TNF-α-Induced Cyclooxygenase-2 Expression in Human Lung Epithelial Cells: Involvement of the Phospholipase C-γ2, Protein Kinase C-α, Tyrosine Kinase, NF-κB-Inducing Kinase, and I-κB Kinase 1/2 Pathway

摘要

TNF-α induced a dose- and time-dependent increase in cyclooxygenase-2 (COX-2) expression and PGE2 formation in human NCI-H292 epithelial cells. Immunofluorescence staining demonstrated that COX-2 was expressed in cytosol and nuclear envelope. Tyrosine kinase inhibitors (genistein or herbimycin) or phosphoinositide-specific phospholipase C inhibitor (U73122) blocked TNF-α-induced COX-2 expression. TNF-α also stimulated phosphatidylinositol hydrolysis and protein kinase C (PKC) activity, and both were abolished by genistein or U73122. The PKC inhibitor, staurosporine, also inhibited TNF-α-induced response. The 12- O -tetradecanoylphorbol 13-acetate (TPA), a PKC activator, also stimulated COX-2 expression, this effect being inhibited by genistein or herbimycin. NF-κB DNA-protein binding and COX-2 promoter activity were enhanced by TNF-α, and these effects were inhibited by genistein, U73122, staurosporine, or pyrolidine dithiocarbamate. TPA stimulated both NF-κB DNA-protein binding and COX-2 promoter activity, these effects being inhibited by genistein, herbimycin, or pyrolidine dithiocarbamate. The TNF-α-induced, but not the TPA-induced, COX-2 promoter activity was inhibited by phospholipase C-γ2 mutants, and the COX-2 promoter activity induced by either agent was attenuated by dominant-negative mutants of PKC-α, NF-κB-inducing kinase, or I-κB (inhibitory protein that dissociates from NF-κB) kinase (IKK)1 or 2. IKK activity was stimulated by both TNF-α and TPA, and these effects were inhibited by staurosporine or herbimycin. These results suggest that, in NCI-H292 epithelial cells, TNF-α might activate phospholipase C-γ2 via an upstream tyrosine kinase to induce activation of PKC-α and protein tyrosine kinase, resulting in the activation of NF-κB-inducing kinase and IKK1/2, and NF-κB in the COX-2 promoter, then initiation of COX-2 expression and PGE2 release.