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/Retinoid X Receptor Pathway
Departments of Environmental Biochemistry (T.K., N.K., S.M., J.N.) and Toxicology (T.N.), Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan
Received October 18, 2004; accepted December 20, 2004
Abstract
Nuclear receptors play important roles in the maintenance of the endocrine system, regulation of organ differentiation, and fetal development. Endocrine disruptors exert their adverse effects by disrupting the endocrine system via various mechanisms. To assess the effects of endocrine disruptors on nuclear receptors, we developed a high-throughput method for identifying activators of nuclear receptors. Using this system, we found that triphenyltin and tributyltin were activators of peroxisome proliferator-activated receptor (PPAR) 
and retinoid X receptor. Because PPAR is a master regulator of adipocyte differentiation, we assessed the effect of organotin compounds on preadipocyte 3T3-L1 cells. We found that organotin compounds stimulated differentiation of 3T3-L1 cells as well as expression of adipocyte marker genes.
; Nishihara et al., 2000; Gray et al., 2001; Sanderson et al., 2002). Although the levels of natural hormones are precisely regulated metabolically, synthetic chemicals elude this regulation to stimulate organs by mechanisms different from those of natural hormones.
The importance of nuclear receptors in endocrine function has been well established by many studies. The human genome contains at least 48 members of the nuclear receptor family (Chawla et al., 2001), and various chemicals bind to nuclear receptors and influence the expression of target genes (Blair et al., 2000; Sultan et al., 2001). To evaluate the effects of numerous synthetic chemicals on many nuclear receptors, we developed the CoA-BAP system, a highthroughput method for identifying nuclear receptor ligands (Kanayama et al., 2003). In the present study, we applied the CoA-BAP system to the evaluation of 16 human nuclear receptors and 40 suspected endocrine disruptors. We found that organotin compounds such as triphenyltin (TPT) and tributyltin (TBT) strongly activated retinoid X receptor (RXR) and PPAR.
Organotin compounds have been used as agricultural fungicides, rodent repellents, and molluscicides and in antifouling paints for ships and fishing nets (Piver, 1973; Fent, 1996). These widespread uses have resulted in the release of increasing amounts of organotins into the environment. Although the toxicity of organotins has been reviewed extensively (Boyer, 1989), the molecular target of organotins has not yet been identified.
Here, we show that TPT and TBT are high-affinity ligands for RXR and PPAR. Organotin compounds act as agonists of both RXR
and PPAR in mammalian reporter gene assays and induce the expression of PPAR target genes. PPAR forms a heterodimer with RXR and binds to a defined DNA sequence in the promoter region of target genes (Mangelsdorf and Evans, 1995). PPAR is activated by a variety of fatty acids and a class of synthetic antidiabetic agents, the thiazolidinediones (Lehmann et al., 1995). PPAR serves as an essential regulator for adipocyte differentiation and lipid storage in mature adipocytes (Tontonoz et al., 1994). In light of these previous findings, we evaluated the effects of TPT and TBT on adipogenesis and found that organotins stimulate the differentiation of preadipocyte 3T3-L1 cells to adipocytes. Our data suggest that organotins exert their toxic effects through activation of the PPAR/RXR signaling pathway.
Materials and Methods
Plasmids. The ligand-binding domains (LBDs) of the human nuclear receptors PPAR (codons 168–468; GenBank accession no. L02932
[GenBank]
), PPAR1 (177–477; L40904
[GenBank]
), PPAR
(139–441; L07592
[GenBank]
), liver X receptor (LXR) (167–447; U22662
[GenBank]
), and LXR
(155–461; U07132
[GenBank]
) were amplified by RT-PCR from human liver mRNA as the template; the LBDs of human farnesoid X receptor (FXR) (193–472; U68233
[GenBank]
) and human estrogen-related receptor (ERR) (194–458; AF094518
[GenBank]
) were amplified similarly from human kidney mRNA and that of human ERR (195–434; AF094517
[GenBank]
) was amplified from human testis mRNA. The DNA sequences of the amplified fragments were confirmed by sequencing after subcloning into pGEX-4T (Amersham Biosciences Inc., Piscataway, NJ). The expression vectors for the human nuclear receptors estrogen receptor (ER)/, thyroid hormone receptor (TR), retinoic acid receptor (RAR)/, RXR/, vitamin D receptor (VDR), and human TIF2 were described previously (Kanayama et al., 2003). For expression in mammalian culture cells, the LBD of hRXR was fused to the C-terminal end of the GAL4 DNA binding domain (amino acids 1–97) in the pBK-CMV expression vector (Stratagene, La Jolla, CA). The expression plasmid of (GAL4-DBD)-PPAR (pM-mPPAR1) and the luciferase reporter plasmid p4xUAS-tk-luc (Kamei et al., 2003) were kind gifts from Dr. Y. Kamei (National Institute of Health and Nutrition, Tokyo, Japan).
