ToxSci Advance Access originally published online on February 19, 2004
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Toxicological Sciences 78, 229-240 (2004)
Toxicological Sciences vol. 78 no. 2 © Society of Toxicology; all rights reserved.
Characterization of the Species-Specificity of Peroxisome Proliferators in Rat and Human Hepatocytes
Molecular Toxicology, Institute of Toxicology, Merck KGaA, 64271 Darmstadt, Germany
Received November 5, 2003; accepted January 2, 2004
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Peroxisome proliferation is a well-defined pleiotropic effect that is mediated by the ligand inducible transcription factor peroxisome proliferator-activated receptor (PPAR)
. Because marked peroxisome proliferation occurs in rodents but not in humans, we aimed to elucidate the molecular and cellular determinants of this species-specificity in hepatocytes. Analysis of peroxisomal marker enzyme activities confirmed that peroxisome proliferators induced acyl-CoA oxidase (ACOX) and to a lesser extent catalase in rat hepatocytes, but not in human hepatoma HepG2 cells. Transient transfection assays revealed that ciprofibrate and Wy 14,643 induced rat but not human PPAR-mediated reporter gene activity in rat FAO and primary hepatocytes on rat but not on human PPAR response elements (PPREs). In contrast, in human HepG2 and primary human hepatocytes, peroxisome proliferators did not induce either human or rat PPAR activity regardless of rat or human PPRE sequences. In addition, no induction of ACOX gene expression was observed in human hepatocytes independent of the expression level of human PPAR. Remarkably, no distinct peroxisome proliferation related responses were observed in human hepatocytes when rat PPAR was transfected, although human hepatocytes were responsive to PPAR-mediated induction of carnitine palmitoyl transferase-1A and 3-hydroxy-3-methylglutaryl-CoA synthase. These results confirmed that PPAR and PPREs are important determinants for the species-specificity of peroxisome proliferation. Nevertheless, our results showed that human hepatocytes limit the extent of peroxisome proliferation regardless of PPAR expression.
Key Words: PPAR; HepG2; FAO; primary hepatocytes; fibrates; Wy 14643.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Peroxisome proliferators are a diverse group of chemicals that cause pleitropic effects including proliferation of peroxisomes that is accompanied by induction of peroxisomal enzyme expression (Reddy and Chu, 1996
). These pleitropic effects are mediated by activation of the peroxisome proliferator-activated receptor (PPAR) , as shown by the phenotypes of PPAR knock-out mice (Corton et al., 2000; Lee et al., 1995; Willson, 2000). PPAR functions as a ligand-inducible transcription factor for genes involved in mitochondrial and peroxisomal metabolism (Corton et al., 2000) and it is well established that marked species differences in response to peroxisome proliferators exist: Rodents show high peroxisomal enzyme induction while humans do not (Vanden Heuvel, 1999).
These species differences in PPAR function have been the focus of several research papers (reviewed in Cattley et al., 1998). The functions of rat and human PPAR are similar and homology of the DNA binding domain and ligand binding domain is high (Goettlicher et al., 1992; Mukherjee et al., 1994; Sher et al., 1993); however, human hepatocytes display only weak induction of marker enzyme activity like acyl CoA oxidase (ACOX). Several determining factors for the different activities of PPAR in rat vs. human hepatocytes have been identified. One factor might be due to the lower expression levels of PPAR in human liver compared to rats (Tugwood et al., 1996) and a second factor might be the existence of an inactive PPAR splice variant in human liver samples (Palmer et al., 1998). Another factor has been shown to be the responsiveness of PPAR regulated genes that is defined by PPAR response elements (PPRE) located within the promoter region of target genes. Both human and rat ACOX gene promoter contain the consensus PPRE half site TGACCT and a second nonconsensus half-site (Tugwood et al., 1992; Varanasi et al., 1996, 1998). Comparison of the rat and human promoter sequences revealed that the human ACOX PPRE did not mediate PPAR activity; however, human PPAR displayed activity on the rat ACOX promoter (Hasmall et al., 2000; Lambe et al., 1999) and was able to induce endogenous genes involved in lipid metabolism (Lawrence et al., 2001).
Our aim was to determine whether PPAR and the PPRE sequences are sufficient to account for these species differences. We, therefore, analyzed the contribution of PPAR, PPRE and in particular how and to what extent the cellular environment may determine PPAR-mediated responses in rat and human hepatocytes. First, marker enzyme inductions in rat primary hepatocytes, the rat hepatoma FAO, and the human hepatoma HepG2 cell line were investigated. In transient transfection studies we then examined whether human or rat PPAR exert activity on a rat or human PPRE derived from the ACOX promoter in rat and human hepatocytes. Subsequently, we measured expression of endogenous peroxisomal marker genes as well as of human PPAR responsive genes in rat and human primary hepatocytes in the presence or absence of transfected rat or human PPAR. This comprehensive analysis revealed that all parameters investigated determined the species-specificity of peroxisome proliferation and that human hepatocytes limited PPAR activity.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Materials and chemicals.
