Carcinogenesis Advance Access originally published online on February 28, 2007
Carcinogenesis 2007 28(6):1171-1177; doi:10.1093/carcin/bgm046
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Hepatocyte-restricted constitutive activation of PPAR
induces hepatoproliferation but not hepatocarcinogenesis
Laboratory of Metabolism, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA
* To whom correspondence should be addressed. Tel: +301 496 9067; Fax: +301 496 8419; Email: fjgonz{at}helix.nih.gov
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Abstract |
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Peroxisome proliferator-activated receptor
(PPAR) is responsible for peroxisome proliferator-induced pleiotropic responses, including the development of hepatocellular carcinoma in rodents. However, it remains to be determined whether activation of PPAR only in hepatocytes is sufficient to induce hepatocellular carcinomas. To address this issue, transgenic mice were generated that target constitutively activated PPAR specifically to hepatocytes. The transgenic mice exhibited various responses that mimic wild-type mice treated with peroxisome proliferators, including significantly decreased serum fatty acids and marked induction of PPAR target genes encoding fatty acid oxidation enzymes, suggesting that the transgene functions in the same manner as peroxisome proliferators to regulate fatty acid metabolism. However, the transgenic mice did not develop hepatocellular carcinomas, even though they exhibited peroxisome proliferation and hepatocyte proliferation, indicating that these events are not sufficient to induce liver cancer. In contrast to the transgenic mice, peroxisome proliferators activate proliferation of hepatic non-parenchymal cells (NPCs). Thus, activation of hepatic NPCs and/or associated molecular events is an important step in peroxisome proliferators-induced hepatocarcinogenesis.
Abbreviations: ACOX, acyl-CoA oxidase; BrdU, 5-bromo-2'-deoxyuridine; CDK, cyclin-dependent kinase; dox, doxycycline; LAP, liver-enriched activator protein; NPC, non-parenchymal cell; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; PP, peroxisome proliferator chemical; PPAR, peroxisome proliferator-activated receptor; tTA, tetracycline-controlled transactivator; (Wt), wild-type
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Introduction |
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Peroxisome proliferator chemicals (PPs) represent a large and structurally diverse group of agents including fibrate hypolipidemic drugs, phthalate ester plasticizers and herbicides, with a high likelihood of clinical, occupational and environmental exposure to humans (1). Exposure of rodents to PPs results in pleiotropic responses including hepatomegaly, proliferation of peroxisomes and induction of genes encoding mitochondrial, peroxisomal and microsomal fatty acid oxidation enzymes (2–5). In addition, although PPs have not been shown to act as genotoxic carcinogens, long-term treatment of rats and mice with PPs results in 100% incidence of hepatocellular carcinomas (5–7).
An increased understanding of the mechanism underlying the effects of PPs was initiated by identification of the nuclear receptor known as peroxisome proliferator-activated receptor (PPAR) (8). Binding and activation of PPAR by PPs are essential for induction of peroxisome proliferation and fatty acid metabolizing enzymes, hepatomegaly and ultimately liver cancer after prolonged exposure of PPs to rodents. PPAR is a member of the nuclear hormone receptor superfamily and three PPAR (, ß and 
) genes were identified that are expressed in different tissues or organs. Among the three, PPAR is mainly expressed in liver, kidney and heart. PPARs function as transcription factors through the classic ligand-dependent nuclear hormone receptor mechanism. Upon binding to their ligands, PPARs undergo conformational changes that allow co-repressor release and co-activator recruitment, heterodimerization with retinoid X receptor, and selective binding to specific DNA sequences termed peroxisome proliferator response elements in the promoters of target genes. PPAR serves a fundamental role in mammals by acting as a central modulator of signaling molecules that mediate changes in gene expression to maintain lipid homeostasis. In addition, PPAR has also been linked to the regulation of genes important in cell growth and differentiation (9,10).
