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Role of the JNK pathway in thrombin-induced ICAM-1 expression in endothelial cells
Narimasa Miho1, Takafumi Ishida, Noriko Kuwaba
1Department of Medicine and Molecular Science, Graduate School of Biomedical Sciences, Hiroshima University, Minami-ku, Japan.
Insights
Thrombin increases ICAM-1 expression via JNK and NF-kappaB signaling pathways. This process involves G-protein coupled receptors, Ras, Rac1, and Src kinases in endothelial cells.
Area of Science:
- Endothelial cell biology
- Molecular signaling
- Inflammation research
Background:
- Thrombin stimulates leukocyte-ICAM-1-mediated adherence to endothelial cells.
- NF-kappaB is a known regulator of ICAM-1, but other pathways may contribute.
- Mitogen-activated protein (MAP) kinases are potential mediators of this process.
Purpose of the Study:
- To investigate the role of MAP kinases (ERK1/2, JNK, p38) in thrombin-induced ICAM-1 expression.
- To elucidate the signaling mechanisms downstream of thrombin in endothelial cells.
Main Methods:
- Western blot analysis of ICAM-1 expression.
- Luciferase assays for promoter activity.
- Pharmacological inhibition of signaling pathways (JNK, MEK, p38, NF-kappaB, Galpha(q)).
- Gene constructs (dominant-negative Ras, Rac1, Rho, betaARK1ct, Csk) and kinase assays for JNK activity.
Main Results:
- JNK inhibition significantly reduced thrombin-induced ICAM-1 expression.
- MEK and p38 inhibition had minimal effects.
- Combined JNK and NF-kappaB inhibition completely blocked ICAM-1 expression.
- Galpha(q) inhibition, dominant-negative Ras/Rac1, betaARK1ct, and Csk all inhibited JNK activation and ICAM-1 expression.
Conclusions:
- JNK, in conjunction with NF-kappaB, regulates thrombin-induced ICAM-1 expression.
- The mechanism is dependent on Galpha(q), Gbetagamma, Ras, Rac1, and Src kinase family members.
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.
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