Nitroalkenes Suppress Lipopolysaccharide-Induced Signal Transducer and Activator of Transcription Signaling in Macrophages: A Critical Role of Mitogen-Activated Protein Kinase Phosphatase 1

Abstract

Nitration products of unsaturated fatty acids are formed via NO-dependent oxidative reactions and appear to be a new class of endogenous antiinflammatory mediators. Nitroalkene derivatives of nitrated linoleic acid (LNO2) and nitrated oleic acid (OA-NO2) alleviate inflammatory responses in macrophages, but the underlying mechanisms remain to be fully defined. Herein we report that LNO2 and OA-NO2 suppress proinflammatory signal transducer and activator of transcription (STAT) signaling in macrophages. In RAW264.7 cells, a murine macrophage cell line, LNO2 and OA-NO2 inhibited the lipopolysaccharide (LPS)-induced STAT1 phosphorylation and the STAT1-dependent transcriptional activity, thereby suppressing expression of its target gene such as iNOS and MCP-1. The nitroalkene-mediated inhibition of STAT1 activity was not affected by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (a NO scavenger), GW9662 (a peroxisome proliferator-activated receptor-γ-specific antagonist) or glutathione (an antioxidant), suggesting an underlying mechanism independent of NO, peroxisome proliferator-activated receptor-γ, or thio-nitralkylation. In contrast, LNO2 or OA-NO2 alone up-regulated both mRNA and protein levels of MAPK phosphatase 1 (MKP-1) and strongly augmented the LPS-induced MKP-1 protein expression. Knockdown of MKP-1 by MKP-1 small interfering RNA enhanced the LPS-induced STAT1 phosphorylation, suggesting that MKP-1 acts as a negative regulator for LPS-induced STAT signaling. In addition, the nitroalkene-mediated inhibitory effects on STAT1 phosphorylation, iNOS expression, and MCP-1 secretion were also largely attenuated by the MKP-1 small interfering RNA approach. Taken together, our data demonstrate that nitroalkenes inhibit proinflammatory STAT signaling through inducting MKP-1 in macrophages.