Accepted Manuscript
Title: Bisphenol A stimulates adrenal cortical cell proliferation via ERß-mediated activation of the sonic hedgehog signalling pathway
Authors: Samantha Medwid, Haiyan Guan, Kaiping Yang
The Journal of Steroid Biochemistry & Molecular Biology
| PII: | S0960-0760(18)30005-0 |
| DOI: | https://doi.org/10.1016/j.jsbmb.2018.01.004 |
| Reference: | SBMB 5100 |
| To appear in: | Journal of Steroid Biochemistry & Molecular Biology |
| Received date: | 5-12-2017 |
| Accepted date: | 4-1-2018 |
Please cite this article as: Medwid S, Guan H, Yang K, Bisphenol A stimulates adrenal cortical cell proliferation via ERß-mediated activation of the sonic hedgehog signalling pathway, Journal of Steroid Biochemistry and Molecular Biology (2010), https://doi.org/10.1016/j.jsbmb.2018.01.004
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Bisphenol A stimulates adrenal cortical cell proliferation via ERß-mediated activation of the sonic hedgehog signalling pathway
Samantha Medwid, Haiyan Guan, and Kaiping Yang
Children’s Health Research Institute & Lawson Health Research Institute, Departments of Obstetrics & Gynaecology and Physiology & Pharmacology, Western University, 800 Commissioners Rd. E., London, Ontario, Canada N6C 2V5
| Running title: | BPA signaling in adrenal cortical cells |
| Keywords: | BPA; cell proliferation; ERß, Shh signaling; adrenal cortical cells |
Correspondence to: Dr. K. Yang Children’s Health Research Institute, Room A5-132 Victoria Research Laboratories
800 Commissioners Road East
London, Ontario, Canada N6C 2V5
Tel: 1-519-685-8500 Ext 55069 Fax: 1-519-685-8186
ACCEP
E-mail: kyang@uwo.ca
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HIGHLIGHTS
· BPA stimulated cell proliferation in the H295A adrenal cortical cell line
· BPA-induced cell proliferation was a result of activation of the Shh pathway
· BPA activated the Shh signaling pathway through an ERß-mediated mechanism
ABSTRACT
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We previously demonstrated that prenatal exposure to bisphenol A (BPA) resulted in increased adrenal gland weight independent of changes in plasma ACTH levels in adult mouse offspring. This finding suggested that BPA exposure likely had a direct effect on adrenal development. Given that (1) sonic hedgehog (Shh) signaling is essential for adrenal development; (2) deletion of the Shh gene in mice results in adrenal hypoplasia; (3) BPA is known to signal through estrogen receptor ß (ERB); and (4) ERß is highly expressed in adrenal glands; we hypothesized that BPA stimulates adrenal cell proliferation via ERß-mediated activation of the Shh pathway. To test this hypothesis, the human adrenal cell line, H295A cells, was used as an in vitro model system. Our main findings were: (1) BPA increased cell number and protein levels of proliferating cell nuclear antigen (PCNA; a universal marker of cell proliferation), cyclin D1 and D2 (key proliferation factors), as well as Shh and its key transcriptional regulator Gli1; (2) cyclopamine, a Shh pathway inhibitor, blocked these stimulatory effects of BPA on cell proliferation; (3) BPA increased the nuclear translocation of ERß; and (4) the ERß-specific agonist DPN mimicked while the ERß-specific antagonist PHTPP abrogated the stimulatory effects of BPA on cell proliferation and Shh signaling. Taken together, these findings demonstrate that BPA
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stimulates adrenal cell proliferation likely through ERß-mediated activation of the Shh signaling pathway. Thus, the present study provides novel insights into the molecular mechanisms underlying our previously reported BPA-induced aberrant adrenal phenotype.
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1. INTRODUCTION
Bisphenol A (BPA) is one of the most well-known and prevalent endocrine disrupting chemicals, and has gained universal attention due to its adverse effects in humans and experimental animal models [1]. BPA is widely used in the production of polycarbonate plastics and epoxy resins, such as food and beverage storage containers and thermal paper receipts [1, 2]. Biomonitoring studies have detected BPA in human saliva, milk, serum and urine collected globally [2]. More alarming is the presence of BPA in human fetal blood, placental tissue and amniotic fluid [2, 3]. This has raised serious concerns about the impact of BPA exposure on the developing fetus during the critical period of organ maturation. Indeed, numerous studies have shown that BPA exerts adverse effects on many fetal organ systems, including the brain [4, 5], lungs [6], liver [7], pancreas [8], heart [9], adrenal gland [10, 11], mammary gland [12, 13], and ovary [14, 15].