Chemical Reagents. Diethyl phthalate, triphenyltin chloride, nitrofen, 4-nonylphenol, octachlorostyrene, permethrin, triphenylmethane, and triphenylethylene were purchased from Kanto Chemical (Tokyo, Japan). Amitrole, 2,4-dichlorophenoxy acetic acid, 1,2-dibromo-3-chloropropane, -hexachlorocyclohexane (lindane), pentachlorophenol, dihexyl phthalate, di-n-pentyl phthalate, dipropyl phthalate, 2,4-dichlorophenol, 4-nitrotoluene, and bisphenol A were purchased from Tokyo Kasei (Tokyo, Japan). Chenodeoxycholic acid, 1,25-dihydroxy cholecalciferol, lithocholic acid, all-trans retinoic acid, 9-cis retinoic acid, and 3,3',5-triiodo-L-thyronine were purchased from Sigma-Aldrich (St. Louis, MO). 15-deoxy-
12,14-Prostaglandin J2, rosiglitazone, and TO-901317 were purchased from Cayman Chemical (Ann Arbor, MI). GW501516 was purchased from Calbiochem (San Diego, CA). All other chemicals were purchased from Wako Pure Chemicals (Osaka, Japan). The 40 chemicals tested and the abbreviations used for them are listed in Table 1.
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Preparation of Proteins. The histidine-tagged fusion protein human TIF2 NID-BAP, in which the nuclear receptor interaction domain of TIF2, was ligated to the bacterial alkaline phosphatase (BAP), was expressed in Escherichia coli BL21 (DE3) cells and purified on Ni-nitrilotriacetic acid agarose resin (QIAGEN, Valencia, CA). Except for LXR/ and FXR, the glutathione S-transferase fusion proteins were expressed in the E. coli BL21 (DE3) pLysS cells; LXR/ and FXR were expressed in E. coli JM109 pRIL cells. The glutathione S-transferase fusion proteins were purified by using glutathione-Sepharose 4B (Amersham Biosciences Inc.).
CoA-BAP System. Detection of ligand-dependent interaction between nuclear receptors and TIF2 was carried out as described previously (Kanayama et al., 2003) but with slight modification. In brief, 2 µg of nuclear receptor protein diluted in 100 µl of carbonate buffer (100 mM NaHCO3, pH 8.4) was incubated in the well of a 96-well polystyrene microtiter plate (MaxiSorp; Nalge Nunc International, Rochester, NY) at 4°C overnight. The plate was washed three times with 120 µl of buffer A (20 mM Tris-HCl, 100 mM KCl, 0.25 mM EDTA, 5% glycerol, 0.5 mM dithiothreitol, and 0.05% Tween 20, pH 7.4), and then 100 µl of TIF2-BAP fusion protein (30 µg/ml) in buffer A was added to a well with the test chemical. After 1-h incubation at 4°C, the plate was washed three times with 120 µl of buffer B (50 mM Tris-HCl, 100 mM KCl, 5 mM MgCl2, and 0.10% Nonidet P-40, pH 7.2). The enzyme reaction was started by the addition of 100 µl of substrate solution (10 mM p-nitrophenyl phosphate in 100 mM Tris-HCl, pH 8.0). After incubation at 37°C for 30 to 90 min, the reaction was stopped by addition of 25 µl of 0.5 N NaOH. Finally, the absorbance at 405 nm was measured with a plate reader (MultiskanJX; Thermo Labsystems, Helsinki, Finland).
