| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Pituitary |
Department of Cell Biology, Physiology and Immunology, University of Córdoba, 14004 Córdoba, Spain
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
and ERß isoforms, as well as those for the truncated ER product (TERP), and the variant ERß2, in pituitaries from ovariectomized rats, i.e., a background devoid of endogenous estrogen. Compared with expression levels at the morning of proestrus, ovariectomy (OVX) resulted in increased pituitary expression of ERß and ERß2 mRNAs, whereas it decreased TERP-1 and -2 levels without affecting those of ER. Administration of estradiol benzoate (as potent agonist for and ß forms of ER) or the selective ER agonist, propyl pyrazole triol, fully reversed the responses to OVX, while the ERß ligand, diarylpropionitrile, failed to induce any significant effect except for a partial stimulation of TERP-1 and -2 mRNA expression levels. To note, the ERß agonist was also ineffective in altering pituitary expression of progesterone receptor-B mRNA, i.e., a major estrogen-responsive target. In all parameters tested, tamoxifen, a selective ER modulator with mixed agonist/antagonist activity, behaved as ER agonist, although the magnitude of tamoxifen effects was significantly lower than those of the ER ligand, except for TERP induction. In contrast, the pure antiestrogen RU-58668 did not modify the expression of any of the targets under analysis. Overall, our results indicate that endogenous estrogen differentially regulates pituitary expression of the mRNAs encoding several ER isoforms with distinct functional properties, by a mechanism that is mostly conducted through ER. Differential regulation of ER isoforms may represent a relevant system for the self-tuning of estrogen responsiveness in female pituitary.
estradiol, estradiol receptor, mechanisms of hormone action, pituitary, progesterone receptor
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
and the more recently cloned ERß [2, 3]. These are ligand-activated transcriptional factors with a common functional organization containing a central DNA-binding domain, a C-terminal ligand-binding domain (LBD), and two transcriptional activation domains (AF1 and AF2). To note, the and ß forms of ER possess different functional properties and show distinct patterns of gene regulation [4–7]. In fact, expression of ER and ERß has been demonstrated in different pituitary cell types, including lactotrophs and gonadotrophs [6]. Thus, the diversity of biological actions of estrogen at the pituitary level may derive, at least partially, from the balance of expression of and ß ER subtypes in those pituitary cell types. Yet, the specific contribution of ER and ERß to the control of female pituitary function has remained elusive so far.
Interestingly, multiple mRNA variants of ER and ERß have been detected in normal tissues and tumor cells (for a review, see [8]). The physiological role of the resulting ER protein isoforms in signaling the biological actions of estrogen is not fully elucidated, but the functional properties of some of these ER variants have been characterized. Among them, a tissue-specific ER variant, termed truncated ER product-1 (TERP-1), has been identified in rat pituitary [8–10]. This form is composed of a unique 5'-end, 31-base pair (bp) sequence fused to exons 5–8 of ER. In addition, lower expression of a TERP-2 variant, similar to TERP-1 except for a 66-bp insertion in front of the ER common sequence, has been reported [9]. From a functional standpoint, TERP-1 is devoid of independent signaling capacity but it is able to modulate the trans-activating activity of ER and ERß [8]. Thus, TERP expression relative to ER and ERß levels may modulate estrogen-regulated transcriptional responses at the pituitary level. To note, the putative function of TERP-2 remains unexplored. Similarly, several ERß variants with altered biological properties have been described in rat and human tissues [8]. The most abundant ERß variant, termed ERß2, contains an in-frame 54-bp insertion in the LBD coding region and is expressed in rat pituitary at relative levels similar to those of the functional ERß [11]. Accordingly, ERß2 can bind ligands with much lower affinity than and ß forms of ER. However, as is the case for TERP-1, ERß2 can competitively inhibit estrogen-dependent transcriptional activation through ER and ERß [8].
