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. 2011 Jul 6;85(4):770–778. doi: 10.1095/biolreprod.111.091926

Does a Nonclassical Signaling Mechanism Underlie an Increase of Estradiol-Mediated Gonadotropin-Releasing Hormone Receptor Binding in Ovine Pituitary Cells?1

Tracy L Davis 1,3, Jennifer D Whitesell 1,4, Jeremy D Cantlon 1, Colin M Clay 1, Terry M Nett 1,2
PMCID: PMC3184292  PMID: 21734267

Abstract

Estradiol-17beta (E2) is the major regulator of GnRH receptor (GnRHR) gene expression and number during the periovulatory period; however, the mechanisms underlying E2 regulation of the GNRHR gene remain undefined. Herein, we find that E2 conjugated to BSA (E2-BSA) mimics the stimulatory effect of E2 on GnRH binding in primary cultures of ovine pituitary cells. The time course for maximal GnRH analog binding was similar for both E2 and E2-BSA. The ability of E2 and E2-BSA to increase GnRH analog binding was blocked by the estrogen receptor (ER) antagonist ICI 182,780. Also, increased GnRH analog binding in response to E2 and the selective ESR1 agonist propylpyrazole triol was blocked by expression of a dominant-negative form of ESR1 (L540Q). Thus, membrane-associated ESR1 is the likely candidate for mediating E2 activation of the GNRHR gene. As cAMP response element binding protein (CREB) is an established target for E2 activation in gonadotrophs, we next explored a potential role for this protein as an intracellular mediator of the E2 signal. Consistent with this possibility, adenoviral-mediated expression of a dominant-negative form of CREB (A-CREB) completely abolished the ability of E2 to increase GnRH analog binding in primary cultures of ovine pituitary cells. Finally, the presence of membrane-associated E2 binding sites on ovine pituitary cells was demonstrated using a fluorescein isothiocyanate conjugate of E2-BSA. We suggest that E2 regulation of GnRHR number during the preovulatory period reflects a membrane site of action and may proceed through a nonclassical signaling mechanism, specifically a CREB-dependent pathway.

Keywords: GnRH receptors, membrane estrogen receptors, ovine, pituitary

Estradiol increases numbers of GnRH receptors in the ovine pituitary via a nonclassical signaling mechanism.

INTRODUCTION

The most dynamic time during the estrous cycle with respect to hypothalamic-pituitary-ovarian interaction is the preovulatory period when serum concentrations of progesterone decline as a result of prostaglandin-F2α (PGF2α)-mediated luteolysis and serum concentrations of estradiol-17β (E2) increase with the development of the preovulatory follicle. This pattern of ovarian hormone secretion leads to important changes at the hypothalamus and anterior pituitary. During the preovulatory period, there is an E2-induced increase in GnRH receptor (GnRHR) numbers in the anterior pituitary [1, 2] followed by an increase in release of GnRH [3]. Thus, both heightened pituitary responsiveness to GnRH and increased GnRH secretion underlie the generation of the ovulatory LH surge. These effects appear to be conserved across most mammalian species, including sheep [1, 3], cattle [2, 4], monkeys [5], mice [6], and rats [7, 8].

The stimulatory effect of E2 on the number of GnRHR is evident in the absence of hypothalamic input and in the primary cultures of ovine [9], rat [10, 11], and murine [6] pituitary cells. Thus, this effect of E2 is mediated directly at the anterior pituitary gland. To determine whether the large number of GnRHR during the preovulatory period is the result of increased expression of the GnRHR gene, several investigators have measured GNRHR mRNA after induction of luteolysis in sheep [12–14]. Concentrations of GNRHR mRNA are increased as early as 12 h after luteolysis and remain high at 24 h [12]. In the early preovulatory period, increased GNRHR gene expression preceded an increase in the number of GnRHR [12], and the maximal number of GnRHR were observed later in the preovulatory period near the onset of the LH surge [1, 14], which occurs approximately 56 h after the administration of PGF2α [1]. These temporal relationships support the hypothesis that increased amounts of GNRHR mRNA lead to greater numbers of GnRHR that in turn maximizes pituitary sensitivity to GnRH in preparation for the LH surge. Thus, the density of GnRHR on gonadotrophs determines their ability to respond to GnRH [15].

To date, virtually nothing is known about the mechanisms underlying the regulation of GnRHR expression by E2. This is troubling because E2 is considered the primary regulator of gene expression and subsequent number of GnRHR on gonadotrophs. Based on the ability of membrane-impermeable E2 conjugates to elicit an acute decrease in LH secretion independent of a hypothalamic site of action [16, 17], we speculated that E2 activation of the GNRHR gene may be initiated at a plasma membrane site of action and increase the number of GnRHR, as determined by GnRH binding, in a nonclassical signaling manner. Herein, we show that E2 conjugated to BSA (E2-BSA) mimics the effects of free E2 on enhancing GnRHR binding in ovine pituitary cells. Furthermore, we suggest that ESR1 (also known as ERα) is the likely candidate for mediating E2 input to the GNRHR gene via a mechanism that is dependent on a member of the cAMP response element binding (CREB) family of transcription factors.

