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Originally published In Press as doi:10.1074/jbc.M106480200 on September 10, 2001

J. Biol. Chem., Vol. 276, Issue 45, 42485-42491, November 9, 2001
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Human Peroxisome Proliferator-activated Receptor alpha  (PPARalpha ) Supports the Induction of Peroxisome Proliferation in PPARalpha -deficient Mouse Liver*

Songtao YuDagger , Wen-Qing CaoDagger , P. KashireddyDagger , Kirstin MeyerDagger , Yuzhi JiaDagger , Douglas E. Hughes§, Yongjun Tan§, Jianchi FengDagger , Anjana V. YeldandiDagger , M. Sambasiva RaoDagger , Robert H. Costa§, Frank J. Gonzalez, and Janardan K. ReddyDagger ||

From the Dagger  Department of Pathology, Northwestern University Medical School, Chicago, Illinois 60611-3008, the § Department of Molecular Genetics, University of Illinois College of Medicine, Chicago, Illinois 60607, and the  Laboratory of Metabolism, NCI, National Institutes of Health, Bethesda, Maryland 20892

Received for publication, July 11, 2001, and in revised form, September 6, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Peroxisome proliferators, which function as peroxisome proliferator-activated receptor alpha  (PPARalpha ) agonists, induce peroxisomal, microsomal, and mitochondrial fatty acid oxidation enzymes, in conjunction with peroxisome proliferation, in liver cells. Sustained activation of PPARalpha leads to the development of liver tumors in rats and mice. The assertion that synthetic PPARalpha ligands pose negligible carcinogenic risk to humans is attributable, in part, to the failure to observe peroxisome proliferation in human hepatocytes. To explore the mechanism(s) of species-specific differences in response to PPARalpha ligands, we determined the functional competency of human PPARalpha in vivo and compared its potency with that of mouse PPARalpha . Recombinant adenovirus that expresses human or mouse PPARalpha was produced and administered intravenously to PPARalpha -deficient mice. Human as well as mouse PPARalpha fully restored the development of peroxisome proliferator-induced immediate pleiotropic responses, including peroxisome proliferation and enhanced expression of genes involved in lipid metabolism as well as nonperoxisomal genes, such as CD36, Ly-6D, Rbp7, monoglyceride lipase, pyruvate dehydrogenase kinase-4, and C3f, that have been identified recently to be up-regulated in livers with peroxisome proliferation. These studies establish that human PPARalpha is functionally competent and is equally as dose-sensitive as mouse PPARalpha in inducing peroxisome proliferation within the context of mouse liver environment and that it can heterodimerize with mouse retinoid X receptor, and this human PPARalpha -mouse retinoid X receptor chimeric heterodimer transcriptionally activates mouse PPARalpha target genes in a manner qualitatively similar to that of mouse PPARalpha .


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Peroxisomes, cytoplasmic organelles of about 0.5 µm in diameter, participate in several important metabolic functions, including simple respiration characterized by H2O2 production and H2O2 degradation, beta -oxidative chain shortening of long chain and very long chain fatty acids, metabolism of glyoxalate, degradation of uric acid, and synthesis of ether phospholipids, cholesterol, and bile acids (1). A profound increase in the size and number of peroxisomes occurs in livers of rats and mice exposed to a broad spectrum of structurally diverse compounds of industrial, pharmaceutical, and agricultural importance designated as peroxisome proliferators (2). These include certain phthalate ester plasticizers, herbicides, leukotriene D4 receptor antagonists, and lipid-lowering drugs, such as clofibrate, ciprofibrate, fenofibrate, gemfibrozil, and Wy-14,643, among others (2, 3). The induction of peroxisome proliferation is mediated by peroxisome proliferator-activated receptor alpha  (PPARalpha ),1 a member of the subfamily of ligand-dependent nuclear transcription factors that regulate the expression of genes associated with lipid metabolism and adipocyte differentiation (4-6). The PPAR subfamily consists of three isotypes (alpha , beta  or delta , and gamma ), which exhibit distinct patterns of tissue distribution and differ considerably in their ligand binding domains and specificities, attesting to the fact that they perform different functions in different cell types (4-6). PPARs contain a central cysteine-rich zinc finger motif DNA binding domain that recognizes DNA sequence elements, designated peroxisome proliferator response elements (PPREs), containing direct repeats of the hexanucleotide sequence AGGTCA separated by one nucleotide present in the 5'-flanking region of target genes (4, 7). PPARs heterodimerize with retinoid X receptor (RXR), a receptor for 9-cis-retinoic acid (7); the ligand-activated PPAR-RXR heterodimer binds the PPRE to transcriptionally activate target genes (4, 5, 7). PPARalpha -dependent induction of peroxisome proliferation in liver is associated with concomitant transcriptional activation of genes encoding for the classical peroxisomal straight chain fatty acid beta -oxidation system, microsomal cytochrome P450 CYP4A isoforms CYP4A1 and CYP4A3, and some of the genes involved in the mitochondrial beta -oxidation among others (1, 8-11). Chronic induction of the morphological phenomenon of hepatic peroxisome proliferation as well as sustained activation of a variety of genes that contain PPREs in their promoters appear to play a role in the development of liver tumors in rodents exposed to synthetic and endogenous PPARalpha ligands (2, 3, 12). Mice lacking PPARalpha (PPARalpha -/-) (13) and those lacking both PPARalpha and AOX of the classical peroxisomal beta -oxidation system (PPARalpha -/- AOX-/-) (14, 15) are refractory to the induction of peroxisome proliferation and PPARalpha -regulated changes in gene expression in livers by synthetic as well as natural PPARalpha ligands, indicating that the PPARalpha isotype is solely responsible for the peroxisome proliferator-induced pleiotropic responses, including hepatic peroxisome proliferation and development of liver tumors.