In the above scenario, it is evident that net estrogen sensitivity in the pituitary critically depends on the balanced expression of several ER subtypes and isoforms with distinct functional properties. Previous evidence indicated that estrogen may participate in the self-regulation of its signaling pathway through modulation of expression of its own receptors [8, 12–15]. However, little is known on the relative contribution of ER and ERß to such a phenomenon. This has been recently approached by the use of knockout mouse models, where either the or ß form of ER is selectively eliminated throughout development (for examples, see [16, 17]). These models are tremendously instrumental but have some physiological limitations, related to potential developmental defects and activation of compensatory mechanisms in the absence of one receptor, that partially hamper interpretation of results. As a complementary approach, novel ER isoform-selective ligands have been recently developed [18, 19], thus allowing specific activation of one ER subtype, in the physiological presence of the other subtype and ER variants [20, 21]. This is especially relevant for the ER pathway, given the proposed role of several ER isoforms, including ERß, as regulators of ER signaling in target tissues.
In addition to pure ER subtype-selective agonists, a number of selective ER modulators (SERMs) have been developed. These are synthetic molecules with mixed agonist/antagonist properties at the ER, depending on the cell type and the biological response considered [22]. Besides obvious pharmacological applications, SERMs may be useful experimental tools to dissect out estrogen-dependent phenomena in several neuroendocrine systems [23–25]. Tamoxifen, the first compound identified with SERM activity, displays the widest spectrum of mixed agonist/antagonist activity at the pituitary level, as it acts as an estrogen antagonist on LH secretion but as an estrogen agonist on PRL secretion and GnRH self-priming [24, 25]. However, the effects of tamoxifen on regulation of ER subtype expression at the pituitary level remain unexplored.
To expand our knowledge on the molecular mechanisms responsible for the homologous regulation of pituitary responsiveness to estrogen, in the present study, we evaluated the ability of different ligands, with distinct agonistic or antagonistic properties at the ER, to modulate the expression of the transcripts encoding ER, TERP, ERß, and ERß2 in female rat pituitary. In detail, the effects of estradiol benzoate (as a potent agonist for and ß forms of ER); the selective ER agonist, propyl pyrazole triol (PPT); the ERß agonist, diarylpropionitrile (DPN); the SERM tamoxifen; and the pure antiestrogen RU-58668 were tested in ovariectomized rats, i.e., a background devoid of endogenous estrogen.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Adult virgin female Wistar rats, weighing 200 ± 10 g, were used. Animals were housed under constant conditions of light (14L:10D, with lights-on at 0700 h) and temperature (22°C), with free access to pelleted food and tap water. Estrous cyclicity was monitored by daily vaginal cytology; only rats with at least two consecutive 4-day estrous cycles were used in the studies. Experimental procedures were approved by the Ethical Committee for animal experimentation of the University of Córdoba and were conducted in accordance with the European Union norm for care and use of experimental animals. Bilateral ovariectomy (OVX) was performed under ether anesthesia at random stages of the estrous cycle.
Treatments
Testing of the effects of different ligands of ER, with distinct agonist/antagonist properties, was conducted in two separate experiments using gonadectomized females, 2 weeks after OVX. In the first setting, rats (n = 5 per group) were daily injected (s.c.) for 3 days with 0.2 ml olive oil (used as vehicle), 25 µg of the synthetic estrogen estradiol benzoate (EB; Sigma Chemical Co., St Louis, MO), 1.5 mg of the selective ER agonist, PPT (Tocris Cookson Ltd., Avonmouth, UK), 1.5 mg of the selective ERß ligand, DPN (Tocris), or PPT + DPN. In the second experiment, 2-wk OVX rats (n = 5 per group) were treated (s.c.) for 3 days with 0.2 ml olive oil, 25 µg EB, 3 mg of the SERM tamoxifen (TX; Sigma), or 0.5 mg of the pure antiestrogen RU58668 (Roussel-Uclaf, Romainville, France). Experimental protocols and doses for the different ER ligands were selected based on previous references, including data from our group [20, 21, 24, 25]. At 1000 h on the day after completion of treatments, the animals were killed by decapitation and pituitaries were immediately collected, frozen in liquid nitrogen, and stored at -80°C until use for RNA analyses. In addition, trunk blood samples were obtained for PRL determination, and the presence of intraluminal fluid accumulation at the uterus (uterine ballooning) and cornified cells in the vaginal smears were recorded. Pituitaries from female rats in proestrus were simultaneously sampled to serve as controls with elevated endogenous estrogen levels.
