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Endocrinology, doi:10.1210/en.2006-0367
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Endocrinology Vol. 147, No. 9 4311-4318
Copyright © 2006 by The Endocrine Society

17ß-Estradiol Modifies Nitric Oxide-Sensitive Guanylyl Cyclase Expression and Down-Regulates Its Activity in Rat Anterior Pituitary Gland

Jimena P. Cabilla, María del Carmen Díaz, Leticia I. Machiavelli, Ariel H. Poliandri, Fernanda A. Quinteros, Mercedes Lasaga and Beatriz H. Duvilanski

Departamento de Química Biológica (J.P.C., M.d.C.D., L.I.M., A.H.P., F.A.Q., B.H.D.), Instituto de Química y Fisico Química Biológicas, Facultad de Farmacia y Bioquímica, and Centro de Investigaciones en Reproducción (M.L.), Facultad de Medicina, Universidad de Buenos Aires, Buenos Aires C1113AAD, Argentina

Address all correspondence and requests for reprints to: Beatriz H. Duvilanski, Ph.D., Departamento de Química Biológica, Instituto de Química y Fisico Química Biológicas, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956, Buenos Aires (C1113AAD), Argentina. E-mail: neuroend{at}ffyb.uba.ar.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Previous studies showed that 17ß-estradiol (17ß-E2) regulates the nitric oxide (NO)/soluble guanylyl cyclase (sGC)/cGMP pathway in many tissues. Evidence from our laboratory indicates that 17ß-E2 disrupts the inhibitory effect of NO on prolactin release, decreasing sGC activity and affecting the cGMP pathway in anterior pituitary gland of adult ovariectomized and estrogenized rats. To ascertain the mechanisms by which 17ß-E2 affects sGC activity, we investigated the in vivo and in vitro effects of 17ß-E2 on sGC protein and mRNA expression in anterior pituitary gland from immature female rats. In the present work, we showed that 17ß-E2 acute treatment exerted opposite effects on the two sGC subunits, increasing {alpha}1 and decreasing ß1 subunit protein and mRNA expression. This action on sGC protein expression was maximal 6–9 h after 17ß-E2 administration. 17ß-E2 also caused the same effect on mRNA expression at earlier times. Concomitantly, 17ß-E2 dramatically decreased sGC activity 6 and 9 h after injection. These effects were specific of 17ß-E2, because they were not observed with the administration of other steroids such as progesterone and 17{alpha}-estradiol. This inhibitory action of 17ß-E2 on sGC also required the activation of estrogen receptor (ER), because treatment with the pure ER antagonist ICI 182,780 completely blocked 17ß-E2 action. 17ß-E2 acute treatment caused the same effects on pituitary cells in culture. These results suggest that 17ß-E2 exerts an acute inhibitory effect on sGC in anterior pituitary gland by down-regulating sGC ß1 subunit and sGC activity in a specific, ER-dependent manner.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
17ß-ESTRADIOL (17ß-E2) IS THE major female sex hormone, mediating the differentiation, proliferation, development, and homeostasis of female reproductive functions. It is an important regulator of the function of the anterior pituitary gland. Alterations in estrogen levels during physiological (puberty, estrous/menstrual cycle, pregnancy, lactation, and ovariectomy/menopause) or pathophysiological conditions are able to modify the secretion of anterior pituitary hormones. 17ß-E2 exerts both genomic and nongenomic effects on target tissues (1). Long-term, genomic effects are mediated by 17ß-E2 binding to its cytoplasmic/nuclear receptor, estrogen receptor (ER), and triggering transcription of target genes. Short-term, nongenomic effects are attributed to a signaling pathway started at the cellular membrane level, which in turn activates multiple signaling pathways leading to posttranslational modifications of important structural and functional proteins. Nevertheless, gene transcription can also result from 17ß-E2 signaling at the membrane, so that both pathways have been described as being integrated and having an impact on overall cell biology (2, 3, 4, 5).

