Cell cross-talk mediates PPAR
null hepatocyte proliferation after peroxisome proliferator exposure
Teresa C. Weglarz and
Eric P. Sandgren1
Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, 2015 Linden Drive, Madison, WI 53706, USA
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Abstract
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Peroxisome proliferator activated receptor
(PPAR
) mediates the liver's responses to peroxisome proliferator compounds. These responses include induction of specific hepatic enzymes, peroxisome proliferation and hepatocyte proliferation. PPAR
null mice, which lack receptor in all cells of the body, do not respond to peroxisome proliferators, indicating that hepatocellular proliferation and other responses require the presence of this receptor in at least some cells. To determine if PPAR
is required specifically in hepatocytes for each response, we used hepatocyte transplantation to generate chimeric livers composed of PPAR
null and positive hepatocytes in PPAR
null or positive hosts. Upon exposure to a peroxisome proliferator, peroxisome proliferation and enzyme induction were restricted to receptor positive hepatocytes, indicating that these responses are cell autonomous with respect to hepatocyte receptor status. However, both PPAR
null and positive hepatocytes in chimeric livers displayed elevated DNA synthesis regardless of host receptor status, as long as at least some hepatocytes contained receptor. These findings indicate that the mitogenic response to peroxisome proliferators does not require PPAR
in all hepatocytes.
Abbreviations: L-PBE, peroxisomal L-bifunctional enzyme; NPC, non-parenchymal cell; PPAR
, peroxisome proliferator associated receptor alpha; TNF
, tumor necrosis factor alpha; uPA, urokinase-type plasminogen activator
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Introduction
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Peroxisome proliferators are a structurally diverse group of compounds to which humans frequently are exposed [reviewed in (1,2)]. They include commonly prescribed hypolipidemic drugs such as clofibrate, used worldwide alone or in combination with HMG-CoA reductase inhibitors (statins) or bile acid sequestrants, to treat hypercholesterolemia. Peroxisome proliferators also include industrial plasticizers such as di(2-ethylhexyl)phthalate, other industrial solvents and herbicides. These compounds are termed peroxisome proliferators because they can induce proliferation of hepatocyte peroxisomes, subcellular organelles that carry out ß-oxidation of fatty acids and cholesterol metabolism. Exposure to these agents also induces transactivation of peroxisomal enzymes, hepatocellular proliferation and hepatocarcinogenesis in sensitive species. Many of these pleiotropic effects are mediated by peroxisome proliferator activated receptors (PPARs), members of the steroid receptor superfamily. There are three isoforms of PPARs,
,
and
. PPAR
is the predominant form in the liver. Following exposure to a peroxisome proliferator, (i) PPAR
is activated, (ii) PPAR
heterodimerizes with retinoid X receptor, then (iii) this dimer binds to the peroxisome proliferator response element in target genes and activates transcription. Targets include genes involved in the ß-oxidation of fatty acids, such as acyl CoA oxidase and peroxisomal L-bifunctional enzyme (L-PBE).
As summarized by Melnick (3) in a recent commentary, despite improved understanding of molecular responses in cells exposed to peroxisome proliferator compounds, there remains controversy regarding the mechanism by which they induce cancer. Estimation of risk is particularly problematic because the response to peroxisome proliferators is species specific. Mice and rats display enzyme induction, peroxisome and hepatocyte proliferation and hepatocarcinogenesis. Thus, peroxisome proliferators had been classified as probable human carcinogens by the United States Environmental Protection Agency. However, human hepatocytes appear to be less responsive, displaying minimal peroxisome and hepatocyte proliferation following exposure (4). Based on these mechanistic considerations, the International Agency for Research on Cancer changed recently the classification of this class of compounds to not classifiable as to its carcinogenicity to humans. Nevertheless, reduced responsivity does not exclude the possibility of cancer risk in humans (3). Specifically, humans display the hypolipidemic effects of peroxisome proliferator compounds via PPAR activation, so a carcinogenic risk should not be excluded until peroxisome proliferation is identified as a necessary step for carcinogenesis, and/or epidemiologic studies conclusively indicate that human exposure to medicinal, industrial or environmental peroxisome proliferators is not associated with increased incidence of cancer. Because of this uncertainty, it is imperative to identify the precise mechanism by which peroxisome proliferators induce carcinogenicity in rodents.
