New Screening Methods for Chemicals with Hormonal Activities Using Interaction of Nuclear Hormone Receptor with Coactivator

Toxicology and Applied Pharmacology 154, 76 – 83 (1999) Article ID taap.1998.8557, available online at http://www.idealibrary.com on

New Screening Methods for Chemicals with Hormonal Activities Using Interaction of Nuclear Hormone Receptor with Coactivator Jun-ichi Nishikawa,* Koichi Saito,*,† Jun Goto,* Fumi Dakeyama,* Masatoshi Matsuo,*,† and Tsutomu Nishihara*,1 *Laboratory of Environmental Biochemistry, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamada-oka, Suita, Osaka 565-0871, Japan, and †Environmental Health Science Laboratory, Sumitomo Chemical Co. Ltd., 1-93, 3-chome, Kasugade-Naka, Konohana-ku, Osaka 554, Japan Received April 3, 1998; accepted August 21, 1998

through their receptors that exist in the cell nucleus and regulate the transcriptional level of target genes in a ligand-dependent manner. Using nuclear hormone receptors, we developed an efficient and relevant assay system to screen endocrine disruptors based on their transcriptional activation mechanism. Nuclear hormone receptors constitute a large superfamily of ligand-inducible transcription factors, which include receptors for steroid hormones, thyroid hormones, vitamin D3, retinoids, prostanoids, and a number of proteins with high sequence homology but as yet unidentified ligands (Mangelsdorf et al., 1995). These receptors display a modular structure, with several regions (A/B, C, D, and E) exhibiting different degrees of evolutionary conservation (Tsai and O’Malley, 1994; Mangelsdorf and Evans, 1995). The highly conserved C region contains two zinc modules responsible for DNA binding and sequencespecific recognition of hormone response elements (HRE) in the proximity of target genes. The E region contains the ligand binding domain (LBD) responsible for selective binding of the cognate ligands with high affinity. Nuclear hormone receptors have two distinct transactivation domains: AF-1, located in the N-terminal A/B region, and AF-2, located in the C-terminal E region. While the AF-1 domain is capable of activating transcription in a hormone-independent manner, the AF-2 domain overlaps the LBD and activates transcription in response to hormones. Further studies suggested that the integrity of a well conserved amphipathic alpha helix located in the extreme C-termini of AF-2 is required for both the ligand-induced conformational change and the ligand-dependent transcription activity (Danielian et al., 1992; Barettino et al., 1994; Durand et al., 1994). Although the mechanisms by which the AF-2 domain transmits ligand binding signals to basal transcription machinery remain poorly understood, several groups have identified proteins that interact with the AF-2 domain of nuclear receptors in a ligand-dependent manner. Those include RIP140 (Cavaille´s et al., 1995) ,TRIP-1/SUG-1 (Lee et al., 1995; vom Baur et al., 1996), TIF1 (Le Douarin et al., 1995), SRC-1 (Onate et al., 1995), TIF2/GRIP-1 (Vogel et al., 1996; Hong et al., 1996), ACTR (Chen et al., 1997), and CBP/p300 (Kamei et al., 1996), which have several LXXLL motifs capable of interacting with nuclear receptors (Torchia et al.,

New Screening Methods for Chemicals with Hormonal Activities Using Interaction of Nuclear Hormone Receptor with Coactivator. Nishikawa, J., Saito, K., Goto, J., Dakeyama, F., Matsuo, M., and Nishihara, T. (1999). Toxicol. Appl. Pharmacol. 154, 76–83. The endocrine system exerts important functions in a multitude of physiological processes including embryogenesis, differentiation, and homeostasis. Xenobiotics may modify natural endocrine function and so affect human health and wildlife. It is necessary, therefore, to understand the degree to which xenobiotics can disrupt endocrine systems. The key targets of endocrine disruptors are nuclear hormone receptors, which bind to steroid hormones and regulate their gene transcription. We have developed relevant assay systems based on the ligand-dependent interaction between nuclear hormone receptor and coactivator. The coactivators used in this study contained CBP, p300, RIP140, SRC1, TIF1, and TIF2. By two hybrid assay in yeast, the interactions of estrogen receptor with RIP140, SRC1, TIF1, and TIF2 were detected and they were completely dependent on the presence of estrogen. Specificity of this assay was assessed by determining the effect of steroids, known estrogen receptor agonists, and phytoestrogens. The pattern of response to chemicals were consistent with estrogenic activity measured by other assay systems, indicating that this assay system is reliable for measuring estrogenic activity. In addition, we carried out in vitro binding studies: GST pull-down assay and surface plasmon resonance analysis. The estrogen receptor also bound to coactivator in response to chemicals depending on their estrogenic activity in vitro. These data demonstrate that the measurement of interaction between steroid hormone receptor and coactivator serves as a useful tool for identifying chemicals that interact with steroid receptors. © 1999 Academic Press