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We recently showed that prenatal exposure to BPA resulted in altered adrenal gland structure and function in adult mouse offspring [10]. Specifically, absolute and relative adrenal gland weight was increased in both male and female adult offspring [10]. Similarly, Panagiotidou et al. reported adrenal hyperplasia in juvenile female rat offspring following exposure to BPA during pregnancy and lactation [11]. Alterations in adrenal weight and structure is normally associated with changes in plasma levels of adrenocorticotrophic hormone (ACTH). However, we did not observe an increase in basal plasma levels of ACTH, and concluded that BPA may directly affect adrenal gland weight independent of plasma ACTH in our prenatally BPA exposed mouse model [10]. BPA has previously been shown to increase cell proliferation in various tissues, including breast cancer [16-18], ovarian cancer [19, 20], neuroblastoma [21], Hela [22], prostate
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cancer [23], seminoma [24] and sertoli cells [25]. However, the effects of BPA on adrenal cortical cell proliferation has never been examined.
Sonic hedgehog (Shh) signaling pathway is a key mediator of embryonic development, as well as cell maintenance and tissue repair in adults [26, 27]. Specifically, the Shh pathway is found to be activated during development, as well as in various forms of cancer due to its role in promoting cell proliferation through direct transcriptional activation of proliferation factors cyclin D1 and cyclin D2 [26]. Shh signaling components (Shh, Gli1, Patched 1) have been detected in human adrenal cortical cell lines, human fetal and adult adrenal glands, as well as both pediatric and adult adrenal tumors [28, 29]. Evidence of Shh involvement in adrenal cell proliferation is demonstrated by the presence of an adrenal cortex hypotrophy phenotype in Shh null mice [30, 31]. Thus, the present study was undertaken to determine (1) if BPA promotes adrenal cell proliferation, which may help explain the increased adrenal gland weight phenotype we reported in our previous study [10]; and (2) if so, whether the stimulatory effects of BPA on adrenal cortical cell proliferation are mediated through ERß-mediated activation of the Shh pathway using a human adrenal cortical cell line as an in vitro model system.
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2. MATERIALS AND METHODS
2.1 Reagents
Bisphenol A was purchased from Sigma-Aldrich Canada Ltd. (CAS 80-05- 7; Oakville, ON) and dissolved in ethanol to prepare 10 mM stock solution, and stored at -20℃. Cyclopamine was purchased from Toronto Research Chemicals (C988400; Toronto, ON), dissolved in ethanol to prepare 10 mM stock solution and stored at -20℃. 2,3-bis(4-Hydroxyphenyl)-propionitrile (DPN) and 4-[2-Phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl]phenol (PHTPP) were purchased from Tocris Bioscience (cat. no. 1494; Minneapolis, MN) and Abcam (ab145148; Toronto, ON), respectively, dissolved in ethanol to a concentration of 100 mM, and stored at - 20°C.
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2.2 Cell Culture
The adrenocortical human cell line NCI-H295 cell line was derived from an adrenal tumor of a 48-year-old female and was first described by Gazdar et al. [32]. The NCI-H295 cell line expresses all steroidogenic enzymes present in the human fetal adrenal glands and is an established model to study adrenal steroidogenesis [33]. The subline, NCI-H295A, was further derived and characterized from the H295R cell line, and is currently the best available model of human fetal adrenal gland cells [34]. H295A cells (generously provided by Dr. Walter L. Miller) were cultured in RPMI 1640 media (Invitrogen) with 2% fetal bovine serum (FBS; Sigma), 0.1% insulin- transferrin-selenium supplement (Sigma I18884) and 100IU penicillin and 100ug/mL were starved in serum-free media 24 h before treatment, and cultured in 0.2% FBS media throughout treatments.