MATERIALS AND METHODS

Materials

Collagenase Type II, hylauronidase Type V, deoxyribonuclease I, fluorescently labeled E2-BSA, E2, and Sephadex G-25 were purchased from Sigma (St. Louis, MO). Estradiol conjugated to BSA was purchased from Steraloids Inc. (batch R288; Newport, RI). Fetal bovine serum (FBS) was purchased from Gemini Bio-Products (West Sacramento, CA). Penicillin/streptomycin and Dulbecco modified Eagle medium (DMEM) were purchased from Mediatech, Inc. (Herndon, VA). Matrigel was purchased from BD Biosciences (San Jose, CA). Glass bottom culture dishes were purchased from MatTek Corp. (Ashland, MA). The estrogen receptor (ER) antagonist (ICI 182,780) and propylpyrazole triol (PPT) were purchased from Tocris Bioscience (Ellisville, MO). The antibody to the estrogen receptor (clone H222) was purchased from Lab Vision Corp. (Fremont, CA). Concanavalin-A conjugated to Alexa 594 was obtained from Molecular Probes (Invitrogen, Carlsbad, CA). All the remaining chemicals were purchased from Sigma or Fisher Scientific (Fairlawn, NJ).

Preparation of the E2 Conjugate and Fluorescently Labeled E2 Conjugate

The E2 conjugated to BSA (E2-BSA; 1,3,5(10)-estratrien-3, 17β-diol-6-one 6-carboxymethyl oxime:BSA) contained six molecules of E2 linked to each molecule of BSA. To remove free E2 from E2 conjugated to BSA, 10 mg of E2-BSA were resuspended in 1 ml PBS. Five ml of diethyl ether were added, and the contents of the tube were vortexed for 1 min and then frozen in a dry-ice methanol bath. The organic phase was decanted and discarded. Extraction of the E2-BSA stock was repeated an additional six times. Based on previous extractions, approximately 20 pg/ml of free E2 remained within the E2-BSA conjugate after seven extractions (Arreguin-Arevalo and Nett, unpublished). As described previously [17], 6-keto-17β-estradiol-6-carboxymethyl oxime (E2-6-CMO; Steraloids Inc.) was conjugated to a 15-amino acid sequence (N-terminus-SGGEVVVDQPMERLY-C-terminus) on the amino group of the serine reside. The conjugation reaction was added to a Sephadex LH20 column to separate E2-6-CMO from the conjugated form (E2-PEP). The presence of the conjugate and absence of free E2-6-CMO in the first peak eluted from the Sephadex LH20 column was confirmed using a Waters QTOF-micro electrospray mass spectrometer with the sample introduced by direct infusion (Macromolecular Resources, Colorado State University, CO). The mass spectrometry confirmed that there was one molecule of E2 linked to each peptide molecule and that the conjugate was devoid of free E2. Fluorescently labeled E2 conjugate (E2-BSA-FITC) was prepared as previously described to remove potentially contaminating free fluorescein isothiocyanate (FITC). To separate FITC from the E2-BSA-FITC, 1 mg of E2-BSA-FITC diluted in PBS was added to a Sephadex G-25 column (0.6 × 11 cm) and eluted with 0.05 M PBS-gelatin [18]. One-ml fractions were collected, and the fraction containing the dark yellow band (fraction 3) was dialyzed at 4°C against 1 L of PBS for 24 h to remove any remaining free FITC.

Dissociation and Culture of Ovine Pituitary Cells

All the procedures involving animals were approved by Colorado State University Animal Care and Use Committee and were in compliance with National Institutes of Health (NIH) guidelines. Sexually mature ewes that had been ovariectomized for at least 30 days prior to pituitary collection were used. Anterior pituitary glands were collected from ewes following anesthesia with sodium pentobarbital and exsanguination. Pituitaries were immediately placed in dissociation medium (137 mM NaCl, 25 mM HEPES, 10 mM glucose, 5 mM KCl; pH 7.4) and transported to the laboratory on ice. Pituitaries were dissociated as previously described [19] with minor modifications. Briefly, pituitary tissue was sectioned into 0.5 mm slices using a Stadie-Riggs tissue slicer. Pituitary slices were washed with dissociation medium and transferred to a flask containing dissociation medium with collagenase Type II (3850 units), hylauronidase Type V (100 units), and deoxyribonuclease I (100 ng). Pituitary tissue was incubated at 37°C in a Dubnoff metabolic shaker for 90 min. Cells were collected by centrifugation and washed with dissociation medium without enzymes; following the final wash, the cells resuspended in phenol red-free DMEM medium containing 10% charcoal-stripped FBS. For all the culture experiments, 2 × 106 cells were plated in 6-well tissue culture dishes at 37°C in an atmosphere of 5% CO2 for at least 36 h prior to addition of the treatments.

