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Molecular Endocrinology, doi:10.1210/me.2004-0397
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Molecular Endocrinology 19 (10): 2502-2516
Copyright © 2005 by The Endocrine Society

Trialkyltin Compounds Bind Retinoid X Receptor to Alter Human Placental Endocrine Functions

Tsuyoshi Nakanishi, Jun-ichi Nishikawa, Youhei Hiromori, Hideaki Yokoyama, Mihoko Koyanagi, Shinri Takasuga, Jun-ichi Ishizaki, Mai Watanabe, Shun-ichi Isa, Naoki Utoguchi, Norio Itoh, Yutaka Kohno, Tsutomu Nishihara and Keiichi Tanaka

Department of Toxicology (T.N., Y.H., H.Y., M.K., S.T., J.I., M.W., S.I., N.I., K.T.) and Laboratory of Environmental Biochemistry (J.N., T.N.), Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka 565-0871; Laboratory of Pharmaceutics (N.U.), School of Pharmaceutical Sciences, Teikyo University, Sagamiko, Kanagawa 199-0195; and Development Division (Y.K.), Fujisawa Pharmaceutical Co., Ltd., Osaka 532-8514, Japan

Address all correspondence and requests for reprints to: Dr. Tsuyoshi Nakanishi, Department of Toxicology, Graduate School of Pharmaceutical Sciences, Osaka University, 1–6, Yamadaoka Suita, Osaka 565-0871, Japan. E-mail: nakanishi{at}phs.osaka-u.ac.jp.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Retinoid X receptor (RXR) is a nuclear receptor that plays important and multiple roles in mammalian development and homeostasis. We previously reported that, in human choriocarcinoma cells, tributyltin chloride and triphenyltin hydroxide, which are typical environmental contaminants and cause masculinization in female mollusks, are potent stimulators of human chorionic gonadotropin production and aromatase activity, which play key endocrine functions in maintaining pregnancy and fetal development. However, the molecular mechanism through which these compounds stimulate these endocrine functions remains unclear. Our current study shows that trialkyltin compounds, including tributyltin chloride and triphenyltin hydroxide, function as RXR agonists. Trialkyltins directly bind to the ligand-binding domain of RXR with high affinity and function as transcriptional activators. Unlike the natural RXR ligand, 9-cis-retinoic acid, the activity of trialkyltins is RXR specific and does not activate the retinoic acid receptor pathway. In addition, trialkyltins activate RXR to stimulate the expression of a luciferase reporter gene containing the human placental promoter I.1 sequence of aromatase, suggesting that trialkyltins stimulate human placental endocrine functions through RXR-dependent signaling pathways. Therefore, our results suggest that activation of RXR may be a novel mechanism by which trialkyltins alter human endocrine functions.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE RETINOID X receptors RXR{alpha}, RXRß, and RXR{gamma}, which are type II nuclear receptors, are thought to be key factors in several nuclear receptor signaling pathways. These molecules specifically bind 9-cis-retinoic acid (9cRA) and thus may be directly involved in the transduction of retinoid signals. In transfected cultured cells, as well as established cell lines, RXRs can act either as homodimers or heterodimeric partners of several other nuclear receptors, including retinoic acid receptors (RAR{alpha}, -ß, and -{gamma}), thyroid hormone receptors (TR{alpha} and -ß), vitamin D receptor (VDR), peroxisome proliferator-activated receptors (PPAR{alpha}, -{gamma}, and -{delta}), and a number of orphan receptors (1, 2, 3). Therefore, RXRs may be central to the modulation of several hormonal signals.

The placenta is a transient, but vital, organ for maintaining pregnancy in mammals. Its functions range from nutrient and gaseous exchange to hormone and growth factor production. Several observations suggest that RXRs play indispensable roles in placental development and physiology. In mice, RXR{alpha} transcripts are strongly expressed in the ectoplacental cone and, at later stages, in giant trophoblastic cells and the labyrinthine zone of the chorioallantoic placenta (4). RXR knock-out experiments in mice have revealed that RXR{alpha} and RXRß are involved in the formation of the chorioallantoic placenta (5, 6). In particular, the inactivation of RXR{alpha} ligand-dependent transcriptional activation function 2, but not ligand-independent transcriptional activation function 1, prevents the formation of labyrinthine trophoblasts and leads to fetal death during the late fetal period or shortly after birth (7). These placental abnormalities are similar to those found in the placentas of vitamin A-deficient rats (8). These observations suggest that the ligand-dependent transcriptional activation of RXR{alpha} is physiologically required for placentation in rodents.

RXR{alpha} mRNA and protein have been detected in human cytotrophoblasts and choriocarcinoma cells (9, 10, 11). Treatment of these cells with 9cRA and synthetic RXR-specific ligands increases the level of mRNA expression of steroidogenic enzymes, such as aromatase and human (h) chorionic gonadotropin (CG) (9, 10, 12, 13, 14, 15). Estrogens and hCG are the principal hormones produced by the placenta during human pregnancy. These hormones are essential for several important events in the establishment and maintenance of pregnancy. Biosynthesis of estrogens requires the catalytic activity of an aromatase enzyme complex, which converts androgenic to estrogenic steroids (16). The human placenta exhibits a high level of aromatase activity and therefore regulates the balance of estrogens in utero (17). Altering aromatase function in utero can cause permanent effects in human embryos; the lack of placental aromatase causes female pseudohermaphroditism, as is seen in patients with aromatase deficiency (16, 18).

hCG is a luteotropic factor and the primary marker of pregnancy in humans. Stimulation by hCG governs not only progesterone production in the corpus luteum during the first trimester (19) but also testosterone production within the fetal testes (20). Given the pivotal functional roles of aromatase and hCG in sexual development and reproduction, the extant retinoid signals of RXR-mediated transcription in the placenta may greatly alter fetal development because of their disruption of these endocrine functions.

Organotin compounds have been used widely as biocides, agricultural fungicides, wood preservatives, disinfecting agents in circulating industrial cooling waters, and antifouling paints for marine vessels (21, 22). There are many reports of the biological effects of organotin compounds, which vary in their toxic effects to eukaryotes. One of the most notable toxicities in sexual development and reproduction is that of tributyltin (TBT)- and triphenyltin (TPT)-mediated endocrine disruption in some species of gastropods (23, 24). This phenomenon is known as "imposex," the superimposition of male genitalia on female. Therefore, these trialkyltin compounds are suspected to cause endocrine-disrupting effects in mammals, including humans. Human exposure to organotin compounds may result from the consumption of organotin-contaminated meat and fish products, occupational exposure during the manufacture and formulation of organotin compounds, or the application and removal of organotin-containing paints (25, 26). The possible exposure of humans to organotins has therefore aroused great concern about potential toxicities.