Endocrine disruptors, compounds that modify natural endocrine function, have emerged as a major public health issue due to their potentially disruptive effects on physiological processes, particularly through interaction directly with steroid hormone receptors (Colborn et al., 1996). In view of this alarming situation, it is necessary to determine if xenobiotics can mimic some steroid effects or modify the activity of these hormones. Most actions of steroid hormones are mediated 1

To whom correspondence should be addressed. Fax: 81-6-879-8244; Email: [email protected]. 0041-008X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.

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1997; Herry et al., 1997). Ligand binding to receptors induces conformational changes in AF-2 and promotes their association with these coactivators (Renaud et al., 1995; Wagner et al., 1995). Therefore, the association of receptor with coactivator is the first event of the ligand-induced transcriptional activation that might be useful for assessing the ability of chemicals to change receptor conformation leading to transcriptional activation. In order to determine the interactions between nuclear receptors and coactivators, we used three systems; yeast twohybrid assay, GST pull-down assay, and surface plasmon resonance analysis. The interactions between receptors and coactivators were caused by the addition of a number of natural and synthetic steroids including phytoestrogens, pharmaceuticals, and commercial chemicals that have been proposed to mimic the steroid hormones through interaction with steroid receptors. These data demonstrate the utility of using receptor– coactivator interaction for assessing chemical interaction with steroid receptors. METHODS Chemicals. b-Estradiol (.97%), estriol (98%), estrone (98%), b-sitosterol (99.5%), and testosterone (.97%) were purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan). 5a-Dihydrotestosterone (100%) was purchased from Sigma Chemical Co. (St. Louis, MO). Bisphenol A (99%), genistein (.99%), p-nonylphenol (technical grade), and stilbesterol (99%) were purchased from Nacalai Tesque (Kyoto, Japan). Molecular biological reagents, unless otherwise stated, were from Toyobo (Osaka, Japan). All other chemicals were reagent grade, obtained from commercial sources, and used without further purification. Plasmids. The yeast expression plasmids, pGBT9 and pGAD424, were purchased from Clontech (Palo Alto, CA). The LBD of rERa (codons 252– 600) was amplified from cDNA (Koike et al., 1987) by PCR. The other LBDs of receptors, AR (605–901), GR(503–795), MR (668 –981), PR (corresponding to mouse PR, 621–923), and TRa (173– 461) were amplified by RT–PCR using total RNA from rat tissues, testis, liver, adrenal, uterus, and liver, respectively. The EcoRI and BamHI sites were introduced in 59 and 39 terminus of amplified fragments and subcloned into EcoRI–BamHI sites of pGBT9 so that they were in the same translational reading frame as the vector’s GAL4 DNA binding domain (GAL4DBD). The receptor interaction domains (RID) of coactivators (CBP, p300, RIP140, TIF1, TIF2, SRC1) were amplified by PCR from cDNAs (Chrivia et al., 1993; Eckner et al., 1994; Cavaille´s et al., 1995; LeDouarin et al., 1995; Voegel et al., 1996; Onate et al., 1995) and subcloned into pGAD424 digested with EcoRI–BamHI for the production of fusion proteins with GAL4 activation domain (GAL4AD). For in vitro interaction studies, the LBD of rERa (252– 600) and RID of hSRC1 (570 –782) were subcloned into pGEX-4T (Pharmacia, Uppsala, Sweden) and pET-28a (Novagen, Madison, WI), respectively. All sequences synthesized by PCR were confirmed by DNA sequencing. Yeast strain. The yeast strain used in this study was Y190 (MATa, ura352, his3-D200, ade2-101, trp1-901, leu2-3, 112, gal4Dgal80D, URA3:::GAL-lacZ, cyhr2, LYS2:::GAL-HIS3), obtained from Clontech (Palo Alto, CA). Yeast cells were transformed with the pGBT9 –receptors and pGAD424 – coactivators using a lithium acetate method and selected by growth on SD medium (lacking tryptophan and leucine). Growth of yeast for receptor assays. Yeast transformants were grown overnight at 30°C with vigorous shaking in 1 ml of selective medium. The 50 ml of overnight culture was then added to 200 ml of fresh medium containing