The inability of synthetic peroxisome proliferators to interact with and damage DNA directly led to the proposal that sustained overproduction of H2O2 and the resulting DNA damage due to disproportionate increases in H2O2-generating oxidases and H2O2-degrading enzyme catalase and liver cell proliferation contribute to liver tumor development in rodents (11, 12, 14, 16-18). While the consequences of sustained PPARalpha activation, in particular the development of hepatocellular carcinomas, in species that manifest a profound degree of hepatic peroxisome proliferation are unequivocally established (2, 12, 17, 19), it is argued that the carcinogenic risk to humans of chronic exposure to PPARalpha ligands is negligible to nonexistent because human liver is assumed to be refractory to peroxisome proliferation (3, 19-21). This refractoriness of human hepatocytes to the inductive effects of PPARalpha ligands, especially of peroxisome proliferation and induction of H2O2-generating peroxisomal beta -oxidation system enzymes, is based primarily, if not exclusively, on data obtained with primary cultures of human hepatocytes and a human hepatocellular carcinoma cell line (21-23). Many factors, such as low levels of PPARalpha expression, expression of a human PPARalpha splice variant that may negatively interfere with PPARalpha function, differences in protein sequence between human and mouse PPARalpha , relative amounts of the three PPAR isotypes in liver cells, competition for the heterodimerization partner RXR, differences in the PPRE of target genes, and possible differences in nuclear receptor coactivators, may indeed account for the purported refractoriness of human hepatocytes to peroxisome proliferation (2, 3, 5, 21-29).

The proposal that humans are resistant to induction of peroxisome proliferation and liver tumor development presupposes (i) that human liver cells in vivo are resistant to peroxisome proliferator-induced pleiotropic responses at the anticipated levels of exposure and (ii) that failure to observe a robust morphological phenomenon of peroxisome proliferative response signifies that changes in the expression patterns of other genes may not be relevant in the development of liver cancer. Widespread exposure to synthetic PPARalpha ligands, nevertheless, raises a potential concern of carcinogenic risk to humans because of the uncertainty regarding the full spectrum of PPARalpha -regulated genes in liver and their role in liver cancer development (30). Systematic analysis of molecular mechanisms underlying tissue and species-specific responses to PPARalpha ligands requires examination of the role of various components of the transcriptional machinery (28, 29) and generation of molecular portraits of gene expression patterns (30). In this study, we examined the functional competency of human PPARalpha on the induction of PPARalpha target genes in PPARalpha -/- mouse liver to determine the role of the species origin of this receptor in peroxisome proliferator-induced immediate pleiotropic responses. Targeted disruption of PPARalpha abolishes the induction of peroxisome proliferation and PPARalpha target genes in mouse liver (10, 13). We demonstrate that introduction of adenoviruses encoding human PPARalpha or mouse PPARalpha into PPARalpha -/- mice fully restores the induction of ligand-mediated responses in liver. These studies establish that human PPARalpha is functionally fully competent in inducing peroxisome proliferation within the context of mouse liver environment, and the human PPARalpha -mouse RXR chimeric heterodimer can transcriptionally activate mouse PPARalpha target genes that contain the PPRE.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Mice and Treatment with Peroxisome Proliferators-- Wild type (C57BL/6J) mice and PPARalpha -/- mice (13), 3-4 months of age and weighing 25-35 g, were used in this study. PPARalpha -/- mice were maintained on powdered diet with or without Wy-14,643 (0.1% w/w), ciprofibrate (0.025% w/w), nafenopin (0.1% w/w), or di(2-ethylhexyl) phthalate (2% w/w) for 10-14 days prior to adenovirus injection and killed on days 2, 3, 4, 5, or 6 after injection as indicated. For dose response of Ad/hPPARalpha to Wy-14,643 the PPARalpha -/- mice were maintained on powdered diet with different doses of Wy14,643 (0% w/w, 0.0125% w/w, 0.025% w/w, 0.05% w/w, and 0.1% w/w, respectively) from 1 day prior to injection and were killed 4 days later. For cell proliferation analysis, mice were given bromodeoxyuridine (0.5 mg/ml) in drinking water for 3 or 5 days after adenovirus injection, and their livers were processed for immunohistochemistry (31). Animal procedures used in this study were approved by the Institutional Review Board for Animal Research of the Northwestern University.