The signaling molecule nitric oxide (NO) is produced by three isoforms of NO synthases (NOS) and acts mainly through its major intracellular receptor, NO-sensitive or soluble guanylyl cyclase (sGC). This enzyme is a heme-containing protein. Many of the NO-dependent cellular signaling events that regulate important physiological processes are mediated through activation of sGC, which is a heterodimeric enzyme, constituted by {alpha}- and ß-subunits. To date, two mammalian isoforms of each subunit ({alpha}1/{alpha}2, ß1/ß2), an inactive {alpha}2 ({alpha}2i), and many splicing variants of both isoforms of variable activity have been identified (7, 8). The most abundant and widely expressed heterodimer appears to be {alpha}1/ß1, which has the greatest basal and NO-stimulated activity. Both subunits are required to form an active, cGMP-producing enzyme, but they are also able to form homodimers ({alpha}1/{alpha}1 and ß1/ß1) that result in inactive forms of sGC (9). Once cGMP has been synthesized, it can alter cell responses by regulating many proteins, such as cGMP-gated ion channels, cGMP-dependent serine/threonine kinases, and cGMP-regulated phosphodiesterases (10, 11, 12).

All the NOS isoforms are present in anterior pituitary (13). Many reports support the regulatory role of 17ß-E2 on the NO/sGC/cGMP pathway as well as on NO and cGMP levels in several tissues. Several authors have reported direct actions of 17ß-E2 on sGC expression and activity. In PC12 cells (14) and uterus (15), 17ß-E2 exerted an inhibitory effect on activity and expression, respectively. On the other hand, 17ß-E2 administration did not modify the expression of the NO receptor in hypothalamus (16); therefore, 17ß-E2 actions depends, to a large extent, on target of action. Previous evidence sustains an inhibitory role of 17ß-E2 on the NO pathway in pituitary gland (17). Ovariectomy increases NOS activity, mRNA, and protein pituitary levels, but 17ß-E2 treatment reverts these effects (18).

Previous studies from our laboratory demonstrate that 17ß-E2 decreases the sensitivity of the pituitary to the inhibitory effect of NO on prolactin release by reducing sGC activity and affecting the cGMP pathway (19).

Because the role of sGC in transducing inter- and intracellular signals elicited by NO is critical, knowledge of the molecular mechanisms involved in sGC regulation may help to elucidate the physiological and pathophysiological significance of this signal transduction pathway in anterior pituitary, in conditions with varied 17ß-E2 levels, such as estrous cycle, pregnancy, lactation, and menopause.

The aim of the present study is to establish whether 17ß-E2 regulates the expression and activity of sGC in anterior pituitary gland from immature rats. Here we report that in vivo 17ß-E2 treatment increases sGC {alpha}1 mRNA and protein expression but exerts an acute inhibitory effect on sGC ß1 mRNA and protein levels and a consequent decrease in cGMP production. This effect was shown to be 17ß-E2 specific, because it is not mimicked by other gonadal steroid treatments and was totally reversed by pure ER antagonist administration, ICI 182,780. The same results are obtained from in vitro experiments using anterior pituitary cells in culture. We demonstrate for the first time that 17ß-E2 acts directly on pituitary sGC and that the mediation of other hormones or factors released from extrapituitary tissues is not required.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
ICI 182,780 was purchased from Tocris Neuramin (Bristol, UK). Leupeptin, pepstatin, phenylmethanesulfonyl fluoride and dithiothreitol were obtained from Alexis-U.S. Biological (Swampscott, MA). Bradford reagent was purchased from Bio-Rad (Hercules, CA). Hydrogen peroxide was purchased from Cicarelli (Buenos Aires, Argentina). Rabbit anti-cGMP polyclonal antiserum was obtained from Chemicon (Temecula, CA). RNA isolation and RT-PCR reagents were obtained from Invitrogen (Carlsbad, CA). All other reagents and antibodies were obtained from Sigma Chemical Co. (St. Louis, MO).