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test chemical. After yeasts were cultured for 4 h at 30°C, b-galactosidase activities were determined. b-Galactosidase assay. Yeast liquid b-galactosidase assay was carried out according to the manufacturer’s protocol (Clontech), with a slight modification. The chemically treated yeasts were collected by centrifugation from 100-ml culture and resuspended in 200 ml of Z buffer (0.1 M sodium phosphate [pH 7.0], 10 mM KCl, 1 mM MgSO4) containing 0.001% SDS. After 4 ml of chloroform was added, samples were incubated for 15 min at 30°C and then enzymatic reaction was started by addition of 40 ml of 4 mg/ml 2-nitrophenylb-D-galactoside (ONPG). When the yellow color developed, 100 ml of 1 M Na2CO3 were added to stop the reaction. b-Galactosidase activity is calculated with the following equation: U510003([OD415]2[1.753OD570]/([t]3[v]3[OD595]), where t 5 time of reaction (min), v 5 volume of culture used in assay (ml), OD595 5 cell density at the start of the assay, OD415 5 absorbance by o-nitrophenol at the end of reaction, and OD570 5 light scattering at the end of reaction. b-Galactosidase activities of the liquid yeast cultures are expressed as the means and standard deviations of the results from three independent transformed yeast colonies. GST pull-down assay. GST–ER fusion protein was expressed in Escherichia coli according to the standard procedure (Pharmacia). Bacteria was disrupted in buffer A (50 mM Tris–HCl [pH 7.5], 0.2 M KCl, 5 mM MgCl2, 0.5 mM EDTA, 10% glycerol) by sonication. The supernatant obtained by centrifugation was used for binding experiments. 35S-Methionine-labeled SRC1 was expressed by in vitro transcription–translation from pET28a-SRC1 following the manufacturer’s instructions (Promega, Madison, WI). 35S-Methionine was purchased from Amersham (Little Chalfont, Buckinghamshire, UK). A crude E. coli extract containing 20 pmol of GST-ER was incubated with 35S-SRC1 in a total volume of 100 ml of buffer B (20 mM Hepes [pH 7.9], 0.15 M KCl, 5 mM MgCl2, 0.05% Triton X-100, 0.05% NP40) on ice for 30 min. Samples were further incubated with 10 ml (packed volume) of glutathione–Sepharose on ice for 30 min. The resin was washed three times with 200 ml of buffer B, and bound protein was eluted with 10 mM glutathione. 35 S-SRC1 was visualized by autoradiography after SDS–PAGE. Surface plasmon resonance analysis. GST–ER fusion was expressed in E. coli and purified according to the standard procedure (Pharmacia). Histidine-tagged SRC1 was expressed in E. coli transformed with pET28a-SRC1 and purified on Ni-nitrilotriacetic acid (NTA) agarose resin (Qiagen, Chatsworth, CA). Binding kinetics were determined by surface plasmon resonance using BIAcore (Pharmacia). Purified histidine-tagged SRC1 was diluted to 150 mg/ml in HBS buffer (10 mM Hepes [pH 7.4], 0.15 M NaCl, 50 mM EDTA, 0.005% Tween20) and was immobilized to a NTA sensor chip according to the manufacturer’s protocol (Pharmacia). Purified GST–ER was diluted to 30 mg/ml in Buffer B and incubated with b-estradiol or test chemical for 30 min in Buffer B on ice prior to the injection over the SRC1-coupled surface at 5 ml/min. Sensorgrams were recorded and normalized to a baseline of 0 resonance units (RU).

