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Endocrinology, doi:10.1210/en.2007-0086
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Endocrinology Vol. 148, No. 7 3371-3382
Copyright © 2007 by The Endocrine Society

Estrogen Receptor-ß Mediates Dihydrotestosterone-Induced Stimulation of the Arginine Vasopressin Promoter in Neuronal Cells

Toni R. Pak, Wilson C. J. Chung, Laura R. Hinds and Robert J. Handa

Department of Biomedical Science, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, Colorado 80523

Address all correspondence and requests for reprints to: Toni R. Pak, Department of Cell Biology, Neurobiology, and Anatomy, Loyola University Chicago, Stritch School of Medicine, 2160 South First Avenue, Maywood, Illinois 60153. E-mail: tpak{at}lumc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Arginine vasopressin (AVP) is a neuropeptide involved in the regulation of fluid balance, stress, circadian rhythms, and social behaviors. In the brain, AVP is tightly regulated by gonadal steroid hormones in discrete regions with gonadectomy abolishing and testosterone replacement restoring normal AVP expression in adult males. Previous studies demonstrated that 17ß-estradiol, a primary metabolite of testosterone, is responsible for restoring most of the AVP expression in the brain after castration. However, 5{alpha}-dihydrotestosterone (DHT) has also been shown to play a role in the regulation of AVP expression, thus implicating the involvement of both androgen and estrogen receptors (ER). Furthermore, DHT, through its conversion to 5{alpha}-androstane-3ß,17ß-diol, has been shown to modulate estrogen response element-mediated promoter activity through an ER pathway. The present study addressed two central hypotheses: 1) that androgens directly modulate AVP promoter activity and 2) the effect is mediated by an estrogen or androgen receptor pathway. To that end, we overexpressed androgen receptor, ERß, and ERß splice variants in a neuronal cell line and measured AVP promoter activity using a firefly luciferase reporter assay. Our results demonstrate that DHT and its metabolite 5{alpha}-androstane-3ß,17ß-diol stimulate AVP promoter activity through ERß in a neuronal cell line.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ARGININE VASOPRESSIN (AVP), a nonapeptide originally described for its role in the regulation of renal fluid balance, is a potent neuromodulator. AVP-expressing neurons are found in a number of brain regions including the bed nucleus of the stria terminalis (BST), medial amygdala (MeA), paraventricular nucleus (PVN), supraoptic nucleus (SON), and suprachiasmatic nucleus (SCN), all of which participate in the regulation of centrally mediated behaviors (1, 2, 3), circadian rhythms (4, 5), and the stress response (6, 7, 8) as well as fluid balance. AVP expression in the BST and MeA is gonadal steroid dependent because gonadectomy of adult male animals abolishes AVP immunoreactivity and testosterone (T) replacement fully restores expression (9, 10).

In the brain, T can be considered a multifunctional precursor hormone. Upon entry into a target cell, T may be converted to 17ß-estradiol (E2) or 5{alpha}-dihydrotestosterone (DHT), depending on the presence of the enzymes, aromatase or 5{alpha}-reductase, respectively. T may also bind and activate androgen receptors (AR) directly. Interestingly, both E2 and DHT are required to restore AVP immunoreactivity to the level seen in intact male rats, with E2 restoring approximately 90% and DHT restoring about 10% of AVP-expressing cells (11). These data have been interpreted to indicate that estrogen receptor (ER) and AR pathways independently mediate AVP expression, yet both are required for full expression. However, the molecular mechanism for the androgenic regulation of AVP has not been demonstrated.

Classically, androgens activate transcription through AR. AR belong to the larger superfamily of nuclear receptors and are classified as class I ligand-activated transcription factors (12). AR are found in AVP-expressing cells in the BST and MeA (13, 14). DHT binds with higher affinity to AR than does T, and both activate transcription by directing the AR to bind to an androgen response element located within the promoter region of androgen-responsive genes.

