Galpha(q) and Gbetagamma regulate PAR-1 signaling of thrombin-induced NF-kappaB activation and ICAM-1 transcription in endothelial cells

Circ Res. 2002 Sep 6;91(5):398-405. doi: 10.1161/01.res.0000033520.95242.a2.

Abstract

As thrombin binding to the G protein-coupled proteinase activated receptor-1 (PAR-1) induces endothelial adhesivity to leukocytes through NF-kappaB activation and intercellular adhesion molecule-1 (ICAM-1) expression, we determined the signaling pathways mediating the response. Studies showed that the heterotrimeric G proteins, Galpha(q), and the Gbetagamma dimer were key determinants of the PAR-1 agonist peptide (TFLLRNPNDK)-induced NF-kappaB activation and ICAM-1 expression in endothelial cells. Cotransfection of RGS3T, a regulator of G-protein signaling that inhibits Galpha(q), or alpha-transducin (Galpha(t)), a scavenger of the Gbetagamma, markedly decreased NF-kappaB activity induced by PAR-1 activation. We determined the downstream signaling targets activated by Galpha(q) and Gbetagamma that mediate NF-kappaB activation. Expression of the kinase-defective protein kinase C (PKC)-delta mutant inhibited NF-kappaB activation induced by the constitutively active Galpha(q) mutant, but had no effect on NF-kappaB activity induced by Gbeta(1)gamma(2). In related experiments, NF-kappaB as well as ICAM-1 promoter activation induced by Gbeta(1)gamma(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 Galpha(q). Cotransfection of the catalytically inactive Akt mutant inhibited the NF-kappaB activation induced by the constitutively active PI 3-kinase mutant as well as that by the activated forms of Galpha(q) and PKC-delta. These results support a model in which ligation of PAR-1 induces NF-kappaB activation and ICAM-1 transcription by the engagement of parallel Galphaq/PKC-delta and Gbetagamma/PI3-kinase pathways that converge at Akt.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Endothelium, Vascular / cytology
  • Endothelium, Vascular / drug effects*
  • Endothelium, Vascular / metabolism
  • Heterotrimeric GTP-Binding Proteins / genetics
  • Heterotrimeric GTP-Binding Proteins / physiology*
  • Humans
  • I-kappa B Kinase
  • Intercellular Adhesion Molecule-1 / genetics*
  • Isoenzymes / metabolism
  • Mutation
  • NF-kappa B / drug effects*
  • NF-kappa B / genetics
  • NF-kappa B / metabolism
  • Oligopeptides / pharmacology
  • Phosphatidylinositol 3-Kinases / metabolism
  • Plasmids / genetics
  • Protein Kinase C / metabolism
  • Protein Kinase C-delta
  • Protein Serine-Threonine Kinases / metabolism
  • Proto-Oncogene Proteins / genetics
  • Proto-Oncogene Proteins / metabolism
  • Proto-Oncogene Proteins c-akt
  • Receptor, PAR-1
  • Receptors, Thrombin / genetics
  • Receptors, Thrombin / physiology*
  • Signal Transduction / drug effects
  • Thrombin / pharmacology*
  • Transcription, Genetic / drug effects

Substances

  • Isoenzymes
  • NF-kappa B
  • Oligopeptides
  • Proto-Oncogene Proteins
  • Receptor, PAR-1
  • Receptors, Thrombin
  • Intercellular Adhesion Molecule-1
  • AKT1 protein, human
  • Protein Serine-Threonine Kinases
  • Proto-Oncogene Proteins c-akt
  • CHUK protein, human
  • I-kappa B Kinase
  • IKBKB protein, human
  • IKBKE protein, human
  • PRKCD protein, human
  • Protein Kinase C
  • Protein Kinase C-delta
  • Thrombin
  • Heterotrimeric GTP-Binding Proteins