Media, serum, supplements, enzymes, and biochemicals were purchased from Sigma (Deisenhofen, Germany) unless otherwise stated. HepG2 and FAO cells were obtained from ECACC (Salisbury, UK). Steroid deprived dextran-coated charcoal stripped fetal bovine serum (DCC/FBS) was from Hyclone (Lot AKD11642A, Perbio Science, Bonn, Germany). Primary male human hepatocytes were obtained from In Vitro Technologies (Order number M00005, Lot MSE, Baltimore, MD). Ciprofibrate, clofibrate, and pirinixic acid (Wy 14,643) were from Sigma-Aldrich (Deisenhofen, Germany).
Cell culture.
HepG2 and FAO cells were cultured in Dulbecco's modified Eagle medium (DMEM)/F12 supplemented with 10% FBS and 5% FBS, respectively, 1 mM sodium pyruvate and antibiotics. Cells were cultured at 37°C/5% CO2 in air in a humidified atmosphere.
Isolation of primary hepatocytes from rat liver.
Primary rat hepatocytes were prepared freshly from male Wistar rats (200–250 g) by the in situ perfusion procedure (Seglen, 1973, 1976). Livers were perfused with 0.5 mg/ml collagenase, the liver capsule removed, and the released hepatocytes filtered through a 250 µm mesh followed by a second filtration step using a 100 µm mesh nitex membrane. The filtered hepatocytes were resuspended in phenol-red free DMEM/F12 supplemented with 10% DCC/FBS and 5 µg/ml insulin. The cell number and viability of the suspension was assessed by trypan blue exclusion. Fifty µl trypan blue was mixed with 50 µl cell suspension and the number of viable cells (cells that were not stained with trypan blue) were determined in a hemacytometer. Primary cell cultures that had a viability of more than 80% were used for the experiments. For better attachment and viability of primary hepatocytes, tissue culture plates were coated with 6 µg/cm2 collagen I. For attachment, rat hepatocytes were cultured at 37°C/5% CO2 in air in a humidified atmosphere for at least 3 h prior to the experiments.
Thawing of cryopreserved human hepatocytes.
Cryopreserved human hepatocytes were thawed according to the procedure provided by the manufacturer (In Vitro Technologies, Baltimore, MD). Briefly, two vials of cryopreserved human hepatocytes were rapidly thawed and poured into 40 ml of phenol-red free DMEM/F12 supplemented with 10% DCC/FBS and centrifuged at 60 x g for 5 min. The cell pellet was resuspended in 5 ml phenol-red free DMEM/F12 supplemented with 10% DCC/FBS and 5 µg/ml insulin. The cell number and viability was assessed as described for rat hepatocytes and primary cell cultures that had a viability of more than 80% were used for the experiments. Primary hepatocytes were seeded on collagen-coated plates. For attachment, human hepatocytes were cultured at 37°C/5% CO2 in air in a humidified atmosphere for at least 3 h prior to the experiments.
Cytotoxicity assay.
For assessing cytotoxicity and cell proliferation induced by the test compounds the WST-1 kit was used according to the protocol provided by the manufacturer (Roche, Mannheim, Germany). This test measures the enzymatic formation of formazan. The formazan formation correlates with cellular enzyme activity and cell number. Therefore, a decrease in formazan formation and hence absorption indicates cytotoxicity. FAO and HepG2 cells were seeded at a density of 0.5 x 105 cells/cm2 while 2 x 105 primary cells/cm2 were seeded in 96-well plates. IC50 values (compound concentration yielding 50% inhibition of formazan formation) were derived by nonlinear curve-fitting of dose-response curves using Origin software (Microcal Software, Northhampton, MA) and are given as mean ± SD of at least three independent experiments.
Protein extraction and determination for enzyme activity assays.
Fifteen hours prior to treatment, cells were seeded in culture medium. FAO and HepG2 cells were seeded at a density of 0.2 x 105 cells/cm2 while 2 x 105 primary cells/cm2 were seeded in tissue culture plates. Then, test compounds were added (vehicle was dimethyl sulfoxide [DMSO] at 0.1% volume per volume (v/v) final concentration). The medium was changed every 24 h during treatment. The cells were harvested in ice cold PBS buffer and lysed in 0.1 % (v/v) Triton X-100 in PBS buffer. Protein concentrations were determined according to Bradford (1976) with bovine serum albumin as standard.
