The FASEB Journal express article 10.1096/fj.01-02-0313fje. Published online August 21, 2002. Low doses of the endocrine disruptor Bisphenol-A and the native hormone 17β-estradiol rapidly activate the transcription factor CREB Ivan Quesada, Esther Fuentes, M. Carmen Viso-León, Bernat Soria, Cristina Ripoll, and Angel Nadal Institute of Bioengineering, Miguel Hernández University, Campus of San Juan, Alicante 03550, Spain Corresponding author: Angel Nadal, Institute of Bioengineering, Miguel Hernández University, San Juan Campus. Carretera Alicante-Valencia Km 87. 03550, Alicante, Spain. E-mail: nadal@umh.es ABSTRACT Endocrine-disrupting chemicals (EDCs) are hormone-like agents present in the environment that alter the endocrine system of wildlife and humans. Most EDCs have potencies far below those of the natural hormone 17β-E2 when acting through the classic estrogen receptors (ERs). Here, we show that the environmental estrogen Bisphenol-A and the native hormone 17β-E2 activate the transcription factor, cAMP-responsive element binding protein (CREB) with the same potency. Phosphorylated CREB (P-CREB) was increased after only a 5-minute application of either BPA or 17β-E2 in a calcium-dependent manner. The effect was reproduced by the membrane-impermeable molecule E2 conjugated to horseradish peroxidase (E-HRP). The increase in P-CREB was not modified by the anti-estrogen ICI 182,780. Therefore, low-dose of BPA activates the transcription factor CREB via an alternative mechanism, involving a non-classical membrane estrogen receptor. Because these effects are elicited at concentrations as low as 10�9 M, this observation is of environmental and public health relevance. Key words: xenoestrogens • estrogen receptors • islets of Langerhans • non-genomic

O

ne of the most common chemicals that behaves as an endocrine disruptor is the compound 4,4�-isopronylidenediphenol, called Bisphenol-A (BPA). BPA was the first synthetic estrogen without a steroid structure, and its estrogenicity was described by

using the ovariectomized rat vaginal cornification assay at the end of 1930s (1). At the same time the same group discovered the potent estrogen diethylestilbestrol or DES (2), whose properties rapidly relegated BPA as a pharmaceutical agent. However, because of its properties as a cross-linking chemical, BPA was chosen by the chemical industry to widely produce plastic polymers, mainly polycarbonates (3, 4). Nowadays, it is used in the manufacture of barrier coatings for inner surfaces of food and beverage cans and is found at high concentrations in extracted food and water from autoclaved cans (5). BPA is used also in dental composites and sealants and as

bioactive bone cement. After a dental treatment, for instance, saliva samples contained amounts of BPA higher than 100 µg (6). Numerous other biological effects attributed to BPA have been described (4). The mechanism proposed for BPA actions is based in its binding to classic ERs (ERα and ERβ), inducing estrogenic signals that modify gene expression (3, 7�10). BPA has an affinity approximately 1:2000 of that of 17β-E2 for ER (7) and therefore it will activate ERs at concentrations within the micromolar level, which are higher than those commonly found in the environment. In addition to the classical pathway described in the above paragraph, BPA can bind to membrane estrogen receptors in order to exert rapid effects (11). Growing evidence suggests that estrogens act through alternative pathways involving membrane estrogen receptors (12). Indeed, a functional non-classical membrane estrogen receptor (ncmER) activated by both 17β-E2 and BPA at picomolar concentrations in the endocrine pancreas was described recently (11, 13). When this receptor is activated, the adenosine triphosphate (ATP)-dependent potassium channels (KATP) at the membrane of β-cells close in a protein kinase G (PKG)-dependent manner, and therefore the plasma membrane depolarizes, enhancing Ca2+ influx through voltage-dependent calcium channels (11, 14, 15). In excitable cells, increases in Ca2+ due to membrane depolarization trigger phosphorylation of CREB (16�19). Moreover, it is known that PKG stimulation also activates CREB (20). Phosphorylation of CREB on Ser133 enables CREB to modulate transcription of genes containing upstream cAMP/Ca2+ response elements (16�19). In here, we demonstrate that 1 nM of 17β-E2 and BPA produced calcium-dependent activation of CREB via a ncmER. Therefore, low-doses of the endocrine disruptor BPA, which can be found in the environment, can modulate gene transcription via an alternative pathway, independent of the classical mechanism involving nuclear ERs. MATERIALS AND METHODS Materials Fura-2 AM were from Molecular Probes (Leiden, Holland); ICI182,780 was a kind gift of Zeneca Pharmaceuticals, Madrid, Spain; Anti P-CREB antibody was from Calbiochem (Madrid, Spain). All other chemicals were from Sigma (Madrid, Spain), unless otherwise stated. Methods Cell isolation and culture Swiss albino OF1 male mice were killed by cervical dislocation. Pancreatic islets of Langerhans were isolated from mice by using collagenase as previously described (21). Isolated islets were dispersed into single cells by enzymatic digestion in the presence of 0.05% trypsin plus 0.02% ethylene diamine tetraacetate (EDTA; Flow Laboratories, Madrid, Spain) for 3 min. Before the trypsin step, we washed the islets three times in Krebs-Ringer containing the following (mM):

