The phenomenon of peroxisome proliferation was first reported by several groups in the mid-1960s where significant increases of hepatic peroxisomes was seen in response to administration of the hypolipidemic drug clofibrate to rats (1). In addition to peroxisome proliferation and hepatomegaly, peroxisomal fatty acid oxidation is induced, and long term administration of this drug causes hepatocarcinogenesis (2). A number of compounds were later identified that share the morphological and biochemical response of clofibrate and deemed “peroxisome proliferators” (PPs).  PPs are a diverse group of chemicals and include the fibrate class of hypolipidemic drugs (clofibrate, ciprofibrate), Wy14,643 (often used as the prototypical peroxisome proliferator), commercially used plasticizers (phthalates, perfluorinated fatty acids) and fatty acids.

During the mid 1980s, it was suggested that the biological effects induced by PPs were mediated by an unidentified cytosolic receptor that modulated gene expression to cause the pleiotropic effect of these chemicals (3). In 1990, a receptor termed peroxisome proliferator-activated receptor (PPAR) was cloned from mouse liver using the estrogen receptor DNA binding domain as a probe. Discovery of PPAR has proven to be pivotal to the understanding of how PPs regulate gene expression and ultimately result in toxicity and cancer (4). Since the initial discovery in mice, PPARs have been cloned in several species, including humans, rodents, amphibians, teleosts and cyclostoma (5). The cloning of three distinct PPARs from Xenopus (6) lead to the realization that a subfamily of these receptors existed. The expression of PPARα, β/δ and γ varies widely from tissue-to-tissue. In numerous cell types from either ectodermal, mesodermal, or endodermal origin, PPARs are co-expressed, although their concentration relative to each other varies widely (7).  PPARα is highly expressed in cells that have active fatty acid oxidation capacity including hepatocytes, cardiomyocytes, enterocytes, and the proximal tubule cells of kidney. PPARβ/δ is expressed ubiquitously and often at higher levels than PPARa and g. PPARg is expressed predominantly in adipose tissue and the immune system and exists as two distinct protein forms γ1 and γ2, which arise by differential transcription start sites and alternative splicing (8).

 PPARs and Toxicology/Cancer.

Long term administration of PPs causes hepatocarcinogenesis in rodent models (2, 9). PPs that cause hepatocarcinogenesis include the fibrate class of hypolipidemic drugs (2), Wy14,643 (10), dehydroepiandrosterone (11), di(2-ethylhexyl) phthalate (DEHP)  (12) and trichloroethylene (13). The incidence of hepatocarcinogenesis in wild-type mice fed Wy14,643 for 11 months is 100% while PPARα-null mice are refractory to this effect (14). Thus, it is clear that the carcinogenic effect of these chemicals is mediated by PPARα, however the specific mechanisms underlying this effect have not been fully elucidated. Further, it is likely that a species difference in sensitivity to PPs exists, with humans appearing to be more resistant to the toxic effects of these compounds. It is important to note that the peroxisome proliferation and regulation of peroxisomal enzymes do not appear to be as highly inducible in the human liver; however, it is not known if these events are causally related to tumorigenesis. Some have suggested that peroxisome proliferators be classified as rodent-specific hepatocarcinogens, although this is an area of intense scientific debate (15). There are several reasons posed for classifying PPs as irrelevant to human cancers including lower PPARα expression and lack of peroxisome proliferation in human hepatocytes. Epidemiological information suggests no increased liver cancer in individuals on long-term fibrate treatment.

Regulation of gene expression by PPAR.

The three PPAR subtypes belong to the nuclear receptor (NR) superfamily and comprise the NR1C subfamily (16). PPARs function in a manner very similar to that of NR1 members such as the vitamin D, retinoic acid and  thyroid hormone receptors (17)(See Figure 1).  Activated PPAR binds to DNA as a heterodimeric complex with retinoid-X-receptor (RXR; NR2B (18)). The PPAR/RXR complex controls gene expression by interacting with specific DNA response elements (peroxisome proliferator response elements, PPREs) located upstream of target genes (19).  Genes containing PPRE motifs include acyl-CoA oxidase (ACO (19)), peroxisomal bifunctional enzyme (PBE or BIF (20)), liver fatty acid-binding protein (L-FABP (21)), and microsomal CYP4A  (22), although many more have been described (23). Members of the RXR-interacting subgroup of NRs typically bind to DNA elements containing two copies of direct repeat arrays spaced by 1-6 nucleotides (DR1-DR6). The idealized consensus binding site (AGGTCA) is similar for most members of this class of NR with the specificity dictated by the number of nucleotides between half-sites as well as the 5' flanking elements (23). In the case of PPAR, a DR1 motif is preferred with PPAR interacting with the 5' repeat and RXR (α, β or γ) binding to the 3' motif (24).  PPREs are similar for all three PPAR subtypes (23-25).

In addition to regulating PPRE-target genes, PPARα can affect gene expression in a non-DNA binding mechanism via protein-protein interactions. Including its heterodimerization partner RXRa, PPARα associates with heat shock protein 70 (hsp70 (26)), hsp90 (27, 28), hepatitis X-protein associated protein (XAP2)(27), liver X receptor (LXR (29)), C/EBPa (30) and GHF-1 (31). These interactions may influence the activity of numerous transcription factors and hence regulate genes that do not contain PPREs. A good example of such a gene is prolactin which is regulated by PPs independent of PPARα’s DNA binding (31).  Our laboratory has been examining the heat shock protein/chaperone complex and its affects on PPARα-dependent gene expression (27, 28).

Most genes previously shown to be regulated by PPARα via PPREs are involved in fatty acid metabolism and do not explain the chemicals’ carcinogenic properties. Recently, we have created a murine SV40-immortalized hepatocytes (MuSH) from wildtype and PPARα null mice that retain the in vivo characteristics of tumor promotion; MuSH wildtype cells undergo increased cell proliferation in response to PPs while the MuSH PPARα null cells are refractory (32). Through the use of gene expression microarray, PPARα regulated genes that are important in cell cycle, differentiation and angiogenesis have been discovered including angiopoietin-like protein 4 (Angptl4), forkhead C2 (FoxC2), glycogen synthase kinase 3 (GSK3), junB and MAPK-phosphatase 1 (MKP-1) (32-34). The mechanism by which these genes are being regulated by PPARα is not fully realized.

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