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2.2 Western Blot Analysis
Levels of various proteins were analyzed using standard western blot analysis, as previously described [35]. Briefly, cells were lysed in SDS gel loading buffer (50mM Tris-HCL, pH 6.8, 2% wt/vol SDS, 10% vol/vol glycerol, 100mM DTT and 0.1% wt/vol bromophenol blue) and equal concentrations of whole cell lysates, or cytosolic and nuclear extracts were loaded on a standard SDS-PAGE gel. Protein was then transferred to a PVDF transfer membrane (Amersham Hybond- P, cat. no. RPN303F, GE Healthcare Lifesciences, Baie D’Urfe, QC), and blocked overnight with 5% milk in TTBS (0.1% vol/vol Tween-20 in TBS). Membranes were then probed with primary antibodies for 1-2 hours at room temperature (Supplemental Table 1). Washing was done with TTBS, 3×10 minutes before labeling with horseradish peroxidase-labeled secondary antibody (Supplemental Table 1), for 1 hour at room temperature. After 3×10 minute TTBS washes, protein were detected using ECL and visualized using a chemiluminescence (cat. no. WBLUR0500, Luminata Crescendo, Western HRP Substrate; Millipore, Etobicoke, ON) and captured on the VersaDoc Imaging System (BioRad). Densitometry was performed using Image Lab Software, comparing levels of proteins expressed as percent of controls.
2.3 Cell Number Assessment
Cells were seeded in 2% FBS-RMPI 1640 culture medium and were incubated overnight. After 24 h serum starvation, the medium was changed to 0.2% FBS RMPI 1640 containing 10 nM BPA. After 72 h incubation, the cells trypsinized, added in equal volumes to trypan blue stain 0.4% (Invitrogen T10282) and counted with Countess Automated cell counter (Invitrogen C10277).
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2.4 Real-time quantitative RT-PCR
The relative abundance of various mRNAs was determined by a two-step real time quantitative RT-PCR (qRT-PCR), as described previously [36], with the following modifications. Briefly, total RNA was extracted from cells using RNeasy Mini Kit (Qiagen Inc., Mississauga, ON) coupled with on-column DNase digestion with the RNase-free DNase Set (Qiagen) according to the manufacturer’s instructions. One microgram of total RNA was reverse-transcribed in a total volume of 20 ul using the High Capacity cDNA Archive Kit (Applied Biosystems, Forest City, CA) following the manufacturer’s instructions. For every RT reaction set, one RNA sample was set up without reverse-transcriptase enzyme to provide a negative control. Gene transcript levels of GAPDH, GLI1 and SHH were quantified separately by pre-designed and validated TaqMan® Gene Expression Assays (Applied Biosystems; Supplemental Table 2) following the manufacturer’s instructions. Briefly, gene expression assays were performed with the TaqMan® Gene Expression Master Mix (Applied Biosystems P/N #4369016) and the universal thermal cycling condition (2 min at 50 ℃ and 10 min at 95 ℃, followed by 40 cycles of 15 s at 95 ℃ and 1 min at 60 ℃) on the ViiATM 7 Real-Time PCR System (Applied Biosystems).
The relative amounts of various gene-specific mRNAs in each RNA sample was quantified by the comparative CT method (also known as 44 CT method) using the Applied Biosystems relative quantitation and analysis software according to the manufacturer’s instructions. For each experiment, gene specific mRNAs were normalized to the housekeeping gene GAPDH. The amount of various gene-specific mRNAs under different treatment conditions is expressed relative to the amount of transcript present in the untreated control.
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2.5 Statistical Analysis
Results are presented as group means ± SEM of four independent experiments, as indicated. Data was analyzed using a Student’s t-test or a one-way ANOVA, followed by a Tukey’s post hoc; statistical significance was set at P<0.05. Statistical analysis was performed using statistical software GraphPad Prism Version 5 Software.
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3. RESULTS
3.1 Time- and concentration-dependent effects of BPA on cell proliferation
As a first step in determining the effects of BPA on cell proliferation, protein levels of PCNA, a universal marker of cell proliferation, were assessed over time. Levels of PCNA protein were unchanged at 24 and 48 h, but were significantly elevated at 72 h following treatment with 10 nM of BPA (Figure 1A). A similar trend of change was observed in cell number following BPA treatment (Figure 1B). We then treated cells with increasing concentrations of BPA (1-1000 nM) for 72 h, and showed that this treatment resulted in a concentration-dependent increase in PCNA protein levels such that the maximal effect was observed at 10 nM BPA (Figure 1C).