Animals and treatments
Immature female Wistar rats (21–23 d old, 35–50 g) were used to avoid both the effects of endogenous 17ß-E2 and surgical procedures such as ovariectomy to remove the main source of endogenous 17ß-E2 production. Animals were kept in controlled conditions of light (12 h light,12 h dark) and temperature (21–24 C). Food and water were supplied ad libitum. All procedures were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Rats were injected sc in the periscapular region with different hormones or with vehicle alone (propylene glycol). The doses were as follows: 17ß-E2 and 17{alpha}-E2, 40 µg/kg body weight; progesterone, 40 mg/kg. For other experiments, animals were injected ip with 2 mg/kg ICI 182,780 30 min before administration of 40 µg/kg 17ß-E2.

Preparation of tissue homogenates for immunoblot analysis
Anterior pituitary glands were removed from decapitated animals and sonicated in lysis buffer containing 10 mM HEPES (pH 7.4), 150 mM NaCl, 10 mM EDTA, 100 µM leupeptin, 350 µM pepstatin, 0.5 mM phenylmethanesulfonyl fluoride, and 0.2 mM dithiothreitol. Sonicates were centrifuged for 20 min at 10,000 x g (4 C), and the post-mitochondrial fraction was used in the immunoblot analysis.

Protein measurement
Protein content of the supernatants was measured by Bradford reagent, using BSA as standard.

Immunoblot analysis
Total protein (20–30 µg) from each sample was boiled for 5 min in Laemmli sample buffer and fractioned on 10% SDS-PAGE. Resolved proteins were transferred to polyvinylidene difluoride membranes and blocked for 2 d at 4 C in blocking buffer (TBS/0.05% Tween 20, 6% nonfat dry milk). Then, membranes were coincubated overnight at 4 C with rabbit antisera anti-sGC {alpha}1 (1:1750) or ß1 (1:700) subunits together with antiactin (1:1000) in blocking buffer. The anti-sGC {alpha}1 and anti-sGC ß1 antibodies were raised against the amino acid residues 673–690 and 189–207 of rat sGC {alpha}1 and ß1, respectively. Therefore, the anti-sGC {alpha}1 antibody recognized the C-terminal sequence of sGC {alpha}1, and the anti-sGC ß1 the N-terminal region of sGC ß1. Blots were washed and incubated for 1 h at room temperature with horseradish-peroxidase-conjugated goat antirabbit IgG (1:2000), followed by detection of immunoreactivity with diaminobenzidine solution containing 0.01% hydrogen peroxide.

Analysis of immunoblot data.
The intensity of immunoblot signals was determined by digital image analysis using the Scion Image (Scion Corp., Frederick, MD) and Gel Pro Analyzer (Media Cybernetics, LP, Silver Spring, MD) software for Windows. To allow statistical comparison of results from different blots, levels were normalized to the value of the actin immunoreactive band in each lane.

Semiquantitative RT-PCR
RNA isolation.
Tissues were removed from decapitated animals and immediately homogenized with TRIZOL reagent. After isolation, total RNA from tissues was spectrophotometrically quantified at 260 nm. RNA integrity was checked in formaldehyde/formamide gel electrophoresis.

RT-PCR.
First-strand cDNA was synthesized with Moloney murine leukemia virus reverse transcriptase in RT buffer containing 5.5 mM MgCl2, 0.5 mM dNTP, 2.5 µM random hexamers, and 3.125 U/µl Moloney murine leukemia virus reverse transcriptase. Reactions were performed in a final volume of 12 µl containing 1 µg RNA. The RT reaction was run at 37 C for 50 min, and reverse transcriptase was inactivated by heating the samples at 70 C for 15 min before the PCR. To check for genomic contamination, the same procedure was performed on samples in a reaction solution lacking reverse transcriptase.