130 NaCl, 5 KCl, 1.1 MgCl2, and 10 N-(2-hydroxyethyl)piperazine-N′-ethanesulfonic acid (HEPES)-NaOH, in the absence of either added calcium or ethyleneglycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA). After the trypsin treatment, the cells were mechanically dispersed by gentle passage through a narrow Pasteur pipette. The cells were centrifuged, re-suspended in culture medium (RPMI 1640) supplemented with 10% (v/v) fetal calf serum, 2 mM L-glutamine, 50 iu/ml penicillin, and 50µg/ml streptomycin, at 37ºC in 5%CO2�95%O2 and plated on glass coverslips. Measuring intracellular calcium in single islet cells Cells were loaded with the calcium-sensitive dye Fura-2 by incubation for 40�60 min at room temperature with 5 µM of the acetoxymethyl derivative, Fura-2 AM. After loading cells were washed with the extracellular solution used during experiments (mM): 120 NaCl, 5 KCl, 25 NaHCO3, 1.1 MgCl2, and 2.5 CaCl2. Fura-2�loaded cells were imaged by using an inverted epifluorescence microscope (Zeiss Axiovert 200, Jena, Germany). A ratio image was acquired approximately every 3 s with an ORCA-100 CCD camera (Hammamatsu Photonics Iberica, Barcelona, Spain) by using a Lambda-10-CS dual filter wheel (Sutter Instrument Company, CA), equipped with 340 and 380 nm, 10 nm bandpass filters, and a range of neutral density filters (Omega opticals, Stanmore, UK). Data were acquired by using the Aquacosmos version 2.0 from Hammamatsu. In all experiments, control solutions contained the same amount of vehicle [dimethyl sulfoxide (DMSO), 10�5%] used for dissolving E2 and BPA. P-CREB immunofluorescence CREB phosphorylation was induced by stimuli application during 5 min; afterwards the stimuli were removed and the cells remained with control medium for 10 min. After this period, cells were fixed with 1% paraformaldehyde (w/v) and were permeabilized with 0.1% Triton X-100 for 10 min. Cells were incubated with and anti-CREB phospho-specific rabbit antibody (1:200) overnight at 4ºC. P-CREB was visualized with fluorescein-conjugated secondary antibodies (1:64) after a 1-h incubation at room temperature and by use of a LSM510 confocal microscope. Nuclei were visualized by using ethidium homodimer-1, 1 µM during 2 min�s incubation at room temperature. In all experiments, control solutions contained the same amount of vehicle (DMSO, 10�5 %) used for dissolving E2 and BPA. RESULTS Membrane depolarization increases P-CREB levels in single islet cells Intracellular calcium was monitored in cultured islet cells with the fluorescent dye Fura-2. Both stimulatory concentrations of glucose and high K+ cause Ca2+ influx in pancreatic β-cells, attributable to membrane depolarization and opening of voltage-dependent Ca2+ channels (21, 22) (Fig. 1A).