RNA Analysis by Final-Time Reverse Transcription-Polymerase Chain Reaction
Total RNA was isolated from pituitary samples of the different experimental groups using the single-step, acid guanidinium thiocyanate-phenol-chloroform extraction method [26]. The expression levels of the transcripts encoding ER, TERP-1, TERP-2, ERß, ERß2, and progesterone receptor (PR) B isoform were assessed by final-time reverse transcription-polymerase chain reaction (RT-PCR), optimized for semiquantitative detection. Such a method is especially useful for highly sensitive detection and discrimination between alternatively spliced species (e.g., ER vs. TERP and ERß vs. ERß2). For amplification of the targets, the primer pairs indicated in Table 1 were used. These sets of primers were designed to span over intronic DNA sequences and were selected based on previous references [6, 11, 15, 27]. To note, assessment of ER mRNA levels was conducted using a specific primer pair that does not allow amplification of TERP messages, while analysis of TERP-1 expression was carried out using a primer pair where the sense primer is located in position nt-11–31 of the specific TERP-1 sequence, thus allowing discrimination from the full-length ER. This primer pair also enables amplification of the TERP-2 variant. Similarly, ERß2 mRNA expression was assessed using a sense primer specific for the 54-bp insert of this isoform. Finally, analysis of PR-B mRNA levels was performed using a primer combination that enables amplification of a 221-bp fragment from the PR unique to the functional B isoform. In addition, as internal control for reverse transcription and reaction efficiency, amplification of a 290-bp fragment of L19 ribosomal protein (RP-L19) mRNA was carried out in parallel in each sample, using the primer pair and conditions indicated in Table 1, as described elsewhere [14, 15, 25].
|
For amplification of the targets, RT and PCR were run in two separate steps. In addition, to enable appropriate amplification in the exponential phase for each target, PCR amplification of specific signals and L19 ribosomal protein transcripts were carried out in separate reactions with different numbers of cycles but using similar amounts of the corresponding cDNA templates, generated in single RT reactions, as previously described [14, 15, 25]. PCR reactions consisted of a first denaturing cycle at 97°C for 5 min, followed by a variable number of cycles of amplification defined by denaturation at 96°C for 30 sec, annealing for 30 sec, and extension at 72°C for 1 min. A final extension cycle of 72°C for 15 min was included. Annealing temperature was adjusted for each target: 58°C for ER, ERß, and ERß2; 57°C for TERP; and 56°C for PR-B and RP-L19. Optimal PCR conditions were defined for each target to allow amplification in the exponential phase and semiquantitative comparison between groups (see Results).
PCR-generated DNA fragments were resolved in Tris-borate buffered 1.5% agarose gels and visualized by ethidium bromide staining. Specificity of PCR products was confirmed by direct sequencing (Central Sequencing Service, University of Córdoba, Spain). In all assays, liquid controls and reactions without RT were included that yielded negative amplification, thus ruling out the possibility of spurious amplification of the signals. Quantification of intensity of RT-PCR signals was carried out by densitometric scanning using an image analysis system (1-D Manager; TDI Ltd., Madrid, Spain). The values for the specific targets were normalized to those of internal RP-L19 controls to express arbitrary units of relative abundance of the transcripts.
