Abstract
The Peroxisome Proliferator Activated Receptor-alpha (PPARα) is a member of the nuclear hormone receptor family which mediates the effects of a distinct class of non-genotoxic carcinogens, the peroxisome proliferators (Issemann and Green, 1990). Peroxisome proliferators cause an increase in size and number of peroxisomes, in addition to hepatomegaly, hyperplasia, altered gene expression and ultimately hepatocarcinogenesis in rodents (Paget 1963; Hess et al, 1965). Humans appear to be a non-responsive species to the tumorigenic properties of peroxisome proliferators (Hanefield et ah, 1980; 1983) despite the existence of transcriptionally active human subtypes of PPARα (Sher et al, 1993). This may be due to species-specific differences in regulation of the receptor by altered expression of messenger ribonucleic acid, interaction with other proteins or post-translational modifications such as phosphorylation. This project was undertaken with a view to understanding the role of phosphorylation in PPARα regulation, with particular emphasis on the role of the Mitogen-Activated Protein Kinase (MAPK) pathway. To this end, novel epitope-tagged variants of the mouse and human PPARα subtypes were constructed for use in studies of PPARα-mediated activation and for detection of receptor protein by Western blotting. A fully functional epitope-tagged human PPARα expression vector was successfully constructed, in addition to a mouse construct which despite encoding a functional PPARα receptor has not retained the epitope-tag sequence. Expression of these constructs by transient transfection in Hepalclc7 cells resulted in significant induction of a reporter gene containing a region of the acyl CoA oxidase promoter in the presence of the peroxisome proliferator Wy-14,643. In order to study the role of the MAPK pathway in PPARα regulation, inhibitors of the upstream kinase of the cascade, Mek, were used in reporter gene assays in Hepalclc7 cells. Addition of Mek inhibitor PD98059 to cells transfected with a PPARα expression vector and a reporter gene containing a region of the acyl CoA oxidase promoter resulted in a significant increase in basal- and ligand-induced reporter gene activity. This provides evidence for a negative role in regulation of PPARα by MAPK, although this is in contradiction with previous work (Shalev et al, 1996; Juge-Aubry et al., 1999). Alternatively the results described herein may be indicative of PD98059 acting as a weak agonist, which has been previously reported for the aryl hydrocarbon receptor (Giddings, PhD thesis, 1999). In comparative studies of PD98059 and another Mek inhibitor U0126, the latter was found to be a more effective inhibitor of MAPK activation than the extensively used PD98059 compound. Preliminary studies descibed herein demonstrate that U0126 may therefore be more effective as a tool in future studies of the regulation of PPARα by the MAPK pathway.