Peroxisome proliferators and fatty acids negatively regulate liver X receptor-mediated activity and sterol biosynthesis

Journal of Steroid Biochemistry & Molecular Biology 77 (2001) 59 – 71 www.elsevier.com/locate/jsbmb

Peroxisome proliferators and fatty acids negatively regulate liver X receptor-mediated activity and sterol biosynthesis Timothy E. Johnson *, Brian J. Ledwith Department of Genetic and Cellular Toxicology, Merck Research Laboratories, WP45 -305, West Point, PA 19486, USA Received 29 June 2000; accepted 20 November 2000

Abstract Peroxisome proliferators (PPs) are potent tumor promoters in rodents. The mechanism of hepatocarcinogenesis requires the nuclear receptor peroxisome proliferator activated receptor-a (PPARa), but might also involve the PPARa independent alteration of signaling pathways that regulate cell growth. Here, we studied the effects of PPs on the mevalonate pathway, a critical pathway that controls cell proliferation. Liver X receptors (LXRs) are nuclear receptors that act as sterol sensors in the mevalonate pathway. In gene reporter assays in COS-7 cells, the basal activity of the LXR responsive reporter gene (LXRE-luc) was suppressed by 10 mM lovastatin and zaragozic acid A, suggesting that this activity was attributed to the activation of native LXRs, by endogenously produced mevalonate products. The potent PP and rodent tumor promoter, pirinixic acid (WY-14643) also inhibited LXR-mediated transcription in a dose related manner (approximate IC50 of 100 mM). As did several other PPs including ciprofibric acid and mono-ethylhexylphthalate. Polyunsaturated and medium to long chain fatty acids at 100 mM were also potent inhibitors; the arachidonic acid analogue eicosatetraynoic acid being the most active (approximate IC50 of 10 mM). Of the PPs and fatty acids tested, there was a strong correlation between the ability of these agents to suppress de novo sterol synthesis in a rat hepatoma cell line, H4IIEC3, and inhibit LXR-mediated transcription in COS-7 cells, but a discordance between these endpoints and PPARa activation and fatty acid acyl-CoA oxidase induction. Taken together, these results suggest that PPs and fatty acids negatively regulate the mevalonate pathway through a mechanism that is not entirely dependent on PPARa activation. Because of the importance of the mevalonate pathway in regulating cell proliferation, the modulation of this pathway by PPs and fatty acids might contribute to their actions on cell growth/differentiation. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: Peroxisome proliferators; Peroxisome proliferator activated receptors; Liver X receptors; Fatty acids; Mevalonate pathway

1. Introduction Peroxisome proliferators (PPs) are a diverse class of chemicals that cause an increase in the number and size of peroxisomes in rodent liver and chronic treatment Abbre6iations: PPs – peroxisome proliferators; PPAR – peroxisome proliferator activated receptor; LXR – liver X receptor; TR – thyroid hormone receptor; RXR – retinoic X receptor; WY-14643 – pirinixic acid; MEHP – mono-ethylhexylphthalate; DHEA-S – dehydroepiandrosterone-sulfate; PUFAs – polyunsaturated fatty acids; ETYA – eicosatetraynoic acid; ZG – zaragozic acid A; TZD – 5-(4-(N-methyl-N(2-pyridyl)amino)ethoxy) benzyl)thiazolidine-2-4dione; FACO – fatty acyl-CoA oxidase; HMG-CoA – 3-hydroxy-3methylglutaryl CoA; AP-1 – activator protein-1; COX – cyclooxygenase; MAP kinase – mitogen activated protein kinase. * Corresponding author. Tel.: + 1-215-652-4323; fax: + 1-215-6524944. E-mail address: timothy – [email protected] (T.E. Johnson).

with some PPs, e.g. pirinixic acid (WY-14643), can result in up to a 100% incidence in liver tumors [1]. PPs are ligands of the peroxisome proliferator activated receptor-a (PPARa, NR1C1), a nuclear receptor that regulates genes involved in fat metabolism [2]. Studies in PPARa null animals indicate that peroxisome proliferation responses and hepatocarcinogenesis induced by PPs require PPARa [3,4]. However, there is a growing body of evidence suggesting that PPs, like other nongentoxic tumor promoters, have growth regulatory effects independent of peroxisome proliferation [5–10]. Thus, although required, PPARa might not be sufficient to induce the full tumorigenic response invoked by PPs. A key pathway that regulates cell growth is the synthesis of mevalonate, isoprenoids and cholesterol. Many studies have shown that inhibition of mevalonate

