R K Seth1, M S Haque, P S Zelenka. 1. National Eye Institute, National Institutes of Health, Building 6/Rm 214, 6 Center Drive, Bethesda, MD 20892-2730, USA.
Abstract
PURPOSE: 12(S)-Hydroxyeicosatetraenoic acid (12(S)HETE), a 12-lipoxygenase metabolite of arachidonic acid, is required for epidermal growth factor (EGF)-dependent DNA synthesis and c-fos induction in lens epithelial cells. The present study was undertaken to identify signal transduction events upstream of c-fos induction that may be regulated by 12(S)HETE. METHODS: The rabbit lens epithelial cell line, N/N1003A, was cultured in serum-free medium, with or without EGF. Activation of PKC and other selected enzymes was examined in the presence of the lipoxygenase inhibitor baicalein and/or exogenous 12(S)HETE. Relative abundance of PKC isoforms in subcellular fractions was determined by immunoblot analysis with isoform-specific antibodies. PKC activity in subcellular fractions was measured by peptide substrate phosphorylation, with and without pseudosubstrate peptide inhibitor. Phosphorylated enzymes were detected by immunoblot analysis. Relative levels of c-fos mRNA were determined by RT/PCR with internal standard. RESULTS: Baicalein blocked EGF-dependent translocation and activation of PKC, without affecting phosphorylation of Erk1/2. Of several PKC isoforms investigated (alpha, betaI, betaII, and gamma), only PKCalpha and betaII were significantly activated by EGF and inhibited by baicalein. 12(S)HETE, in combination with EGF, countered the effect of lipoxygenase inhibitors on PKC activation, and 12(S)HETE in the absence of EGF stimulated PKC translocation. Also of note, 12(S)HETE alone activated PKCgamma, an isoform that was not significantly activated by EGF. Inhibiting PKC activation with GF109203X blocked induction of c-fos by EGF but did not affect EGF-stimulated phosphorylation of Erk1/2, indicating that the effect of PKC on c-fos induction is independent of the Erk1/2 pathway. CONCLUSIONS: In lens epithelial cells, 12(S)HETE-dependent activation of PKCalpha and betaII acts in concert with other EGF-dependent signals to induce c-fos mRNA.
PURPOSE:12(S)-Hydroxyeicosatetraenoic acid (12(S)HETE), a 12-lipoxygenase metabolite of arachidonic acid, is required for epidermal growth factor (EGF)-dependent DNA synthesis and c-fos induction in lens epithelial cells. The present study was undertaken to identify signal transduction events upstream of c-fos induction that may be regulated by 12(S)HETE. METHODS: The rabbit lens epithelial cell line, N/N1003A, was cultured in serum-free medium, with or without EGF. Activation of PKC and other selected enzymes was examined in the presence of the lipoxygenase inhibitor baicalein and/or exogenous 12(S)HETE. Relative abundance of PKC isoforms in subcellular fractions was determined by immunoblot analysis with isoform-specific antibodies. PKC activity in subcellular fractions was measured by peptide substrate phosphorylation, with and without pseudosubstrate peptide inhibitor. Phosphorylated enzymes were detected by immunoblot analysis. Relative levels of c-fos mRNA were determined by RT/PCR with internal standard. RESULTS:Baicalein blocked EGF-dependent translocation and activation of PKC, without affecting phosphorylation of Erk1/2. Of several PKC isoforms investigated (alpha, betaI, betaII, and gamma), only PKCalpha and betaII were significantly activated by EGF and inhibited by baicalein. 12(S)HETE, in combination with EGF, countered the effect of lipoxygenase inhibitors on PKC activation, and 12(S)HETE in the absence of EGF stimulated PKC translocation. Also of note, 12(S)HETE alone activated PKCgamma, an isoform that was not significantly activated by EGF. Inhibiting PKC activation with GF109203X blocked induction of c-fos by EGF but did not affect EGF-stimulated phosphorylation of Erk1/2, indicating that the effect of PKC on c-fos induction is independent of the Erk1/2 pathway. CONCLUSIONS: In lens epithelial cells, 12(S)HETE-dependent activation of PKCalpha and betaII acts in concert with other EGF-dependent signals to induce c-fos mRNA.
Authors: Jennifer Yeung; Patrick L Apopa; Joanne Vesci; Victor Kenyon; Ganesha Rai; Ajit Jadhav; Anton Simeonov; Theodore R Holman; David J Maloney; Olivier Boutaud; Michael Holinstat Journal: Mol Pharmacol Date: 2011-12-09 Impact factor: 4.436