RNA Analysis by Real-Time RT-PCR
To verify changes in gene expression observed with final-time RT-PCR, real-time RT-PCR was performed with the same experimental samples using the iCycler iQ Real-Time PCR detection system (Bio-Rad Laboratories, Hercules, CA). Reverse transcription of total RNA was conducted as described in detail elsewhere [14]. The synthesized cDNAs were further amplified (1:10) in triplicate by PCR using SYBR green I as fluorescent dye and 1x iQ Supermix containing 50 mM KCl, 20 mM Tris-HCl, 0.2 mM dNTPs, 3 mM MgCl2, and 2.5 U iTaq DNA polymerase (Bio-Rad) in a final volume of 25 µl. The PCR cycling conditions were as follows: initial denaturation and enzyme activation at 95°C for 5 min, followed by 40 cycles of denaturation at 95°C for 15 sec, annealing for 15 sec, and extension at 72°C for 1 min. Specific annealing temperatures were selected for each target as described above. Product purity was confirmed by dissociation curves and random agarose gel electrophoresis. No template controls were included in all assays, yielding no consistent amplification.
Calculation of relative expression levels of each target was conducted based on the cycle threshold (CT) method [28]. The CT for each sample was calculated using the iCycler iQ Real-Time PCR detection system software with an automatic fluorescence threshold (Rn) setting. Efficiency of PCR reactions was assessed for each target by amplification of serial dilutions (over five orders of magnitude) of cDNA fragments of the transcripts under analysis. Depending on specific PCR conditions, efficiencies ranged from 90% to 120%. Accordingly, fold expression over OVX values were calculated for each target by the equation 
, where 
CT is determined by subtracting the corresponding RP-L19 CT value (internal control) from the specific CT of each target and experimental condition, and CT is obtained by subtracting the CT of each experimental sample from that of the OVX sample (taken as reference value 100). Note that no significant differences in CT values were observed for RP-L19 between the treatment groups.
PRL Measurement by RIA
Serum PRL levels in the experimental groups were measured in duplicate by RIA using a double-antibody method with a kit supplied by the National Institutes of Health (Bethesda, MD) and a previously described microassay method [25]. Rat PRL-I-6 was labeled with 125I by the chloramine T method and serum PRL concentrations were expressed as nanograms/milliliter of the reference preparation PRL-rat-RP-3. Intraassay coefficient of variation was 9%, and assay sensitivity was 10 pg/tube. All samples from the different experimental groups were measured in the same assay.
Presentation of Data and Statistics
Final-time and real-time RT-PCR analyses were carried out in duplicate from at least four independent RNA samples of each experimental group. Statistical analysis of semiquantitative data from final-time RT-PCR assays was conducted after normalization of absolute optical densities of the specific targets to that of RP-L19 signals. In addition, statistical analysis of data from real-time RT-PCR assays was performed upon relative levels of expression obtained using the CT method (see above). Expression levels in 2-wk OVX females were taken as 100% and the other values were normalized accordingly. Semiquantitative RNA data from real-time RT-PCR assays are presented as mean ± SEM. Results were analyzed for statistically significant differences using ANOVA, followed by Student-Newman-Keuls multiple range test (SigmaStat 2.0 software; Jandel Corporation, San Rafael, CA). P 
0.01 was considered significant.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Expression analyses of target mRNAs were initially conducted in pituitary samples using final-time RT-PCR. Optimization of assays for semiquantitative analysis was carried out for each target by testing different numbers of PCR cycles that were correlated with the intensity of the amplicons. Such a procedure revealed a linear relationship for amplification between cycles 22–36 for ER and TERP, cycles 26–38 for ERß and ERß2, cycles 20–32 for PR-B, and cycles 20–30 for RP-L19, with correlation coefficients r2 
0.98 for all targets. Linearity of amplification for such cycle conditions was further confirmed by real-time PCR assays (see below). Accordingly, and considering previously published protocols [14, 15, 25, 27], 30 (ER and TERP), 33 (ERß and ERß2), 28 (PR-B), and 24 (RP-L19) PCR cycles were chosen for further analysis.