Effect of E2 on Percent Change of GnRH Receptors

An initial experiment was conducted to determine if a short duration of exposure to E2, after which the E2 was removed from the culture and the cells were incubated for an additional 12 h, would increase the number of GnRH analog binding to GnRHR in ovine pituitary cells compared to vehicle-treated cells. Anterior pituitary glands were collected from ovariectomized ewes during the month of May and prepared as previously described. Pituitary cells were treated for 15, 30, or 60 min at 37°C with either vehicle (0.01% ethanol), E2 (40 nM), or E2-BSA (40 nM). The concentration of E2-BSA was based on the molecular weight of BSA (66 kDa). After 15-, 30-, and 60-min treatments, the medium was removed, cells were washed twice with PBS, and E2-free medium was added to the cells. Cells were then cultured until 12 h posttreatment, which has been shown to be the time of maximal increase in number of GnRHR in ovine pituitary cells [9]. An additional treatment was included in which pituitary cells were cultured with E2 (40 nM) or E2-BSA (40 nM) for the entire 12 h incubation. Relative number of GnRHR was determined by radioreceptor assay [20]. Data are presented as GnRH analog binding as a percent of control for three pituitaries.

Because the previous data were obtained using a single, rather high, concentration of E2 and E2-BSA, an experiment was conducted using various concentrations (0.1 pM to 10 nM) of E2 or E2-BSA. Vehicle (0.01% ethanol) or the various concentrations of E2 or E2-BSA were added to the cells and incubated for 12 h. The relative number of GnRHR was determined by radioreceptor assay in triplicate for each treatment. Data are presented as GnRH analog binding as a percent of control and replicated three times using anterior pituitary glands collected from ovariectomized ewes during the months of January and February. Because of the controversy of using E2-BSA conjugate and the question of removal of all the free E2, an additional study was conducted following the experiments described above using pituitary cells treated with various concentrations (0.1 pM to 10 nM) of E2-PEP for 12 h. The relative number of GnRHR was determined by radioreceptor assay. Data are presented as GnRH analog binding as a percent of control and replicated four times using anterior pituitary glands collected from ovariectomized ewes during the anestrous period (month of July).

To determine if the time course for increasing the number of GnRHR in gonadotrophs was similar for E2 and E2-BSA, pituitary cells were cultured with vehicle (0.01% ethanol), 0.1 nM E2, or 10 nM E2-BSA for 3, 6, 9, or 12 h before the end of the incubation. Relative number of GnRHR was determined in duplicate by radioreceptor assay and reported as GnRH analog binding as a percent of control. The experiment was replicated four times using anterior pituitary glands collected from ovariectomized ewes during the months of January and February.

To determine if the effects of the estrogens could be inhibited, cells were cultured in the presence or absence of an ER antagonist, ICI 182,780. Pituitary cells were cultured for 12 h with 0.1 nM E2 or 10 nM E2-BSA with or without 10 μM ICI 182,780, and then the relative number of GnRHR was determined in triplicate for each treatment. Data were replicated four times using anterior pituitary glands collected from ovariectomized ewes during the months of January and February and are presented as percent change of GnRH analog binding.

Effect of a Dominant-Negative ESR1 Mutant on E2 and an ESR1 Agonist-Stimulated Increase in GnRH Receptors

To determine if the increase in GnRH analog binding was mediated through ESR1, a dominant-negative mutant for ESR1 (L540Q) and PPT, a selective ESR1 agonist, were used. The dominant-negative ESR1 mutant was kindly provided by Dr. J. Larry Jameson (Northwestern University, Feinberg School of Medicine, Chicago, IL). This form of ESR1 has a mutation within the COOH-terminal activation domain and exhibits similar binding affinity for E2 as the wild-type receptor [21]. Further, it has been suggested that this mutant ESR1 functions as a dominant-negative protein by its inability to recruit the steroid receptor coactivator proteins [22]. An adenovirus was constructed using a bacterial recombination of the L540Q dominant-negative mutant in pAdTrackCMV with pAd-Easy in BJ5183 Escherichia coli as described by He et al. [23]. Pituitary cells were infected for 24 h with the adenoviral vector containing the L540Q dominant-negative mutant form of ESR1. Twenty-four hours postinfection, pituitary cells were treated with either 4 nM E2 or 0.82 nM PPT for 12 h, and GnRH analog binding was determined by radioreceptor assay. The experiment was replicated three times.

Effect of a Dominant-Negative Inhibitor of CREB on E2-Stimulated Increase in GnRH Receptors

To determine if the CREB pathway was involved in the estrogen-stimulated increase in numbers of GnRH receptors, a dominant-negative inhibitor of CREB (A-CREB) was used. The dominant-negative inhibitor, A-CREB, was kindly provided by Charles Vinson (Laboratory of Biochemistry, National Cancer Institute, National Institutes of Health, Bethesda, MD). The dominant-negative form of A-CREB prevents the basic region of wild-type CREB from binding to DNA [24]. The coding sequence of A-CREB including the N-terminal FLAG epitope was amplified from pRc/CMV, and a Kpn1 site was engineered immediately before the start codon. The PCR product was digested by Kpn1 and HindIII and inserted into pAdTrackCMV at these sites. Adenovirus was constructed using bacterial recombination of A-CREB in pAdTrackCMV with pAd-Easy in BJ5183 E. coli using standard procedures [23]. Cells were infected for 24 h with adenoviral vectors containing A-CREB or GFP, which was used as a control. Cells were treated with 0.1 nM E2 for 12 h, and GnRH analog binding was determined by radioreceptor assay. The experiment was replicated five times.

