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Endocrinology Vol. 143, No. 6 2208-2215
Copyright © 2002 by The Endocrine Society

RECEPTORS

Endocrine Disrupter Bisphenol A Induces Orphan Nuclear Receptor Nur77 Gene Expression and Steroidogenesis in Mouse Testicular Leydig Cells

Kwang-Hoon Song, Keesook Lee and Hueng-Sik Choi

Hormone Research Center, Chonnam National University, Kwangju 500-757, Republic of Korea

Address all correspondence and requests for reprints to: Hueng-Sik Choi, Ph.D., Hormone Research Center, Chonnam National University, Kwangju, 500-757, Republic of Korea. E-mail: . hsc{at}chonnam.ac.kr


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The orphan nuclear receptor Nur77 (NR4A1) is a member of the nuclear receptor superfamily, which plays an important role in the regulation of LH-mediated steroidogenesis in testicular Leydig cells. The aim of the current study was to investigate the potential role of bisphenol A (BPA) on orphan nuclear receptor Nur77 gene expression and steroidogenesis. Northern blot analysis demonstrated that BPA transiently induced Nur77 mRNA expression, and protein kinase inhibitor H-89 and PD98059 strongly inhibited the induction of BPA-mediated Nur77 gene expression in mouse Leydig tumor cell line, K28. Moreover, BPA increased the activation of mitogen-activated protein kinase. Transient transfection assay demonstrated that BPA increased Nur77 gene promoter activity and Nur77 transactivation, whereas BPA did not significantly affect the interaction of Nur77 with its corepressor. Furthermore, BPA increased progesterone biosynthesis in K28 cells, which was suppressed by overexpression of dominant negative Nur77. Finally, BPA injection to prepubertal mice revealed that the expression of Nur77 mRNA was elevated, and this induction was correlated with increased concentration of testicular T in vivo. Taken together, these results demonstrated that BPA induces Nur77 gene expression and subsequently alters the steroidogenesis in testicular Leydig cells. These observations provide a novel mechanism by which BPA acts as an endocrine disrupting chemical.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ENDOCRINE DISRUPTING CHEMICALS (EDCs) have been implicated to alter the development as well as the reproductive and endocrine systems (1, 2, 3, 4, 5). Bisphenol A (4,4'-isopropylidenediphenol; BPA), one of the well-known EDCs, is a monomer in polycarbonate plastics and a constituent of epoxy and polystyrene resins that are used extensively in the food-packaging industry and dental sealants (6, 7, 8). It has been shown that the in vivo effects of BPA mimic those of E2 in rodents, including the vaginal cornification (9), growth, and differentiation of the mammary gland (10); a decreased cholesterol level in serum (11), an increased PRL level (12); and an increased c-fos mRNA level in the uterus and vagina (9). Moreover, it has been well characterized that BPA is weakly estrogenic in vitro and in vivo. BPA leaching from autoclaved polycarbonate flasks competed with [3H]-estradiol for binding to ER from the rat uterus. It also induced PRs and promoted cell proliferation in the cultured human mammary cancer cell (13). More recently, weak estrogenicity of BPA was confirmed with approximately 15,000 times less potency than 17ß-estradiol (14). Furthermore, BPA binds to ER{alpha} and ERß with low affinity and transactivates the estrogen response element-driven reporter gene in vitro (14). Although EDCs have the potential to alter reproductive function and endocrine system, actual mechanism of their action have not been identified thoroughly.

The nuclear receptor superfamily is a large group of related transcription factors that regulate physiological homeostasis by direct interaction with a specific cis-element on their target genes (15, 16). Although many nuclear receptors in the group are well characterized, there is a large and growing number of receptors with unknown functions or ligands, called orphan nuclear receptors (15). Among orphan nuclear receptors, PXR (pregnane X receptor) (17, 18) and constitutive androstane receptor (19, 20) recognize many classes of xenobiotic chemicals and activate a response that results in the expression of the xenobiotic metabolizing enzyme, providing a link between the internal and external environment (17). EDCs, phthalic acid, and nonylphenol enhance PXR-mediated transcription through the interaction of PXR with coactivator protein (21), and more recently it has been reported that EDCs block proteasome-mediated degradation of PXR, which may result in the up-regulation of the PXR level (22). These observations suggest that EDCs may play an important role in the endocrine functions by altering the action of orphan nuclear receptors.