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synthesis by 3-hydroxy-3-methylglutaryl CoA (HMGCoA) reductase inhibitors, e.g., lovastatin inhibit cell cycle progression and cell proliferation [11] Important sensors in the mevalonate pathway are the liver X receptors (LXRs). LXRs are in a subfamily of steroid hormone nuclear receptors that include PPARs, thyroid hormone receptor (TR), farnesyl X receptor and pregnane X receptor. These receptors bind to their heterodimeric partner retinoic X receptor (RXR) and upon activation by ligand, interact with response elements on target promoters in the nucleus and regulate gene transcription. Two LXR subtypes have been identified; LXRa (NR1H3) which is present predominantly in liver and LXRb (NR1H2) which is ubiquitously expressed [12– 15]. LXRs are activated by oxysterols with 22(R)-hydroxycholesterol and 24(S), 25 epoxycholesterol being the most active[16–19]. A role for LXRa in the mevalonate pathway was demonstrated in LXRa null mice. These mice had no apparent phenotype when fed normal chow, but when given a cholesterol rich diet, they accumulated massive amounts of cholesterol in their liver’s [20]. Also, Cholesterol 7a-hydroxylase, a gene that is regulated by LXRa, and encodes for the rate limiting enzyme in the conversion of cholesterol into bile acids was markedly downregulated. Intriguingly, when LXRa null mice were fed normal chow, the expression levels of other genes that control mevalonate metabolism e.g., HMGCoA synthase and HMG-CoA reductase were upregulated. These observations suggest that LXRa can have positive and negative actions on the mevalonate pathway. Even though LXRb was expressed, it could not compensate for the lack of LXRa, indicating it may have a different function. However, the similar activation profile of LXRb by oxysterols and recent evidence from LXRa/LXRb double knockouts [21] suggests that LXRb is involved in regulating mevalonate metabolism under certain conditions. LXRb could also potentially play a role in regulating sterol synthesis in extrahepatic tissues, where LXRa is absent. There is evidence to support an association between the fatty acid b-oxidation and mevalonate pathways. Acetyl-CoA, a byproduct of b-oxidation, can be used to synthesize mevalonate and many of the enzymes involved in fatty acid and mevalonate metabolism are located in the peroxisomes [22]. In addition, PPARa and LXRa can form heterodimers with each other [23]. Interestingly, in vivo, the PP clofibrate was reported to inhibit cholesterol synthesis and conversely, mevalonin (lovastatin) was shown to cause peroxisome proliferation [24,25]. Because of potential interactions between these pathways, we examined the effect of PPs on LXR-mediated transcription and de novo sterol synthesis. We found that several PPs and fatty acids inhibited LXR-mediated activity in gene reporter assays in COS7 cells. Furthermore, of the PPs and fatty acids tested,

we found a strong correlation between the ability of these agents to suppress LXR-mediated transcription in COS-7 cells and inhibit sterol biosynthesis in a rat hepatoma cell line (H4IIEC3). Surprisingly, a poor correlation was found between these endpoints and PPARa activation or fatty acyl-CoA oxidase (FACO) activity.

2. Materials and methods WY-14643 was purchased from ChemSyn Sciences, Lenexa, KA. Ciprofibrate was provided from Sterling Winthrop, Inc., Rensselaer, NY. Mono-ethylhexylphthalate (MEHP) was obtained from TCI, Portland Oregon and clofibrate, dehydroepiandrosterone-sulfate (DHEA-S), mevalonolactone and all fatty acids were from Sigma Chemical Company, St. Louis, MO. Lovastatin, zaragozic acid A (ZG) and L-631,033 were obtained from the Merck Chemical Collection and L-165,461 was kindly provided by Dr. Conrad Santini at Merck. All test agents were prepared as stocks in dimethylsulfoxide (DMSO), methanol or ethanol and diluted at 500– 1000 fold in culture medium. The final concentration of these solvents in medium did not exceed 0.2%.

2.1. Cell culture Monkey kidney epithelium derived COS-7 cells and H4IIEC3 rat hepatoma cells were obtained from ATCC, Rockville, MD. Each cell type was routinely cultured in Dulbecco’s Modified Eagles Medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 mg/ml streptomycin and 2 mM L-glutamine (all Gibco, Grand Island, NY). Cells were cultured in a humidified atmosphere, 5% CO2, 95% air at 37°C.

2.2. Gene reporter transcription assays Cells were plated in 96-well plates (Costar, Cambridge, MA) at 8× 103 cells/well/0.2 ml of growth medium. The receptor and reporter gene constructs have been described previously [23,26]. Briefly, the reporter plasmid LXRE-luc contained 3 copies of the native LXRE from MMTV-LTR, which was cloned in the BglII site of the enhancerless SV40 promoter/luciferase expression vector pGL2 (Promega, Madison, WI). The MMTV-luciferase was the plasmid pJA358, in which the firefly luciferase gene is regulated by an inducible MMTV promoter that contains tandem repeats of a glucocorticoid hormone response (GR) element. The chimeric receptor constructs GR/PPARa, GR/LXRb were under the expression control of the SV40 promoter and contained the amino terminal do-

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main and the DNA-binding domain of GR fused to the ligand binding domains of PPARa and LXRb, respectively. A calcium phosphate precipitate containing 10 mg/ml of the receptor plasmid, where appropriate, and 10 mg/ml of the reporter plasmid, was diluted 100 fold and then added to the cells. About 24 h after transient transfection, the cells were washed twice with Hanks Balanced Salt Solution (HBSS) and then refed with culture medium containing 2% activated charcoal stripped serum (Hyclone, Logan, UT). The cells were then treated with test agents for 48 h. At harvest, the cell monolayers were washed twice with HBSS and then lysed in luciferase lysis buffer (Promega) directly in the 96-well plate. Extracts were assayed using the luciferase assay system (Promega). Samples were analyzed in a Microlumat LB 96 P luminometer (Wallac). Transfection efficiency was monitored in representative experiments through the cotransfection of pCH110, a constitutively active b-galactosidase expression plasmid followed by detection using the Galacton Plus assay kit (Tropix, Bedford, MA). Representative experiments are shown. Each experiment was repeated two-ten times, with similar results obtained in each experiment. The treatments at the concentrations used in these studies caused no apparent change in cellular morphology or reduction in monolayer confluence over the 48-h treatment period as compared with the concurrent control.

