Ca2+ entry via TRPC channels is necessary for thrombin-induced NF-kappaB activation in endothelial cells through AMP-activated protein kinase and protein kinase Cdelta.
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.
Bair
Angela M
AM
Department of Pharmacology and Center for Lung and Vascular Biology, College of Medicine, University of Illinois, Chicago, Illinois 60612 and Institut fur Pharmakologie und Toxikologie, Universitat des Saarlandes, 66421 Homburg, Germany.
Thippegowda
Prabhakar B
PB
Department of Pharmacology and Center for Lung and Vascular Biology, College of Medicine, University of Illinois, Chicago, Illinois 60612 and Institut fur Pharmakologie und Toxikologie, Universitat des Saarlandes, 66421 Homburg, Germany.
Freichel
Marc
M
Department of Pharmacology and Center for Lung and Vascular Biology, College of Medicine, University of Illinois, Chicago, Illinois 60612 and Institut fur Pharmakologie und Toxikologie, Universitat des Saarlandes, 66421 Homburg, Germany.
Cheng
Ni
N
Department of Pharmacology and Center for Lung and Vascular Biology, College of Medicine, University of Illinois, Chicago, Illinois 60612 and Institut fur Pharmakologie und Toxikologie, Universitat des Saarlandes, 66421 Homburg, Germany.
Ye
Richard D
RD
Department of Pharmacology and Center for Lung and Vascular Biology, College of Medicine, University of Illinois, Chicago, Illinois 60612 and Institut fur Pharmakologie und Toxikologie, Universitat des Saarlandes, 66421 Homburg, Germany.
Vogel
Stephen M
SM
Department of Pharmacology and Center for Lung and Vascular Biology, College of Medicine, University of Illinois, Chicago, Illinois 60612 and Institut fur Pharmakologie und Toxikologie, Universitat des Saarlandes, 66421 Homburg, Germany.
Yu
Yanni
Y
Department of Pharmacology and Center for Lung and Vascular Biology, College of Medicine, University of Illinois, Chicago, Illinois 60612 and Institut fur Pharmakologie und Toxikologie, Universitat des Saarlandes, 66421 Homburg, Germany.
Flockerzi
Veit
V
Department of Pharmacology and Center for Lung and Vascular Biology, College of Medicine, University of Illinois, Chicago, Illinois 60612 and Institut fur Pharmakologie und Toxikologie, Universitat des Saarlandes, 66421 Homburg, Germany.
Malik
Asrar B
AB
Department of Pharmacology and Center for Lung and Vascular Biology, College of Medicine, University of Illinois, Chicago, Illinois 60612 and Institut fur Pharmakologie und Toxikologie, Universitat des Saarlandes, 66421 Homburg, Germany.
Tiruppathi
Chinnaswamy
C
Department of Pharmacology and Center for Lung and Vascular Biology, College of Medicine, University of Illinois, Chicago, Illinois 60612 and Institut fur Pharmakologie und Toxikologie, Universitat des Saarlandes, 66421 Homburg, Germany. Electronic address: tiruc@uic.edu.
D055372:AMP-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002118:Calcium; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D006490:Hemostatics / Q000494:pharmacology*; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D051745:Protein Kinase C-delta / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics; D050052:TRPC Cation Channels / Q000235:genetics / Q000378:metabolism*; D013917:Thrombin / Q000494:pharmacology*; D013927:Thrombosis / Q000235:genetics / Q000378:metabolism; D051996:Transcription Factor RelA / Q000235:genetics / Q000378:metabolism*
D006490:Hemostatics; C498143:RELA protein, human; C498144:Rela protein, mouse; D050052:TRPC Cation Channels; C489571:TRPC4 ion channel; D051996:Transcription Factor RelA; C096351:transient receptor potential cation channel, subfamily C, member 1; C497259:Prkcd protein, mouse; C497258:PRKCD protein, human; D051745:Protein Kinase C-delta; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D055372:AMP-Activated Protein Kinases; D013917:Thrombin; D002118:Calcium