Inhibition of activator protein-1 transcription factor activation by omega-3 fatty acid modulation of mitogen-activated protein kinase signaling kinases

Abstract

BACKGROUND: Lipopolysaccharide (LPS)-stimulated macrophages (Mphi) produce excess tumor necrosis factor (TNF), and the direct inhibition of IkappaB phosphorylation and its subsequent separation from the nuclear factor kappaB (NFkappaB)-IkappaB complex has been experimentally supported as a mechanism for omega-3 fatty acid (FA) inhibition of this TNF response. However, TNF production is a "late" event in the LPS-induced Mpsi inflammatory cascade, and in addition to NFkappaB-associated pathways, a separate transcription factor, activator protein-1 (AP-1) is an important pathway for Mpsi proinflammatory cytokine production. The mitogen-activated protein kinase (MAPK) cascade regulates both NFkappaB-IkappaB--and AP-1-associated gene transcription through several cross-amplifying phosphorylation kinases, specifically p44/42 [ie, extracellular signal-regulated kinase (ERK) 1/2], p38, and c//jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK). The activation of these kinases occurs in the proximal MAPK cascade and activation modulates AP-1 activation. In this set of experiments, it was hypothesized that inhibition of MAPK signaling phosphorylation kinases by omega-3 fatty acids in a model of LPS-stimulated Mphi(s) would alter the activation of the proinflammatory cytokine transcription factor AP-1. METHODS: RAW 264.7 cells were pretreated with a sterile, commercially available, pharmaceutical grade omega-3 FA emulsion, equivalent grade omega-6 FA emulsion, or Dulbecco's modified eagles medium (media alone) for 4 hours. Cells were washed twice and exposed to LPS for 15 minutes. Total cell lysates were collected, and both total and phosphorylated portions of the p44/42, p38, and JNK/SAPK proteins were determined by Western blotting. AP-1 nuclear translocation was determined by electromobility shift assay. RESULTS: Phosphorylation of p44/42 and JNK/SAPK proteins of the MAPK pathways in LPS-stimulated Mpsi(s) was significantly reduced by omega-3 FA treatment compared with Mphi treated with omega-6 FA or media alone. In contrast, phosphorylation of p38 was not inhibited in the presence of omega-3 or (omega-6 FA treatment compared with media alone. Omega-3 FA pretreatment inhibited AP-1 activation. CONCLUSIONS: omega-3 FA inhibited p44/42 and JNK/SAPK phosphorylation; however, p38 remained unchanged. Phosphorylation of p44/42 and JNK/SAPK are the immediate prior steps in AP-1 activation. Attenuated AP-1 activation and subsequent attenuated gene-level proinflammatory cytokine elaboration is anticipated after inhibition of these MAPK intermediates and is confirmed by the reduction in AP-1 activity. These results provide further evidence for the transcriptional level regulation in the elaboration of proinflammatory cytokines by omega-3 FA in this Mphi model.

Clinically, ω-3 fatty acids have been demonstrated to have anti-inflammatory properties. To further define the mechanism(s) for these clinically noted effects, the role of ω-3 fatty acids on the activation of MAPK cascade intermediates and resultant transcription factor AP-1 were examined. In this study, inhibition of macrophage MAPK-associated signal transduction and AP-1 activation by ω-3 fatty acids was observed, further supporting the potential clinical benefits for ω-3 FA as nutritional anti-inflammatory agents.