Epidemiological and clinical studies have demonstrated beneficial effects of n-3 polyunsaturated fatty acids (PUFA) in reducing the risks of cardiovascular and inflammatory diseases. The molecular and cellular bases for the beneficial effects are not well understood. Results from our clinical and in vitro studies demonstrated that the expression of the inducible cyclooxygenase (COX-2) is suppressed by n-3 PUFA intake in lipopolysaccharide (LPS)-stimulated monocytes. Similarly, other investigators have demonstrated that n-3 PUFAs suppress the expression of proinflammatory cytokines in monocytes. These effects cannot be explained based on the alteration of substrate availability in eicosanoid biosynthesis , the prevailing hypothesis for the beneficial effects of n-3 PUFA intake. Induction of early response gene (COX-2 cytokines) expression does not require intervening protein synthesis, implying that suppressed expression of COX-2 by dietary n-3 PUFAs is mediated by modulating the signaling pathways. Results from studies by us and others suggest that Lyn (the major Src kinase in monocytes) and p21Ras (Ras) are the upstream components in signaling pathways leading to the expression of COX-2 and TNF-alpha in LPS-stimulated macrophages. The functional activation of Src kinases and Ras requires localization to the plasma membrane, which in turn requires palmitoylation (more accurately acylation by fatty acids) of the signaling molecules. Hence, our hypothesis is that dietary n-3 PUFAs suppress COX-2 expression by modulating membrane localization and functional activation of the signaling molecules;
Specific aims are: 1) to determine whether Lyn and Ras are the upstream components in the LPS-induced signaling pathways leading to the expression of COX-2 in the monocytes cell line transfected with Lyn or Ras mutant cDNAs. 2) To determine whether enriching cell membrane phospholipids with n-3 PUFAs affects membrane localization of Lyn and/or Ras, and in turn modulates the activation of downstream effectors and COX-2 expression in the cells stably transfected with Lyn or Ras mutant cDNAs. 3) To determine whether Lyn and Ras are acylated by fatty acids other than palmitic acid and whether the palmitoylation of Lyn and Ras is altered by n-3 PUFAs. Altered activity of these signaling molecules by n-3 PUFAs will lead to the modulation of downstream events and ultimately cellular responses to extracellular stimuli. Results from these studies can unveil significant new insight toward understanding the mechanism for the suppressed expression of COX-2 and perhaps some cytokines by n-3 PUFAs. Furthermore, results from these studies can provide exciting new information that helps elucidate the molecular and cellular mechanisms(s) by which different dietary fatty acids modify the risks of various chronic diseases.
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