Coronary artery disease (CAD) remains the leading cause of death and disability in our society. Of all known risk factors promoting CAD, a high serum level of low-density lipoprotein (LDL)-cholesterol is one of the most important risk factors. The plasma level of this lipoprotein is strongly influenced by genetic factors and the amount of dietary fat and cholesterol; dietary modification remains the cornerstone of CAD prevention. Our knowledge of the regulatory mechanism(s) controlling cholesterol homeostasis in response to dietary cholesterol is limited, as is the role of signaling pathways initiated by alterations in cholesterol levels. Also, the interrelationships between these signaling pathways and sterol response element-binding proteins (SREBPs), as well as the mechanism linking SREBP processing and cholesterol levels are not clear. The emerging picture from our work is that regulation of hepatic LDL receptors, crucial for cholesterol homeostasis, results from the activity of a few interlinked regulatory signaling pathways. We have previously shown, for the first-time, involvement of specific isoforms of protein kinase C (PKC) in the regulation of LDL receptor expression in cultured hepatic cells, possibly through direct modulation of activity by cholesterol. Based on our recent results, we propose a central role for B-isoform of PKC (PKCb) in regulating cholesterol homeostasis via modulating expression of selected genes crucial for this process.
In Specific Aim 1, the role of PKCb in cholesterol homeostasis will be established by evaluating the effect of knockout of this ldnase on overall cholesterol homeostasis. Initial studies with PKC(-deficient mice strongly support a central role of this kinase in controlling the responsiveness of plasma cholesterol to the changes in the dietary cholesterol content. Cholesterol metabolism will be compared in detail between normal and PKCb mutants fed special diets.
The Specific Aim 2 will compare expression of the hepatic genes critical for cholesterol homeostasis in the above animals.
In Specific Aim 3, mechanisms by which PKCb affects SREBP-2 expression and its proteolytic processing, possibly by regulating INSIG- 1 phosphorylation will be examined. Finally, the nature of the isoform-specific interaction between cholesterol and PKCb will be examined in the Specific Aim 4. The photoactive cholesterol probe will be used to define at the molecular level structure(s) and amino acids that produce isoform-specific binding of PKCb to cholesterol. The proposed studies will not only establish the role of PKCb in diet-induced hypercholesterolemia, but will also identify genes regulated by this kinase, to correlate regulatory mechanisms to animal physiology. They will also help understand the complex interactions between environment and genetics leading to atherosclerosis. Accomplishment of the above aims will unravel a central signaling component that may act as a sensor to respond to dietary cholesterol; modulation of its activity may be the preferable mode for the treatment of lipid disorders in the 21st century.
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