Acyl-CoA oxidase (ACOX) activity.
Palmitoyl-CoA oxidase activity was assayed by determining the production of hydrogen peroxide using a modified photometric detection method (Small et al., 1985). The reaction medium contained 11 mM phosphate buffer and 40 mM aminotriazol buffer at pH 7.4, 0.8 mg horseradish peroxidase IV-A, 0.05 mM leuco-dichlorodihydro-fluoresceine diacetate and cell homogenate containing 50 to 200 µg of protein in 300 µl. The reaction was started by the addition of 10 µl of 3 mM palmitoyl-CoA lithium salt solution in 11 mM phosphate buffer pH 7.4. The enzyme kinetic was measured for 10 min at 490 nm.
Catalase activity.
The catalase activity was determined by a photometric method (Hübl and Breitschneider, 1964). The assay was performed at 4°C. The reaction mixture (200 µl) contained 20 mM imidazole-HCl buffer pH 7, 0.1% weight per volume (w/v) delipidated bovine serum albumin, and 1.5 mM hydrogen peroxide. The reaction was started by the addition of the cell homogenate (5–40 µg protein/ml) and stopped with 100 µl of 2 M sulfuric acid containing 0.125 % (w/v) titanium oxysulfate. The remaining hydrogen peroxide was measured at 412 nm by the absorption of peroxytitanium sulfate.
Carnitine-acyltransferase activity.
Carnitine-acyltransferase (CAT) activity was determined according to (Bieber and Markwell, 1981) with slight modifications. The reaction volume was 200 µl containing 125 mM Tris-HCl buffer pH 8.0, 2.5 mM ethylenediaminetetraacetic acid, 0.5 mM dithionitrobenbenzoeacid (DTNB), 0.2 mM acetyl-CoA, and 20–500 µg protein. The reaction was started by addition of 5 mM L-carnitine. The DTNB complex was measured for 10 min at 412 nm.
Generation and characteristics of expression and reporter plasmids.
The mammalian expression plasmids for human and rat PPAR (pSG5-hPPAR and pSG5-rPPAR, respectively) were kindly provided by W. Wahli (IBA, Geneva, Switzerland). The mammalian expression plasmid for human RXR (pcDNA4-hRXR) was generated using standard cloning procedures (Ausubel et al., 1998). The full-length cDNA of human RXR (GenBank accession number NM_002957; nucleotide positions 55–1559) was cloned into pcDNA4-HisMAX (Invitrogen, Karlsruhe, Germany) using the TOPO TA cloning kit (Invitrogen, Karlsruhe, Germany) and total RNA isolated from human liver samples. The correct sequence of the cDNA was verified by sequencing. Firefly luciferase reporter plasmids were generated by placing three copies of the rat (Tugwood et al., 1992) or human (Varanasi et al., 1996, 1998) PPAR response element derived from the ACOX promoter (rat ACOX PPRE: TGACCTTTGTCCT and human ACOX PPRE: AGGTCAGCTGTCA) in the multiple cloning site of the pGL3-SV40 reporter plasmid (Promega, Mannheim, Germany) and named pGL3-SV40-3xrPPRE and pGL3-SV40-3xhPPRE, respectively. The luciferase reporter containing the full-length rat ACOX promoter (pGL3-rACOX) was kindly provided by J. Tugwood (AstraZenca, UK; (Tugwood et al., 1992)).
Transient transfection and transactivation assay.
HepG2 and FAO cells were seeded on 24-well plates 15 h prior to transfection in phenol-red free DMEM/F12 supplemented with 10 and 5% DCC/FBS, respectively. 0.2 x 106 viable primary hepatocytes were dispensed per well in collagen-coated 24-well plates in phenol-red free DMEM/F12 supplemented with 10% DCC/FBS and 5 µg/ml insulin as described above. The plasmids were transfected in phenol-red free DMEM/F12 supplemented with 5% DCC/FBS using Fugene 6 (Roche, Mannheim, Germany) according to the protocol provided by the manufacturer. Each well received 0.1 µg empty pSG5, pSG5-hPPAR or pSG5-rPPAR, 0.1 µg of pcDNA4-hRXR, 0.5 µg of firefly luciferase reporter and 0.01 µg pRL-CMV (Renilla luciferase for normalization; Promega, Mannheim, Germany). After transfection, cells were treated with test compounds (final concentration of vehicle DMSO 0.5 % v/v) for 22 h in medium supplemented with 10 % DCC/FBS as described above. Luciferase assays were performed using the Dual-Luciferase Reporter Assay System (Promega, Mannheim, Germany) according to the protocol provided by the manufacturer. Each value was normalized for cell number and transfection efficiency to the luciferase control (pRL-CMV) and each data point obtained represents the average of duplicate determinations. All experiments were repeated at least three times. EC50 values (ligand concentration yielding half-maximal activation) were derived by nonlinear curve-fitting of transactivation curves using Origin software (Microcal Software, Northhampton, MA) and are given as mean ± SD of at least three independent experiments. Control transfections were performed using 0.5 µg empty pGL3-basic (Promega, Mannheim, Germany) instead of reporter vector per well. Transfection efficiencies were highest in HepG2 cells (average of four tests: 20,000 light units of pRL-CMV per well) and were in FAO approximately 25 % compared to HepG2 (average of four tests: 5000 light units of pRL-CMV per well). Primary rat hepatocytes displayed transfection efficiencies of 3000 light units of pRL-CMV per well (average of four tests), and primary human hepatocytes displayed transfection efficiencies of 2000 light units of pRL-CMV per well (average of three tests).
