Addition of Peroxisome Proliferator-Activated Receptor to Guinea Pig Hepatocytes Confers Increased Responsiveness to Peroxisome
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[CANCER RESEARCH 59, 4776–4780, October 1, 1999] Advances in Brief Addition of Peroxisome Proliferator-activated Receptor a 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 b-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 b guinea pigs appear refractory to these adverse effects. The peroxisome eration, and peroxisomal -oxidation enzyme activity is weak (12) proliferator (PP)-activated receptor a (PPARa) 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 b-oxidation by PPs weak (15) or absent such as acyl-CoA oxidase. Hepatic expression of PPARa 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 PPARa. To test this hypothesis, risk of liver cancer in patients receiving fibrate PP drug therapy (17). a a we introduced mouse PPAR and its heterodimerization partner, RXR , The PPARa 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 PPARa in trans- a 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 mM nafenopin in the absence or presence of the RXR ligand, 9-cis- Activated PPARa binds to DNA as a heterodimer with the RXR at retinoic acid (5 mM) gave only a 1.7 6 1.5- or 3.3 6 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 mPPARa and RXR gave a strong induction of repeats spaced by 1 bp, termed PPREs (25, 26). PPREs have been 6 a 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 a 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 PPARa 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 PPARa 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 hPPARa, gpPPARa, and mPPARa 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 PPARa 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 b-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- PPARa may be depleted due to expression of alternatively spliced tional enzyme and thiolase, as well as genes of the cytochrome PPARa mRNA lacking exon 6 that leads to a truncated, inactive P4504A family (8–10). In contrast, species such as guinea pigs and PPARa (30, 38). This suggests that the quantity of functional PPARa 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 PPARa 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 PPARa was verified by species- CTL.astrazeneca.com. specific PCR for mouse PPARa and by reporter gene assay using a rat 2 The abbreviations used are: PP, peroxisome proliferator; ACO, acyl-CoA oxidase; PPARa, peroxisome proliferator-activated receptor a; RXR, retinoid X receptor; PPRE, ACO PPRE promoter-luciferase reporter construct (9, 32). Key ex- PPARa response element; HNF-4a, hepatocyte nuclear factor 4a; COUP-TF, chicken periments were repeated with hPPARa and gpPPARa to determine ovalbumin upstream promotor-transcription factor; CIPCO, cyanide-insensitive palmitoyl a CoA oxidase; DMF, dimethyl formamide; hPPARa, human PPARa; mPPARa, mouse whether the ability of PPAR to confer increased responsiveness was PPARa; gpPPARa, guinea pig PPARa; CMV, cytomegalovirus. dependent on the species origin of the receptor. The data presented 4776 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1999 American Association for Cancer Research. SPECIES DIFFERENCES IN PPARa suggest that species differences in quantity of PPARa 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-ml aliquot was resuspended in N-tris(hydroxy- carcinogens. methyl)methyl-2-aminoethanesulfonic acid, disrupted by sonication, and stored at 270°C for CIPCO and protein assays. The pellet from the 200-ml m z 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,N9,N0-tetraacetic acid, 5% 2 b 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 mM CoA, 370 mM NAD1,94mM nicotinamide, was purchased from Sigma. Galacto-Light Plus b-galactocidase assay reagent 2.8 mM DTT, 2 mM KCN, 12.5 mg/ml BSA (fatty acid free), 100 mg/ml was from Tropix Inc. Luciferin luciferase assay reagent was from Promega. All flavin adenosine dinucleotide, 50 mg/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 b-galactocidase expression plasmid pCMV- instructions. b-Galactosidase and luciferase activity were assayed as de- .LacZ was obtained from Clontech (Basingstoke, United Kingdom). pcDNA3 scribed previously (40). Briefly, b-galactosidase activity was determined was from R1D Systems (Oxon, United Kingdom). hPPARa cDNA was a gift by incubating 10 ml of cell extract with Galacto-Light Plus reagent (Tropix from Dr. F. Gonzalez (National Cancer Institute, Bethesda, MD), and mRXRa Inc.) according to the manufacturer’s instructions. Luciferase activity was cDNA was a gift from Prof. Pierre Chambon. The plasmids pCMV.mPPARa, determined by incubating a 40-ml aliquot of cell extract with luciferin pAco(2581/2471).G.Luc (30), pCMV.gpPPARa, pCMV.hPPARa (34), and reagent (Promega) according to the manufacturers instructions. Luciferase pCMV.mRXRa (40) have been described previously.