Addition of Peroxisome Proliferator-Activated Receptor to Guinea Pig Hepatocytes Confers Increased Responsiveness to Peroxisome

[CANCER RESEARCH 59, 4776–4780, October 1, 1999] Advances in Brief

Addition of Peroxisome Proliferator-activated Receptor ␣ to Guinea Pig Hepatocytes Confers Increased Responsiveness to Peroxisome Proliferators

Neil Macdonald, Peter R. Holden, and Ruth A. Roberts1 AstraZeneca Central Toxicology Laboratory, Alderley Park, Macclesfield SK10 4TJ, United Kingdom

Abstract humans are considered to be nonresponsive to the adverse effects of PPs associated with increased ␤-oxidation and peroxisome prolifera- The fibrate drugs, such as nafenopin and fenofibrate, show efficacy in tion (3, 7, 11, 12). In guinea pigs, there is no increased DNA synthesis hyperlipidemias but cause peroxisome proliferation and liver tumors in or liver enlargement and only a small increase in peroxisome prolif- rats and mice via nongenotoxic mechanisms. However, humans and ␤ guinea pigs appear refractory to these adverse effects. The peroxisome eration, and peroxisomal -oxidation enzyme activity is weak (12) proliferator (PP)-activated receptor ␣ (PPAR␣) mediates the effects of even at very high PP concentrations (13). Similarly, cultured human PPs by heterodimerizing with the retinoid X receptor (RXR) to bind to hepatocytes are refractory to the adverse effects of PPs (14) because DNA at PP response elements (PPREs) upstream of PP-regulated genes, the induction of peroxisomal ␤-oxidation by PPs weak (15) or absent such as acyl-CoA oxidase. Hepatic expression of PPAR␣ in guinea pigs (15), and PPs cannot induce DNA synthesis (reviewed in Ref. 11) or and humans is low, suggesting that species differences in response to PPs suppress apoptosis (16). In addition, there appears to be no increased may be due at least in part to quantity of PPAR␣. To test this hypothesis, risk of liver cancer in patients receiving fibrate PP drug therapy (17). ␣ ␣ we introduced mouse PPAR and its heterodimerization partner, RXR , The PPAR␣ was originally cloned from mouse liver (18) and was into guinea pig hepatocytes by transient transfection and determined shown to mediate the pleiotropic effects of PPs in rodents, such as responsiveness to the PP nafenopin by cyanide-insensitive palmitoyl-CoA enzyme induction, peroxisome proliferation, and hepatocarcinogen- oxidation (CIPCO). Expression of the mRNA for mouse PPAR␣ in trans- ␣ fected guinea pig hepatocytes was verified using species-specific PCR. In esis (8, 9, 19–22). PPAR is activated by hypolipidemic drugs but guinea pig hepatocytes transfected with control plasmids and treated with also by natural ligands, such as fatty acids and eicosanoids (23, 24). 50 ␮M nafenopin in the absence or presence of the RXR ligand, 9-cis- Activated PPAR␣ binds to DNA as a heterodimer with the RXR at retinoic acid (5 ␮M) gave only a 1.7 ؎ 1.5- or 3.3 ؎ 1.5-fold induction in direct repeat 1 (DR1) elements (degenerate AGGTCA direct repeats CIPCO, respectively. However, addition of ligands to hepatocytes co- spaced by 1 bp) that comprise two degenerate direct AGGTCA transfected with both mPPAR␣ and RXR gave a strong induction of repeats spaced by 1 bp, termed PPREs (25, 26). PPREs have been ␣ ؎ CIPCO (14.8 8.6-fold). Mouse, human, and guinea pig PPAR showed identified in the promoter regions of a number of genes that are ␣ equivalent function in the CIPCO assays. Thus, quantity of PPAR plays transcriptionally regulated by PPs (8, 9, 19, 25, 27–30). However, the a significant role in the lack of response to PPs in guinea pigs. In humans, presence of an active PPRE in the promoter region of a particular