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Biology of Reproduction 63, 259-266 (2000)
© 2000 Society for the Study of Reproduction, Inc.

Regular article

Inhibition of Rainbow Trout (Oncorhynchus mykiss) Estrogen Receptor Activity by Cadmium1

R. Le Guével2,a, F.G. Petita, P. Le Goffa, R. Métiviera, Y. Valotairea, and F. Pakdela

a Equipe d'Endocrinologie Moléculaire de la Reproduction, UPRES-A CNRS 6026, Université de Rennes I, France

ABSTRACT

This study was conducted to determine if the cadmium-mediated inhibition of vitellogenesis observed in fish collected from contaminated areas or undergoing experimental exposure to cadmium correlated with modification in the transcriptional activity of the estrogen receptor. A recombinant yeast system expressing rainbow trout (Oncorhynchus mykiss) estradiol receptor or human estradiol receptor was used to evaluate the direct effect of cadmium exposure on estradiol receptor transcriptional activity. In recombinant yeast, cadmium reduced the estradiol-stimulated transcription of an estrogen-responsive reporter gene. In vitro-binding assays indicated that cadmium did not affect ligand binding to the receptor. Yeast one- and two-hybrid assays showed that estradiol-induced conformational changes and receptor dimerization were not affected by cadmium; conversely, DNA binding of the estradiol receptor to its cognate element was dramatically reduced in gel retardation assay. This study provides mechanistic data supporting the idea that cadmium is an important endocrine disrupter through a direct effect on estradiol receptor transcriptional activity and may affect a number of estrogen signaling pathways.

estradiol, estradiol receptor, female reproductive tract, hormone action, oocyte development

INTRODUCTION

Environmental contamination by toxic heavy metal ions such as cadmium, copper, zinc, and mercury from various sources (e.g., volcanic activity, weathering of rocks, and industrial, mineral mining, and agriculture activities) has been a problem for decades [1]. These metals are not eliminated from ecosystems. Cadmium enters the food chain through environmental contamination and concentrates within organisms because of its relatively long half-life. The main process for biological detoxification of heavy metals is binding to metallothioneins, which are a group of heavy-metal-binding proteins isolated from several tissues of many vertebrates [2].

The toxic effects of cadmium to aquatic organisms, the prime target of environmental pollutants, have been well-documented [3–5]. Some fish, such as salmonids, are particularly sensitive to the presence of cadmium in their environment [1, 4, 5]. Cadmium toxicity has been associated with interference of enzymatic processes [6] and essential-metal-ion imbalance [7, 8]. Furthermore, cadmium is frequently associated with reproductive disorders in mammals [9–12] and in fish. Cadmium induces alteration of hormonal balance in rainbow trout (Oncorhynchus mykiss) testes in vitro [13], and it impairs control of ovarian maturation and steroidogenesis by pituitary gonadotropic hormone in female common carp (Cyprinus carpio) [14]. Eggs obtained from rainbow trout exposed to cadmium fail to develop to the fry stage, and in brown trout (Salmo trutta), oogenesis appears to be delayed [4]. Exposure of fish embryos to Cd++ also promotes premature hatching, retarded growth, increased mortality, and developmental abnormalities [15, 16].

In all oviparous vertebrates, the liver is one of the main targets for 17ß-estradiol (E2). Therefore, this organ is often used as a model to study the regulation of the expression of estrogen-dependent genes. The action of E2 is mediated by estrogen receptor (ER), which is a member of the nuclear receptor superfamily that functions as ligand-activated transcription factors. Upon E2 binding, ER dimerizes so that it may bind to specific DNA sequences called estrogen-responsive elements (EREs) and may stimulate the transcription of specific genes. The ER possesses a modular structure in which various aspects of receptor function are associated with specific domains (i.e., A, B, C, D, E, and F domains) of the peptide sequence [17, 18]. The C domain bears the DNA-binding function of nuclear receptors. This DNA domain is thought to coordinate two zinc ions with cysteine residues into "zinc finger" motifs and to adopt a conformation appropriate for DNA binding. Two well-characterized transcriptional activation functions (AFs) have been described in ER: the ligand-independent function AF-1, which is located in the N-terminal A/B domain; and the ligand-dependent function AF-2, which is located in the E domain [19, 20].