Cellular Localization of Estrogen Receptors

Dissociated pituitary cells were plated on Matrigel-coated 35 mm glass bottom culture dishes. After overnight incubation, the pituitary cells were washed twice with PBS and incubated at room temperature for 2 min with 10 nM E2-BSA-FITC in PBS. Cells were washed three times with PBS after E2-BSA-FITC labeling and incubated 10 min with 2% paraformaldehyde. Cells were washed following fixation, and the plasma membranes were labeled with concanavalin-A conjugated to Alexa 594. To inhibit binding of E2-BSA-FITC to the plasma membrane, cells were pretreated with E2 (10 μM), an antibody directed against the ligand-binding domain of the ER (H222 clone; 1 μg), or an ER antagonist (ICI 182,780; 10 μM) at room temperature for 10 min. With the pretreatments remaining in the PBS, 10 nM E2-BSA-FITC was added and incubated at room temperature for 2 min. Confocal images were acquired using a 488–594-nm argon ion laser.

Radioreceptor Assay

After treatment, cells were washed with PBS and gently scraped from the culture dish wells, pelleted by centrifugation (750 × g), and resuspended in 200 μl GnRH receptor assay buffer (10 mM Tris-HCl, 1 mM CaCl2, 0.1% BSA, pH 7.4). One million cells were incubated with 0.2 nM [125I]-D-Ala6-GnRH-ethylamide at 4°C for 4 h. Nonspecific binding was determined for each treatment well by incubating 1 × 106 cells with 0.2 μM unlabeled D-Ala6-GnRH-ethylamide. After incubation, 3 ml ice-cold GnRH receptor buffer were added, and the cells centrifuged for 15 min at 750 × g. Supernatants were decanted, and radioactivity in the cell pellet was quantified. Specific binding was calculated by subtracting nonspecific binding from the total binding.

Statistical Analyses

The relative changes in number of GnRHR caused by treatment was reported as a change of percentage of the specific binding of [125I]D-Ala6-GnRH-ethylamide in E2-treated cells compared to percentage of the specific binding of [125I]D-Ala6-GnRH-ethylamide in untreated control cells (control = 100% binding). Differences in GnRH analog binding were determined by ANOVA using the general linear model of SAS. Differences in GnRH analog binding between treated and untreated cells were separated using the least-significant-difference test. Data are presented as mean ± SEM.

RESULTS

E2 and E2 Conjugates Increase GnRH Analog Binding in Ovine Pituitary Cells

Estradiol at 0.1 pM to 10 nM increased the percent of GnRH analog binding above the control (Fig. 1A). Although a greater concentration was required (10 nM), treatment with E2-BSA also led to a significant increase in the percent of GnRH analog binding and to the same total increase as that induced by E2. Estradiol conjugated to the 15-amino acid peptide (E2-PEP) increased the percent of GnRH analog binding above the control in a dose response similar to that of E2 (Fig. 1B). An increase was observed with E2-PEP treatment at 1 pM to 10 nM, albeit at a lesser extent than when the E2 and E2-BSA study was conducted, presumably due to the sheep being in anestrus with fewer pituitary estrogen receptors [25] and decreased responsiveness (Davis and Nett, unpublished results).

FIG. 1.

FIG. 1.

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Effect of dose of E2 and E2-BSA (A) and E2-PEP (B) on relative number of GnRH receptors as indicated by GnRH analog binding in pituitary cells. Treatments were added 12 h before the end of the incubation. Binding is represented as a percent of the control (100%). Values are the mean ± SEM for three replicates (A) and four replicates (B). *P < 0.05.

A Similar Time Course of Increased GnRH Analog Binding Is Evident for Pituitary Cells Treated with Either E2 or E2-BSA

If E2 and E2-BSA are working through a similar mechanism to elicit enhanced GnRH analog binding in pituitary cells, then a similar time course of response should be evident. Consistent with this, 0.1 nM E2 or 10 nM E2-BSA increased GnRH analog binding at 6, 9, or 12 h but not at either 0 or 3 h (Fig. 2A). Also, we sought to determine whether a short-term exposure (15 min) to E2 (40 nM) followed by removal of E2 and a 12 h incubation is equivalent to exposure of pituitary cells with E2 for an entire 12 h. Thus, primary cultures of ovine pituitary cells were cultured with E2 for 0, 15, 30, 60 min or 12 h. Consistent with a relatively rapid signaling event, a brief 15 min exposure to E2 followed by removal of the E2 for the remaining 12 h incubation period was sufficient to increase GnRH analog binding to the same extent as a 12 h exposure (Fig. 2B).