Cell Culture. Mouse 3T3-L1 (Dainippon Pharmaceutical, Osaka, Japan) and mouse NIH-3T3 (clone 5611, JCRB0615; Japanese Cancer Research Resources Bank, Osaka, Japan) fibroblasts were maintained at 37°C in 5% CO2 in Dulbecco's modified Eagle's medium (DMEM) (Nissui, Tokyo, Japan) supplemented with 10% calf serum (MP Biomedicals, Aurora, OH). Mouse F9 embryonic carcinoma cells were maintained in 5% CO2 at 37°C in DMEM supplemented with 10% fetal bovine serum (FBS) (MP Biomedicals).
Transient Transfection Assays. One day before transfection, 1 x 105 cells were plated in a 35-mm dish containing phenol red-free minimum Eagle's medium (Nissui) supplemented with 10% charcoal/dextran-treated FBS. The cells were transfected by lipofection using FuGENE 6 transfection reagent (Roche Diagnostics, Indianapolis, IN) with pBK-CMV-GAL4-hRXR or pM-mPPAR1 (300 ng/dish), p4xUAS-tk-luc (600 ng/dish), and RSV-gal (100 ng/dish). Fresh medium with or without test chemical was added the day after transfection. After incubation for 24 h, cells were harvested and assayed for luciferase and -galactosidase activity.
Adipocyte Differentiation Assays. Mouse 3T3-L1 preadipocyte cells were used for the differentiation experiments. The day after the cells reached confluence, the medium was replaced with DMEM containing 10% FBS, 10 µg/ml insulin, 0.5 mM 3-isobutyl-1-methylxantine (IBMX), and 1 µM dexamethasone (Dex). At the same time, the cells were treated with a test chemical (rosiglitazone, 9-cis retinoic acid, or an organotin compound). After 60 h, the medium was replaced with DMEM containing 10% FBS, 5 µg/ml insulin, and the test chemical. After 6 days, cells were fixed with 4% paraformaldehyde and stained with 0.5% Oil Red O. The amount of triglyceride was determined by Triglyceride E Test (Wako Pure Chemicals).
RNA Isolation, Northern Blotting, and RT-PCR Analyses. The 3T3-L1 cells were grown in DMEM containing 10% calf serum. The day after the cells became confluent, they were treated with vehicle (dimethyl sulfoxide) only, rosiglitazone (Rosi), TPT, or TBT in DMEM containing 10% FBS and 5 µg/ml insulin. The cells were harvested at various times after treatment, and total RNA was isolated using TRIzol (Invitrogen, Carlsbad, CA). For Northern blot analyses, 25 µg of total RNA was electrophoresed through a 1% agarose gel containing 2% formaldehyde and then transferred to a Hibond-N+ nylon membrane (Amersham Biosciences Inc.). The filter was hybridized with each probe, which was labeled with [-32P]dCTP by using a random labeling kit (TaKaRa, Shiga, Japan). For RT-PCR, cDNA was synthesized using ReverTra Ace (Toyobo, Osaka, Japan), and polymerase chain reaction was performed using Ampli-Taq Gold (Applied Biosystems, Foster City, CA). The primers used for amplification of the aP2 gene (a marker for adipocyte differentiation) were 5'-AAAATGTGTGATGCCTTTGTGGG-3' and 5'-TCATGCCCTTTCATAAACTCTTGTGG-3'.
Results
Application of CoA-BAP System to Endocrine Disruptors. Reproductive abnormalities in wildlife can be associated with exposure to environmental pollutants capable of mimicking the action of natural hormones. Because the nuclear receptors of intrinsic hormone systems are likely to be targets of industrial chemicals, information on their ability to bind these chemicals is valuable for environmental risk assessment. To determine whether suspected endocrine disruptors can bind to members of the nuclear receptor family, we constructed assay systems for human nuclear receptors, including ER/, RAR/, TR, VDR, RXR/, PPAR//, FXR, LXR/, and ERR/, on the basis of the previously described CoA-BAP system (Kanayama et al., 2003). The cognate ligand for each nuclear receptor enhanced alkaline phosphatase activity in a dose-dependent manner (Fig. 1). In the ERR systems, 4-hydroxy tamoxifen-dependent dissociations between ERR and coactivator were observed, as reported previously (Coward et al., 2001; Tremblay et al., 2001).