Use of the Ppar-null mouse model revealed that PPAR is responsible for PP-induced pleiotropic responses, including the development of hepatocellular carcinoma in mice, as these null mice were refractory to all known PP-induced pleiotropic responses including peroxisome proliferation, hepatomegaly and hepatocellular carcinomas (11,12). While carcinogenesis is a complicated multistage process, the induction of oxidative stress and lipid peroxidation, increase in hepatocyte proliferation and/or decrease in apoptosis are central components of the cascade of molecular events leading to liver tumors in rodents fed PPs (5). However, the link between PPAR and hepatocarcinogenesis in rodents at the molecular and cellular level is not fully understood. To clarify whether activation of PPAR only in hepatocytes is sufficient to the induction of hepatic neoplasia and whether non-parenchymal hepatic cells contribute to the carcinogenesis process, the potent viral transcriptional activator VP16 was fused to the mouse PPAR cDNA to create a transcription factor that constitutively activates PPAR-responsive genes in the absence of ligands. Transgenic mice were produced whereby inducible expression of the VP16PPAR transgene was targeted to hepatocytes using the tetracycline regulatory system under the control of the liver-enriched activator protein (LAP or cEBPß) promoter (13,14) to determine whether activation of PPAR in hepatocytes can produce the same hepatic responses as PPAR ligand treatment in mouse. In comparison with wild-type (Wt) mice treated with the potent PPAR ligand Wy-14,643, LAP-VP16PPAR mice exhibited significantly decreased serum triglycerides and free fatty acids, peroxisome proliferation and marked induction of PPAR target genes encoding fatty acid ß-oxidation enzymes. However, the VP16PPAR transgenic mice at 1 year of age exhibited no gross or microscopic evidence of either preneoplastic hepatic lesions or hepatocellular neoplasia.
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Materials and methods |
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Generation of transgenic mice
The tetracycline response element promoter-driving expression of the VP16PPAR
fusion protein were recently generated (15). Briefly, the potent viral transcriptional activator VP16 was fused to the mouse PPAR cDNA to create a transcription factor that constitutively activates PPAR-responsive genes in the absence of ligand. The single-transgenic mice were generated with the VP16PPAR fused to the tetracycline response element (TREVP16PPAR) (14). Subsequently, double-transgenic (LAP-VP16PPAR) mice were produced by breeding with LAP/tetracycline-controlled transactivator (tTA) transgenic mice expressing the tTA transgene under the control of the LAP or cEBPß promoter (13,14) to reconstitute the tetracycline-responsive regulatory system (16,17). TREVP16PPAR mice and LAP/tTA transgenic mice behaved similar to Wt mice throughout the study. Therefore, mice with these three genotypes were grouped together as control littermates unless otherwise specified. Mice expressing both transgenes were subsequently bred into the 129/Sv strain background for at least four generations. The transgenic animals were screened by Southern blot analysis or polymerase chain reaction (PCR) of tail DNA. The primers for the transgene VP16PPAR, tTA and mouse endogenous PPAR were described previously (14,15,18).
Treatment
Mice were maintained under a standard 12 h light–dark cycle with water and chow provided ad libitum. Handling was in accordance with animal study protocols approved by the National Cancer Institute Animal Care and Use Committee. Some mice were administered doxycycline (dox; 200 mg/kg; Bio-Serv, Frenchtown, NJ) in the diet to regulate expression of the transgene. To compare the effects caused by the transgene and PPAR ligands, Wt mice (129/Sv background) were also administered Wy-14,643 [0.1% (w/w); Bio-Serv] in the diet.
Serum lipids
For serum analysis, mice were deprived of food for 16 h, blood collected and then returned to the appropriate diet for an additional 3 days before they were killed. Serum total triglycerides and free fatty acid were measured using a commercial kit (Sigma, St Louis, MO).
Palmitoyl-CoA oxidation
The liver homogenates in 20% sucrose were centrifuged at 600g for 10 min. The resulting supernatant was centrifuged at 10 000g for 10 min to yield a subcellular fraction containing primarily mitochondria, peroxisomes and lysosomes. Palmitoyl-CoA oxidation was measured in this subcellular fraction as the reduction of NAD+ at 340 nm in the presence of KCN as an inhibitor of mitochondrial ß-oxidation (2,19).