Within the orphan nuclear receptor family of transcription factors, Nur77, also known as NGFI-B and TR3, is one of the immediate-early response genes originally identified by virtue of its rapid activation by nerve growth factor in PC12 pheochromocytoma cells (23); serum also induces this receptor gene expression in fibroblast (24). Nur77 binds to its cognate DNA sequence, which consists of a classical half-site preceded by two adenine nucleotides called NGFI-B (Nur77)-binding response elements (NBREs) or Nur response elements (NurREs) and constitutively activates gene expression (25, 26, 27, 28). In addition to its gene induction, Nur77 activity appears to be regulated by posttranslational modification (29, 30, 31, 32) and is rapidly modified via phosphorylation, and the extent of phosphorylation is dependent on the types of stimuli. It has been reported that Nur77 plays an important role in the hypothalamus-pituitary-adrenal axis (33, 34) and T-cell receptor-mediated apoptosis (35, 36, 37). Moreover, Nur77 has been proposed to influence adrenocortical steroidogenesis (33), and our recent report demonstrated that Nur77 is involved in LH-mediated steroidogenesis in mouse testicular Leydig cells (38).

In the present study, we have demonstrated that BPA induces orphan nuclear receptor Nur77 gene expression via PKA and MAPK signaling pathways, and the induction of Nur77 is correlated with steroidogenesis in mouse testicular Leydig cells. Taken together, these results suggest that BPA may disrupt endocrine homeostasis not only by directly binding to nuclear receptor as previously suggested but also by inducing expression of orphan nuclear receptor Nur77.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Hormone, reagents, and animals
Ovine LH (LH-s-26; 2300 IU/mg) was obtained from the National Hormone and Pituitary Distribution Program, NIDDK, NIH (Baltimore, MD). BPA, 4,4'-cyclohexylidenebisphenol (bisphenol E), Bis(4-hydroxyphenol)methane (bisphenol F), 4,4'-(1,3-phenylenedisopropylidene)bisphenol (bisphenol M), 4,4'-(1,4-phenylenedisopropylidene)phenol (bisphenol P), 4,4'-sulfonyldiphenol (bisphenol S), and 4,4'-cylcohexylidenebisphenol (bisphenol Z) were purchased from Sigma-Aldrich Corp. (Milwaukee, WI). H-89 and PD98059 were purchased from Calbiochem (San Diego, CA). Male ICR mice were purchased from Daehan Laboratories (Chungbuk, Korea). Eighteen-day-old mice received a single ip injection of BPA (125 mg/kg), and testes were obtained at a different time interval for Northern blot analysis and RIA.

Cells
The mouse Leydig tumor cell line, K28 cell line, was maintained in DMEM containing 15% FBS (38, 39, 40). For BPA treatment, the exponentially growing cells in the 100-mm dish was split into an appropriate number of 60-mm dishes (3 x 105 cells/dish) and cultured for 2 d in DMEM supplemented with 15% FBS. Cells were washed with PBS, and 4 ml serum-free DMEM containing low density lipoprotein (5 µg/ml) were added to the cells. After cultivating the cell for the designated time points, culture medium and cells were taken for RIA and total RNA preparation, respectively. When cells were incubated with BPA, the concentration of ethanol in the media was 0.1% (vol/vol).

Plasmids
The mouse Nur77 cDNA, dominant negative Nur77 cDNA, Nur77 promoter-luciferase reporter, NurRE 3copy-POMC-Luc reporter, and NBRE-tk-Luc reporter construct were described previously (27, 37, 38), and the pcDNA3-c-jun, pcDNA3-c-fos, LexA-SMRT-D, and B42-Nur77 constructs were from Dr. JaeWoon Lee (POSTECH, Kyoungbuk, Republic of Korea).