gene however, lack of PPAR␣ may be only one factor dictating lack of response because recent data show that the human acyl-CoA oxidase gene lacks a from one species does not necessarily infer the presence of an active functional PP response element. PPRE in the equivalent gene in all species (31). For example, the rat ACO promoter contains an active PPRE (9), but the human ACO Introduction promoter displays sequence differences and lacks activity (32, 33). Although human and guinea pig hepatocytes are refractory to the PPs2 are a class of nongenotoxic rodent hepatocarcinogens that adverse effects of PPs, cDNAs encoding for a functional, full-length includes industrial plasticizers; fibrate hypolipidemic drugs, such as PPAR␣ have been isolated both from guinea pig (34, 35) and human nafenopin; and certain chlorinated solvents (1–5). In mice and rats, liver (10, 36). The hPPAR␣, gpPPAR␣, and mPPAR␣ display com- treatment with PPs results in hepatic peroxisome proliferation, in- parable activity in reporter gene assays using a minimal PPRE from creased hepatocyte DNA synthesis, suppression of hepatocyte apo- the rat ACO promoter (34). However, humans and guinea pigs show ptosis, liver enlargement, and hepatocarcinoma (reviewed in Refs. 3, around 10-fold lower hepatic expression of PPAR␣ when compared 6, and 7). In addition, PPs up-regulate transcription of enzymes with responsive species, such as rats and mice (Refs. 34, 37, and 38; involved in the ␤-oxidation of long-chain fatty acids, such as ACO, reviewed in Ref. 39), and, at least in humans, the pool of active enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase bifunc- PPAR␣ may be depleted due to expression of alternatively spliced tional enzyme and thiolase, as well as genes of the cytochrome PPAR␣ mRNA lacking exon 6 that leads to a truncated, inactive P4504A family (8–10). In contrast, species such as guinea pigs and PPAR␣ (30, 38). This suggests that the quantity of functional PPAR␣ may represent an important aspect of species differences in response. Received 3/29/99; accepted 8/17/99. The costs of publication of this article were defrayed in part by the payment of page To test this, we used transient transfection to increase the level of charges. This article must therefore be hereby marked advertisement in accordance with PPAR␣ and RXR in guinea pig hepatocytes in vitro and determined 18 U.S.C. Section 1734 solely to indicate this fact. responsiveness to the PP nafenopin, using CIPCO as an end point. 1 To whom requests for reprints should be addressed, at Cancer Biology Group, AstraZeneca Central Toxicology Laboratory, Alderley Park, Macclesfield SK10 4TJ, CIPCO is an established and robust indicator of peroxisome prolifer- United Kingdom. Phone: 1625-516413; Fax: 1625-582897; E-mail: ruth.roberts@ ation. Expression of functional PPAR␣ was verified by species- CTL.astrazeneca.com. specific PCR for mouse PPAR␣ and by reporter gene assay using a rat 2 The abbreviations used are: PP, peroxisome proliferator; ACO, acyl-CoA oxidase; PPAR␣, peroxisome proliferator-activated receptor ␣; RXR, retinoid X receptor; PPRE, ACO PPRE promoter-luciferase reporter construct (9, 32). Key ex- PPAR␣ response element; HNF-4␣, hepatocyte nuclear factor 4␣; COUP-TF, chicken periments were repeated with hPPAR␣ and gpPPAR␣ to determine ovalbumin upstream promotor-transcription factor; CIPCO, cyanide-insensitive palmitoyl ␣ CoA oxidase; DMF, dimethyl formamide; hPPAR␣, human PPAR␣; mPPAR␣, mouse whether the ability of PPAR to confer increased responsiveness was PPAR␣; gpPPAR␣, guinea pig PPAR␣; CMV, cytomegalovirus. dependent on the species origin of the receptor. The data presented 4776