Determination of liver lipid content
Liver triglyceride and cholesterol contents were determined using the procedures described previously (20).
Hepatocyte proliferation and apoptosis
For determination of cell proliferation using 5-bromo-2'-deoxyuridine (BrdU), mice were anesthetized with 2.5% avertin and implanted subcutaneously with an osmotic pump (Alzet model 2001, 1 µl/h DURECT Corporation, Cupertino, CA) containing 200 µl of 16 mg/ml BrdU (Sigma). After 1 week of receiving the respective diet, the mice were killed by overexposure to carbon dioxide, livers removed and weighed and sections from both the left and median lobes were obtained and fixed in 10% phosphate-buffered formalin (Fisher scientific, Fair Lawn, NJ). Sections from the duodenum were also obtained and fixed in phosphate-buffered formalin. The duodenum sample was used to verify uniform flow of BrdU up to the time the animals were killed. Apoptosis in hepatocytes was analyzed by using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (Promega Corporation, Madison, WI) as described previously (14).
Histological analyses
Mice were killed by overexposure to carbon dioxide and the livers were excised, fixed in 10% neutral-buffered formalin (Fisher Scientific), embedded in paraffin and 4–6 µm sections were prepared. Sections were stained with hematoxylin and eosin (H & E), and were evaluated by light microscopy.
Immunochemistry
Immunostaining for the VP16 tag was performed using polyclonal antibodies (Abcam, Cambridge, MA) and detected by use of the ABC-kit (Vector Laboratory, Burlingame, CA). Immunostaining for BrdU (DakoCytomation, Carpinteria, CA) was performed using monoclonal antibody, which was labeled with biotin by ARK kit (DakoCytomation) prior to application to tissues. In brief, sections were washed in phosphate-buffered saline (PBS) and incubated in 0.3% hydrogen peroxide in 100% methanol for 30 min at room temperature. The sections were then incubated in citrate buffer (pH 6.0) at 100°C for 10 min. After washing in PBS, the sections were blocked in PBS containing 5% skim milk at room temperature for 30 min. The sections were then rinsed and incubated sequentially with primary antibody (diluted 1:100 in PBS containing 1% bovine serum albumin) for 2 h at room temperature, biotinylated goat anti-rabbit immunoglobulin G (diluted 1:50 in PBS containing 1% bovine serum albumin) for 30 min (when polyclonal antibody was used as the primary antibody) and avidin–biotinylated peroxidase complex (Vector Laboratory) in PBS for 30 min. The bound antibody was visualized by 3,3'-diaminobenzidine as a peroxidase substrate. Sections were rinsed in water, counterstained with hematoxylin (Sigma), dehydrated and mounted in permanent mounting medium. The BrdU labeling index was determined by counting at least 1500 nuclei per slide (at random high-power fields; magnification 300x) and calculated as 100% x (number of stained hepatocyte nuclei/total number of stained + unstained hepatocyte nuclei).
Northern blot analysis
Total RNA was isolated by mechanical disruption of liver tissue in Trizol reagent (Invitrogen, Carlsbad, CA) following the manufacturer's protocol. The concentration of RNA was determined by spectrophotometry. Northern blot analysis was carried out as described previously (21). Briefly, 10 µg of total RNA was electrophoresed on a 1.0% agarose gel containing 0.22 M formaldehyde, transferred to a nylon membrane and cross-linked by ultraviolet light exposure. Membranes were hybridized in ULTRAhyb buffer (Ambion, Austin, TX) with random primer 32P-labeled cDNA probes (21,22) following the manufacturer's protocol, and washed with salt/detergent solution using standard procedures.
