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Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267
Address all correspondence and requests for reprints to: Scott M. Belcher, Ph.D., Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, 231 Albert Sabin Way, P.O. Box 670575, Cincinnati, Ohio 45267-0575. E-mail: scott.belcher{at}uc.edu.
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Introduction |
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and ERß that act as transcriptional transactivators to regulate estrogen-responsive gene expression. As in other tissues, rapid actions of E2 and chemicals with estrogen-like properties can also impact directly intracellular signaling in the brain (1, 2, 3, 4). The ERK1/2 are components of a MAPK signaling cascade that regulates a variety of important cellular events and in some cell types is rapidly responsive to E2 (1). These signaling intermediaries are expressed throughout the brain and play important roles in the regulation of neuronal proliferation, differentiation, viability, and postsynaptic information processing (5, 6, 7, 8, 9). In the brain, rapidly acting estrogen-dependent mechanisms can facilitate ERK-dependent enhancement of hippocampal long-term potentiation (10), protect primary cortical and hippocampal neurons from excitotoxicity (11, 12), influence midbrain dopaminergic neuron and astrocyte function (13, 14), and impact the development and viability of cerebellar neurons (15).
Xenoestrogens are man-made environmental contaminants defined by their ability to act synergistically with or antagonize the activity of endogenous estradiol. The prototypical xenoestrogen bisphenol A (BPA) is structurally similar to the potent nonsteroidal synthetic estrogen diethylstilbestrol, can bind ERs with low affinity to mimic or block some actions of endogenous E2, and has been linked to certain hormone-responsive cancers (16, 17, 18, 19). Along with acting through the ERs to influence E2-responsive gene expression, some environmental estrogens can also activate rapid intracellular signaling mechanisms that are normally activated by endogenous E2 (20, 21, 22, 23). BPA is produced in large amounts for use as a monomer in the production of polycarbonate plastics and epoxide resins that are used as coatings for food cans and plastic packaging, dental sealants, and water pipes. As a result, there is extensive human exposure to BPA that has been estimated to range from 2–20 µg/kg/d (24). During pregnancy, BPA is detectable in maternal (0.3–18.9 ng/ml) and fetal (0.2–9.2 ng/ml) serum, demonstrating ready passage of BPA through the placenta (25). Compared with other tissues, BPA is concentrated approximately 5-fold in amniotic fluid during early pregnancy, further indicating significant fetal exposure during important periods of human development (26). The levels of BPA to which adult and fetal humans are exposed negatively impacts reproductive function, neurodevelopment, and hippocampal synapse formation in rodents (27, 28, 29, 30, 31, 32). However, little is known about the impact of environmental estrogens on rapid estrogen-induced signaling during nervous system development.
Unlike more widely studied E2-sensitive neurons from the forebrain (e.g. the telencephalonic cereberal cortex and hippocampus and the diencephalonic thalamus and hypothalamus), the hindbrain developmental lineage of the metencephalon-derived cerebellum is unrelated to reproductive endocrine function of the brain. Because of the disconnect between the sexual/reproductive actions of E2, and current understanding of cerebellar development and function, the idea that the cerebellum is a primary E2-sensitive brain region has remained controversial. This controversy persists despite numerous in vitro and in vivo studies that clearly demonstrate ERs are expressed and functional in the developing and adult cerebellum (15, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45). Because there is little evidence supporting sexual differentiation of the cerebellum, we reasoned that during cerebellar development, E2 is involved with general developmental effects that represent a subset of E2 responses mediated through regulation of growth factor-like intracellular signaling. With the recent elucidation of the pattern of ERK activity during cerebellar ontogeny (9), we are now able to test the hypothesis that E2 rapidly modulates in vivo ERK signaling in the cerebellum and that low doses of the xenoestrogen BPA impact normal E2-regulated signaling functions in the developing brain. In the present study we examined the rapid effects that E2 and BPA, alone or in combination, have on ERK1/2 signaling in cells of the developing cerebellar cortex in vivo. Analysis of our results revealed that E2 and BPA, at low and comparable concentrations, could rapidly modulate ERK signaling in a dose-dependent fashion. Additionally, BPA was found to act as a highly potent disruptor of rapid E2-induced actions in the developing cerebellum. Those results suggest that low doses of BPA, and potentially other environmental estrogens, alter rapid actions of endogenous estrogens with potentially negative consequences to the developing brain.