RNA extraction and isolation.
The RNA was isolated according to the instructions given by the manufacturer (QIAGEN RNeasy® Mini Kit, Hilden, Germany) using additionally a QIAshredder spin column (QIAGEN, Hilden, Germany) and DNA digestion (QIAGEN DNase Kit, Hilden, Germany).
Real-time quantitative PCR.
The purified RNA was subjected to reverse transcription using random hexamers and TaqMan® reverse transcription reagents (Applied Biosystems, Weiterstadt, Germany) according to the protocol provided by the manufacturer. The primer and probes used for the real time PCR assay were designed using the PrimerExpress software (Applied Biosystems, Weiterstadt, Germany) and oligonucleotides were synthesized by Applied Biosystems (Weiterstadt, Germany). Target genes with sequences of primer and probes used are listed in Table 1. The probes were all labeled with the fluorescent dyes FAM (6-carboxy-fluorescein) and TAMRA (6-carboxy-tetramethyl-rhodamin) at the 5'- and 3'-end, respectively (Applied Biosystems, Weiterstadt, Germany). Real-time PCR was performed on an Applied Biosystems ABI Prism 7000 Sequence Detection System. The primer concentration was optimized prior to use with a fixed probe concentration of 200 nM in a TaqMan® Universal PCR buffer (Applied Biosystems, Weiterstadt, Germany) in 25 µl reaction volume per well. All samples were run in triplicates and a standard curve from 2.5 to 20 ng cDNA was generated for each experiment. Ten ng of sample cDNA was used and normalized to 18S ribosomal RNA (rRNA) control (for human and rat 18S rRNA pre-developed TaqMan assay reagents [PDAR] were used; Applied Biosystems, Weiterstadt, Germany). Forty-one cycles were run with the following parameters: 2 min at 50°C, 10 min at 95°C and for each cycle 15 s at 95°C for denaturation and 1 min at 60°C for transcription. Two different negative controls were performed, one omitting the reverse transcription step and one omitting target RNA. Assays were evaluated only when the negative controls did not show any amplification products.
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Statistical analysis.
Statistical analyses were performed with the XLStat software (Addinsoft, Paris, France). Significant differences of treated samples compared to vehicle control (Figs. 1–6) were determined using ANOVA followed by Dunnett's test (n = 3, p > 0.05). Statistical significance (n = 3, p > 0.05) based on pair-wise comparison in Figures 3 and 4 were determined using ANOVA followed by Tukey's test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cytotoxicity
We investigated the species specificity of peroxisome proliferators in rat and human hepatocytes as well as in rat hepatoma FAO and human hepatoma HepG2 cells. For the assessment of cytotoxicity, we determined IC50 values in FAO, HepG2, and primary rat and human hepatocytes treated with peroxisome proliferators (Table 2). Wy 14,643 was more cytotoxic than the fibrates and the primary hepatocytes were less sensitive to the cytotoxic effects of peroxisome proliferators than FAO and HepG2 cells (Table 2).
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Peroxisome Proliferators Induced Marker Enzyme Activity in Rat Hepatocytes But Not Human HepG2 Cells
A hallmark physiological response in rodent peroxisome proliferation is the induction of the peroxisomal enzymes ACOX and carnitine-acyltransferase (CAT). Catalase, the enzyme that detoxifies peroxide produced by ACOX is usually induced to a lower extent in rodents and rat hepatocytes (Reddy and Chu, 1996
). We therefore analyzed whether clofibrate and ciprofibrate induced a distinct enzyme induction pattern in primary rat hepatocytes, rat FAO, or human HepG2 cells (Fig. 1; Duclos et al., 1997; Perrone et al., 1998; Stangl et al., 1995). We first performed dose-response and time-course experiments with clofibrate and ciprofibrate (data not shown). Maximum inductions were observed with 250 µM fibrates treated for 72 h and are shown in Figure 1. As expected, fibrates strongly induced ACOX and CAT in primary rat hepatocytes whereas FAO cells showed lower but distinct induction of ACOX and CAT (Figs. 1A and 1B). In contrast, catalase was weakly induced (maximum of 2.2-fold induction in rat hepatocytes when treated with ciprofibrate; see Fig. 1C). The human HepG2 cells exerted only slight inductions of ACOX and CAT activity that reached significance for ciprofibrate treatment (Fig. 1).
