Abstract
In Syrian hamster embryo (SHE) fibroblasts, epidermal growth factor receptor (EGFR) tyrosine kinase activity regulates the metabolism of endogenous linoleic acid to (13S)-hydroperoxyoctadecadienoic acid (13S)-HPODE). (13S)-HPODE stimulates EGF-dependent mitogenesis in a SHE cell phenotype, which expresses tumor suppressor genes (supB+), but was not effective in a variant that does not express these suppressor genes (supB-). In the present study, we have investigated the potential effects of this lipid metabolite on the EGFR signaling pathways in these two SHE cell lines. Treatment of quiescent SHE cells with EGF produced a rapid, transient increase in the tyrosine phosphorylation of EGFR. Dependence on EGF concentration for EGFR tyrosine phosphorylation was similar in both SHE cell lines, but a more prolonged phosphorylation was detected in the supB- variant. Incubation of supB+ cells with (13S)-HPODE and EGF increased EGFR autophosphorylation and tyrosine phosphorylation on several signaling proteins with Src homology-2 domains including GTPase-activating protein. The lipid metabolite did not significantly alter EGF-dependent tyrosine phosphorylation in the supB- variant. Tyrosine phosphorylation of mitogen-activated protein (MAP) kinase was also measured. The addition of (13S)-HPODE increased the extent and duration of MAP kinase tyrosine phosphorylation in supB+ cells but not in the supB- variant. MAP kinase activity in supB+ cells, as measured in immunoprecipitates from cells after the addition of EGF, was increased by the presence of (13S)-HPODE. The addition of (13S)-HPODE did not directly alter EGFR kinase activity or the internalization of the EGFR. However, the addition of (13S)-HPODE to supB+ cells extended the tyrosine phosphorylation of the EGFR in response to EGF. The dephosphorylation of the EGFR was measured directly, and a slower rate was observed in the supB- compared with the supB+ cells. Incubation of the supB+ cells with (13S)-HPODE attenuated the dephosphorylation of the EGFR. Thus, (13S)-HPODE stimulates EGF-dependent mitogenesis and up-regulation of EGF-dependent tyrosine phosphorylation by inhibiting the dephosphorylation of the EGFR. This study shows that a metabolite of an essential dietary fatty acid, linoleic acid, can modulate tyrosine phosphorylation and activity of key signal transduction proteins in a growth factor mitogenic pathway.
Highlights
From the Eicosanoid Biochemistry Section, Laboratory of Molecular Carcinogenesis, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709 and ‡Division of Basic Medical Sciences, Mercer University School of Medicine, Macon, Georgia 31207
The (13R)-hydroxyoctadecadienoic acid (HODE) enantiomer and the analogous 15lipoxygenase metabolites of arachidonic acid, (15S)-HPETE/15HETE, were not active [11]. This finding suggests a specific interaction of (13S)-HPODE with either a receptor or with a intracellular signal transduction protein of the EGF signaling pathway. These results indicate that the formation of (13S)-HPODE/ (13S)-HODE is a component of the EGF signaling pathway and this lipid metabolite potentiates EGF-dependent mitogenesis in the supBϩ cell line
EGF-stimulated Tyrosine Phosphorylation in Syrian hamster embryo fibroblasts (SHE) Cells— The addition of EGF to serum-starved SHE cells stimulated a rapid autophosphorylation of the epidermal growth factor receptor (EGFR) (170 kDa) as measured by Western analysis using an antibody for phosphorylated tyrosine residues (anti-Tyr(P))
Summary
Materials—Bovine serum albumin (BSA), IBR modified Dulbecco’s modified Eagle’s medium (IBR), phosphate-buffered saline (PBS), trypsin, and gentamicin were from Life Technologies, Inc. The medium was aspirated, and the cells were rinsed with 10 ml of PBS containing 0.1% BSA and 2 mM sodium vanadate and lysed with 0.8 ml of ice-cold lysis buffer: 1% Triton X-100, 50 mM Tris, pH 8.5, 150 mM NaCl, 5 mM EDTA, 5 mM PMSF, 50 mM NaF, 2.0 mM sodium vanadate, 20 g/ml leupeptin, 20 g/ml aprotinin. The reaction was stopped by the addition of 5% trichloroacetic acid, vortexed, and maintained on ice. The samples were centrifuged for 5 min at 15,000 ϫ g and the supernatant spotted on P-81 phosphocellulose chromatography paper. MAP Kinase Activity Assay—Treated cells were washed twice with ice-cold PBS and lysed in ice-cold lysis buffer: 50 mM HEPES, pH 7.4, 50 mM sodium pyrophosphate, 50 mM sodium fluoride, 50 mM sodium chloride, 5 mM EDTA, 5 mM EGTA, 2 mM sodium orthovanadate, 0.5 mM PMSF, 10 g/ml leupeptin, and 0.01% Triton X-100. Gels were loaded with lysate from 4 ϫ 105 cells
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