Previously, we reported that both tributyltin chloride (TBTCl) and triphenyltin hydroxide (TPTOH) enhance hCG secretion and aromatase activity in human choriocarcinoma cells. In addition, these compounds cause dose-related increases in the steady-state mRNA levels of both hCGß and aromatase in human choriocarcinoma Jar cells after their exposure to nontoxic concentrations (27). These results suggest that these trialkyltin compounds are potent stimulators of human placental hCG production and aromatase activity in vitro and act as endocrine disruptors, the effects of which might alter local hCG and estrogen concentrations in pregnant women. However, the molecular mechanism underlying trialkyltin-induced alterations of human placental endocrine functions remains unclear. To extend our knowledge of the correlation between the structure of organotin compounds and their endocrine-disrupting effects, we assessed the effects of 17 tin compounds on hCG secretion, aromatase activity, and the mRNA levels of hCG and aromatase in Jar cells. We found that the effects of organotin compounds are related to both the number and length of their alkyl chains, suggesting that organotin compounds might interact with a target molecule in a fashion similar to that by which environmental estrogenic chemicals interact with estrogen receptors (28, 29, 30, 31, 32, 33, 34). Further, the promoter sequences of both human placental hCGß and aromatase have several common half-site sequences (T/AGGTCA), of which nuclear receptor response elements typically are composed (13, 14). In addition, expression of both human placental hCG and aromatase is induced by specific RXR ligands (9, 10, 13, 14). In light of all of these results, we hypothesize that organotin compounds interact with RXRs to alter placental endocrine functions. Here we demonstrate that trialkyltin compounds bind to RXRs with high affinity and stimulate transcription through these receptors to alter endocrine functions in human choriocarcinoma cells.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Effects of Organotin Compounds on hCG Production and Aromatase Activity in Human Choriocarcinoma Cells
Previously, we reported that both TBTCl and TPTOH enhance hCG secretion and aromatase activity in human choriocarcinoma cells (27). To extend our knowledge of the correlation between the structures of organotin compounds and their endocrine-disrupting effects, we assessed the effects of 17 tin compounds (Fig. 1Go) on hCG secretion from, and aromatase activity in, Jar choriocarcinoma cells after their exposure to nontoxic concentrations of these compounds, which were determined from the results of [3H]thymidine uptake assays (data not shown). The most active compounds were TBT or TPT derivatives (Fig. 2Go, Group III). Exposure to ≥ 10 nM of each of these trialkyltin compounds caused statistically significant increases in hCG production by Jar cells. Aromatase activity also increased significantly as the concentrations of the TBT derivatives increased beyond 10 nM or those of the TPT derivatives increased in excess of 30 nM. Among the other trialkyltin compounds (Group I), tripropyltin chloride (TPrTCl) and tricyclohexyltin hydroxide (TChTOH) were active. Like the TBT and TPT compounds, TPrTCl stimulated both hCG production and aromatase activity, whereas TChTOH stimulated hCG production but not aromatase activity. Among the butyltin and phenyltin derivatives (Group II), neither of the mono-alkyltin compounds altered hCG production or aromatase activity. Dibutyltin dichloride (DBTCl2) stimulated aromatase activity at 30 nM but failed to induce hCG production at any of the concentrations tested. In contrast, diphenyltin dichloride (DPTCl2) stimulated hCG production at 30 nM but not aromatase activity at any tested concentration.



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  		Fig. 1. Structures of the Tin Compounds Used in This Study

The abbreviation for, and purity of, each compound used are indicated in parentheses.

 


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  		Fig. 2. Effects of Tin Compounds on hCG Secretion (A, C, and E) and Aromatase Activity (B, D, and F) in Jar Cells

Cells were treated with various nontoxic concentrations of tin compounds for 48 h. A nontoxic concentration of a tin compound was defined as a concentration at which the uptake of [3H]thymidine was ≥ 80% that for the vehicle alone (data not shown). Results are expressed as mean ± 1 SD of triplicate cultures. Group I (A and B): comparison of different lengths of alkyl chains in trialkyltin compounds. Group II (C and D): comparison of different numbers of alkyl chains in butyltin and phenyltin compounds. Group III (E and F): comparison of different fourth function groups on the tin of TBT and TPT. The hCG production and aromatase activity in vehicle-only cells, calculated from all experiments, were 290.0 ± 85.3 mIU/well·24 h and 4.08 ± 0.91 pmol/well·4 h, respectively.

 
There were no significant differences in hCG production and aromatase activity among the TBT and TPT derivatives (Group III), suggesting that the ligand on the trialkyltin (as long as it is not an alkyl group) is relatively unimportant to the stimulation of these endocrine functions. However, the presence of a fourth alkyl group on the tin atom decreased the stimulus potency of the alkyltin compounds on hCG production and aromatase activity, because both tetrabutyltin (TeBT) and tributylvinyltin (TBVT) failed to stimulate these placental functions at doses less than 100 nM (Fig. 2Go, Group III). These results suggest that the potency of the effects induced by alkyltin compounds is related to both the number and length of the alkyl groups.

In addition, we investigated the tin compound-induced mRNA expression of hCGß and aromatase at either the concentration that elicited the greatest response in each endocrine function or the maximal nontoxic concentration. The changes in hCGß and aromatase mRNA expression were almost parallel to those in hCG secretion and aromatase activity (Fig. 3Go). These results indicate that the observed alkyltin-induced alterations in these placental functions are both caused by regulation at the mRNA level. Such overt correlation between the mRNA expression induced by alkyltin compounds and their structure led us to hypothesize that alkyltin compounds may interact with a nuclear receptor to alter placental endocrine functions; a similar mechanism has been demonstrated for environmental estrogenic chemicals that interact with estrogen receptors (28, 29, 30, 31, 32, 33, 34).



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  		Fig. 3. Effects of Tin Compounds on the mRNA Expression of hCG ß (A, C, and E) and Aromatase (B, D, and F) in Jar Cells

Total RNA isolated from Jar cells was treated with tin compounds for 24 h (open bars) and 48 h (solid bars). The concentrations of each compound were: 10 µM of TOTH, SnCl4, MBTCl3, and TBVT; 3 µM of MPTCl3 and TeBT; 1 µM of 9cRA and TMTCl; 300 nM of DPTCl2; 100 nM of TETBr, TBTCl, TPTOH, TPTCl, TBTBr, and TBTH; and 30 nM of TPrTCl, TChTOH, and DBTCl2. The relative hCGß and aromatase mRNA levels for each condition were determined by quantitative RT-PCR three times for each of the three independent cultures (see Materials and Methods). Results are expressed as mean ± 1 SD of three independent cultures. Groups I (A and B), II (C and D), and III (E and F) correspond to the groups described in the legend for Fig. 2Go. *, P < 0.05; **, P < 0.01; and ***, P < 0.005 vs. vehicle.

 
Interaction of Alkyltin Compounds with the Hormone-Binding Domain of RXRs
In human placental cells, both hCG production and aromatase activity are controlled by cAMP-dependent intracellular signal pathways. However, in our previous study, neither TBTCl nor TPTOH exerted any effect on cAMP production (27). After a literature search to identify a signaling pathway common to the mRNA expression of both hCG and aromatase, we arrived at the hypothesis that alkyltin compounds act as ligands of RXRs to activate the transcription of hCG and aromatase, because these placental factors are both induced by specific ligands of RXRs (9, 10, 13, 14). In fact, we were able to confirm that a natural RXR ligand, 9cRA, and a synthetic RXR-specific ligand, LG100268, induced mRNA expression of both hCGß and aromatase in Jar cells (Fig. 4Go).



