinase kinase β downstream of the Ca2+ influx or knockdown of the downstream Ca2+/calmodulin-dependent protein kinase kinase β target kinase, AMPK, also prevented NF-κB activation. Further, we observed that AMPK interacted with PKCδ and phosphorylated Thr505 in the activation loop of PKCδ in thrombin-stimulated endothelial cells. Expression of a PKCδ-T505A mutant suppressed the thrombin-induced but not the TNF-α-induced NF-κB activation. These findings demonstrate a novel mechanism for TRPC channels to mediate NF-κB activation in endothelial cells that involves the convergence of the TRPC-regulated signaling at AMPK and PKCδ and that may be a target of interference of the inappropriate activation of NF-κB associated with thrombosis.