FIG. 2.

FIG. 2.

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Effect of duration of E2 treatment on relative number of GnRH receptors as indicated by GnRH analog binding. A) E2 (0.1 nM) or E2-BSA (10 nM) were added for 0, 3, 6, 9, or 12 h prior to the end of the incubation. Pituitary cells for all the points were cultured for 12 h. Values are the mean ± SEM for four replicates. B) Pituitary cells were incubated for 15, 30, or 60 min with 40 nM E2, washed with PBS, and incubated in medium without steroid for the remaining 12 h. As a positive control for GnRH analog binding, cells were incubated in the presence of E2 (40 nM) for 12 h. Binding is representative as a percent of the control (100%). Values are the mean ± SEM for three replicates. *P < 0.05.

E2 and E2-BSA Increase in GnRH Analog Binding Is Sensitive to Blockage by ICI 182,780

Next we sought to determine if the ability of E2 and E2-BSA to increase GnRH analog binding is mediated by one of the classical forms of ER (ESR1 or ESR2, also known as ERα and ERβ, respectively). Pituitary cells were cultured with either 0.1 nM E2 or 10 nM E2-BSA in the presence of the pure ER antagonist ICI 182,780. The inclusion of ICI 182,780 effectively blocked the increase in GnRH analog binding in response to either E2 or E2-BSA (Fig. 3). Thus, it is likely that the ability of E2 to enhance pituitary number of GnRHR is mediated via one or both of the classical ER isoforms rather than via novel ER proteins such as GPR30 [26, 27].

FIG. 3.

FIG. 3.

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Effects of an ER antagonist on E2 stimulated an increase in GnRH receptors. Pituitary cells were cultured with E2 (0.1 nM) or E2-BSA (10 nM) for 12 h in the presence or absence of ICI 182,780 (10 μM). Values are the mean ± SEM for four replicates. *P < 0.001.

Adenoviral-Mediated Overexpression of Dominant-Negative ESR1 Blocks Both E2- and PPT-Mediated Increase in GnRH Analog Binding

Based on the previous data, it appears that the ability of E2 to enhance GnRH analog binding is sensitive to antagonism of one of the classical isoforms of ER. Given that E2-mediated negative feedback on secretion of LH is dependent on ESR1 as determined by the use of ESR1 knockout mice [28–32], we reasoned that the likely ER subtype mediating the increase in GnRH analog binding is ESR1. To test this possibility, we examined the impact of a dominant-negative form of ESR1 (L540Q) on the ability of both E2 and ESR1 agonist (PPT) to increase GnRH analog binding. The dominant-negative L540Q mutant effectively blocked both E2- and PPT-enhanced GnRH analog binding in primary cultures of ovine pituitary cells (Fig. 4).

FIG. 4.

FIG. 4.

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Expression of a dominant-negative ESR1 mutant inhibits E2- and ESR1 agonist (PPT)-mediated increase in GnRH receptors. Pituitary cells were infected for 24 h with adenovirus encoding a dominant-negative mutant form of ESR1 (L540Q). At 24 h postinfection, cells were treated with 4 nM E2 or 0.82 nM PPT for 12 h. Relative numbers of GnRH receptors are indicated as GnRH analog binding as a percent of the control (100%). Values are the mean ± SEM for three replicates. *P < 0.05.

Adenoviral-Mediated Overexpression of A-CREB Blocks E2-Induced GnRH Analog Binding

Cumulatively, our data support the notion that E2 regulation of the GnRHR number may reflect a membrane-initiated signaling event or a nonclassical signaling mechanism mediated via ESR1. The ability of membrane forms of ESR1 to integrate signaling through both AP-1 [33, 34] and CREB is well established [35, 36]. Furthermore, recent work has established that E2 leads to rapid phosphorylation of CREB in ovine gonadotrophs [37]. To determine if this pathway contributes to E2 regulation of GnRHR number, we applied an experimental paradigm based on overexpression of a dominant-negative form of CREB termed A-CREB. Consistent with a potential role for CREB family members in E2 activation of the GnRHR gene, the ability of 0.1 nM E2 to enhance GnRH analog binding was blocked by adenoviral-mediated overexpression of A-CREB but not GFP (included as a control for infection) (Fig. 5).

FIG. 5.

FIG. 5.

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Expression of a dominant-negative CREB blocks E2-mediated increase in GnRH receptors. Pituitary cells were infected for 24 h with adenovirus encoding green fluorescent protein (GFP, control for cytotoxic effects) or dominant-negative CREB. At 24 h postinfection, cells were treated with 0.1 nM E2 for 12 h. Cells that were not infected and treated with vehicle for 12 h served as a binding control (100%). Values are the mean ± SEM for five replicates. *P < 0.05.