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3.2 Effects of BPA on the expression of key cell proliferation factors
To further examine the effects of BPA on cell proliferation, protein levels of the two key proliferation factors, cyclin D1 and cyclin D2, were determined. Although levels of both cyclin D1 and cyclin D2 proteins were unchanged after 48 h of BPA treatment (Figure 2A), they were significantly increased after 72 h of BPA treatment (Figure 2B).
3.3 Effects of BPA on selected components of the Shh signaling pathway
Shh signaling is known to be essential for adrenal development and proliferation. Adrenal specific Shh knockout mice display severe adrenal hypoplasia, specifically an underdeveloped cortex in fetal and adult mice [30, 37]. To explore the role of Shh signaling in mediating BPA-induced cell proliferation, changes in key Shh signaling pathway components were examined. Levels of Shh
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mRNA, but not Gli1 mRNA, were increased at 48 h post BPA treatment (Figure 3A&B). In contrast, protein levels of both Shh and Gli1 were elevated following 48 h of BPA treatment (Figure 3C&D), which returned to control levels at 72 h (data not shown).
3.4 Effects of BPA on activity of the Shh signaling pathway
Activation of the Shh signaling pathway involves translocation of the Shh transcription factor Glil from cytoplasm to the nucleus where it acts as an activator of Shh target genes [38, 39]. To determine if BPA activates the Shh signaling pathway, we measured Gli1 protein levels in cytosolic and nuclear fractions following treatment with BPA for 48 h. BPA treatment significantly increased Gli1 protein in nuclear but not cytosolic fraction (Figure 4A&B). To ascertain if BPA activation of the Shh signaling pathway is ligand-dependent, we used cyclopamine (Cyc), which blocks the Shh pathway at the SMO receptor. We treated cells with BPA in the presence and absence of Cyc, and examined changes in Gli1 protein. We found that Cyc prevented BPA-induced increases in Gli1 protein levels (Figure 4℃).
3.5 Effects of Shh pathway inhibition on BPA-induced cell proliferation
Shh signaling is known to induce cell proliferation in a variety of tissues [26, 40, 41]. Specifically, the transcription factor Glil is known to directly stimulate the transcription of cyclin D1 and D2 genes, CCND1 and CCND2 [26]. To provide functional evidence for the involvement of the Shh signaling pathway in mediating BPA-induced cell proliferation, we assessed changes in protein levels of PCNA, cyclin D1 and D2 following treatment with BPA in the presence and absence of
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the Shh pathway inhibitor Cyc. Cyc completely blocked BPA-induced increases in levels of PCNA (Figure 5A), as well as cyclin D1 and D2 (Figure 5B) protein.
3.6 Effects of BPA on estrogen ß expression and activity
The translocation of ER from cytosol to the nucleus is essential for transcriptional activation of estrogen target genes [42-44]. To examine if BPA activates ERß, we measured protein levels of ERß in total cell lysates as well as cytosolic and nuclear fractions following BPA treatment for 48 h. Although BPA treatment did not alter total ERß protein levels (Figure 6A), it decreased cytosolic while increasing nuclear levels of ERß protein (Figure 6B&C).
3.7 Effects of DPN and PHTPP on BPA-induced cell proliferation
We then investigated the involvement of ERß in BPA-induced cell proliferation using the ERB specific agonist DPN and the ERß specific antagonist PHTPP. Treatment with DPN significantly increased protein levels of PCNA (Figure 7A) as well as cyclin D1 and cyclin D2 (Figure 7C) at 72 h. Furthermore, pretreatment with PHTPP completely prevented BPA-induced increases in levels of PCNA (Figure 7B), cyclin D1 and cyclin D2 (Figure 7D) proteins.
3.8 Effects of DPN and PHTPP on BPA-induced activation of the Shh signaling pathway
ERa has been shown to increase Shh activity in breast [45, 46] and gastric [47] cancer cells. However, this effect has yet to be shown with ER. Therefore, we tested the hypothesis that BPA acts through ERß to activate the Shh signaling pathway. We showed that the ERß specific agonist DPN increased protein levels of both Shh and Glil after 48 h treatment (Figure 8A&C). Importantly, the ERß specific antagonist PHTPP completely blocked BPA-induced increases in both Shh and Gli1 protein levels (Figure 8B&D).