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  		Fig. 4. Effects of Various Nuclear Receptor Agonists on the mRNA Expression of hCG ß (A) and Aromatase (B) in Jar Cells

Total RNA isolated from Jar cells was treated with tin compounds for indicated time periods. The concentrations of each compound were: 10 µM of CDCA; 100 nM of 9cRA, atRA, rosiglitazone, or 15dPGJ2; and 100 nM of LG100268 or AM580. The relative hCGß and aromatase mRNA levels for each condition were determined by quantitative RT-PCR three times for each of the three independent cultures (see Materials and Methods). Results are expressed as mean ± 1 SD of three independent cultures. *, P < 0.05; **, P < 0.01; and ***, P < 0.005 vs. vehicles.

 
Accordingly, we examined the dose-response effectiveness of TBTCl and TPTOH in stimulating RXR activity by using human choriocarcinoma JEG-3 cells cotransfected with a human RXR{alpha} expression plasmid and a luciferase (LUC) reporter plasmid containing an RXR response element (RXRE). RXR homodimers bind to direct repeat (DR)1 motifs (1, 2, 3). We constructed and used pTALDR1, which contains two DR1 sequences, as a reporter plasmid. Treatment with 1–100 nM 9cRA resulted in a 3- to 10-fold increase in LUC activity, whereas similar treatment with LG100268 led to a 3.3- to 4.5-fold increase (Fig. 5AGo). In addition, 100 nM 9cRA slightly stimulated the expression of LUC in JEG-3 cells transfected with a control plasmid, because JEG-3 cells express endogenous RXR{alpha} (10). Treatment of RXR{alpha}-transfected JEG-3 cells with 1–100 nM TBTCl stimulated LUC expression 1.5- to 9-fold, and exposing the cells to the same concentrations of TPTOH induced LUC expression 1.8- to 19-fold; these results suggest that low doses of these trialkyltin compounds activate RXR.



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  		Fig. 5. Ability of TBTCl and TPTOH to Activate RXR and RAR

A, JEG-3 cells were cotransfected with 10 ng of either pSVhRXR{alpha} or pSVSPORT1 in addition to 0.1 µg pTALDR1 (see Materials and Methods) and then treated with various concentrations of 9cRA, LG100268, TBTCl, or TPTOH. B, JEG-3 cells were cotransfected with 10 ng each of pSVhRXR{alpha} and pSV40hRARß in addition to 50 ng pTALDR2 or pTALDR5 and then treated with 100 nM of atRA, 9cRA, LG100268, TBTCl, or TPTOH. pRL-TK (2 ng) was cotransfected as the control for normalization (see Materials and Methods). The results are expressed as average fold activation ± 1 SD after normalization to Renilla LUC activity. *, P < 0.05; **, P < 0.01; and ***, P < 0.005.

 
To identify the region of RXR involved in activation by alkyltin compounds, we used a chimeric receptor consisting of the DNA-binding domain of the yeast transcription factor GAL4 and the ligand-binding domain (LBD) of RXR{alpha} (GAL-RXR). Consistent with the results in Fig. 5AGo, the activity of the LUC reporter construct in JEG-3 cells cotransfected with the GAL-RXR chimeric receptor and the UAS-LUC reporter increased in a dose-dependent manner after incubation with not only 9cRA but also either TBTCl or TPTOH (Fig. 6Go). We then investigated the effect of the other tin compounds on activation of GAL-RXR chimeric receptors after exposure of the cells to nontoxic concentrations. TPrTCl, TeBT, and TBVT, as well as the TBT and TPT derivatives, activated transcription through GAL-RXR. TChTOH also activated transcription though GAL-RXR markedly, but the level of activation was only slight compared with that induced by the TBT and TPT derivatives. The level of GAL-RXR activation induced by these tin compounds was almost parallel to the increase in mRNA expression of hCGß or aromatase in JEG-3 cells treated with these compounds.



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  		Fig. 6. Ability of Tin Compounds to Activate GAL-RXR

JEG-3 cells were cotransfected with 10 ng p4xUAS-tk-luc, 5 ng pBK-CMV-GAL4-hRXR{alpha}, and then treated with 9cRA or each of the tin compounds. The doses of each compound were: 0, 1, 10, or 100 nM of 9cRA, TETBr, TBTCl, TPTOH, TPTCl, TBTH, or TBTBr; 0, 0.1, 1, or 10 µM of TOTH, SnCl4, MBTCl3, or TBVT; 0, 1, 10, or 30 nM of TPrTCl, TChTOH, or DBTCl2; 0, 0.1, 1, or 3 µM of MPTCl3 or TeBT; 0, 10, 100, or 300 nM of DPTCl2; and 0, 10, 100, or 1000 nM of TMTCl. pRL-TK (2 ng) was cotransfected as the control for normalization (see Materials and Methods). The results are expressed as average fold activation ± 1 SD after normalization to Renilla LUC activity. Groups I, II, and III correspond to the groups described in Fig. 2Go. *, P < 0.05; **, P < 0.01; and ***, P < 0.005.

 
RXR serves as a common heterodimerization partner for several receptors (1, 2, 3). In addition to RXR homodimer, RXR-specific ligands can activate two types of complex. One type is the permissive heterodimers, such as PPAR-RXR (35, 36, 37, 38, 39) and farnesoid X-activated receptor (FXR)-RXR (40), which can be fully activated by a ligand of either RXR or its partner receptor and are activated synergistically in the presence of both ligands; the other is the conditionally permissive heterodimer RXR-RAR, which can be conditionally activated by RXR ligands only in the presence of an RAR agonist (38, 41). To identify the complexes involved in alkyltin-induced mRNA expression of both placental factors, we assessed the effects of the ligands of various RXR partners. The PPAR ligand 15-deoxy-{Delta}12,14-prostaglandin J2 (15dPGJ2) and the PPAR{gamma}-specific ligand rosiglitazone induced mRNA expression of hCGß, but not aromatase, in Jar cells. Chenodeoxycholic acid (CDCA), an FXR agonist, failed to increase the mRNA expression of either placental factor. However, a natural RAR ligand, all-trans-retinoic acid (atRA), and a synthetic RAR{alpha}-specific ligand, AM580, induced expression of the mRNA of both hCGß and aromatase in human placental cells, as previously described (15). Furthermore, the natural RXR ligand 9cRA also can function as an RAR agonist to transactivate RXR-RAR heterodimer (1, 2, 3, 42). Accordingly, we examined whether typical trialkyltin compounds, which function as RXR agonists, can activate RXR-RAR transcription, as does 9cRA, by using LUC reporter plasmids containing an RAR response element (DR2 or DR5). Cells simultaneously transfected with human RXR{alpha} and RARß expression vectors dramatically respond to atRA and 9cRA (Fig. 5BGo). Although TBTCl and TPTOH also stimulated the expression of LUC, the effectiveness of these organotin compounds was comparable to that of LG100268, which does not transactivate RXR-RAR heterodimer (Fig. 5BGo). In addition, we examined the effect of TBTCl and TPTOH on activation of RAR{alpha}, -ß, and -{gamma} by using chimeric receptors in which the GAL4 DNA binding domain was fused to the LBD of RARs (GAL-RARs) instead of RXR{alpha}. Neither alkyltin compound activated transcription through GAL-RARs (data not shown). These results suggest that the alkyltin compounds, which activate transcription of the reporter construct through GAL-RXR, interact with the LBD of RXR and transactivate RXR homodimer, but not RXR-RAR heterodimer, unlike 9cRA. Taken together, these results suggest that RXR homodimer may be involved in the alkyltin-induced mRNA expression of both placental factors, provided that the hCGß induced also involves PPAR-RXR heterodimers.