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Materials and Methods |
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The heads of anesthetized animals were secured in a stereotaxic apparatus; neonatal animals were fixed in the stereotaxic apparatus equipped with a small animal adaptor (46). Fasciae and cervical musculature were dissected to expose the occipital bone, which was separated from the surrounding bones of the calvaria dorsally along the interparietooccipital suture and bilaterally along the dorsal half of the temporooccipital suture. Bilateral separation lines were directed ventromedially crossing the dorsal condyllar fossa to the dorsal arch of the foramen magnum just above the occipital condyles. The occipital bone was separated from the first cervical vertebra with a transverse cut of the dorsal atlantooccipital membrane. The occipital bone was removed and the dura mater and arachnoidea were cut along the midsagittal plane exposing the dorsocaudal cerebellar surface. In adults, injections were made through a small hole drilled into the midline dorsal surface of the skull 12.3 mm behind the bregma.
With a 5-µl Hamilton syringe attached to the stereotaxic apparatus, injections were directed into cerebellar folia 6 and 7. The needle was vertically lowered in the midsagittal plane to the suprafastigial region and pulled back 0.5 mm to create a cavity for the injected material. Cyclodextrin-encapsulated 17ß-estradiol or BPA (Sigma Chemical Co., St. Louis, MO) at each concentration (10–12 to 10–6 M) was injected (3 µl) into the cerebella of P4–10 animals, with older animals receiving 5 µl. For each age, noninjected, mock-injected, and equal molar vehicle-injected animals (2-hydroxypropyl-ß-cyclodextrin in PBS or PBS/0.001% dimethylsulfoxide; Sigma) were used as controls. Estradiol was also coinjected with either BPA or the selective MEK inhibitor UO126 (1 µM; Promega, Madison, WI). Six minutes after the initiation of injection, animals were rapidly decapitated and brains dissected and placed in ice-cold fixative [4% paraformaldehyde/3% acrolein in 0.1 M phosphate buffer (PB), pH 7.4]. Brains were fixed overnight and postfixed in 4% paraformaldehyde until tissue processing.
Immunohistochemistry
Immunohistochemical protocols for staining cerebellar sections with rabbit anti-phospho-p44/42 MAPK antiserum (1:400) (catalog no. 9101; Cell Signaling Technology, Inc., Beverly, MA) were described previously (9). Briefly, 40-µm-thick sections were cut and rinsed three times for 10 min in PB. To eliminate unbound aldehydes, sections were incubated for 20 min in 1% sodium borohydride, followed by six 7-min washes in PB. Free-floating sections were incubated overnight with primary antiserum at 4 C and then extensively washed with PB. Bound antibodies were visualized with nickel-intensified diaminobenzidine by the avidin-biotin peroxidase complex method following standard protocols (Vectastain ABC Kit; Vector Laboratories, Burlingame, CA). Digital images were captured with a SpotRT (Bridgewater, NJ) CCD camera using Image Pro Plus version 4.5 software. Final graphics were generated and labeled using Photoshop version 6.01 (Adobe, San Jose, CA) software.
Quantitative analysis
In neonatal animals, E2/BPA-induced changes in pERK-IR were generally confined to the internal granular layer (IGL) and corpus medullare of the posterior lobe; therefore, folium IX of the cerebellum was selected for OD measurements. Gray-scale digital images of one midsagittal section, at the mediolateral level of the injection site, per animal were analyzed using Image Pro Plus version 4.5. To correct for possible fluctuations in light intensity during image capture, and to ensure the accuracy of measurements, image brightness was normalized to background levels. The area of the IGL, including the corpus medullare, was outlined in folium IX, and the mean OD was determined with Image Pro Plus version 4.5 using Area/Mean OD function yielding OD values normalized to the calculated area expressed as arbitrary OD units. Preliminary studies comparing manual counts of immunopositive cells with OD measurements revealed a 1:1 correlation between the calculated OD and immunopositive cell number. Statistical analysis and graphic representation of data were prepared using Prism version 4.0 (GraphPad) software. The level of significance between groups was determined by one-way ANOVA with posttest comparison using Tukey-Kramer multiple comparison test. At P10, the density values for the different treatments (E2 and BPA) and concentrations were analyzed for significant differences by a two-factor ANOVA with specific group differences identified using Bonferroni’s posttest for multiple comparison (P < 0.05). A quantitative assessment of E2’s action was not performed for adults because of the highly focal nature of the observed changes in pERK-IR.
