Hepatocellular proliferation in response to a peroxisome proliferator does not require TNF
signaling
Steven P. Anderson1,2,
Corrie S. Dunn,
Russell C. Cattley,3 and
J.Christopher Corton,4
CIIT Centers for Health Research, Research Triangle Park, NC 27709;
1 Graduate Program in Comparative Biomedical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC, USA
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Abstract
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Rodents exposed to peroxisome proliferator xenobiotics respond with marked increases in hepatocellular replication and growth that results in tumor formation. Recently, tumor necrosis factor-
(TNF
) was proposed as the central mediator of this maladaptive response. To define the role of TNF
signaling in hepatocellular growth induced by peroxisome proliferators we administered three daily gavage doses of the potent peroxisome proliferator, Wy-14 643, to mice nullizygous for TNF-receptor I (TNFR1), TNFR2, or both receptors. We demonstrate here that regardless of genotype the mice responded with almost identical increases in liver to body weight ratios and hepatocyte proliferation. Lacking evidence that TNF
signaling mediates these effects, we then examined the possible contribution of alternative cytokine pathways. Semi-quantitative, reverse transcriptase polymerase chain reaction analysis revealed that wild type mice acutely exposed to Wy-14 643 had increased hepatic expression of Il1ß, Il1r1, Hnf4, and Stat3 genes. Moreover, hepatic adenomas from mice chronically exposed to Wy-14 643 had increased expression of Il1ß, Il1r1, Il6, and Ppar
1. Expression of Il1
, Tnf
, Tnfr1, Tnfr2, Ppar
, or C/ebp
was not altered by acute Wy-14 643 exposure or in adenomas induced by Wy-14643. These data suggest that the hepatic mitogenesis and carcinogenesis associated with peroxisome proliferator exposure is not mediated via TNF
but instead may involve an alternative pathway requiring IL1ß and IL6.
Abbreviations: APP, acute-phase proteins; C/ebp, CCAAT enhancer binding protein; Hnf, hepatic nuclear factor; Il, interleukin; LPS, lipopolysaccharide; Nf
B, nuclear factor kappa B; PP, peroxisome proliferator; Ppar, Peroxisome proliferator-activated receptor; RT-PCR, reverse transcriptase-polymerase chain reaction; Stat, signal transducer of activated transcription; Tgf, tumor growth factor; Tnf, tumor necrosis factor; WY, Wy-14 643
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Introduction
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In the livers of rodents, chronic exposure to members of a diverse group of xenobiotics or endogenous fatty acids leads to increases in the quantity and size of single-membrane organelles called peroxisomes (1). Xenobiotic peroxisome proliferators (PP) include several commonly used hypolipidemic drugs, phthalate ester plasticizers, herbicides, and synthetic fatty acids. Mice and rats exposed to these compounds quickly manifest an adaptive hepatic response, consisting of hepatocellular hypertrophy and hyperplasia, that progresses to overt hepatic neoplasia (2). The carcinogenic properties of PP in rodents, coupled with relatively high exposure potential, have generated concern about potential adverse human health effects (3).
Although the mechanism of rodent carcinogenesis is largely unknown, several lines of evidence suggest that the tumors arise as a result of perturbations in homeostatic gene expression in the liver. First, PP generally lack evidence of genotoxicity as measured by classic initiation, DNA adduct, or short-term mutagenicity assays (3). Second, hepatic responses to PP exposure, including gene induction (4) and tumorigenesis (5), are mediated through ligand activation of a nuclear receptor transcription factor known as the peroxisome proliferator-activated receptor
(PPAR
). Third, proliferative hepatic lesions induced by PP regress by a mechanism involving decreased cell proliferation and increased apoptosis shortly after exposure is discontinued (68), a phenomenon not reported for genotoxic hepatocarcinogens. Collectively, these observations argue persuasively that PP-induced hepatocellular tumors are a maladaptive consequence of altered hepatic gene expression.
