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

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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.