Compounds which result in the proliferation of peroxisomes represent a diverse group of chemicals which have important biological and toxicological effects in mammalian cells. Peroxisome proliferators include certain hypolipidemic drugs, phthalate ester plasticizers and various herbicides and pesticides. Recent studies have shown that many genes affected by peroxisome proliferators are regulated by a novel member of the steroid hormone receptor superfamily, the peroxisome proliferator- activated receptor (PPAR). The role of this protein in maintaining normal cellular physiology is not known, although many lines of evidence suggest that it is involved in lipid metabolism and possibly growth regulation. The present grant proposal will explore how the function of this potentially important steroid hormone receptor is regulated. First, the overall response of cells to peroxisome proliferators may be regulated by the concentration of their cognate receptor. The mechanism for control of PPAR expression has not been thoroughly examined, although studies in our laboratory have shown that peroxisome proliferators as well as factors affected by nutritional status regulate the accumulation of the messenger RNA for the predominant form of PPAR found in rodent liver (PPARalpha). The factors that effect PPARalpha expression will be examined in rat hepatoma cells to determine possible endogenous and exogenous modes of regulation. Based on preliminary studies, the induction of PPARalpha mRNA by peroxisome proliferators is predominantly a post-transcriptional event. The effect of peroxisome proliferators on PPARalpha mRNA stability will be examined and possible mechanisms explored. Second, based on studies with closely related steroid hormone receptors, numerous protein-protein interactions may affect the process of PPAR-mediated gene regulation. Novel proteins which interact with PPARalpha will be examined using interaction cloning, a technique which examines the binding of a bacterial-expressed PPARalpha to a rat hepatoma cDNA expression library. The binding of PPARalpha to these novel proteins will be characterized to determine the strength of binding and the domain of PPARalpha involved. Finally, studies will be implemented to determine the result of the protein-protein interactions on peroxisome proliferator-dependent gene regulation to determine if PPARalpha heterodimers are involved in DNA binding, receptor phosphorylation or cell growth. Taken together these studies enhance our knowledge of the molecular mechanisms of peroxisome proliferator-dependent gene regulation and will greatly enhance our understanding of the role played by PPAR in mediating cellular responses to this important group of rodent carcinogens.
