Role of PPAR-gamma Isoforms in Regulation of Macrophage apoE & LPL Expression

描述（由申请人提供）：已鉴定出四（4）种新的PPAR-gamma（g）转录物，使PPAR-g转录物同种型总数达到7种。这7种转录物共同编码3种不同的PPAR-g蛋白同种型。PPAR-g是多功能蛋白质转录因子，负责调节许多不同的基因和各种生物学功能。已经清楚地证明，PPAR-g2在脂肪形成中起主要作用。同样地，目前的兴趣是鉴定有助于巨噬细胞介导的动脉粥样硬化形成的PPAR-g同种型。拟定的研究将直接确定单个PPAR-g亚型对巨噬细胞分泌的2种蛋白质载脂蛋白E（apoE）和脂蛋白脂酶（LPL）的影响。这两种蛋白质在动脉粥样硬化中起关键作用，并且在其基因的调节区域中具有PPAR-regulatory element（PPRE）。ApoE是抗动脉粥样硬化的，而LPL促进动脉粥样硬化形成。其中单个PPAR-g蛋白同种型过度表达或被抑制的THP-1巨噬细胞将被遗传工程化。对于蛋白质的过表达，将用含有特异性PPAR-g同种型的全长cDNA的慢病毒感染细胞。为了减少蛋白质的表达，将使用与特定PPAR-g同种型互补的小干扰RNA（siRNA）来破坏PPAR-g同种型特异性转录物。将确定单个PPAR-g蛋白同种型对apoE和LPL转录调节的影响。转录水平的任何变化都与巨噬细胞功能的改变相关，包括LPL促进的细胞胆固醇摄取和apoE介导的胆固醇流出。这些研究将清楚地确定哪些PPAR-g蛋白亚型是有益的或有害的。PPAR-g蛋白同种型在它们的NH 2-末端序列上不同。这可能会改变蛋白质折叠和配体结合。将测定每种PPAR-g蛋白同种型与各种PPAR-g特异性配体（包括类二十烷酸和噻唑烷二酮）结合并被其活化的特异性和亲和力。PPAR-g的激活将使用报告基因测定法建立，以在过氧化物酶体增殖物调节元件的控制下诱导荧光素酶表达。与公共卫生的相关性：PPAR-g与许多人类疾病有关，包括动脉粥样硬化、糖尿病、肥胖和某些癌症。它们是用于治疗II型糖尿病的一类胰岛素增敏剂的分子靶标。人们越来越关注PPAR-g活化的有害副作用。关于个体PPAR-g蛋白异构体的配体特异性及其特异性基因靶点的信息将允许选择性诱导期望的基因和抑制有害的基因。

DESCRIPTION (provided by applicant): Four (4) new PPAR-gamma (g) transcripts have been identified, bringing the total PPAR-g transcript isoforms to 7. Together, the 7 transcripts encode for 3 different PPAR-g protein isoforms. PPAR-g are multifunctional protein transcription factors responsible for the regulation of many different genes and various biological functions. It has been clearly demonstrated that PPAR-g2 plays a major role in adipogenesis. Likewise, the current interest is in identifying the PPAR-g isoforms contributing to macrophage mediated atherogenesis. The proposed studies will directly determine the effects of individual PPAR-g isoforms on 2 proteins secreted by macrophages, apolipoprotein E (apoE) and lipoprotein lipase (LPL). Both proteins play a pivotal role in atherosclerosis and have a PPAR-regulatory element (PPRE) in the regulatory regions of their genes. ApoE is anti-atherosclerotic whereas LPL promotes atherogenesis. THP-1 macrophages in which individual PPAR-g protein isoforms are either over-expressed or suppressed will be genetically engineered. For over-expression of proteins, cells will be infected with lentiviruses containing full length cDNA for specific PPAR-g isoforms. For reducing the expression of proteins, small interfering RNA (siRNA) complementary to specific PPAR-g isoforms will be used to destroy PPAR-g isoform-specific transcripts. The effect of individual PPAR-g protein isoforms on the regulation of apoE and LPL transcription will be established. Any change in transcript levels will be correlated to alteration of macrophage function including LPL-promoted cellular cholesterol uptake and apoE-mediated cholesterol efflux. These studies will clearly establish which PPAR-g protein isoforms are beneficial or harmful. PPAR-g protein isoforms differ in their NH2-terminal sequences. This may alter protein folding and ligand binding. The specificity and affinity for each PPAR-g protein isoform to bind to and be activated by various PPAR-g-specific ligands including eicosanoids and thiazolidinediones will be determined. Activation of PPAR-g will be established using reporter gene assays for the induction of luciferase expression under the control of the peroxisome proliferator regulatory element. Relevance to Public Health: PPAR-g are implicated in many human diseases including atherosclerosis, diabetes, obesity and certain cancers. They are the molecular targets of a class of insulin-sensitizing agents used for the treatment of Type II diabetes. There is growing concern about the harmful side-effects of PPAR-g activation. Information about ligand specificities of individual PPAR-g protein isoforms and their specific gene targets will allow for the selective induction of desirable genes and repression of harmful ones.