Linoleic acid-induced expression of inducible nitric oxide synthase and cyclooxygenase II via p42/44 mitogen-activated protein kinase and nuclear factor-κB pathway in retinal pigment epithelial cells

Abstract

High linoleic acid (LA) intake is known to correlate with age-related macular degeneration (AMD), but the molecular mechanisms remain unclear. This study was conducted to investigate the effects of LA on expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase II (COX-2) and their associated signaling pathways in human retinal pigment epithelial (RPE) cells. ARPE-19 cells were treated with different concentrations of LA. Expressions of iNOS and COX-2 were examined using semiquantitative reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis. Concentrations of nitric oxide (NO) and prostaglandin E2 (PGE2) in the culture medium were determined by enzyme-link immunosorbent assay (ELISA). Activation of p42/44, p38, JNK mitogen-activated protein kinase (MAPK) and nuclear factors (NF)-κB were evaluated by Western blot analysis and electrophoretic mobility shift assay (EMSA). We found that LA induced expression of iNOS and COX-2 in RPE cells at the mRNA and protein levels in a time-and dose-dependent manner. Upregulation of iNOS and COX-2 resulted in increased production of NO and PGE2. Moreover, LA caused degradation of IκB and increased NF-κB DNA binding activity. Effects of LA-induced iNOS and COX-2 expression were inhibited by a NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC). LA activated p42/44, but not p38 or JNK MAPK. Inhibition of p42/44 activity by PD98059 significantly reduced LA-induced activation of NF-κB. Linoleic acid-induced expression of iNOS and COX-2 as well as PGE2 and NO release in RPE cells were sequentially mediated through activation of p42/p44, MAPK, 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.