Quantitative real-time PCR
Real-time PCR reactions were carried out using SYBR Green PCR master mix (AB Applied Biosystems, Warrington, UK) and the ABI PRISM 7900 HT Sequence Detection System (AB Applied Biosystems). The sequence and GenBank accession number for the forward and reverse primers used to quantify mRNA were as follows: acyl-CoA oxidase (ACOX) (AB340914
[GenBank]
) forward: 5'-CCAGTCTGAAATCAAGAGAAGCGAG-3' and reverse: 5'-AAAGTGGAAGGCATAGGCGGTG-3'; cyclin D1 (NM_007631
[GenBank]
) forward: 5'-ACCCTGACACCAATCTCCTCAAC-3' and reverse: 5'-TGGATGGCACAATCTCCTTCTG-3' and cyclin-dependent kinase 4 (CDK4) (NM_009870
[GenBank]
) forward: 5'-TTGTGCAGGTAGGAGTGCTG-3' and reverse: 5'-TGCCAGAGATGGAGGAGTCT-3'. The following conditions were used for PCR: 95°C for 15 s, 94°C for 10 s, 60°C for 30 s and 72°C for 30 s in 45 cycles. Relative expression levels of mRNA were normalized to GAPDH and analyzed for statistical significance with the Student's t-test.
Immunoblot analysis
Immunoblot analysis was carried out on liver homogenates. Proteins were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and immunoblotting using monoclonal anti-catalase (Sigma) or rabbit anti-peroxisomal membrane protein 70 polyconal antibodies (Abcam) as primary, anti-rabbit immunoglobulin G horseradish peroxidase as secondary antibodies (Sigma) and an enhanced chemiluminescence detection kit (Pierce, RockFord, IL). Immunoblotting with goat anti-ß-actin or anti-GAPDH antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) were used as loading controls.
Data analysis
All data are presented as the mean ± standard error of mean. The differences between groups were assessed by analysis of variance. Differences were considered statistically significant at P < 0.05.
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Results |
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Generation and characterization of the LAP-VP16PPAR
transgenic miceNorthern blot analysis of liver samples revealed the expression of both endogenous PPAR
and transgenic LAP-VP16PPAR mRNA in adult liver samples. The high molecular weight bands corresponding to the endogenous mouse PPAR were detected in all samples, whereas the mRNA corresponding to the transgenic VP16PPAR only appeared in LAP-VP16PPAR mice in the absence of dox (Figure 1A). As expected, dox repressed the expression of VP16PPAR in LAP-VP16PPAR mice as noted by the absence of VP16PPAR mRNA in dox-treated LAP-VP16PPAR mice. Immunohistochemical analysis of liver sections showed that VP16PPAR proteins (detected by a VP16 antibody) were expressed only in the hepatocytes of LAP-VP16PPAR mice in the absence of dox (Figure 1B), in contrast to the absence of VP16PPAR proteins in Wt or LAP-VP16PPAR mouse liver in the presence of dox (data not shown). Thus, by employing the tetracycline regulation system, constitutive expression of the LAP-VP16PPAR transgene in mouse hepatocytes was achieved.
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Hepatomegaly is one of the pleiotropic effects caused by PPs in rats and mice. Hepatomegaly was observed in LAP-VP16PPAR
mice in the absence of dox as revealed by the increased liver to body weight ratio compared with Wt mice. As expected, dox treatment abolished this effect, indicating that it is due to expression of the transgene (Figure 1C). However, the extent of hepatomegaly in LAP-VP16PPAR mice was lower than the hepatomegaly induced by 2 weeks Wy-14,643 treatment in Wt mice. Histological examination of liver sections revealed that Wt mice treated with Wy-14,643 for 2 weeks had hepatocyte hypertrophy with obvious eosinophilic cytoplasm (Figure 1D). In contrast, no hypertrophy and eosinophilic cytoplasms were observed in LAP-VP16PPAR mice in the absence of dox (Figure 1D). In addition, lipid accumulation was observed in the liver of LAP-VP16PPAR mice in the absence of dox (Figure 1D). Analysis of liver lipid content indicated that the increased lipid accumulation is due to triglycerides, but not cholesterol (Figure 1E). As expected, dox treatment abolished this phenotype, indicating that it is due to expression of the VP16PPAR transgene.