suggest that species differences in quantity of PPAR␣ plays a role in aliquot prior to pelleting cells by centrifugation at 2000 rpm for 2 min. The the lack of response to the PP class of nongenotoxic rodent hepato- pellet from the 800-␮l aliquot was resuspended in N-tris(hydroxy- carcinogens. methyl)methyl-2-aminoethanesulfonic acid, disrupted by sonication, and stored at Ϫ70°C for CIPCO and protein assays. The pellet from the 200-␮l ␮ ⅐ Materials and Methods aliquot was resuspended in 100 l of lysis buffer (25 mM Tris phosphate, 2mM DTT, 5 mM 1,2-diaminocyclohexane N,N,NЈ,NЉ-tetraacetic acid, 5% Ϫ ␤ Reagents. Nafenopin was a gift from Ciba-Geigy (Basel, Switzerland). glycerol, 0.01% Triton X-100 in dH2O) and stored at 70°C for -galac- N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammoniummethyl sulfate and tocidase and luciferase assays. CIPCO assays were carried out as described S-palmitoyl-CoA were from Roche Molecular Biochemicals. Trypsin was previously (43) with some modifications. The assay medium contained 60 from Life Technologies, Inc. (Paisley, United Kingdom). 9-cis-Retinoic acid mM Tris-HCl, pH 8.3, 50 ␮M CoA, 370 ␮M NADϩ,94mM nicotinamide, was purchased from Sigma. Galacto-Light Plus ␤-galactocidase assay reagent 2.8 mM DTT, 2 mM KCN, 12.5 ␮g/ml BSA (fatty acid free), 100 ␮g/ml was from Tropix Inc. Luciferin luciferase assay reagent was from Promega. All flavin adenosine dinucleotide, 50 ␮g/ml palmitoyl-CoA. Protein concen- tissue culture plastics were from Nunc. All other materials were purchased tration in the CIPCO assay aliquot of each sample was assessed using the from Flow, Life Technologies, Inc., or Sigma. Bradford protein assay reagent (Bio-Rad) following the manufacturer’s Plasmid Constructions. The ␤-galactocidase expression plasmid pCMV- instructions. ␤-Galactosidase and luciferase activity were assayed as de- .LacZ was obtained from Clontech (Basingstoke, United Kingdom). pcDNA3 scribed previously (40). Briefly, ␤-galactosidase activity was determined was from RϩD Systems (Oxon, United Kingdom). hPPAR␣ cDNA was a gift by incubating 10 ␮l of cell extract with Galacto-Light Plus reagent (Tropix from Dr. F. Gonzalez (National Cancer Institute, Bethesda, MD), and mRXR␣ Inc.) according to the manufacturer’s instructions. Luciferase activity was cDNA was a gift from Prof. Pierre Chambon. The plasmids pCMV.mPPAR␣, determined by incubating a 40-␮l aliquot of cell extract with luciferin pAco(Ϫ581/Ϫ471).G.Luc (30), pCMV.gpPPAR␣, pCMV.hPPAR␣ (34), and reagent (Promega) according to the manufacturers instructions. Luciferase pCMV.mRXR␣ (40) have been described previously. activity for each plate was determined and normalized for ␤-galactosidase Culture and Transient Transfection of Primary Hepatocytes. Hepato- activity. CIPCO activity was determined per plate and expressed as nmol of ϩ cytes were isolated from male Dunkin-Hartley guinea pigs by collagenase NAD reduced/min/mg of protein, normalized for ␤-galactosidase activity. perfusion as described previously (41). Viability was determined by trypan Data points are the mean of three determinations. blue exclusion. All cell preparations had a viability of 70–95% on isolation. Freshly isolated hepatocytes were diluted to 2 ϫ 106 cells/ml in FCS-free L-15 Results and Discussion ␮ medium supplemented with 10% tryptose phosphate broth, 10 g/ml insulin, ␤ 1mM hydrocortisone, 2 mML-glutamine, 100 units/ml penicillin, 100 units/ml The PP Nafenopin Induces -Oxidation of Long-Chain Fatty streptomycin, and 340 ␮M vitamin C. Hepatocytes (2 ϫ 106) were transfected Acids in Rat but not in Guinea Pig Primary Hepatocytes. Fig. 1 in