Thus far, PPAR
binding sites have not been identified in regulatory regions of any known growth control genes, indicating that PP may induce perturbations in hepatocyte growth indirectly (9). Recent studies suggest that several cytokines with mitogenic and inflammatory potential are attractive candidate mediators of the hepatic mitogenesis that follows PP exposure. Indirect evidence that cytokine signaling is altered after PP exposure comes from our studies on a group of inflammatory response genes known as acute-phase proteins (APP). Exposing rats or mice to various PP, including Wy-14 643 (WY), gemfibrozil, di-n-butyl phthalate, or di-(2-ethylhexyl) phthalate, results in altered hepatic mRNA levels for APP. Subchronic exposure times ranging from 5 to 13 weeks reduces APP expression (10), while hepatic adenomas from rats exposed to PP for 78 weeks have increased APP expression compared with untreated animals (11). APP gene expression is primarily regulated at the level of transcription through interaction between cis-acting sequences in the regulatory regions of APP genes and trans-acting nuclear transcription factors (12,13). Many of these transcription factors are regulated by cytokines. For example, type 1 APP are induced by tumor necrosis factor
(TNF
) (and IL1) activation of NF
B and C/EBP transcription factors, while type 2 APP are induced by IL6 (and IL1) activation of C/EBP and STAT transcription factors.
Hepatic TNF
, IL1, and IL6 are important modulators of hepatocyte proliferation following partial hepatectomy (14,15) or chemical-induced necrosis (16). Three recent findings suggest that TNF
mediates the effects of PP on hepatocyte growth (17,18) and thus may play a role in the events that lead to cancer. First, inactivation of Kupffer cells, which can secrete TNF
and other cytokines, decreases PP-induced hepatocyte proliferation (19). Second, antibodies against TNF
injected into rats prior to PP treatment diminishes hepatocyte proliferation (17). Third, Tnf
gene expression is increased in the livers of PP-treated rats under some conditions (17,20), although not consistently (21,22). However, it is unclear if a change in Tnf
expression in rodents treated with PP drives the increased hepatocellular proliferation, or if this alteration is merely a bystander event.
Here, we report experimental results designed to directly test the hypothesis that TNF
is required for PP-induced hepatocyte growth. Using mice with genetically disrupted TNF
signaling we show that this pathway is not required for PP to induce hepatocyte proliferation. Examination of other cytokines capable of regulating cell growth leads us to propose that an alternative pathway involving IL1ß, and perhaps IL6, is activated after PP exposure and is required for PP-induced hepatocyte growth in rodents.
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Materials and methods
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Animals
These studies were conducted under federal guidelines for the use and care of laboratory animals and were approved by the Institutional Animal Care and Use Committee of the CIIT Centers for Health Research (CIIT). Control and treated mice and rats were provided with NIH-07 rodent chow (Zeigler, Gardners, PA) and deionized, filtered water ad libitum. Lighting was on a 12-h lightdark cycle. In Study I, 6- to 7-week-old male C57BL/6-TNFR-1 (TNFrsflatm1Imx), C57BL/6-TNFR-2 (Tnfrsflbtm1Imx), or C57BL/6-TNFR-1/TNFR-2 (Tnfrsflatm1Imx/Tnfrsflbtm1Imx) as well as C57BL/6 wild-type mice (Jackson Laboratories, Bar Harbor, ME) were acclimated for 12 days. In Study I, mice were given a single gavage dose of Wy-14643 (50 mg/kg body weight) in methyl cellulose vehicle, or vehicle alone each day for three consecutive days. In Study II, B6C3F1 male mice (Charles River, Raleigh, NC) 10.5 weeks old were given a gavage dose of WY (50 mg/kg body weight) or vehicle as in Study I every day for up to 3 days. Groups of mice were euthanized at 2, 4, 6, 9, 12, 24, 48, and 72 h after the initial dosing. In Study III, male F344 rats (Charles River) ~9 months of age were given a single intravenous injection of lipopolysaccharide (TCA-extracted Salmonella typhimurium, Sigma Chemical) at 1.0 mg/kg body weight in 0.9% NaCl or an equivalent volume of 0.9% NaCl alone. The rats were euthanized 2 h after injection. In Study IV, male wild type SV129 mice from the CIIT Ppar
-null mouse breeding colony were housed two per cage and fed the NIH-07 diet containing 0.1%WY w/w for 12 months. Mice in Studies I and II were administered BrdU as previously described (23).
Necropsy
Animals were deeply anesthetized with pentobarbital injection and killed by exsanguination. Portions of the livers were rapidly minced, snap-frozen in liquid nitrogen and stored at 70°C until analysis. Liver tumors (>5 mm) were dissected away from non-lesion liver prior to freezing. Slices of livers and liver tumors were fixed in 10% neutral buffered formalin, routinely processed, and stained with hematoxylin and eosin. H&E stained liver sections were examined by light microscopy, and tumors were diagnosed as adenomas or carcinomas using standard criteria (8).