In oviparous vertebrates, vitellogenesis is an important event allowing oocyte growth through incorporation of vitellogenin (VTG). Production of VTG by the liver is tightly controlled by E2 levels [21]. This glycolipophosphoprotein is transported in the blood to the ovaries, where it is then incorporated and processed into oocytes to form the major yolk protein used for feeding of the embryo [22]. Obviously, all events affecting the vitellogenesis process can reduce the overall reproductive success. Impaired VTG production and uptake have been demonstrated in winter flounder (Pleuronectes americanus) populations collected from contaminated areas [23] or undergoing experimental exposure to cadmium during early vitellogenesis [24]. In rainbow trout, injection of cadmium with E2 inhibits transcription and translation of VTG [25].

Regarding the importance of salmonids as a source of food, but also as an experimental model for environmental studies related to metal contamination [26], it is important to elucidate at the molecular level why salmonids, particularly during the reproductive stages, are highly sensitive to cadmium. The present study was performed to test the hypothesis [25] that cadmium-induced inhibition of VTG mRNA synthesis in rainbow trout could result from a modification of the biological activity of rainbow trout estradiol receptor (rtER). For this purpose, a recombinant yeast system expressing rtER or human estradiol receptor (hER) and an estrogen-responsive reporter gene was used to determine if this action was a direct effect on ER and at what level cadmium inhibits ER-mediated gene induction. Determination of transcriptional activity in recombinant yeast as well as dimerization, ligand-binding, and DNA-binding properties of ER were performed to investigate the influence of cadmium on the different steps implicated in the estradiol response.

MATERIALS AND METHODS

Chemicals

Amino acids, E2, charcoal dextran, protease inhibitors, o-nitrophenyl-ß-D-galactopyranoside, lyticase, and heavy metal chloride were from Sigma Chemical Co. (St. Louis, MO). Yeast nitrogen base and dropout were from Difco (Elancourt, France). The [3H]estradiol and [32P]dCTP were from NEN (Boston, MA), and the Poly(dI-dC) was from Boehringer Mannheim (Meyland, France).

Full-Length Rainbow Trout, hER, and Truncated rtER Expression Vectors

Expression vectors YEpucG and YEphER (HEGO wild-type hER expression vector) [27] were gifts from B.S. Katzenellenbogen [28]. To construct YEprtER, the 3.3-kilobase (kb) EcoRI fragment of rtER cDNA [29] was inserted into the unique BamHI site of YEpucG [30]. Truncated rtER, rtER-{Delta}EF (rtER {Delta}234–575), and rtER-{Delta}AB (rtER {Delta}1–140) were constructed by polymerase chain reaction (PCR) [31]. The truncated rtER were inserted into the BamHI site of YEpucG and, respectively, named YEprtER-{Delta}EF and YEprtER-{Delta}AB expression vectors.

Yeast Strains and Transformation

The yeast strains used in this study were BJ-ECZ (containing the URA3-2ERE-CYC1-LacZ reporter gene) [28], BJ2168 (Yeast Genetic Stock Center, Berkeley, CA), and Y187 (Clontech Laboratories, Palo Alto, CA). Yeast strains were transformed by the lithium acetate method as described by Petit et al. [30] and selected by growth on complete minimal medium (CM medium: 0.13% dropout powder lacking uracil and tryptophan, 0.67% yeast nitrogen base, 1% dextrose, 39 mM [NH4]2SO4). BJ-ECZ was transformed by the YEprtER and YEphER expression vectors [30]. BJ2168 was transformed by the YEprtER-{Delta}EF and YEprtER-{Delta}AB expression vectors and by the pLR{Delta}21-U3ERE (URA-3ERE-URA3-lacZ) reporter vector [31]. The yeast strain Y187 was transformed by the PCL1 full-length Gal-4 expression vector (Clontech).