Membrane Fluorescence Is Evident on Ovine Pituitary Cells Incubated with FITC Conjugated to E2-BSA

To determine if plasma membrane-associated E2 binding sites are detectable, ovine pituitary cells were incubated with E2-BSA-FITC (Fig. 6). Importantly, membrane fluorescence was attenuated when cells were preincubated with either free E2, antibody directed against the ligand binding domain of ESR1 (clone H222), or ICI 182,780 (Fig. 6).

FIG. 6.

FIG. 6.

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Fluorescently labeled E2-BSA (E2-BSA-FITC) staining associated with the plasma membrane of ovine pituitary cells. Pituitary cells were incubated with E2-BSA-FITC alone (A) or in the presence of 1000-fold excess of E2 (B), an antibody specific for the ligand-binding domain of the ER (H222 clone) (C), or an ER-specific antagonist (ICI 182,780) (D). Confocal images of E2-BSA-FITC are shown in the left panel. The plasma membrane was identified by concanavalin A conjugated to Alexa 594 (middle panel). Colocalization of E2-BSA-FITC and concanavalin A appears yellow in the merged view (right panel).

DISCUSSION

The historical paradigm of E2 action posits that the hormone diffuses across both the plasma and nuclear membrane where it interacts with high-affinity ER. While there is little doubt that this model appropriately describes E2 regulation of many of its target genes, it has become very apparent that this hormone is capable of eliciting changes in gene expression that are initiated by interacting with nonnuclear receptors located at the plasma membrane [38] or even intracellular membrane compartments such as the smooth endoplasmic reticulum [39]. This is especially evident in ER knockout and knock-in mice that contain a mutated ER that cannot bind to ERE (estrogen response element); therefore, E2-stimulated gene expression and subsequent protein translation is via a nonclassical pathway, and it has been shown that ERE-independent E2 genomic effects can occur independently of ER directly binding to DNA [40]. This is likely the mechanism that occurs with the GNRHR gene promoter because an ERE has not been identified in the GNRHR gene promoter from any species [41–45], and it is exquisitely clear that E2 has profound effects on the regulation of this gene. Herein we have explored the possibility that this effect of E2 may reflect a mechanism mediated via an ER on the plasma membrane through the nonclassical signaling pathway. Our interest in this possibility was stimulated by previous work examining the impact of membrane-impermeable E2 conjugates on LH secretion in sheep [16]. In that study, E2, E2-PEP, and E2-BSA led to a rapid suppression of LH secretion that persisted for approximately 6 h. The rapidity of this response (within 30 min) is not consistent with an exclusively genomic mechanism. Following the transient suppression of LH secretion, a preovulatory-like surge of LH was evident at approximately 12 h post-E2 infusion. In contrast, the E2-PEP and E2-BSA conjugates did not induce a preovulatory-like surge of LH; however, the mean LH measured during the surge window was significantly greater than that measured during the preinfusion period, suggesting that sensitivity of the pituitary gland may have increased at that time even through there was no increase in GnRH secretion [46].

The E2-induced preovulatory LH surge is due to at least two components: first, an increase in GnRHR numbers in the pituitary [1] and second, a large and temporally delayed, prolonged release of GnRH from the hypothalamus [3]. The absence of a full LH surge in animals treated with the BSA-conjugated E2 [16] likely reflects the loss of the second component as a result of either an inability of the E2 conjugates to cross the blood-brain barrier or the lack of E2 membrane signaling to GnRH neurons [46]. In contrast, if E2 activation of membrane-signaling events are involved in the synthesis of GnRHR, it would explain the discrete increase in LH secretion observed during the expected preovulatory-like surge of LH in sheep treated with membrane-impermeable E2-BSA [16]. Consistent with this notion, we find that E2-BSA stimulates an increase in GnRH analog binding in primary cultures of ovine pituitary cells in a time course similar to that measured for free E2. Also, because gonadotrophs are the only ovine pituitary cell type thought to express E2 receptors [47], detection of displaceable membrane binding of a fluorescent E2 conjugate supports the hypothesis that membrane-associated binding sites for E2 are present on the same cells that express GnRHR in the ovine anterior pituitary.

If a membrane site of action underlies the ability of E2 to enhance the number of GnRHR, then at issue is the identity of the ER. Since the first characterization of rapid, nongenomic signaling induced by E2, much effort has been devoted to isolating and characterizing the protein that mediates these effects. It is clear that this issue is complex and likely involves both the classical ER (ESR1 and ESR2) as well as novel proteins such as GPR30 that are not members of the superfamily of nuclear receptors. Certainly much evidence has been generated that supports the concept that ER are transported to and localized in the plasma membrane, which is dependent on posttranslational modification and association with other proteins [48–53]. Thus, the ability of E2 to signal through a membrane site of action does not require invoking the presence of novel ER isoforms. Consistent with this, we find that the ability of both E2 and E2-BSA to elicit increased GnRH analog binding is completely blocked by inclusion of the ER antagonist ICI 182,780. Because this compound has not been shown to block E2 signaling through GPR30, it seems likely that the operative receptor is either ESR1 or ESR2. Several additional lines of evidence seem to best support ESR1 rather than ESR2 as the key receptor. First, the selective ESR1 agonist PPT mimics the effects of E2 on GnRH analog binding. Second, overexpression of a dominant-negative ESR1 (L540Q) effectively blocks both E2 and PPT increase in GnRH analog binding. Third, we have previously shown that responsiveness of the gonadotrophs to E2 is primarily mediated via ESR1 when cells or animals are treated with PPT [54].