suspension using 12 ␮gofN-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl- shows that the PP nafenopin (50 ␮M) gave a strong induction of ammoniummethyl sulfate and a plasmid cocktail containing 6.0 ␮gof ␤-oxidation of long-chain fatty acids in rat but not in guinea pig pCMV.PPAR␣ or pcDNA3, 6.0 ␮g of pCMV.mRXR␣ or pcDNA3, 1.0 ␮gof hepatocytes. In the rat, a 20-fold induction was seen as determined pCMV.LacZ, and 1.0 ␮g of pAco(Ϫ581/Ϫ471)G.Luc. After transfection, by CIPCO, a convenient and robust marker of peroxisome prolif- 2 hepatocytes were placed in 25-cm flasks containing 3 ml of L-15 medium eration and associated ␤-oxidation. In contrast, in hepatocytes supplemented as described above plus 10% fetal bovine serum. Untransfected from the guinea pig, there was only a small induction of CIPCO control hepatocytes were placed in culture immediately after dilution. Cultures activity (2.6 Ϯ 0.6-fold). This is consistent with previous reports, were maintained at 37°C in a humidified atmosphere, and the medium was in which PPs cause a marked induction of CIPCO in rat liver in changed after 5 h. After 24 h, fresh medium was added containing either DMF, 5 ␮M 9-cis-retinoic acid, 50 ␮M nafenopin, or 5 ␮M 9-cis-retinoic acid/50 ␮M vivo but only a small induction in guinea pig liver at equivalent nafenopin. Cells were cultured in the presence of ligands for a further 72 h, and plasmatic levels of the hypolipidemic PP ciprofibrate (44). These the medium was changed every 24 h. In all of these experiments, the trans- data confirm species differences in CIPCO reported in vivo and fection efficiency for guinea pig hepatocytes, as estimated by 5-bromo-4- confirm our in vitro system as a useful model for testing the chloro-3-indolyl-␣-D-galactopyranoside staining for ␤-galactosidase activity hypothesis that addition of PPAR␣ can confer increased respon- (see Ref. 42), was between 7% and 12% (data not shown). siveness to PPs. Species-specific PCR Detection of Transfected mPPAR␣. Total RNA was isolated from transfected and untransfected guinea pig hepatocytes 24 h after transfection using Total RNA Isolation Reagent (Advanced Biotechnol- ogies, Epsom, Surrey, United Kingdom). To eliminate transfected plasmid DNA from the RNA samples, poly(A)ϩ RNA was isolated from the total RNA samples using Miltenyi Biotec mRNA isolation kit following the manufacturer’s instructions. Fifty ng of each poly(A)ϩ RNA sample, as well as 50 ng of mouse hepatocyte total RNA, were reverse-transcribed using the Amersham Pharmacia Biotech First Strand cDNA synthesis kit (33-␮l reaction) following the manufacturer’s instructions. Three ␮l of each cDNA synthesis reaction, 10 ng of poly(A)ϩ RNA that had not undergone reverse transcription, and a RNA free reverse transcription reaction mixture were used as PCR templates with primers that were species specific for mPPAR␣ [sense, 5Ј-CGCCAGCACG GACGAGT-3Ј (bases 261–277 of the mPPAR␣ coding region); antisense, 5Ј-AAAAGGCGGGTTGTTGC-3Ј (bases 701–717 of the mPPAR␣ coding region)]. PCR conditions were as follows: 50 pmol of each primer, 200 ␮M ␮ dNTPs, 3 mM MgCl2, 1 unit of Taq (Promega); reaction volume, 50 l. Cycles were as follows: 94°C for 1 min; 35 cycles of 94°C for 1 min, 50°C for 1 min, and 72°C for 1 min; and 72°C for 3 min. Ten ␮l of each PCR reaction were run on a 2% agarose gel containing ethidium bromide. Gels were photographed under UV illumination using the Imager (Appligene) gel documentation system. ␤ ␮ Reporter Gene and CIPCO Assays. Medium was discarded and the Fig. 1. Induction of -oxidation by 50 M nafenopin in rat but not in guinea pig hepatocytes. Long-chain fatty acid ␤-oxidation is measured by CIPCO activity per mg of monolayers washed once with 2 ml of PBS. Cells were scraped into 1 ml hepatocyte protein. Data are expressed as fold induction over solvent (DMF) control. of ice-cold PBS that was subsequently split into a 200-␮l and an 800-␮l Columns, mean of triplicate results; bars, SD. 4777

