Cytochrome P450(CYP) 2E1 is responsible for the metabolism of a variety of endogenous agents and xenobiotics, including carcinogens, therapeutic agents and toxicants. Pathophysiological conditions (e.g., diabetes, fasting, high fat diets, etc.) elevate CYP2E1 mRNA and protein levels, causing oxidative cellular stress and increased bioactivation of substrates into toxic or carcinogenic products. Preliminary results show that glucocorticoid receptor (GR) agonists (Dex) and antagonists (RU486) increase and decrease, respectively, CYP2E1 mRNA levels in a concentration- and time-dependent manner. Preliminary data from RNA gel shift assays designed to examine protein-RNA interactions, revealed the formation of a glucocorticoid concentration-dependent CYP2E1 mRNA-protein complex. Thus, the hypothesis of this proposal is that Dex increases CYP2E1 mRNA levels by enhancing CYP2E1 mRNA stability via a specific RNA-binding protein(s).
The specific aims of this proposal are: 1.) to determine the effects of glucocorticoids on CYP2E1 mRNA turnover and transcription using mRNA stability and nuclear run-on assays; 2.) to determine whether Dex and GR alter the mRNA-protein binding levels, and whether this affects storage and translation of CYP2E1 mRNA in the cytosolic compartment, which will be determined using RNA gel shifts and polyribosomal distribution assays, respectively; and 3.) to identify the specific mRNA sequence(s) required for protein(s) binding to CYP2E1 mRNA using by reverse ligation-mediated PCR. It is anticipated that results generated from this proposal will provide a better understanding of CYP2E1 gene regulation in response to GR agonists, which are administered to suppress inflammation in chronic inflammatory diseases such as asthma, rheumatoid arthritis, and autoimmune diseases.