2.3. Protein isolation and Western hybridization Cells were washed with cold PBS and lysed in SDS lysis buffer (1% SDS, 1 mM EDTA, 1 mM EGTA, 20 mM Tris –HCl, pH 7.5 and 1 mM PMSF). Fifty micrograms of cell lysate was electrophoresed on a 4– 20% SDS-polyacrylamide gel and transferred to ImmobilonP membranes (Millipore, Bedford, MA). The membranes were blocked at room temperature in 5% nonfat milk in TBST (10 mM Tris– HCl, pH 8.0, 150 mM NaCl and 0.1% Tween 20). PPARa protein was detected using the MS1128 anti-PPARa peptide IgG at 2 mg/ml in 0.1% milk/TBST. This antibody cross-reacts with mouse, rat and human PPARa, but not with PPARg or PPARd (unpublished data). The secondary antibody used was horseradish peroxidase conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA) at a 1:8000 dilution in 5% milk/TBST. Reactive proteins were detected with enhanced chemiluminescence (Amersham Life Sciences Inc., Arlington Heights, IL).

2.4. 14C-acetate incorporation into digitonin precipitable sterols H4IIEC3 cells were plated in 6-well plates (Corning, Corning, NY) and grown until about 80– 90% conflu-

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ence in DMEM containing 10% serum. The cells were then washed and switched to Opti-MEM medium (Gibco) supplemented with 0.1% serum for 48 h. Cells were refed with fresh medium and treated with the test agents for usually 24 h; 1 mM sodium acetate (cold) and 1 mCi/ml of 14C-acetate (NEN, Specific activity of 54 mCi/mmol) was added for the last 3 h of incubation. The cells were then washed twice in HBSS and lysed in 0.1 N NaOH. A fifty microliter aliquot was removed for protein determination and the remaining lysate was used for analysis. 14C-acetate incorporation into digitonin precipitable sterols was done as described previously, with minor modifications [27]. Briefly, cell lysates were saponified in 2 N NaOH (prepared in 50% ethanol) for 1 h at 70°C under nitrogen. The nonsaponifiable lipids were then extracted three times with hexane and the pooled extracts were washed with 20 mM NaOH, then dried in a Turbo Vap (Zymark Corp, Hopkinton, MA) at 40°C under nitrogen for about 30 min. The dried extracts were dissolved in 50% ethanol and then a 1% digitonin solution (prepared in 50% ethanol) and a 100 ml of a 2 mg/ml cholesterol carrier solution were added. Two hundred microliters of water was then added, the samples were immediately vortexed, and digitonin bound sterols were precipitated overnight. The digitonides were then pelleted at 3000 rpm for 15 min, dissolved in glacial acetic acid and then counted in a scintillation counter (Beckman Instruments, Fullerton, CA). The results were normalized to protein. There was no consistent difference in the amount of total protein between treated and control cells indicating that none of the treatments induced significant toxicity in these cells. Representative experiments are shown. Each experiment was repeated 2–5 times with similar results obtained in each experiment.

2.5. Analysis of fatty acyl-CoA oxidase induction FACO activity was assayed by measuring lauroyl CoA-dependent H2O2 production fluorometrically according to the method of [28]. Twenty-five micrograms of total cell lysate was incubated at 37°C for 5 min in 500 ml of a reaction mixture containing 60 mM potassium phosphate, 0.02% Triton X-100 (pH 7.4), 400 U/ml of horseradish peroxidase, 2 mM flavin adenine nucleotide, 100 mM p-hydroxy-phenylacetic acid and 500 mg bovine serum albumin (BSA). Ten microliters of a 5-mM lauroyl CoA solution was added to the appropriate tube every 10 s. After a total incubation time of 10 min, the reaction was stopped by adding 2 mM potassium cyanide prepared in 100 mM sodium carbonate buffer (pH 10.5). FACO activity was measured in a fluorometer at an excitation of 320 nm and emission of 405 nm. The specific activity was defined as nM H2O2/ mg/min and was calculated from a standard curve. A Representative experiment is shown. The experiment

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was repeated twice, with similar results obtained in each experiment.

2.6. Protein determination Protein was measured using the BCA protein kit (Pierce Chemical) using the microwell protocol. BSA was used as the standard.

