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C.-H. Yang, I.-M. Fang, C.-M. Yang, M.-S. Chen; Linoleic Acid Induces Expression of Monocyte Chemoattractant Protein 1 via PI3K, PKC, IKK, and NF-b Pathway in Retinal Pigment Epihelial Cells. Invest. Ophthalmol. Vis. Sci. 2009;50(13):2337.
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High linoleic acid (LA) intake is known to correlate with age-related macular degeneration, but the molecular mechanisms remain unclear. Our previous studies have shown LA could induce inflammatory genes expression in human retinal pigment epithelial (RPE) cells. Therefore, this study was conducted to investigate the effects of LA on expression of monocyte chemoattractant protein 1(MCP-1) and its associated signaling pathways in human RPE cells.
ARPE-19 cells were treated with different concentrations of LA. Expressions of MCP-1 were examined using semiquantitative reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis. Activation of phosphatidylinositol 3 kinase (PI3K), protein kinase C (PKC), I-ΚB kinase (IKK) and nuclear factors (NF)-ΚB were evaluated by Western blot analysis, electrophoretic mobility shift assay (EMSA) and luciferase reporter gene assay.
LA induced expression MCP-1 in RPE cells at the mRNA and protein levels and phosphoylation of PKCΔ and IKK, but not Akt, MAPK p38 and JNK, in a time-and dose-dependent manner, determined by RT-PCR, Western blotting and ELISA. Effects of LA induced MCP-1 expression were attenuated by inhibitor of PI3K (LY294002), PKCΔ (staurosporine), IKK (wedelactone) and NF-ΚB (BAY11-7082 and PDTC) or transfection of RPE cells with siRNA for PI3K, PKCΔ, and IKK. Moreover, LA increased NF-ΚB DNA binding activity confirmed by EMSA and luciferase reporter gene assay which was done by transfection of RPE cells with wild-type and NF-ΚB binding site mutated MCP-1 promoter plasmid.
LA-induced expression of MCP-1 in RPE cells were sequentially mediated through activation of PI3K, PKCΔ, IKK and then NF-ΚB. These results may provide new insights into both mechanisms of LA action on RPE cells and pathogenesis of age-related macular degeneration.
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