Novel Active Repressor Model of Human LDL-Receptor Regulation

描述(申请人提供)：低密度脂蛋白受体(LDL-R)是一系列膜相关受体中的一员，这些受体共同结合了40多个配体。人的低密度脂蛋白受体与载脂蛋白B和载脂蛋白E密切结合，载脂蛋白B和载脂蛋白E将大部分循环中的胆固醇运送到肝脏、动脉壁、大脑和类固醇生成器官。经典的低密度脂蛋白受体调节模型需要通过类固醇反应元件结合蛋白(SREBP)的方式在低水平的类固醇作用下主动上调。其中，基础基因的表达被认为是一个被动的无刺激过程。相反，基于试点数据，我们提出了低密度脂蛋白-受体基因调控概念的典型性转变，其中Krueppel-like factor超家族中的转录蛋白主动沉默基础低密度脂蛋白-受体基因转录。其中一个因子KLF13可以在猪卵巢和昆虫SL2细胞中抑制和激活猪的低密度脂蛋白受体启动子。同样的蛋白质上调红系珠蛋白、淋巴细胞RANTES和SM22-α，下调CYP1A1和KLF13启动子。这些数据构成了KLF13在激活的低密度脂蛋白受体基因抑制和去抑制之间的分子开关的新论断。R21的探索性和发展性是为了在肝脏和血管平滑肌细胞(VSMCs)中测试这一独特的模型，VSMCs介导大量低密度脂蛋白胆固醇的流动。了解KLF13的作用对于从药理学上靶向这种转录多肽的活性，从而上调肝脏的低密度脂蛋白受体基因的表达，下调血管平滑肌的低密度脂蛋白受体基因的表达，从而在原则上阻断两个关键控制点的动脉粥样硬化循环具有重要意义。为此，我们确定了这里所述的两个特定目的作为假设：目的一、KLF13主动抑制基础低密度脂蛋白受体基因的表达；目的二、低固醇激活低密度脂蛋白受体启动子需要逆转基因沉默。解决这些问题的技术策略包括高效的病毒转染HepG2和血管平滑肌(VSM)细胞；RNA干扰以消除转录因子信息；染色质免疫沉淀(ChIP)低密度脂蛋白-R启动子结合蛋白；以及建议的辅助调节因子SL2昆虫细胞表达。综上所述，R21提案旨在测试一种新的低密度脂蛋白-受体基因调控范例，其中必须克服对基础转录的强烈抑制，以允许启动子激活。结果有可能揭示一种新的干预策略，释放肝脏中的低密度脂蛋白受体表达，并沉默动脉壁中的低密度脂蛋白受体表达。外行人总结说，动脉粥样硬化的部分原因是动脉壁中过度的胆固醇沉积和肝脏清除胆固醇的相对失败。我们已经在卵巢细胞中发现了一种基因，出人意料地可以增加和减少细胞对胆固醇含量高的低密度脂蛋白颗粒的摄取。该项目测试该基因的产物是否可以减少动脉肌肉中的低密度脂蛋白沉积，并增加肝脏对低密度脂蛋白的清除。如果是这样的话，靶向这种基因产物可能会将胆固醇积累从动脉转移到肝脏。这将为动脉粥样硬化性疾病患者提供一种新的治疗方法。

DESCRIPTION (provided by applicant): The low-density lipoprotein receptor (LDL-R) is a member of an array of membrane-associated receptors that collectively bind more than 40 ligands. The human LDL-R avidly binds to apolipoproteins B and E, which carry the majority of circulating cholesterol to the liver, arterial wall, brain and steroidogenic organs. The classic LDL-R regulatory model entails active upregulation by low sterol by way of a sterol response- element binding protein (SREBP). Basal gene expression is therein viewed as a passive process of nonstimulation. In contrast, based upon pilot data, we propose a paradigmatic shift in the concept of LDL-R gene regulation, wherein transcriptional proteins in the superfamily of Krueppel-like factors (KLF's) actively silence basal LDL-R gene transcription. One of the factors, KLF13, can both repress and activate the pig LDL-R promoter in swine ovarian and insect SL2 cells. The same protein upregulates the erythroid globin, lymphocyte RANTES and SM22-alpha, and downregulates the CYP1A1 and KLF13 promoters. These data frame the novel thesis that KLF13 serves as a molecular switch between active LDL-R gene repression and derepression. The exploratory and developmental nature of the R21 is to test this unique model in liver and vascular smooth muscle cells (VSMCs), which mediate large fluxes in LDL cholesterol. The significance of understanding the role of KLF13 arises in targeting the activity of this transcriptional peptide pharmacologically, so as to upregulate LDL-R gene expression in the liver and downregulate the same in VSM, thereby in principle interrupting the atherogenic cycle at two key loci of control. To this end, we identify 2 Specific Aims stated here as Hypotheses: Aim I. KLF13 actively inhibits basal LDL-R gene expression; and Aim II. Activation of the LDL-R promoter by low sterol requires reversal of gene silencing. Technical strategies to address these issues include high-efficiency viral transfection of hepG2 and vascular smooth muscle (VSM) cells; RNA interference to knock down transcriptional-factor messages; chromatin immunoprecipitation (ChIP) of LDL-R promoter-bound proteins; and SL2 insect-cell expression of proposed coregulators. In summary, this R21 proposal is designed to test a novel paradigm of LDL-R gene regulation, wherein strong repression of basal transcription must be overcome to allow promoter activation. Outcomes have the potential to unmask a new interventional strategy to unleash LDL-R expression in the liver and silence LDL- R expression in the arterial wall. Layman's summary Atherosclerosis is driven in part by excessive cholesterol deposition in the arterial wall and reciprocal failure of cholesterol removal by the liver. We have identified a gene in ovarian cells that, unexpectedly, can both increase and decrease cellular uptake of cholesterol-laden LDL particles. This project tests whether the product of this gene could decrease LDL deposition in arterial muscle and increase LDL removal by the liver. If so, targeting this gene product could potentially shift cholesterol accumulation away from arteries toward the liver. This would represent a new treatment approach for patients with atherosclerotic disease.