Analysis and fitting of E2 and BPA data
The biphasic effects of E2 and BPA at the low-dose concentration range (10–12 to 10–9 M) were assumed to act at two binding sites, one stimulatory with a high affinity and one with a lower affinity that inhibits the effect of the first site. Based on the observed similarity in effect at each dose and the fairly low resolution of the experiments, it was also assumed that neither of the two sites distinguishes between E2 and BPA. Thus, data points were fit with the Hill-type equation:
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Primary cultures of cerebellar neurons
Primary cerebellar granule cell cultures were prepared from male or female Sprague-Dawley rats at P7–9 and were maintained under serum-free conditions without the addition of exogenous steroids as previously described (15, 47). Quantitative Western blot analysis of rapid BPA-induced ERK phosphorylation in these primary cultures of cerebellar cells followed previously published protocols (15). Presented Western blot results are representative of four independent BPA dose-response experiments.
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Results |
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At P8, injection of E2 at 10–12 to 10–11 M and 10–7 to 10–6 M significantly increased pERK-immunopositive cell numbers in the IGL and the corpus medullare of the posterior cerebellar lobe. The effects of E2 were most pronounced in folium IX (Fig. 1
, A–D). The results of the in vivo E2 dose-response analysis indicated a bimodal dose dependency with no influence on ERK signaling at intermediate concentrations of E2 (e.g. Fig. 2A). These results were reminiscent of previous in vitro dose-response results obtained with a homogenous population of primary cultured cerebellar granule cell neurons (15).
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, A and E) (9). E2 injection further stimulated ERK phosphorylation in granule cells in the IGL and in cells of the corpus medullare (Fig. 1, E–H). The sensitivity to E2 exposure was spread to all lobes of the cerebellar cortex (Fig. 1, F and H, arrowheads). A bimodel dose response to E2 was again observed with 10–10 and 10–6 M being most efficacious at increasing pERK-IR cells numbers (Fig. 2B). In cerebella exposed to intermediate concentrations of E2 (10–9 to 10–8 M), the pattern and density of pERK-immunostained cells was comparable to controls (Fig. 2B). Coinjection of 10–10 M E2 with the selective MEK inhibitor U0126 completely blocked rapid E2-dependent increases in pERK-IR cell numbers (Fig. 3, A, B, and G).
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, D–F). Comparison of the in vivo dose-response curves for E2- and BPA-mediated effects on pERK-IR cell numbers reveals a striking similarity between the bimodal dose dependency of E2 and BPA (Fig. 3G). At low doses, E2 and BPA were equally potent and efficacious. The calculated EC50 for their low-dose effects were 7.46 and 3.25 pM, respectively. The low-dose data for E2 and BPA (10–12 to 10–9 M) fit well to two Hill-type components. The EC50 value for the stimulatory component was 8 pM, and the EC50 of the second inhibitory component was 0.4 nM (Fig. 3H). The Hill coefficients for the stimulatory and inhibitory components were 0.4 and 2, respectively. At high concentrations, BPA was significantly less efficacious (Fig. 3G), with average high-dose EC50 values of 126 nM for E2 and 539 nM for BPA.
The ability of BPA to inhibit rapid E2 actions was tested by coinjection of different concentrations of BPA with 10–10 M E2. This dose inhibition analysis revealed that essentially all rapid estrogenic effects of either estrogenic compound were blocked by an equal molar mixture of the maximally active concentrations (10–10 M) of E2 and BPA (Fig. 3, C and H). This blockade was also observed with a 1:1 mixture of 10–11 M E2 and BPA (not shown). Whereas 10–12 M of either BPA or E2 alone was able to significantly increase levels of pERK-IR (Fig. 3G), when presented as a mixture, 10–12 M BPA was able to block approximately 50% the pERK-stimulating activity of 10–10 M E2 (Fig. 3H).