In the first setting, the effects of in vivo administration of estradiol benzoate, the selective ER ligand, PPT, the potency-selective agonist of ERß, DPN, and the combined administration of PPT + DPN were assessed on an OVX background. Taking proestrous values as controls, OVX resulted in a significant increase (P 0.01) in pituitary expression of ERß and ERß2 mRNAs, whereas it decreased TERP-1 and TERP-2 levels without affecting those of ER. As anticipated for a well-known estrogen-responsive gene [27], PR-B mRNA expression was significantly decreased (P 0.01) after OVX. In this model, administration of 25 µg EB fully reversed the responses to OVX in terms of TERP, ERß, and PR-B expression. Similarly, in vivo treatment with 1.5 mg PPT significantly enhanced (P 0.01) pituitary expression of TERP-1, TERP-2, and PR-B mRNAs, while it decreased the relative mRNA levels of ERß and ERß2 transcripts. The effects of PPT were similar in magnitude to those of EB administration. In contrast, DPN failed to induce any significant effect except for a partial stimulation of TERP-1 and TERP-2 mRNA expression levels, which was statistically significant (P 0.01). Note that the ERß agonist was also ineffective in altering pituitary expression of PR-B mRNA. Finally, coadministration of PTT and DPN in vivo resulted in changes in mRNA expression over OVX values that were statistically similar to those induced by EB or PPT alone (Fig. 1).
|
In addition, the actions of EB, the selective ER modulator TX, or the pure antiestrogen RU-58668 were monitored in an independent setting. In this experiment, OVX and EB treatment resulted in changes in pituitary expression levels of TERP, ERß, ERß2, and PR-B targets similar to those observed in the first setting. In all parameters assayed, TX behaved as ER agonist, although the magnitude of responses to TX was significantly lower (P 0.01) than those of PPT, except for induction of TERP-1 and TERP-2 mRNA expression. In contrast, the pure antiestrogen did not modify the expression levels of any of the targets under analysis in pituitaries from OVX rats (Fig. 2).
|
Pituitary ER-Isoform mRNA Expression: Real-Time RT-PCR Analysis
To verify changes in gene expression observed by final-time RT-PCR, real-time RT-PCR was performed in the same experimental groups. Relative levels of expression of the targets were calculated using the CT method, and comparative analyses between groups were conducted considering OVX data as reference values. Real-time PCR analyses fully confirmed our initial final-time RT-PCR data. Thus, EB and PPT completely reversed the responses to OVX, while DPN did not induce any significant effect except for partial stimulation of TERP levels (P 0.01). The effects of coadministration of PTT and DPN were statistically similar to those induced by EB or PPT alone. The SERM TX induced responses similar to EB and PPT, although to a lower potency except for TERP levels, while the pure antiestrogen RU-58668 did not modify the expression of any of the targets (Table 2). A compilation of relative expression levels of ER, TERP-1, TERP-2, ERß, ERß2, and PR-B mRNAs in the different experimental groups, calculated using the CT method [28], is provided in Figure 3.