Whole-Cell Extract and Estradiol-Binding Assays

Yeast cells were grown in 50 ml of CM medium with or without of 20 µM Cd2+ during 4 or 20 h at 30°C under vigorous shaking (300 rpm) to an absorbance of 1.5–2.0 at 600 nm. The cells were harvested, the cell walls removed using lyticase, and the spheroplasts lysed according to the method described by McDonnell et al. [32] to obtain a whole-cell extract. Estradiol-binding assays were performed using a modification of the procedure described by Pakdel and Katzenellenbogen [33]. For the E2-binding assay, 90 µg of whole-cell extract protein (1 mg/ml) from yeast were incubated at 4°C for 16 h with 20 nM [3H]estradiol (NEN) in Tris buffer (10 mM Tris-HCl [pH 7.4] 10% glycerol) in the presence of increasing concentrations of cadmium (from 0- to 10 000-fold excess over [3H]estradiol). For Scatchard analysis, 45 µg of whole cell extract protein from yeast treated by 20 µM Cd++ during 4 and 20 h (described earlier) were incubated at 4°C for 16 h with [3H]estradiol at increasing concentrations from 0.25 to 20 nM [30]. For this experiment, cadmium was not added to the incubation media. Nonspecific binding was determined in the presence of a 150-fold excess of unlabeled ligand. Free and specifically bound [3H]estradiol were separated by the addition of an equal volume of dextran-coated charcoal (0.5% charcoal, 0.05% dextran, 10 mM Tris-HCl [pH 7.5]). Affinity of rtER for estradiol was determined by the Scatchard method [34].

ß-Galactosidase Assays

The ß-galactosidase assays were performed according to the method described by Petit et al. [30]. Induction of ß-galactosidase activity was strictly dependent on the presence of rtER and estrogens. The E2 concentration necessary to achieve maximal activation was 10 nM. For the transactivation assays, heavy metal salts were added to the CM medium in the presence or absence of estradiol during 4 h at 30°C. The ß-galactosidase assay was performed by hydrolysis of the NpGal (o-nitrophenyl-ß-D-galactopyranoside; Sigma) substrate in Z buffer (60 mM Na2HPO4, 10 mM KCl, 1 mM MgSO4, 50 mM ß-mercapto ethanol [pH 7]), and the colored product was measured at 420 nm (A420) using a spectrophotometer. The cell density was measured at 600 nm (A600), and the ß-galactosidase activity was expressed in Miller units [35].

One- and Two-Hybrid Assays

The rtER cDNA was subcloned in the BamHI cloning site of the pAS2-1 or the pACT2 vectors (Clontech), generating the fusion proteins rtER-Gal4DBD and rtER-Gal4AD, respectively. The fusion between the Gal-4 DBD and the rtER domains C through F was constructed by inserting the rtER1–140 cDNA (rtER{Delta}AB) generated by PCR in the BamHI cloning site of the pGBT10 vector (Clontech). All these fusions were verified by sequencing.

Y190 yeast cells (URA3: GAL1UAS-GAL1TATA-LacZ reporter gene) were transformed using the lithium-acetate method, and transformants were selected by growth on CM medium including 1 µM E2 or ethyl alcohol and 25 mM 3-aminotriazole that inhibits the slight endogenous activity of the his-3 gene. In parallel, transformation efficiency was evaluated by plating a fraction of the transformation mixture on CM plates. Then, the eventual transactivation activity was first tested by growth on the selective media. To confirm this activity, resulting from a protein-protein interaction in the two-hybrid or an endogenous transactivation ability in the one-hybrid test, ß-galactosidase activity was tested using a filter-lift assay by transferring the colonies on Whatman filter. Filters were frozen for 15 sec in liquid nitrogen, thawed at room temperature, and incubated on Whatman 3MM soaked with Z Buffer containing 0.33 mg/ml X-Gal for 3 to 24 h at 30°C. Quantification of ß-galactosidase activity was performed as described earlier using NpGal substrate [30]. Activity was expressed in Miller units [35].