3. Results

3.1. Peroxisome proliferators and fatty acids inhibit li6er X receptor-mediated gene transcription Because LXRs act as sterol sensors, we measured LXR-mediated transcription as an indicator of PPs action on the mevalonate pathway. We transiently transfected the LXRE-luc reporter gene, which contains three LXR binding sites, into COS-7 cells. By Northern analysis, these cells express LXRb but no apparent LXRa mRNA (data not shown). As shown in Fig. 1A,

cells transfected with LXRE-luc had a high basal level of reporter activity that could be substantially suppressed by about 75% with the HMG-CoA reductase inhibitor lovastatin at 10 mM, suggesting that endogenous mevalonate products are activating native LXRs (predominately LXRb) and stimulating reporter activity. This result is similar to that reported by Forman et al. in CV-1 cells, the parental cell line of COS-7 [16]. Giving further support to this hypothesis, we also observed that the squalene synthase inhibitor ZG at 10 mM suppressed LXR-dependent transcription (Fig. 1A). Interestingly, the prototypical PP and potent rodent tumor promoter, WY-14643 suppressed LXR-mediated transcription by about 90%, not at the low dose (10 mM) but at a high dose (250 mM), which we had previously shown to be associated with cell growth regulatory changes. Other PPs were also tested for their ability to inhibit LXR-mediated transcription. At 500 mM, ciprofibrate ester and MEHP markedly suppressed LXRE-luc reporter activity while the PP and steroid DHEA-S had little or no effect (Fig. 1B). Similar results were obtained when DR4-luc, a reporter gene

Fig. 1. Peroxisome proliferators inhibit LXR-mediated transcription in COS-7 cells. (A) Cells were transiently transfected with the LXRE-luc reporter gene and then treated with the test agents at the indicated concentrations for 48 h. (B) Cells were transfected as described in (A) and than treated with the indicated PPs at the indicated concentrations for 48 h. (C) Cells were transfected as described in (A) and then treated with WY-14643 at increasing for 48 h. (D) Cells were transiently transfected with MMTV-luc and GR/PPARa and then treated with WY-14643 at increasing concentrations for 48 h. Cells were analyzed for luciferase activity as described in Section 2. The experiments shown are representative of 2– 10 independent experiments. Shown is the mean activity 9 SEM of four replicate wells for each treatment and 8 – 16 replicates for the control.

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Fig. 2. Effect of fatty acids on LXR-mediated transcription and PPARa activation in COS-7 cells. (A) Cells were transiently transfected with LXRE-luc and then treated with the indicated PUFAs at 100 mM (50 mM for ETYA) for 48 h. Inset, cells were transfected as described in (A) and then treated with ETYA at increasing concentrations for 48 h. (B) Cells were transiently transfected with MMTV-luc and GR/PPARa and then treated with PUFAs as described in (A). (C) Cells were transfected as described in (A) and treated with the indicated fatty acids at 100 mM (50 mM for ETYA) for 48 h. (D) Cells were transfected as described in (B) and then treated with the indicated fatty acids at 100 mM (50 mM for ETYA) for 48 h. Cells were analyzed for luciferase activity as described in Section 2. Experiments shown are representative of 2 – 3 independent experiments. Shown is the mean activity 9SEM, for each treatment and 16 replicates for the control.

that is also stimulated by LXRs, was used, but no effect was seen with the pGL2 control plasmid that lacked LXR binding sites (data not shown). Further studies with WY-14643 revealed that LXR-mediated activity was inhibited in a dose related manner with an approximate EC50 (based on several experiments) of about 100 mM (Fig. 1C). To determine what the PPARa activation profile was at these high doses, in a parallel experiment, we cotransfected the GR/PPARa chimeric receptor in the presence of the MMTV-luc reporter gene (contains GR binding sites). This strategy uses the concept that the ligand binding domains of nuclear receptors can be interchanged to form functional chimeric receptors that are ligand-dependent transcriptionally activated, similar to their wild type counterparts [29,30]. As expected, WY-14643 was a strong activator of the chimeric GR/PPARa receptor even at concentrations of 100 mM and above which markedly reduced LXRE-luc activity (compare Fig. 1C with Fig. 1D). Since PPs appear to mimic some of the actions of fatty acids, we examined the effects of a variety of saturated and unsaturated fatty acids of different chain lengths on LXR-mediated activity. Fatty acids are also ligands of PPARa [31,32], so in parallel experiments, we compared the ability of fatty acids to activate

GR/PPARa. All the polyunsaturated fatty acids (PUFAs) tested at 100 mM suppressed LXR-mediated activity to roughly the same extent (Fig. 2A). The medium to long chain fatty acids myristic (C14:0), palmitic (C16:0) and oleic (C18:1) also suppressed LXR-luc activity, while the short chain capric (C10:0) and lauric (C12:0) acids and the very long chain erucic (C22:1) acid had no inhibitory effect (Fig. 2A and C). As expected all the fatty acids activated GR/PPARa to various degrees (Fig. 2B and D), but there did not appear to be a correlation between the ability of these fatty acids to activate GR/PPARa and inhibit LXRmediated activity (compare Fig. 2A with Fig. 2B and Fig. 2C with Fig. 2D). A closer examination of the dose relation showed that the PUFAs arachidonic acid, linolenic acid, EPA and DHA generally had a more pronounced inhibition of LXR-mediated activity at 100 than at 10 mM (data not shown). In contrast, the synthetic molecule eicosatetraynoic acid (ETYA) was a more potent inhibitor with an approximate EC50 of about 10 mM (Fig. 2A inset). The concentrations of PUFAs and fatty acids used here are in the same concentration range known to modulate hepatic gene expression [33,34]. These results indicate that some PPs and fatty acids inhibit LXR-mediated transcription in COS-7 cells.

