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Low doses of the endocrine disruptor bisphenol-A and the native hormone 17ß-estradiol rapidly activate transcription factor CREB¹

Institute of Bioengineering, Miguel Hernández University, Campus of San Juan, Alicante 03550, Spain

²Correspondence: Institute of Bioengineering, Miguel Hernández University, San Juan Campus. Carretera Alicante-Valencia Km 87. 03550, Alicante, Spain. E-mail: nadal{at}umh.es

SPECIFIC AIM

The aim of the present work was to investigate the effect of a low dose of the endocrine disruptor chemical bisphenol-A (BPA) in activating the transcription factor CREB. We demonstrate that an equal dose of BPA and 17ß-estradiol (17ß-E2) activates CREB in a calcium-dependent manner via a nonclassical membrane estrogen receptor. Since these effects are elicited at concentrations as low as 10^-9 M, this observation has environmental relevance.

PRINCIPAL FINDINGS

1. Membrane depolarization increases P-CREB levels in single islet cells
In pancreatic ß cells, stimulatory concentrations of glucose and extracellular high K⁺ cause Ca²⁺ influx due to membrane depolarization and opening of voltage-dependent Ca²⁺ channels.

In other excitable cells, depolarization-induced Ca²⁺ entry couples to phosphorylation of CREB on Ser133 (P-CREB), which is the activated form of CREB.

To study CREB phosphorylation in islet cells, cells in primary culture were treated with either a stimulatory glucose concentration 8 mM glucose or 50 mM KCl for 5 min; cells were analyzed 10 min later for P-CREB by immunostaining with an antibody specific for CREB phosphorylated on Ser133. Cells were also stained with ethidium to identify the total number of nuclei.

In the absence of a stimulatory glucose concentration (3 mM), few nuclei displayed P-CREB staining (Fig. 1 ). Membrane depolarization with 50 mM KCl caused a maximal increase in the percentage of nuclei with P-CREB staining (Fig. 1) . Depolarization induced by 8 mM glucose, the physiological stimulus for ß cells, increased P-CREB labeled nuclei, which was prevented in the absence of extracellular Ca²⁺ (Fig. 1) .

View larger version (25K): [in this window] [in a new window]   Figure 1. 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+). CREB activation was determined in pancreatic islet cells from mice in primary culture. CREB phosphorylation was induced by stimuli application for 5 min; the stimuli were then removed and cells remained in the control medium for 10 min. Cells were incubated with an anti-CREB phospho-specific rabbit antibody and P-CREB was visualized with fluorescein-conjugated secondary antibodies using an LSM510 confocal microscope. Nuclei were visualized using ethidium homodimer-1. In all experiments, control solutions contained the same amount of vehicle (DMSO, 10-5 %) used for dissolving E2 and BPA. Numbers are expressed as percentage ± SE of cells positive for nuclear P-CREB. Results are representative of at least 150 cells in 3 different experiments. *P < 0.05, Student’s t test comparing each condition with 8G.

2. BPA and 17ß-E2 activate CREB in a Ca²⁺-dependent manner via an alternative mechanism
Bisphenol-A and 17ß-estradiol elevate [Ca²⁺]_i at concentrations as low as 1 nM; this increase in Ca²⁺ is due to a depolarization-induced Ca²⁺ influx. Since depolarization-induced Ca²⁺ entry couples to phosphorylation of CREB on Ser133 (P-CREB) (Fig. 1) , we sought to investigate whether BPA and 17ß-E2 activate CREB.

When BPA and 17ß-E2 were applied at 1 nM for 5 min, the number of nuclei labeled with the anti P-CREB antibody was increased (Fig. 2 A). Remarkably, 1 nM BPA potentiates CREB activation induced by 8 mM glucose by 35% (Fig. 2A ), a percentage similar to that induced by 1 nM BPA on [Ca²⁺]_i elevations. CREB phosphorylation induced by 17ß-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. 2A ).