Specific primers for both subunits of sGC were designed from published sequences (15) with Oligo Perfect designer software (Invitrogen). These primers were sGC {alpha}1 subunit (forward, 5'-ACACAATATGCATCTCCGATGG-3', and reverse, 5'-GCTCTCTATACTCGCTTTGACCAA-3') and sGC ß1 subunit (forward, 5'-CCCGTGGAAACTGATGTCAA-3', and reverse, 5'-CGGGACCTAGTAGTCACGCA-3'). The amplified products spanned from nucleotide position bases 1971–2054 (C-terminal region of sGC {alpha}1), and from 714–823 (N-terminal region of sGC ß1). Actin was used as an endogenous control (forward, 5'-ACCACAGCTGAGAGGGAAATCG-3', and reverse, 5'-AGAGGTCTTTACGGATGTCAACG-3'). Actin primers were designed to detect amplification of DNA contamination. Then, samples were thermocycled for PCR amplification (Mastercycler; Eppendorf, Hamburg, Germany). The reaction mixture contained GoTaq PCR buffer, 1.5 mM MgCl2, 200 µM of each dNTP, 0.625 U GoTaq polymerase, and 300 nM of each primer. We used RT-PCR methods to determine relative changes in mRNA expression. Reactions were subjected to a varying number (n = 16–40) of cycles of PCR amplification (melting phase at 94 C for 30 sec, annealing at 55 C for 30 sec, and extension at 72 C for 1 min) to find out the optimal cycle number within the linear range for PCR amplification. Amplified products collected at various cycles were analyzed by electrophoresis in 1.5% agarose-ethidium bromide gels, and the optimal cycle number was 24 cycles for sGC {alpha}1, sGC ß1, and ß-actin.

Analysis of semiquantitative PCR data.
The intensity of PCR product signals was determined by digital image analysis using the Gel Pro Analyzer software for Windows. To allow statistical comparison of results from different experiments, sGC {alpha}1 and ß1 levels were normalized to the value of the ß-actin amplified band in each lane.

Intracellular cGMP determination
Anterior pituitary glands were quickly removed and placed on dry ice. Subsequently, they were sonicated in warm 50 mM sodium acetate (pH 6.2), boiled for 10 min, and centrifuged at 10,000 x g for 10 min. Supernatants were stored at –70 C until cGMP determination. cGMP was assayed as previously described (20) by specific RIA using rabbit anti-cGMP polyclonal antiserum and acetylated cGMP as standard. Total protein content in the pellets was measured as described above.

Cell culture
Animals were killed by decapitation, and the anterior pituitary glands removed. The cells were obtained from the glands by enzymatic (trypsin/deoxyribonuclease I) and mechanical dispersion (extrusion through a Pasteur pipette) as described previously (21). Cells were cultured for 3 d (37 C, 5% CO2 in air) in DMEM supplemented with 10% carbon-dextran-adsorbed fetal bovine serum, 10 µl/ml MEM amino acids, 2 mM glutamine, 5.6 µg/ml amphotericin B, and 25 µg/ml gentamicin. Cells were seeded onto 24-well tissue culture plates (0.1 x 106 cells per well). In the inhibitor assays, cells were preincubated with 10 µM ICI 182,780 for 30 min before 17ß-E2 treatment.

Statistical analysis
Results are expressed as mean ± SEM and evaluated by one- or two-way ANOVA followed by Dunnett’s or Student-Newman-Keuls’ test, respectively. Differences between groups were considered significant if P < 0.05. Results were confirmed by at least three independent experiments.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
17ß-E2 acute treatment increased sGC {alpha}1 and decreased sGC ß1 protein expression
We investigated the influence of a physiological dose of 17ß-E2 on protein expression levels of sGC {alpha}1 and ß1 using Western blot. As expected, the sGC {alpha}1 and sGC ß1 immunoreactive bands were coincident with relative molecular masses of approximately 80 and 70, respectively. Figure 1Go shows that in vivo administration of 17ß-E2 to immature rats resulted in an increase of sGC {alpha}1 protein levels and, concurrently, a decrease in ß1 protein levels. These opposite effects began to be evident as soon as 3 h after 17ß-E2 administration (20% decrease in ß1 levels compared with control) and were maximal 9 h after injection (40% decrease). At this time, {alpha}1 levels had augmented significantly (80% increase vs. control). By 16 h after injection, {alpha}1 and ß1 protein expression levels tended to return to control values (0 h after injection). Thus, 17ß-E2 acute treatment differentially affected sGC {alpha}1 and ß1 protein expression levels.