One important mechanistic consideration concerns the dependence of phenotypic response on the presence of PPAR
in the target cell. Thurman and colleagues have determined that Kupffer cells (liver macrophages) are activated upon exposure to peroxisome proliferators (5) and release TNF
and other mitogenic cytokines (68), and inactivation of Kupffer cells in rats administered a peroxisome proliferator blocks hepatocyte proliferation and TNF
production but not peroxisome proliferation, as measured by acyl CoA oxidase activity (7). They therefore proposed that peroxisome proliferators induce hepatocyte proliferation via activation of Kupffer cells (9,10). However, Kupffer cells do not contain measurable PPAR
(9), yet PPAR
null mice display neither hepatocyte nor peroxisome proliferation (11), indicating the dependence of these responses on the receptor. Similarly, Hasmall et al. (12) using an in vitro system demonstrated that co-cultures of mouse non-parenchymal cells (NPC) and PPAR
positive (but not PPAR
negative) hepatocytes supported hepatocyte DNA synthesis in response to peroxisome proliferator, regardless of whether the NPC were derived from PPAR
positive or null mice, yet receptor positive hepatocytes did not proliferate following identical treatment when cultured without NPC. PPAR
null hepatocytes did not proliferate with or without NPC. Collectively, these findings suggested that Kupffer cell induction of hepatocyte proliferation might require PPAR
in hepatocytes.
To identify unequivocally the cell type within which PPAR
is necessary and sufficient to induce hepatocyte proliferation in an intact animal, we generated mice that were chimeric for receptor status (+/+ versus -/-) in liver. We then exposed these mice to a potent peroxisome proliferator and correlated hepatocyte PPAR
status with peroxisome proliferation, enzyme induction and hepatocyte proliferation.
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Materials and methods
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Animals
PPAR
heterozygous null (+/-) mice in the SV/129 background were obtained from Dr Frank Gonzalez, National Cancer Institute, Bethesda, MD. These mice were backcrossed at least six generations into the C57BL/6 strain. PPAR
null mice have been described (11), and are identified using PCR (11). We described recently transgenic C57BL/6 strain mice carrying a major urinary protein-urokinase-type plasminogen activator (MUP-uPA) construct (13,14). These mice are identified using PCR, as described (13). Hepatocyte-targeted expression of uPA is hepatotoxic (15). However, individual hepatocytes can delete or otherwise stably inactivate expression of the transgene, and subsequently proliferate following release from uPA-mediated toxicity, to repopulate the liver with healthy, transgene-deficient parenchyma (15). Similarly, livers in young uPA-expressing transgenic mice support extensive repopulation by transplanted donor hepatocytes (13,14,16). By 2 months post-transplant, MUP-uPA recipient mouse livers are chimeric: they contain donor hepatocyte clones derived from the healthy transplanted hepatocytes, and endogenous hepatocyte clones derived from host hepatocytes that inactivated transgene expression (13). MUP-uPA transgenic mice of the C57BL/6 strain were used as recipients for transplanted syngeneic hepatocytes in the studies described below, and the extent of repopulation generally ranged from 20 to 80%. All donor cells were marked by a human placental alkaline phosphatase (hPAP) transgene to permit unequivocal identification (17). Transgenic mice used in these studies have been assigned the following genetic designations: MUP-uPA line 350-2, TgN(MUPPlau)1Eps; R26-hPAP line 952-1, TgN(R26ALPP)8Eps. Mice were housed and maintained in accordance with the Guide for the Care and Use of Laboratory Animals. All experimental procedures were approved by the Institutional Animal Care and Use Committee of the University of Wisconsin-Madison.
Liver cell isolation and transplantation
Donor hepatocytes carrying the R26-hPAP transgene (17) were isolated via two-step EDTA/collagenase perfusion as described (13). Recipient mice were 24 weeks of age at the time of hepatocyte transplant. We transplanted two classes of donor hepatocytes, PPAR
positive and PPAR
null, into spleens of two classes of recipient transgenic mice, PPAR
positive and PPAR
null. Using this approach,
105 transplanted hepatocytes, or 0.1% of adult hepatocyte number, seed liver via the portal circulation (13). Donor hepatocytes then undergo 1012 cell doublings during repopulation, but contaminating donor non-parenchymal cells are not subject to growth stimulus and therefore would not be amplified. All recipient mice were maintained for at least 16 weeks to allow for repopulation by and reorganization of donor hepatocytes before treatment.