The first evidence for nongenomic signaling by steroid hormones are based on the ability of these molecules to elicit biochemical and cellular events that are simply too rapid to reflect a requirement for biosynthesis of new mRNA and protein. The ability of E2, E2-PEP, and E2-BSA to rapidly suppress GnRH-induced LH secretion [16, 17] clearly fits with this line of evidence. In contrast, the most distal mechanism underlying the effects of E2 on GnRHR expression is, almost certainly, transcriptional. In support of this notion, it is well established that E2 elicits coordinate changes in GnRHR mRNA and GnRHR protein [9, 14, 55–59]. While these data do not eliminate the possibility of a posttranscriptional mechanism such as mRNA stabilization, the ability of actinomycin D to block E2 up-regulation of GnRHR number in ovine pituitary cells does not support this theory [56]. Thus, the most likely mechanism is that E2 activates GNRHR gene expression. Consistent with this notion, we have established E2 responsiveness of the ovine GNRHR gene promoter in multiple lines of transgenic mice [60]. Furthermore, as has been demonstrated in sheep, this effect of E2 is evident in the absence of GnRH input [9, 56, 61]. Thus, E2 is exerting a direct pituitary effect to increase GNRHR promoter activity in both sheep and in the transgenic mouse model. Based on these data, one might logically predict the presence of high-affinity binding sites for ER within the ovine GNRHR gene promoter. This, however, is not the case. A canonical ERE is not present in the ovine GNRHR gene [43], and E2 responsiveness is not detectable using standard in vitro analyses of promoter regulation [60]. Thus, it appears that E2 leads to an increase in GNRHR gene expression through a noncanonical pathway. At issue then is the identity of this pathway.

It is interesting to note that the presence of a cAMP response element (CRE) is one of the most conserved structural features in the proximal promoter of the GNRHR gene from multiple species, including sheep and humans. In the ovine promoter, the CRE is capable of binding CREB and mediating enhanced transcriptional activity in response to cAMP and forskolin [62]. Although the ability of E2 to module transcriptional activity of target genes through cAMP, protein kinase A, and CREB pathways has been well described in neurons [63–69], recently a 30-min exposure to E2 was shown to increase levels of phosphorylated CREB in gonadotrophs in the ovine pituitary gland [37, 47]. Based on these date, we were intrigued with the possibility that E2 may influence transcriptional activity of the GnRHR gene and subsequent protein synthesis through a CREB/ATF-dependent mechanism. Consistent with this possibility, adenoviral-mediated overexpression of a dominant-negative form of CREB prevented E2-induced increase of GnRH analog binding in sheep pituitary cells. Importantly, this result was not due to a nonspecific effect of infection because an equivalent dose of adenovirus expressing GFP did not attenuate the increase in GnRH binding in response to E2. These data are the first to clearly implicate a candidate intracellular pathway that underlies E2 regulation of the GNRHR gene and subsequent protein synthesis. We stress the term candidate in this context because of the finding that an A-CREB molecule in which the basic amino acids comprising the DNA-binding domain have been replaced with acidic amino acids nevertheless retains the capacity to dimerize but is incapable of DNA binding [24]. As such, A-CREB exerts its dominant-negative effects by effectively sequestering wild-type partners in a nonproductive dimer. What is important here is that CREB does not simply homodimerize but is capable of heterodimerization with other CREB/ATF family members as well as members of the AP-1 family of transcription factors [70]. Thus, at present we cannot conclude that CREB is the operative player in mediating E2 input to the GNRHR gene. Ultimately, this issue will likely have to be resolved by a combination of DNA-protein binding and chromatin immunoprecipitation assays. Unfortunately, given the diversity of endocrine cells types in the anterior pituitary gland, this will not be a trivial undertaking. Finally, as mentioned above, the ovine GNRHR gene promoter contains a functional CRE. Certainly we speculate that this site may be important in mediating E2 input; however, because transgenic mice represent the only reliable model in which E2 responsiveness of the GNRHR gene promoter is recapitulated, it will be neither quick nor easy to directly test this hypothesis.

