Calcium signaling mediates nuclear factor-kappa B activation in endothelial cells

Thrombin ligation of the protease-activated receptor (PAR-1) signals the activation of the transcription factor NF-kappaB in endothelial cells (EC) that contributes to initializing and amplifying innate immunity and tissue inflammation. We show that Ca2+ influx via Ca 2+ store depletion-activated TRPC channels and the resultant activation of AMP-activated protein kinase (AMPK) are essential in mediating thrombin-induced NF-kappaB activation. NF-kappaB activation was markedly reduced following knockdown of the TRPC isoform, TRPC1, in human EC, and also was reduced in EC obtained from TRPC4 knockout mice (a TRPC1-related channel). Inhibition of Ca2+/Calmodulin-dependent protein kinase kinase beta (CaMKKbeta) downstream of Ca2+ influx or knockdown of the downstream CaMKKbeta target, AMPK, also prevented NF-kappaB activation. We further demonstrated that Ca2+-independent isoform of protein kinase C, PKCdelta, is downstream of AMPK and is required for NF-kappaB activation. The results indicate that Ca2+ influx via TRPC1 is a critical feed-forward pathway responsible for TRPC1 expression. Further, we identified the critical NF-kappaB binding sites on the TRPC1 5' regulatory region that are required for thrombin-induced TRPC1 expression. These data demonstrate that Ca 2+ influx via TRPC is responsible for NF-kappaB activation in response to thrombin exposure in EC. Additionally we illustrate a novel relationship between the coagulation cascade and the activation of NF-kappaB mediated by the thrombin-induced Ca2+ influx via TRPC1 and the activation of the AMPK/PKCdelta pathway.