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3.2. Peroxisome proliferators indirectly suppress li6er X receptor-mediated acti6ity Previous studies have found that the effects of HMGCo A reductase inhibitors on a variety of cellular processes including LXR-mediated activity can be reversed by mevalonate [16,35]. Furthermore, the PP ciprofibric acid was reported to inhibit HMG-CoA reductase activity [36]. Thus, we examined the ability of mevalonate to counteract the suppression of LXRE-luc by WY-14643. As shown in Fig. 3A, both lovastatin and WY-14643 inhibited LXR-mediated transcription. While having no effect on its own at doses up to 1000 mM, mevalanolactone (the lactone form of mevalonate) completely reversed the effect of 10 mM lovastatin, but had no effect on the suppression of LXR-mediated transcription by 250 mM WY-14643. Similar results were seen with 100 mM WY-14643 (data not shown).

These data indicate that WY-14643 inhibits LXR-mediated activity through a mechanism that is downstream of mevalonate. PPs and fatty acids could suppress LXR-mediated transcription by interfering directly with LXR transactivation or alternatively by inhibiting the synthesis of endogenous LXR ligands (mevalonate products). To test for a direct effect, we tested the ability of LXR to be activated by oxysterols in the presence and absence of WY-14643. Because of the high basal activity of LXRE-luc in transfected COS-7 cells, we could not further stimulate this reporter in the presence of exogenous oxysterols. Therefore, we transiently transfected COS-7 cells with the GR/LXRb receptor and the MMTV-luc reporter gene. This chimeric receptor is activated by oxysterols, similar to that of the native LXRa and LXRb receptors (unpublished observations). As expected, treatment with 10 mM 25-OHC and 22(R)-OHC activated GR/LXRb (Fig. 3B). Although there was some variability between the quadruplicate treatments, there was no apparent modulation of GR/ LXRb transactivation by oxysterols in the presence of 250 mM WY-14643. Interestingly, in the absence of exogenous oxysterols, there is some activation of GR/ LXRb, presumably by endogenous ligands, which was inhibited by more than half in the presence of WY14643. No effect of WY-14643 on MMTV-luc was seen in the absence of GR/LXRb (data not shown). These results suggest that WY-14643 does not interfere with the ability of ligands to bind to the LXRb ligand binding domain, but does not exclude the possibility of a direct interaction with other domains of the receptor.

3.3. Peroxisome proliferators and fatty acids inhibit sterol synthesis in H4IIEC3 cells

Fig. 3. Mechanism of inhibition for LXR-mediated transcription by WY-14643 in COS-7 cells. (A) Cells were transfected with LXRE-luc and then treated with DMSO (solid circles) 10 mM lovastatin (open circles) or 250 mM WY-14643 (solid triangles), in the presence or absence of increasing concentrations of mevalonolactone, for 48 h. (B) Cells were transiently transfected with MMTV-luc and GR/LXRb and then treated with 10 mM 25-OHC or 22(R)-hydroxycholesterol (black bars), in the presence or absence of 250 mM WY-14643 (white bars), for 48 h. Experiments shown are representative of two independent experiments. Shown is the mean activity 9 SEM of four replicate wells for each treatment and eight replicates for controls. In (A) standard errors (not shown) were 910%.

The results obtained from the gene reporter experiments support the hypothesis that PPs and fatty acids suppress LXR-mediated activity by inhibiting synthesis of mevalonate products. Since oxysterols are known LXR ligands, the synthesis of cholesterol and other sterols appear to be important for maintaining LXRmediated transcriptional activity in the liver. Therefore, we examined the effect of PPs and fatty acids on de novo sterol synthesis in H4IIEC3 liver hepatoma cells. These parental cells and a subclone FaO have been routinely used to study the effect of peroxisome proliferators on fatty acid metabolism. They express many liver differentiation markers including several classes of P450 enzymes and like hepatocytes they can undergo peroxisome proliferator responses, e.g., FACO, cytochrome P4504A1 induction and peroxisome proliferation after PP treatment [37–41]. Therefore, despite being immortalized, H4IIEC3 cells are an appropriate model in which to study the effects of PPs and fatty acids.

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De novo sterol synthesis was assessed in H4IIEC3 cells by the technique of measuring the incorporation of 14 C acetate into digitonin precipitable sterols. Preliminary experiments indicated that in the presence of 1 mM cold sodium acetate, the amount of 14C-acetate incorporation into digitonin precipitable sterols was linear from 1–6 h (data not shown). As expected, a 24 h treatment with 1 mM lovastatin inhibited de novo sterol synthesis by about 80% (Fig. 4A). Similarly, WY-14643 led to a dose dependent inhibition of 14Cacetate incorporation. Furthermore, Wy-14643 and lovastatin suppressed de novo sterol synthesis with the same temporal kinetics i.e., maximal inhibition with both compounds occurred within 3 h after the beginning of treatment (Fig. 4B). Other PPs and fatty acids were also good inhibitors (Fig. 4C) and of the agents that were tested in both assays, there was a strong correlation between the ability of PPs and fatty acids to suppress sterol synthesis in H4IIEC3 cells and their ability to inhibit LXR-mediated activity in COS-7 cells (compare Fig. 1B and Fig. 4C). For example WY14643, MEHP, arachidonic acid and EYTA strongly inhibited both sterol synthesis and LXR-mediated activity, while DHEA-S was a relatively poor inhibitor of both these endpoints. Collectively, these findings strongly suggest that PPs and fatty acids negatively regulate LXR-mediated activity by suppressing the synthesis of mevalonate products.