Mouse PPAR␣ mRNA Is Expressed and Can Activate a PPRE Reporter Plasmid Co-transfected into Guinea Pig Hepatocytes. Constitutively, guinea pig hepatocytes express only a fraction of the PPAR␣ seen in the mouse or rat (34), suggesting that low levels of PPAR␣ may explain their lack of response to the adverse effects of PPs, such as peroxisome proliferation and ␤-oxidation. We wished to test this hypothesis by introducing a mouse PPAR␣ expression vector into guinea pig hepatocytes and evaluating the effects on the expression of endogenous ␤-oxidation genes. First, we established that mouse PPAR␣ mRNA was being expressed after transfection of the mouse PPAR␣ expression into guinea pigs hepatocytes (Fig. 2). Species-specific PCR for mPPAR␣ detected a strong band at the expected size (456 bp) in cDNA from mouse hepatocytes and from mPPAR␣-transfected guinea pig hepatocytes but not in untransfected guinea pig hepatocytes. Next, we wished to determine that the vector we were introducing was expressing sufficient PPAR␣ to give increased PP-dependent PPRE-mediated gene expression using the rat ACO minimal PPRE reporter construct. Fig. 3a shows that the PPAR␣ ligand nafenopin was unable to activate transcription in control transfections but caused a 6-fold increase in reporter gene activity after addition of mPPAR␣. Introduction of mPPAR␣/RXR into Guinea Pig Hepatocytes Confers Increased PP Responsiveness in Assays of ␤-Oxidation. Having established introduction of sufficient PPAR␣ to activate reporter gene expression, we examined the induction of ␤-oxidation by nafenopin in guinea pig hepatocytes with and without transfected mPPAR␣ and its heterodimerization partner, RXR (Fig. 3b). The ␤-oxidation of long-chain fatty acids was determined by CIPCO activity. In the absence of both receptor expression plasmids, the addition of nafenopin (column 5), 9-cis-retinoic acid (column 2), or both ligands (column 6) had only a weak effect on CIPCO. When RXR was transfected alone, either with or without 9-cis-retinoic acid, Fig. 3. Addition of mPPAR␣ to guinea pig hepatocytes activates transcription of the rat again there was no effect or only a weak effect on CIPCO upon acyl-CoA-luciferase reporter plasmid and confers increased CIPCO activity. a, ligand- dependent transcriptional activation of the rat ACO reporter plasmid by mPPAR␣. prACO addition of nafenopin (columns 8 and 7, respectively). When PPAR␣ (Ϫ581/Ϫ471).G.Luc reporter plasmid (1.0 ␮g) was transfected into 2 ϫ 106 guinea pig was transfected alone, there was some induction by nafenopin both hepatocytes together with pcDNA3.LacZ (1.0 ␮g) as a transfection control in either the presence or absence of receptor expression vector (pCMV.mPPAR␣;6␮g) and 50 ␮M nafenopin (naf). Data are shown as fold induction over DMF/pcDNA3 control of luciferase, corrected for transfection efficiency. b, ligand-dependent increase in CIPCO activity by addition of mPPAR␣ in the presence of mRXR␣. pCMV.PPAR␣ (6.0 ␮g) and/or pCMV.mRXR␣ (6.0 ␮g) was transfected into 2 ϫ 106 guinea pig hepatocytes together with pcDNA3.LacZ (1.0 ␮g) in either the presence or absence of 50 ␮M nafenopin (naf) and/or 9-cis-retinoic acid (5 ␮M). Data are shown as fold induction over DMF/pcDNA3 control (column 1) of CIPCO, corrected for transfection efficiency. Columns, mean of triplicate results; bars, SD.