RESULTS

Ligand-Dependent Interaction between ER LBD and Coactivator in Yeast The GAL4DBD–ER LBD and GAL4AD– coactivator fusion proteins were expressed from their expression plasmids in yeast. Because the yeast strain Y190 harbors a GAL4 binding site upstream of a lacZ reporter gene, GAL4DBD-ER binds to the regulatory region of the lacZ gene. If GAL4DBD-ER interacts with GAL4AD-coactivator, GAL4AD recruits the basal transcriptional machinery to the promoter region of the

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FIG. 1. Ligand-dependent interaction of estrogen receptor with various coactivators in yeast. Yeast strain Y190 was transformed with GAL4 activation domain fused to coactivator (shown) and GAL4 DNA binding domain fused to ligand-binding domain of estrogen receptor. 17b-estradiol was added to yeast cultures in doses ranging from 10210 to 1025 M. Following 4 h incubation the cultures were then assayed for b-galactosidase activity. The values are represented as the rate of b-galactosidase activity divided by the cell density. Means 6 SD (n 5 3) are shown.

lacZ gene, resulting in production of b-galactosidase. Therefore, the b-galactosidase activity corresponds to the strength of interaction between ER and coactivator. The protein–protein interactions between ER and coactivators were strictly dependent on the presence of 17b-estradiol (Fig. 1). The ER showed 17b-estradiol-dependent interaction with coactivators in the following order: TIF2 . SRC1 . RIP140 . TIF1 . p300 . CBP (Fig. 1). Specificity of this assay was assessed by determining the effect of steroids, known ER agonists, and phytoestrogens. 17b-estradiol and estrone showed the same level of b-galactosidase induction. The synthetic estrogen, diethylstilbesterol (DES), was as effective as 17b-estradiol at inducing b-galactosidase activity, followed by estriol, genestein, pnonylphenol, and bisphenol A. Dihydrotestosterone (DHT) was active at a high concentration, while testosterone and b-sitosterol were inactive in this assay (Fig. 2). There were slight differences in patterns of response with different coactivators. Ligand-Dependent Interaction between ER LBD and Coactivator in Vitro We next sought to address whether the ER interacted with coactivator directly in vitro. SRC1 was expressed and la-

FIG. 2. Dose–response curves for a variety of estrogenic compounds. Chemicals were added to yeast cultures in doses ranging from 10210 to 1025 M. Following 4 h incubation the cultures were then assayed for b-galactosidase activity. The values are represented as the rate of b-galactosidase activity divided by the maximum activity induced by b-estradiol. Means 6 SD (n 5 3) are shown.

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GST–ER LBD and SRC1 was not observed at this concentration (1026 M). The ability of ER to interact with SRC1 was also investigated in real time using a biosensor. The RID of SRC1 was expressed as a histidine fusion protein and immobilized on a NTA sensor chip. The binding of ER to SRC1 was monitored upon its introduction to the sensor surface. The purified GST–ER was incubated with 17b-estradiol for 30 min prior to assay. A strong binding response was observed under these conditions, but was not seen in the absence of preincubation with 17b-estradiol or in the GST control (data not shown). The effect of preincubation with various chemicals is shown in Fig. 4. 17b-Estradiol, estrone, and DES showed the same level of enhancing effect on the interaction, followed by estriol, genestein, p-nonylphenol, and bisphenol A. b-Sitosterol, testosterone, and DHT were inactive in this assay. Application to Other Nuclear Receptors

FIG. 3. Ligand-dependent interaction between estrogen receptor and coactivator in vitro. In vitro translated and 35S-labeled SRC1 was incubated with a crude E. coli extract containing GST–ER (top) or GST (bottom) in the presence of various chemicals (1026 M) and then glutathione–Sepharose was added. Labeled peptides retained on the beads after extensive washing were analyzed by SDS–PAGE and visualized by autoradiography. Lane 1, 20% of the translated products used in the incubation; lane 2, solvent control; lane 3, b-estradiol; lane 4, estrone; lane 5, estriol; lane 6, genistein; lane 7, b-sitosterol; lane 8, stilbestrol; lane 9, p-nonylphenol; lane 10, bisphenol A; lane 11, DHT; and lane 12, testosterone.