Adenoviral Gene Transfer-- Construction of recombinant adenovirus Ad/hPPARalpha was as follows. Human PPARalpha cDNA (32) was cloned into pCMV expression cassette at the SalI site, and the entire cassette was cut out with ClaI and BamHI and cloned into the EcoRV and BglII sites of pShuttle vector (Quantum Biotechnologies, Inc.). The linearized shuttle vector and AdEasy vector (Quantum Biotechnologies, Inc.) were then co-transformed into Escherichia coli strain BJ5183. Positive recombinant plasmid Ad/hPPARalpha was selected (Fig. 1). The linearized Ad/hPPARalpha was then transfected with LipofectAMINE2000 (Life Technologies, Inc.) into the packaging cell HEK293A. Virus was purified with CsCl banding and stored at -70 °C. The mouse PPARalpha (mPPARalpha ) cDNA (33) was cloned into pShuttle vector; the generation and manipulation of Ad/mPPARalpha was as described for Ad/hPPARalpha . Adenoviral construct of Ad/LacZ was the generous gift of Dr W. El-Deiry (University of Pennsylvania, Philadelphia) and has been described previously (34). Mice were intravenously injected (tail vein) in a volume of 200 µl with 1 × 1011 virus particles of Ad/LacZ, Ad/hPPARalpha , or Ad/mPPARalpha . Mice injected with PBS served as controls. For dose response of Ad/hPPARalpha or Ad/mPPARalpha to Wy-14,643 the PPARalpha -/- mice were injected with 1 × 1011 virus particles of Ad/hPPARalpha or Ad/mPPARalpha as described above. Mice treated with 0.1% Wy14,643 and injected with PBS served as controls. Livers were separated and quickly fixed or frozen and used for the desired experiments.


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  		Fig. 1.   Recombinant adenovirus PPARalpha . hPPARalpha or mPPARalpha was cloned downstream of the cytomegalovirus (CMV) promoter, and the SV40 poly(A) sequence was located downstream of PPARalpha cDNA. Ad5, adenovirus serotype 5; LITR, left inverted terminal repeat; RITR, right inverted terminal repeat; Delta E1, deletion of E1 gene; Delta E3, deletion of E3 gene.

Northern and Immunoblot Procedures-- Total RNA was isolated from liver using Trizol reagent (Life Technologies, Inc.). After glyoxylation, samples were electrophoresed on an 0.8% agarose gel, transferred to a nylon membrane, and then hybridized at 42 °C in a 50% formamide hybridization solution using 32P-labeled cDNA probes as described previously (31). Liver extracts were subjected to 7.5 or 10% SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and immunoblotted as described previously (10, 15, 31). Some of the Western blot signals were quantified by scanning densitometry as previously described by using the NIH Image software (10, 31). The values from PBS-injected mice were assigned the number 1.0.

Morphology-- beta -Galactosidase activity was visualized by incubating liver sections in PBS containing 5 mM potassium ferricyanate, 2 mM MgCl2, and <FR><NU>1</NU><DE>20</DE></FR> volume of 20 mg/ml 5-bromo-4-chloro-3-indolyl beta -D-galactoside in dimethylformamide at 37 °C. Tissue fixed in 10% neutral buffered formalin was embedded in paraffin using standard procedures. Sections (4-µm thick) were cut and stained with hematoxylin and eosin. Immunohistochemical localization of human PPARalpha (hPPARalpha ), L-PBE, proliferating cell nuclear antigen, and bromodeoxyuridine was performed as described previously (31). For cytochemical localization of peroxisomal catalase, tissues were fixed in 1.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) for 4 h at 4 °C and incubated in alkaline 3',3'-diaminobenzidine (DAB) substrate as described previously (14). Semithin sections, without counterstain, were examined by light microscopy. Ultrathin sections for electron microscopy were contrasted with uranyl acetate and lead citrate.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