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Using these systems, we evaluated 40 suspected endocrine disruptors (Table 1) recognized by various organizations (e.g., World Health Organization and Ministry of the Environment in Japan). The effects of the tested chemicals on the interaction between nuclear receptors and TIF2 (Fig. 2) suggest that several compounds possess agonistic activities for multiple receptors simultaneously. Butyl benzyl phthalate, hexachlorocyclohexane, maneb, mancozeb, and alkylphenols were weakly agonistic for multiple receptors, including ER. One intriguing finding was that the effect of TBT on RXR was as strong as that of its endogenous ligand, 9-cis retinoic acid (Fig. 3), and the agonist effect of TPT on PPAR was as strong as that of its well known ligand, Rosi (Fig. 3). The EC50 values of TBT on RXR (7.4 x 10–8 M) and TPT on PPAR (9.5 x 10–8 M) were almost the same as those of 9-cis retinoic acid (4.3 x 10–8 M) and Rosi (1.1 x 10–7 M), respectively. Because triphenylmethane and triphenylethylene were not agonistic for RXR and PPAR, the tin moiety was important for activity (Fig. 3).
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Organotin Compounds Potentiated Transactivation by RXR and PPAR. The observations that organotin compounds enhanced the protein-protein interaction between the coactivator TIF2 and RXR or PPAR suggested that these compounds activate transcription via these receptors. To confirm the results we obtained from the CoA-BAP system, we performed a reporter gene assay in mammalian culture cells using an expression vector for (GAL4-DBD)-RXR or (GAL4-DBD)-PPAR and a reporter plasmid containing the luciferase gene along with GAL4 upstream activating sequence. Both TPT and TBT induced the transactivation function of RXR or PPAR in a dose-dependent manner (Fig. 4). The effectiveness of these organotin compounds was comparable with that of known ligands. In addition, dibutyltin chloride, a TBT metabolite in vivo, also activated reporter activity in the PPAR system (data not shown).
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Induction and Promotion of Adipocyte Differentiation by Organotin Compounds in 3T3-L1 Cells. Recent studies indicate that PPAR plays a central role in adipocyte gene expression and differentiation (Tontonoz et al., 1994). PPAR is abundantly expressed in adipocytes, and its ligands induce the efficient conversion of fibroblastic cells to adipocytes, as measured by induction of adipocyte-specific genes and lipid accumulation (Lehmann et al., 1995). If organotin compounds can function as activators for PPAR/RXR in vivo, these compounds probably induce adipocyte differentiation. To investigate this possibility, we treated 3T3-L1 cells with TPT or TBT in two types of differentiation medium, a complete differentiation medium that contained the inducers IBMX, Dex, insulin, and FBS and an incomplete differentiation medium that lacked IBMX and Dex. Although insulin is not always necessary for induction of differentiation, it efficiently enhances adipocyte development. Adipocyte differentiation was confirmed by staining with Oil Red O for lipid droplet accumulation. As expected, treatment of 3T3-L1 cells with either TPT or TBT in complete differentiation medium promoted adipocyte differentiation as well as did Rosi (Fig. 5, a–d). Even in incomplete differentiation medium, addition of organotin compounds induced adipocyte differentiation in contrast with the lack of induction after treatment with vehicle only (Fig. 5, e–h). Moreover, mRNA expression of the adipocyte differentiation marker aP2 was induced in a dose-dependent manner by addition of organotin compounds (Fig. 6a). PPAR mRNA also was induced during the differentiation process (Fig. 6a), in agreement with the results of a previous study (Tontonoz et al., 1994). Induction of aP2 mRNA expression occurred late in adipogenesis (Fig. 6b), and organotin-treated cells demonstrated accumulation of triglyceride (Fig. 6c). Together, these data provide strong evidence that the organotin compounds TPT and TBT can function as inducers of adipocyte differentiation through PPAR.