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4. DISCUSSION
Proper adrenal gland development is essential for adrenal steroidogenesis, particularly glucocorticoid production in later-life. We recently demonstrated that prenatal exposure to BPA resulted in abnormal adrenal gland development and function in adult mouse offspring, including increased adrenal gland weight independent of plasma ACTH levels [10]. However, the molecular mechanisms underlying the BPA-induced increase in adrenal gland weight remain unknown. Therefore, the present study was designed to address this important question using the best available model of fetal adrenal cortical cells, the H295A cell line. We have demonstrated that BPA stimulates adrenal cell proliferation via ERß-mediated activation of the Shh signaling pathway. Thus, our present findings reveal a plausible molecular mechanism by which BPA influences adrenal gland development and function.
The concentration of BPA used in this study (10 nM) is in line with those used in previous in vitro studies [48]. Importantly, this concentration (equivalent to 2.28 ng/ml) is well within the range previously reported in plasma (0.5-22.3 ng/ml) [49] and urine (0.16-43.42 ng/ml) [50] of pregnant women in North American.
BPA has been shown to influence cell proliferation in both in vivo and in vitro models. In experimental animal models, prenatal exposure to BPA led to increased cell proliferation in fetal liver [7], prostate [51], pancreas [52], and pituitary gland [53]. In contrast, offspring of rats exposed to BPA during pregnancy and lactation showed decreased proliferation in neural stem cells of the hypothalamus and sub-ventricular zone [54]. In several in vitro models, BPA increases cell proliferation at various concentrations [16-25]. Interestingly, in sertoli cells, nanomolar concentrations of BPA induced cell proliferation, while micromolar concentrations decreased cell proliferation, suggesting that the effect of BPA on cell proliferation is concentration-dependent
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[55]. To the best of our knowledge, we are the first to demonstrate that BPA, at environmentally relevant concentrations, significantly increases cell number as well as the expression of PCNA, cyclin D1 and D2, three key markers of cell proliferation, in adrenal cortical cells. This indicates that BPA stimulates adrenal cortical cell proliferation. Thus, our present study provides a plausible cellular mechanism by which prenatal BPA exposure results in increased adrenal gland weight in adult mouse offspring [10].
Activation of the Shh signaling pathway is known to increase the transcription of genes encoding both cyclin D1 and D2 genes, leading to increased cell proliferation [26]. Recently, BPA has been shown to increase levels of microRNA-107 (miRNA-107), which inhibits the expression of suppressor of fused homolog (SUFU) and GLI family zinc finger 3 (Gli3) in human endometrial cancer in RL95-2 cells [56]. Both SUFU and Gli3 are repressors of the Shh signaling pathway, thus BPA-induced suppression of these proteins may potentially lead to the activation of Shh signaling and consequently increased proliferation in endometrial cells [56]. Therefore, we investigated the possibility that the BPA-induced adrenal cortical cell proliferation may be mediated via activation of the Shh signaling pathway. As a first step in examining this possibility, we determined the effects of BPA on Shh expression, and found that levels of both Shh mRNA and protein were increased after 48 hours of BPA treatment, which preceded the increase in cell proliferation we observed at 72 hours.
An increase in Shh protein and secretion results in its binding to the transmembrane receptor Patched 1 (Ptch1), which prevents Ptch1 from inhibiting another transmembrane protein smoothened (SMO) [38, 39]. SMO can then be released from the plasma membrane into the cytoplasm, leading to the release of a complex containing the transcription factors Gli1-3, allowing them to translocate to the nucleus to regulate transcription of target genes [38, 39]. Specifically,
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the nuclear translocation of the positive transcriptional regulator Gli1, is considered a marker of Shh signaling activation [38, 39]. Therefore, we investigated the potential for BPA to alter Gli1 protein and mRNA levels. We found that although BPA did not alter Gli1 mRNA, it increased Gli1 protein levels at 48 hours. The regulation of Glil at post-transcriptional level is well established and could be a result of changes in translation and phosphorylation efficiency [57, 58]. Furthermore, BPA significantly increased Gli1 protein levels in the nuclear fraction without altering those in the cytosolic fraction, suggesting that BPA enhanced nuclear translocation of Gli1, and consequently the activity of the Shh signaling pathway. Given the observed increase in Gli1 protein levels in total cell lysates, the relatively minor and non-significant decrease seen in cytosolic Gli1 levels is consistent with our notion of an enhanced Glil nuclear translocation following BPA treatment. It is known that activation of the Shh signaling pathway is mediated through either the ligand-dependent or the ligand-independent pathway [59]. To determine if BPA acts through the ligand-dependent Shh signaling pathway, we examined the effects of BPA on Glil protein levels in the presence and absence of cyclopamine. Cyclopamine is a potent inhibitor of the Shh signaling pathway by preventing release and translocation of the SMO receptor. In the present study, we showed that cyclopamine blocked the effects of BPA on Glil protein levels, indicating that BPA activates the Shh pathway through the ligand-dependent pathway.