PPAR, PPRE, and the Cellular Environment Determine the Different Transactivation in Rat Compared to Human Hepatocytes
We further examined whether the species-specific activity of peroxisome proliferators were due to rat or human PPAR or the promoter sequences. For this purpose, FAO, HepG2, as well as rat and human primary hepatocytes were transfected with either rat or human PPAR expression vectors. As reporter genes we used either a rat (Tugwood et al., 1992) or human (Varanasi et al., 1996, 1998) ACOX PPRE or the rat full-length ACOX promoter (Tugwood et al., 1992). Control transfections were performed with a reporter vector lacking PPRE sequences that did not show any induced activity (data not shown). Wy 14,643 as well as ciprofibrate induced a dose-dependent increase of full-length rat ACOX and rat ACOX PPRE reporter gene activity in rat primary hepatocytes and rat FAO cells, but not in primary human hepatocytes or HepG2 cells (data not shown). The potencies (EC50) of Wy 14,643 and ciprofibrate to induce PPAR were comparable in rat hepatocytes and FAO on both rat ACOX PPRE (3xPPRE) and the full-length ACOX promoter (Table 3).
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We then compared the basal normalized activities of transfected rat or human PPAR
on the rat ACOX PPRE in the different cell lines (Fig. 2). Transfection of rat PPAR yielded a distinct—although statistically not significant—4.2-fold induction of the rat ACOX PPRE in FAO and a strong and significant 20-fold induction in rat primary hepatocytes in the absence of ligand compared to transfection of empty vector (Fig. 2). No significant effects of either rat or human PPAR were observed in human HepG2 and human primary hepatocytes (Fig. 2). Comparable results were obtained with the full-length rat ACOX promoter, whereas the human ACOX PPRE showed no increased normalized activity when either rat or human PPAR were transfected in all cell lines tested (data not shown).
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For comparison of the induction of rat vs. human ACOX PPRE by either rat or human PPAR
in the presence of ligand, the maximum normalized fold inductions compared to the vehicle control in FAO (Fig. 3A) and HepG2 (Fig. 3B) are shown. The rat PPAR induced only the rat PPRE or the rat ACOX promoter in FAO cells, whereas the human PPAR did not produce any significant transactivation on the human PPRE in HepG2 cells when treated with peroxisome proliferators (Fig. 3). The human PPAR was only slightly active on either human (1.6 ± 0.4 fold induction; Fig. 3A) or rat PPRE (1.8 ± 0.4 fold induction; Fig. 3A). Induction of the full-length ACOX promoter was significantly induced by Wy 14,643 in the presence of transfected rat PPAR but not human PPAR compared to endogenous PPAR (Fig. 3A). No distinct PPAR-mediated activity was observed in HepG2 cells, regardless of PPAR or PPRE used (Fig. 3B).
Since the effects observed in FAO and HepG2 cells could be due to the transformed cell type and may not reflect the physiology in hepatocytes, we performed the same experiments in primary rat and human hepatocytes (Fig. 4). The rat primary hepatocytes facilitated marked peroxisome proliferator induced activity of rat PPAR on rat ACOX PPRE or the full-length ACOX promoter only (Fig. 4A). In contrast to the results obtained in FAO cells (Fig. 3A), induction of the full-length ACOX promoter was not increased by transfection of rat or human PPAR into rat primary hepatocytes (Fig. 4A), which is likely due to high level of endogenous PPAR expression (see below) and/or the high basal activity of transfected rat PPAR in primary rat hepatocytes (Fig. 2). In agreement with the results obtained in HepG2 cells (Fig. 3B), we did not observe any distinct PPAR-mediated activity in human hepatocytes (Fig. 4B). Surprisingly, transfected rat PPAR did not increase the induction of rat ACOX PPRE or ACOX promoter by Wy 14,643 or ciprofibrate in human hepatocytes (Fig. 4B), although the transfection efficiencies of rat and human PPAR were comparable for primary rat and human hepatocytes (see Materials and Methods section).