3.4. The effects of PPs on LXR-mediated acti6ity and sterol synthesis do not appear entirely dependent on PPAR acti6ation Because PPs and fatty acids are ligands for PPARa, we were interested to determine if the observed effects of these agents on LXR-mediated transcription was mediated by this receptor. Northern analysis of Poly A+ RNA showed that COS-7 cells expressed PPARg and PPARd/hNUC-1 mRNA, but not PPARa (data no shown). Furthermore, the lack of PPARa protein by Western analysis confirms that PPARa is not expressed in these cells (Fig. 5A). Moreover, as described earlier, there was a poor correlation between the effect of PPs and fatty acids on LXR-mediated activity and GR/ PPARa activation in COS-7 cells (Figs. 1 and 2). Taken together, these results indicate that the effects of PPs and fatty acids on LXR-mediated activity appear unlikely to be mediated through PPARa. However, since both PPARg and PPARd are expressed in COS-7, the effects seen could be controlled by one or both of these receptors. Thus, we tested the effect of PPARg and PPARd agonists on LXR-mediated transcription. As seen in Fig. 5B, WY-14643 significantly inhibited LXRmediated transcription. In contrast, neither the PPARg selective ligand 5-(4-(N-methyl-N(2-pyridyl)amino)eth-

Fig. 4. Effects of PPs on sterol synthesis in H4IIEC3 cells. (A) Cells were treated with WY-14643 or lovastatin at the indicated concentrations in Opti-MEM containing 0.1% serum for 24 h. (B) Cells were grown as described in (A) and treated with lovastatin (1 mM) WY14643 (250 mM) for the indicated times. 14C-acetate/cold acetate (Section 2) was added for the last 3 h of incubation for each time point. (C) Cells were grown as described in (A) and treated with the indicated PPs at the indicated concentrations for 24 h. 14C-acetate incorporation into digitonin precipitable sterols was measured as described in Section 2. The experiments shown are representative of 2 – 3 independent experiments. Shown is the amount of 14C-acetate incorporation, normalized to protein and expressed as a percentage of control, of two replicate wells for each treatment and four replicates for the control. Standard errors are shown.

oxy) benzyl)thiazolidine-2-4-dione (TZD), the PPARd selective antagonist L-631,033 nor the dual PPARg/ PPARd agonist L-165,461 had any significant effect on inhibiting LXR-mediated activity, at doses that activated their respective receptors [42,43].

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We also explored the involvement of PPARa in mediating the effects of PPs on de novo sterol synthesis in liver cells. PPARa expression has been reported in H4IIEC3 cells [38]. In addition, there is a high amount of PPARa protein expressed in these cells, comparable to that seen in mouse liver or rat hepatocytes (Fig. 5A). To test the involvement of PPARa activation, we compared the effect of PPs on sterol synthesis with the ability of these agents to induce FACO enzymatic activity in H4IIEC3 cells. Cells were treated with several doses of WY-14643 or DHEA-S for 48 h and then sterol synthesis and FACO induction were assessed in the same experiment, i.e. from the same cell lysates. Similar to previous results, WY-14643 inhibited 14C acetate incorporation into digitonin precipitable sterols in a dose-related manner with significant inhibition seen at 250 mM (Fig. 5C). In contrast, maximal induction of

FACO activity (about a five-fold increase) occurred at 50 mM (Fig. 5D), a dose that had no effect on sterol synthesis. Being a weaker PP, DHEA-S at 500 mM induced about a two-fold stimulation in FACO activity, but interestingly did not suppress sterol production. These results indicate that even though PPARa is present in H4IIEC3 cells, the effects of PPs on sterol synthesis do not appear to be entirely dependent on PPARa activation.

4. Discussion The focus of this work was to investigate the effects of PPs on the mevalonate pathway. We found using gene reporter assays in COS-7 cells that several PPs and fatty acids inhibited LXR-mediated activity. Consistent

Fig. 5. Effects of PPs and fatty acids on LXR-mediated transcription and sterol synthesis are not entirely dependent on PPAR activation. (A) Endogenous protein expression of PPARa in mammalian cells. Total protein was isolated and fractionated (50 mg/lane) as described in Section 2. PPARa was detected with MS1128 anti-PPARa peptide IgG in the various cell types. (B) Effect of PPAR subtype selective ligands on LXR-mediated transcription. COS-7 cells were transfected with LXRE-luc and then treated with the PPAR agonists at the indicated concentrations for 48 h. The experiment is representative of two independent experiments. Shown is the mean luciferase activity of four replicate wells for each treatment and eight replicates for the control. (C) Effect of PPs on sterol synthesis in H4IIEC3 cells. Cells were treated with WY-14643 or DHEA-S at the indicated concentrations for 48 h in Opti-MEM containing 0.1% serum. Sterol synthesis was measured as described in Section 2. Shown is the amount of 14C-acetate incorporation 9SEM, normalized to protein and expressed as a percentage of control of two replicate wells for each treatment and four replicates for the control (D) Effect of PPs on FACO activity. Lysates were obtained from cells treated in (C) and FACO activity was measured as described in Section 2. Shown is the mean FACO activity (nM H2O2/mg/min), normalized to protein and expressed as a percentage of control for duplicate wells for each treatment and quadruplicates for the control. Standard errors are shown. The experiments shown are representative of two independent experiments.

