Epidemiology studies have documented a relationship between dietary cholesterol absorption efficiency with plasma cholesterol level and risk of atherosclerosis. However, very little information is currently available on the mechanism regulating the expression of genes important for this absorption process. This application will address the possible role of specific nutrients and intestinal hormones on the expression of pancreatic cholesterol esterase, a protein that catalyzes cholesterol absorption through the digestive tract. Preliminary data have indicated that acute phase activation of cholesterol esterase biosynthesis is mediated by intestinal hormones and results in increase cholesterol esterase mRNA translation. In contrast, chronic feeding of rats with a high fat/high cholesterol diet increases cholesterol esterase mRNA level, due to nutrient activation of its gene transcription.
Specific Aim 1 is designed to determine the molecular mechanism regulating cholesterol esterase mRNA translation. Preliminary results have shown the presence of a cholesterol esterase-specific translation inhibitory factor in control cells that reduces cholesterol esterase mRNA translation in vitro. This inhibitory factory was present in guanadinium thiocyanate/phenol/chloroform extracts, suggesting that it is an RNA. Experiments are planned to isolate and characterize this specific translational control RNA (tcRNA). Standard size fractionation and gel electrophoresis techniques will be utilized to isolate the tcRNA for sequence analysis. The mechanism of translational regulation will be explored by determining the role of the tcRNA on cholesterol esterase polypeptide chain initiation and elongation.
Specific Aim 2 will study the mechanism by which fat and cholesterol increase cholesterol esterase gene transcription. The role of specific types of dietary fat on cholesterol esterase gene transcription will also be explored. The regulatory domain in the cholesterol esterase gene will be identified by the ability of various sequences to confer cholesterol responsiveness of a reporter gene in transfected AR42J cells. Suspected regions for the putative cholesterol responsive elements will be evaluated by DNA footprinting and gel retardation assays. A limited number of transgenic mice experiments will be performed with the chimeric constructs to verify dietary responsiveness of gene transcription and translation of mRNA. Moreover, the results will contribute to our understanding on mechanism that controls dietary cholesterol absorption.
