CYP2E1 levels are elevated in response to disease (e.g., diabetes), obesity, and nutritional status. As CYP2E1 catalyzes the conversion of low molecular weight environmental toxicants and procarcinogens, as well as therapeutic agents, to reactive toxic and/or carcinogenic products, is active in the metabolism of eicosanoids, and is capable of increasing cellular oxidative stress, there is an increase disease risk associated with elevated CYP2E1 levels and/or xenobiotic exposure. In general, there is an increased incidence of liver disease and cancer in obese individuals, chronic alcoholics, and diabetic individuals. Recent experiments have shown that insulin regulates CYP2E1 mRNA and protein levels in primary cultured hepatocytes in a manner mechanistically identical to that in vivo. Low insulin levels (1 nM) elevate CYP2E1 mRNA and protein levels about 10-fold relative to high insulin concentrations (1 microM). Mechanistic studies implicate the Src kinase, PI 3-kinase, Akt kinase/Protein kinase B, p70 S6 kinase signaling cascade in regulation of CYP2E1 expression and polyribosomal distribution, and mRNA EMSA studies incriminate cytoplasmic storage and translation in CYP2E1 mRNA stabilization. The hypothesis of this research is that the Src kinase, PI 3-kinase, Akt kinase/Protein kinase B, p70 S6 kinase signaling pathway regulates CYP2E1 gene expression via post-transcriptional events affecting cytoplasmic mRNA storage and turnover. Thus, the specific objectives of this research are: 1) To examine the effects of Src kinase, PI 3-kinase, Akt/Protein kinase B and p70 S6 kinase inhibitors and PP1, PP2A, and PP2B phosphatase inhibitors on CYP2E1 transcription rates and mRNA turnover (stability) in the presence and absence of insulin; 2) To definitively assess with dominant negative Src kinase, PI 3-kinase, and Akt/Protein kinase B constructs the role of Src kinase, PI 3-kinase, and Akt/Protein kinase B on CYP2E1 mRNA levels in primary cultured hepatocytes; 3) To assess the role of kinase signaling in protein binding to CYP2E1 mRNA constructs; 4) To examine whether altered kinase, phosphatase activity affects cytoplasmic CYP2E1 mRNA storage. The experiments will provide a mechanistic understanding of the seminal observation that Src kinase, PI 3-kinase, Akt kinase/Protein kinase B, p70 S6 kinase signaling regulates CYP2E1 expression. The results of this research will provide valuable information on how hormonal status affects the elevation of gene products that increase the risk of disease associated with either xenobiotic exposure or with the conversion of endogenous fatty acids, eicosanoids or steroids to physiologically active modulators of cell and organ function.
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