|
|
Effects of ER Ligands on Vaginal Cornification, Uterine Ballooning, and Serum PRL Levels
The effects of the different ER ligands were also tested and recorded on the day of sampling (Table 3) in terms of induction of vaginal cornification and intraluminal fluid accumulation at the uterus (uterine ballooning), as well as PRL responses, which were assayed in trunk blood samples. Vaginal smears from females in the morning of proestrus (P1000) displayed typical proestrous epithelial nucleated cells. Two-week OVX rats displayed vaginal smears infiltrated by leukocytes and did not show uterine ballooning. In contrast, OVX rats injected with EB, PPT, or PPT + DPN had abundant estrous cornified cells in vagina and intense proestrous-like uterine ballooning. These responses were absent in OVX rats treated either with DPN or the antiestrogen RU-58668, whereas injection of TX induced vaginal cornification but failed to cause uterine ballooning. In OVX rats, serum PRL levels were significantly decreased from control proestrous values, while treatment with EB, PPT, PPT + DPN, and, to a lesser extent, TX significantly enhanced serum PRL concentrations (P 0.01). In contrast, PRL levels remained unaltered in OVX rats treated with DPN or RU-58668.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
is the major signal transducing ER form at the pituitary [8, 18, 19]. However, dramatic changes in circulating estrogen levels (i.e., OVX vs. OVX + EB) did not result in concomitant changes in ER mRNA expression, in line with previous reports [8, 13]. In contrast, clear-cut opposite changes were detected for TERP-1 and TERP-2 mRNA levels, that were decreased by OVX and up-regulated by estrogen, and ERß and ERß2 transcripts, that were elevated by OVX and downregulated by estrogen. Aiming at dissecting out the contribution of and ß forms of ER in such regulatory responses, we used a pharmacological approach based on two recently developed ER subtype-selective ligands: the potent ER agonist, PPT, and the ERß-ligand, DPN. To note, PPT displays 400-fold higher affinity binding for ER and it is virtually unable to activate ERß [16]. In contrast, DPN is a potency-selective agonist for ERß, with 70-fold higher binding affinity for ERß and over 170-fold higher biopotency at ERß than at ER [17]. By using such ER-selective ligands, the above estrogen responses were mostly attributed to ER activation, as PTT, but not DPN, was able to completely mimic the effects of the synthetic estrogen EB. Moreover, comodulatory effects of ERß on ER-mediated effects were not observed in our experimental settings, as coadministration of DPN failed to alter any of the responses of PPT alone.
Although the above data may suggest a negligible role of ERß signaling in adult female pituitary, our results also show that activation of ERß via the potency-selective ligand DPN induced a clear-cut significant elevation in TERP-1 and TERP-2 mRNA levels over OVX values. These truncated ER variants are predominantly expressed in lactotrophs, a pituitary cell type that expresses ERß also [6]. However, a recent study using ER and ERß knockout mouse models indicated that estrogen induction of TERP expression is solely conducted via ER [19]. A tentative explanation for such apparently conflictive results is that, in our experiments, DPN might be inducing a marginal activation of ER, thus resulting in increased TERP mRNA levels. This possibility, however, appears extremely unlikely as, at the dose used, DPN failed to elicit any typical ER-mediated effect in terms of vaginal cornification, uterotropic response, and PRL secretion (see Table 3). Moreover, DPN was unable to increase the expression of a major estrogen-responsive target at the pituitary [27], namely PR-B (see Fig. 3). Indeed, DPN biopotency at the ERß is over 170-fold higher than at ER [21], whereas DPN induction of TERP mRNA levels was as high as 25%–40% of that of PPT. Taken together, these facts cast doubts on the possibility that DPN might induce TERP expression via ER, and support a specific physiological role of ERß in signaling some of the pituitary actions of estrogen.
In contrast with ER variants, hormonal regulation of ERß isoform expression at the pituitary remains poorly characterized. Our results indicate that pituitary ERß2 mRNA levels are finely regulated by estrogen, as OVX increases and estrogen replacement decreases ERß2 mRNA expression; a profile that is strikingly similar to that of ERß mRNA levels. To note, estrogen inhibition of ERß and ERß2 expression is conveyed through activation of ER, but not ERß. Overall, concomitant changes in expression of both ERß isoforms following manipulation of endogenous estrogen tone strongly suggest a cooperative role of these variants in signaling/modulating estrogen actions at the pituitary. Noteworthy, not only ERß2, but also ERß, has been reported to inhibit ER signaling in different target tissues [29, 30]. A similar pattern of regulation of ERß and ERß2 mRNAs by estrogen has been very recently reported in rat uterus [31].