General Approach-- The availability of PPARalpha -/- mice enabled us to determine the immediate effects of transient expression of human PPARalpha on the induction of peroxisome proliferator-mediated pleiotropic responses in PPARalpha -/- mouse hepatocytes in vivo. Since PPARalpha -/- mice are nonresponsive to peroxisome proliferators, as they fail to manifest hepatic peroxisome proliferation and overexpression of genes regulated by PPARalpha (10, 13), we used recombinant adenovirus to express human PPARalpha in PPARalpha -/- mouse liver and evaluated the immediate pleiotropic effects of PPARalpha agonists. As expected, PPARalpha -/- mice maintained on Wy-14,643-containing diet failed to show peroxisome proliferation and overexpression of PPARalpha target genes in liver as evidenced by observations in mice given PBS or Ad/LacZ intravenously. To assess the ability of adenovirally expressed recombinant human PPARalpha to support peroxisome proliferation in PPARalpha -/- mouse hepatocytes, we infected PPARalpha -/- mice maintained on a diet containing Wy-14,643 with 6.125 × 109-4 × 1011 infectious viral particles by tail vein injection and determined the expression levels of L-PBE, the second enzyme of the inducible classical peroxisomal beta -oxidation system (1). Immunoblots of liver samples obtained 4 days after infection revealed maximum induction with 1 × 1011 infectious viral particles (Fig. 2A). At the higher viral load (4 × 1011), no further increase in L-PBE expression was noted despite increased expression of human PPARalpha . Peroxisomal catalase level did not change in these livers after Ad/hPPARalpha (Fig. 2A) as this gene is not regulated by PPARalpha (12). To investigate the time period between recombinant viral infection and the induction of human PPARalpha to effectuate induction of PPARalpha -responsive genes in PPARalpha -/- liver, mice were killed at 2, 3, 4, 5, and 6 days after a single tail vein injection of 1 × 1011 viral particles. As illustrated in Fig. 2B, hPPARalpha was barely detectable until day 3, although the expression of L-PBE began to manifest on day 2, suggesting that PPARalpha target genes are transcriptionally activated as soon as the expression of this receptor ensues. After day 3, the L-PBE levels increased perceptibly with maximum expression between 4 and 6 days (Fig. 2B). PPARalpha -/- mice that did not receive Wy-14,643 failed to show L-PBE induction when infected with Ad/mPPARalpha (Fig. 2C, lane 2) or Ad/hPPARalpha (Fig. 2C, lane 7) suggesting that the natural/biological ligands are insufficient to activate the introduced mPPARalpha or hPPARalpha as these ligands can be effectively metabolized by the peroxisomal AOX (14, 18). We determined the dose response to ascertain the comparative sensitivity of normalized expression of human and mouse PPARalpha in PPAR-null liver to Wy-14,643 (Fig. 2, C and D). The data indicate that both mouse and human PPARalpha respond similarly to a given dose of Wy-14,643 as determined by the expression of L-PBE.


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  		Fig. 2.   Inducibility of PPARalpha target gene expression. A, determination of effective viral dose. PPARalpha -/- mice maintained on Wy-14,643-containing diet for 10 days were infected with 4 × 1011 (group 1), 1 × 1011 (group 2), 2.5 × 1010 (group 3), 1.25 × 1010 (group 4), and 6.125 × 109 (group 5) infectious Ad/hPPARalpha particles by tail vein injection and killed 4 days later while still on Wy-14,643. Group 6 received 4 × 1011 Ad/LacZ viral particles. Representative immunoblot analysis (two mice in each group) of hPPARalpha , L-PBE (PPARalpha -responsive gene), and catalase (nonresponsive gene) reveals optimal L-PBE induction with 1 × 1011 (group 2). B, time course of induction of PPARalpha -responsive L-PBE gene after viral injection. PPARalpha -/- mice (two in each group) maintained on Wy-14,643 were infected with 1 × 1011 Ad/hPPARalpha viral particles and killed 2, 3, 4, 5, and 6 days after infection. PPARalpha -/- mice injected with PBS or Ad/LacZ were killed 6 days after the injection. Liver samples were immunoblotted for PPARalpha , L-PBE, and catalase. C and D, dose-response studies in PPARalpha -null mice expressing the same amount of human or mouse PPARalpha in liver. Wy-14,643 was administered in the diet at the concentrations indicated, and mice were injected with the same amount of mouse (lanes 2-6) or human (lanes 7-11) adenoviral particles containing PPARalpha (1 × 1011). Lanes 2 and 7 represent mouse and human controls, respectively. Lane 1 represents PPAR-null mouse given PBS and no PPAR-containing virus while maintained on Wy-14,643, and the values are used as a baseline for comparison (black box). PPARalpha -injected mice given PBS and no drug were used to assess the effect if any of endogenous ligands. L-PBE and catalase expression in liver was determined by immunoblot analysis (C), and the data was quantitated (D). triangle  and  represent L-PBE levels obtained with mouse and human PPAR, respectively. × and  represent catalase levels obtained with mouse and human receptor, respectively. d, days.