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with these observations, these molecules also suppressed de novo sterol synthesis in H4IIEC3 liver cells. These results suggest that PPs and fatty acids are negative regulators of the mevalonate pathway.

4.1. Effects of peroxisome proliferators and fatty acids on li6er X receptor-mediated gene transcription and sterol synthesis When the LXRE-luc reporter gene was transiently transfected into COS-7 cells and then analyzed 48 h later, a high basal level of luciferase was seen. This observation is similar to a report by Forman et al. showing that when LXRa and RXR plasmid constructs were co-transfected along with a LXR-responsive reporter gene in CV-1 cells, the reporter gene had an elevated basal activity which could be suppressed by lovastatin and mevastatin [16]. In their experiments with wild type LXRa, some basal reporter activity was seen in the absence of exogenously added RXR, but its addition markedly boosted the response. They concluded from these studies, that the high basal activity of the reporter gene was a result of endogenous mevalonate metabolites activating the LXRa/RXR heterodimer. In our studies using COS-7 cells, a clonal derivative of CV-1, we found that transfection of the reporter gene (LXRE-luc) by itself resulted in an elevated basal luciferase activity. This might reflect differences in the endogenous expression of RXR between the two cell lines. The inhibition of LXRE-luc by lovastatin seen in our studies suggests that the suppression of basal reporter activity is being mediated analogous to that seen Forman et al. i.e., through inhibition of mevalonate metabolites that act as activators of endogenous LXRs (primarily LXRb) present in COS-7 cells. The similar response of the DR4-luc reporter gene, which is also bound by LXRs [23] gives further support to this hypothesis. ZG, an inhibitor of squalene synthase, also suppressed LXR-mediated activity at 10 mM. Although this is about 100-fold higher than the IC50 for inhibiting the enzyme in vitro [44], it is well known that there are marked differences in doses at which ZG inhibits sterol synthesis in rat hepatocytes vs. other cell types. ZG is to taken up into hepatocytes via a specific transporter, i.e. apparently absent in other cell types. In addition, ZG also binds avidly to serum proteins, which also limits cellular exposure [45]. Since COS-7 cells lack the transporter and the transcription experiments are done in the presence of serum, it is not surprising that higher doses would be required to inhibit LXR-mediated activity. These observations suggest that cholesterol-lowering drugs like lovastatin and ZG inhibit LXR-mediated activity by suppressing the synthesis of endogenous mevalonate products that act as LXR ligands.

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Intriguingly, several PPs including the potent rodent tumor promoter WY-14643 also suppressed LXR-mediated transcription. A notable exception was DHEA-S. Although this steroid is a peroxisome proliferator in rodents, the mechanism appears to be different than other PPs. For example, even though DHEA-S fails to induce peroxisome proliferator responses in PPARa knockout mice, it cannot transactivate any of the PPAR subtypes in ligand dependent transcription assays using either wild type or chimeric receptors [46], suggesting that it causes peroxisome proliferation through an indirect mechanism, i.e. not by directly activating PPARa. The effect on LXR-mediated activity was not limited to synthetic agents, because many naturally occurring fatty acids including PUFAs and medium to long chain fatty acids were also potent inhibitors. The most active was the synthetic arachidonic acid analogue ETYA indicating that fatty acids by themselves and not a metabolite were responsible for the negative regulation of LXR-mediated transcription. Similar to the effect of cholesterol lowering agents, the suppression of LXR-mediated transcription by PPs and fatty acids appears to be controlled by the ability of these agents to suppress the synthesis of endogenous mevalonate products and not by a direct interference of the LXR receptor. This conclusion is based on the following observations: First, WY-14643 failed to inhibit the transactivation of the chimeric GR/LXRb receptor by oxysterols. However, the basal activity of the MMTV-luc reporter gene, resulting from the activation of GR/LXRb by endogenous ligands, could be markedly suppressed by WY-14643. Second PPs and fatty acids inhibited de novo sterol synthesis in H4IIEC3 liver cells and of the agents that were tested in both assays, there was a strong correlation between their effect on sterol synthesis and their actions on LXR-mediated activity.

4.2. The effects of PPs and fatty acids on the me6alonate pathway are not entirely dependent on PPARh acti6ation Despite being PPARa ligands, we could find no evidence that this receptor was directly responsible for the effects of PPs and fatty acids on LXR-mediated transcription. No PPARa was detectable at either the mRNA or protein level in COS-7 cells. Furthermore, the ability of PPs and fatty acids to inhibit LXR-mediated transcription poorly correlated with their ability to transactivate the chimeric receptor, GR/PPARa. The lack of an effect of the PPARg selective ligand TZD and the PPARd selective agonist L-631,033 and the dual PPARg/PPARd agonist L-165,461 on LXR-mediated activity suggest that activation of PPARg and PPARd is also probably not involved. However, since these PPAR subtypes are expressed in COS-7, further

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studies would be required to exclude their involvement. The effect of PPs and fatty acids on de novo sterol synthesis in H4IIEC3 liver cells was also not entirely dependent on PPARa activation, even though these cells expressed PPARa protein, because there was no correlation between the inhibition of sterol synthesis and the stimulation of FACO, an enzyme induced by PPARa.