RNA isolation and quantitation
RNA was isolated as previously described (11) and DNase-treated (Promega, Madison, WI). First-strand cDNA was generated from 5 µg of total RNA according to the manufacturer's recommendation (SuperScript II reverse transcriptase (RT) GIBCO/BRL, Gaithersburg, MD) using random hexamer primers. Polymerase chain reaction (PCR) was performed on 1 µl of the cDNA reaction using commercially available (Clontech; Ambion, Austin, TX) or custom-designed (Primer ExpressTM, PE BioSystems, Foster City, CA), gene-specific primers and Amplitaq (PE Biosystems) or Advantage 2 DNA Polymerase (Clontech), according to the manufacturer's recommendations. Glyceraldehyde phosphate dehydrogenase (Gapdh) (Clontech) or ribosomal 18S (Ambion) genes were PCR amplified for endogenous controls. The optimal number of cycles required for detection of products in the linear range of amplification was determined for each of the cDNA-primer pair combinations in preliminary experiments. Each target gene was amplified a minimum of three separate times. PCR products were resolved on 1.5% agarose gels containing ethidium bromide. Photographs of the gels were scanned using a flatbed scanner and analyzed by NIH Image as previously described (11). The mRNA transcript levels were determined by taking the ratio of the PCR product for the target gene to the PCR product of the endogenous control. Statistical test of significance was ANOVA post-hoc testing performed using the TukeyKramer test using a P-value of
0.05 (JMPTM, SAS Institute, Research Triangle Park, NC).
Determination of hepatocyte proliferation
Nuclei that incorporated BrdU were identified by immunohistochemistry (8). The hepatocytes were analyzed using the object recognition system (CHRIS), (Sverdrup Medical/Life Sciences Imaging Systems, Fort Walton Beach, FL) using a Nikon MicrophotTM microscope with a Dage CCD color video camera at a magnification of 700x. A minimum of 1000 cells were counted for each animal. The labeling index was calculated by dividing the number of labeled hepatocyte nuclei by the total number of hepatocyte nuclei counted, and the results were expressed as a percentage.
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Results
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Liver weight and DNA synthesis in Tnf
-receptor (Tnfr)-null mice
TNF
signaling pathways play important roles in stimulating acute phase protein gene expression (12,13) and also in promoting hepatocellular regeneration following either partial hepatectomy (14,15) or exposure to certain hepatotoxins (24,25). TNF
elicits responses through binding to one of two receptors. High concentrations of TNF
induce responses through the TNF receptor 1 (TNFR1, p55), which transmits signals promoting growth inhibition and cell death, while low concentrations stimulate responses through the TNF receptor 2 (TNFR2, p75), which stimulates cell proliferation (26). Paradoxically, only the TNFR1 is required for normal hepatic regeneration (16). Recent reports suggest that TNF
is also a central mediator in the hepatic mitogenesis associated with PP (17,18). If so, mitogenesis induced by PP should be absent or greatly diminished in mice lacking one or both of these receptors. To test this prediction, we evaluated hepatomegaly and DNA synthesis following PP treatment of wild-type mice and in mice nullizygous for Tnfr1, Tnfr2, or both receptors (Tnfr1/Tnfr2). Livers from wild type and nullizygous mice constituted roughly 5% of the body weight in untreated mice (Figure 1A
). Administration of WY (50 mg/kg body weight) by daily gavage for 3 days resulted in modest but significant hepatomegaly (to ~6% of body weight) regardless of Tnfr status. Hepatocyte proliferation assessed by BrdU-positive nuclei revealed a 3-fold increase after WY treatment in wild type, Tnfr1, and Tnfr1/Tnfr2 mice (Figure 1B
). Tnfr2 mice administered WY had twice the number of labeled hepatocytes (~8%) as did wild type, WY-treated mice (~4%). These results demonstrate that the mitogenic effects of PP are not mediated through TNF
activation of either TNFR1 or TNFR2.
Cytokine gene expression during hepatic mitogenesis induced by WY
Several converging lines of evidence suggest that, in addition to TNF
, other cytokine signaling pathways play important roles in hepatic regeneration following partial hepatectomy. Cytokines and cytokine receptors that might stimulate hepatocellular growth include IL1
, IL1ß, IL1R1, and IL6. Those thought to inhibit this process, primarily through negative regulation of Tnf
expression, include IL4 and IL10 (14,15). Therefore, it seemed plausible that these pathways might also modulate the hepatic mitogenesis associated with exposure to PP or other non-genotoxic hepatocarcinogens.
