MECHANISMS AND REGULATION OF INTESTINAL CHOLESTEROL TRANSPORT

描述（由申请人提供）： 抑制肠道尼曼-匹克型C1样1（NPC 1 L1）转运蛋白，肠道胆固醇吸收的重要介质，已成为高胆固醇血症管理的一个重要目标。尽管在治疗方式上取得了这些进展，但将冠心病高危患者的血浆胆固醇降低到低水平仍然很差。因此，鉴定靶向NPC 1 L1的新机制对于更有效地治疗高胆固醇血症是必要的。然而，NPC 1 L1的抑制触发了适应性细胞途径的激活，如固醇反应元件结合蛋白-2（SREBP 2），这可能会阻碍降胆固醇药物的效率。在设计更有效的高胆固醇血症治疗方法时，也应考虑抑制这些代偿机制。我们的努力集中在研究NPC 1 L1抑制的新机制以及肠道SREBP 2在体内胆固醇稳态中的作用。我们的初步数据表明，蛋白酶体和溶酶体依赖的途径参与NPC 1 L1蛋白的正常营业额。此外，MAPK途径的抑制通过降低其蛋白质稳定性而导致NPC 1 L1的细胞水平降低。我们的研究结果表明，DNA甲基化是一个主要的表观遗传决定因素参与控制NPC 1 L1在肠上皮细胞的表达。为了理解代偿途径的作用，我们最近产生了具有组成型活性SREBP 2的肠特异性过表达的新型转基因小鼠（称为ISR 2小鼠）。我们的数据显示在ISR 2小鼠中LDL和VLDL级分中血浆胆固醇水平的增加，表明单独的肠SREBP 2的活化足以增加血浆胆固醇。我们假设MAPK通路的抑制通过蛋白酶体和/或溶酶体降解降低NPC 1 L1蛋白的稳定性，并且DNA甲基化参与NPC 1 L1基因沉默。我们进一步假设肠脂质合成/吸收的增加和活性肠SREBP 2产生的乳糜微粒导致血浆胆固醇的增加。我们的研究旨在利用体外和体内模型来解决这些假设。针对特定目的1设计的研究将研究通过MAPK依赖性途径调节NPC 1 L1蛋白稳定性的机制，并确定所涉及的NPC 1 L1结构域。具体目标2将系统地研究NPC 1 L1基因沉默的DNA甲基化机制。针对特定目标3设计的研究将集中于研究具有活性SREBP 2的肠特异性过表达的小鼠（ISR 2小鼠）中高胆固醇血症的代偿机制。我们提出的研究对于阐明肠道在维持胆固醇稳态中的关键作用以及阐明更有效治疗高胆固醇血症的潜在靶点非常重要。

DESCRIPTION (provided by applicant): Inhibition of intestinal Niemann-Pick type C1 Like 1 (NPC1L1) transporter, an essential mediator of intestinal cholesterol absorption, has emerged as an important target for management of hypercholesterolemia. Despite such advances in the therapeutic modalities, reducing plasma cholesterol to low levels in patients with high risk for coronary heart disease remains poor. Therefore, identifying novel mechanisms targeting NPC1L1 is warranted for more effective treatment of hypercholesterolemia. The inhibition of NPC1L1, however, triggers the activation of adaptive cellular pathways such as the Sterol Response Element Binding Protein-2 (SREBP2) that could hinder the efficiency of cholesterol lowering drugs. Suppressing these compensatory mechanisms should also be taken into consideration when designing more effective treatment of hypercholesterolemia. Our efforts are focused at investigating the novel mechanisms involved in the inhibition of NPC1L1 as well as delineating roles of intestinal SREBP2 in body cholesterol homeostasis. Our preliminary data showed that proteasomal and lysosomal-dependent pathways are involved in the normal turnover of NPC1L1 protein. Also, inhibition of MAPK pathway caused a decrease in the cellular level of NPC1L1 by decreasing its protein stability. Our findings showed that DNA methylation is a major epigenetic determinant involved in controlling NPC1L1 expression in intestinal epithelia cells. To understand the role of compensatory pathways, we have recently generated novel transgenic mice with intestine- specific overexpression of constitutively active SREBP2 (designated as ISR2 mice). Our data showed an increase in levels of plasma cholesterol in the LDL and VLDL fractions in ISR2 mice indicating that the activation of intestinal SREBP2 alone is sufficient to increase plasma cholesterol. We hypothesized that inhibition of MAPK pathway decreases NPC1L1 protein stability via proteasomal and/or lysosomal degradation, and that DNA methylation is involved in NPC1L1 gene silencing. We further hypothesize that the increase in intestinal lipid synthesis/absorption and chylomicron production by active intestinal SREBP2 lead to an increase in plasma cholesterol. Our studies are designed to address these hypotheses utilizing both in vitro and in vivo models. Studies designed for Specific Aim 1 will investigate the mechanisms involved in the regulation of NPC1L1 protein stability by the MAPK dependent pathway and determine the structural domains of NPC1L1 involved. Specific Aim 2 will systematically investigate mechanisms of NPC1L1 gene silencing by DNA methylation. Studies designed for Specific Aim 3 will focus on investigating the compensatory mechanisms underlying hypercholesterolemia in mice with intestine specific-overexpression of active SREBP2 (ISR2 mice). Our proposed studies are important to elucidate the critical role of the intestine in the maintenance of cholesterol homeostasis and to unravel potential targets for more effective treatment of hypercholesterolemia.