In other excitable cells, depolarization-induced Ca2+ entry couples to phosphorylation of CREB on Ser133 (P-CREB), which is the activated form of CREB (18). To study CREB phosphorylation in islet cells, we treated single cells in primary culture with either 8 mM glucose or 50 mM KCl for 5 min; we analyzed the cells 10 min later for P-CREB by immunostaining with an antibody specific for CREB phosphorylated on Ser133 (Fig. 1B, C). Cells were also stained with ethidium to identify nuclei (Fig. 1B, C). In the absence of a stimulatory glucose concentration (3G) few nuclei displayed P-CREB staining (Figs. 1B and 2). Membrane depolarization with 50 mM KCl caused a maximal increase in the percentage of nuclei with P-CREB staining (Figs. 1C and 2). Depolarization induced by 8 mM glucose, the physiological stimulus for β-cells, increased P-CREB labeled nuclei, which was prevented in the absence of extracellular Ca2+ (Fig. 2). Activation of CREB phosphorylation by 17β-E2 and BPA We first investigated the role of BPA and 17β-E2 in modifying [Ca2+]i levels, in cultured pancreatic islets cells. Both elevated [Ca2+]i at 1 nM concentration in the presence of 8 mM glucose (Fig. 3), due to depolarization-induced Ca2+ influx as previously described (11, 14). To explore CREB activation, P-CREB was examined in single cells in the presence of 8 mM glucose, which already activated CREB (Fig. 4A). Both 17β-E2 and BPA applied at 1 nM concentration for 5 min increased the number of labeled nuclei (Fig. 4B and 5). Remarkably, 1 nM BPA potentiates CREB activation induced by 8 mM glucose by about 35% (Figs. 4C and 5), a similar percentage to that induced by 1 nM BPA on [Ca2+]i elevations. CREB phosphorylation induced by 17β-E2 and BPA was abolished in the absence of extracellular calcium, consistent with the idea that activation of calcium influx by membrane depolarization is necessary for activation of CREB (Fig. 5A). 17β-E2 and BPA induced increases in P-CREB are independent of classical ERs Rapid effects of 17β-E2 are mediated by either a classical intracellular ER located at the cytosol or at the plasma membrane or a non-classical membrane estrogen receptor. The existence of both receptor types can be distinguished by their sensitivity to the anti-estrogen ICI 182,780. The effects mediated by a classical membrane receptor are blocked by ICI 182,780, whereas non-classical estrogen membrane receptors are insensitive to the antiestrogen (13, 23�25). The experiment of Figure 5B demonstrates that the mentioned anti-estrogen did not affect 17β-E2 and BPA-induced activation of CREB. Moreover, the rapid effects described should be triggered at the plasma membrane because they are rapid in onset and are reproduced by the membrane-impermeable E2 conjugated to horseradish peroxidase (E-HRP) (Fig. 5B). Therefore, the effects described should be mediated by a non-classical ER located at the plasma membrane. DISCUSSION This report demonstrates for the first time that equal dose of the endocrine disruptor BPA and the hormone 17β-E2 activate the transcription factor CREB via a membrane receptor. Activation of CREB should provoke the transcriptional activation of CREB-responsive genes. The finding that CREB activation is observed at a concentration 10-9 M gives this observation an environmental relevance.

The stimulus-secretion coupling process in pancreatic β-cells involves glucose metabolism, which provokes the closure of KATP channels responsible for the resting membrane potential. As a result, the plasma membrane potential depolarizes, opening the L-type voltage-dependent calcium channels and increasing [Ca2+]i , which triggers insulin secretion (26,27). In neurones, calcium influx through L-type calcium channels, but not through N- and P-/Q type, caused CREB phosphorylation (28). We have shown in figure 1 that depolarization induced by high extracellular KCl induced a rise in [Ca2+]i in mouse pancreatic islets cells. This [Ca2+]i rise was already demonstrated to be a calcium influx through L-type calcium channels, the only type of voltage gated calcium channel present in mouse β-cells (21,27). Depolarization of the membrane using a stimulatory glucose concentration also produced CREB phosphorylation, abolished in the absence of extracellular Ca2+. These experiments suggest that Ca2+ influx is necessary for the activation of CREB by membrane depolarization. Both 17β-E2 and BPA applied at 1nM potentiate glucose-induced Ca2+ signals as shown in figure 3. This rise in Ca2+ was demonstrated before to be a consequence of a decrease in KATP channel activity and the subsequent membrane depolarization (11,14,15). The Ca2+ entry provoked by 17β-E2 and BPA triggers CREB phosphorylation, as demonstrated by the absence of effect when extracellular Ca2+ was removed. Although Ca2+ is necessary for CREB activation, we can not discard a role for PKG in this process, since activators of PKG increased CREB phosphorylation in several cellular systems (17, 20). We have demonstrated previously that 17β-E2 rapidly activates PKG in pancreatic β- and α-cells (13, 15), and therefore a role for PKG in CREB phosphorylation is possible. Most developmental effects of estrogens are mediated by their binding to an intracellular steroid receptor protein, either ERα or ERβ (12). Reports of environmental estrogen affecting sexual development and health of wildlife suggested that EDC function as estrogens (29). BPA is not an exception; it has been demonstrated that it binds to ER by using a rat uterine bioassay (6, 7). BPA also compete for 17β-E2 to ERβ (9), and its binding to ERs results in a list of effects of genomic nature (4, 10). The affinity of BPA for the classical ER is approximately 1/2000 that of the natural estrogen 17β-E2 (7). This relatively low potency seems to be a general rule for most EDC, which have potencies 1/50th to 1/10000th those of 17β-E2 (30, 31). This fact has opened an ongoing debate about whether the concentration of EDC commonly found in the environment can indeed cause health disorders (8, 32�34). We demonstrate here, however, that BPA and 17β-E2 are equally potent in activating the transcription factor CREB, and they do so at concentrations as low as 10�9M. The difference is that in the present work, BPA acts via a membrane receptor rather that via a nuclear ER. Until now, few reports have described the mimicry of estrogens and EDC at the membrane level. It has been shown that EDC, others that BPA, modulate K+ and Ca2+ channels (35), the xenoestrogen DES produces rapid prolactin release (36), and that pesticides provoke testicular androgen production (37). Plenty of evidence indicates the existence of membrane estrogen receptors, either with a structure identical to the classical ERs, related to the classical ERs or