The ability of BPA to stimulate ERK signaling in cerebellar granule cells was confirmed by quantitative Western blot analysis of pERK levels in primary cultures of neonatal cerebellar granule cells (Fig. 4, A and B). Compared with control, significant increases in levels of pERK1 and pERK2 were detected after 10-min exposures to 10–10, 10–8, and 10–6 M BPA. In response to 10–10 M BPA, the 4.2-fold pERK1 stimulation relative to vehicle-treated control was significantly greater than the 2.75-fold induction by 10–8 M BPA. Similar bimodality in efficacy was not detectable for ERK2 activation, suggesting that BPA may differentially influence rapid ERK1 and ERK2 signaling.
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, I–L, and 2C). The decrease in pERK-IR was most apparent at 10–6 M E2 (Figs. 1L, 2C, and 5A) with smaller, yet significant decreases observed with 10–11, 10–10, and 10–7 M E2 (Fig. 2C). In contrast to the overall reduction in the number of pERK-immunopositive cells at P12, E2 stimulated nuclear localized pERK-IR in scattered Golgi-like neurons (Fig. 5A, black arrowheads). In interneurons between P12 and P19, the pERK-stimulating effects of E2 progressively increased to the levels observed in adults (not shown).
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, inset), Golgi-like interneurons, basket cells, stellate cells, globular Lugaro-like cells (48) (Fig. 1, N and P, inset, and Fig. 5), and focally in clusters of Bergmann glia (Fig. 1P). In contrast to the pERK-stimulatory effects, E2 eliminated pERK-IR from the axon hillock and ventrolateral perikaryal regions of Purkinje cells, an effect not seen outside of the injected cerebellar folium (Fig. 5, B1–B2). |
Discussion |
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Many of the actions of E2 are mediated through the transcriptional activity of the intracellular ERs; however, E2 and estrogen-like chemicals can also rapidly influence intracellular signaling independent of classical ER-mediated transcriptional mechanisms (1, 11, 15, 51, 52, 53). Throughout the developing brain, activation of the ERK1/2 signaling cascade is involved in proliferation, differentiation, and neuronal plasticity (54, 55, 56, 57). The impact of these estrogenic compounds on ERK signaling in the developing cerebellum is likely to influence cerebellar development and neurotransmission in the mature cerebellum. Because rapid signaling actions of E2 are necessary for full expression of ER-mediated transactivation of estrogen-responsive gene expression (58), the ability of BPA to influence the rapid actions of endogenous E2 in the cerebellum at concentrations in the range of human fetal exposures (25) has especially profound implications regarding the ability of endocrine disrupting chemicals to modify the normal neurodevelopmental actions of endogenous estrogens.
Experiments using systemic injection of high concentrations of E2 into ERKO, ßERKO, and the double ßERKO knockout mice demonstrated that rapidly E2-induced cAMP signaling was dependent on the ERs normally expressed in a given brain region (59). In the medial preoptic nucleus, a brain region where ER and ERß are coexpressed (60, 61), E2 could also rapidly stimulate ERK signaling, with the loss of either ER blocking the rapid E2-induced increases in ERK phosphorylation observed in wild-type medial preoptic nucleus cells (59). Given this critical dependence on classical ER expression in rapidly responsive regions of the mouse brain, it is likely that in the cerebellum, where only ERß is expressed at any appreciable level (39, 40, 41, 50), the rapid effects of E2 on ERK phosphorylation are mediated through ERß. Additional evidence suggesting that ERß mediates rapid E2 actions in the cerebellum is the close correlation between the ontogeny of ERß expression in specific cell types (50) and the onset of detectable rapid actions of E2 on pERK-IR in those cells.