Yeast Nuclear Extracts and DNA-Binding Assays

Nuclear extracts were performed using a modification of the method described by Ponticelli and Struhl [36]. Briefly, yeast cells were incubated with 1500 U of lyticase and then lysed in ice-cold lysis buffer. Spheroplasts were incubated in CM medium containing 1 M sorbitol for 30 min and lysed in ice-cold lysis buffer with Potter-Elvehjem homogenizer. Cell debris and unlysed spheroplasts were removed by centrifugation. Nuclei were harvested by centrifugation and resuspended in ice-cold 0.1 M Tris/acetate buffer (pH 7.9). Nuclear proteins were extracted by ammonium sulfate precipitation. The ammonium sulfate precipitate was then recovered by centrifugation, and the pellet was suspended in 200 µl of ice-cold Hepes buffer (20 mM Hepes [pH 7.6], 10 mM MgSO4, 10 mM EGTA, 20% glycerol). After dialysis against Hepes buffer for 12 h at 4°C, insoluble material was eliminated by centrifugation [31]. For the gel retardation assay, nuclear extract of yeast expressing rtER, hER (2.5 µg), or Gal4 (4 µg) was incubated in the binding buffer (10 mM Tris-HCl [pH 7.5]; 100 mM KCl; 10% glycerol; 100 µg/ml BSA; 5 µg/ml each of the protease inhibitors aprotinin, leupeptin, pepstatin A; and 1 mM PMSF) with 1 µg of Poly(dI-dC) with or without heavy metals (i.e., Cd, Zn, Co, and Ni) for 15 min at room temperature. The untransformed yeast nuclear extract was used as a control. Then, samples were incubated for 15 min at room temperature with 60 000 cpm of the 32P-labeled, double-stranded oligonucleotide EREc (6-6) (5'-tcgaggatccAGGTCAcagTGACCTc-3') for ER protein extracts or the 32P-labeled oligonucleotide Gal-4-binding site (5'-tcgagCGGAAGACTCTCCTCCGgtcacagtgacc-3'). After incubation, the reactions were loaded onto a 5% polyacrylamide gel and electrophoresed at 4°C for 2–3 h at 200 V in 0.5x Tris-Borate-EDTA buffer. After electrophoresis, the gels were dried, the bound and free radiolabeled probe quantified using an Instantimager (Packard, Rungis, France), and the gels exposed to Kodak Biomax film (Kodak, Rochester, NY).

Statistics

Raw data were compared within groups using ANOVA. When significant, the Fisher protected least significant difference test was performed for the post hoc comparisons of the groups (2 x 2).

RESULTS

ER Transcriptional Activity Is Decreased by Cadmium but Not Other Heavy Metals in a Recombinant Yeast System

The previously developed yeast system, expressing rtER [30] and containing an estrogen-responsive reporter gene, was used to determine the direct effect of cadmium on the estrogenic response. In this expression system, transcription of the Lac-Z reporter gene is strictly dependent on the presence of rtER, and no ß-galactosidase activity is detected in ER-deficient yeast cells, even in the presence of a high E2 concentration. However, a basal transcription of LacZ in yeast expressing rtER was detected in the absence of E2. (This induction represents ~15% of the maximum activity obtained with 10 nM E2.) This ligand-independent transactivation may be attributable to the AF-1 of the receptor [30]. To determine the ability of cadmium and other selected heavy metal divalent ions to affect the transcriptional activity of rtER, yeast cells were incubated 4 h with 10 µM metal ions, with or without 10 nM E2. Results are shown in Table 1 as the percentage of E2-stimulated ß-galactosidase activity (i.e., maximal activity). Cadmium affected the E2-dependent transcriptional activity of rtER but not the ligand-independent activity. The other metals tested (i.e., Cu2+, Zn2+, Mn2+, Ni2+, and Co2+) were ineffective in altering the E2 response significantly.


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  		TABLE 1. Effect of heavy metals on transcriptional activity of rtER expressed in yeast.a

rtER and hER Activities in Recombinant Yeast Are Differentially Altered by Cadmium

We next assessed the dose effect of cadmium on rtER, hER, and the yeast Gal-4 activator transcriptional activities in the presence of E2. An increasing concentration of cadmium markedly decreased the estradiol-mediated transcriptional activity of both rtER and hER (Fig. 1A). However, a difference in cadmium sensitivity was found between rtER and hER, producing a shift in the cadmium concentration-response curve to a higher concentration for hER. These data indicate a lower sensitivity of the human receptor to cadmium (IC50s: hER, 600 µM; rtER, 20 µM). On the other hand, cadmium did not disturb the basal transcriptional activity of rtER or Gal-4, indicating that cadmium did not interfere with the general cellular transcriptional and translational machinery. Because cadmium affected the biological activity of ER, experiments were performed to determine if, in yeast, cadmium could compete with other essential divalent ions such as Ca2+, Mg2+, and Zn2+ and, therefore, disrupt some general ion-mediated cellular mechanisms indirectly implicated in the estradiol response [37–42]. To verify this point, a cadmium dose-response curve was performed both with or without a 10-fold molar excess of calcium, zinc, or magnesium over cadmium (Fig. 1B). Data showed that none of these ions could reverse the inhibitory effect of cadmium on rtER transcriptional activity. This experiment demonstrates that cadmium may act at the level of rtER by a mechanism distinct from a competition with the three other essential divalent ions in yeast.