with (column 14) and without (column 13)9-cis-retinoic acid, although there was no induction of CIPCO on addition of 9-cis-retinoic acid in the absence of nafenopin (column 10), suggesting the presence of a small amount of 9-cis-retinoic acid but very little natural PPAR␣ ligand (24). When RXR was co-transfected with PPAR␣, the level of CIPCO was quite high, even in the absence of nafenopin (column 12), but there was little stimulation of CIPCO on addition of PPAR␣ ligand alone (column 14). However, in the presence of both receptors and both ligands (column 16), there was a large induction of CIPCO activity. Thus, the addition of mPPAR␣ to guinea pig hepatocytes in the presence of appropriate co-factors confers on guinea pig hepatocytes the ability to respond to PPs, as monitored by endogenous enzyme activity. This pattern of ligand dependent PPAR␣ and RXR effects is comparable to that seen in rACO.PPRE reporter gene assays using cell lines (26). The nafenopin-dependent CIPCO induction seen in transfected Fig. 2. Expression of mouse PPAR␣ mRNA in guinea pig hepatocytes after transfection. Mouse PPAR␣-specific PCR of reverse-transcribed poly(A)ϩ RNA from transfected guinea pig hepatocytes was marked under optimal conditions but was (T; lane 2) and untransfected (UT; lane 1) guinea pig hepatocytes. Ϫ (lane 3) depicts the not as consistent nor as strong as that seen in untransfected rat control (no RNA reverse transcription), and con (lane 4) depicts the same poly(A)ϩ RNA as in lane 2 (T) but with the reverse transcription step omitted. Mu (lane 5) depicts the hepatocytes (Fig. 1). This may be because the guinea pig hepatocytes positive control (reverse-transcribed mouse total RNA). can mount only a partial response to PPs even in the presence of 4778

gpPPAR␣, and hPPAR␣, co-transfected with RXR␣, to increase nafenopin-dependent induction of ␤-oxidation in guinea pig hepatocytes over that seen in control transfected hepatocytes. Again, all three PPAR␣s gave similar increases in ligand-dependent ␤-oxidation induction in guinea pig hepatocytes (Fig. 4b). It has been suggested that the lack of an adverse response to PPs in hepatocytes from nonresponsive species may be due to insufficient levels of PPAR␣. However, some human genes clearly can respond to PPs because the activation of PPAR␣ by the hypolipidemic fibrate PPs forms the basis of their clinical use (29, 45, 46). Thus, human and guinea pig hepatocytes may express sufficient PPAR␣ to activate the genes associated with hypolipidemia but insufficient PPAR␣ to activate the full response seen in rat and mice, such as peroxisome proliferation, ␤-oxidation, growth perturbation, and cancer. Here, we have shown that transient transfection of additional PPAR␣ along with its dimerization partner RXR␣ into guinea pig hepatocytes is sufficient to increase PP responsiveness as assayed by CIPCO activity. In addition, mPPAR␣, gpPPAR␣, and hPPAR␣ were equally able to activate genes controlled by PPREs, either in the context of a simple reporter plasmid or within a more complex native response element. These data provide evidence that, at least in guinea pigs, quantity of PPAR␣ plays a significant role in the reduced ␤-oxidation response to PPs. Although quantity of PPAR␣ may also contribute to the lack of response in humans, the PPRE in the promoter region of the ACO gene is inactive in humans (32, 33). Because this is the first and rate-limiting enzyme in the ␤-oxidation pathway, human hepatocytes would continue to be refractory to PP induction of ␤-oxidation even after overexpression of PPAR␣. Fig. 4. Addition of mPPAR␣, hPPAR␣, or gpPPAR␣ to guinea pig hepatocytes As well as variations in levels of PPAR␣ and the structure of activates transcription of the rat acyl-CoA-luciferase reporter plasmid and confers in- PPREs, there may be additional factors that contribute to species creased CIPCO activity. a, ligand-dependent transcriptional activation of the rat ACO reporter plasmid by mPPAR␣, hPPAR␣, or gpPPAR␣ under conditions found to be differences in response to PPs. The promoter regions of PP-responsive optimal for mPPAR␣ activity (see Fig. 3). The mPPAR␣, hPPAR␣, or gpPPAR␣ receptor genes can contain negative regulatory elements that are indirectly expression vectors (6.0 ␮g) were transfected into 2 ϫ 106 guinea pig hepatocytes together ␣ Ϫ Ϫ ␮ ␮ switched off by ligand dependent PPAR activation (31). Also, other with prACO( 581/ 471).G.Luc reporter plasmid (1.0 g), pcDNA3.LacZ (1.0 g), and ␣ pCMV.mRXR␣ (6 ␮g) in the presence of 9-cis-retinoic acid (5 ␮M) and in the presence nuclear receptors, such as HNF-4 and COUP-TF, are able to bind to or absence of 50 ␮M nafenopin. Data are shown as fold induction over DMF/pcDNA3 the rat ACO PPRE and suppress PP-dependent gene activation (47). control of luciferase, corrected for transfection efficiency. b, ligand-dependent increase in Specifically, HNF-4␣ shares the ability of PPAR␣ to bind to the rat CIPCO activity caused by addition of mPPAR␣, gpPPAR␣, or hPPAR␣ in the presence of mRXR␣. pCMV.PPAR␣ (6.0 ␮g) and/or pCMV.mRXR␣ (6.0 ␮g) was transfected into ACO PPRE (47) but gives only a fraction of the reporter gene 2 ϫ 106 guinea pig hepatocytes together with pcDNA3.LacZ (1.0 ␮g) in the presence of expression. In addition, both COUP-TF and HNF-4␣ show saturable 9-cis-retinoic acid (5 ␮M) and in the presence or absence of 50 ␮M nafenopin (naf). Data are shown as fold induction over DMF/pcDNA3 control (column 1) of CIPCO, corrected dose-dependent suppression of activation of PPRE reporter plasmid for transfection efficiency. Columns, mean of triplicate results; bars, SD. by PPAR␣ in the presence of PPs (47). Thus, species differences both in the hypolidemic and in the adverse effects of PPs may be dictated in part by levels of PPAR␣, but also by diversity in the PPREs of sufficient receptor. However, it seems more likely that the lower level specific genes, and by expression of other direct repeat 1 (DR1) of CIPCO in transfected guinea pig hepatocytes reflects the low binding nuclear receptors, such as HNF-4␣ and COUP-TF. Suppres- transfection efficiency seen in primary hepatocytes. sion by negative regulatory elements together with competition with PPAR␣ from Nonresponsive Species also Increase PP Respon- other nuclear receptors for PPRE binding may constitute the molec- siveness of Guinea Pig Hepatocytes. Having shown that increasing ular basis of a threshold for PPAR␣ activation at PPREs. the quantity of PPAR␣ expressed in guinea pig hepatocytes altered In summary, the data presented here suggest that species differ- increased their PP responsiveness, we next determined whether there ences in quantity of PPAR␣ plays a role in the lack of an adverse were qualitative differences between PPAR␣ cloned from PP nonre- response in guinea pigs to the PP class of nongenotoxic rodent sponsive and responsive species played in response to PPs. First, we hepatocarcinogens. In humans, lower expression of PPAR␣ provides compared the ability of mPPAR␣, gpPPAR␣, and hPPAR␣ to in- one explanation for a lack of response, in addition to the lack of crease ligand-dependent induction of a PPRE reporter plasmid (Fig. activity of PPREs upstream of key genes, such as ACO (32, 33). 4a) under the optimal conditions (co-addition of RXR and 9-cis- retinoic acid) defined previously. In the absence of any PPAR␣ References expression plasmid, the addition of nafenopin had no effect. However, 1. Reddy, J. K., Azarnoff, D. L., and Hignite, C. E. Hypolipidaemic hepatic peroxisome as seen previously, the addition of nafenopin in the presence of proliferators form a novel class of chemical carcinogens. Nature (Lond.), 283: mPPAR␣ gave a 3-fold induction of reporter gene activity. Similarly, 397–398, 1980. transfection of PPAR␣ from either guinea pig or human into guinea 2. Reddy, J. K., and Lalwani, N. D. Carcinogenesis by hepatic peroxisome proliferators. 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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1999 American Association for Cancer Research. Addition of Peroxisome Proliferator-activated Receptor α to Guinea Pig Hepatocytes Confers Increased Responsiveness to Peroxisome Proliferators

Neil Macdonald, Peter R. Holden and Ruth A. Roberts

Cancer Res 1999;59:4776-4780.

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