Alternatively, DHT can be converted to 5{alpha}-androstane-3ß,17ß-diol (3ßAdiol), or its stereoisomer 3{alpha}Adiol, by a variety of steroid-metabolizing enzymes, including 17ß-hydroxysteroid dehydrogenase (17ßHSD), 3{alpha}-hydroxysteroid oxidoreductase (3{alpha}HSD), and 3ß-hydroxysteroid oxidoreductase (3ßHSD) (15, 16). Although 3{alpha}Adiol maintains bidirectional catalysis with DHT, the conversion to 3ßAdiol is irreversible. Notably, recent reports have shown that despite the androgenic origin of 3ßAdiol, it preferentially binds and activates ERß and has a low binding affinity for AR (17). Furthermore, we have previously established that 3ßAdiol’s ability to activate transcription through ERß is mediated by an estrogen response element (ERE) (18). Together, these data raise the possibility that DHT increases AVP expression indirectly, by metabolism to a compound that acts through an ER- and not AR-mediated pathway.

There are two subtypes of ER, termed ER{alpha} and ERß, which are encoded by distinct genes but have highly homologous DNA-binding domains (97%) (19). Furthermore, the binding affinities of ER{alpha} and ERß for E2 are relatively similar despite only 57% homology in their ligand-binding domains (19, 20). At least three splice variants of ERß (also called ERß1) have been identified in rodents since its first detection in 1996. These include variants lacking the third exon ({delta}3), lacking the fourth exon ({delta}4), or with an insert between exons 5 and 6 (ß2). Combinations of these three result in a number of different variant mRNAs (21, 22, 23, 24). Although a functional significance for these variant forms of ERß has yet to be elucidated, they are found in numerous tissues including many brain areas (22, 23, 25).

The present study addressed two central hypotheses: 1) that androgens directly modulate AVP promoter activity and 2) the effect is mediated by an ER or AR pathway. To that end, we overexpressed AR, ERß, and ERß splice variants in a neuronal cell line and measured AVP promoter activity using a firefly luciferase reporter assay. Our results demonstrate that DHT and its metabolite 3ßAdiol stimulate AVP promoter activity through ERß in a neuronal cell line.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Transient transfections
Cell culture.
The human neuroblastoma-derived cell line SK-N-SH (American Type Culture Collection, Manassas, VA), was used for all transient transfections. Cells were maintained in MEM with Earle’s salts, containing 4.5% glucose and L-glutamine (Invitrogen Inc., Carlsbad, CA) supplemented with 1x nonessential amino acids and 10% fetal bovine serum (Gemini Bioproducts, Woodland, CA). Cells were grown to 70% confluency, and all transient transfection experiments were performed within 10 passages.

Expression vectors.
Plasmid expression vectors for ER included a pcDNA 3.0 vector (Invitrogen, Carlsbad, CA) containing a cDNA insert coding for rat ERß1, rat ERß1{delta}3, and rat ERß2 (generously provided by Dr. Tom Brown, Pfizer Corp, Cambridge, MA). The GRIP-1 (NR box) expression vector consisted of the amino acid sequence 629–760 of the glucocorticoid receptor interacting protein-1 (GRIP1) subcloned into the pM vector (generously provided by Dr. Donald P. McDonnell and Julie M. Hall, Duke University, Durham, NC). The AR expression vector consisted of a pCMV vector containing a cDNA insert coding for full-length rat AR (generously provided by Drs. Cynthia L. Jordan and Douglas A. Monks, Michigan State University, Lansing, MI). Upon receipt, the full-length AR was digested from the pCMV vector at the BglII and BamHI restriction sites and subsequently subcloned into the pcDNA 3.1 expression vector (Invitrogen). The pcDNA/AR expression vector was validated by transient transfection into SK-N-SH cells followed by 15 h of 10 nM DHT treatment. AR protein was detected using immunocytochemistry and was determined to be functional if DHT induced a translocation of AR immunoreactivity from the cell cytoplasm to the nucleus (see Fig. 1Go).