Figure 1
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  		FIG. 1. Acute 17ß-E2 treatment increases sGC {alpha}1 protein expression but decreases ß1 levels in immature rats. Animals (three to five per group) were injected with 17ß-E2 (40 µg/kg body weight) and killed at different time lapses. A, Representative Western blot from one experiment; B, bars represent the mean ± SEM of sGC {alpha}1 or ß1 densitometric values normalized to actin as a percentage of control (n = 4). ANOVA followed by Dunnett’s test: *, P < 0.05; **, P < 0.01 vs. respective controls.

 
17ß-E2 administration increased sGC {alpha}1 and decreased sGC ß1 mRNA expression
Because these changes in sGC subunit protein levels could be the result of modifications in mRNA expression, we studied next the action of acute 17ß-E2 treatment on {alpha}1 and ß1 mRNA expression by semiquantitative RT-PCR, starting from earlier times. mRNA levels of both subunits were analyzed at different cycles from the exponential phase of PCR amplification. Relative quantification was not possible at high cycle numbers because of the signal saturation. For subsequent studies, we chose cycle 24 to compare differences between treatments. At this cycle number, all the analyzed mRNAs were still at the linear range of amplification. By 3 h after 17ß-E2 administration, ß1 mRNA levels were reduced (20% decrease vs. control), whereas {alpha}1 mRNA levels remained similar to control values (data not shown). By 6 h after 17ß-E2 injection, we observed a considerable rise in {alpha}1 mRNA levels (50% stimulation vs. control; Fig. 2Go, A and B) and, concurrently, a sustained decrease in ß1 mRNA levels (60% inhibition respect to control; Fig. 2Go, C and D). This tallies with the effects at the protein level and considering that the 17ß-E2 effect on ß1 expression was earlier in time, we can affirm that the variations in the levels of {alpha}1 and ß1 mRNAs are responsible for the changes in protein expression.


Figure 2
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  		FIG. 2. 17ß-E2 acute treatment raises sGC {alpha}1 mRNA and decreases ß1 levels. Animals (three to five per group) were injected with 17ß-E2 (40 µg/kg body weight) and killed after 6 h. Average densitometric values are shown. Bars represent mean ± SEM of sGC {alpha}1 and ß1 PCR amplification products at cycle 24 normalized to actin with control and 17ß-E2 treatments (n = 3). ANOVA followed by Dunnett’s test: *, P < 0.05 vs. control.

 
17ß-E2 acute treatment diminished cGMP production
Because both isoforms are required in a 1:1 stoichiometry to form an active, cGMP-producing enzyme and considering the contrary effect of 17ß-E2 on both sGC subunits, we sought to investigate whether 17ß-E2 treatment affected the global activity of sGC. cGMP production significantly decreased 6 h after injection (70% decrease vs. control), when the opposite effect on both subunits’ expression became more evident (control, 682 ± 79 fmol cGMP/µg protein; 6 h after 17ß-E2, 223 ± 36; 9 h after 17ß-E2, 220 ± 76; P < 0.01 for 6- 9-h time points vs. control, ANOVA followed by Dunnett’s test). Hence, 17ß-E2 acute treatment reduced sGC global activity, which coincides with the down-regulation of the ß1 subunit.

17ß-E2 actions on protein and RNA expression of sGC subunits and cGMP production were specific
To further demonstrate that the effect on sGC expression was 17ß-E2 specific, 17ß-E2 inactive isomers and another nonrelated steroid hormone were used. Animals were injected with 17{alpha}-E2 (a 17ß-E2 stereoisomer that binds poorly to ER) or with progesterone and killed after 6 h. Figure 3Go shows that neither 17{alpha}-E2 nor progesterone treatment was able to significantly modify {alpha}1 or ß1 mRNA (Fig. 3AGo) or protein levels (Fig. 3Go, B and C) compared with control. Furthermore, neither 17{alpha}-E2 nor progesterone administration could modify cGMP production vs. control [control, 536 ± 41 fmol cGMP/µg protein; 17ß-E2, 223 ± 36 (P < 0.01 vs. control, ANOVA followed by Dunnett’s test); 17{alpha}-E2, 587 ± 47; progesterone, 498 ± 33; n = 3]. In sum, these results demonstrate that the modification of {alpha}1 and ß1 expression and sGC activity is a specific consequence of 17ß-E2 acute treatment.