To test the hypothesis that hepatic mitogenesis induced by PP is driven by alterations in signaling by cytokines other than TNF
, we examined hepatic expression of several cytokines and their receptors at seven time points following daily gavage doses of WY in B6C3F1 mice. Hepatocellular proliferation, measured by BrdU incorporation, was minimal up to 24 h following the initial WY gavage. Thereafter, the rate of cell proliferation sharply increased at 48 h and was maintained at the 72 h time point (Anderson et al., unpublished data). We postulated that cytokines influencing the cell cycle status in the liver would exhibit altered expression preceding or coincident with the 48 h time point. The relative mRNA abundance of each of these factors, as well as the cytokine-induced inflammatory mediators Cox2 and iNos, was determined by RT-PCR because preliminary experiments revealed that many of the cytokines were not detectable using northern blot, ribonuclease protection, or competitive RT-PCR assays (S.P.Anderson, unpublished data). Transcript abundance was normalized to ribosomal 18S RNA expression and the results were expressed as ratios.
Hepatic IL6 and TNF
mRNAs were expressed at very low levels at all time points and could only be consistently amplified after 35 (TNF
) or 40 (IL6) cycles of amplification using a high-sensitivity, high-fidelity DNA polymerase (Advantage cDNA PolymeraseTM, Clontech). The paucity of transcripts for these cytokines in mouse liver is consistent with observations in human liver (27). Furthermore, expression of these cytokines was not altered throughout any of our experiments. In contrast, Il1ß showed a statistically significant increase in expression that began at 9 h, peaked at 12 h (~2.5-fold increase), and was elevated at 24, 48, and 72 h (Figure 2
). Il1r1, the major receptor for both IL1
and IL1ß, showed slight but significant increases at 12 and 48 h. Tnfr1 showed slight, but significant increases at 24 and 72 h. The expression of Il1
and Tnfr2 was not altered at any of the time points. As expected, the expression of ApoAI, a gene that is down regulated by PP in mouse liver (28), was decreased after PP treatment. Il4, Il10, Cox2, and iNos were undetectable at any of the time points (data not shown).

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Fig. 2. Hepatic cytokine expression in B6C3F1 mice receiving daily gavage doses of WY (50 mg/kg body weight) for 3 days. (A) RT-PCR was performed as described in Materials and methods, using total RNA from three individual animals per treatment group. Equal loading was assessed by expression of ribosomal 18S. ApoA1 expression was examined as a positive control. (B) Densitometric analysis. mRNA transcript levels are expressed as ratios of the mean cytokine band intensities (n = 3) to the mean 18S band intensities (n = 3). Statistically significant changes are discussed in the text and were determined using ANOVA followed by TukeyKramer test (P < 0.05).
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As positive controls, hepatic cytokine expression was evaluated by RT-PCR in animals killed 2 h following a single intravenous dose of bacterial lipopolysaccharide (LPS). As expected, there was mild induction of Il6, moderate induction of Il1ß, and marked induction of Tnf
, Cox2, and iNos (Figure 3
).

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Fig. 3. Hepatic cytokine expression 2 h following intravenous injection (1.0 mg/kg body weight) of bacterial lipopolysaccharide (LPS). Expression was determined by densitometric analysis of RT-PCR products as described for Figure 2 .
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Transcription factor gene expression during hepatic mitogenesis induced by WY
All mammals, including rodents and humans, exhibit a stereotypical systemic response to localized inflammatory insults termed the acute-phase response. An integral component of this response is increased hepatic synthesis of a family of serum proteins known as the APP (12,13). Previously, we found that hepatic APP expression was altered following subchronic and chronic exposure of rodents to PP of variable potency (10,11). Many of these genes have response elements for transcription factors with known or suspected roles in regulating hepatocellular growth and differentiation (29). To test the hypothesis that altered expression of hepatic acute-phase genes is an indicator of dysregulated hepatic transcription factor homeostasis, we examined the relative mRNA abundance of several transcription factors with roles in the regulation of hepatic growth regulation as well as in inducing expression of inflammatory mediators. Hepatocyte nuclear factor-4 (Hnf4) was induced ~2-fold at 6 h following a single gavage dose of WY and ~2.5-fold at 12 h. Thereafter, mRNA levels gradually decreased and achieved basal levels by 72 h (Figure 4
). Signal transducer and activator of transcription-3 (Stat3) was induced ~2-fold at 12 h and ~1.5-fold at 48 and 72 h. Stat5ß showed a slight but significant induction (~1.7-fold) at 6 and 9 h that peaked at 12 h and gradually declined to ~1.5-fold at 48 h. Ppar
, Ppar
(isoform 1), and CCAAT enhancer binding protein-
(C/ebp
) levels were unaltered by WY treatment.