4.3. Biological actions of peroxisome proliferators and fatty acids on hepatic me6alonate metabolism and growth regulation The effects of PPs on LXR-mediated activity and sterol synthesis in cell cultures parallel those findings reported in vivo. Studies have found that the PPs clofibrate and ciprofibrate inhibit cholesterol plasma levels in rats in vivo and sterol synthesis and HMGCoA reductase activity in hepatocytes isolated from PP-treated rats [24,47]. Curiously, the PP gemfibrozil was reported to enhance cholesterol levels and HMGCoA-reductase activity in rats in vivo but inhibited these same endpoints in cultured rat hepatocytes [48,49]. The mechanism of suppression by ciprofibric acid appears to be through inhibition of HMG-CoA reductase [36]. However, other PPs have no effect on this protein and may inhibit enzymes in the mevalonate pathway, distal to HMG-CoA reductase [50,51]. The failure of mevalonate to counteract the inhibitory activity of WY-14643 on LXR-mediated transcription suggests that this PP acts on enzyme (s) downstream of HMG-CoA reductase. Interestingly, mevalonate also failed to reverse the effect of gemfibrozil on inhibiting HMG-CoA reductase in cultured rat hepatocytes [52] indicating that some PPs can modulate this enzyme not by a direct effect, but through a mechanism that might involve negative feedback from downstream mevalonate products. Thus, the effect of PPs on sterol synthesis is complex and may occur at several points in the mevalonate pathway. The inhibition of LXR-mediated transcription seen in our studies suggests that PPs might alter the expression of LXR-controlled genes due to the lack of endogenous LXR ligands which would effect both LXR subtypes. This hypothesis is supported by the observation that PPs e.g., WY-14643 and clofibrate modulates CYP-7A activity [47,53,54], a gene that appears regulated in part by LXRa [18]. A recent report found that PPs and certain fatty acids upregulated LXRa gene expression [55], which would appear to contradict our results, that these agents suppress LXR-mediated activity. However, our results in COS-7 cells were based primarily on LXRb activity, which was not affected in the Tobin et al. study. Furthermore, the increase in LXRa gene expression could be the result of a feedback mechanism due to the suppressed LXR transcriptional

activity on downstream target genes. The well-known inhibition of fibrates on CYP-7A transcriptional activity is more consistent with PPs and fatty acids having an overall negative effect on cholesterol and bile acid biosynthesis. The high doses of PPs required to inhibit LXR-mediated gene transcription and sterol synthesis are in the same concentration range that we and others have previously shown to affect cell growth regulation. For example WY-14643 and other PPs at high doses (100– 1000 mM) can induce the expression of immediate early genes e.g., activator protein-1 (AP-1), and cyclooxygenase-2 (COX-2), activate mitogen activated protein kinase (MAP kinase) signaling and regulate S-phase progression in immortalized liver cell lines that express little or no PPARa and do not undergo peroxisome proliferation [5– 8]. Interestingly, in these studies DHEA-S also had no effect on any of these growth regulatory endpoints. Several studies have found a lack of correlation between cell growth and peroxisome proliferation induced by PPs and suggest that higher doses may be more relevant to the carcinogenic effect of these agents [9,56–60]. Furthermore, plasma levels in carcinogenicity studies in rodents treated with clofibrate and ciprofibrate can reach as high as 1.4 mM with likely higher levels achieved in liver [61,62]. These results support the hypothesis that high doses of PPs may have growth regulatory effects that are not entirely dependent on PPARa activation, which would occur at much lower doses. This is supported by the observations that the MEK inhibitor, PD098059, prevented the induction of DNA synthesis and the suppression of TGF-b induced apoptosis by the PP nafenopin in primary hepatocytes, but had no effect on the induction of peroxisome proliferation [10]. However, it is clear that PPARa is required for the formation of mouse liver tumors. Thus, it is likely that the tumorigneic response induced by PPs involves both PPARa dependent and independent events. Our observation that PPs negatively regulate the mevalonate pathway is provocative since this pathway is important for cell growth. For example, cholesterol is necessary for the generation of new membranes in dividing cells and for the synthesis of steroid hormones. Moreover, the prenylation of signaling proteins by isoprenoids are critical to regulating key growth regulatory pathways e.g., signal transduction pathways and cytoskeletal organization [63]. Modulation of some or all of these pathways could potentially contribute to the growth altering effects of PPs. In conclusion, we found that PPs and fatty acids are negative regulators of the mevalonate pathway. This is in stark contrast to their positive effects on fatty acid metabolism. Although the actions of these molecules on fatty acid b-oxidation has been adequately shown to be dependent on PPARa, we have provided several pieces

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of evidence suggesting that the negative effects of these molecules on LXR-mediated activity and sterol synthesis are not entirely dependent on PPARa activation. The action of PPs and fatty acids on the mevalonate pathway could have important implications on their ability to regulate hepatic gene transcription and control cell growth/differentiation.

Acknowledgements We would like to thank our Merck Collaborators: Dr. Azriel Schmidt and John Menke for providing us with reporter and receptor plasmids; Drs. Sam Wright, Joel Berger and John Woods for giving us the PPARa antibody and Gary Dysart and Laural Ludlow for their technical expertise and for performing the FACO and Western analysis, respectively.

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