Binding of Tin Compounds to RXR
To characterize the binding affinities of these tin compounds to RXR directly, we performed analyses of the saturation binding of [14C]TPTOH and [3H]9cRA to chimeric receptors, which consisted of glutathione S-transferase (GST) fused to the LBD of human RXRs (GST-RXRs). The binding of 9cRA to GST-RXRs was specific and saturative (Fig. 7Go). Scatchard analyses of the binding of [3H]9cRA to GST-RXR{alpha}, -ß, and -{gamma} yielded dissociation constant (Kd) values of 11.7, 15.5, and 8.66 nM, respectively. These Kd values were similar to those previously reported (3, 42), suggesting that this system is useful for determining the binding affinity of alkyltin compounds to RXRs. Scatchard analyses of the binding of [14C]TPTOH to RXR{alpha}, -ß, and -{gamma} yielded Kd values of 55.5, 241, and 95.3 nM, respectively (Fig. 7Go). Although the Kd values of TPTOH for RXRs were approximately 5- to 15-fold higher than those for 9cRA, our results indicate that TPTOH binds to the RXRs with high affinity in a saturable and specific manner.



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  		Fig. 7. Saturation Kinetics for the Binding of [3H]9cRA and [14C]TPTOH to hRXR{alpha}, -ß, and -{gamma}

Specific binding (solid square) is defined as total binding (solid circle) minus nonspecific binding (solid triangle). Scatchard analysis was performed on specific binding data (triplicates at each point) to yield the indicated dissociation constants (Kd value) for each receptor.

 
To further test which of the other tin compounds might bind to RXR as a ligand, we performed competitive ligand-binding assays. We measured the ability of [3H]9cRA or [14C]TPTOH to compete with the tin compounds for binding to GST-RXR{alpha}. The TBT and TPT derivatives all competed with [3H]9cRA for binding to GST-RXR{alpha} in a concentration-dependent manner (Fig. 8Go). Consistent with the Kd value of TPTOH for RXR{alpha}, the IC50 value of TPTOH for binding [3H]9cRA was approximately 5-fold higher than that of unlabeled 9cRA. The IC50 values of the other TBT and TPT derivatives for binding [3H]9cRA were almost the same as that of TPTOH, demonstrating that these trialkyltin compounds bind directly to RXR. In contrast to the results of competitive assay for [3H]9cRA, 9cRA failed to completely out compete [14C]TPTOH for binding to GST-RXR{alpha}, whereas the TBT and TPT derivatives successfully competed for binding (Fig. 8BGo). The IC50 value of 9cRA for binding [14C]TPTOH was more than 10-fold higher than those of TBTs and TPTs (Table 1Go). Further, TPrTCl bound to GST-RXR{alpha} as well as did the TBT and TPT derivatives, because its IC50 values for binding [3H]9cRA and [14C]TPTOH were almost the same as those of the TBT and TPT derivatives (Fig. 8Go and Table 1Go). However, TChTOH, TeBT, and TBVT failed to compete with [3H]9cRA for binding to GST-RXR{alpha}, despite their ability to activate RXR{alpha}. Although TChTOH out competed [14C]TPTOH for binding to GST-RXR{alpha}, TeBT only slightly out competed [14C]TPTOH, and TBVT failed to compete with [14C]TPTOH for binding to GST-RXR{alpha} (Table 1Go). By contrast, triethyltin bromide (TETBr), which was unable to activate transcription through an RXR, bound weakly to GST-RXR{alpha}. The IC50 values of TETBr for binding [3H]9cRA and [14C]TPTOH were approximately 25- to 50-fold higher than those of the TBT and TPT derivatives (Table 1Go). The remaining tin compounds (trimethyltin chloride, TMTCl; trioctyltin hydride, TOTH; butyltin trichloride, MBTCl3; phenyltin trichloride, MPTCl3; DBTCl2; DPTCl2; and SnCl4) did not compete successfully with either [3H]9cRA or [14C]TPTOH for binding to GST-RXR{alpha}, suggesting that they were unable to bind to RXRs.



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  		Fig. 8. Competition by 9cRA and Alkyltin Compounds with [3H]9cRA (A) and [14C]TPTOH (B) for Binding to the LBD of hRXR{alpha}

The LBD of hRXR{alpha} protein was incubated with increasing concentrations of unlabeled 9cRA or alkyltin compounds as competitors in the presence of [3H]9cRA or [14C]TPTOH as ligand. Results are expressed as percent specific binding. Each experiment was performed at least twice, and representative curves are shown.

 

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  		Table 1. IC50 of Tin Compounds for Competition of [3H]9cRA and [14C]TPTOH Binding to hRXR{alpha}, and EC50 of Tin Compounds for GAL-RXR Reporter Assay

 
Trialkyltin Compounds Stimulate the Expression of an LUC Construct Containing the Human Placental I.1 Sequence of Aromatase via Activation of RXR{alpha}
To determine whether trialkyltin compounds, which bind to and activate RXRs, induce aromatase expression in the human placenta via ligand-dependent activation of RXRs, we assessed the dose-response effectiveness of TBTCl and TPTOH by using JEG-3 cells cotransfected with a human RXR{alpha} expression plasmid and a LUC reporter plasmid containing the promoter sequence (–2295 to +107 bp) of exon I.1, which is the major promoter of human placental aromatase (Fig. 9AGo; and Refs.16, 43 , and 44). As expected, LG100268 stimulated the expression of LUC by 2.4- to 3.5-fold. These results suggest that the aromatase promoter is regulated by ligand-bound RXR (13). In addition, TBTCl and TPTOH stimulated the expression of LUC in a dose-dependent manner to levels approaching those induced by 9cRA and LG100268.



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  		Fig. 9. Ability of TBTCl and TPTOH to Increase Transcription of an LUC Reporter Gene Containing the Human Placental Promoter I.1 Sequence of Aromatase via the Activation of RXR but not RAR

A, JEG-3 cells were cotransfected with 10 ng of either pSVhRXR{alpha} or pSVSPORT1 in addition to 50 ng PGVArom and then treated with various concentrations of 9cRA, LG100268, TBTCl, or TPTOH. B, JEG-3 cells were cotransfected with 10 ng pSVhRXR{alpha} or pSV40hRARß, or both, in addition to 50 ng PGVArom, and then treated with 100 nM of atRA, 9cRA, LG100268, TBTCl, or TPTOH. pRL-TK (2 ng) was cotransfected as the control for normalization (see Materials and Methods). The results were expressed as average fold activation ± 1 SD after normalization to Renilla LUC activity. *, P < 0.05; **, P < 0.01; and ***, P < 0.005. NS, Not significant (P ≥ 0.05).