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Fig. 4. Hepatic transcription factor expression in B6C3F1 mice receiving daily gavage doses of WY (50 mg/kg body weight) for 3 days. Analysis was as described in Figure 2 . (A) RT-PCR. (B) Densitometric analysis. Statistically significant changes are discussed in the text.
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Cytokine gene expression during hepatocarcinogenesis induced by WY
Since the hepatic mitogenesis induced after acute exposure to WY was preceded by transcriptional induction of several cytokine signaling factors, it seemed plausible that the chronic mitogenesis driving the production of hepatocellular tumors was stimulated by transcriptional induction of one or more of these factors. To test this hypothesis, we examined hepatic cytokine gene expression in the WY-induced hepatocellular adenomas and the adjacent, non-tumor tissue in four different mice, and compared expression to age-matched control mice. Expression patterns of several genes affecting cell cycle control and proliferation during mitogenesis induced by PP were examined as positive controls (30). As expected, Cyclin D1, p21, Tgfß, and c-Myc, were induced from 2- to 5-fold in non-tumor hepatic parenchyma in animals receiving chronic WY exposure compared with controls (Figure 5
). c-Myc was induced even higher (up to 4.5-fold) in WY-induced tumors compared with surrounding tissue.

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Fig. 5. Hepatic cell cycle regulatory gene expression during hepatocarcinogenesis induced by feeding wild-type mice WY (1000 p.p.m.) for 52 weeks. Analysis was as described in Figure 2 . (A) RT-PCR. (B) Densitometric analysis. *Statistical differences from control.
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Analysis of cytokine mRNA levels revealed that Il1ß was induced >2-fold in the treated, non-tumor liver, and this induction persisted in the adenomas (Figure 6
). Il6 was significantly induced in the adenomas but not in the non-tumor tissue. Expression of Il1r1, Il1
, Tnf
, Tnfr1, and Tnfr2 was unchanged. These results suggest that hepatocarcinogenesis induced by PP is associated with increased IL1, and possibly IL6, signaling and occurs independently of TNF
pathways.

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Fig. 6. Hepatic cytokine gene expression during hepatocarcinogenesis induced by feeding wild-type mice WY (1000 p.p.m.) for 52 weeks. Analysis was as described in Figure 2 . (A) RT-PCR. (B) Densitometric analysis. *Statistical differences from control.
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Transcription factor gene expression during hepatocarcinogenesis induced by WY
We also examined tumor-bearing and control livers for changes in mRNAs encoding several transcription factors involved in regulating the expression of cytokine or PP-responsive genes (Figure 7
). Ppar
1 was induced ~2-fold in non-tumor tissue compared with controls, and was further induced in the adenomas up to ~3-fold in relation to control liver. Expression of Hnf4, Ppar
, Stat3, or Stat5ß was not altered in any of the groups.

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Fig. 7. Hepatic transcription factor expression during hepatocarcinogenesis induced by feeding wild-type mice WY (1000 p.p.m.) for 52 weeks. Analysis was as described in Figure 2 . (A) RT-PCR. (B) Densitometric analysis. *Statistical differences from control.
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Discussion
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Although the development of PP-induced liver tumors in mice depends on PPAR
(5), more proximate mediators of the carcinogenesis have not been identified. However, some evidence suggests that cytokines play important roles. First, compensatory liver regeneration following partial hepatectomy of rodents is associated with increased transcription of Il1ß, Il6, and Tnf
(1316), and regeneration is defective in mice lacking the Tnfr1 gene (16,31). Second, rodents treated with PP have altered hepatic expression of the cytokine-regulated acute-phase protein genes (10,11). TNF
is a direct hepatic mitogen (18) and is reportedly induced, albeit weakly, in the liver following PP exposure (17,19). Therefore, it was suggested that the hepatic growth stimulating effects of PP in rodents are due to increased production and secretion of TNF
from hepatocytes, most likely following induction of NF
B in Kupffer cells (32). Contrary to this model, preliminary studies from our laboratory revealed no difference in Tnf
mRNA levels in the livers of rodents treated with PP (21). To resolve this discrepancy, we sought to more definitively test the hypothesis that TNF
or perhaps other cytokines mediate the rodent hepatocellular proliferation induced by PP.