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Discussion
Our study was designed to evaluate the effects of suspected endocrine disruptors on various nuclear receptors. The data show that several compounds have simultaneous effects on multiple nuclear receptors. In particular, organotin compounds (e.g., TBT and TPT) showed strong effects on RXR or PPAR, at levels comparable with those of 9-cis retinoic acid, an endogenous RXR ligand, and rosiglitazone, a known agonist of PPAR. In CoA-BAP systems, TBT showed strong effect on protein-protein interaction between RXR and TIF2, but TPT showed slight effect (Fig. 3a). TPT showed strong effect on protein-protein interaction between PPAR and TIF2, but TBT did not (Fig. 3b). On the contrary, when tested in the transactivation assay, both TBT and TPT activated not only RXR but also PPAR (Fig. 4). This discrepancy might reflect the diversity of coactivators. To date, many coactivators have been identified as nuclear receptor-interacting proteins. These coactivators are supposed to have cell- or tissue-specific functions in vivo (Smith and O'Malley, 2004). In addition, PPAR reportedly changes its interaction partners depending on ligands (Kodera et al., 2000). We used only TIF2 in CoA-BAP system, whereas cells used for transactivation assays have many coactivators. The discrepancy of results from CoA-BAP systems and transactivation assays might be explained by this difference of coactivators. Because in vitro screening methods tend to produce false positive or false negative results like this, positive compounds should be further examined by other studies in a physiological context. Therefore, we examined the effects of organotin compounds on transcriptional regulation and adipogenesis, which is a famous physiological event related to PPAR/RXR pathway.
Exposure of rats in utero to TBT induces a dramatic increase in the incidence of low-birth-weight fetuses because of maternal hypothyroidism (Adeeko et al., 2003). Furthermore, the RXR agonist bexarotene causes clinically significant hypothyroidism in patients with cutaneous T-cell lymphoma (Duvic et al., 2001), and experimental exposure of rats to LG100268 (a selective RXR agonist) induces the acute phase of hypothyroidism (Liu et al., 2002). The similarities between the toxicities of TBT and selective RXR agonists suggested to us that at least some of the toxic effects of organotin compounds are mediated by RXR.
Most of the toxic effects of organotin compounds on sexual development and reproductive function have been documented in mollusks (Matthiessen and Gibbs, 1998). In gastropods, TBT and TPT cause imposex (Morcillo and Porte, 1999), an irreversible syndrome in which male genital tracts (mainly a penis and a vas deferens) are imposed on female organisms (Smith, 1971). Although the physiological functions of organotin compounds have been studied extensively, the molecular target of organotin compounds had been unclear. To this end, we found that TPT and TBT were agonists for RXR and PPAR. It has been thought that the sexual toxicity of organotin compounds results from increased androgen levels because of inhibition of the aromatase enzyme complex that catalyzes conversion of androgen to estrogen. This enzyme complex consists of microsomal CYP19 and the reduced form of the flavoprotein nicotinamide adenine dinucleotide phosphate reductase. TBT-induced imposex in neogastropods reportedly is mediated by inhibition of aromatase (Bettin et al., 1996), and TBT inhibits the catalytic activity of aromatase derived from transfected cells (Heidrich et al., 2001; Cooke, 2002). However, the effective concentrations of enzyme inhibition were relatively high (above 10–6 M). In this study, we found that TBT and TPT induced the transactivation function of RXR and PPAR at 10–8 M. It is reasonable that the effective concentration on gene expression was different from that on enzyme inhibition. In consistent with this, Nakanishi et al. (2004) demonstrated that 10–8 M TBT or TPT induced hCG or aromatase activity along with mRNA expression in placental cells (Nakanishi et al., 2002). In ovarian granulose cells, 20 ng/ml (about 6 x 10–8 M) TBT or TPT suppresses the P450aroma gene expression (Saitoh et al., 2001). We have to consider the toxicities of organotin compounds in distinguishing the low-dose effect from high-dose effect. Recently, we reported that RXR plays an important role in the development of gastropod imposex, by showing the cloning of RXR homolog from marine gastropod, binding of organotins to that receptor, and imposex induction by injection of RXR ligand 9-cis retinoic acid (Nishikawa et al., 2004). Gastropod imposex is known to be typically induced by very low concentrations of TBT and/or TPT (Bryan et al., 1986; Gibbs and Bryan, 1986; Horiguchi et al., 1997). Although it has been theorized that organotins increases androgen levels through inhibition of aromatase activity and/or a suppression of androgen excretion, the inhibitory concentration of organotins is not low enough for explaining imposex induction. The low-dose effects are likely to be mediated by receptors. However, the study of organotin effects in mammals is still important, because the compositions of nuclear receptor family members are very different between vertebrates and invertebrates (Escriva et al., 1997; Laudet, 1997). For example, there are no known homologs of steroid hormone receptors in the Drosophila melanogaster or Caenorhabditis elegans genomes, and the group members of TR, RAR, VDR, and PPAR seem to be late acquisitions during the evolution of the superfamily. Therefore, we examined the effects of suspected endocrine disruptors on human nuclear receptor family members. As a result, PPAR was identified as a new target molecule of organotin compounds in addition to RXR. This finding might introduce new insights in physiological functions of organotin compounds in mammals.