PPAR mRNA Is Highly Abundant in Rat But Not in Human Hepatocytes
Since rat but not human hepatocytes showed PPAR-mediated activity (Figs. 2 and 4), we analyzed the basal mRNA expression levels of endogenous PPAR in the rat and human cell lines used (Table 4). As expected, the basal expression level of endogenous PPAR in rat FAO cells was more than 4-fold higher compared to human HepG2 cells. Rat primary hepatocytes showed the highest basal expression levels of PPAR whereas HepG2 and human primary hepatocytes had similar, low basal expression levels of human PPAR (Table 4).
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Human Hepatocytes Remain Refractory to Peroxisome Proliferator Induced Gene Expression Independent of Rat or Human PPAR
Expression LevelIn order to investigate PPAR
-mediated endogenous gene expression by peroxisome proliferators, we quantified mRNA expression of ACOX, catalase, and the proliferation marker PCNA by TaqMan® analysis. The promoter region of the genes coding for ACOX and catalase contain PPREs (Girnun et al., 2002; Tugwood et al., 1992; Varanasi et al., 1996), but it is not known whether the PCNA promoter contains a PPRE. We first analyzed the effects of endogenous PPAR in the cell lines investigated when treated with noncytotoxic doses of Wy 14,643 and ciprofibrate that yielded maximum inductions in transactivation assays (Figs. 3 and 4). Gene expression was analyzed at 2, 24, and 72 h and normalized to 18S rRNA. Consistent with the enzyme induction data (Fig. 1), no significant catalase induction was observed in HepG2 or human hepatocytes (data not shown). In primary rat hepatocytes and FAO cells, catalase was induced about 2-fold and the proliferation marker PCNA was not induced at all in either rat or human cells tested (data not shown). In contrast, the well-defined marker gene for peroxisome proliferation ACOX was highly induced in FAO and rat hepatocytes after 24 and 72 h, respectively, upon treatment with Wy 14,634 or ciprofibrate (Fig. 5) consistent with the enzyme induction data (Fig. 1) and the transactivation experiments (Figs. 3 and 4).
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ACOX expression was weakly, but significantly induced in HepG2 cells treated with Wy 14,643 and primary human hepatocytes treated with Wy 14,643 and ciprofibrate for 24 h compared to the vehicle control (Fig. 6). All other treatments did not induce any significantly increased expression of ACOX in HepG2 cells or in human hepatocytes compared to the vehicle control (Fig. 6).
It is well established that rat hepatocytes respond to peroxisome proliferators by induction of ACOX whereas human hepatocytes remain refractory (Lawrence et al., 2001). We have therefore extended our study and investigated the effects of both endogenous PPAR and transfected rat or human PPAR on the expression of ACOX and other rat and human PPAR responsive genes, i.e., 3-hydroxy-3-methylglutaryl-CoA synthase (HMGCS), carnitine palmitoyl transferase-1A (CPT1A), and apolipoprotein CIII (apo CIII; Brandt et al., 1998; Hsu et al., 2001; Lawrence et al., 2001; Rodriguez et al., 1994; Staels et al., 1995). Based on the time-course experiments of ACOX expression (Figs. 5 and 6), we treated FAO, HepG2, primary rat and human hepatocytes for 24 h with 100 µM Wy 14,643 and analyzed PPAR target gene expression (Figs. 7–9). Transfection of rat and human hepatocytes as well as hepatoma cells with human or rat PPAR cDNA yielded a distinct increase of human or rat PPAR mRNA expression, respectively (Figs. 7 and 8). Furthermore, transfected rat PPAR increased basal ACOX expression levels in FAO (Fig. 7A) but not in primary rat hepatocytes (Fig. 8A), which is likely due to the higher endogenous PPAR expression in primary rat hepatocytes compared to FAO cells (Table 4). Wy 14,643 distinctively induced ACOX gene expression in mock and rat PPAR transfected FAO as well as in rat hepatocytes (Figs. 7A and 8A). Increased human PPAR mRNA expression—up to similar levels as observed for rat PPAR in rat hepatocytes (Figs. 7 and 8)—as well as overexpression of rat PPAR mRNA did not distinctively increase the susceptibility to ACOX induction by Wy 14,643 in HepG2 or human hepatocytes (Figs. 7B and 8B).