To ascertain whether BPA-induced activation of the Shh signaling pathway leads to increased cell proliferation, we treated cells with BPA in the presence and absence of cyclopamine. We found that cyclopamine completely abrogated the stimulatory effects of BPA on cell proliferation, as indicated in protein levels of PCNA, cyclin D1 and D2. Taken together, these results demonstrate the involvement of the Shh signaling pathway in BPA-induced adrenal cortical cell proliferation.
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It is well known that BPA acts as an ERß agonist, with a higher affinity for ERß than ERa [60-62]. Furthermore, ERß is the dominant estrogen receptor expressed in human H295R adrenal cortical cells [63]. Therefore, we then investigated the role of ERß in BPA-induced cell proliferation and Shh activation. Given that a key step in ERß activation is its nuclear translocation upon binding to its ligand [64], we determined the effects of BPA on ERß translocation at 48 h. This time point was chosen based on the BPA-induced increase in Shh mRNA at 48 h. We found that levels of ERß protein were increased in the nuclear fraction but decreased in the cytosolic fraction following BPA treatment, indicating that BPA enhanced translocation of ER to the nucleus in H295A cells. However, it is likely that the BPA-induced increase in ERß nuclear translocation may have occurred earlier than 48 h.
Although estrogen has previously been shown to increase adrenal cell proliferation in both animal models [65] and the H295R cell line [63], the estrogen receptor subtype involved remains unknown. We then sought to determine if activation of ERß stimulates adrenal cell proliferation using the ERB selective agonist DPN. We showed that DPN increased protein levels of the three key proliferation markers, PCNA, cyclin D1 and D2, indicating that the activation of ERß by DPN led to increased cell proliferation. To provide evidence for the involvement of ERß in mediating BPA-induced cell proliferation, we treated cells with BPA in the presence and absence of the ERß- specific antagonist PHTPP. We found that PHTPP completely blocked the stimulatory effects of BPA on PCNA, cyclin D1 and D2 protein. Taken together, these results demonstrate that that ERß mediates BPA-induced proliferation in adrenal cells.
The ability of estradiol to activate the Shh signaling pathway has previously been demonstrated in ERa positive breast and gastric cancer cells [45-47], however it remains unknown if a similar effect can be observed through ERß. Therefore, to determine if ERß activates the Shh
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signaling pathway in adrenal cells, we examined the effects of ERß specific agonist DPN on expression of the two key proteins in the Shh signaling pathway. We found that DPN increased both Shh and Gli1 protein levels, indicating a novel link between ERß and Shh activation. We then determined if the activation of ER by BPA leads to activation of the Shh signaling pathway. We treated cells with BPA in the presence and absence of ERß-specific antagonist PHTPP, and found that PHTPP abrogated the stimulatory effects of BPA on Shh and Gli1 protein levels. Collectively, these results indicate that BPA stimulates adrenal cell proliferation via ERß-induced activation of the Shh signaling pathway.
In conclusion, the present study demonstrates for the first time that BPA acts on ERß to activate the Shh signaling pathway, which in turn leads to increased proliferation in H295A cells (Figure 9). Thus, our present study reveals a novel BPA-induced cell proliferation signalling pathway that may underlie the increased adrenal gland weight phenotype we reported previously in prenatally BPA exposed adult mouse offspring.
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FUNDING SOURCES
This work was supported by the Canadian Institutes of Health Research (Operating Grant MOP- 111158).