unrelated to them (25). Rapid CREB activation has been described in the nervous system, likely via a membrane estrogen receptor (38, 39). Rapid effects of estrogens via an ER concentrated in caveolae, pharmacologically similar if not identical to the nuclear ERα, have recently been demonstrated to activate MAPK, AP-1 and CREB (40). Recently, we described the existence of a common receptor for 17β-E2 and xenoestrogens, including BPA, in pancreatic β-cells (11). This receptor is unrelated to ERα and ERβ. and it is insensitive to the antiestrogens ICI 182,780 and tamoxifen. We have named it non-classical membrane estrogen receptor (ncmER) (11, 13). In the present report, we demonstrate that the receptor involved in BPA-induced CREB activation is unaffected by ICI 182,780. Moreover it should be a membrane receptor because the effects of both 17β-E2 and BPA are rapid (5 min�s exposure is sufficient) and are reproduced by the membrane-impermeable molecule E-HRP. These results show that low dose of BPA may alter gene expression via a new pathway that involves CREB activation after binding to a ncmER. This represents a new mechanism that should help to understand the differences found in the estrogenicity of endocrine disruptors. ACKNOWLEDGMENTS The authors thank A. Pérez for technical assistant and Ana B. Ropero for her help in several aspects of the project. M.C.V.L. is a recipient of a research studentship from Instituto de Salud Carlos III. This work was supported by a grant from Instituto de Salud Carlos III, FISss. REFERENCES

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Received April 18, 2002; accepted June 27, 2002.

Fig. 1

Figure 1. Membrane potential depolarization caused an elevation in [Ca2+]i in single islet cells, loaded with the calcium fluorescent dye Fura-2. Cells in the presence of 8 mM glucose were exposed to 30 mM KCl to depolarize the membrane and cause opening of voltage-dependent L-type calcium channels. B) In the presence of a basal glucose concentration of 3 mM, a scarcity of cells was stained with P-CREB antibody (9±3%, 174 cells in three different experiments) (left panel), nuclei were stained in red with ethidium (middle panel). Transmitted image (right panel) (C) Membrane depolarization induced by KCl produced staining of P-CREB in 83±3% of the cells tested (248 in three different experiments). All nuclei were stained with ethidium (middle panel).

Fig. 2

Figure 2. Summary of the effects of membrane depolarization with either KCl, 8 mM glucose (8G) or 8 mM glucose in the absence of extracellular Ca2+ (0Ca2+). Numbers are expressed as percentage ± SEM of cells positive for nuclear P-CREB. Results are representative of at least 150 cells in three different experiments.

Fig. 3

Figure 3. Bisphenol-A (1 nM) mimics 1 nM 17β-E2 in producing elevations of [Ca2+]i in mouse pancreatic β-cells loaded with the calcium sensitive dye Fura-2. Cells are in the presence of 8 mM glucose (30% of the cells tested, n = 80).

Fig. 4

Figure 4. Glucose (8 mM) triggers CREB phosphorylation in more that 30% of the cells (left panel). Ethidium staining in red (middle panel). Transmitted image (right panel) (B) 1 nM 17β-E2 potentiates CREB activation induced by 8 mM glucose by a 38% (right panel). Ethidium staining (middle panel). C) BPA (1 nM) potentiates CREB activation induced by 8 mM glucose by a 38% (right panel). Ethidium staining (middle panel).

Fig. 5

Figure 5. Summary of the effects of treatment: Glucose (8 mM, 8G); 17β-estradiol (1 nM, E2); 17β-estradiol (1 nM) in the absence of extracellular Ca2+ (E2+0Ca2+); Bisphenol-A (1 nM, BPA); BPA (1 nM) in the absence of extracellular Ca2+ (BPA+ 0Ca2+), all stimuli are in the presence of glucose (8 mM). B) Summary of the effects of treatment: glucose (8 mM, 8G); 17β-estradiol (1 nM, E2); E2 (1 nM) conjugated to horseradish peroxidase (E-HRP); 17β-E2 (1 nM) in the presence of ICI182,780 (1 µM, E2+ICI); Bisphenol-A (1 nM, BPA); Bisphenol-A (1 nM) in the presence of ICI182,780 (1µM, BPA+ICI); all stimuli are in the presence of glucose (8 mM). Numbers are expressed as percentage ±SEM of cells positive for nuclear P-CREB. Results are representative of at least 150 cells in at least three different experiments. *P<0.05, student t-test comparing each condition with 8G.