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P. Hu, M. Dwyer, A. Wielgus, S. Cousins, G. Malek; Investigating the Effect of Poly-unsaturated Fatty Acids on PPAR activity in ARPE19 Cells. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1863.
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Dysregulated turnover of extracellular matrix (ECM) molecules play a role in age-related macular degeneration (ARMD). Epidemiological studies have shown an increased risk associated with dietary intake of omega-6 vs -3 poly-unsaturated fatty acids (PUFA). We investigated the endogenous activity of PUFA ligand-activated nuclear hormone receptors, PPAR alpha, delta/beta and gamma in ARPE19 cells in the presence or absence of fatty acids.
ARPE19 cells were chronically fed arachidonic acid (omega-6), docosahexanoic acid (omega-3) PUFAs or fatty acid free control medium for at least three weeks. Transcription factor DNA binding activity was determined in nuclear extracts. Luciferase reporter construct DR1-LUC, pCMV-beta-galactosidase normalization plasmid and mammalian expression vectors for human PPARs were transfected in ARPE19 cells in the presence or absence of PUFAs, or the receptor ligands clofibrate, GW7845, GW647 or carbaprostacyclin to measure PPAR activity. For knockdown, cells were transfected with siRNA oligos to each of the PPARs. Controls included scramble siRNA oligos and mock transfection. RNA and protein cellular expression of ECM molecules fibronectin, collagen IV, vitronectin and laminin in transfected cells was determined.
Treatment of ARPE19 cells with PUFAs did not affect PPAR DNA binding activity. Endogenous activity of PPARs in ARPE19 cells was as follows: alpha>delta/beta>gamma. Activity of PPAR was 2.5 fold higher in arachidonic acid treated cells vs. control. 90% knockdown of PPAR alpha and delta/beta and 78% knockdown of PPAR gamma was seen with after treatment with specific RNAi oligomers. Over 40-70% decrease in RNA expression for fibronectin and collagen and 30-50% decrease in expression for laminin and vitronectin was seen in cells treated with either PUFA-PPAR alpha or delta knockdown, respectively.
All three PPARs are endogenously active in ARPE19 cells, which increases following treatment with an omega-6 PUFA. Knockdown of PPAR alpha and delta more so than gamma in PUFA treated ARPE19 cells results in decreased expression of ECM molecules tested, suggesting that endogenous PPARs may be involved in ECM expression. We hyopothesize that PPARs may alter ECM turnover by their regulatory actions on intermediary profibrogenic growth factors like TGF beta.
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