Northern blot analysis
BPA-treated K28 cells and 18-d-old mouse testis total RNA were isolated, and Northern blot analysis was carried out as described previously (38).

Western blot analysis
BPA-treated K28 cells were resuspended in lysis buffer (50 mM Tris-HCl, pH 7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EGTA, 1 mM phenylmethylsulfonyfluroide, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 mM NaF), incubated on ice for 10 min, and then lysed by homogenizer. The lysates were centrifuged to remove cell debris, and the supernatant was collected and frozen at -80 C until further use. Protein concentrations were estimated using bicinchoninic acid protein assays (Pierce Chemical Co., Rockford, IL). Protein lysates (50 µg) were boiled for 5 min in denaturing sample buffer and loaded onto 10% continuous gradient SDS-polyacrylamide gel, and protein were transferred to nitrocellulose membrane (Amersham Pharmacia Biotech, Arlington Heights, IL). The membrane was blocked with TBST buffer (10 mM Tris-buffered isotonic saline, pH 7.0, 0.1% merthiolate, and 0.1% Tween-20) containing 5% nonfat dry milk for 30 min at room temperature with shaking and followed by incubation with primary antibody (mouse anti-pp42/44MAPK mAb, New England Biolabs, Inc., Beverly, MA) in TBST buffer for 24 h at 4 C with gentle shaking. Membrane was washed twice with TBST for 10 min and the appropriate horseradish peroxidase-conjugated secondary antibody (dilution of 1:2000; Amersham Pharmacia Biotech). Bound antibodies were visualized using ECL (Amersham Pharmacia Biotech) according to the manufacturer’s protocol. Membrane was stripped by incubation with 0.1 M glycine, 0.1 M NaCl (pH 2.5) for 30 min at room temperature and reprobed for the detection of unphosphorylated p42/44MAPK.

Transient transfection and ß-galactosidase (ß-gal) assay
Twenty-four hours before transfection, K28 cells were plated in 24-well culture dishes at a density of 2.5 x 104 cells/well. Transfection was performed using LipofectAMINE PLUS reagent (Life Technologies, Inc., Rockville, MD) with Nur77 promoter-Luc, NBRE-Luc, NurRE-Luc, and pCMV-ß-gal as an internal control as described previously (38). The luciferase activities were normalized to the ß-gal activity expressed from the cotransfected pCMV-ß-gal plasmid and reported as means ± SE in relative luciferase units. All transfection experiments were performed at least five times in duplicate.

Yeast two-hybrid protein interaction assay
The interaction between SMRT and Nur77 in yeast was measured by activation of the lacZ reporter constructs as detected by ß-gal assays. Yeast strain EGY48 (p80p-lacZ) (CLONTECH Laboratories, Inc., Palo Alto, CA) was transformed with the appropriate plasmids encoding the LexA-SMRT-D fusion protein and B42-Nur77 proteins (41). The colonies were selected on synthetic medium lacking L-uracil, L-histidine, and L-tryptophan (SC-UHW) at 30 C for 3 d, and ß-gal activity in extracts prepared from liquid culture was determined. Five independent colonies from each plate were grown overnight in 2 ml SC-UHW with or without indicated concentration of BPA. The cells were harvested and assayed for ß-gal activity as described previously (21, 42).