Cumulatively, our data on the regulation of ER isoform expression by estrogen and ER-subtype-selective ligands suggest that estrogen is likely able to self-modulate its own pituitary responsiveness mostly, but not exclusively, through activation of ER. Although estrogen has been shown to repress ER at the protein level [8], our present results demonstrate, however, that these regulatory effects, at the mRNA level, are not conducted by changes at the ER, but instead through modulation of the relative levels of expression of TERP-1, TERP-2, ERß, and ERß2 transcripts. This appears to be the case in some physiological situations, as reported very recently for pregnancy and lactation [32]. Overall, it is tempting to postulate that, depending on the pituitary cell type, interaction of ER protein with its own ligands not only activates signaling but also turns on different mechanisms for the fine tuning of pituitary responsiveness to estrogen. These would include repression of inhibitory ERß forms, which may be essential for the full expression of estrogen effects at the ER, as well as induction of dominant negative isoforms, as TERP-1, which may participate in the auto-limitation of estrogen effects. In addition, although activation of ERß failed to modify its own expression levels, it did induce a significant stimulation of TERP-1 and TERP-2 mRNA levels. This may constitute an additional, novel mechanism whereby ERß is able to repress ER signaling activity at the pituitary. The molecular mechanisms for such a regulatory cross-talk between ER and ERß isoforms remain to be elucidated.
Besides analysis of the effects of ER-subtype-selective ligands, we evaluated the ability of TX to regulate mRNA expression levels of different ER variants. Our current data indicate that, in terms of regulation of ER-isoform expression, TX behaves as an ER-selective ligand, as TX elevated TERP-1, TERP-2, and PR-B mRNA levels and decreased ERß and ERß2 mRNA expression; responses that are similar to those elicited by the ER agonist PPT, albeit to a lower magnitude. These results extend our previous findings indicating that, at the pituitary, TX carries out a combined action with antagonistic effects on LH secretion and agonistic actions on PRL release, GnRH self-priming, and ER isoform mRNA regulation [24, 25, and present results]. Further demonstration of the specificity of the agonistic effects of TX comes from the fact that the pure estrogen antagonist RU-58668 failed to induce any significant change in pituitary levels of TERP-1, TERP-2, ERß, ERß2, and PR-B mRNAs.
In conclusion, the results presented herein indicate that endogenous estrogen differentially regulates pituitary expression of the mRNAs encoding several ER isoforms with distinct functional properties, including TERP-1, TERP-2, ERß, and ERß2, through a mechanism that is mostly conducted through ER. In addition, ERß-mediated induction of TERP-1 and -2 mRNA expression might contribute also to the modulation of pituitary sensitivity to estrogen. Overall, differential regulation of ER isoforms by different receptor ligands may represent a pivotal system for the self-tuning of estrogen responsiveness in female pituitary.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
2 Correspondence: Manuel Tena-Sempere, Physiology Section, Department of Cell Biology, Physiology and Immunology, Faculty of Medicine, University of Córdoba, Avda. Menéndez Pidal s/n, 14004 Córdoba, Spain. FAX: 34 957 218 288; fi1tesem{at}uco.es
Received: 19 July 2003.
First decision: 13 August 2003.