The effect of ontogeny on rapid actions of E2 and BPA
In the developing rat cerebellum, E2 rapidly modulated ERK phosphorylation after the first postnatal week. At P8, E2 increased the number of pERK-IR cells in the posterior lobe of the cerebellum. During cerebellar development, P8 is marked by a slowing from peak granule cell mitosis, the settlement of postmigratory granule cell neurons, and the establishment of mossy fiber afferents on the settled granule cells, processes that occur first in posterior cerebellar folia (62, 63). The established excitatory mossy fiber synapses on granule neurons must undergo further maturation before becoming fully functional around P12, and inhibitory Golgi cell efferents are established on granule cells only after P11 (63). Thus, during the time E2 stimulates pERK signaling in granule cells, the neural connectivity of granule cells is composed solely of immature excitatory synapses. Because both E2 and ERK signaling play important functional roles in the regulation of glutamatergic neurotransmission, excitatory synaptic dynamics, and assembly of the postsynaptic cytoskeleton (64), it is possible that the observed E2-induced increases in the number of pERK-IR cells reflect transient involvement with modulating the maturation and/or activity of immature excitatory synapses.
Intracerebellar injection of E2 at P12 rapidly decreased the number of pERK1/2-IR granule cells and stimulated pERK-IR in Golgi-like inhibitory neurons. Developmentally at P12, the maturation of granule cell excitatory afferents is completed. In contrast to earlier times during development, abundant inhibitory Golgi terminals are established on granule cell neurons and on the Golgi cells themselves. The correlation between formation of this inhibitory network and E2 decreasing the number of pERK-IR granule cells suggests that increased inhibitory synaptic transmission plays a cell-specific role in regulating the rapid responsiveness to estrogens in excitatory granule cells and Golgi interneurons. Although the nature of the developmental cues responsible for the difference in granule cell responsiveness are unknown, in maturing granule cells, between P8 and P12, there is a developmental switch in the estrogenic signaling mechanism linking the activated E2/ER complex to the ERK signaling cascade that results in blockade of baseline ERK signaling upstream of ERK1/2 and/or increased phosphatase activity in response to E2. At the level of ERK1/2, the result of those changes is an alteration in the balance between E2-induced kinase and dephosphorylating activities on the ERK signaling cascade.
At later times during postnatal cerebellar development, the effect of E2 on pERK signaling became progressively more adult-like. In the cerebellar cortex of adult rats, E2 activated the ERK pathway in inhibitory interneurons (stellate-, basket-, Lugaro-, and Golgi-like neurons) that are differentially responsible for the final shaping of Purkinje cell function. Stellate cells form inhibitory synapses on the stem of Purkinje cell dendritic branches, which selectively isolates dendritic branch excitation from the soma of Purkinje cells. Basket neurons block Purkinje cell output at the ventrolateral soma and adjacent axon hillock area of Purkinje cells, whereas Lugaro cells terminate on stellate, basket, and Golgi neurons (63). To synchronize their function, these three types of inhibitory cerebellar neurons synapse with each other, forming an intrinsic inhibitory network. Because E2 activates the ERK pathway in these inhibitory interneurons but eliminates pERK-IR from the axon hillock area of Purkinje cells, it is possible that E2 activation of ERK signaling in these interneurons is reflected in the dephosphorylation of pERK in Purkinje cells. Additional studies are underway to determine the changing functional role of E2-modulated ERK activation in cerebellar neurotransmission and plasticity.
Pharmacology and mechanism of rapid actions of E2 and BPA
The concentration-response data for rapid ERK actions of E2 and BPA in the cerebellum resulted in distinctly bimodal dose-response curves that were characterized by an inverted U-shaped low-dose effect from 10–12 to 10–10 M, and a high-dose effect at 10–7 to 10–6 M. Such atypical inverted U-shaped and bimodal dose-response curves describing the actions of hormones have begun to receive more widespread appreciation, with the differential actions that low and high concentrations and mixtures of estrogenic compounds have on the growth of the prostrate and breast cancer cells standing out as well known examples (65, 66, 67). As asserted previously (15), the differences between the efficacy of BPA and E2 at the high range, a distinction not apparent at low concentrations, suggest that the rapid ERK-signaling effects of low and high doses of these estrogens are mediated through different mechanisms.