Over the past decade. it has become increasingly clear that E2 signaling involves both membrane- and nuclear-mediated mechanisms. Based on the present data, the ability of E2 to regulate GNRHR gene expression may be added to the expanding list of biological responses to E2 in which the initial signal is propagated at the plasma membrane, probably through a nonclassical signaling mechanism. This does not seem to be an unreasonable hypothesis considering the number of hormones that signal via G-protein-coupled receptors at the plasma membrane of cells to alter gene expression and subsequent protein synthesis in cells. Estrogen receptors associated with the plasma membrane have been shown to activate G-proteins [71–74], indicating that this steroid hormone receptor may be considered a nontraditional G-protein-coupled receptor. Furthermore, it has been shown that GnRH itself acting through the GnRHR, a G-protein-coupled receptor, regulates the GNRHR gene [75]. There is now relatively extensive evidence supporting a membrane-initiated, nonclassical signal underlying the ability of E2 to rapidly and transiently suppress GnRH-induced LH secretion. As such, it is tempting to speculate that the E2 signals pathways that lead to acute suppression of LH secretion, and, subsequently, increased GNRHR gene expression may result from a common membrane site of action. These two actions of E2 may be involved with 1) decreasing secretion of LH prior to the ovulatory surge to permit a buildup of LH stores in the anterior pituitary and 2) increasing sensitivity of the anterior pituitary to GnRH, both of which might contribute to maximizing secretion of LH during the ovulatory surge.

The use of E2 conjugated to BSA to study the effects of E2 at the membrane has been quite controversial. The linkage of the large BSA molecule to the C-6 position of E2 has been shown to have differential effects in cells relative to free E2 [76, 77]; however, in the present study, it was shown that E2-BSA increased GnRHR numbers in a manner similar to free E2. Other concerns have been raised that the bulkiness of BSA could alter the ability of binding of E2-BSA to the ER and alter the biological outcome [46, 76]. That a greater concentration of E2-BSA was required to increase the numbers of GnRHR is not surprising because binding of E2-BSA to the soluble E2 receptor is only about 7% that of E2 (Arreguin-Arevalo and Nett, unpublished results). Further, if the ER is tethered in the plasma membrane, it is likely to be much less able to interact with E2-BSA than soluble receptor. Thus, the need for a greater concentration of E2-BSA to induce the same increase in GnRHR as E2 would be expected. It should also be noted that 10 nM E2-BSA was used in the present study, and this is the commonly used concentration of free E2 for in vitro studies, which is much greater than circulating concentrations of E2 in vivo. Based on previous experiments conducted by Arreguin-Arevalo and Nett (unpublished results), there is approximately 20 pg/ml of free E2 remaining in the E2-BSA conjugate following the extraction procedure. Based on this concentration, approximately 3.0 fM of free E2 was added to cells receiving E2-BSA. This concentration is well below that required for a physiological response based on our dose-response experiments. Two other major concerns of using conjugated E2-BSA include release of free E2 or proteolytic degradation of the BSA, which would allow smaller amino acids attached to E2 to gain access to nuclear receptors [78]. Our laboratory has conducted studies to refute these claims. Administration of E2-BSA to ewes causes a rapid nongenomic effect as observed by a decrease in secretion of LH; however, the preovulatory surge release of LH that is considered to be a genomic effect is not observed [16, 46]. The results from these studies provide evidence that the diethyl ether extraction method removes free E2 from the conjugated form and that E2-BSA is stable in vivo for a number of hours and does not appear to be degraded by proteolysis because genomic effects were not observed. Finally, a classical study of administration of E2 and E2-BSA to rodents to measure the effect on uterine weight was conducted (Clay et al., unpublished results) in which 0.1 μg E2 or 250 μg E2-BSA was administered to the animals. Only 0.1 μg E2 increased uterine weight, and there was no difference in uterine weight between controls and E2-BSA treated animals, again providing further evidence that our method of removing free E2 from the conjugated form is effective and that E2-BSA is stable in vivo such that free E2 is not available for classical genomic effects.

We should emphasize that while our data support a role for membrane-associated ESR1 in GNRHR gene regulation, we do not reject a role for the nuclear receptor. What is intriguing in this regard is that the ESR1 molecule containing the L540Q mutation clearly blocks the E2-induced increase in GnRH analog binding. However, there is no evidence we are aware of that this particular mutation in L540Q affects the ability of E2 to signal through a membrane site of action. Rather, the presence of this single amino acid conversion appears to alter the ability of ER to recruit coactivators to an E2 responsive promoter [22]. As such, enhanced transcription of the GNRHR gene to E2 may be initiated by a membrane form of ESR1 but requires participation of nuclear ER for the ultimate transcriptional response. The latter almost certainly does not involve direct binding of ER to the GNRHR gene but rather may reflect interactions of ER with primary DNA-binding proteins (e.g., AP-1 and CREB) and the formation of a ternary complex that then acts to recruit key coactivators. If correct, then E2 signaling to the GNRHR gene may well reflect an important physiological model of the concept of parallel pathways as originally proposed by Vasudevan and Pfaff [35].

Footnotes

1

Supported by the National Research Initiative Competitive Grant no. 2005-35203-15376 from USDA Cooperative State Research, Education, and Extension Service; a grant from the Colorado State University Experiment Station; NIH Training Grant T32HD07031-29 (T.L.D.); and NIH HD0655943. T.M.N. has previously consulted for Boehringer Ingelheim Pharmaceuticals, Inc., and Mylan Laboratories, Inc., and has received lecture fees from the Society of Toxicology. He holds an equity position in Gonex, Inc.

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