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Inhibition of activator protein‐1 transcription factor activation by omega‐3 fatty acid modulation of mitogen‐activated protein kinase signaling kinases
Author(s) -
Babcock TA,
Kurland A,
Helton WS,
Rahman A,
Anwar KN,
Espat NJ
Publication year - 2003
Publication title -
journal of parenteral and enteral nutrition
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.935
H-Index - 98
eISSN - 1941-2444
pISSN - 0148-6071
DOI - 10.1177/0148607103027003176
Subject(s) - kinase , p38 mitogen activated protein kinases , mapk/erk pathway , protein kinase a , mitogen activated protein kinase kinase , proinflammatory cytokine , phosphorylation , microbiology and biotechnology , mitogen activated protein kinase , activator (genetics) , activating transcription factor 2 , chemistry , biology , biochemistry , inflammation , gene expression , immunology , promoter , receptor , gene
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.