Interferon-gamma-induced epithelial ICAM-1 expression and monocyte adhesion. Involvement of protein kinase C-dependent c-Src tyrosine kinase activation pathway.
Interferon-gamma (IFN-gamma) induced intercellular adhesion molecule-1 (ICAM-1) expression in human NCI-H292 epithelial cells, as shown by enzyme-linked immunosorbent assay and immunofluorescence staining. The enhanced ICAM-1 expression resulted in increased adhesion of U937 cells to NCI-H292 cells. Tyrosine kinase inhibitors (genistein or herbimycin), Src family inhibitor (PP2), or a phosphatidylinositol-phospholipase C inhibitor (U73122) attenuated the IFN-gamma-induced ICAM-1 expression. Protein kinase C (PKC) inhibitors (staurosporine or Ro 31-8220) also inhibited IFN-gamma-induced response. 12-O-Tetradecanoylphorbol-13-acetate (TPA), a PKC activator, stimulated ICAM-1 expression; this effect was inhibited by tyrosine kinase or Src inhibitor. ICAM-1 promoter activity was enhanced by IFN-gamma and TPA in cells transfected with pIC339-Luc, containing the downstream NF-kappaB and gamma-activated site (GAS) sites, but not in cells transfected with GAS-deletion mutant, pIC135 (DeltaAP2). Electrophoretic gel mobility shift assay demonstrated that GAS-binding complexes in IFN-gamma-stimulated cells contained STAT1alpha. The IFN-gamma-induced ICAM-1 promoter activity was inhibited by tyrosine kinase inhibitors, a phosphatidylinositol-phospholipase C inhibitor, or PKC inhibitors, and the TPA-induced ICAM-1 promoter activity was also inhibited by tyrosine kinase inhibitors. Cotransfection with a PLC-gamma2 mutant inhibited IFN-gamma- but not TPA-induced ICAM-1 promoter activity. However, cotransfection with dominant negative mutants of PKCalpha or c-Src inhibited both IFN-gamma- and TPA-induced ICAM-1 promoter activity. The ICAM-1 promoter activity was stimulated by cotransfection with wild type PLC-gamma2, PKCalpha, c-Src, JAK1, or STAT1. An immunocomplex kinase assay showed that both IFN-gamma and TPA activated c-Src and Lyn activities and that these effects were inhibited by staurosporine and herbimycin. Thus, in NCI-H292 epithelial cells, IFN-gamma activates PLC-gamma2 via an upstream tyrosine kinase to induce activation of PKC-alpha and c-Src or Lyn, resulting in activation of STAT1alpha, and GAS in the ICAM-1 promoter, followed by initiation of ICAM-1 expression and monocyte adhesion.
Chang
Ya-Jen
YJ
Department of Pharmacology, College of Medicine, National Taiwan University, No. 1 Jen-Ai Road, 1st Section, Taipei 10018, Taiwan.
Holtzman
Michael J
MJ
Chen
Ching-Chow
CC
D000818:Animals; D002448:Cell Adhesion; D002467:Cell Nucleus / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D005799:Genes, Dominant; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000096:biosynthesis*; D007371:Interferon-gamma / Q000378:metabolism* / Q000494:pharmacology; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D009000:Monocytes / Q000166:cytology*; D009154:Mutation; D051966:Phospholipase C gamma; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D014407:Tumor Cells, Cultured; D010738:Type C Phospholipases / Q000378:metabolism; D020298:U937 Cells; D019061:src-Family Kinases / Q000378:metabolism
D004791:Enzyme Inhibitors; D007527:Isoenzymes; D011994:Recombinant Proteins; D018799:Intercellular Adhesion Molecule-1; D007371:Interferon-gamma; D008156:Luciferases; D011505:Protein-Tyrosine Kinases; D016392:Proto-Oncogene Proteins pp60(c-src); C060902:lyn protein-tyrosine kinase; D019061:src-Family Kinases; D011493:Protein Kinase C; D010738:Type C Phospholipases; D051966:Phospholipase C gamma