Prostaglandins as Negative Regulators Against Lipopolysaccharide, Lipoteichoic Acid, and Peptidoglycan-Induced Inducible Nitric Oxide Synthase/Nitric Oxide Production Through Reactive Oxygen Species–Dependent Heme Oxygenase 1 Expression in Macrophages

Although prostaglandins (PGs) were reported to exert proinflammatory and anti-inflammatory effects in macrophages, their action mechanisms remain unclear. The effects of PGs including PGJ2 (J2), Δ12-PGJ2 (Δ12), 15-deoxy-Δ12,14 PGJ2 (15d), PGE2 (E2), and PGF2α (F2α) on lipopolysaccharide (LPS)–, lipoteichoic acid (LTA)–, and peptidoglycan (PGN)–induced inducible nitric oxide (NO) synthase (iNOS)/NO production by RAW264.7 macrophages were investigated. First, we found that induction of cyclooxygenase 2 (COX-2) protein occurred at a time earlier than that of heme oxygenase 1 (HO-1) protein, and the addition of the COX-2 inhibitor NS398 reduced HO-1 protein expression in LPS-, LTA-, and PGN-treated RAW264.7 macrophages. Incubation of RAW264.7 macrophages with the indicated PGs showed that J2, Δ12, and 15d significantly induced HO-1 protein expression; however, E2 and F2α did not. Heme oxygenase 1 protein induced by J2, Δ12, and 15d was inhibited by the transcriptional inhibitor, actinomycin (Act) D; the translational inhibitor, cycloheximide; and the antioxidant, N-acetyl cysteine (NAC). Increases in intracellular peroxide levels by J2, Δ12, and 15d were detected via a 2’,7’™-dichlorofluorescein diacetate (DCFH-DA) analysis, and they were prevented by the addition of NAC. In addition, J2, Δ12, and 15d produced significant inhibition of LPS-, LTA-, and PGN-induced iNOS protein and NO production by RAW264.7 cells, in accordance with increased HO-1 protein expression. Reductions of LPS-, LTA-, and PGN-induced phosphorylated c-Jun N-terminal kinase, c-Jun protein, and activator protein 1 luciferase activity by J2, Δ12, and 15d were identified, and the addition of the HO-1 inhibitor, tin protoporphyrin, reversed the inhibitory effects of Δ12 and 15d on LPS- and LTA-induced iNOS/NO, phosphorylated c-Jun N-terminal kinase, and c-Jun protein expressions by macrophages. Knockdown of HO-1 protein expression by HO-1 small interfering RNA blocked Δ12 and 15d inhibition of LPS- and LTA-induced events. Moreover, the compound, cyclopentenone (CP), which mimics the CP moiety of 15d, and its analog cyclohexenone were used, and cyclohexenone showed more potent induction of the HO-1 protein with effective inhibition of LPS-, LTA-, and PGN-induced iNOS/NO production than CP in macrophages. Reactive oxygen species–dependent HO-1 protein expression by PGs, which inhibited LPS-, LTA-, and PGN-induced iNOS/NO production, was identified in macrophages. ABBREVIATIONS Act D—actinomycin D AP-1—activator protein-1 BCIP—5-bromo-4-chloro-3-indolyl phosphate CH—cyclohexenone CHX—cycloheximide COX—cyclooxygenase CP—cyclopentenone DCFH-DA—2’,7’-dichlorofluorescein diacetate HO-1—heme oxygenase 1 iNOS—inducible nitric oxide synthase LPS—lipopolysaccharide LTA—lipoteicoic acid MTT—3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide NAC—N-acetyl cysteine NO—nitric oxide PGD2—prostaglandin D2 PGE2—prostaglandin E2 PGJ2—prostaglandin J2 PGF2α—prostaglandin F2α PGN—peptidoglycan ROS—reactive oxygen species siRNA—small interfering RNA SnPP—tin protoporphyrin