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We then analyzed the expression of the well-characterized human PPAR
responsive CPT1A, apo CIII, and HMGCS genes, to verify that the human hepatocytes used are responsive to PPAR agonists (Fig. 9). Apo CIII expression levels did not change distinctively in rat or human primary hepatocytes treated with Wy 14,643 independent of the expression level of human or rat PPAR (data not shown). Although several reports showed that apo CIII expression was down-regulated by peroxisome proliferators in human liver and HepG2 cells (Clavey et al., 1999; Lawrence et al., 2001; Staels et al., 1995), other reports failed to show a PPAR-mediated regulation of apo CIII expression in human HepG2 cells (Hsu et al., 2001). This apparent discrepancies have been attributed to the culture conditions of hepatocytes, in particular the differences of cell culture media and characteristics of the supplemented serum (Clavey et al., 1999). Nevertheless, Wy 14,643 strongly induced CPT1A and HMGCS in both rat and human primary hepatocytes in the presence and absence of transfected human or rat PPAR, supporting that human PPAR is functional in the human hepatocytes used (Fig. 9). |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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It is well established that peroxisome proliferators exert distinct effects in rodents in contrast to humans. A number of studies have been published that analyzed various aspects of this species-specificity (for reviews see, e.g., Cattley et al., 1998
; Vanden Heuvel, 1999), and we aimed to provide a comprehensive analysis of the involvement of PPAR, PPRE, and in particular the cellular context in the species-specificity of PPAR agonists.
To this end, we first analyzed induction of the marker enzymes ACOX and CAT as well as catalase in rat and human hepatocytes. Confirming earlier reports (Duclos et al., 1997; Perrone et al., 1998; Stangl et al., 1995), fibrates induced strongly ACOX and CAT but to a lesser extent catalase in rat hepatocytes whereas human HepG2 cells were refractory. To investigate whether the observed distinct enzyme induction is due to differences in the PPRE sequence of the ACOX gene (Lambe et al., 1999), the PPAR (Lawrence et al., 2001), or the cellular environment of rat vs. human, we analyzed the activity of either rat or human PPAR on a rat or human ACOX-derived PPRE (Tugwood et al., 1992; Varanasi et al., 1996, 1998) in rat and human hepatocytes. These experiments confirmed previously published reports (Hasmall et al., 2000; Lambe et al., 1999) that the human PPAR does not activate a human PPRE derived from the ACOX promoter in either rat FAO or human HepG2 cells when treated with PPAR agonists. Human PPAR was not able to significantly activate the rat ACOX PPRE or the full-length ACOX promoter in human hepatocytes; an observation that was also evident for rat PPAR and may reflect the nonresponsiveness of human hepatocytes as discussed in more detail later. In addition, human PPAR did not activate the rat ACOX PPRE in rat FAO or primary hepatocytes. In contrast, others showed that human PPAR was active on a rat ACOX promoter in murine cells (Hasmall et al., 2000; Sher et al., 1993). We also observed induction of the rat ACOX promoter in rat hepatocytes in the presence of human PPAR, although this activity could not be discriminated from endogenous rat PPAR. Taken together, overexpressed human PPAR exerted some activity on the rat ACOX promoter; however, in our hands, this activity was lower than that of rat PPAR at comparable mRNA expression levels. Lambe and colleagues (1999) compared the identical human and rat PPRE sequences used in this study in transactivation assays using the mouse PPAR. They showed that the human PPRE conferred no activity to mouse PPAR whereas the rat PPRE was responsive to peroxisome proliferators (Lambe et al., 1999). We confirmed that rat PPAR did not activate through the human ACOX PPRE but distinctively induced the rat ACOX PPRE and the full-length rat ACOX promoter. The published results and our study showed that both human PPAR and human PPRE sequence limit the activation of peroxisome proliferation related gene transactivation in hepatocytes.
Next we investigated whether the expression level of human PPAR could account for the observed lack of peroxisome proliferation related effects in human hepatocytes. Human PPAR is expressed at low levels in liver compared to rodents (Braissant et al., 1996; Lemberger et al., 1996; Palmer et al., 1998; Tugwood et al., 1996) and we confirmed these different expression levels in the cell lines used for this study. Nevertheless, HepG2 and primary human hepatocytes had significant levels of PPAR reaching up to 25% mRNA expression level compared to the rat FAO cells. In addition, overexpression of human PPAR up to a level comparable to that observed for rat PPAR in primary rat hepatocytes did not increase the induction of ACOX in human hepatocytes. Our observation was confirmed by other studies that showed that overexpression of human PPAR in HepG2 cells did not increase the responsiveness to peroxisome proliferators with regard to ACOX mRNA expression and enzyme activity (Hsu et al., 2001; Lawrence et al., 2001). In contrast to these studies and our data are results obtained in hepatocytes of guinea pigs, a species that is not responsive to peroxisome proliferators (Macdonald et al., 1999). In guinea pig hepatocytes, transfection of human or guinea pig PPAR increased nafenopin-induced peroxisome proliferation related effects (Macdonald et al., 1999). It is therefore likely that regulation of the peroxisomal marker enzyme palmitoyl-CoA oxidase may be differently regulated in guinea pig hepatocytes compared to human hepatocytes. Taken together, the lack of human PPAR activity in human hepatocytes is unlikely to be due to the low level of human PPAR expression. Palmer and colleagues (1998) showed that splice variants of human PPAR exist that lack activity and are dominant-negative (Gervois et al., 1999). Our analysis of mRNA PPAR expression was done with a TaqMan® probe that is located upstream of the spliced exon 6 of the inactive PPAR variant (Palmer et al., 1998). The observed amounts of human PPAR mRNA in human hepatocytes correspond therefore to the total amount of wild-type and inactive human PPAR variant. This inactive PPAR splice variant could therefore contribute to the lack of peroxisome proliferation related effects observed in human hepatocytes.