 
Because RAR agonists induced mRNA expression of aromatase (Fig. 4BGo), we examined the effectiveness of cotransfection with a human RARß expression plasmid in stimulating the LUC activity by these alkyltin compounds to identify whether RAR is involved in alkyltin-induced aromatase expression (Fig. 9BGo). In the presence of the natural RAR ligands, atRA or 9cRA, RAR statistically significantly increased the expression of LUC (P < 0.05 or P < 0.01), whereas RAR had no effect on the LUC activity induced by LG100268, TBTCl, or TPTOH (Fig. 9BGo). These results suggest that the expression of human placental aromatase induced by these alkyltin compounds is involved in a ligand-dependent signaling pathway of RXRs but not in transactivation of RXR-RAR.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Although our previous study of human placental cells showed that TBTCl and TPTOH enhance hCG secretion and aromatase activity with an accompanying increase in the mRNA expression of each factor (27), the underlying molecular mechanism had remained unclear. In our previous study, we examined the effect of TBTCl and TPTOH on cAMP concentrations in human choriocarcinoma cells, because hCG production and aromatase activity in the human placenta are both well known to be controlled by cAMP-dependent intracellular signal pathways (16, 17, 45, 46). However, neither of these trialkyltin compounds altered cAMP production (27). We then speculated that activation of RXRs is a common signaling pathway of alkyltin-stimulated hCG production and aromatase activity, because both of these events are induced by specific ligands of RXRs (Fig. 4Go and Refs.9, 10, 13 , and 14). In our present study, we provide evidence that trialkyltin compounds stimulate the transcription of RXRs because of a high-affinity interaction with the LBD of the receptor. In addition, trialkyltin compounds stimulate the expression of an LUC reporter construct containing the human placental promoter I.1 sequence of aromatase via a ligand-dependent signaling pathway of RXR.

In humans, the tissue-specific expression of aromatase is strictly regulated. Human aromatase is a single-copy gene composed of 10 exons: exons II through X encode the aromatase protein as well as the 3'-untranslated region of mRNA common to all estrogen-producing tissues (16). There are a number of variants of exon I. These encode the 5'-untranslated regions of various aromatase mRNAs, which are selectively expressed in some tissues by alternative splicing (16, 43, 44). The tissue-specific expression of aromatase in humans appears to be mediated by tissue-specific promoters lying upstream of the respective exon I sequences and by the binding of transcription factors to specific regions of each promoter. In the placenta, aromatase is driven by the placental major promoter (I.1), and the transcript contains exon I.1. In contrast to our results, Saitoh et al. (47) recently reported that TBT inhibits aromatase activity and decreases mRNA levels in ovarian granulosa cells. They also suggested that TBT-induced suppression of aromatase in the cells is partly regulated at the transcriptional level because of association with the cAMP-protein kinase A pathway or regulation by the steroidogenic tissue-specific transcriptional factor adrenal 4 binding protein/steroidogenic factor 1. In contrast to those in the placenta, ovarian transcripts of aromatase contain a 5' sequence immediately upstream of the translation start site, because expression of the gene in the ovary uses a proximal promoter (II) that is strongly regulated by adrenal 4 binding protein/steroidogenic factor 1 and cAMP. In addition, RXR and PPAR{gamma} ligands suppress the mRNA expression of aromatase in ovarian granulosa cells (48, 49). Therefore, in light of our findings, TBT-induced suppression of aromatase in ovarian granulosa cells may involve RXR activation.

The RXRs stand out as unique members of the type II nuclear receptor subfamily and play a dual role in nuclear receptor signaling. On one hand, they can bind to their own response element (DR1) as a homodimer and activate transcription in response to their ligands, and, on the other hand, they serve as partners for other nuclear receptors (1, 2, 3). Trialkyltin compounds bind to RXRs to induce their transcription. In turn, the expression of hCG and aromatase induced by these compounds may involve either RXR-homodimer or -heterodimer, or both. The existence of three types of heterodimers—nonpermissive, conditionally permissive, and fully permissive—has been described. Nonpermissive heterodimers include RXR-TR and RXR-VDR, which cannot be activated by RXR agonist regardless of the presence (or absence) of the agonist of its partner receptor; formation of the heterodimer is thought to preclude the binding of ligand to RXR (50, 51). RXR-ligand-dependent transcription in promoter I.1 of human aromatase is reported to be regulated by RXR-VDR heterodimers, owing to binding to the imperfect palindromic sequence located from –183 to –172 bp upstream of the transcriptional start site (13). However, within these complexes, RXR acts as a silent partner, as described earlier (50). In addition, we used a GAL-VDR chimeric receptor to confirm that TBT and TPT could not activate transcription of VDR (data not shown). Consequently, both alkyltin-induced aromatase activity and mRNA expression may not involve the association of RXR-VDR. Although RXR-TR heterodimer generally is believed to be nonpermissive (51), Castillo et al. (52) recently demonstrated that RXR-TR heterodimer can function as a permissive heterodimer to allow 9cRA-induced stimulation of prolactin gene transcription in rat pituitary cells. Accordingly, we used a synthetic DR4 reporter gene to examine whether TBTCl and TPTOH stimulate transcription of RXR-TR heterodimer. However, these trialkyltin compounds had no effect on transactivation of these complexes in the presence or absence of T3. We then used a GAL-TR chimeric receptor to confirm that TBT and TPT could not activate transcription of TR (data not shown). These results suggest that these trialkyltin-induced transcriptional activities also do not involve the association of RXR-TR.

As an example of the second type of heterodimer, the RXR-RAR heterodimer exhibits conditional permissivity because full response to RXR agonist occurs only in the presence of an RAR agonist (38, 41). Our results showed TBT and TPT function as RXR agonists but not RAR agonist. Although RXR-RAR heterodimer generally is believed to be nonpermissive in the absence of RAR agonist, LG100754, which binds to RXRs but not RARs, strongly transactivates this heterodimer pair (53). In addition, the mRNA expression of both hCG and aromatase is also induced by RAR-specific agonist (Fig. 4Go), and it is possible that in light of the result in Fig. 9BGo, the transcription from promoter I.1 of human aromatase may be regulated by RXR-RAR heterodimers. However, TBTCl and TPTOH failed to transactivate RXR-RAR heterodimer on either RAR response element (DR2 and DR5) reporter elements or promoter I.1 elements of human aromatase (Figs. 5BGo and 9BGo). These results suggest that, unlike the effect of LG100754, these trialkyltin-induced transcriptional activities do not involve the association of RXR-RAR.

The third type is the permissive heterodimers, such as PPAR-RXR and FXR-RXR, which exhibit dual ligand permissivity, because they can be activated by the agonists of either RXR or its partner receptor, or both, in a more-than-additive fashion (35, 36, 37, 38, 39, 40). The PPAR ligand 15dPGJ2, PPAR{gamma}-specific ligand rosiglitazone, and FXR ligand CDCA all failed to increase mRNA expression of aromatase in Jar cells (Fig. 4BGo), suggesting that neither PPAR-RXR nor FXR-RXR heterodimers are involved in organotin-induced aromatase expression in the human placenta and that RXR homodimer may be required for organotin-induced aromatase expression. By contrast, PPAR agonists, in addition to RXR and RAR agonists, stimulate mRNA expression of hCGß, as previously described (Fig. 4Go and Refs.9, 10, 12 , and 14). These findings indicate that organotin-induced hCGß expression might involve either PPAR-RXR heterodimers or RXR homodimer.

