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Mechanistic Evaluation of PPAR
-Mediated Hepatocarcinogenesis: Are We There Yet?
Department of Veterinary and Biomedical Sciences and Center for Molecular Toxicology and Carcinogenesis, 312 Life Sciences Building, The Pennsylvania State University, University Park, Pennsylvania 16802
1 For correspondence via fax: (814) 863-1696. E-mail: jmp21{at}psu.edu.
Received September 14, 2007; accepted September 26, 2007
Key Words: peroxisome proliferators; peroxisome proliferator-activated receptor-
; liver cancer; humanized mouse models.
The fibrate class of hypolipidemic drugs have been used for many years as therapeutic agents to reduce serum triglycerides, increase serum high density lipoprotein cholesterol, and in doing so, improve the health of individuals with dyslipidemias. Long before the actual mechanism of action was delineated, it was postulated that these drugs functioned by activating a nuclear receptor. Indeed, the identification and cloning of the peroxisome proliferator–activated receptor- (PPAR) (Issemann and Green, 1990
) led to a plethora of research that definitively confirmed that PPAR was the central mediator for the hypolipidemic effects of fibrates. But the story actually became significantly more complicated over the years because it turned out that there were many other chemicals, collectively termed "peroxisome proliferators" that activated this receptor. More importantly, the seminal work by Reddy and colleagues showing that long-term administration of PPAR agonists caused liver cancer (Reddy et al., 1980) has raised serious concern regarding the safety of the collective class of compounds that activate PPAR, that humans are exposed to. There has been considerable research undertaken over the past 25 years examining the effect of peroxisome proliferators, including comparative analysis between a variety of species indicating that there is a species difference in the response to peroxisome proliferators (reviewed in Klaunig et al. [2003]; Peters et al. [2005]). The paper by (Yang et al. 2007) in this issue of Toxicological Sciences provides another turning point in this field.
The mode of action for peroxisome proliferator–induced hepatocarcinogenesis has been studied extensively in rodents (reviewed in Klaunig et al. [2003]; Peters et al. [2005]). In response to ligand activation, PPAR mediates increased expression of proteins required for lipid catabolism including peroxisomal acyl-CoA oxidase and other mitochondrial fatty acid catabolizing enzymes. The increased expression of acyl-CoA oxidase may or may not be involved in contributing to oxidative damage. While this pathway is critical for modulating serum lipids and is required for daily variation in fatty acid catabolism in both humans and rodents, prolonged or potent ligand activation of PPAR also causes significant mitogenic effects in rodent liver. This increased signaling for cell proliferation, and inhibition of apoptotis is thought to promote growth of DNA-damaged cells and ultimately liver tumors. However, while proteins that regulate the cell cycle are increased in a PPAR-dependent mechanism, the direct PPAR target genes that caused this effect are not known. There is a large body of evidence from multiple models including comparisons of human versus rodent cells, comparisons of rodents to nonhuman primates, and limited epidemiological and human studies suggesting that humans do not respond to peroxisome proliferators by increasing hepatocyte growth and/or liver cancer. Unfortunately, the mechanisms proposed to account for the species differences have been met with reasonable criticism (reviewed in Klaunig et al. [2003]; Peters et al. [2005]). For example, it has been suggested that human liver contains significantly less PPAR as compared to rodent liver, but there are observations inconsistent with this viewpoint (reviewed in Klaunig et al. [2003]; Peters et al. [2005]). Enter the PPAR-humanized mouse.
The first PPAR-humanized mouse was generated using a cDNA expression vector driven by a repressible tet-off response element (Cheung et al., 2004). The transgenic mice expressing the human PPAR isoform were generated on a mouse PPAR-null background, allowing for examination of potential species differences in an in vivo model. Results from analysis using this mouse line demonstrated that while peroxisome proliferators can effectively induce the hypolipidemic effects via activation of the human PPAR, the mitogenic and hepatocarcinogenic effect of peroxisome proliferators do not occur (Cheung et al., 2004; Morimura et al., 2006). Thus, despite conclusive evidence that the mouse PPAR mediates the hepatocarcinogenic effect of peroxisome proliferators (Hays et al., 2005; Peters et al., 1997), there are clear differences in biological responses mediated by the mouse versus human PPAR. More recently, it was shown that the mechanism by which peroxisome proliferators lead to a mitogenic effect in mouse liver is due to PPAR-dependent downregulation of let7c microRNA, which causes upregulation of c-myc, an effect that does not occur in the humanized PPAR mice (Shah et al., 2007). These groundbreaking observations suggest that the differences in species response (e.g., both respond by modulating lipid catabolism, but only rodents exhibit liver hyperplasia) may be due to this species-specific regulation of a microRNA. While further work is required to clarify this model using other human model systems, it set the stage for the paper in this issue where a second PPAR-humanized mouse was characterized (Yang et al., 2007). The mice described in this study were generated using a PAC clone containing a large coding sequence of the human PPAR gene, rather than a cDNA encoding the receptor. This allows for expression of the human isoform via transcriptional regulator molecules that might function differently as compared to mice, but more importantly and in contrast to the earlier humanized mouse line, the human PPAR is expressed in other tissues in addition to liver. Results from this study clearly demonstrate the suitability of this model for examining the species differences in response to peroxisome proliferators. The PAC PPAR-humanized mice exhibit essentially the same phenotype in liver as the original liver-specific PPAR-humanized mouse, as shown by the induction of lipid catabolism and lowering of serum triglycerides upon ligand treatment, as well as the lack of a mitogenic response. Further, the differences in let7c microRNA expression leading to increased expression of c-myc were also noted between the wild-type and PAC PPAR-humanized mice.
So why should we be excited about this model, as it is similar to the original cDNA PPAR-humanized mouse? There are a several reasons. It provides a novel tool for examining species differences in their response to peroxisome proliferators, and results from such studies can be applied to risk assessment. Additionally, while the liver-specific PPAR-humanized mouse can modulate expression of the human PPAR and potentially model human levels, expression of PPAR in the PAC PPAR-humanized mouse is regulated by elements found in the human gene. Most notably, since there are a number of other toxicities associated with administration of peroxisome proliferators, or PPAR agonists, including effects induced by phthalate mono-esters, extrahepatic tumors (e.g., the tumor triad), toxicities associated with perfluorinated compounds and others, this new mouse line provides an excellent model for determining whether there are species differences in these toxicities. While findings from such studies may not conclusively demonstrate a species difference, they can certainly move the field forward and provide stronger foundations for future studies to confirm any potential finding.
It is worth noting that the evidence to date has not demonstrated that peroxisome proliferators (PPAR agonists) are not a human health hazard due to differences in the function of PPAR. However, there is a large body of evidence from multiple observations that support this idea, in particular for relatively low-affinity compounds such as phthalate mono-esters and possibly the fibrates. Skeptics may indicate that the PPAR-humanized mouse does not model humans precisely because there could be different coactivators, corepressors, target gene response elements; the list could go on and on. But this is something we will have to contend with, as with the limitations of many model systems. Clearly, the recent findings using the PPAR-humanized mice have significantly advanced our understanding. So, are we there yet? One cannot tell at this point, but the PAC PPAR-humanized mouse is a step in the right direction. This new mouse model will have extensive application in the field of toxicology and human risk assessment.
ACKNOWLEDGMENTS
The author would like to acknowledge Michael, Elizabeth, and Timothy Peters for providing technical assistance with the title.
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