We were surprised to find that organotin compounds were high-affinity ligands for RXR and PPAR. Until recently, it had been thought that among synthetic compounds, only hormone analogs could bind hormone receptors, because the relationships between hormones and their cognate receptors are very specific. However, some industrial chemicals do have unexpected effects on hormone receptors. Nuclear receptors are the likely targets, because their intrinsic ligands are fat-soluble, low-molecular-weight agents, as are the environmental pollutants. In fact, organotin compounds promote the adipocyte differentiation as agonists for PPAR/ RXR. The ligands of PPAR and RXR are expected for antidiabetic agents, but they have some side effects at the same time (Mukherjee et al., 1997; Yaki-Jarvinen, 2004). Although they may be good medicines when used under a doctor's control, wildlife are exposed to synthetic chemicals in uncontrolled manner. It is possible that TBT and TPT cause adverse health effects on the organisms by disturbing the endocrine process mediated by PPAR/RXR.
Acknowledgements
We are grateful to Dr. Y. Kamei (National Institute of Health and Nutrition, Japan) for providing the GAL4-reponsive reporter plasmid and pM-mPPAR1.
Footnotes
This work was supported by grants from the Ministry of the Environment, and Health, Labor, and Welfare, Japan.
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
ABBREVIATIONS: BAP, bacterial alkaline phosphatase; TPT, triphenyltin; TBT, tributyltin; RXR, retinoid X receptor; PPAR, peroxisome proliferator-activated receptor; LBD, ligand-binding domain; LXR, liver X receptor; RT-PCR, reverse transcription-polymerase chain reaction; FXR, farnesoid X receptor; ERR, estrogen-related receptor; ER, estrogen receptor; TR, thyroid hormone receptor; RAR, retinoic acid receptor; VDR, vitamin D receptor; TIF2, transcriptional intermediary factor 2; hRXR, human retinoic acid receptor; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; IBMX, 3-isobutyl-1-methylxanthine; Dex, dexamethasone; Rosi, rosiglitazone; LG100268, 6-(1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)cyclopropyl)pyridine-3-carboxylic acid; TO-901317, N-(2,2,2-Trifluoroethyl)-N-[4-(2,2,2-trifluoro-1-hydroxy-1-trifluoromethylethyl)phenyl]benzenesulfonamide; GW501516, 2-methyl-4-((4-methyl-2-(4-trifluoromethylphenyl)-1,3-thiazol-5-yl)-methylsulfanyl) phenoxy-acetic acid.
Address correspondence to: Dr. Jun-ichi Nishikawa, Department of Environmental Biochemistry, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamada-oka, Suita, Osaka 565-0871, Japan. E-mail: nisikawa{at}phs.osaka-u.ac.jp
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1 to 10 times as much as cognate ligand; yellow, 
) showed strong agonistic activity for hRXR
). B, TPT (
) showed strong agonistic activity to hPPAR
) and TPE (
) did not show any agonistic activity. Activity of the vehicle control (dimethyl sulfoxide) only is shown by 
.