To address these possibilities, we constructed a LUC reporter plasmid containing the promoter sequence (–455 to +365 bp) of hCGß5, which is the predominant hCGß subunit expressed in the human placenta, and assessed the effectiveness of TBTCl and TPTOH in stimulating LUC activity by using JEG-3 cells cotransfected with a human RXR{alpha} expression plasmid and the hCGß5-LUC reporter plasmid. However, trialkyltin compounds, RXR, and PPAR ligands failed to stimulate LUC expression (data not shown), whereas cAMP analogs stimulated gene expression, as previously described (45). Furthermore, Tarrade et al. (14) reported that ligand-dependent mRNA expression of hCGß is transcriptionally controlled by PPAR{gamma}-RXR heterodimers, which bind to DR1 as well as does RXR homodimer. However, like us, they failed to detect expression of reporter gene constructs containing imperfect DR1 motifs in the regulatory region of the hCGß gene. Transcriptional regulation in promoter I.1 of human aromatase and the hCGß promoter is not yet fully understood, and neither the PPAR response element nor the RXRE involved in both promoter activation by RXR ligands has been identified. Further studies are needed to clarify the precise mechanism of action of RXRs in the expression of human placental aromatase and hCG, because the ligand-dependent signaling pathways of RXRs appear intricate.

We assayed 15 tin compounds, in addition to TBTCl and TPTOH, for their ability not only to induce hCG production, aromatase activity, and mRNA expression of both factors but also to activate RXR through binding to the LBD of the receptor. hCG production and aromatase activity did not differ significantly among the TBT and TPT derivatives. In addition, the abilities to bind to the LBD of RXR and activate the receptor were similar among these compounds, because they all competed with both [3H]9cRA and [14C]TPTOH for binding to RXR approximately as well as did TBTCl and TPTOH. These results suggest that the exact identity of the ligand on the trialkyltin (as long as it is not another alkyl group) is relatively unimportant for binding to RXR.

By contrast, approximately 50- to 100-fold higher concentrations of tetraalkyltin compounds such as TeBT and TBVT were needed to elicit a response, compared with those of the TBT and TPT derivatives. In addition, although the tetraalkyltin compounds stimulated transcription through RXR, they hardly competed with [3H]9cRA for binding to the LBD of RXR. This observation may indicate that the tetraalkyltin compounds were metabolically converted to the active form in the cells. This hypothesis is supported by the general trend of the previous results showing that organotin compounds undergo dealkylation by the microsomal monooxygenase system, which is dependent on cytochrome P450 in the liver and other organs (54, 55, 56). The presence of a fourth alkyl group on the tin atom may interfere with the binding of alkyltin compounds to RXR, and activation of the receptor by these tetraalkyltin compounds may be the result of their metabolic conversion in cells to the active dealkylated form (e.g. TBT). Such events are reminiscent of an early observation that atRA could activate RXR in cells because of its metabolic conversion to the high-affinity ligand 9cRA (57). Although the dialkyltin compounds neither bind to nor activate RXR, DBTCl2 and DPTCl2 induced expression of the mRNA of aromatase and hCGß, respectively. It remains unclear why these dialkyltin compounds induced expression of the mRNA of aromatase or hCGß, but the induction appears to be caused by a mechanism other than activation of RXRs.

Among the trialkyltin compounds other than TBT and TPT derivatives, TPrTCl was most active. TPrTCl activated transcription of RXR as well as did 9cRA and, like TBTCl and TPTOH, completely out competed both [3H]9cRA and [14C]TPTOH for binding to RXR. TETBr bound weakly to RXR, but we were unable to detect TETBr-induced transcription of RXR and mRNA expression of hCGß and aromatase. The fact that TETBr is cytotoxic at concentrations greater than 300 nM, according to the result of the [3H]thymidine uptake assay (data not shown), may render TETBr-stimulated RXR activation undetectable. TChTOH, which activated transcription of RXR, completely out competes [14C]TPTOH for binding to the LBD of RXR, whereas it cannot out compete [3H]9cRA at all. This difference in the ability of TChTOH to compete with [3H]9cRA and [14C]TPTOH may be caused by differences in their ligand-protein contacts (see following paragraph). These results suggest that the affinity of tin compounds to RXR is related to both the numbers and lengths of their alkyl groups.

RXR has been characterized as a nuclear receptor that demonstrates a highly restricted substrate specificity. Until recently, 9cRA was defined as the most potent RXR activator. However, various fatty acids, such as docosahexaenoic acid (DHA) and phytanic acid, and methoprene acid, a synthetic juvenile hormone analog used as an insect regulator, have been identified as RXR ligands (58, 59, 60, 61, 62). In addition, several retinoids with RXR selectivity have been developed (63), because retinoids are important therapeutic agents in the treatment of cancer and proliferative diseases of the skin. Although fatty acid- or methoprene acid-induced RXR activation requires greater than 1000-fold higher concentrations than that induced by 9cRA (58, 59, 60, 61, 62), the protein-ligand interactions of almost all RXR ligands share several common characteristics. Recently, Egea et al. (64) have obtained some interesting findings through analyzing the crystal structures of RXR with DHA, the synthetic ligand BMS649, and 9cRA. For example, RXR ligands contain a carboxylate group, which is important in their ability to be buried stably in the predominantly hydrophobic pocket. This functional group is involved in an ionic interaction with the strictly conserved basic residue R316 of helix H5 and forms a hydrogen bond with the backbone carbonyl amide group of the ß-turn residue A327. Furthermore, ligand atoms C14–C22 of DHA and the tetrahydrotetramethylnaptho group of BMS649 occupy the same hydrophobic cavity (delineated by helices H3, H7, and H11), in which the ß-ionone ring of 9cRA is buried stably. However, although trialkyltin compounds bind RXR with high specificity and induce RXR activation at doses similar to those of 9cRA, they lack a carboxylate group. Further, except for the TPT derivatives and TChTOH, RXR-stimulating trialkyltin compounds also lack sufficiently long fatty acid and cyclic functional groups, both of which might be buried in the ß-ionone binding subpocket. In the competitive ligand-binding assay, RXR-stimulating trialkyltin compounds, except for TChTOH, completely out competed both [3H]9cRA and [14C]TPTOH, whereas 9cRA could not completely out-compete [14C]TPTOH for binding to RXR. In addition, TChTOH out competed [14C]TPTOH but not [3H]9cRA. Together, these results suggest that the protein-ligand interaction of trialkyltin compounds and RXR is very different from those seen with other RXR ligands. Indeed, previous studies have reported that, despite the overall similarity of protein-ligand interactions, RXR ligands differ, especially within the L-shaped binding pocket. Although further studies are necessary to clarify which amino acid of RXR is important to the binding of trialkyltin compounds to the ligand-binding pocket, the ligand-protein contacts of these trialkyltin compounds are probably unique to them.