RIA
For BPA treatment, the exponentially growing K28 cells in the 100-mm dish were split into an appropriate number of 60-mm dishes (3.5 x 105 cells/dish) and cultured for 24 h in DMEM supplemented with 15% FBS. Transfection was performed using LipofectAMINE PLUS reagent (Life Technologies, Inc.) with dominant negative Nur77 expression vector (1 µg) or pcDNA3 empty vector (1 µg) and internal control pCMV-ß-gal. After 24-h transfection, cells were washed twice with Dulbecco’s PBS, and medium were replaced and incubated for 24 h. Cells were washed twice with Dulbecco’s PBS and 4 ml serum-free DMEM containing LDL (5 µg/ml) were added to the cells. After cells incubated with BPA (1 µM) for the designated time point, the cell culture media were taken for RIA. Culture media were assayed directly without further purification. Total T from the BPA-injected mouse testis were extracted by ethyl ether and used for further experiments. A general assay procedure was used as described previously (38, 43). Progesterone and T concentrations were calculated with the RIAsmart program (Packard, Downer’s Grove, IL). Between- and within-assay coefficients of variation for progesterone were 9.4% and 9.2%, and those for T were 7.7% and 8.2%, respectively. The lower limits of assay sensitivity for progesterone and T were 12.5 pg and 5 pg, respectively. Transfection efficiency was normalized by ß-gal activity. Each treatment group contained duplicate culture, and each experiment was repeated at least three times.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
EDC, BPA, induces the orphan nuclear receptor Nur77 gene expression
It has been reported that Nur77 is involved in steroidogenesis (33, 38) and that BPA inhibits hCG-stimulated steroidogenesis in cultured mouse Leydig tumor cells (44). To investigate whether BPA action on steroidogenesis is mediated through orphan nuclear receptor Nur77, the effects of BPA on Nur77 gene expression were analyzed in testicular Leydig cell, K28. As shown Fig. 1Go, A and B, the expression of Nur77 mRNA was transiently induced by BPA. This induction was maximal at 30 min, returning to basal level within 2 h, and recurrent induction was observed at 24 h. BPA induced Nur77 mRNA level in a dose-dependent manner, reaching a saturation level at 1 µM (Fig. 1Go, C and D).



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  		Figure 1. BPA induces Nur77 gene expression in Leydig tumor cell line. Mouse testis Leydig cell line, K28, was cultured in serum-free conditions for 24 h, and quiescent cells were then treated with BPA (1 µM) at designated time points (A) or with different concentrations of BPA for 30 min (C). Total RNA (20 µg) was analyzed by Northern blot analysis using Nur77 cDNA as a probe. The migration distances of 28S and 18S rRNA (left) and Nur77 transcript (right) are indicated (A, C). The expression of glyceraldehyde-3-phosphate dehydrogenase was used as an internal control. The Nur77 mRNA was quantified using a phosphorimager and normalized for GAPDH RNA levels in each sample (B, D). Data were representative of two independently performed experiments.

 
To gain a better understanding of the specificity of this induction, we tested a series of related bisphenolic compounds for their effects on Nur77 gene expression. Although these chemicals have similar structures, the response was specifically observed only with BPA (Fig. 2AGo). It is interesting that the induction level of Nur77 by BPA was about one-third of that by LH (Fig. 2BGo). These results suggest that BPA specifically induces orphan nuclear receptor Nur77 gene expression in a time- and dose-dependent manner.



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  		Figure 2. BPA-specific induction of Nur77 gene expression. K28 cells were incubated as described in Fig. 1Go in the presence of ethanol control (C) or LH (100 ng/ml), BPA (1 µM), 4,4'-cyclohexylidenebisphenol (bisphenol E; 1 µM), bis(4-hydroxyphenol)methane (bisphenol F; BPF; 1 µM), 4,4'-(1, 3-phenylenedisopropylidene)bisphenol (bisphenol M; BPM; 1 µM), 4,4'-(1, 4-phenylenedisopropylidene)phenol (bisphenol P; BPP; 1 µM), 4,4'-sulfonyldiphenol (bisphenol S; BPS; 1 µM), or 4,4'-cyclohexylidenebisphenol (bisphenol Z; BPZ; 1 µM) (A, B). Total RNA (20 µg) was analyzed by Northern blot analysis. The migration distances of 28S and 18S rRNA (left) and Nur77 transcript (right) are indicated (A, B). The expression of GAPDH was used as an internal control. Data were representative of two independently performed experiments.