Accepted: 27 October 2003.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
and ß. Endocrinology 1997 138:863-870 and ERß at AP1 sites. Science 1997 277:1508-1510 and ß in the rat pituitary gland. Endocrinology 1998 139:3976-3983 and ß messenger ribonucleic acid expression in rat testis during postnatal development. J Endocrinol 2000 165:345-357[Abstract] and ß in GH3 cells. Endocrinology 1999 140:2651-2658 protein and estrogen responsiveness. Endocrinology 2000 141:2174-2184 (ER), but not ERß, modulates estrogen stimulation of the ER-truncated variant, TERP-1. Endocrinology 2002 143:4196-4202 and estrogen receptor ß knockout mice. Neuroendocrinology 2003 77:324-333[CrossRef][Medline]-selective agonists. J Med Chem 2000 43:4934-4947[CrossRef][Medline] and ERß, in estrogen target tissues in vivo through the use of an ER-selective ligand. Endocrinology 2002 143:4172-4177 activity by ERß in the uterus. Endocrinology 2003 144:3159-3166 transcriptional activity and is a key regulator of the cellular response to estrogens and antiestrogens. Endocrinology 1999 140:5566-5578-regulated gene transcription, supporting a "Ying-Yang" relationship between ER and ERß in mice. Mol Endocrinol 2003 17:203-208This article has been cited by other articles:
|
|
 |
|
  A. Gordon, J. C Garrido-Gracia, R. Aguilar, C. Bellido, J. A G. Velasco, Y. Millan, M. Tena-Sempere, J. Martin de las Mulas, and J. E Sanchez-Criado The ovary-mediated FSH attenuation of the LH surge in the rat involves a decreased gonadotroph progesterone receptor (PR) action but not PR expression J. Endocrinol., March 1, 2008; 196(3): 583 - 592. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. A. Arreguin-Arevalo, T. L. Davis, and T. M. Nett Differential Modulation of Gonadotropin Secretion by Selective Estrogen Receptor 1 and Estrogen Receptor 2 Agonists in Ovariectomized Ewes Biol Reprod, August 1, 2007; 77(2): 320 - 328. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Lopez, C. J. Lelliott, S. Tovar, W. Kimber, R. Gallego, S. Virtue, M. Blount, M. J. Vazquez, N. Finer, T. J. Powles, et al. Tamoxifen-Induced Anorexia Is Associated With Fatty Acid Synthase Inhibition in the Ventromedial Nucleus of the Hypothalamus and Accumulation of Malonyl-CoA. Diabetes, May 1, 2006; 55(5): 1327 - 1336. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J E Sanchez-Criado, J M. de las Mulas, C Bellido, V M Navarro, R Aguilar, J C Garrido-Gracia, M M Malagon, M Tena-Sempere, and A Blanco Gonadotropin-secreting cells in ovariectomized rats treated with different oestrogen receptor ligands: a modulatory role for ER{beta} in the gonadotrope? J. Endocrinol., February 1, 2006; 188(2): 167 - 177. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. A Taylor, P. Cowin, J. F. Couse, K. S. Korach, and G. P. Risbridger 17{beta}-Estradiol Induces Apoptosis in the Developing Rodent Prostate Independently of ER{alpha} or ER{beta} Endocrinology, January 1, 2006; 147(1): 191 - 200. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. A. Blake, L. M. Brown, M. W. Duncan, S. W. Hunsucker, and S. M. Helmke Estrogen Regulation of the Rat Anterior Pituitary Gland Proteome Experimental Biology and Medicine, December 1, 2005; 230(11): 800 - 807. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J Varayoud, J G Ramos, L Monje, V Bosquiazzo, M Munoz-de-Toro, and E H Luque The estrogen receptor {alpha} {Sigma}3 mRNA splicing variant is differentially regulated by estrogen and progesterone in the rat uterus J. Endocrinol., July 1, 2005; 186(1): 51 - 60. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G.-S. Lee, H.-J. Kim, Y.-W. Jung, K.-C. Choi, and E.-B. Jeung Estrogen Receptor {alpha} Pathway Is Involved in the Regulation of Calbindin-D9k in the Uterus of Immature Rats Toxicol. Sci., April 1, 2005; 84(2): 270 - 277. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. M. Navarro, J. M. Castellano, R. Fernandez-Fernandez, M. L. Barreiro, J. Roa, J. E. Sanchez-Criado, E. Aguilar, C. Dieguez, L. Pinilla, and M. Tena-Sempere Developmental and Hormonally Regulated Messenger Ribonucleic Acid Expression of KiSS-1 and Its Putative Receptor, GPR54, in Rat Hypothalamus and Potent Luteinizing Hormone-Releasing Activity of KiSS-1 Peptide Endocrinology, October 1, 2004; 145(10): 4565 - 4574. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK |