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FIGURE LEGENDS
Figure 1: Time- and concentration-dependent effects of BPA on cell proliferation. H295A cells were treated with 10 nM of BPA for various times (24-72 h) or increasing concentrations (1-1000 nM) of BPA for 72 h. At the end of treatment, levels of PCNA (a universal marker of proliferation) (A, C) and cell number (B) were determined by western blotting and cell counting, respectively. Data are presented as mean ± SEM (*P<0.05, *** P<0.001 vs. control; n=4 independent experiments).
Figure 2: Effects of BPA on key cell proliferation factors. H295A cells were treated with 10 nM of BPA for 48 h (A) or 72 h (B). At the end of treatment, levels of the two key cell proliferation factors, cyclin D1 and cyclin D2 were determined by western blotting. Data are presented as mean ± SEM ( ** P<0.01 vs. control; n=4 independent experiments).
Figure 3: Effects of BPA on selected components of the Shh signaling pathway. H295A cells were treated with 10 nM of BPA for 48 h. At the end of treatment, levels of Shh mRNA (A) and and Glil mRNA (B) were determined by qRT-PCR. Levels of Shh protein (C) and Glil protein (D) were determined by western blotting. Data are presented as mean ± SEM (*P<0.05, ** P<0.01, vs. control; n=4 independent experiments).
Figure 4: Effects of BPA on the activity of the Shh signaling pathway. H295A cells were treated with 10 nM BPA for 48 h. At the end of the treatment, levels of Gli1 protein in cytosolic (A) and nuclear (B) extracts were determined by western blotting. Alternatively, H295A cells were treated with either 10 nM BPA, 10 uM cyclopamine (Cyc) or both for 48 h. At the end of treatment, levels of Gli1 protein were determined by western blotting (C). Data are presented as mean ± SEM (*P<0.05 vs. control; different letters indicate statistically significant differences among groups; n=4 independent experiments).
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Figure 5: Effects of Shh inhibition on BPA-induced cell proliferation. H295A cells were treated with 10 nM BPA, 10 uM cyclopamine (Cyc) or both for 72 h. At the end of treatment, levels of PCNA (A), cyclin D1 and cyclin D2 (B) were determined by western blotting. Data are presented as mean ± SEM (Different letters indicate statistically significant differences among groups; n=4 independent experiments).
Figure 6: Effects of BPA on estrogen receptor B expression and activity. H295A cells were treated with 10 nM of BPA for 48 h. At the end of treatment, levels of ERß protein in total (A) cytosolic (B) and nuclear (C) extracts were subjected to western blotting. Data are presented as mean ± SEM ( ** P <. 0.01 vs. control; n= 4 independent experiments).
E.
Figure 7: Effects of DPN and PHTPP on BPA-induced cell proliferation. H295A cells were treated with 10 nM DPN, 10 nM BPA, 100 nM PHTPP, or both PHTPP and BPA for 72 h. At the end of treatment, levels of PCNA protein (A&B), cyclin D1 and cyclin D2 protein (C&D) were determined by western blotting. Data are presented as mean ± SEM ( ** P<0.01, *** P<0.001 vs. control; different letters indicate statistically significant differences among groups; n=4 independent experiments).
Figure 8: Effects of DPN and PHTPP on BPA-induced Shh pathway activation. H295A cells were treated with 10 nM DPN, 10 nM BPA, 100 nM PHTPP, or both PHTPP and BPA for 48 h. At the end of treatment, levels of Shh protein (A&B) and Glil protein (C&D) were determined by western blotting. Data are presented as mean ± SEM ( ** P<0.01 vs. control; different letters indicate statistically significant differences among groups; n=4 independent experiments).
Figure 9: A schematic representation of the postulated molecular pathway by which BPA stimulates cell proliferation in human adrenal cells. BPA readily crosses the cell membrane into the cytoplasma where it binds to and activates ERB. The activated ERß translocates to the nucleus
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where it promotes transcription of the Shh gene, leading to increased Shh mRNA and protein. Shh is secreted, acts in an autocrine/paracrine fashion and binds to Patched 1 (Ptch1) receptor, preventing Ptch1 from inhibiting smoothened (SMO). SMO is then released from the plasma membrane into the cytoplasma, leading to the release of a complex containing the key Shh transcription factor Gli1. Glil translocates to the nucleus where it binds to the promoters of key proliferation factors CCNDI and CCND2, enhancing their transcription, and ultimately leading to increased cell proliferation.