beled with 35S-methionine, and the LBD of ER was expressed in E. coli as a fusion protein with glutathione S-transferase. GST pull-down assays were performed in the presence of chemicals used in the yeast system (Fig. 3). The interaction of 35S-SRC1 with GST–ER LBD was not observed in the absence of added compound (Fig. 3, top, lane 2). The presence of 17b-estradiol significantly enhanced the interaction between 35S-SRC1 and GST–ER LBD (Fig. 3, lane 3). The estrogenic compounds (estrone, estriol, and DES) were as effective as 17b-estradiol at enhancing interaction (Fig. 3, lanes 4, 5, and 8) followed by genestein (Fig. 3, lane 6). The effect of p-nonylphenol (Fig. 3, lane 9) or bisphenol A (Fig. 3, lane 10) on the interaction between

The yeast two-hybrid assay can easily be adapted for other receptors by exchanging the ER portion of GAL4DBD fusion with other receptors. In fact, when ER LBD was changed to AR LBD, the b-galactosidase activity of yeast transformed by pGBT9 –AR LBD and pGAD424 –SRC1 showed androgen-dependent increase (Fig. 5). Testosterone and DHT were equally active and 17b-estradiol was active at high concentration, while p-nonylphenol and bisphenol A were inactive in the androgen receptor assay (Fig. 5). We next adapted this system to other nuclear receptors including PR, GR, MR, and TR. Tests in the yeast two-hybrid system indicated that coactivators interacted specifically with all six nuclear receptor LBDs. These interactions occurred in the presence but not in the absence of a cognate ligand (Fig. 6). Based on their strength of interaction (data not shown), we chose the following combinations: ER–TIF2, AR–SRC1, PR–TIF2, GR–SRC1, MR–SRC1, and TR–TIF2. Because the sensitivity is slightly different depending on the kind of receptor, we used the following concentrations of ligands: b-estradiol (1028 M), 5a-dihydrotestosterone (1027 M), progestrone (1028 M), corticosterone (1026 M), aldosterone (1027 M), T3 (1027 M), p-nonylphenol (1025 M), and bisphenol A(1024 M). b-Estradiol did not show activity on AR at this concentration (1028 M) consistent with liquid assay. All hormonal activities can be assessed simultaneously on a filter that has yeast clones harboring receptor and coactivator expression plasmids. DISCUSSION

So far, numerous kinds of man-made chemicals have been released into the environment regardless of their potential disruptive effects for endocrine systems. As a result, there are numerous examples of reproductive anomalies in wildlife in areas contaminated with chemicals that have hormone-like

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FIG. 4. Ligand-dependent interaction of estrogen receptor with SRC1 measured by a biosensor employing SPR detection. Purified GST–ER treated with various chemicals were injected over a sensor chip with immobilized SRC1, as detailed in Methods.

activity. In order to avoid these risks, we have to determine whether man-made chemicals are capable of perturbing the normal hormonal milieu in humans and other animals. In this

paper we demonstrate the utility of interaction assays between hormone receptors and coactivators for assessing the potential hormone-like activities of chemicals.

FIG. 5. Yeast two-hybid assay for androgen receptor activity. Chemicals were added to yeast cultures in doses ranging from 10210 to 1025 M. Following 4 h incubation the cultures were then assayed for b-galactosidase activity. The values are represented as the rate of b-galactosidase activity divided by the cell density. Means 6 SD (n 5 3) are shown.

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FIG. 6. Simultaneous detection of various hormonal activities. Yeasts harboring receptor (shown in left side) and coactivator expression plasmids were grown on a Whatman #1 filter layered over SD (2Leu, 2Trp) agar plate. The filter were then lifted out and placed on a filter presoaked in a SD medium containing an appropriate hormone (shown in top). Following 4 h incubation the yeasts were broken by freeze and thaw using liquid nitrigen. The X-gal was used as a substrate for b-galactosidase activity. ER, estrogen receptor; AR, androgen receptor; PR, progesterone receptor; GR, glucocorticoid receptor; MR, mineral corticoid receptor; and TR, thyroid hormone receptor.