Human PPARalpha -directed Induction of Peroxisome Proliferation in PPARalpha -/- Mouse Liver-- To fully evaluate the effect of human PPARalpha in the PPARalpha -null mouse liver, we injected 1 × 1011 viral particles intravenously and analyzed livers of mice killed on days 4 and 6 after infection for peroxisome proliferation and peroxisome proliferator-induced immediate pleiotropic responses (Figs. 3 and 4). Nearly 60% of hepatocytes stained positively for beta -galactosidase activity in PPARalpha -/- mice injected with Ad/LacZ and killed 4 or 6 days after injection (Fig. 3A). Likewise ~60-70% of hepatocytes expressed hPPARalpha transgene at immunohistochemically detectable levels between 4 and 6 days postinjection (Fig. 3B). As expected, control PPARalpha -/- livers revealed no PPARalpha expression (Figs. 2, 3C, and 4). No significant inflammatory infiltrate was detected in any of the livers infected with Ad/hPPARalpha during the 6-day duration of this study. Hepatic peroxisome proliferation in PPARalpha -/- mice that were fed either control diet or Wy-14,643-containing diet and infected with Ad/hPPARalpha transgene was evaluated by immunohistochemical staining for L-PBE (Fig. 3, D and E). Intense granular staining of hepatocyte cytoplasm was evident in Wy-14,643-fed PPARalpha -/- mice expressing human PPARalpha transgene (Fig. 3D) when compared with PPARalpha -/- mice maintained on Wy-14,643 but given PBS or Ad/LacZ intravenously (Fig. 3E). PPARalpha -/- mice that were injected with Ad/hPPARalpha but maintained on normal chow without the ligand failed to show peroxisome proliferation (not illustrated). Peroxisome proliferation was also evaluated by light microscopic (Fig. 3, F and G) and electron microscopic (not illustrated) evaluation of sections of liver processed to visualize peroxisomal catalase. Marked increases in the number and size of peroxisomes in the liver of the Wy-14,643-fed PPARalpha -/- mouse were clearly seen 6 days after Ad/hPPARalpha infection (Fig. 3F) as compared with the control PPARalpha -/- mouse (Fig. 3G). Liver cell proliferation, as assessed by bromodeoxyuridine incorporation or by staining for proliferating cell nuclear antigen in Wy-14,643-fed PPARalpha -/- mice 5 days after Ad/hPPARalpha (Fig. 3H) or Ad/LacZ (Fig. 3I) infection, was not significantly different from that observed in Wy-14,643-treated wild type mice (Fig. 3J), implying that human PPARalpha can support the initial wave of liver cell proliferation occurring after exposure to peroxisome proliferators (35). These observations clearly establish that the presence of exogenous synthetic ligand alone in the PPARalpha -/- liver cells or the expression of exogenous human PPARalpha in the absence of synthetic PPARalpha ligand such as Wy-14,643 is not sufficient to induce peroxisome proliferation and liver cell proliferation.


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  		Fig. 3.   A, beta -galactosidase staining of 6-day Ad/LacZ PPARalpha -/- liver. B and C, PPARalpha immunohistochemical staining of PPARalpha -/- mouse liver 6 days after Ad/hPPARalpha (1 × 1011 particles) (B) and Ad/LacZ infection (C). D and E, L-PBE immunohistochemical staining of PPARalpha -/- mouse liver 6 days after Ad/hPPARalpha (D) and Ad/LacZ (E) infection. F and G, histochemical staining of PPARalpha -/- mouse liver for peroxisomal catalase 6 days after Ad/hPPARalpha (F) and Ad/LacZ (G) infection. H-J, proliferating cell nuclear antigen immunohistochemical staining of PPARalpha -/- mouse liver 5 days after Ad/hPPARalpha (H) and Ad/LacZ infection (I) and wild type mouse fed Wy-14,643 for 5 days (J). PPARalpha -/- mice were maintained on Wy-14,643-containing diet for 10 days before viral infection and until they were killed.

