Stress–induced Modulation of Cyclooxygenase–3 (COX–3) Gene Expression In Human Retinal Pigment Epithelial Cells

Abstract

Abstract: : Purpose: Cyclooxygenase (COX) enzymes catalyze the cyclooxygenation and endoperoxidation of arachidonic acid that results in prostaglanidn H₂, the common precursor of prostaglandins and thromboxane. This study examined COX–1, COX–2 and COX–3 (the most recently described member of this gene family) RNA and protein levels in ARPE–19 cells exposed to stress–inducing cytokines and proinflammatory mediators including IL–1ß, TNFα, H₂0₂, and A2E. A2E (N–retinylidene–N–retinylethanolamine) accumulates in RPE from abcr –/– mice and in Stargardt's disease. Macular degeneration in this disease may result from A2E–triggered toxicity to the RPE. Methods: ARPE–19 cells were grown to 70% confluence in DMEM–F12 medium supplemented with 10% FBS. Cells were serum starved for 8 hrs in DMEM–F12 at which times IL–1ß, (10 ng/ml), TNFα (10 ng/ml), H₂0₂ (0.6 uM), TNFα/H₂0₂ and A2E (10 uM) were added. Total RNA and whole cellular proteins were isolated at 8 hrs. Cyclooxygenase–1, –2 and –3 RNA and protein levels were quantitated using RT–PCR and Western analysis, respectively. Results: IL–1ß, H₂O₂, TNFα, TNF/H₂O₂ or A2E induced COX–2 mRNA 2.5–, 3.0–, 2.1–, 5.4–, and 6.3–fold over controls, however, no significant effects were seen in COX–1 or COX–3 RNA abundance. Interestingly, H₂O_2, TNFα/H₂O₂ or A2E increased COX–3 protein 1.3–, 1.8– or 1.9–fold over controls, respectively. Conclusions:COX–2 expression was induced in cytokine, peroxide and A2E stressed ARPE–19 cells. COX–1 and COX–3 have equivalent gene promoters (since COX–3 RNA is derived from alternate splicing of the COX–1 transcript) and no significant changes in COX–1 or COX–3 RNA levels were observed. Lack of induction of COX–1 or COX–3 RNA message by H₂O_2, TNFα/H₂O₂ or A2E, coupled to increases in COX–3 protein under identical treatment conditions, suggests that significant post–transcriptional or post–translational controls may modulate COX–3 gene expression in the RPE. These events may regulate COX–3 gene expression during pro–inflammatory and/or pro–oxidative stresses in RPE cells, during retinal degenerations and other retinal diseases.

Keywords: inflammation • oxidation/oxidative or free radical damage • retinal pigment epithelium