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  		FIG. 1. Cadmium-dose effect of on ER transcriptional activity in yeast expressing the full-length rtER, hER, or the yeast Gal-4 transcription factor. Cadmium was added in culture medium with or without 10 nM estradiol (A). Yeast strain expressing the full-length rtER was treated by cadmium with a 10-fold molar excess of calcium, zinc, or magnesium salts over cadmium (B). After 4 h of exposure, cells were lysed and the ß-galactosidase activity quantified. Results are expressed as a percentage of maximal estradiol induction without metal and represent the mean ± SEM of independent clones (A: rtER, n = 8; hER, n = 5; Gal4, n = 4. B: n = 4 for competition with Ca, Mg and Zn.)

Cadmium Does Not Affect Ligand Binding to rtER

One possible mechanism implicated in the cadmium effect could be an inhibition of estradiol binding to the hydrophobic hormone-binding pocket of ER protein. To verify this point, a hormone-binding assay and Scatchard analysis were performed with rtER from whole-yeast-cell extract. Results indicated that the [3H]estradiol binding to its receptor was not affected by cadmium up to a 10 000-fold excess over [3H]estradiol (data not shown). Incubation of yeast cells with 20 µM cadmium (a concentration sufficient to inhibit 50% of the estradiol-mediated LacZ induction; Fig. 1A) during 4 or 20 h had no effect on the hormone/receptor dissociation constant of rtER from protein yeast extract (Kd = 1.02 and 1.07 nM vs. 0.95 nM without cadmium, respectively). These results showed that cadmium-mediated inhibition of rtER biological activity was not caused by an inhibition of estradiol binding to its receptor.

Cadmium Represses the AF-2-Related Activity of rtER

As previously shown with the full-length rtER, a basal transcription of LacZ in yeast expressing rtER was detected in the absence of ligand, and E2 induced a higher activity of the reporter gene expression (Fig. 2A). As with the BJECZ yeast strain containing the 2ERE-CYC1-lacZ reporter gene, cadmium was able to inhibit the E2-induced lacZ gene expression in the BJ2168 yeast strain containing the 3ERE-URA3-lacZ reporter gene but was unable to affect the basal activity of rtER (Fig. 2A). Moreover, the transcriptional activity of the yeast Gal-4 transcription factor, used as a control, was unaffected by cadmium (Fig. 2B). This confirms the specificity of cadmium action on the estradiol-mediated rtER transcriptional activity. The constitutive ß-galactosidase activity of the truncated rtER-{Delta}EF, which lacks the hormone binding domain and its E2-dependent AF-2 (Fig. 2C), represent 28% (7.6 ± 1.5 Miller units) of the maximal E2-induced activity (27.5 ± 0.5 Millers units) obtained with the full-length receptor (Fig. 3A). This transcriptional activity of the rtER-{Delta}EF truncated receptor, reflecting the AF-1 ligand-independent activity of the A/B region of rtER, was not inhibited by 50 µM cadmium (Fig. 2C). This result indicates that cadmium does not affect the AF-1 of rtER.



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  		FIG. 2. Yeast expressing full-length rtER (A), rtER-{Delta}EF (C), and rtER-{Delta}AB (D) truncated receptors were treated for 4 h by 50 µM cadmium with or without 10 nM E2. The rtER-{Delta}EF, truncated on the C-terminal region and, thus, lacking the hormone-binding domain (domain E/F), was constitutively active (C). The rtER-{Delta}AB, truncated in the N-terminal region and, thus, lacking the A/B domain, was inducible by E2 (D). The Gal-4 transcription factor, expressed in yeast, was constitutively active and used as a control (B). Results are expressed as a percentage of maximal estradiol induction without metal and represent the mean ± SEM of six independent clones. The ß-galactosidase activity expressed in Miller units is indicated above each column. Statistical analysis was performed as described in Materials and Methods. Significant differences are indicated by a different letter (P < 0.001)



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  		FIG. 3. The rtER fusion with the Gal-4 DBD (rtER-Gal4DBD; A) was introduced in yeast containing the LacZ reporter gene placed under the control of 3 Upstream Activator Sequence for Gal-4 (UASG). Yeast transformants containing the rtER-Gal4DBD construct were treated with 10 or 100 µM Cd2+ with or without E2 (B). Results are expressed in Miller Units and represent the mean ± SEM of nine independent clones

We next examined the effect of cadmium on the AF-2 of the rtER-{Delta}AB truncated receptor. In the absence of E2, the rtER-{Delta}AB truncated receptor activity was low (0.38 ± 0.03 Miller units). This truncated receptor was inducible by E2, and the induced ß-galactosidase activity represented 14% (3.8 ± 0.4 Miller units) of the maximal activity of the full-length receptor (Fig. 2D). This activity of rtER-{Delta}AB reflects the AF-2 ligand-dependent activity of the E domain of rtER. Cadmium was able to inhibit the AF-2-related transcriptional activity of this truncated receptor, and the magnitude of inhibition was higher than that with the full-length rtER receptor (64% vs. 44%) at 50 µM cadmium.

