CYP2El catalyzes the bioactivation of low molecular weight environmental toxicants, procarcinogens, and therapeutic agents to reactive toxic and/or carcinogenic products and is capable of increasing cellular oxidative stress. Elevated CYP2El levels are associated with increased disease risk, especially in conjunction with xenobiotic exposure. CYP2El mRNA and protein levels are elevated in response to disease (e.g., diabetes), obesity, nutritional status, and alcoholism, and an increased incidence of liver disease and cancer has been reported in chronic alcoholics, and in obese and diabetic individuals. It is therefore of interest to examine the regulation of CYP2El expression by endogenous agents such as hormones, growth factors, and nutritional components. Recent experiments have shown that epidermal growth factor (EGF) negatively regulates CYP2El expression in primary cultured rat hepatocytes, a system in which individual growth factors or hormones can be studied free of the homeostatic control and other confounding endogenous agents present in vivo. EGF (1 - 50 ng/ml) decreased CYP2El mRNA levels by up to 85%, and preliminary studies suggest that a Src kinase, PI 3-kinase signaling pathway mediates the regulation of CYP2El expression. Mechanistic studies suggest that gene transcription and mRNA turnover are involved in the negative regulation of CYP2El by EGF, and gel shift studies implicate RNA-protein binding in CYP2El mRNA stabilization. The hypothesis of this research is that an EGF receptor-activated Src kinase, PI 3-kinase signaling pathway regulates CYP2El expression through transcriptional and post-transcriptional events, affecting mRNA storage and turnover. Thus, the specific aims of this research are 1) to examine the effects of Src kinase, PI 3-kinase system and p7O S6 kinase inhibitors on CYP2El transcription rates and mRNA turnover (stability) in the presence and absence of EGF and HGF in primary cultured rat hepatocytes; 2) to definitively assess, with dominant negative constructs of Src kinase, PI 3-kinase and Akt/Protein kinase B, the role of each of these kinases in the basal and EGF/HGF-mediated regulation of CYP2El expression in primary cultured hepatocytes; 3) to examine the role of cytosolic protein binding to CYP2El mRNA as a mechanism of EGF-mediated destabilization of CYP2El mRNA; and 4) to examine, by microarray analysis, the expression pattern of hepatic genes following treatment of primary cultured rat hepatocytes with EGF/HGF, and alterations in gene expression following treatment with EGF/HGF in the presence of the Src kinase, PI 3-kinase, and p7O S6 kinase inhibitors, and dominant negative kinase constructs. These experiments will hopefully provide a mechanistic understanding of the growth factor-mediated regulation of CYP2El, a gene product which increases the risk of disease associated with xenobiotic exposure.
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