Experimental design
Five experimental groups were administered peroxisome proliferator (Figure 1): (I) PPAR
-positive MUP-uPA transgenic mice that received PPAR
-positive donor hepatocytes; (II) PPAR
null MUP-uPA transgenic mice that received PPAR
null donor hepatocytes; (III) PPAR
-positive MUP-uPA transgenic mice that received PPAR
null donor hepatocytes; (IV) PPAR
-null MUP-uPA transgenic mice that received PPAR
-positive donor hepatocytes; and (V) PPAR
null mice. Additional experimental groups received vehicle without peroxisome proliferator (VIX). At 16 or more weeks post-transplant, recipient mice were implanted with a mini-osmotic pump (Alzet model 2001, Alza Pharmaceuticals, Palo Alto, CA) containing 0.2 ml of 16 mg/ml of the nucleotide analog 5-bromo-2'-deoxyuridine (BrdU), which is incorporated into DNA of cells undergoing DNA synthesis (14). All were administered either 50 mg/kg of the peroxisome proliferator WY-14,643 [4-chloro-6-(2,3-xylidino)pyrimidinylthio-acetic acid; ChemSyn Laboratories, Lenexa, KS] in 2% methylcellulose vehicle (Sigma) or methylcellulose only by oral gavage for 7 consecutive days.
On day 7, mice were killed. Liver was fixed in Carnoy's fixative for 3060 min at 4°C, transferred to 70% EtOH, and embedded in paraffin, then 6 µm liver sections were cut. Liver sections were (i) treated histochemically to detect hPAP, which marked donor cells, and/or (ii) treated immunohistochemically to detect BrdU, using a rat monoclonal anti-BrdU antibody (Accurate Scientific) (14), or to detect L-PBE, using a rabbit anti-L-PBE antiserum (generously provided by Dr J.Reddy) (18). Separate pieces of liver were frozen in Histoprep compound (Fisher), sectioned at 10 µm, then treated histochemically to detect hPAP or to detect peroxisomes using a catalase reaction (18). The BrdU labeling index of both endogenous and donor hepatocytes was determined by examining approximately 1000 hepatocyte nuclei per slide for each category of hepatocyte, and expressed as the percentage of BrdU-labeled hepatocyte nuclei. Hepatocytes immediately adjacent to a donor/host boundary were not included in the counting. The MannWhitney test was used to compare different populations of cells and treatment groups.
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Results
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Control chimeric mice with identical receptor status in both host and donor cells responded as expected to a peroxisome proliferator (19). WY-14,643 administration increased proliferation (as measured by the % hepatocyte nuclei that incorporated BrdU) in both host and donor PPAR
positive hepatocytes in chimeric mice (Figure 1, Group I and Figure 2A, +/+ host and +/+ donor) relative to the comparable cell type in vehicle-treated chimeric mice (see Figure 2F, Groups VI and VII). When both host and donor cells were PPAR
null, proliferation was not increased by WY-14,643 treatment (Figure 2B, Group II versus Groups VIII and IX). We did observe increased proliferation of WY-14,643-treated endogenous null hepatocytes in chimeric transgenic mice versus null hepatocytes in non-transgenic mice (Figure 2B and E, respectively, Group II versus Group V; P = 0.02), mimicking the increase in BrdU labeling in unmanipulated adult MUP-uPA versus adult non-transgenic mouse liver following a single injection of BrdU (P = 0.02). The data point to a transgene effect and indicate that appropriate comparisons must be between chimeric mouse hepatocytes.
In striking contrast, the response was different in chimeric mice discordant for receptor status (Figure 1, Groups III and IV; Figure 2C and D, respectively). Note that in experimental Group III some hepatocytes are PPAR
null but remaining hepatocytes and all other cells in the body are PPAR
positive, whereas in experimental Group IV the converse is true. In each group of these mice, proliferation of both PPAR
positive and null hepatocytes increased in response to WY-14,643. The increase was equivalent to that in Group I mice that were PPAR
positive in all cells, and proliferation of PPAR
null hepatocytes in Groups III and IV were significantly elevated relative to the respective Group II control hepatocytes (P < 0.017). Thus, hepatocyte proliferation in response to a peroxisome proliferator does not require the presence of the receptor in proliferating cells. Additionally, although PPAR
must be present in at least some hepatocytes to induce proliferation, as expected it was not required in NPC (Group IV). Because of inter-animal variability we were not able to determine if the magnitude of any response correlated with the extent of hepatic repopulation. However, our data suggest that induction of proliferation in receptor negative hepatocytes can occur when as few as 20% of hepatocytes possess receptor.
We also evaluated the relationship between PPAR
status and both peroxisome proliferation and induction of peroxisome proliferator-responsive genes. We measured the former by histochemical detection of catalase activity, and the latter by immunohistochemical detection of L-PBE. In contrast to hepatocyte proliferation, there was a precise correspondence between catalase activity or L-PBE induction and hepatocyte PPAR
status (Figure 3 and data not shown) in livers from all WY-14,643-treated chimeric mice (Figure 1, Groups IIV). The data indicate that peroxisome proliferation and enzyme induction in vivo require the presence of receptor in the responding hepatocyte.