That cadmium did not inhibit the transcriptional activity of Gal-4 transcriptional factor (Figs. 1A and 2B) allowed use of rtER and the Gal-4 DNA-binding domain (DBD) or Gal-4 activation domain (AD) fusion proteins to study cadmium action on ER. Yeast transformants containing the rtER-Gal4DBD construct (Fig. 3A) were treated with 10 or 100 µM Cd2+ in absence or the presence of E2. As shown in Figure 3B, this protein had no transcriptional activity in the absence of ligand. In the presence of 1 µM E2, induction of the ß-galactosidase activity was measured. Such an E2-induced activity has been shown previously for hER in a similar fusion protein [43]. In this fusion protein, the AF-2 of ER is not functional, and the transcriptional activity is thought result from a ligand-induced conformational change in the protein, allowing exposition of the AF-1 [43]. Therefore, it was interesting to study the effect of cadmium on this putative conformational change. Cadmium to a concentration of 100 µM had no effect on this E2-induced activity (Fig. 3B). Thus, cadmium may not affect the exposition of AF-1, reflecting an estradiol-induced tridimensional change in the rtER-Gal4DBD fusion protein.

ER Dimerization Is Not Affected by Cadmium

The two-hybrid system was used to examine the effect of Cd2+ on the dimerization property of rtER in the absence or the presence of ligand. Because of the endogenous activity of rtER-Gal4DBD in the presence of estradiol (Fig. 3B), this construct was used only when studying the ligand-independent dimerization of rtER. Cotransformation of yeast by this fusion protein and rtER-Gal4AD (Fig. 4A) led to a low but significant ß-galactosidase activity (3 Miller units) corresponding to the homodimerization of rtER in the absence of ligand and the Gal4AD-mediated induction of the reporter gene (Fig. 4B). Cadmium treatment at 10 or 100 µM did not affect this ligand-independent receptor dimerization.



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  		FIG. 4. A) Schematic representation of fusion proteins used in the two-hybrid system. Construction of these fusions is described in Materials and Methods. Yeast cells, cotransformed with the indicated constructs, were exposed for 4 h to ethanol, 1 µM E2, and 10 or 100 µM cadmium with or without E2. B) Coexpression of rtER-Gal4DBD and rtER-Gal4AD constructs. C) Expression of rtER{Delta}AB-Gal4DBD construct alone (+/-) and coexpression of rtER{Delta}AB-Gal4DBD and rtER-Gal4AD constructs (+/+). Results are expressed in Miller units and represent the mean ± SEM of nine independent clones

Another fusion protein (i.e., rtER{Delta}AB-Gal4DBD) was constructed by fusion of the Gal-4 DBD with the C-terminal region of rtER (domains C–F; Fig. 4A). When this construct was introduced alone in yeast, no transcriptional activity was detected either in the presence or the absence of 1 µM estradiol. Cadmium had no effect on the activity of this fusion protein (Fig. 4C). This result reflects the fact that the AF-2 of rtER is not functional in such a construction, as previously revealed with the rtER-Gal4DBD construct (Fig. 3B). In addition, this observation confirms that rtER-Gal4DBD activity is mediated by the AF-1 of rtER (Fig. 3B). When rtER{Delta}AB-Gal4DBD and rtER-Gal4AD were coexpressed for the two-hybrid assay, no transcriptional activity was observed in the absence of E2, showing that these two proteins dimerize very poorly without ligand. When rtER{Delta}AB-Gal4DBD and rtER-Gal4AD recombinant yeast were treated for 4 h by 1 µM E2, a ß-galactosidase activity was revealed. This activity demonstrated that the two constructs interact in a hormone-dependent manner in this yeast system. Cadmium treatment to a concentration of 100 µM did not affect this E2-induced dimerization (Fig. 4C). These data imply that the inhibitory effect of cadmium on the transactivation properties of rtER is not mediated by a reduced receptor dimerization.

