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Ranjana K. Seth, M. S. Reza Haque, Peggy S. Zelenka; Regulation of c-fos Induction in Lens Epithelial Cells by 12(S)HETE-Dependent Activation of PKC. Invest. Ophthalmol. Vis. Sci. 2001;42(13):3239-3246.
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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
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
results. Baicalein blocked EGF-dependent translocation and activation of PKC,
without affecting phosphorylation of Erk1/2. Of several PKC isoforms
investigated (α, βΙ, βII, and γ), only PKCα and βΙΙ
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
PKCγ, 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 PKCα and βΙΙ acts in concert with other EGF-dependent
signals to induce c-fos mRNA.
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