Figure 1
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  		FIG. 1. AR expression in the SK-N-SH neuronal cell line. A, Photomicrograph of RT-PCR product for AR mRNA after 1.0% agarose gel electrophoresis and staining with ethidium bromide. Total RNA was isolated from the human prostate tumor-derived cell line LNCaP (control) or the human neuroblastoma-derived cell line SK-N-SH. Presence of 279-bp band indicates the presence of human AR mRNA in representative cell line. B, Transient transfection of SK-N-SH cells with an empty expression vector or vector containing insert for full-length AR. Cells were treated with vehicle (0.009% EtOH) or 10 nM DHT for 15 h. After treatment, cells were processed by immunocytochemistry for presence of AR protein. Panels I and II indicate an absence of AR protein. Panel III shows primarily cytoplasmic localization of AR protein. Panel IV shows nuclear localization of AR protein. Inset in panels III and IV show a higher magnification of selected cells from that same panel.

 
Reporter constructs.
The full-length rat AVP promoter (5446 bp) was subcloned into the promoterless luciferase vector (pGL2 basic; generously provided by Drs. Daniel M. Dorsa and Robert A. Shapiro, OR Health & Science University, Portland, OR). Truncations were made by restriction enzyme digestion upstream from the transcription start site at position –1370 (AVP1.3), –740 (AVP740), and –306 (AVP306) and subsequently subcloned into the pGL2 basic promoterless luciferase vector. The pJ3 reporter construct (generously provided by Dr. Rosalie Uht, University of Virginia, Charlottesville, VA), expressing ß-galactosidase under the control the sv40 promoter, was used as an internal control for calculating plasmid transfection efficiency.

Transfections.
Cells were plated at a density of 0.5 x 105 cells per well in 24-well plates for 48 h before transfection to achieve a final confluency of 70–80%. All constructs were transfected in triplicate wells within each assay, and each transfection assay was repeated a minimum of six times. Each experiment was performed using a minimum of three different preparations for each plasmid. Transfections were carried out using a lipid-mediated transfection reagent (Fugene6; Roche Molecular Biomedical, Indianapolis, IN) according to the manufacturer’s instructions. Cells were incubated with transfection media complex overnight followed by replacement with MEM containing 10% dextran-charcoal-stripped fetal bovine serum (Hyclone Laboratories, Logan, UT) to ensure steroid-free culture conditions. Twenty-four hours after transfection, cells were incubated with media containing hormone treatments for 15 h and then lysed for luciferase analysis. All treatments were performed in MEM containing 10% dextran-charcoal-stripped fetal bovine serum to ensure depletion of all endogenous steroid hormones. In addition, each transfection experiment was performed a minimum of three times with phenol red-free media, and no differences were observed from the transfections performed with media containing phenol red. Luciferase activity was measured using 20 µl cell lysate added to 100 µl luciferin substrate (Promega Corp., Madison, WI). ß-Galactosidase activity was measured using 40 µl cell lysate added to 200 µl galacton substrate (Tropix–GalactoLight kit; Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions. Relative light units were measured using a 20/20 TD luminometer (Turner Designs, Sunnyvale, CA).

Hormone treatments.
The following compounds were diluted in 95% ethanol (EtOH) (vehicle control) and used at a final concentration of 100 nM in 0.001% EtOH: 3ßAdiol, E2, and DHT (Steraloids, Newport, RI). Each compound was added to MEM with Earle’s salts supplemented with 10% dextran-charcoal-stripped fetal bovine serum (Hyclone Laboratories, Logan, UT).

Site-directed mutagenesis.
The putative ERE located at position –995/–984 of the AVP promoter was deleted using the QuikChange II XL kit according to manufacturer’s instructions (Stratagene, La Jolla, CA). Briefly, the primer sequence 5'-GCTATGTAGTAAAGCCTGGACTAAACGACTGC-3' was designed to target the desired region of the AVP promoter. A standard PCR was performed on a thermal cycler using the AVP1.3-luciferase reporter construct as a template. After the reaction, the parent plasmid was digested using the DpnI restriction enzyme and the daughter plasmid, containing the desired mutation, was transformed into XL-10 Ultragold competent cells and amplified. The presence of the site-directed mutation was confirmed by DNA sequencing (Retrogen Inc., San Diego, CA).