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Discussion
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Our findings demonstrate the dependence of several peroxisome proliferator-induced hepatic phenotypes on the presence of PPAR
in specific cell types. PPAR
null mice, which lack receptor in all cells of the body, do not respond to peroxisome proliferators, indicating that hepatocellular proliferation and other responses require the presence of this receptor in at least some cells (11). PPAR
null hepatocytes in chimeric livers lack peroxisome proliferation and transcriptional activation of a peroxisome proliferator responsive gene, indicating that these responses require PPAR
in the responding cell (Figure 3). These events therefore are cell autonomous with respect to receptor status. In contrast, induction of hepatocyte proliferation is not cell autonomous. This response merely requires PPAR
in some hepatocytes (Figure 1, Groups III and IV). Interestingly, PPAR
null hepatocyte proliferation is normal when induced by the hepatomitogen TCPOBOP (20) or by partial hepatectomy (21), indicating that these stimuli activate different growth signaling pathways than peroxisome proliferators.
Our observations support a model for peroxisome proliferator-mediated induction of hepatocyte proliferation in vivo that is consistent with findings of Thurman and colleagues (5,7,10,22) and Hasmall et al. (12). Previous reports by these groups and others indicated that both Kupffer cells and PPAR
-positive hepatocytes were necessary to support hepatocyte proliferation; neither alone was sufficient. Peroxisome proliferators can activate Kupffer cells directly, possibly via stimulation of oxidant production (23), even in PPAR
null mice (5). The significance of Kupffer cell activation is emphasized by the finding that conditioned medium from WY-14,643-treated Kupffer cells produced a slight (2-fold) increase in rat hepatocyte DNA synthesis in vitro (24). Whether DNA synthesis in this experimental system requires PPAR
in the responding hepatocytes was not addressed. However, direct Kupffer cell activation (and corresponding cytokine release) cannot be sufficient to induce hepatocyte replication in vivo, since hepatocytes in peroxisome proliferator-treated PPAR
null mice do not proliferate (11).
The data still do not explain the mechanism underlying the dependence of hepatocyte proliferation on cross talk between hepatocytes and Kupffer cells. In one model, Kupffer cell and hepatocyte activation by peroxisome proliferators would be independent processes. Kupffer cells would be stimulated to release a pro-mitogenic substance like TNF
or IL-1ß via a PPAR
-independent mechanism, while hepatocytes would undergo PPAR
-dependent replication priming and become competent to respond to the mitogenic cytokine. Interestingly, TNF
is not necessary for this response, as hepatocyte replication is observed in TNF
null and TNF
receptor null mice following peroxisome proliferation administration (8,25). In a second model, Kupffer cell and hepatocyte activation remain separate processes, but receptor positive hepatocytes produce a signal (secreted or passed via intercellular junctional complexes) that renders all hepatocytes responsive to mitogens. In a third model, signaling would be linear: activation of receptor positive hepatocytes produces a signal that instructs Kupffer cells to release key mitogenic substances (of course, receptor-independent Kupffer cell activation may be required to render Kupffer cells competent to respond to this signal). Our findings argue against the first model for regulation of peroxisome proliferator-mediated hepatocyte replication. In a PPAR
null host, the presence of some PPAR
positive hepatocytes is sufficient (and necessary) to induce replication in all hepatocytes, regardless of receptor status, in response to a peroxisome proliferator. Thus, hepatocyte replication is non-cell autonomous with respect to hepatocyte PPAR
status. Future investigations must continue to address the hepatocyte- and Kupffer cell-derived signals that are active in this pathway.
This model has several implications. If carcinogenicity depends on peroxisome proliferation and/or transcriptional activation of PPAR
-responsive genes, chimeric mice chronically exposed to a peroxisome proliferator will develop only PPAR
-positive liver tumors. If hepatocyte proliferation alone induces cancer, both PPAR
-positive and null tumors should develop in chimeric mice. As noted by Melnick, this information would be important for evaluating the carcinogenic risk of human exposure (3). In addition, intervention strategies to reduce cancer risk in exposed individuals would vary considerably depending on the mechanism of peroxisome proliferator-associated carcinogenesis.
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Notes
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1 To whom correspondence should be addressed Email: sandgren{at}svm.vetmed.wisc.edu 
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Acknowledgments
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We thank Dr Frank Gonzalez for providing PPAR
null mice, Dr Janardan Reddy for helpful discussions and for the L-PBE antibody, and Meg Bowden, Renee Szakaly and Kristin Wentworth for technical assistance. This study was supported by funding from Glaxo-Wellcome to EPS, and by NIH grants RO1-ES07671 to EPS and 2-T32-ES7015 to T.C.W.
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Received March 14, 2003;
revised September 2, 2003;
accepted September 12, 2003.