Role of the JNK pathway in thrombin-induced ICAM-1 expression in endothelial cells.
OBJECTIVE
Thrombin induces leukocyte adherence to endothelial cells via increased expression of intercellular adhesion molecule-1 (ICAM-1). Although ICAM-1 expression is regulated by NF-kappaB, recent studies have suggested that additional signaling mechanisms may also be involved. The goal of this study was to determine whether mitogen-activated protein (MAP) kinases, including extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), and p38 MAP kinase (p38), mediate thrombin-induced ICAM-1 expression in endothelial cells.


METHODS
Western blot analysis using anti-ICAM-1 antibody and luciferase assays were performed in cultured endothelial cells after addition of signal transduction inhibitors or transfection of various gene constructs. JNK kinase activity was determined by a kinase assay using c-Jun as a substrate or by Western blot analysis with anti-phospho-JNK antibody.


RESULTS
Treatment of endothelial cells with the JNK-specific inhibitors, SP600125 or JNK inhibitory peptide 1 (JNKI1), resulted in a significant decrease in thrombin-induced ICAM-1 expression as demonstrated by Western blot analysis (67 +/- 3% and 72 +/- 7%, respectively). In contrast, inhibitors of MEK and p38 had only minimal effect. The combination of SP600125 and the NF-kappaB inhibitor, BAY11-7082, resulted in complete inhibition of thrombin-induced ICAM-1 expression. The Galpha(q) inhibitor, YM-254890, inhibited thrombin-induced JNK activation and ICAM-1 expression. Dominant-negative Ras and Rac1, but not Rho, inhibited thrombin-induced JNK activation and ICAM-1 promoter activity. Finally, thrombin-induced JNK activation and ICAM-1 promoter activity were inhibited by betaARK1ct (a Gbetagamma subunit scavenger) and Csk.


CONCLUSIONS
These data suggest that, in concert with NF-kappaB, JNK regulates thrombin-induced ICAM-1 expression by a mechanism that is dependent on Galpha(q), Gbetagamma, Ras, Rac1 and the Src kinase family.
Department of Medicine and Molecular Science, Graduate School of Biomedical Sciences, Hiroshima University, Minami-ku, Japan.
Narimasa
N
Miho
Takafumi
T
Ishida
Noriko
N
Kuwaba
Mari
M
Ishida
Keiko
K
Shimote-Abe
Kumiko
K
Tabuchi
Tetsuya
T
Oshima
Masao
M
Yoshizumi
Kazuaki
K
Chayama
D000818:Animals; D001013:Aorta, Thoracic; D015153:Blotting, Western / Q000379:methods; D002417:Cattle; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000378:metabolism*; D015870:Gene Expression; D047468:Immunoprecipitation; D018799:Intercellular Adhesion Molecule-1 / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D048670:MAP Kinase Kinase 4 / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000502:physiology*; D013917:Thrombin / Q000378:metabolism*; D018631:ras Proteins / Q000378:metabolism; D019061:src-Family Kinases / Q000378:metabolism
D016328:NF-kappa B; D018799:Intercellular Adhesion Molecule-1; D019061:src-Family Kinases; D048670:MAP Kinase Kinase 4; D013917:Thrombin; D018631:ras Proteins