Recently, several coactivators were shown to be involved in transcriptional activation through nuclear hormone receptors (reviewed in Horwitz et al., 1996). On the other hand, the yeast estrogen screen (YES) has been widespread by virtue of its ease of manipulation and ability to handle large sample numbers quickly and inexpensively (Arnold et al., 1996; Gaido et al., 1997). However, the mechanism of transcriptional activation is different in different species. Especially, the endocrine system is not necessary for unicellular organisms such as yeast. Because yeast does not have nuclear hormone receptors, the coactivators that mediate signals from receptors to the basal transcriptional machinery might differ from yeast to vertebrates. In YES, transcriptional activation of ER might occur by spurious interaction of ER with unrelated factors existing in yeast, resulting in unreliableness. In order to overcome this problem, we used the two-hybrid assay. In the two-hybrid assay, the interactions of steroid hormone receptors with coactivators are genuine, because they are derived from mammals. Furthermore, by using the activation domain derived from yeast transactivator GAL4, we can obtain strong b-galactosidase activity. Another advantage of the yeast two-hybrid assay is ease of application for other receptors. Although estrogenic activity is attracting much attention, endocrine

disruptors may bind to another receptor or multiple receptors resulting in destruction of the endocrine system. In order to examine multiple hormonal activities simultaneously, we constructed many receptor assay systems including ER, AR, PR, GR, MR, and TR. These assay systems serve as a useful tool for identifying hormone-like chemicals in a short time. Multiple proteins have been identified which interact with ER in a ligand-dependent manner (reviewed in Horwitz et al., 1996). It may be possible that different agonists and antagonists differently influence interactions of ER with coactivators. By these means, different coactivators may be selected by different chemicals. In Fig. 2, p-nonyphenol appears as active as genistein with TIF2, yet barely active with TIF1. This result is possible to be explained by different ligand selectivity of ER for coactivator binding. It is currently difficult to choose one coactivator for assessing estrogenic activity, because very little is known about which of coactivators are actually involved in the transcriptional activation of steroid hormone receptors in intact cells. The sensitivity of the yeast two-hybrid based estrogen assay seems to be lower than the previous described YES system (Arnold et al., 1996; Gaido et al., 1997). This is due to short-term incubation of chemicals with recombinant

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yeasts (4 h vs overnight). With an overnight incubation, we were able to observe an increase in the activity of b-galactosidase (fivefold enhancement at 1 nM) and a left shift of the dose–response curve (data not shown). The detectable and saturant levels of 17b-estradiol were shifted from 1 nM to 10 pM and from 10 mM to 1 nM, respectively (data not shown). If it is necessary to detect an extreamly weak estrogenic activity, it is achieved by simply prolonging the incubation time. However, the longer exposure of xenobiotics may affect the growth or viability of yeast, resulting in causing complexity of interpretation. When applying the screening system, it should be considered that the permeability of xenobiotics through the cell membrane can differ due to their divergent chemical structures. The differences between in vivo and in vitro study (Fig. 2 and Fig.4) may reflect different permeabilities of chemicals. In addition, if chemicals are toxic for yeast, the yeast assay cannot be used. The toxicity of the test compound can be evaluated by the value of OD595 after 4 h incubation with yeast. If chemicals are toxic, in vitro studies are useful. A combination of these methods is best for determining the hormonal activity of xenobiotics. Studies of interaction between hormone receptors and coactivators are beneficial because of their specificity, sensitivity, and ease of manipulation. Moreover, it is worth noting that this interaction is critical in the transcription activation pathway through the hormone receptor. It is postulated that endocrine disruptors may bind to hormone receptors and activate their target genes inappropriately. Therefore, these interaction assays are relevant to the function of hormones. While useful as tools for screening, these assays, or any in vitro assay alone, cannot completely address accumulation, distribution, and metabolism of the compound. We are planning to test numerous chemicals and environmental samples by using these in vitro assay systems. Subsequently, positive compounds should be further examined by in vivo study. ACKNOWLEDGMENTS We are grateful to Dr. Muramatsu for providing rat ER cDNA. This work was supported in part by grants from the New Energy and Industrial Technology Development Organization and the Japanese Standards Association.

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