 
Involvement of PKA and MAPK signaling pathway in BPA-mediated Nur77 induction
To determine the signaling pathways involved in BPA-mediated Nur77 gene expression, K28 cells were treated with various inhibitors of diverse signaling pathways in combination with BPA. Northern blot analysis indicated that the induction of Nur77 by BPA treatment was 40% decreased by PKA inhibitor, H89 (10 µM), and 45% by MAPK inhibitor, PD98059 (10 µM), whereas there were no significant effects of PKC inhibitor, bisindolylmaleimide I (100 nM) and PI3K inhibitor, wortmannin (10 nM), suggesting that both PKA and MAPK are involved in BPA-mediated Nur77 gene induction (Fig. 3AGo). Moreover, c-jun and c-fos mRNA was concurrently induced with that of Nur77 (Fig. 3BGo), and cotransfection with Nur77 promoter reporter and c-jun increased Nur77 promoter activity followed by BPA treatment, whereas c-fos did not show significant synergistic effect on Nur77 promoter activity (Fig. 3CGo).



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  		Figure 3. BPA-mediated Nur77 gene expression is regulated by PKA and MAPK signaling. K28 cells were cultured in serum-free conditions for 24 h, and these quiescent cells were then first treated with various protein kinase inhibitors, H-89 (10 µM), bisindolylmaleimide I (GFX; 100 nM), PD 98059 (PD; 10 µM), Wortmannin (WM; 10 nM), for 1 h, followed by BPA (1 µM) treatment for 30 min (A). Twenty micrograms total RNA were analyzed by Northern blot analysis. Data were representative of three independently performed experiments. The blot used at Fig. 1AGo was rehybridized with probe from the mouse c-jun and c-fos (B). The migration distances of 28S and 18S rRNA (left) and Nur77, c-jun (Jun), and c-fos (Fos) transcripts (right) are indicated (A, B). The expression of GAPDH was used as an internal control. Cotransfection of Nur77 promoter reporter and c-jun and/or c-fos in the presence or absence of BPA (C). K28 cells were transfected with indicated Nur77 promoter luciferase reporter plasmids along with pcDNA3-c-jun (200 ng) and/or pcDNA3-c-fos (200 ng) and pCMV-ß-gal plasmid as an internal control. Transfected cells received vehicle (EtOH), or BPA as indicated concentration, and assayed for luciferase activity after 36 h. Luciferase activity was normalized by ß-gal activity to determine the transfection efficiency. The data shown represent the mean of three independent experiments. Data are reported as fold activation relative to the control. Effect of BPA on p42/44MAPK phosphorylation (D) is shown. Quiescent K28 cells were stimulated with BPA (1 µM) for the indicated time periods. After the indicated

 
Because BPA-mediated Nur77 gene induction was reduced by PD98059, we examined whether BPA leads to the activation of MAPK. The activity of the p42/44MAPK was determined by immunoblot analysis with a specific antibody that recognized the phosphorylated forms of the MAPK in cell extracts obtained at different times. The dual phosphorylation of p42/44MAPK was stimulated 5 min after the addition of 1 µM BPA and reached a maximum level at 10 min. The dual phosphorylation of p42/44MAPK has decreased to near the basal level at 1 h after addition of BPA (Fig. 3DGo). Taken together, these results indicate that BPA induces Nur77 gene expression via PKA and MAPK signaling pathways.

BPA induces Nur77 gene promoter and its target sequence reporter activity
To further confirm whether the Nur77 gene expression is regulated at a transcriptional level by BPA treatment, transient transfection assays were carried out with Nur77 gene promoter-driven luciferase reporter. Nur77 gene promoter activity was increased approximately 2.5- to 3.5-fold (Fig. 4AGo), indicating that the response of the Nur77 gene promoter to BPA was similar to that of mRNA induction. To determine whether BPA-mediated Nur77 induction represents the enhancement of Nur77 transactivation, Nur77 DNA binding site-driven luciferase reporters were examined with BPA. As indicated in Fig. 4BGo, the activity of reporter-containing Nur77 monomer binding site (NBRE-Luc) was increased by the addition of BPA, albeit no significant response was observed at high doses. Interestingly, there was no response of NurRE-Luc-containing homo/heterodimer-binding site (Fig. 4CGo).