Induction of PPAR target genes and fatty acid oxidation in the LAP-VP16PPAR transgenic mice
To examine whether expression of the VP16PPAR transgene induces PPAR target genes, the expression of known hepatic PPAR target genes were examined through northern blot analysis. Compared with Wt control, the induction of genes encoding enzymes involved in peroxisomal (ACOX and bifunctional enzyme), mitochondrial (long chain acyl-CoA dehydrogenase and very long chain acyl-CoA dehydrogenase) and microsomal CYP4A3 fatty acid catabolism was observed in LAP-VP16PPAR mice in the absence of dox, and the extent of induction was comparable with Wt mice treated with Wy-14,643 for 2 weeks (Figure 2A). Administration of dox to LAP-VP16PPAR mice abolished these effects, indicating the role of VP16PPAR expression in the phenotypes observed. As these genes contain peroxisome proliferator response element binding sites in their promoter regions (23), this result demonstrated that expression of the VP6PPAR transgene in LAP-VP16PPAR mice functions in the same manner to regulate hepatic target gene expression as PPAR activated by ligand in Wt mice.
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To examine whether induction of these genes changed fatty acid metabolism, the palmitoyl-CoA oxidation activity was examined. Compared with Wt control, the induction of palmitoyl-CoA oxidation was observed in LAP-VP16PPAR
mice in the absence of dox, and the extent of induction was comparable with Wt mice treated with Wy-14,643 for 2 weeks (Figure 2B). Administration of dox to LAP-VP16PPAR mice abolished this effect, indicating that it is due to the expression of VP16PPAR. As a result, in the absence of dox, LAP-VP16PPAR mice also had significantly decreased serum triglycerides (Figure 2C) and free fatty acids (Figure 2D) compared with Wt mice. These effects were comparable with Wt mice treated with Wy-14,643 for 2 weeks (Figure 2C and D). Both effects were also abolished upon dox treatment. This indicated that expression of the VP16PPAR transgene in mouse hepatocytes is sufficient to reduce serum lipids.
Induction of peroxisome proliferation in the LAP-VP16PPAR transgenic mice
The hallmark feature of rodents administered with PPs is peroxisome proliferation. To determine whether peroxisome proliferation occurred by expression of the transgene VP16PPAR, the protein levels of the major peroxisomal membrane protein 70, a marker of peroxisome proliferation, were examined by western blot analysis. Compared with Wt control mice, a robust induction of peroxisomal membrane protein 70 was observed in LAP-VP16PPAR mice in the absence of dox; the extent of induction was similar to that observed in the Wt mice treated with Wy-14,643 for 2 weeks (Figure 3A). As expected, dox treatment abolished this induction, indicating that it was due to expression of the VP16PPAR transgene. In combination with the induction of genes encoding the peroxisomal ACOX and bifunctional enzyme in LAP-VP16PPAR mice (Figure 2A), this result indicated that activation of PPAR only in hepatocytes is sufficient to induce peroxisome proliferation. As peroxisome proliferation is always associated with an induction in expression of catalase, catalase protein levels were determined by western blot analysis. An increase in catalase was observed in Wt mice treated with Wy-14,643 as compared with untreated Wt controls (Figure 3B). However, no changes were observed in LAP-VP16PPAR mice in the absence of dox compared with control, suggesting that activation of PPAR only in hepatocytes is not sufficient to induce catalase expression.
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Induction of hepatocyte proliferation in the LAP-VP16PPAR
transgenic miceHepatocyte proliferation is considered as a key step in PP-induced hepatocellular carcinomas. Hepatocyte proliferation was determined by measuring BrdU incorporation into hepatocyte nuclei. Immunohistochemical analysis revealed a high degree of incorporation of BrdU in livers of Wt mice after 2 weeks Wy-14,643 treatment (Figure 4A) with a labeling index average of 20.5% compared with 1.6% in untreated Wt controls (Figure 4B). In the absence of dox, LAP-VP16PPAR
mice also had a high degree of incorporation of BrdU (Figure 4A) with an average labeling index of 20.8% compared with 1.0% in LAP-VP16PPAR mice in the presence of dox (Figure 4B). As expected, dox treatment abolished this induction, indicating that it is due to expression of the VP16PPAR transgene. Interestingly, non-parenchymal cell (NPC) proliferation was not observed in LAP-VP16PPAR mice in the absence of dox in contrast to the dramatic proliferation of these cells in Wy-14,643-treated Wt mice (Figure 4A). In addition, no significant increases in numbers of apoptotic cells were observed in the Wt mice treated with Wy-14,643 or in LAP-VP16PPAR mice in the absence of dox as determined by the transferase-mediated dUTP nick end-labeling assay (data not shown). These data demonstrated that administration of PPs to mice induced both hepatocyte and NPC proliferation and activation of PPAR only in hepatocytes induce hepatocyte proliferation with no evidence for NPC proliferation.