Figure 3
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  		FIG. 3. 17ß-E2 acute treatment decreases sGC global activity. Animals (three to five per group) were injected with 17ß-E2 (40 µg/kg body weight), 17{alpha}-E2, an inactive isomer of 17ß-E2 (40 µg/kg), or progesterone (Prog, 40 mg/kg) and killed after 6 h. A, Average densitometric values from semiquantitative RT-PCR. Bars represent the mean ± SEM of sGC {alpha}1 or ß1 mRNA densitometric values at cycle 24 normalized to actin mRNA and expressed as percentage of control (n = 3). B, Representative Western blot from one experiment. C, Average densitometric values from three independent experiments. Bars represent the mean ± SEM of sGC {alpha}1 or ß1 protein densitometric values normalized to actin and expressed as percentage of control. ANOVA followed by Dunnett’s test: *, P < 0.05; **, P < 0.01 vs. control.

 
17ß-E2-mediated effect on sGC protein, mRNA expression, and activity required the specific activation of ER
To prove that the effect of 17ß-E2 on sGC depends on ER activation, animals were pretreated with the pure ER antagonist ICI 182,780 30 min before 17ß-E2 injection. Figure 4AGo shows that competitive inhibition of 17ß-E2 binding to ER exerted by ICI 182,780 completely prevented the 17ß-E2 effect on {alpha}1 and ß1 mRNA expression. Equally, ICI 182,780 administration completely avoided the increase in {alpha}1 and decrease in ß1 protein expression when administered before 17ß-E2 alone and had no effect on sGC expression when injected by itself (Fig. 4Go, B and C). Moreover, antagonist injection restored sGC activity to basal levels when administered with 17ß-E2 and did not alter sGC activity when administered alone [control, 480 ± 42 fmol cGMP/µg protein; 17ß-E2, 245 ± 29 (P < 0.01 vs. control, ANOVA followed by Dunnett’s test); ICI 182,780, 502 ± 46; ICI 182,780 plus 17ß-E2, 450 ± 35; n = 3]. These results suggest that 17ß-E2 effects on sGC protein and mRNA expression as well as on cGMP production are mediated by ER-specific activation.


Figure 4
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  		FIG. 4. The pure ER antagonist ICI 182,780 (ICI) reverts 17ß-E2 effects on activity and mRNA and protein expression of sGC. Animals (three to five per group) were injected with 17ß-E2 (40 µg/kg body weight) and/or with ICI 182,780 (2 mg/kg) 30 min before 17ß-E2 injection and were killed after 6 h. A, Average densitometric values from semiquantitative RT-PCR at cycle 24. Bars represent the mean ± SEM of sGC {alpha}1 or ß1 mRNA densitometric values normalized to actin mRNA and expressed as percentage of control (n = 3). B, Representative Western blot from one experiment. C, Average densitometric values from Western blot. Bars represent the mean ± SEM of sGC {alpha}1 or ß1 protein densitometric values normalized to actin and expressed as percentage of control (n = 3). ANOVA followed by Student-Newman-Keuls’ test: *, P < 0.05; **, P < 0.01 vs. respective controls; #, P < 0.05 vs. 17ß-E2.

 
17ß-E2 acute treatment increased sGC {alpha}1 and decreased sGC ß1 mRNAs and protein levels in vitro in an ER-dependent fashion
To compare the E2 effects in vivo and in vitro, pituitary cell cultures from immature rats were incubated with 10–9 M 17ß-E2 for 6 h or coincubated with 10 µM ICI 182,780. In this time, 17ß-E2 was able to raise sGC {alpha}1 mRNA expression as well as reduce sGC ß1 mRNA (Fig. 5AGo). Accordingly, protein levels of both subunits were also modified by E2 treatment (Fig. 5Go, B and C). Antagonist administration fully reverted these effects on mRNA and protein expression when coincubated together with 17ß-E2 and had no effect by itself. Because in cell cultures 17ß-E2 exerted the same actions on protein and mRNA expression as those previously seen in vivo, these effects would not require other extrapituitary factors. Considering that ICI 182,780 totally blocked 17ß-E2 actions, they would therefore depend on ER activation.