Human PPARalpha -directed Gene Expression in PPARalpha -/- Mouse Liver-- To investigate whether human PPARalpha is sufficient for the mediation of peroxisome proliferator-induced immediate pleiotropic responses in PPARalpha -/- mouse liver, we evaluated the mRNA levels of several well known PPARalpha -regulated genes such as AOX, L-PBE, peroxisomal 3-ketoacyl-CoA thiolase, CYP4A1, and CYP4A3 by Northern blotting (Fig. 4A). As expected, administering Wy-14,643 to PPARalpha -/- mice failed to induce overexpression of PPARalpha -regulated genes (see Ad/LacZ and PBS groups). When human PPARalpha transgene was introduced by adenoviral approach, a marked overexpression of target gene mRNAs was discerned in Wy-14,643-fed PPARalpha -/- mouse livers 4 and 6 days postinjection (Fig. 4A). In the absence of ligand, human PPARalpha transgene failed to induce overexpression of these target genes (data not shown). We also evaluated the induction of fatty acid-metabolizing enzymes in the liver of PPARalpha -/- mice expressing human PPARalpha by immunoblotting (Fig. 4, B and C). Levels of hepatic peroxisomal fatty acid beta -oxidation enzymes in the Wy-14,643-treated PPARalpha -/- mouse liver were similar to the constitutive levels of expression as reported previously (10, 13), but the human PPARalpha -expressing PPARalpha -/- mouse livers exhibited significant increases in Wy-14,643-mediated AOX, L-PBE, and peroxisomal 3-ketoacyl-CoA thiolase (Fig. 4B). A modest increase in very long chain acyl-CoA dehydrogenase and medium chain acyl-CoA dehydrogenase, the enzymes involved in mitochondrial lipid metabolism, was noted (Fig. 4B). Hepatic levels of catalase and urate oxidase, two peroxisomal enzymes not known to be regulated by PPARalpha , did not change in Wy-14,643-treated PPARalpha -/- mice irrespective of the PPARalpha status.


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  		Fig. 4.   A, Northern blot showing the expression levels of different genes in PPARalpha -/- mouse liver 4 and 6 days after PBS, Ad/LacZ, or Ad/hPPARalpha (1 × 1011 particles) tail vein injection. All mice were on Wy-14,643-containing diet (see legends for Figs. 2 and 3). B, immunoblot showing the expression of hPPARalpha and selected proteins involved in fatty acid metabolism. Liver homogenates (20 µg) from Wy-14,643-fed PPARalpha -/- mice killed 6 days after PBS, Ad/LacZ, or Ad/hPPARalpha tail vein injection were subjected to SDS-polyacrylamide gel electrophoresis and immunoblotted (two mice for each group). Two AOX polypeptides, A and B, are shown. C, the Western signals in B were then quantified by scanning densitometry. Each bar represents the average of two immunoblots of PBS, Ad/LacZ, and Ad/hPPARalpha , respectively. The PBS group value is assigned a number of 1.0. cat, catalase; d, days; PTL, peroxisomal 3-ketoacyl-CoA thiolase; D-PBE, peroxisomal D-3-hydroxyacyl-CoA dehydratase/D-3-hydroxyacyl-CoA dehydrogenase bifunctional protein; UOX, urate oxidase; VLCAD, very long chain acyl-CoA dehydrogenase; MCAD, medium chain acyl-CoA dehydrogenase; SCAD, short chain acyl-CoA dehydrogenase.

Recently, using cDNA microarray, we identified several genes that are up-regulated in the Wy-14,643-treated wild type mouse liver, and none of these encode peroxisomal proteins (30). These include CD36, monoglyceride lipase, Ly-6D, cell death-inducing DNA fragmentation factor alpha , pyruvate dehydrogenase kinase-4, C3f, and others (27). Up-regulation of these genes in mouse liver was shown to be dependent upon peroxisome proliferation vis à vis PPARalpha as no induction was noted in PPARalpha -/- mice treated with peroxisome proliferators (27). To determine whether human PPARalpha can also mediate the expression of these newly identified genes we assessed the levels of expression of six genes, namely CD36, Ly-6D, Rbp7, monoglyceride lipase, C3f, and pyruvate dehydrogenase kinase-4, in Wy-14,643-treated PPARalpha -/- mouse liver expressing human PPARalpha (Fig. 5). Human PPARalpha expression resulted in the up-regulation of these genes in Wy-14,643-treated PPARalpha -/- mouse liver (Fig. 5), indicating that human PPARalpha is also capable of regulating these genes in livers in response to synthetic PPARalpha ligands.


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  		Fig. 5.   Northern blot analysis to assess the Wy-14,643-induced expression of nonperoxisomal genes (CD36, Ly-6D, Rbp7, monoglyceride lipase (mLipase), C3f, and pyruvate dehydrogenase kinase-4 (PDK4)) in PPARalpha -/- mouse liver 4 and 6 days after PBS, Ad/LacZ, or Ad/hPPARalpha (1 × 1011 particles) tail vein injection. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is used as control. d, days.