TNF
is not required for WY-induced hepatocyte proliferation
Compared with wild-type mice, mice lacking Tnfr1, Tnfr2, or both genes, exhibit no difference in hepatocellular proliferation or hepatomegaly after treatment with the potent PP, WY. Consistent with these findings, we (unpublished studies) and others (33) have observed no difference in hepatocellular proliferation in Tnf
-null mice compared to wild-type mice after an acute PP administration. Contrary to the prevailing view, these results demonstrate that TNF
signaling is not required for WY-induced hepatocellular proliferation in rodents.
Possible explanations for this discrepancy between our results and the prevailing hypothesis are numerous but unconvincing. For example, it is possible that the developmental programs of Tnfr-null and Tnf
-null mice have been altered in a manner that compensates for defective TNF
signaling. If so, one might expect the null mice to have an altered pattern of hepatic cytokine gene expression. Thus far, using ribonuclease protection assays, we have not observed any such differences between control or WY-treated Tnfr-null mice and wild type mice (unpublished observations). Furthermore, compared with wild type mice, Tnfr-null mice actually have decreased rates of hepatocellular proliferation following partial hepatectomy, or treatment with carbon tetrachloride (16,31,34), or fumonisin B1 (35). Thus, one would have to hypothesize that the Tnfr-null mice have selectively compensated for WY-induced cell proliferation but not proliferation induced by other treatments; an unlikely scenario. It should be noted that we have observed clear differences in WY induction of acyl-CoA oxidase protein between wild type and Tnfr-null mice (Stauber et al., manuscript in preparation) demonstrating that the Tnfr-null mice exhibit some differences in responses to a peroxisome proliferator. Another explanation for the disparity could be that Tnfr-null mice do show cell proliferation rates equivalent to wild-type mice after WY treatment but that the onset of maximum replication is simply delayed. However, wild-type mice showed increased hepatocellular proliferation that peaked between 48 and 72 h under our experimental conditions (Anderson et al., manuscript in preparation). In our studies with the null mice, hepatocyte proliferation was examined <1 day after the peak in hepatocyte proliferation. If the Tnf
-null mice do have delays in hepatocyte proliferation, the proliferation recovers to or exceeds wild-type levels in <1 day. This appears unlikely as Tnfr1-null mice exhibited significant decreases in the level, but not the time of maximum DNA synthesis after partial hepatectomy (16,34).
Central to the current model for PP-induced hepatocellular proliferation is increased hepatic expression of Tnf
. We show here that expression of Tnf
, Tnfr1, and Tnfr2 not only remains constant both prior to and during the period of maximum mitogenesis (48 h), but that expression of these genes does not change in liver tumors induced by chronic exposure to WY. Furthermore, we have been unable to detect TNF
protein in the livers of control and WY-treated mice with a commonly used commercial antibody specific to that protein in the mouse (unpublished data). These results confirm and extend our previous observations that TNF
levels are not altered during hepatocarcinogenesis induced by WY in rats (21). The Tnf
induction observed by others could be related to liver cytotoxicity induced by the higher doses of PP used in these studies. Animals were exposed to doses of WY 2-fold higher than our conditions (17,20) and thus, increased TNF
production may have been a nonspecific response to cell death. Consistent with this, LPS-treated mice express higher levels of Tnf
and have more extensive liver damage when pretreated with WY at this high dose (36). Taken together, our results and the results of others (22,24,25,33), show that PP-induced hepatic mitogenesis in rodents occurs independently of changes in Tnf
expression levels.