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A
PCNA
GAPDH
200
PONGAPCH [% of control]
Control
150-
BPA
…
100-
T
50-
0
24 h
48 h
72 h
B
500
3 400-
Cel Number [% of control)
Control
BPA
*
300-
200-
T
100
0
24 h
48 h
72 h
C
PCNA
GAPDH
180
PONAGAPCH [% of control]
120-
60-
0
0
1
10
100
1000
BPA concertraton (nM)
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A
B
Cyclin D1
Cyclin D1
Cyclin D2
Cyclin D2
B-tubulin
[% of control) Cydin D2/B-tubulin Cyclin D1/3-tubulin (% of control)
B-tubulin
150
[% of control) Cydin De/B-tubun Cyclin D1/-tubulin .. (% of control]
T
200
**
100
150
100
50
50
0
C
BPA
0
150
200
C
BPA
100
150
100
50
60
0
C
BRA
0
C
BPA
48 h
72h
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A
sth/GAPCH [% of control)
200
B
150-
150
GI1/GAPCH [% of control]
100
100
50
50
0
C
BPA
O
C
BPA
c
shh
D
Glit
B-tubulin
B-tubulin
shhj’-tubulin [% of control)
200
150
Ginti-tubulin [% of control)
250
200
100
150
50
100
50
0
BRA
0
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A
B
Gi1
Gli1
-
GAPDH
Lamin 81
Glit/GAPOH (% of contra)
150
Git/Lamin 81 (% of control)
300
100
200
60-
100
0
Č
BPA Cytosolic
0
C
BPA Nuclear
C
Gli1
GAPOH
GI1/GAPCH (% of control)
200
b
150
a
a
100
a
60
0
Č
Cyc BPA Cyc
.BPA
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A PCNA
GAPDH
PCNA/GAPCH [% of control)
200
b
150
100
a
8
a
50-
O
C
Cyc
BPA
Cyc +BPA
B
Cyclin D1
Cyclin D2
B-tubulin
Oyclin D1.1-Lbulin (% of control)
160
b
120
a
a
a
80
40
0
C
Cyc
BPA
Cyc
Cydn De-tutun (% of control)
200
+BPA
b
150
ab
100-
a
a
60-
0
C
Cyc BPA Cyc
+BOA
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A
ERB
GAPDH
BRIVGAPCH (5% of control)
150
100
50
0
C
BPA
B
ERB
GAPDH
BRAGAPOH (% of control)
150
100
50-
0
C
BPA
Cytosolc
c
ER3
Lamin B1
ERIVLamin B1 (% of control)
250
₾ 200
**
-
150
100
50
0
C
BRA
Nuclear
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A
B
PCNA
PCNA
GAPDH
GAPDH
PCNAGAPCH (% of control)
200
PCNA/GAPOH (% of control]
250-
150
200
b
100
150
ab
T
a
a
50-
100
50-
O
C
DPN
0
C
PHTPP
BPA
PHTPP
c
D
+BPA
Cyclin D1
Cyclin D1
Cyclin D2
Cyclin D2
ß-tubulin
B-tubulin
Cyclin D1/stubulin [% of control)
200
Cyclin D1/-tubulin [% of control)
200-
b
150
150-
ab
a
a
100
100
50
50
0
C
DẪN
0
C
Cyclin D2/1-tubulin [% of control)
Cyclin D2/1-tubulin [% of control)
PHTPP BPA PHTPP
200
200-
b
+BPA
150
**
150
100
100-
a
a
a
50
50-
0
C
DẪN
0
C PHTPP BPA PH
PHTPP +BPA
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A
B shh
shh
B-Tubulin
B-tubulin
shh/1-tubulin (% of control)
200
shh’s-tubulin (% of control)
200
150
b
150
a
ab
8 T
100
100
50
50
0
Č
DPN
O
C
PHTPP BPA PHTPP
.BPA
C
G11
D
·
Gli1
GAPDH
GAPDH
150-
**
250
b
GITIGAPOH [% of control)
GI1/GAPOH [% of control]
200
T
100
150
a
&
50
100
a
50
0
C
DPN
O
C
PHTPP BPA PHTPP
+BPA
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ahh
SMO
Translocation
0#1
DPA
SMO
ERŞ
Glit
shh
Trarsioceton
Tratalocation
Transcription 0/11
Transcription
ERD
KXXX
CENDE COND2
XXXXCO SMM
Nucleus
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