Peroxisomes are subcellular organelles that are involved in a variety of metabolic processes. The proliferation of, and the activities within peroxisomes appears to be directly effected by a variety of chemicals including hypolipidemic agents and rodent hepatocarcinogens. The recent identification of a member of the steroid receptor superfamily, a family of ligand-induced transcription factors, that can be activated by a variety of peroxisomal proliferators offers a primary avenue for understanding the mechanism(s) by which peroxisome proliferators might be able to regulate metabolic processes such as lipogenesis, while at the same time creating an environment for the development of hepatocellular carcinomas. The specific goals of this project are: i) to fully characterize the structure of a group of orphan intracellular receptor- like cDNA clones that were isolated on the basis of their homology to the rat peroxisome proliferator activated receptor (rPPAR); ii) to evaluate the expression parameters for these clones and how they relate to the parental rPPAR gene, and; iii) to identify primary ligand(s) for rPPAR and its family members. To accomplish these goals, the primary structure for unique rPPAR-like cDNA clones will be deduced from DNA sequence analysis data, and this sequence information will subsequently be used to create: i) probes for Southern blot analyses and defining the genomic integrity of these genes, ii) RNase protection assay probes for studying tissue distribution profiles for the putative receptors, and iii) mammalian expression constructs for the examination of ligand and response element specificities. The ability of these orphan receptors to transactivate luciferase reporter constructs in the presence of a variety of classical DNA response elements and peroxisome proliferators (transient co-transfection assay) will be used to establish ligand and response element specificities for newly identified orphan receptors. Finally, of critical importance will be the establishment of primary ligands for rPPAR and its family members. This will be accomplished by the organic extraction of components from sera or tissues, isolation of ligand(s) by physical separation techniques or affinity chromatography, and analysis of these components in co-transfection and ligand binding assays. The results of these studies should further our understanding of peroxisome involvement in lipogenesis as well as hepatocarcinogenesis, and provide avenues for the development of novel lipid lowering agents as well as an estimate of their health risk to humans.