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  		Figure 4. BPA increases Nur77 gene promoter activity and its transactivation. K28 cells were transfected with indicated luciferase reporter plasmids and pCMV-ß-gal plasmid as an internal control, as described in Materials and Methods. Transfected cells received no additional treatment (C), or BPA as indicated concentration, and assayed for luciferase activity after 36 h. Luciferase activity was normalized by ß-gal activity to determine the transfection efficiency (A, B, C). Effect of BPA on the interaction between Nur77 and its corepressor protein (D) is shown. Yeast expressing the LexA-SMRT-D and B42-Nur77 or empty vector (vehicle) was grown for 24 h at 30 C in the presence of BPA (1 µM) or ethanol vehicle. The interaction between Nur77 and SMRT-D was assessed in a ß-gal assay.

 
It has been reported that PXRs interact with nuclear receptor coactivator proteins in the presence of phthalic acid or nonylphenol in the yeast two-hybrid protein interaction assay (21). To examine whether BPA affects interaction between Nur77 and a corepressor protein, silencing mediator of retinoid and thyroid receptor (SMRT), which has been previously characterized as a Nur77 corepressor (41), a yeast two-hybrid protein interaction assay was performed. As illustrated in Fig. 4DGo, Nur77 interacted with SMRT in the absence or presence of BPA, suggesting that BPA has no significant effect on the interaction between Nur77 and SMRT.

BPA stimulates steroidogenesis in vitro and in vivo
To examine the in vitro effects of BPA on steroidogenesis, progesterone synthesis was determined by RIA in Leydig cells, K28. BPA treatment stimulated progesterone synthesis gradually up to 12 h, and this increase was sustained up to 24 h. Interestingly, overexpression of dominant negative Nur77, which encodes a Nur77 protein lacking the N-terminal transactivation domain and inhibits the transactivation activity of Nur77 (29, 37, 38, 45), decreased progesterone synthesis induced by BPA by approximately 25%, suggesting that orphan nuclear receptor Nur77 is involved in the BPA-mediated enhancement of progesterone synthesis (Fig. 5AGo).



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  		Figure 5. Involvement of Nur77 in BPA-mediated progesterone biosynthesis. K28 cells were cultured in the absence ({blacksquare}) or presence of BPA (1 µM; {circ}) and dominant negative Nur77 (DN-Nur77; {bullet}) at the designated time points, and then the cultured medium was collected and concentration of progesterone in medium was measured by RIA as described in Materials and Methods. Each time point represents the average amount of progesterone per cell number in three independent experiments (A). The blot used at Fig. 1AGo was rehybridized with probe from the mouse StAR, P450SCC/CYP11A (CYP11A), and 3ß-HSD (B). The expression of GAPDH was used as an internal control.

 
To confirm BPA-mediated progesterone synthesis, we investigated several kinds of steroidogenic enzyme expression. Northern blot analysis revealed that BPA increased the level of steroidogenic acute regulatory (StAR) mRNA at 30 min, sustained until 3 h, returned to basal level at 6 h, and again reached maximal level at 24 h. Moreover, BPA increased cholesterol side chain cleavage enzyme (P450SCC/CYP11A) mRNA at 1 h, and this increase of mRNA level was sustained up to 24 h. Finally, 3ß-hydroxysteroid dehydrogenase (3ß-HSD) mRNA was increased at 30 min of BPA treatment, remained its highest level for 2 h, then returned to basal, and again increased at 12 h (Fig. 5BGo), whereas no significant effect of BPA was observed on 20{alpha}-hydroxysteroid dehydrogenase mRNA level (data not shown).

We confirmed the effects of EDC BPA on the steroidogenesis and Nur77 gene expression in vivo using prepubertal mice (18 d old). Northern blot analysis revealed that BPA injection (125 mg/kg) dramatically increased Nur77 mRNA levels after 1 h and returned to its basal level after 6 h at 18-d-old mouse testis (Fig. 6Go, A and B). The level of Nur77 gene expression was 6-fold higher at 1 h after BPA injection than control (vehicle only). Moreover, BPA treatment significantly increased T synthesis (2-fold), and the time pattern of this increment was consistent with that of Nur77 induction (Fig. 6Go, A and C). Taken together, these results suggest that BPA-mediated Nur77 gene expression results in the increased steroidogenesis both in vitro and in vivo.