RT-PCR
RNA isolation.
Total RNA was isolated from SK-N-SH cells using the guanidinium thiocyanate method (26). Briefly, cells were trypsinized, washed in PBS, and resuspended in 250 µl GIT buffer [4 M guanidinium thiocyanate, 25 mM sodium citrate (pH 7.0), and 0.5% (wt/vol) N-laurylsarcosine, and 0.1 M ß-mercaptoethanol]. RNA was extracted with phenol/chloroform and EtOH precipitated, and the pellet was resuspended in nuclease-free water. Quantification of RNA was performed with a Beckman DU 530 spectrophotometer (Beckman Coulter Inc., Fullerton, CA).

Reverse transcription.
Total RNA (2 µg) was combined with 0.5 µg oligo d(T), heated to 65 C, and rapidly cooled on ice. The RNA-primer mix was combined with M-MLV buffer [50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2], 10 mM dithiothreitol, 0.5 mM dNTP, and 0.5 mM M-MLV reverse transcriptase (Invitrogen, Carlsbad, CA). The reverse transcriptase reaction was performed by incubating for 10 min at room temperature, 50 min at 42 C, and then 95 C for 5 min to terminate the reaction.

PCR.
PCR was performed using FastStart DNA Master SYBR Green I kit according to manufacturer’s instructions (Roche). Optimal MgCl2 concentration was experimentally determined for each set of primers. Five picomolar of forward and reverse primer was added per reaction. Master mix containing MgCl2, SYBR Green, and primer pairs were aliquotted into capillary tubes (Roche) followed by the addition of 1/20th of the RT reaction, except control, which received DNA-free water of the same volume. In a Roche Light Cycler, capillary tubes were heated to 95 C for 6 min, then a repeated cycle specific to each primer (see below) with florescence detection at the end of each 72 C step, and then melted with continuous florescence detection to 95 C. PCR products were resolved on a 2% agarose gel and compared with a DNA ladder of known size (Fisher Scientific, Pittsburgh, PA; Exactgene 50-bp ladder) to confirm product size, and to verify specificity, the products were subjected to a thermal melting curve analysis to determine whether the melting temperature of the product was consistent with the calculated theoretical melting temperature based on sequence.

The following primer sequences targeting the human sequence for the gene indicated and specific repeated cycle conditions are as follows: 17ßHSD forward, 5'-CCGGGAGCGTGGGAGGATTGAT 3', and reverse, 5'-TCGGTGGTGAAGTAGCGCAGGG-3' (95 C for 2 sec, 68 C for 5 sec, and 72 C for 15 sec); 3ßHSD forward, 5'TCATCCACACCGCCTGTATCAT-3', and reverse, 5'-TTTCAGATTCCACCCGTTAGCC-3' (95 C for 2 sec, 64 C for 5 sec, and 72 C for 12 sec); AR forward, 5'-GACAACAACCAGCCCGACTCCT-3', and reverse, 5'-GCTGTACATCCGGGACTTGTGC 3' (95 C for 2 sec, 67 C for 5 sec, and 72 C for 12 sec); and ER-ß1 forward, 5'-CCTGACCAAGTTGGCCGACAAG-3', and reverse, 5'-CTGGGAGGAGATGCCGCTCTT-3' (95 C for 2 sec, 66 C for 5 sec, and 72 C for 19 sec).

Immunocytochemistry
Cells were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer for 10 min. The cells were 1) rinsed with 0.1 M PBS, 2) blocked and permeabilized with 0.1% BSA/0.3% Triton X/PBS for 1 h, 3) incubated overnight at 4 C with the polyclonal rabbit androgen receptor antibody PG-21 at 1:1000 dilution (provided by Dr. G. Prins), 4) rinsed with PBS, 5) incubated with biotinylated goat antirabbit (Vector Laboratories, Burlingame, CA), 6) rinsed with PBS, 7) incubated with biotin-coupled horseradish peroxidase and avidin (ABC Elite, Vector) at 1:800 dilution, 8) rinsed with 0.05 M Tris-buffered-saline, and 9) finally reacted with 0.05% diaminobenzidine/0.25% nickel ammonium sulfate/0.01% H2O2/Tris-buffered saline for 10 min.