We then scrutinized whether the above-described factors are sufficient to determine the species-specificity of peroxisome proliferation related effects. Hsu and colleagues showed that human hepatocytes, although refractory to peroxisome proliferation related effects, are susceptible to PPAR-mediated expression of CPT1A and HMGCS, genes that are involved in lipid metabolism in human liver (Hsu et al., 2001). Here, we demonstrated that the primary human hepatocytes used maintained their PPAR responsiveness with regard to CPT1A and HMGCS expression. In addition, Lawrence et al. showed that overexpressed human or murine PPAR increased expression of HMGCS but not ACOX in HepG2 cells (Lawrence et al., 2001). Consistent with this report, overexpression of rat PPAR did not increase ACOX induction in human hepatocytes, which is likely due to the lack of responsiveness of the PPRE in the human ACOX promoter (Lambe et al., 1999).
Intriguingly, we could not observe a distinct induction of rat or human ACOX PPRE or the full-length rat ACOX promoter in human hepatocytes when either human or rat PPAR was overexpressed to mRNA levels comparable to that observed in primary rat hepatocytes. Macdonald et al. showed that nafenopin induced activity of human and murine PPAR on a rat ACOX PPRE in guinea pig hepatocytes (Macdonald et al., 1999). However, others showed that Wy 14,643 induced weakly—about 2-fold and less compared to the vehicle control—the rat ACOX PPRE in HepG2 cells in the presence of overexpressed human PPAR (Hsu et al., 2001) consistent with our results. Hsu et al. demonstrated that a murine PPAR mutant with an increased sensitivity for ligand-induced activation retained responsiveness to peroxisome proliferators in HepG2 cells (Hsu et al., 2001). However, wild-type murine as well as human PPAR displayed no distinct responsiveness to peroxisome proliferators on a rat ACOX PPRE in HepG2 cells (Palmer et al., 1998) similar to our results. Since the rat PPAR expression plasmid used in our study yielded similar transfection efficiencies and also comparably high mRNA expression levels in both rat and human hepatocytes and hepatoma cell lines, we conclude that human hepatocytes impede PPAR-mediated induction of both rat and human ACOX reporter gene activity. However, depending on the properties of PPAR, the target gene promoter or the potency of the PPAR ligand, this unfavorable environment for peroxisome proliferation in human hepatocytes may be overcome.
In conclusion, our results confirmed that the differences in promoter sequences of PPAR target genes and the PPAR contribute to the species-specificity of peroxisome proliferators in hepatocytes. However, these factors are not sufficient to determine the species-specificity of peroxisome proliferation, since human hepatocytes hampered a marked induction of peroxisome proliferation marker genes and PPAR activity. It is therefore conceivable that hepatocytes may lack or overexpress co-regulators in a species-specific manner that might facilitate or inhibit PPAR-mediated gene expression. Identification of these specific factors, which consequently may also be involved in the tumorigenic effects of PPAR agonists in the rodent liver, would further enhance our understanding of the molecular mechanisms of the species-specificity of peroxisome proliferators.
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ACKNOWLEDGMENTS |
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The authors wish to thank Drs. G. Blaich, Abbott Laboratories, and P.-J. Kramer, Merck KGaA for supporting this study. We gratefully acknowledge Ms. Katherine Beckett and Dr. J. Tugwood (AstraZeneca, Macclesfield, UK) as well as Dr. W. Wahli (IBA, Lausanne, Switzerland) for kindly providing us with plasmids used in these studies. We thank Ms. Margret Kling for help in establishing the enzyme activity assays and Dr. H. K. Kinyamu (NIEHS, USA) for editing the manuscript.
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NOTES |
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1 Present address: GenPharmTox Biotech AG, 82152 Martinsried, Germany.
2 To whom correspondence should be addressed. Fax: +49-6151-72 91 8517. E-mail: stefan.o.mueller{at}merck.de
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