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Cyclins and CDKs regulate the transit of cells through the cell cycle. These proteins were found previously to be markedly up-regulated in Wt mice treated with Wy-14,643 (24). Consistent with this finding, a 2 week Wy-14,643 treatment was shown to cause a marked induction in the expression of various genes involved in cell-cycle control (proliferating cellular nuclear antigen, CDK1, CDK4 and cyclin D1) in the livers of Wt mice (Figure 4C and D). As expected, the expression of these genes was also up-regulated in LAP-VP16PPAR
mice in the absence of dox and dox treatment abolished this induction. This indicated that up-regulation of cell-cycle control genes may contribute to the induction of hepatocyte proliferation by activation of PPAR only in hepatocytes.
Absence of liver tumors in aged LAP-VP16PPAR transgenic mice
After long-term feeding with Wy-14,643 in the diet, grossly visible lesions were observed in 11-month-old Wt mice as expected from earlier studies (11). However, grossly visible lesions were not found in LAP-VP16PPAR mice in the absence of dox aged over 1 year. Microscopic examination of liver sections was consistent with the gross findings, as hepatocellular carcinomas and hepatic lesions were observed in the long-term Wy-14,643-treated Wt mice, but not in >20 LAP-VP16PPAR mice at the age of over 1 year in the absence of dox (Figure 5A). Expression of the VP16PPAR transgene in these aged mice maintained induction of the ACOX mRNA to a similar extent as Wt mice under long-term treatment with Wy-14,643 (Figure 5B). Furthermore, although aged mice have a lower levels of hepatocyte proliferation, LAP-VP16PPAR mice and Wt mice after long-term treatment with Wy-14,643 had a similar induction of hepatocyte proliferation (Figure 5C and D). These results demonstrate that constitutive activation of PPAR in mouse hepatocytes induces hepatocyte proliferation, but is not sufficient to induce liver tumors.
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Discussion |
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Expression of the VP16PPAR
transgene in mouse hepatocytes provides a useful strategy for examining PPAR mediated effects in mouse liver. This approach has previously been used to uncover the biological functions of PPAR in skin and mammary gland development (15,25). LAP-VP16PPAR mice exhibited various peroxisome proliferator-induced responses including significantly decreased serum triglycerides and free fatty acids, peroxisome proliferation and marked induction of PPAR target genes encoding fatty acid ß-oxidation enzymes. This indicates that activation of PPAR only in hepatocytes is sufficient to induce these effects. However, in contrast to a high degree of hepatomegaly and eventual liver tumors after long-term Wy-14,643 feeding, LAP-VP16PPAR mice exhibited only a moderate degree of hepatomegaly and no liver tumors when aged over 1 year. This demonstrates that activation of PPAR in mouse hepatocytes is not sufficient to induce liver tumors.