The mechanisms responsible for the observed low-dose inverted U-shaped dose dependency of rapid ERK signaling in cerebellar neurons are unknown. However, if the rapid actions of E2 are mediated through ERß1 (Kd for E2, 0.14 nM) (68), the primary ERß isoform expressed in the cerebellum (49), some of the pharmacology underlying E2’s low-dose effects can be explained simply by the fact that detectable rapid effects, like most hormone actions, occur at agonist concentrations well below those necessary to saturate the available receptors. However, the relative binding affinity of BPA at ERß1 is approximately 15 times lower than for E2 (68). The ability of BPA to act as an equally potent agonist of rapid ERK phosphorylation and to inhibit the rapid actions of E2 at very low concentrations is inconsistent with rapid activation of ERK1/2 being dependent simply on the occupancy of the primary ERß1 ligand-binding site. Furthermore, the lack of responsiveness to E2 or BPA at concentrations above 10–10 M cannot be explained by increased occupancy and saturation of the ERß ligand-binding site alone. The finding that rapid low-dose agonist activity of both BPA and E2 were completely blocked when each was presented in a 1:1 mixture suggests that E2 and BPA are acting in an even more complicated fashion.
The pharmacological characteristics of the rapid low-dose agonist actions and the inhibitory actions of E2 and BPA suggest a model in which the rapid-acting ER complex contains a high-affinity (picomolar) stimulatory binding site and a lower-affinity (nanomolar) inhibitory binding site. The resulting concentration-response data fit well with a Hill-type equation that assumed a high-affinity stimulatory binding site and a lower-affinity site that inhibited the effect of the first site (Fig. 3H). The resulting stimulatory and the inhibitory Hill coefficients were 0.4 and 2, respectively. Because the Hill coefficient gives a quantitative measure of the degree of cooperativity between binding sites, a Hill coefficient of 0.4 for the low-dose ERK-stimulatory actions is suggestive of negative cooperativity. The existence of multiple intramolecular ligand-binding sites within the ligand-binding domain of classical nuclear ERs is supported by studies that have identified two tamoxifen-binding sites in ER (69, 70) and by the identification of a second E2-binding site within the ligand-binding domain of both ER and ERß (71, 72).
To explain the paradoxical ability of 1 pM BPA to stimulate ERK signaling in the absence of E2 and to also disrupt the same rapid action of E2 when presented in a mixture, it is proposed that there exists an additional high-affinity BPA-binding site that becomes available only upon ligand binding at the high-affinity site of the rapid ERK-stimulating receptor. Binding of BPA, or lower-affinity E2 binding, at this additional site stimulates an inhibitory activity. Based on our recent finding that along with rapidly stimulating ERK phosphorylation, E2 also rapidly stimulates a protein phosphatase 2A-like ERK-dephosphorylating activity in cerebellar granule cells (73), it is proposed that binding of E2 at the high-affinity site results in conformational changes to reveal a latent intermolecular BPA-binding site. The binding of BPA at that site acts to stimulate phosphatase activity via an independent signaling cascade. Although the proposed model represents a simplified working hypothesis, it is important to note that numerous factors such as the coexpression of multiple isoforms of ERß (e.g. ERß1 and ERß2) with differences in ligand-binding affinities, the potential influence of ERß homodimers and ERß1/ß2 heterodimers, posttranslational modifications of the receptors, localization of the receptor at the membrane, and the changeable nature of the receptor signaling complex all likely contribute to the complex pharmacological properties of the rapid actions of these estrogens.
In summary, we showed that E2 was able to rapidly regulate ERK signaling in the developing cerebellum in vivo and that there are cell-specific developmental changes in the sensitivity of excitatory and inhibitory neurons to the rapid effects of estrogens. The ability of the endocrine-disrupting chemical BPA to mimic and also inhibit the rapid actions of E2 highlights the potential for environmentally relevant concentrations of xenoestrogens to adversely effect development and function throughout brain.
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Acknowledgments |
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Footnotes |
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First Published Online August 25, 2005
Abbreviations: BPA, Bisphenol A; E2, estradiol; ER, estrogen receptor; GCP, granule cell precursor; IGL, internal granular layer; P4, postnatal d 4; PB, phosphate buffer.
Received May 10, 2005.
Accepted for publication August 16, 2005.
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References |
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6 per group); #, values for E2 and BPA at 10–6 M are significantly different (P < 0.05); @, difference between values for 10–10 M E2/U0126 are not significantly different from control values and are significantly different from the 10–10 M E2 treatment group (P < 0.001).