ER DNA-Binding Activity Is Inhibited by Cadmium

One possible explanation for the inhibitory effect of cadmium on ER biological activity is an alteration of its DNA binding properties. Gel-shift analysis showed that cadmium markedly reduced the DNA-binding activity of ER (Fig. 5, A and B), whereas Gal-4 DNA binding was not affected by cadmium (Fig. 5C). Nevertheless, although the rtER DNA-binding activity was clearly diminished, hER binding at 50 µM cadmium was poorly affected. However, at 100 µM cadmium, a significant inhibition of DNA binding was observed for hER (Fig. 5B). These results show that cadmium can inhibit both rtER and hER DNA-binding activities, but that rtER is more sensitive than hER to this inhibition. This observation agrees with the greater sensitivity of rtER to cadmium compared to hER observed in the yeast system. The other metals tested (i.e., Zn, Co, and Ni) did not produce any significant effect on DNA binding, except for Zn2+, which seemed to increase the binding of both rtER and hER.



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  		FIG. 5. Radiolabeled ERE and the Gal-4 DNA-binding site were used as probes and incubated with nuclear extracts from yeast expressing rtER (A), hER (B), or Gal-4 (C), with or without cadmium, zinc, cobalt, and nickel. The probe was also incubated with 2.5 µg of protein from yeast nuclear extract expressing no receptor for the determination of unspecific binding. The binding activity of proteins to the radiolabeled probe was determined by counting the bound cpm. The total count obtained without metal was used as a reference. Results are expressed as a percentage of this reference after subtraction of nonspecific binding and represent the mean of three independent experiments ± SEM. Statistical analysis was performed as described in Materials and Methods. Significant differences are indicated by a different letter (P < 0.001). The Photoshop-scanned autoradiography of one representative experiment is shown above each graph

DISCUSSION

In the present study, we tested the influence of several essential (i.e., Cu2+, Co2+, Mn2+, and Zn2+) and nonessential metal ions (i.e., Cd2+ and Ni2+) on the ER transactivation functions. These metals were designated as being insidious environmental industrial pollutants and could form metal complexes with organic ligands that involve bidentate or tetradentate coordination with O, N, or S electronic donor atoms of macromolecules [39]. Several groups have reported that these metals can interact with enzymes [6, 39], transcription factors [40–42], and the steroid-binding property of nuclear receptors [38] and, therefore, can affect the biological properties of these proteins. Among the heavy metals tested in our experiments, only cadmium inhibited the transcriptional activity of E2-activated rtER in recombinant yeast. This inhibitory action was dose-dependent and specific for ER, because no effect was observed on the yeast Gal-4 transcription factor. Moreover, hER was also sensitive to the inhibitory action of cadmium, but at a lower level. In addition, our results demonstrate that cadmium-induced inhibition of ER transcriptional activity is not mediated by an inhibition of estradiol binding to rtER, which contrasts with the report of Simons et al. [38], in which Cd++ blocked steroid binding to the glucocorticoid receptor (GR) by interacting with the vicinal thiol implicated in the hormone binding [38].

In ER, two activation functions have been described [19, 20]. The A/B domain possesses a ligand-independent activation function (i.e., AF-1), and the E domain possesses a ligand-dependent activation function (i.e., AF-2). In the full-length ER activated by E2, these functions act synergistically [20]. Two truncated rtER receptors, rtER-{Delta}EF and rtER-{Delta}AB, were used to determine whether cadmium could affect both AF-1 and AF-2. The rtER-{Delta}EF was truncated in the C-terminal region, lacked the hormone-binding domain, and was constitutively active. The rtER-{Delta}AB was truncated in the N-terminal region, lacked the A/B domain, and was inducible by E2. Both truncated rtER-{Delta}EF and rtER-{Delta}AB possess the DNA-binding domain (i.e., domain C) and can induce the reporter gene in yeast.

In yeast expressing rtER, the ligand-independent transcription function (i.e., basal activation) was not affected by cadmium. Similarly, the activity of the rtER-{Delta}EF truncated receptor, displaying only the ligand-independent AF-1, was not inhibited by cadmium. On the other hand, the full-length receptor and the rtER-{Delta}AB truncated receptor (displaying only the ligand-dependent AF-2) were inhibited by cadmium. These results demonstrate that the putative site of cadmium action is localized in the C-terminal domain of ER, which is a domain containing important regions for ligand binding, dimerization, DNA binding, and transactivation.

