Rates of efflux of free cholesterol (FC) and phospholipid (PL) play major regulatory roles in lipid homestasis in vascular cells. Foam cell formation may represent, at least in part, a failure of lipid efflux. The ABC1 transporter protein and caveolin, the structural protein of FC-rich cell surface caveolae are two important determinants of cell lipid levels. Caveolin forms a scaffold whose association with signal transduction proteins is regulated by FC. ABC 1 catalyses the transport of cellular PL to apolipoprotein A-1 (apo A-1) the major high density lipoprotein (HDL) protein.
In Specific Aim 1 the hypothesis is tested that caveolae can also serve as sensors reporting changes in membrane FC content via internally generated signals that among other effects, transcriptionally regulate caveolin expression, the organization of caveolae at the cell surface, and FC transport between the cell surface and internal FC pools. Transfer of cell surface FC to ABC1-generated apo A-1/PL complexes will be used to perturb FC levels in caveolae. Effects on signal transduction and nuclear response to FC efflux will be determined.
In Specific Aim 2, paradoxical findings on the effect of oxysterols on cell FC homeostasis, FC efflux and caveolin expression will be investigated. The hypothesis will be tested that 7-ketocholesterol, a major oxysterol of human atherosclerotic lesions, downgrades normal FC-dependent signaling from caveolae thereby promoting inappropriate FC accumulation. ABC1 deficiency is associated with human Tangier disease, where normal HDL is absent from the circulation. ABC1 levels are usually up regulated by FC; but regulation in response to oxysterols and cAMP differs markedly among different vascular cells. The molecular basis of the transcriptional regulation of ABC1 expression in response to FC, oxysterols and cAMP will be determined in Specific Aim 3. PL efflux by ABC1 is dependent upon extracellular apo A-1 and, it has been proposed, on cell surface phosphatidylserine distribution.
In Specific Aim 4, systematic mutagenesis of apo A-1 domains and individual charged amino acids will be carried out to identify sequences essential for ABC1 activity. Each of these studies has significant relevance to the basis of FC accumulation in atherosclerosis.
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