To our knowledge, ours is the first study to clarify the molecular mechanism of trialkyltin-induced endocrine-disrupting effects in the human placenta. Through RXR activation, trialkyltin compounds may be potent endocrine disruptors of other human tissues, because these compounds alter the endocrine functions, differentiation, and other processes of several human cell types (21, 47, 65, 66, 67). Furthermore, we demonstrated that trialkyltin compounds function as RXR ligands with novel structures, which bind to the LBD of RXR with high affinity and stimulate transcription of the receptor. We believe that our results provide information useful in the design of novel RXR ligands.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Chemicals and Cell Culture
TBTCl, TPTOH, TOTH, and DBTCl2 were obtained from Tokyo Kasei Kougyo (Tokyo, Japan). SnCl4, TMTCl, TETBr, TChTOH, MBTCl3, MPTCl3, DPTCl2, TPTCl, TBTH, TBTBr, TeBT, and TBVT were obtained from Aldrich Chemicals (Milwaukee, WI). 9cRA, atRA, AM580, and CDCA were obtained from Sigma Chemical Co. (St. Louis, MO). 15dPGJ2 and rosiglitazone were obtained from Cayman Chemical (Ann Arbor, MI). TPrTCl was obtained from Merck (Darmstadt, Germany). LG100268 (>95% pure) was synthesized in the Medical Chemistry Laboratories of Fujisawa Pharmaceutical (Osaka, Japan). Human choriocarcinoma cell lines Jar and JEG-3 were obtained from American Type Culture Collection (ATCC; Manassas, VA). Jar cells (ATCC No. HTB-144) were cultured in RPMI 1640 medium with 2 mM L-glutamine, 1 mM pyruvate, 4.5 g/liter glucose, and 10% fetal calf serum (FCS). JEG-3 cells (ATCC no. HTB-36) were cultured in MEM with 2 mM L-glutamine, 0.1 mM MEM nonessential amino acid solution (Invitrogen, Carlsbad, CA), and 10% FCS. To determine the effect of tin compounds on hCG secretion, aromatase activity, and mRNA expression of Jar cells, the cells were seeded and precultured for 24 h and then treated with either various concentrations of tin compounds in 0.1% dimethyl sulfoxide (DMSO) or vehicle alone (0.1% DMSO) for an additional 24 or 48 h. In control experiments, 0.1% DMSO did not alter hCG secretion, aromatase activity, mRNA expression of hCGß and aromatase, or the results of reporter gene assays in any of the cell lines examined.

Determination of hCG Production in Culture Media
hCG production was assessed as previously described (27). Jar cells were seeded in 48-well plates (4 x 104 cells per well) in regular culture medium supplemented with 5% charcoal-stripped FCS instead of 10% normal FCS. After 24 h, cells were treated with various tin compounds for 48 h. To determine hCG production, the cells were then washed and cultured in fresh medium for another 24 h. Culture supernatant was collected, and hCG concentration was determined by ELISA. Microtiter ELISA plates were coated with 5 µg/ml rabbit polyclonal antibody against intact hCG in 0.05 M sodium bicarbonate, 0.02 M sodium carbonate buffer (pH 9.6) overnight at 4 C. They were blocked for 2 h at room temperature with 1% (wt/vol) gelatin in PBS, washed with 0.05% (vol/vol) Tween 20 in PBS (TPBS), and incubated for 2 h at 37 C with 50 µl collected test samples. After being washed three times with TPBS, the plates then were incubated for 2 h at 37 C with 1:1000 mouse monoclonal antibody against the ß-subunit of hCG. After being washed with TPBS, the plates were incubated for an additional 2 h at 37 C with 1:1000 rabbit antimouse IgG1 antibody conjugated with horseradish peroxidase (Zymed Laboratories, Inc., South San Francisco, CA). The plates then were washed with TPBS and developed using 2.5 mM 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (Sigma) in 0.1 M citrate buffered solution containing 0.015% H2O2. The reaction was stopped by the addition of 0.1% NaN3, after which the plates were read at a wavelength of 415 nm in a microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA). The level of hCG in the culture supernatant was calculated from a standard curve by using Microplate Manager III software (Bio-Rad). The standardized hCG was a kind gift from Teikoku Hormone Manufacturing (Tokyo, Japan).

Aromatase Assay
Aromatase activity was determined as previously described (27). Jar cells were seeded in 12-well plates (1.5 x 105 cells per well) in regular culture medium supplemented with 5% charcoal-stripped FCS instead of 10% normal FCS. After 24 h, cells were treated with various tin compounds for 48 h. At the end of each treatment, cells were washed three times with PBS. Aromatase activity then was determined by tritium release assay. This method measures production of 3H2O, which forms as a result of aromatization of the substrate [1ß-3H]androst-4-ene-3,17-dione (New England Nuclear, Boston, MA). Serum-free medium containing [1ß-3H]androst-4-ene-3,17-dione solution (54 nM) was prepared, and 0.5 ml of this solution was added to each well. In addition, wells containing media alone were tested to provide control values for aromatase activity. After incubation for 1 h, each plate was placed on ice, and 200 µl of culture medium was withdrawn from each well. The medium was extracted with 500 µl chloroform, vortexed, and then centrifuged for 1 min at 9000 x g. A 100-µl aliquot of the aqueous phase was mixed with 100 µl of a 5% wt/vol charcoal 0.5% wt/vol dextran T-70 suspension, vortexed, and then incubated for 10 min. After centrifugation of the solution for 5 min at 9000 x g, a 150-µl aliquot was removed to measure the level of radioactivity by liquid scintillation.

Quantitative RT-PCR
Jar cells were seeded in 100-mm tissue culture plates (1 x 106 cells) and treated with various compounds in regular culture medium supplemented with 5% charcoal-stripped FCS instead of 10% normal FCS, after which total RNA was extracted from them using TRIzol reagent (Invitrogen). mRNA expression of hCGß and aromatase in Jar cells was determined by quantitative RT-PCR, as previously described (27). Total RNA (5 µg) extracted from Jar cells was reverse transcribed using SuperScript II reagent (Invitrogen) and oligo-(dT) as primer. The reaction was performed by incubation for 1 h at 42 C. After termination of cDNA synthesis, reaction mixtures were diluted with 4 volumes of Tris-EDTA. Aliquots (2 µl) of diluted reverse transcribed products were amplified in a reaction mixture containing 2x buffer from the QuantiTect SYBR Green PCR kit (QIAGEN, Valencia, CA) and 0.5 µM of each primer by using LightCycler (Roche Diagnostics, Mannheim, Germany). After preincubation at 95 C for 15 min, PCR was performed with 35–40 cycles of denaturation at 95 C for 15 sec, annealing at 65 C for 30 sec, and elongation at 72 C for 10 sec. Primers used were as follows: human aromatase, 5'-CCGGCCTTGTTCGTATGGTCA-3' and 5'-CAACACGTCCACATAGCCCGA-3'; hCGß, 5'-CCGTGTGCATCACCGTCAACA-3' and 5'-GTTGCACACCACCTGAGGCAG-3'; and human ß-actin, 5'-CTACGAGCTGCCTGACGGC-3' and 5'-GCCACAGGACTCCATGCCC-3'.