View larger version (32K): [in this window] [in a new window]   Figure 2. A) Summary of the effects of treatment with 8 mM glucose (8G), 1 nM 17ß-estradiol (E2), 1 nM 17ß-estradiol in the absence of extracellular Ca2+ (E2+0Ca2+), 1 nM bisphenol-A (BPA), 1 nM BPA in the absence of extracellular Ca2+ (BPA+ 0Ca2+); all stimuli were in the presence of 8 mM glucose. B) Summary of the effects of treatment with 8 mM glucose (8G), 1 nM 17ß-estradiol (E2), 1 nM E2 conjugated to horseradish peroxidase (E-HRP), 1 nM 17ß-E2 in the presence of 1 µM ICI 182,780 (E2+ICI), 1 nM bisphenol-A (BPA), 1 nM bisphenol-A in the presence of 1 µM ICI 182,780 (BPA+ICI); all stimuli were in the presence of 8 mM glucose. Numbers are expressed as percentage ± SE of cells positive for nuclear P-CREB. Results are representative of at least 150 cells in at least 3 different experiments. *P < 0.05, Student’s t test comparing each condition with 8G.

Both 17ß-estradiol- and bisphenol-A-induced activation of CREB were independent of classic ERs, since the specific ER blocker ICI 182,780 was without effect (Fig. 2B ). The membrane-impermeable molecule estradiol conjugated to peroxidase (E-HRP) mimics their effects, suggesting the involvement of a membrane receptor (Fig. 2B ).

CONCLUSIONS

Most developmental effects of estrogens are explained by their binding to an intracellular estrogen receptor protein, either ER or ERß. Reports of environmental estrogen affecting sexual development and health of wildlife suggested that endocrine disruptors function as estrogens. BPA is not an exception, it has been demonstrated to bind to both ER and ERß, inducing different effects of genomic nature.

However, the affinity of BPA for the classic ER is 1/2000th that of the natural estrogen 17ß-E2. This relatively low potency, seems to be a general rule for most endocrine disruptors, which have potencies from 1/50th to 1/10000th those of 17ß-E2. This has opened an ongoing debate about whether the concentration of endocrine disruptors commonly found in the environment can indeed cause health disorders.

We demonstrate here, however, that BPA and 17ß-E2 are equally potent in activating the transcription factor CREB, and they do so at concentrations as low as 10^-9 M. The difference is that in the present work BPA acts via a membrane receptor rather than via a nuclear ER.

We propose that BPA and 17ß-E2 act by binding to a common receptor in the plasma membrane of pancreatic ß cells. This receptor is pharmacologically different from ER and ERß and we have named it "nonclassical membrane estrogen receptor" (ncmER) (Fig. 3 ). The binding of either BPA or 17ß-E2 produces the closing of ATP-dependent potassiumchannels in a PKG-dependent manner and subsequent potentiation of calcium signals. The increase in [Ca²⁺]_i provokes the phosphorylation of CREB (Fig. 3) , which should modulate the transcription of genes containing upstream cAMP/Ca²⁺ response elements.

View larger version (91K): [in this window] [in a new window]   Figure 3. Proposed model for the action of BPA and 17ß-E2. Binding of BPA or 17ß-estradiol to the nonclassical membrane estrogen receptor (ncmER) activates a guanylyl cyclase and protein kinase G (PKG), closing KATP channels. The subsequent depolarization (Vm) opens L-type calcium channels, inducing Ca2+ influx. As a result, CREB is phosphorylated. Activation of CREB will modulate the transcription of genes containing upstream cAMP/Ca2+ response elements. A stimulatory glucose concentration is needed to produce these effects.

These findings show that a low dose of bisphenol-A 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 us to understand the differences found in the estrogenicity of endocrine disruptors.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0313fje; to cite this article, use FASEB J. (August 19, 2002) 10.1096/fj.02-0313fje

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