Role of PKCbeta in Diet-induced Hypercholesterolemia

描述（由申请人提供）：冠状动脉疾病（CAD）仍然是我们社会中导致死亡和残疾的主要原因。在所有已知的促进冠心病的危险因素中，高水平的低密度脂蛋白(LDL)-胆固醇是最重要的危险因素之一。该脂蛋白的血浆水平受遗传因素和膳食脂肪和胆固醇含量的强烈影响；饮食调整仍然是预防冠心病的基石。我们对膳食胆固醇控制胆固醇稳态的调节机制的了解是有限的，正如胆固醇水平改变引发的信号通路的作用一样。此外，这些信号通路与甾醇反应元件结合蛋白（SREBP）之间的相互关系，以及SREBP加工与胆固醇水平之间的联系机制尚不清楚。从我们的工作中得出的新结论是，对胆固醇稳态至关重要的肝脏LDL受体的调节是由一些相互关联的调节信号通路的活动引起的。我们之前首次表明，蛋白激酶C （PKC）的特定亚型参与了培养肝细胞中LDL受体表达的调节，可能是通过胆固醇对其活性的直接调节。根据我们最近的研究结果，我们提出PKC的b -异构体（PKCb）通过调节对这一过程至关重要的选定基因的表达，在调节胆固醇稳态中发挥核心作用。在Specific Aim 1中，将通过评估敲除该酶对总体胆固醇稳态的影响来确定PKCb在胆固醇稳态中的作用。对PKC缺陷小鼠的初步研究强烈支持该激酶在控制血浆胆固醇对膳食胆固醇含量变化的反应性方面的核心作用。将详细比较喂食特殊日粮的正常和PKCb突变体的胆固醇代谢。特异性目的2将比较上述动物中对胆固醇稳态至关重要的肝脏基因的表达。在Specific Aim 3中，我们将研究PKCb影响SREBP-2表达及其蛋白水解过程的机制，可能是通过调节insg - 1的磷酸化。最后，胆固醇和PKCb之间的异构体特异性相互作用的性质将在Specific Aim 4中进行检查。光活性胆固醇探针将用于定义在分子水平上的结构(s)和氨基酸产生异构体特异性结合PKCb到胆固醇。拟建的研究不仅将确定PKCb在饮食诱导的高胆固醇血症中的作用，还将确定受该激酶调控的基因，以将调节机制与动物生理学联系起来。它们还将有助于了解导致动脉粥样硬化的环境和基因之间复杂的相互作用。上述目标的实现将揭示一个可能作为对膳食胆固醇反应传感器的中心信号组件；调节其活性可能是21世纪治疗脂质紊乱的首选模式。

DESCRIPTION (provided by applicant): 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.