Thrombin induces proteinase-activated receptor-1 gene expression in endothelial cells via activation of Gi-linked Ras/mitogen-activated protein kinase pathway.
We addressed the mechanisms of restoration of cell surface proteinase-activated receptor-1 (PAR-1) by investigating thrombin-activated signaling pathways involved in PAR-1 re-expression in endothelial cells. Exposure of endothelial cells transfected with PAR-1 promoter-luciferase reporter construct to either thrombin or PAR-1 activating peptide increased the steady-state PAR-1 mRNA and reporter activity, respectively. Pretreatment of reporter-transfected endothelial cells with pertussis toxin or co-expression of a minigene encoding 11-amino acid sequence of COOH-terminal Galphai prevented the thrombin-induced increase in reporter activity. Pertussis toxin treatment also prevented thrombin-induced MAPK phosphorylation, indicating a role of Galphai in activating the downstream MAPK pathway. Expression of constitutively active Galphai2 mutant or Gbeta1gamma2 subunits increased reporter activity 3-4-fold in the absence of thrombin stimulation. Co-expression of dominant negative mutants of either Ras or MEK1 with the reporter construct inhibited the thrombin-induced PAR-1 expression, whereas constitutively active forms of either Ras or MEK1 activated PAR-1 expression in the absence of thrombin stimulation. Expression of dominant negative Src kinase or inhibitors of phosphoinositide 3-kinase also prevented the MAPK activation and PAR-1 expression. We conclude that thrombin-induced activation of PAR-1 mediates PAR-1 expression by signaling through Gi1/2 coupled to Src and phosphoinositide 3-kinase, and thereby activating the downstream Ras/MAPK cascade.
Ellis
C A
CA
Department of Pharmacology, College of Medicine, University of Illinois, Chicago, Illinois 60612, USA.
Malik
A B
AB
Gilchrist
A
A
Hamm
H
H
Sandoval
R
R
Voyno-Yasenetskaya
T
T
Tiruppathi
C
C
D000595:Amino Acid Sequence; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*; D004789:Enzyme Activation; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D008969:Molecular Sequence Data; D037342:Pertussis Toxin; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D044463:Receptor, PAR-1; D018179:Receptors, Thrombin / Q000235:genetics*; D013917:Thrombin / Q000494:pharmacology*; D015533:Transcriptional Activation; D010566:Virulence Factors, Bordetella / Q000494:pharmacology; D018631:ras Proteins / Q000378:metabolism*
D012333:RNA, Messenger; D044463:Receptor, PAR-1; D018179:Receptors, Thrombin; D010566:Virulence Factors, Bordetella; D037342:Pertussis Toxin; D019869:Phosphatidylinositol 3-Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D013917:Thrombin; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D018631:ras Proteins