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

Ni Cheng,Stephen M. Vogel,Richard D. Ye,Chinnaswamy Tiruppathi,Marc Freichel,Asrar B. Malik,Prabhakar B. Thippegowda,Angela M. Bair,Veit Flockerzi,Yanni Yu

doi:10.1074/jbc.m803984200

Abstract

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-kappaB 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 Cdelta (PKCdelta) to mediate NF-kappaB activation in endothelial cells. We observed that thrombin-induced p65/RelA, AMPK, and PKCdelta 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 beta downstream of the Ca2+ influx or knockdown of the downstream Ca2+/calmodulin-dependent protein kinase kinase beta target kinase, AMPK, also prevented NF-kappaB activation. Further, we observed that AMPK interacted with PKCdelta and phosphorylated Thr505 in the activation loop of PKCdelta in thrombin-stimulated endothelial cells. Expression of a PKCdelta-T505A mutant suppressed the thrombin-induced but not the TNF-alpha-induced NF-kappaB activation. These findings demonstrate a novel mechanism for TRPC channels to mediate NF-kappaB activation in endothelial cells that involves the convergence of the TRPC-regulated signaling at AMPK and PKCdelta and that may be a target of interference of the inappropriate activation of NF-kappaB associated with thrombosis.

Highlights

Activation of the transcription factor nuclear factor-␬B (NF␬B)3 is a double-edged sword
Expression of a PKC␦-T505A mutant suppressed the thrombin-induced but not the TNF-␣-induced NF-␬B activation. These findings demonstrate a novel mechanism for transient receptor potential canonical (TRPC) channels to mediate NF-␬B activation in endothelial cells that involves the convergence of the TRPC-regulated signaling at AMPK and PKC␦ and that may be a target of interference of the inappropriate activation of NF-␬B associated with thrombosis
Thrombin-induced PKC␦ Phosphorylation at Thr505 Is Mediated Ca2ϩ Influx-dependent AMPK Activation Leads to Throm- by AMPK and Not by PDK1—To confirm the relationship bin-induced PKC␦ Activation—Since we observed that throm- between AMPK and PKC␦ in thrombin-induced NF-␬B activabin-induced p65/RelA activation was prevented in both TRPC1 tion in endothelial cells, first, we determined thrombin-inand AMPK␣ knockdown human pulmonary arterial endothelial cells (HPAECs), we investigated the possi- duced association of AMPK with PKC␦ by immunoprecipitability that thrombin-induced Ca2ϩ influx signal is a prerequisite tion

Summary

Introduction

Activation of the transcription factor nuclear factor-␬B (NF␬B)3 is a double-edged sword. In contrast to control cells, thrombin-induced I␬B␣ degradation and p65 nuclear translocation were prevented in TRPC1 knockdown cells (Fig. 1D), suggesting that Ca2ϩ influx signaling is required for thrombininduced p65/RelA activation in HPAECs. Impairment of p65/RelA Nuclear Translocation in Trpc4Ϫ/Ϫ

Results

Conclusion