Figure 5
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  		FIG. 5. In vitro 17ß-E2 treatment increases sGC {alpha}1 but decreases ß1 mRNA and protein levels in pituitary cells in an ER-dependent manner. Anterior pituitary cell cultures from immature rats were incubated with 1 nM 17ß-E2 and/or 10 µM ICI 182,780 (ICI) for 6 h. A, Average densitometric values from semiquantitative RT-PCR at cycle 24. Bars represent the mean ± SEM of sGC {alpha}1 or ß1 mRNA densitometric values normalized to actin mRNA and expressed as percentage of control (n = 3); B, representative Western blot from one experiment; C, average densitometric values from Western blot. Bars represent the mean ± SEM of sGC {alpha}1 or ß1 protein densitometric values normalized to actin and expressed as percentage of control (n = 3). ANOVA followed by Student-Newman-Keuls’ test: *, P < 0.05; **, P < 0.01 vs. respective controls; #, P < 0.05 vs. 17ß-E2.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
E2 actions on NO receptor
The results we are presenting demonstrate that in vivo 17ß-E2 administration differentially affects sGC subunits, increasing {alpha}1 levels and reducing ß1 levels. Also, sGC activity was dramatically reduced by 17ß-E2. Several hypotheses may explain these findings. First, both subunits are known to be necessary, in a strict 1:1 stoichiometry, to form an active enzyme. In most tissues, the basal levels of {alpha}1 and ß1 sGC are not expressed equally. In rat anterior pituitary, the {alpha}1 subunit is supposed to be the limiting factor in the formation of sGC (22). In these results presented now, despite the increase in {alpha}1 expression, the reduction in ß1 levels could be enough to down-regulate global sGC activity.

Second, we need to bear in mind that this rise in {alpha}1 expression could be a result of other {alpha}1 species. The {alpha}1 splicing products lack different N-terminal regions, present in the {alpha}1 sequence, and are predicted to result in the formation of N-terminally truncated {alpha}1 protein with significantly reduced activity (7). It is important to mention that the anti-sGC {alpha}1 antibody used for immunoblot studies as well as the primers used to amplify sGC {alpha}1 mRNA are directed to the C-terminal sequence of rat sGC {alpha}1. Therefore, we cannot differentiate {alpha}1 species. Additional molecular studies are being performed to confirm the mechanism of this increase in {alpha}1 expression. Considering that 17ß-E2 treatment also raises {alpha}1 mRNA expression, it seems more likely that the increase on this subunit protein level is a consequence of the stimulatory action on {alpha}1 expression. In addition, the {alpha}1 rise could also lead to {alpha}1/{alpha}1 homodimer formation. These nonfunctional structures might contribute, independently of the ß1 decrease, to down-regulate sGC global activity (9, 23).

In the present work, we were interested in evaluating 17ß-E2 effects on both sGC subunits, because changes in only one of the subunits, broadly described in the literature, could lead to incorrect interpretations. We demonstrate for the first time an opposite effect of 17ß-E2 on the two sGC subunits. For this reason, we must consider the regulation of {alpha}1 and ß1 gene expression. Although they are closer together in the same chromosome, their promoter regions have different regulatory sequences and response elements (24). Even considering that they lack estrogen response elements, a wide spectrum of 17ß-E2-activated factors could be mediating these 17ß-E2 actions on pituitary sGC expression. More studies are now being done to elucidate 17ß-E2 mechanisms.