Activation of PPAR is causally related to the induction of liver tumors in susceptible rodent species as revealed by the finding that Ppar-null mice are refractory to hepatocarcinogenesis after feeding prototypical PPs such as Wy-14,643 and bezafibrate (11,12). The increased oxidative stress and increased hepatocyte proliferation and/or decreased apoptosis have been proposed as key mediators of the hepatocarcinogenic effects by PPs (26). Production of reactive oxygen species caused by sustained overproduction of H2O2 by increased expression and activity of the peroxisomal enzyme ACOX in response to activation of PPAR was the earliest proposed response to be implicated in the mechanism of carcinogenesis by PPs (27). LAP-VP16PPAR mice also exhibited a robust expression of ACOX and accompanied peroxisome proliferation and palmitoyl-CoA oxidation, but had no changes in catalase protein expression in contrast to the robust induction of catalase by Wy-14,643 in Wt mice. This suggests that the oxidative stress induced by increased expression of ACOX and peroxisome proliferation is not directly responsible for the development of liver cancer. Indeed, the degree of peroxisome proliferation observed in rodents fed PPs does not always correlate with tumorigenity (28). Development of liver tumors in mice with a disrupted Acox1 gene suggested the existence of other potential sources oxidative stress (29). Thus, peroxisome proliferation and induction of ACOX that occurs in response to treatment with PPs appears more associative than causative in the hepatocarcinogenesis response.
Increased hepatocyte proliferation and inhibition of apoptosis were also proposed to mediate the hepatocarcinogenic effects by PPs (26). Quite surprisingly, the BrdU incorporation study revealed that similar to Wt mice treated with Wy-14,643, LAP-VP16PPAR mice also had marked induction of hepatocyte proliferation. However, no significant changes of apoptosis were observed in both cases. In addition, induction of the cell-cycle genes such as CDK1, CDK4 and cyclin D1 by Wy-14,643 in Wt mice, which was not observed in Ppar-null mice (30), was also observed in LAP-VP16PPAR mice. This demonstrates that activation of PPAR in hepatocytes only is sufficient to induce hepatocyte proliferation in vivo. To our knowledge, both of these cell-cycle genes are not direct PPAR target genes and thus further investigation is required to define how activation of PPAR in hepatocytes induce these genes and hepatocyte proliferation.
The increased liver weight after administration of PPs to rodents is probably the result of both hypertrophy and hyperplasia of hepatocytes. The typical hepatocyte hypertrophy and prominent eosinophilic cytoplasm induced by PPs in Wt mice were not observed in LAP-VP16PPAR mice. Most interestingly, induction of proliferation of NPCs was only observed in Wt mice upon Wy-14,643 treatment, but not in LAP-VP16PPAR mice. These results suggest that the differences in hepatocyte morphology and in tumor induction between LAP-VP16PPAR mice and Wt mice treated with Wy-14,643 may be associated with the differences in activation of NPCs.
Hepatic NPCs include Kupffer cells, hepatic stellate cells and sinusoid endothelial cells. However, studies on the involvement of NPCs in peroxisome proliferator-induced liver tumor are limited. NPCs have been implicated in the hepatic proliferative response to PPs. DNA synthesis induced by nafenopin was prevented by the removal of NPCs from normal primary hepatocyte cultures and was restored by returning NPCs to the purified hepatocytes (31,32). Kupffer cell inhibitors such as methyl palmitate or glycine could prevent the activation of NF
B by reactive oxygen species and induction of hepatocyte proliferation in response to Wy-14,643 (33–35). This suggests the involvement of activation of NFB by Kupffer cells in hepatocyte proliferation caused by PP treatment in vivo. In the present study, induction of hepatocyte proliferation upon expression of activated PPAR only in hepatocytes indicates that hepatocyte proliferation induced by PPs does not require the activation of Kupffer cells, although the existence of Kupffer cells appears to be necessary to support hepatocyte proliferation. On the other hand, PPs appear to directly activate Kupffer cells in vivo (36,37). The oxygen radicals produced by activated Kupffer cells may contribute to the oxidative stress that in turn may be responsible for the initiation of hepatocarcinogenesis.
Understanding the mechanism of PP-induced carcinogenicity in rodents will allow the evaluation and comparison with possible mechanisms in humans that will be of great value in human risk assessment. The LAP-VP16PPAR mouse model described in this study provides a tool to investigate the carcinogenic mechanisms that act through PPAR. In addition, this mouse model should also facilitate examination of the pleiotropic effects induced by PPs on various physiological responses during development.
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Acknowledgments |
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This study was supported by the Intramural Research Program of the National Cancer Institute, National Institutes of Health.
Conflict of Interest Statement: None declared.
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