In the one-hybrid assay, the rtER-Gal4DBD fusion had no transcriptional activity in the absence of ligand, but an induction of the ß-galactosidase activity was measured in the presence of E2. This activity may be attributable to a ligand-induced conformational change in the protein, allowing exposition of the AF-1 [43]. Cadmium had no effect on this E2-induced conformational change, because no transcriptional inhibition was seen.

Results obtained with the two-hybrid assay demonstrate that cadmium does not affect the ligand-independent and ligand-dependent dimerization properties of rtER, and they lead to the conclusion that the inhibitory effect of cadmium on the transactivation properties of rtER is not mediated by reduced dimerization of the receptor. Nevertheless, our results clearly show that cadmium inhibits ER binding to specific DNA sequences, whereas Zn2+ increases ER binding, particularly with rtER. Consequently, the effect of cadmium on the AF-2, as revealed with the rtER-{Delta}AB truncated receptor, could be indirect and mediated by an inhibition of DNA binding. This inhibitory action on DNA binding could reflect a disruption of zinc binding in the zinc finger motif of the C domain of ER by competition with cadmium. This hypothesis is consistent with previous observations that cadmium could replace Zn2+ in the zinc finger motifs of ER [44] or GR [45, 46] in vitro. However, when Zn2+ was substituted by Cd2+ in zinc finger motifs, the DNA-binding property of ER was preserved [44], whereas in our results, cadmium induced a decrease in DNA-binding activity. Moreover, ER DNA-binding assays performed with Cd2+ in the presence or the absence of a 10-fold molar excess of Zn2+ showed no differences from observations with cadmium alone (data not shown). These observations indicate that cadmium-inhibition of ER DNA binding probably does not result from a substitution of zinc by cadmium in the receptor zinc fingers. This conclusion is in accordance with Zn2+ binding in the zinc finger motif being quite strong and resistant to chelators such as EDTA and 1,10-phenanthroline in physiological condition [46]. In addition, native nuclear receptors seem to naturally co-ordinate Zn2+ instead of Cd2+ in vivo [46].

That only the E2-activated receptor was affected by cadmium in our experiments suggests that the activated and unactivated receptors display some structural differences in the DBD itself. Unactivated nuclear receptors are recovered from cells in heterocomplexes containing both heat shock proteins (hsp) and immunophilins [47]. Some components of receptor heterocomplexes are proteins with an established chaperone function (e.g., hsp90 and hsp70). One event after E2 activation is the transformation of ER to a DNA-binding form by conformational changes that are followed by dissociation of the heterocomplexes. In our particular case, the site (or sites) implicated in cadmium action could be inaccessible in the unactivated receptor or protected by chaperone proteins. During the activated state, this site would become unmasked, and cadmium could exert its inhibitory action by diminishing the interaction of activated ER with DNA, thus reducing ER transcriptional activity. A putative site of cadmium action could be the DBD domain of ER. Effectively, the ER C-terminal region of the DBD domain interacts with hsp90 and is involved in the masking of the DNA-binding function of unactivated ER [48]. This characteristic could explain why cadmium can display inhibitory properties only when ER is activated by E2.

In conclusion, we have demonstrated that cadmium has a direct effect on ER biological activity, thus explaining some vitellogenesis defects observed in fish after cadmium contamination. Our report provides mechanistic data supporting the idea that cadmium is an important endocrine disrupter that may alter a broad range of genetic programs controlled by estrogen.

ACKNOWLEDGMENTS

We are grateful to Dr. B.S. Katzenellenbogen for providing the yeast expression vector YEpucG. We also thank Dr. G. Salbert and Dr. Y. Le Dréan for a critical reading of the manuscript and Dr. T. Bailhache for help with the statistical analysis of data.

FOOTNOTES

First decision: 14 December 1999.

1 Supported by the Centre National de la Recherche Scientifique and the European Economic Community. Back

2 Correspondence: R. Le Guével, Équipe d'Endocrinologie Moléculaire de la Reproduction, UPRES-A CNRS 6026, Université de Rennes I, Campus de Beaulieu, 35042 Rennes Cedex, France. FAX: 33 299 28 67 94; remy.le-guevel{at}univ-rennes1.fr Back

Accepted: February 29, 2000.

Received: November 4, 1999.

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