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  		Figure 6. Induction of Nur77 mRNA and alteration of steroidogenesis stage by BPA treatment at prepubertal stage. Northern blot analysis and RIA were performed as described in Materials and Methods. Twenty micrograms testis total RNA from BPA-injected mouse (n = 5) was analyzed by Northern blot analysis. The migration distances of 28S and 18S rRNA (left) and Nur77 transcript (right) are indicated (A). The expression of GAPDH was used as an internal control. The Nur77 mRNA was quantified using a phosphorimager and normalized for GAPDH RNA levels in each sample (B). BPA-injected mouse testis was used for RIA (C). Data were representative of three independently performed experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The EDCs have been demonstrated to alter endocrine function through changing steroid hormone receptor-mediated protein synthesis, interference with membrane receptor binding, and steroidogenesis or synthesis of other hormones (13, 44, 46, 47). Especially phthalic acid, alkyphenols, BPA, and DDT have been shown to disrupt via binding to estrogen or androgen receptor by mimicking estrogenic action (48, 49). However, other cellular mechanisms still remain to be determined. Here we report a transient and rapid induction of orphan nuclear receptor Nur77 gene expression and alteration of steroidogenesis in BPA-treated mouse testis and testicular Leydig cell line.

To extend our knowledge of effects of EDCs on nuclear receptors, we have tested the effects of BPA on several nuclear receptor gene expressions in K28 cells, but among them only Nur77 was shown to have a significant responsiveness to BPA. We have previously shown that LH induces Nur77 in mouse testicular Leydig cells (38). Interestingly, we found that BPA-mediated Nur77 gene expression peaked earlier and declined faster than that of LH mediation. Moreover, Nur77 gene induction was observed again at 24 h after BPA treatment, suggesting that BPA-mediated cellular signaling is characterized as a biphasic induction pattern. In contrast to Nur77, Nurr-1 and NOR-1, subfamily of Nur77, the inductions were not detected by BPA treatment (data not shown). Interestingly, although bisphenolic compounds have a similar chemical structure, Nur77 gene induction was solely affected by BPA, indicating that the methyl group of BPA may play a critical role in determining the specificity of Nur77 gene induction.

It has been reported that the cellular signaling pathways involved in Nur77 induction are diverse, depending on stimuli (38, 50). In contrast to our previous report on PKA-, PKC-, and PI3K-mediated Nur77 gene induction by LH (37), BPA-mediated Nur77 expression was reduced by PKA inhibitor, H-89, and MAPK inhibitor, PD98059, suggesting that BPA and LH may induce Nur77 gene expression through different signaling pathways. Moreover, c-jun and c-fos genes were also induced by BPA and the time pattern of inductions were similar to that of Nur77, suggesting that MAPK might be a common signaling pathway for BPA-mediated c-jun, c-fos, and Nur77 gene expression. Furthermore, it has been reported that acute carbon monoxide intoxication induces NGFI-B (Nur77), c-fos, and c-jun mRNA expression in mouse brain (51), and immediate-early expression of Nur77 is mediated by multiple AP-1-like elements, which can interact with products of immediate-early genes such as Fos/Jun (52), suggesting that c-jun and c-fos may be involved in the BPA-mediated Nur77 gene induction. Moreover, it has been recently reported that JunD is implicated in activation of Nur77 promoter by PGF2{alpha} (53). Along with this, our result that cotransfection of c-jun significantly increased Nur77 promoter activity followed by BPA treatment suggests the involvement of Jun family members in BPA-mediated Nur77 gene expression. Signaling pathways involved in the endocrine disrupter have been poorly characterized, and here we show for the first time that BPA induces Nur77 gene expression via activation of MAPK, suggesting a link between the signaling cascade and the action of endocrine disrupters.