Plasmid Construction
Human RXR{alpha} and RARß cDNAs were amplified by RT-PCR using total RNA from JEG-3 cells. The amplified RXR{alpha} fragment was cloned into pSVSPORT1 (Invitrogen), whereas the RARß fragment was cloned into a simian virus 40 promotor-containing expression vector. The resulting RXR{alpha} and RARß expression vectors were termed pSVhRXR{alpha} and pSV40hRARß, respectively. A 2.4-kb promoter I.1 sequence of the human aromatase gene (–2295 to +107 bp) was PCR amplified from JEG-3 genomic DNA. KpnI and SmaI sites were introduced into the 5'- and 3'-termini, respectively, of the amplified fragment, which then was subcloned into the KpnI–SmaI site of PGVB2 (Nippon Gene, Tokyo, Japan); the resulting reporter construct was termed PGVArom.

To construct a reporter plasmid containing an RXRE and RAR response element, response elements were cloned into the SmaI site of pTAL-Luc (CLONTECH Laboratories, Inc., Palo Alto, CA); response elements with the underlined consensus hexanucleotide sequence were as follows: DR1 x 2 (5'-AGGTCA a AGGTCA a AGGTCA a AGGTCA-3'); DR2 x 2 (5'- aa AGGTCA aa AGGTCA ccatcccgggaaa AGGTCA aa AGGTCA cc-3'); DR5 x 2 (5'-aa AGGTCA ccgaa AGGTCA ccatcccgggaaa AGGTCA ccgaa AGGTCA cc-3'); the resulting reporter constructs were termed pTALDR1, pTALDR2, and pTALDR5, respectively. The LBDs of hRXR{alpha} (codons 201–693), hRXRß (codons 275–534), and hRXR{gamma} (codons 172–455) were amplified by RT-PCR using mRNA from human liver and kidney and subcloned into pGEX-4T (Amersham Biosciences, Piscataway, NJ). These constructs were used for generation of glutathione S-transferase (GST)-hRXR fusion proteins. For chimeric receptor assay, the LBD of hRXR{alpha} was fused to the C-terminal end of GAL4-DNA binding domain (amino acids 1–147) in the pBK-CMV expression vector (Stratagene, La Jolla, CA) to yield pBK-CMV-GAL4-hRXR{alpha}. All sequences synthesized by PCR were confirmed by DNA sequencing. The plasmid p4xUAS-tk-luc, a LUC reporter construct containing four copies of the GAL4 binding site [upstream activating sequence (UAS) of GAL1] followed by a thymidine kinase promoter, was a kind gift from Dr. Y. Kamei (National Institute of Health and Nutrition, Japan).

Transient Transfection Assay
Transfection was performed with Lipofectamine regent (Invitrogen) in accordance with the manufacturer’s instructions. JEG-3 cells (3 x 104 cells) were seeded in 24-well plates 24 h before transfection with the optimal dose of each DNA construct. At 18 h after transfection, various compounds were added to the transfected cells, which were then cultured in regular culture medium supplemented with 1% charcoal-stripped FCS instead of 10% normal FCS. The cells were harvested 30 h later, and extracts were prepared and assayed for LUC activity by using the dual-LUC reporter assay system (Promega Corp., Madison, WI) in accordance with the manufacturer’s instructions. To normalize LUC activity for transfection and harvesting efficiency, the Renilla LUC control reporter construct pRL-TK (Promega) was cotransfected as an internal standard in all reporter experiments. The results are expressed as the average relative LUC activity of at least quadruplicate samples.

Ligand Binding Assay
The GST-RXR fusions were expressed in Escherichia coli DH5{alpha} cells and purified according to the manufacturer’s (Amersham Biosciences) instructions. The purified proteins (30 µg/ml) were incubated with increasing concentrations of either [3H]9cRA (1.63 tBq/mmol, Amersham Biosciences) or [14C]TPTOH (2.04 gBq/mmol, Amersham Biosciences) with or without a 100-fold molar excess of each unlabeled compound. After incubation at 4 C for 1 h, specific binding was determined by hydroxyapatite binding assay as described elsewhere (68). Binding in the presence of a 100-fold molar excess of unlabeled ligand was defined as nonspecific binding; specific binding was defined as total binding minus nonspecific binding. Similarly, tin compounds were used to compete for [3H]9cRA and [14C]TPTOH in this assay to determine the binding preferences of RXRs.

Statistics
Data were analyzed by the two-tailed unpaired Student’s t test by using SPSS software (SPSS, Inc., Chicago, IL). Control and treatment group data were always obtained from equal numbers of replicate experiments. Values with P < 0.05 were considered statistically significant.


   ACKNOWLEDGMENTS
 
We thank Dr. Y. Kamei (National Institute of Health and Nutrition, Tokyo, Japan) for providing the plasmid p4xUAS-tk-luc and Teikoku Hormone Manufacturing (Tokyo, Japan) for the standardized hCG for ELISA.


   FOOTNOTES
 
This work was supported in part by grants from a Grant in Aid for Scientific Research (nos. 13470499 and 15201012) from the Ministry of Education, Science, Sports, and Culture of Japan; Industrial Technology Research Grant Program in 2001 from New Energy and Industrial Technology Development Organization of Japan; Health and Labor Sciences Research Grants (Research on Advanced Medical Technology) from the Ministry of Health, Labor and Welfare of Japan; and The Long-Range Research Initiative, Japan.

First Published Online June 7, 2005

Abbreviations: atRA, All trans-retinoic acid; CDCA, chenodeoxycholic acid; CG, chorionic gonadotropin; 9cRA, 9 cis-retinoic acid; 15dPGJ2, 15-deoxy-{Delta}12,14-prostaglandin J2; DBTCl2, dibutyltin dichloride; DHA, docosahexaenoic acid; DMSO, dimethyl sulfoxide; DPTCl2, diphenyltin dichloride; DR, direct repeat; FCS, fetal calf serum; FXR, farnesoid X-activated receptor; GST, glutathione S-transferase; LBD, ligand-binding domain; LUC, luciferase; MBTCl3, butyltin trichloride; MPTCl3, phenyltin trichloride; PPAR, peroxisome proliferator-activated receptor; RAR, retinoic acid receptor; RXR, retinoid X receptor; RXRE, RXR response element; TBT, tributyltin; TBTBr, tributyltin bromide; TBTCl, tributyltin chloride; TBTH, tribulytin hydride; TBVT, tributylvinyltin; TChTOH, tricyclohexyltin hydroxide; TeBT, tetrabutyltin; TETBr, triethyltin bromide; TMTCl, trimethyltin chloride; TOTH, trioctyltin hydride; TPBS, Tween 20-PBS; TPrTCl, tripropyltin chloride; TPT, triphenyltin; TPTCl, triphenyltin chloride; TPTOH, triphenyltin hydroxide; TR, thyroid hormone receptor; VDR, vitamin D receptor.

Received for publication October 7, 2004. Accepted for publication June 1, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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NURSA Molecule Pages Link:

Nuclear Receptors:   RXRα  |  RXRβ  |  RXRγ
Ligands:   LGD 100268  |  Am 580  |  9-cis-Retinoic acid  |  15-Deoxy-Δ12 14 prostaglandin  |  Rosiglitazone



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