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Pranab K. Mukherjee, John Cefalu, Nicolas G. Bazan; The p38 Kinase Involved In Interleukin-1β/lipopolysaccharide-mediated Cyclooxygenase-2 Induction Is Selectively Inhibited By Neuroprotectin D1 (NPD1) In RPE Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(14):6586.
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Cyclooxygenase-2 (COX-2) catalyzes the synthesis of prostaglandins and is a pro-inflammatory effector as well as a regulator of central nervous system functions. Several eye diseases, including glaucoma and age-related macular degeneration, involve pro-inflammatory signaling. An 830 bp-containing COX-2 promoter construct linked to a luciferase reporter gene is upregulated by interleukin (IL)-1β, and a 50 nM concentration of the lipid mediator neuroprotectin D1 (NPD1) inhibits COX-2 promoter gene expression (Mukherjee et al, Proc Natl Acad Sci, 2004). In the present study, we investigated the bioactivity of NPD1 (50 nM) on the p38 MAP kinase (MAPK) in the induction of the pro-inflammatory gene COX-2 in a human retinal pigment epithelial cell line, ARPE-19.
ARPE-19 cells were grown for 72 h, serum-starved for 8 h, and exposed to either NPD1 or p38 MAPK inhibitor SB203085 before IL-1β or LPS treatment for 8 h. Cells were harvested and COX-2 levels were measured by Western blot analysis using COX-2 specific antibody. In some experiments, ARPE-19 cells were exposed to lipoxin A4 or 15-epi-lipoxin A4. Fugene-6 was used to transfect ARPE-19 cells. A promoterless β-galactosidase was used as transfection control.
ARPE-19 cells, pre-exposed to the p38 MAPK inhibitor SB203085 or NPD1, did not lead to COX-2 induction induced either by IL-1β or LPS, implicating that p38 MAPK is a mediator. Also, NPD1 and SB203085 inhibited the IL-1β- and LPS-induced COX-2 expression in 830bp COX-2 promoter linked to luciferase reporter gene-transfected RPE cells. In addition, two other anti-inflammatory lipid mediators derived from arachidonic acid (lipoxin A4 and 15-epi-lipoxin A4) inhibited the expression of COX-2 at a low concentration (100 nM). However, at a very high concentration (500 nM), lipoxin A4 and 15-epi-lipoxin A4 were able to inhibit COX-2 expression by nearly 50%. Moreover, NPD1 and SB203085 inhibited the p38 MAPK phosphorylation induced by IL-1β and LPS.
These results identify a novel site of NPD1 action on p38 MAPK phosphorylation mediated by IL-1β and LPS in ARPE-19 cells. As a consequence, COX-2 expression is modulated. These observations have implications on the modulation of pro-inflammatory signaling and other inflammatory resolution events relevant for RPE cell integrity.
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