It is well established that the Nur77 is a transcription factor, and it has been shown to bind DNA and activate transcription as a monomer and homodimer or heterodimer (25, 26, 27, 28, 33). In our current study, BPA induced Nur77 gene expression and its transcriptional activity, suggesting that BPA-induced Nur77 translocates to the nucleus where it presumably regulates the transcription of its target gene. Unexpectedly, in contrast to LH-mediated Nur77 transactivation, which confers higher responsiveness on NurREreporter containing Nur77 dimer-binding element than NBRE-reporter containing Nur77 monomer-binding element (38), NurRE-reporter was not significantly activated by BPA treatment, suggesting that BPA-mediated Nur77 function may not be required for dimerization or BPA treatment may prevent Nur77 dimerization.

In contrast to the interaction of PXR with its coactivator in the presence of EDCs (21, 54), BPA did not show any significant effects on the interaction between Nur77 and its corepressor, SMRT. Although we could not examine the interaction between Nur77 and its coactivator in the presence of BPA because of the lack of information of Nur77 coactivator, BPA-mediated Nur77 transactivation is not owing to the dissociation of corepressor, SMRT, by BPA. However, there still remains a possibility that BPA may increase the interaction of Nur77 with unknown coactivator as known in EDC-mediated PXR transactivation or dissociation of other uncharacterized corepressor. In addition, BPA is unlikely to act as a ligand for Nur77 because there was no significant effect of BPA on Nur77 transactivation in yeast-based receptor assay (14) (data not shown).

To investigate the causal relationship between BPAmediated Nur77 induction and steroidogenesis, we tested the effects of BPA on steroidogenesis in cultured mouse testicular Leydig cell line, K28, by measuring the progesterone production. In contrast to a previous report from another laboratory (44), we found that BPA increased the expression of steroidogenic enzymes, StAR, cholesterol side chain cleavage enzyme (P450SCC/CYP11A), and 3ß-HSD, and progesterone synthesis. Apparent discrepancies between our current study and that of previous reports (44) may be because of the difference in experimental approaches. Nikula et al. (44) preincubated BPA for 48 h and then examined hCG-stimulated steroidogenesis of Leydig cells, whereas we tested intact BPA effect on steroidogenesis in testicular Leydig cells. Moreover, overexpression of dominant negative Nur77 suppressed BPA-mediated progesterone synthesis suggesting BPA-mediated Nur77 gene induction is involved in steroidogenesis. In addition, in vivo BPA injection also increased T production, and the induction of T production was correlated with Nur77 gene induction, strongly supporting that BPA mediates both Nur77 gene induction and steroidogenesis. Because steroidogenesis of 18-d-old mice is not significantly active, alteration of steroidogenesis by BPA may result in premature puberty. Furthermore, it has been reported that prenatal treatment with BPA significantly reduced the number of days between vaginal opening and first vaginal oestrus (55), suggesting the possibility of alteration on BPA-mediated steroidogenesis.

To our knowledge, this is the first report that an EDC stimulates orphan nuclear receptor Nur77 gene expression, which is linked to steroidogenesis in Leydig cells. Our current study also suggests that the endocrine-disrupting effect of BPA is engaged not only in the binding of this chemical to nuclear receptor but also in the induction of orphan nuclear receptor gene expression. Further investigation on both aspects of BPA effects is necessary to fully understand the role of BPA as an endocrine disruptor.


   Acknowledgments
 
The authors are thankful to Dr. JaeWoon Lee for providing DNA constructs and critical reading of this manuscript, Dr. Jaemog Soh and Mr. Jason Lee for critical reading of this manuscript, and Dr. Ryun-Sup Ahn for technical assistance.


   Footnotes
 
This work was supported by Hormone Research Center Grant (HRC-G0201) and in part by Korean Andrological Society (to H.S.C.).

Abbreviations: BPA, Bisphenol A; EDC, endocrine disrupting chemical; ß-gal, ß-galactosidase; 3ß-HSD, 3ß-hydroxysteroid dehydrogenase; NBRE, Nur77-binding response element; NurRE, Nur response element; PXR, pregnane X receptor; StAR, steroidogenic acute regulatory.

Received November 2, 2001.

Accepted for publication February 12, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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