We have also demonstrated here that this acute inhibitory effect on sGC is 17ß-E2 specific, because other steroid hormones such as progesterone or 17{alpha}-E2 had no effect on subunit expression or sGC activity in the time course studies. Furthermore, this inhibition of sGC was ER dependent, because all 17ß-E2 actions were entirely abolished by pretreatment with the ER antagonist ICI 182,780. Thus, 17ß-E2 could be up-regulating the expression of {alpha}1 species through ER and also activating the expression of certain factors that down-regulate ß1 expression in a direct or indirect fashion (25). It is also plausible that 17ß-E2 could repress the transcriptional activity of the genes responsible for the expression of ß1 sGC. 17ß-E2 has been reported to repress the transcription of other genes through ER activation and may involve coactivators and/or repressors or the inhibition of transcription factors (26, 27, 28, 29).

We have shown that acute 17ß-E2 treatment has the same dual effect on sGC mRNA expression, raising {alpha}1 mRNA levels and lowering ß1 levels. Recent reports demonstrated that sGC expression is subject to posttranscriptional regulation. The elav-like mRNA-binding protein human-antigen R (HuR) stabilizes sGC mRNA (30). cGMP- and cAMP-eliciting agonists decrease HuR expression in rat aortic tissue and concomitantly decrease expression of sGC {alpha}1 and sGC ß1 subunits (31). We do not know whether HuR is present in pituitary gland, but sGC mRNA expression in this gland could also be posttranscriptionally regulated in a HuR-dependent or -independent manner.

We find it striking that we have obtained the same results with in vitro pituitary cell cultures. We could therefore state that this 17ß-E2-produced response on sGC does not depend on the secondary release of other hormones or factors from extrapituitary tissues.

E2 actions on NO pathway
Previous reports have shown that 17ß-E2 affects the NO pathway indirectly, via hypothalamic factors, in pituitary (32). We are reporting, for the first time, that 17ß-E2 acutely and directly affects mRNA, protein, and activity of sGC in pituitary gland in vivo and in vitro. The mechanisms by which long-term exposure to NO down-regulates sGC expression and activity are well documented, but we do not know whether a decrease in NO levels affects sGC expression. Previous studies from our laboratory showed that pituitaries from ovariectomized and estrogenized rats with high levels of prolactin release showed no changes in prolactin levels when incubated with NO donors or cGMP analogs (19). Therefore, 17ß-E2 treatment seemed to desensitize pituitary to NO actions. Thus, it seems unlikely that the 17ß-E2 effect on sGC expression could be mediated by 17ß-E2-dependent NOS down-regulation. Additional research is required to elucidate this mechanism.

A number of authors have reported the participation of 17ß-E2 in the NO/sGC/cGMP pathway in many tissues. Depending on multiple factors, 17ß-E2 is capable of exerting stimulatory or inhibitory effects. Previous data have described the negative effect of gonadal steroids on the NO/sGC/cGMP pathway in anterior pituitary gland. Changes of neuronal NOS protein levels during the estrous cycle in rats have been reported (32). Furthermore, NOS seems to be negatively regulated by gonadal steroids (18, 33, 34). In both estrogen-treated pituitaries with prolactin cell hyperplasia and in GH3 tumors, estrogen has been shown to down-regulate neuronal NOS mRNA and protein. Evidence presented by ourselves and others proves that 17ß-E2 affects the NO pathway at multiple points. The results presented now help to explain how 17ß-E2, by acting on sGC, could exert its inhibitory effects on the NO pathway in addition to its inhibitory effect on NOS. The NO pathway in pituitary not only regulates hormonal release but is also involved in cell viability and gland remodeling (6, 35, 36, 37). Because 17ß-E2 has multiple targets of action and is involved in a plethora of processes, understanding the mechanisms by which it affects them will contribute to discover targets for treatment of many physiological and pathophysiological processes.


   Footnotes
 
This work was supported by research grants from the Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT), Universidad de Buenos Aires, and Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET).

Disclosure summary: all authors have nothing to declare.

First Published Online June 1, 2006

Abbreviations: 17ß-E2, 17ß-Estradiol; ER, estrogen receptor; HuR, human-antigen R; NOS, NO synthase; sGC, soluble guanylyl cyclase.

Received March 21, 2006.

Accepted for publication May 25, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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