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-Δ^12,14 PGJ₂ (15d), PGE₂ (E ₂), 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, Δ, 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 Δ 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.