The long term objective is to determine whether dietary n-3 polyunsaturated fatty acids (PUFA) modulate the expression of key enzymes involved in the receptor mediated signal transduction, and in turn suppress the expression of key enzymes involved in eicosanoid biosynthesis. Results from these studies will provide crucial information in understanding the mechanism of some of biological actions of dietary n- 3 PUFA that can not be explained on the basis of the alteration of substrate availability for eicosanoid biosynthesis. Furthermore, results from these studies can open exciting possibility that modifying the composition of dietary fatty acids (and perhaps other nutrients) can be used as a supplemental means to ameliorate certain chronic diseases for which altered expression of key enzymes (i.e. cyclooxygenase (COX)) involved in synthesizing inflammatory mediators is beneficial.
The specific aims are: 1) to determine whether dietary n-3 PUFA suppress the expression of the rate limiting enzyme (COX) in prostaglandin synthesis, and 5-lipoxygenase (5-LO) in blood monocytes of humans consuming dietary n-3 or n-6 PUFA. 2) To determine whether suppressed expression of the enzymes by n-3 PUFA is mediated by altered activity and/or expression of protein kinase C (PKC) in cells in culture. (The hypothesis is that decreased expression of these enzymes by n-3 PUFA is mediated by suppressed activity and/or expression of PKC, which in turn results from reduced level of arachidonic acid (PKC activator) released during the signal transduction). 3) To determine whether or not altered expression of COX, 5-LO or PKC by dietary n-3 PUFA results from specific change in the level of transcription of corresponding gene instead of altered degradation of mRNA or enzyme proteins. The expression of both constitutive and inducible COX and 5-LO will be assessed in stimulated human monocytes or in macrophages and smooth muscle cells (SMC) (derived from rats fed n-3 PUFA) by determining: i) levels of mRNA by reverse transcription and polymerase chain reaction ii) the rate of enzyme protein synthesis by metabolic labeling and immunoprecipitation using specific antibodies iii) enzyme activities. In animal studies, comprehensive time courses for the expression of the enzymes and translocation of PKC will be performed in macrophages and SMC cultured in the media containing homologous serum. Nuclear runoff transcription assay and pulse chase experiments will be carried out to assess the level of transcription and the half lives of de novo synthesized enzyme.
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