Our long-term goal is to identify dietary and genetic factors regulating the expression of HDL binding protein-1 (HBP-1). This protein appears to be responsible for the active excretion of cholesterol from cells. We hypothesize that when cells become loaded with cholesterol in vivo via diet and/or genetic abnormalities, HBP-1 would serve to protect against atherogenesis through reverse cholesterol transport processes. This project has four specific aims which are to: (1) characterize the molecular and genetic factors involved in dietary regulation of HBP-1 expression; (2) characterize the gene for HBP-1; (3) generate transgenic mice overexpressing HBP-1; and (4) generate mice unable to express functional HBP-1 protein using gene targeting technology.
The first aim will be accomplished by screening mouse strains for differences in structures and quantitative levels of HBP-1 protein and mRNA. The expression of HBP-1 has already been shown to be altered by cellular cholesterol content in cell culture studies.
This aim will establish this behavior in vivo and determine modes of inheritance and regulation for HBP-1. In the second aim, mouse genomic clones will be obtained by conventional techniques using human cDNA already available. One purpose is to generate appropriate genomic clones for use in aim #4, and the other is to identify specific regulatory regions based on genomic sequences. In the third aim, mice overexpressing HBP-1 will be generated and used to determine whether overexpression will prevent or retard atherogenesis in diet-induced, atherosclerosis-susceptible mice of strain C57BL/6. Further, transgenic mice will be fed semi-synthetic diets designed to alter cellular cholesterol content for functional studies of HBP-1.
The fourth aim uses homologous recombination technology to generate mice in which the HBP-1 gene is dysfunctional. We will determine whether the absence of functional HBP-1 will promote atherogenesis in otherwise resistant mice. The experimental procedures described in this proposal represent a comprehensive strategy to define the importance of HBP-1 in whole animal lipoprotein metabolism and its relevance to protection against atherosclerosis. Our eventual goal is to design specific dietary interventions to protect against atherosclerosis.
