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

PPAR-γ 亚型在巨噬细胞 apoE 调节中的作用

• 批准号: 7071490
• 负责人: JHEEM D MEDH
• 金额: $ 21.45万
• 依托单位: CALIFORNIA STATE UNIVERSITY NORTHRIDGE
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-05-01 至 2009-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7071490
• 关键词: apolipoprotein E; atherosclerosis; blood lipoprotein transport; cholesterol; lipoprotein lipase; macrophage; molecular pathology; pathologic process; peroxisome proliferator activated receptor; protein isoforms; protein structure function; receptor binding; receptor expression; reporter genes; small interfering RNA; tissue /cell culture; transcription factor; transfection

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.

描述（由申请人提供）：已经确定了四（4）个新的PPAR-GAMMA（G）转录本，使总PPAR-G转录本同工型达到7。在一起，共同编码了3种不同的PPAR-G蛋白质同工型。 PPAR-G是负责调节许多不同基因和各种生物学功能的多功能蛋白转录因子。已经清楚地证明，PPAR-G2在脂肪形成中起主要作用。同样，当前的兴趣是识别造成巨噬细胞介导的动脉粥样硬化的PPAR-G同工型。拟议的研究将直接确定单个PPAR-G同工型对巨噬细胞，载脂蛋白E（APOE）和脂蛋白脂肪酶（LPL）分泌的2种蛋白质的影响。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调控元件（PPRE）。 APOE是抗动脉粥样硬化的，而LPL促进了动脉粥样硬化。单个PPAR-G蛋白同工型过表达或抑制的THP-1巨噬细胞将进行基因设计。为了过表达蛋白质，将用含有全长cDNA的慢病毒感染细胞，用于特定的PPAR-G同工型。为了减少蛋白质的表达，将使用对特定PPAR-G同工型的小干扰RNA（siRNA）来破坏PPAR-G同工型特异性转录本。将建立单个PPAR-G蛋白同工型对APOE和LPL转录调节的影响。转录水平的任何变化都将与巨噬细胞功能的改变有关，包括LPL促进的细胞胆固醇摄取和APOE介导的胆固醇外排。这些研究将清楚地确定哪种PPAR-G蛋白同工型是有益或有害的。 PPAR-G蛋白同工型的NH2末端序列有所不同。这可能会改变蛋白质折叠和配体结合。将确定每个PPAR-G蛋白同工型的特异性和亲和力，以结合并被包括eicosanoids和噻唑烷二酮在内的各种PPAR-G特异性配体激活。 PPAR-G的激活将使用报告基因测定法建立，以控制过氧化物酶体增殖物调节元件的诱导荧光素酶表达。与公共卫生：PPAR-G相关的是许多人类疾病，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敏化剂的分子靶标。人们对PPAR-G激活的有害副作用越来越担心。有关单个PPAR-G蛋白同工型配体特异性及其特定基因靶标的配体特异性的信息，将允许选择性诱导理想的基因和抑制有害基因。