G a q and G bg Regulate PAR-1 Signaling of Thrombin-Induced NF- k B Activation and ICAM-1 Transcription in Endothelial Cells

—As thrombin binding to the G protein–coupled proteinase activated receptor-1 (PAR-1) induces endothelial adhesivity to leukocytes through NF- k B activation and intercellular adhesion molecule-1 (ICAM-1) expression, we determined the signaling pathways mediating the response. Studies showed that the heterotrimeric G proteins, G a q , and the G bg dimer were key determinants of the PAR-1 agonist peptide (TFLLRNPNDK)-induced NF- k B activation and ICAM-1 expression in endothelial cells. Cotransfection of RGS3T, a regulator of G-protein signaling that inhibits G a q , or a -transducin (G a t ), a scavenger of the G bg , markedly decreased NF- k B activity induced by PAR-1 activation. We determined the downstream signaling targets activated by G a q and G bg that mediate NF- k B activation. Expression of the kinase-defective protein kinase C (PKC)- d mutant inhibited NF- k B activation induced by the constitutively active G a q mutant, but had no effect on NF- k B activity induced by G b 1 g 2 . In related experiments, NF- k B as well as ICAM-1 promoter activation induced by G b 1 g 2 were inhibited by the expression of the dominant-negative mutant of 85-kDa regulatory subunit of PI 3-kinase; however, the expression of this mutant had no effect on the response induced by activated G a q . Cotransfection of the catalytically inactive Akt mutant inhibited the NF- k B activation induced by the constitutively active PI 3-kinase mutant as well as that by the activated forms of G a q and PKC- d . These results support a model in which ligation of PAR-1 induces NF- k B activation and ICAM-1 transcription by the engagement of parallel G a q/PKC- d and G bg /PI3-kinase pathways that converge at Akt. ( Circ Res . 2002;91: lll - lll .) 8 PAR-1 activation induces endothelial adhesivity toward human neutrophils (PMNs) and mono-cytes. 1,9 The coupling of PAR-1 with G q , G i , and G12/13 in endothelial cells is required for the activation of multiple cellular responses. 10 In the present study, we addressed the pathways by which PAR-1 signals NF- k B activation and ICAM-1 transcription in endothelial cells. The results demonstrate that ligation of PAR-1 mediates NF- k B activation and ICAM-1 transcription through the heterotrimeric G protein, G a q , and the released G bg dimer. The downstream signaling involves activation of parallel protein kinase C (PKC)- d and PI 3-kinase pathways, which converge at Akt to induce NF- k B–dependent ICAM-1 transcription. confluence. Using this protocol, we achieved a transient transfection efficiency of 21 6 2% (mean 6 SD; n 5 3). Cell extracts were prepared and assayed for reporter gene activity using the Promega Biotech Dual Luciferase Assay System. Firefly ( Pho-tinus pyralis ) luciferase activity was normalized to sea pansy ( Renilla reniformis ) luciferase activity and expressed as relative light units (RLU)/ m g cell protein or fold increase. Protein content was determined using the Bio-Rad protein determination kit. its coupled heterotrimeric G proteins, G i , G q , and G binding for p85, p101. dominant-negative NF- k B