Abstract
The signaling pathway involved in tumor necrosis factor-alpha (TNF-alpha)-induced intercellular adhesion molecule-1 (ICAM-1) expression was further studied in human A549 epithelial cells. TNF-alpha- or 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ICAM-1 promoter activity was inhibited by a protein kinase C (PKC) inhibitor (staurosporine), tyrosine kinase inhibitors (genistein and herbimycin A), or an Src-specific tyrosine kinase inhibitor (PP2). TNF-alpha- or TPA-induced IkappaBalpha kinase (IKK) activation was also blocked by these inhibitors, which slightly reversed TNF-alpha-induced but completely reversed TPA-induced IkappaBalpha degradation. c-Src and Lyn, two members of the Src kinase family, were abundantly expressed in A549 cells, and their activation by TNF-alpha or TPA was inhibited by the same inhibitors. Furthermore, the dominant-negative c-Src (KM) mutant inhibited induction of ICAM-1 promoter activity by TNF-alpha or TPA. Overexpression of the constitutively active PKC or wild-type c-Src plasmids induced ICAM-1 promoter activity, this effect being inhibited by the dominant-negative c-Src (KM) or IKKbeta (KM) mutant but not by the nuclear factor-kappaB-inducing kinase (NIK) (KA) mutant. The c-Src (KM) mutant failed to block induction of ICAM-1 promoter activity caused by overexpression of wild-type NIK. In co-immunoprecipitation and immunoblot experiments, IKK was found to be associated with c-Src and to be phosphorylated on tyrosine residues after TNF-alpha or TPA treatment. Two tyrosine residues, Tyr188 and Tyr199, near the activation loop of IKKbeta, were identified as being important for NF-kappaB activation. Substitution of these residues with phenylalanines abolished ICAM-1 promoter activity and c-Src-dependent phosphorylation of IKKbeta induced by TNF-alpha or TPA. These data suggest that, in addition to activating NIK, TNF-alpha also activates PKC-dependent c-Src. These two pathways converge at IKKbeta and go on to activate NF-kappaB, via serine phosphorylation and degradation of IkappaB-alpha, and, finally, to initiate ICAM-1 expression.
Highlights
Effect of Inhibitors of protein kinase C (PKC), Tyrosine Kinase, or Src Kinase on the Induction of intercellular adhesion molecule-1 (ICAM-1) Promoter Activity by tumor necrosis factor (TNF)-␣ or TPA in A549 Cells—In our previous study [17], we found that PKC and tyrosine kinase were involved in TNF-␣-induced ICAM-1 expression
The PKC-dependent tyrosine kinase activation pathway is involved in TNF-␣-induced nuclear factor-B (NF-B) activation and ICAM-1 expression in A549 alveolar epithelial cells and in causing monocytes to adhere to these cells [17]
TNF-␣- and TPA-induced ICAM-1 promoter activity were both inhibited by PKC, tyrosine kinase, and Src kinase inhibitors, indicating the possible involvement of the Src tyrosine kinase family downstream of PKC activation in the induction of ICAM-1 expression
Summary
Overexpression of the constitutively active PKC␣ or wild-type c-Src plasmids induced ICAM-1 promoter activity, this effect being inhibited by the dominant-negative c-Src (KM) or IKK (KM) mutant but not by the nuclear factor-Binducing kinase (NIK) (KA) mutant. The intracellular signaling pathways by which TNF-␣ and IL-1 cause ICAM-1 expression in A549 human alveolar epithelial cells have been explored and found to involve the sequential activation of protein kinase C␣ (PKC␣), protein-tyrosine kinase, nuclear factor-B-inducing kinase (NIK), and IB kinase  (IKK) [16, 17].
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