Interleukins and Stats in WY-induced hepatocarcinogenesis
Lacking evidence that TNF
signaling pathways significantly contribute to hepatocellular proliferation induced by PP, we examined mRNA levels of other mitogenic pathways including cytokines, their receptors and transcription factors that mediate their downstream effects. Our data suggest a role for IL1ß, and possibly IL6, in WY-induced liver cancer. IL1ß, IL1
, and IL6, like TNF
, are hepatic mitogens elaborated by Kupffer cells in response to a variety of stimuli as well as partial hepatectomy (14,15,18,37). Here, we demonstrate that IL6 levels are increased in WY-induced hepatic adenomas, but are unchanged at earlier timepoints. Although an earlier study found that IL6 inhibited the growth of cultured normal and transformed rat liver cells (38), a more recent study found that co-expression of IL6 and the soluble IL6r in double-transgenic mice led to nodular regenerative hyperplasia and adenomas of the liver (39). IL6 is reported to stimulate the growth of several other tumor types, including human colon carcinoma cells (40), non-tumorigenic rat urothelial cells (41), normal and neoplastic human cervical epithelial cells (42), and human bladder carcinoma cells (43). Thus, although Il6 mRNA levels do not change prior to adenoma formation in our studies, IL6 may play a role in stimulating or maintaining WY-induced tumors.
In contrast to the expression pattern of Il6, hepatic levels of Il1ß mRNA significantly increased 12 h following WY gavage, and remain elevated throughout the period of maximum cell replication (48 h) and adenoma formation. IL1ß is an attractive candidate mediator of cell proliferation. Recent studies have shown that IL1ß can act as a direct mitogen in cultures of primary hepatocytes (44). As shown in our studies upregulation precedes stimulation of cell cycle regulatory genes. IL1ß, like IL1
, elicits responses via the IL1R1. This receptor, similar to the TNFR1 receptor, activates several mitogenic signaling pathways involving NF
B and JNK family kinases (45). To our knowledge, PP activation of hepatic Jnk expression has not been reported, while activation of NF
B has (32,46) and has not (24) been observed after PP exposure. While IL1ß usually induces hepatic APP gene expression by activating NF
B, IL1ß and IL6 also activate STAT3 (47). Our results demonstrate that the mitogenesis induced by PP is preceded by increases in hepatic mRNA levels of Stat3, Stat5ß and transcriptional targets of STAT3. STAT3 and STAT5ß are induced in hepatocytes by hepatocyte growth factor, epithelial growth factor, and insulin or insulin-mimetics (4852). Stat3 is also induced in regenerating liver (29), which may simply be a non-specific, stress-induced response (53), and its activity decreases during hepatocyte differentiation (54). Ligand-dependent activation of STATs often leads to differentiation and growth regulation, while constitutive, or ligand-independent, activation promotes growth dysregulation. Similarly, constitutively active STAT5ß promotes cell proliferation in certain cell lines (55). Many tumor cell lines and human cancers have activated STATs, most often STAT3 (56). STAT3 can serve as an oncogene in hepatoma cells, where constitutive activation leads to transformation (57). Why STAT3 is oncogenic is unknown, but several genes involved in regulating the cell cycle, including the Cyclin D1, c-Myc, and p21 genes examined here, are downstream targets of STAT3 (57). Among other STAT3 targets is the anti-apoptotic gene Bcl-XL, a gene we previously reported to be overexpressed in the great majority of WY-induced hepatic adenomas in mice (58). Collectively, our results are consistent with IL1ß- (and possibly IL6-) dependent induction of Stat3 playing a role in PP-induced hepatocyte proliferation and hepatocarcinogenesis.
Hepatocyte nuclear factor-4 is induced after an acute dose of WY
HNF4, another hepatic transcription factor important in regulating APP gene expression, as well as hepatocyte growth and differentiation, is transiently induced prior to the onset of mitogenesis but not in the tumors. Hnf4 induction is accompanied by a decrease in ApoAI, a gene negatively regulated by HNF4 (28,59). HNF4 and HNF1 are the only liver-enriched transcription factors known whose expression is strictly correlated with hepatic differentiation in cultured rat hepatoma cells (60), but whether or not this correlation is true in vivo is uncertain. One study reported that only a fourth of chemically induced liver tumors had decreased expression of Hnf1
and Hnf4 (61), while other studies found expression was markedly reduced (62), or varied extensively (63). Moreover, treating rats with the PP Medica 16 or bezafibrate for 6 days leads to a downregulation of Hnf4 mRNA, and HNF4 protein decreased at 3 days but not at 1 or 2 days (64). Taken as a whole, these results indicate that Hnf1 and Hnf4 are unlikely candidates for genes significantly affecting hepatocarcinogenesis induced by PP. In our studies, the early induction of Hnf4 after PP exposure may simply reflect a commitment to the liver cell phenotype by rapidly replicating hepatocytes.