Comparative Response of Human and Mouse PPARalpha to Structurally Different Peroxisome Proliferators-- We sought to assess the comparative ability of ciprofibrate, di(2-ethylhexyl) phthalate, nafenopin, and Wy-14,643 to activate human and mouse PPARalpha in PPARalpha -/- mice. PPARalpha -/- mice were fed a diet containing one of these peroxisome proliferators, and the expressions of three PPARalpha target genes (AOX, L-PBE, and CYP4A3) in liver was analyzed 4 days after intravenous injection of 1 × 1011 particles of Ad/hPPARalpha , Ad/mPPARalpha , or Ad/LacZ (Fig. 6A). In Northern blots, under the hybridization conditions used, hPPARalpha did not recognize mPPARalpha ; likewise the mPPAR cDNA probe failed to recognize hPPARalpha (Fig. 6A). All four peroxisome proliferators used in this experiment activated both human and mouse PPARalpha in a qualitatively similar manner as evidenced by the levels of mRNA expression of target genes (Fig. 6A). The catalase mRNA level in liver served as a loading control in all groups. We also assessed the constitutive and inducible levels of AOX and L-PBE by immunoblot analysis (Fig. 6, B and C). Both human and mouse PPARalpha caused marked increases in the protein content of AOX as well as L-PBE, the first two enzymes of the inducible peroxisomal beta -oxidation system in PPARalpha -/- mouse liver in response to structurally different PPARalpha ligands.


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  		Fig. 6.   Comparative response of hPPARalpha and mPPARalpha to different peroxisome proliferators. PPARalpha -/- mice were fed a diet containing Wy-14,643, ciprofibrate, di(2-ethylhexyl) phthalate (DEHP), or nafenopin. A, Northern blot showing the expression of three PPARalpha target genes in liver of the PPARalpha -/- mouse 4 days after intravenous injection of (1 × 1011 particles) Ad/hPPARalpha (H), Ad/mPPARalpha (M), or Ad/LacZ (LacZ). PBS was injected as nonviral control. The catalase mRNA level in liver served as a loading control in all groups. B, immunoblot showing the expression of L-PBE, AOX, and catalase in the liver of mice used for Northern blot analysis (as in A). C, the AOX immunoblots shown in B were quantified by scanning densitometry. The values from PBS groups were assigned a number of 1.0. H, Ad/hPPARalpha -injected; M, Ad/mPPARalpha -injected.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Several nuclear receptors such as PPARalpha (4), Ahr (36), constitutive androstane receptor (37, 38), and steroid and xenobiotic receptor/pregnenolone X receptor (39) regulate the transcriptional activation of specific target genes in liver in response to xenobiotic ligands. Inducible gene expression, resulting from exposure to xenobiotic chemicals that transcriptionally activate genes by a receptor-mediated mechanism, presents many uncertainties when extrapolating the toxicological implications from one species to the other and in assessing risk to humans. Evidence suggests that some of these receptors function as species-specific xenosensors (39), and they may differ in their content or protein composition to account for species differences in the expected responses (24). Species differences in the magnitude of xenobiotic-induced hepatic peroxisome proliferation, especially between rodents and humans, are documented, but the mechanisms remain largely speculative. A close concordance with the magnitude of peroxisome proliferation and hepatocarcinogenesis in rats and mice (2, 3, 19) is used as the basis for the assertion that since humans appear to be refractory to hepatic peroxisome proliferation they are resistant to the inductive effects of PPARalpha ligands including liver cancer risk (19-21). Humans possess a functional PPARalpha , but it is has been shown to be weaker in response to Wy-14,643 when compared with that of rat and mouse PPARalpha s in trans-activation assays (6, 24, 25, 27, 32). The low levels of PPARalpha expression in human liver and the presence of PPARalpha splice variants have also been suggested to contribute to the resistance of human hepatocytes to peroxisome proliferation (22-25). Increasing the expression of PPARalpha in HepG2 cells to levels found in mouse liver has not been found sufficient to confer peroxisome proliferator-induced responsiveness (22, 23). These results imply that HepG2 cell lines may have acquired many epigenetic alterations, such as methylation of genes, in passage over the years as they also lack expression of CYP4A11 and possibly other genes. These considerations as well as differences in the levels of PPARalpha and its heterodimerization partner RXR and in the PPRE of target genes or other components of transcriptional machinery may contribute to the nonresponsiveness of human hepatocytes (28, 29, 40). The assumption that human AOX and possibly the entire panoply of peroxisomal beta -oxidation system genes and CYP4A omega -oxidation genes in human have evolutionarily been altered appears somewhat farfetched and is not based on sound rationale (23, 41). Human PPARalpha has been found to differ at some amino acid positions in the ligand binding domain from rat and mouse receptor (27, 32), but the pharmacological relevance of these changes has not been established. The data we present in this study on the response to different ligands and to varying doses of Wy-14,643 suggest that these differences do not alter the sensitivity of the human PPARalpha .

