The applicants have shown that macrophages (Mp) from old mice produce more LPS-stimulated PGE2 than those from young mice due to increased activity of the enzyme cyclooxygenase (COX) which a result of higher expression of the inducible form of COX, COX-2. Vitamin E (E) reduces this age-related increase in PGE2 production by decreasing the activity, but not expression, of COX-2. The E-induced decrease in PGE2 contributes to E's enhancement of T cell function in the aged.
The specific aims of this proposal are: 1) To determine the mechanism of age-associated increase in COX-2 mRNA level by examining transcription rate and mRNA stability in young and old Mp. Since Mp from old mice have higher ceramide levels than those of young mice and ceramide has been shown to augment expression of COX-2, the applicants will 2) Determine if ceramide is involved in the regulation of age-related increases in COX-2 mRNA. This will be tested by examining the effect of age on the level of sphingomyelin, ceramide and sphingosine, and the activity of the enzyme neutral sphingomyelinase (NSMase). Furthermore, the effect of increasing young Mp ceramide levels on PGE2 production and COX-2 mRNA expression will be determined. Since GSH, at physiological concentration, inhibits NSMase, they will determine the mechanism of increased ceramide level in old Mp by measuring GSH, NSMase activity, PGE2, COX activity, and COX-2 mRNA levels in the presence or absence of GSH or the GSH synthesis inhibitor, BSO. 3) To determine the signaling mechanism of age-associated increases in COX-2 mRNA expression. Since ceramide has been shown to regulate mitogen-activated protein kinase (MAPK) pathways (SAPK/JNK, p38, ERK), which have been implicated in regulation of COX-2, the applicants propose that the age-related increase in ceramide levels will alter MAPK-mediated signal transduction and the nuclear transcription factor(s) activity regulated by them, resulting in higher expression of COX-2. This will be tested by comparing MAPK specific activity between young and old Mp, and confirming the roles of particular pathways by using specific inhibitors. A link with ceramide will be determined using exogenously added membrane-permeable ceramide analogues. The relevance of particular pathway(s) to age-associated upregulation of COX-2 will be investigated by transient transfection studies. 4) To determine the mechanism of E-induced decrease in COX activity. Since Mp from old mice have higher NO production and NO and its metabolite peroxy nitrite (ONOO) have been shown to increase activity of COX without affecting COX expression, and because E decreases NO production in old Mp, the applicants propose that E decreases COX activity of old mice through reduction of NO and thus ONOO. The applicants will test this by comparing the level of NO and O2 in Mp from young and old mice fed 30 or 500 ppm E. The effect of NO and ONOO generators and inhibitors on PGE2 production and COX activity of Mp from old mice fed 30 or 500 ppm E will be determined. Since higher levels of PGE2 have been implicated in the pathogenesis of immune and inflammatory diseases, the results from these studies are anticipated to aid in developing interventions to reverse and/or delay age-associated dysregulation of immune and inflammatory responses.
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