Role of PPAR
in WY-induced hepatocarcinogenesis
WY is a very potent PP and is used mainly because of its efficacy as a PPAR
agonist. In addition, WY also activates PPAR
isoforms (9). Previously, we reported that Ppar
mRNA is upregulated in non-tumorous hepatic parenchyma and also in hepatic adenomas from rats treated with WY (23). Here, we report that Ppar
1 is induced during chronic exposure to WY and is further induced in hepatocellular adenomas. PPAR
1 modulates several critical aspects of development and homeostasis, including adipocyte differentiation, glucose metabolism, and macrophage development and function (65). A role for PPAR
1 in PP-induced rodent liver cancer has not been previously reported, but increased expression has been reported in mouse liver tumors induced by griseofulvin (66), and in rat colon tumors induced by azomethane (67). Also, administering PPAR
1 ligands to C57BL/6J-APCMin/+ mice, a strain genetically predisposed to intestinal neoplasia, leads to a dramatic increase in tumor incidence compared to untreated C57BL/6J-APCMin/+ mice (68,69). The mechanisms underlying the effects of chronic Ppar
1 activation in PP-exposed animals are unknown, but some PPAR
agonists are capable of activating Ppar
1 and Ppar
in Ppar
-null mice, possibly by autoregulation (70). Because Ppar
1 is normally expressed at quite low levels in the liver, activation of this receptor may induce expression of other, normally quiescent genes controlling cell growth. For example, colons from C57BL/6J-APCMin/+ mice treated with the PPAR
agonist, BRL 49 653, over-express ß-Catenin, a protein implicated in tumorigenesis (69). Significantly, ß-Catenin mutation and nuclear protein accumulation is frequently observed during several types of liver tumors in mice and in humans (7174), but similar changes have not been observed in liver tumors induced by PP. Currently, we are examining whether WY induces cancer in rodents via Ppar
1-mediated dysregulation of cell growth.
Model of Il-1ß induction hepatocyte proliferation
As a summary of the data presented here, we propose a molecular model for the increased hepatocellular proliferation observed in rodents following PP administration (Figure 8
). In this model, PP stimulate hepatic Kupffer cells (or perhaps hepatocytes) to upregulate NF
B dependent or independent on PPAR
, which leads to increased synthesis and secretion of Il1ß. IL1ß acts in a paracrine fashion to stimulate IL1R1 on neighboring hepatocytes, which then upregulates Stat3. STAT3 stimulates or represses genes involved in hepatocellular proliferation or apoptosis, respectively. Several variations of this model are possible. As one example, IL1ß also induces the production of reactive oxygen species through NADPH oxidase-mediated activation of NF
B (75), and mice lacking the catalytic subunit of NADPH oxidase are resistant to hepatocellular proliferation after PP exposure (46). Conceivably, then, NF
B activation could result in a decrease in the level of hepatocyte apoptosis typically observed during acute exposure to PP (45,76), setting the stage for additional events, such as increased expression of Ppar
1, to trigger tumor formation. Future work will be aimed at determining the components of the IL1ß signaling pathway required for PP-induced hepatocyte growth.
 |
Notes
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2 Current address: GlaxoSmithKline Research and Development, Five Moore Drive, Research Triangle Park, NC 27709, USA 
3 Current address: Experimental Pathology, Amgen, Thousand Oaks, CA, USA 
4 To whom correspondence should be addressed at: CIIT Centers for Health Research, P.O. Box 12137, Six Davis Drive, Research Triangle Park, NC, 27709, USA. Email: corton{at}ciit.org 
 |
Acknowledgments
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This work was recently published in abstract form (77) as part of the proceedings for the annual meeting of the Society of Toxicology 2000. We thank Li-Qun Fan, Otis Lyght, Mary Morris, Anja Stauber, Cynthia Swanson, and Delorise Williams for expert technical assistance with necropsies; Drs Richard Miller and Leslie Recio for comments on the manuscript; Dr Barbara Kuyper for editorial assistance; and Sadie Leak for typing the manuscript. This work was partly supported by grants from the National Institutes of Environmental Health Sciences (NIEHS) to S.P.A. and J.C.C.
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Received May 14, 2001;
revised May 14, 2001;
accepted July 20, 2001.