Our results clearly show that human PPARalpha is fully capable of transcriptionally activating peroxisomal beta -oxidation system genes as well as several nonperoxisomal genes, and this receptor is as effective as mouse PPARalpha under in vivo conditions within the context of intact liver. These studies demonstrate the restoration of peroxisome proliferator-induced pleiotropic responses in PPARalpha -/- mouse liver with recombinant adenovirus that expresses human PPARalpha or mouse PPARalpha . In the absence of exogenous ligands, the mouse or human PPARalpha failed to elicit significant alterations in the expression of PPARalpha target genes (Fig. 2C, lanes 2 and 7), implying that the endogenous ligands such as very long chain fatty acids and/or their acyl-CoAs do not exert any inductive effects as these are effectively metabolized by AOX (1, 14). Human PPARalpha activated a full spectrum of target genes in mouse liver, and these include many well characterized genes such as AOX, L-PBE, peroxisomal 3-ketoacyl-CoA thiolase, CYP4A1, and CYP4A3 that possess PPREs in their 5'-flanking regions (2, 4). The overexpression of these genes in PPARalpha -/- liver with human PPARalpha establishes that human PPARalpha heterodimerizes with mouse RXR in mouse liver in vivo, and the chimeric hPPARalpha -mRXR heterodimers recognize the PPRE in mouse target genes. Also of interest is that hPPARalpha in PPARalpha -/- mouse liver enhanced the expression of several newly identified genes found to be overexpressed in mouse liver with peroxisome proliferation in a PPARalpha -dependent fashion (30). These include CD36 (42), Ly-6D (43), pyruvate dehydrogenase kinase-4 (44), monoglyceride lipase (45), Rbp7 (46), and C3f (47). CD36 and Ly-6D are cell surface/membrane-associated proteins whose functions in peroxisome proliferator-induced pleiotropic responses remain to be explored. CD36, a scavenger receptor known to interact with a large variety of ligands including oxidized low density lipoproteins, is up-regulated in Wy-14,643-treated mouse livers with peroxisome proliferation (48) and has been shown to play a role in foam cell conversion of macrophages (48). Because of the induction in liver of many nonperoxisomal genes by synthetic PPARalpha ligands and since hPPARalpha is functionally capable of mediating this induction, we consider the assumption that human liver cells are refractory to the induction of peroxisome proliferator-induced pleiotropic responses including liver cancer development somewhat premature.

For many xenobiotics, there is no reliable system other than in humans to assess the toxicological significance directly and quantitatively (39). Recently a "humanized" animal to establish the role of steroid and xenobiotic receptor/pregnenolone X receptor activation in xenoprotection was developed to demonstrate that species origin of the receptor, rather than the promoter structure of CYP3A genes, dictates the species-specific pattern of CYP3A inducibility (39). The adenoviral-controlled expression of human PPARalpha provided valuable information on the ability of this receptor to support peroxisome proliferator-induced immediate pleiotropic responses, but this approach cannot be used for long term studies or for effective quantitative titration of hPPARalpha expression that is identical to the mPPARalpha normally expressed in mouse liver so that extrapolation of risk between rodents and humans can be made as to the relative levels of PPARalpha expressed in these susceptible and resistant species. Development of humanized PPARalpha -/- mouse strains that express different levels of human PPARalpha under the control of the albumin promoter in liver will be useful for such comparison and also for long term carcinogenic studies. More importantly, mouse liver repopulated with human hepatocytes (49) may prove to be immensely informative to evaluate the immediate and delayed pleiotropic responses induced by PPARalpha ligands in intact human liver cells.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants GM23750 (to J. K. R.) and CA84472 (to M. S. R.), a merit review grant from the Department of Veterans Affairs (to A. V. Y.), and the Joseph L. Mayberry, Sr. Endowment Fund.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

|| To whom correspondence should be addressed: Dept. of Pathology, Northwestern University Medical School, 303 East Chicago Ave., Chicago, IL 60611-3008. Tel.: 312-503-8144; Fax: 312-503-8249; E-mail: jkreddy@northwestern.edu.

Published, JBC Papers in Press, September 10, 2001, DOI 10.1074/jbc.M106480200

    ABBREVIATIONS

The abbreviations used are: PPAR, peroxisome proliferator-activated receptor; PPRE, peroxisome proliferator response element; AOX, straight chain fatty acyl-CoA oxidase; L-PBE, peroxisomal enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase bifunctional protein; CYP4A1 and CYP4A3, genes encoding microsomal cytochrome P450 fatty acid omega -hydroxylases; h, human; m, mouse; PBS, phosphate-buffered saline; RXR, retinoid X receptor; CD36, cluster of differentiation 36; Ly-6D, lymphocyte antigen 6 complex locus D; Rbp7, retinoid-binding protein 7.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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