Statistics
Data were analyzed by two-way ANOVA (hormone treatment x plasmid concentration) followed by Tukey’s honestly significant difference test. Differences were considered significant when P < 0.05. All transfection data are represented as percent change compared with vehicle-treated, empty vector controls.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characterization of AR expression in the SK-N-SH neuronal cell line
For these studies, we used the neuroblastoma-derived cell line SK-N-SH as a model system. Cells were initially tested for the presence of endogenous AR. Real-time RT-PCR indicated an absence of AR mRNA in this cell line (Fig. 1AGo). Next, SK-N-SH cells were transiently transfected with the AR expression vector to determine whether the exogenously transfected AR plasmid construct produced functional AR protein. These results showed that cells transfected with an empty vector control (pcDNA3.1) showed no AR immunoreactivity irrespective of hormone treatment (Fig. 1BGo, panels I and II). In contrast, after transient transfection with an AR-containing plasmid, AR immunoreactivity was detected and was primarily localized to the cytoplasm of vehicle-treated cells (Fig. 1BGo, panel III; see inset). After treatment with 10 nM DHT, there was a hormone-dependent translocation of AR immunoreactivity to the nucleus (Fig. 1BGo, panel IV, see inset). Together, these results validated the efficacy of our transfection system and the functionality of our AR expression vector.

Effects of DHT on AR-mediated AVP promoter-luciferase activity
To determine whether androgens directly modulate AVP promoter activity through an AR-mediated mechanism, we cotransfected SK-N-SH cells with an expression vector containing rat AR and with a luciferase reporter construct containing the full-length (5.5 kb) rat AVP promoter (Fig. 2Go). Cells were then treated with either vehicle or DHT for 15 h. Overexpression of AR, at plasmid concentrations of 0.5 and 1.0 µg, significantly reduced full-length (5.5 kb) AVP promoter-luciferase activity in cells treated with vehicle alone (Fig. 3AGo). However, these same concentrations of plasmid AR also significantly reduced luciferase activity in cells cotransfected with AR and the promoterless luciferase reporter construct (data not shown). There was no significant difference in the AR-induced reduction of luciferase activity between the promoterless and the AVP promoter-containing luciferase reporter constructs, suggesting that overexpression of AR in these cells induced a generalized squelching of transcription that was not specific to the AVP promoter. Similar effects were observed when the AVP promoter was truncated at 1.3 kb (Figs. 2Go and 3BGo).


Figure 2
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  		FIG. 2. AVP promoter and response elements. The diagram depicts the full-length AVP promoter (5.5 kb) and approximate location of documented response elements. Below the full-length promoter are diagrams of the promoter constructs that were truncated by restriction enzyme digestion at positions –1.3, –740, and –306 kb upstream from the transcription start site. The promoter construct designated AVP1.3{delta}ERE describes the 1.3-kb promoter containing a site-directed mutation of a putative ERE located at position –995/–984.

 

Figure 3
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  		FIG. 3. Effects of AR overexpression and DHT treatment on AVP promoter activity. Cotransfection of SK-N-SH cells with the full-length (5.5 kb) (A) or the 1.3-kb AVP promoter-luciferase reporter construct (B) and an expression vector containing 1.0 µg/well of AR. After transfection, cells were treated with 0.009% EtOH (vehicle) or 10 nM DHT for 15 h. Data are represented as percent change in relative light units from vehicle-treated empty vector control ± SEM. *, Significant difference from empty vector controls (P < 0.05).

 
Treatment with 10 nM DHT significantly reduced full-length AVP (5.5 kb) promoter-luciferase activity in cells transfected with 0.1 µg plasmid AR (Fig. 3AGo). This ligand-dependent effect was abolished with the truncated AVP promoter (1.3 kb), suggesting that the site of AR-mediated inhibition by DHT lies upstream of the 1.3-kb region of the AVP promoter (Fig. 3BGo).

Characterization of androgen metabolic enzymes in the SK-N-SH neuronal cell line
The nonaromatizable androgen DHT can be further metabolized intracellularly to 3ßAdiol and its stereoisomer 3{alpha}Adiol (Fig. 4Go). Because 3ßAdiol has been shown to have a high affinity for binding ERß, we next determined whether the SK-N-SH neuronal cell line endogenously expressed the enzymes necessary to convert DHT to 3ßAdiol. The primary enzymes involved in the conversion process have been identified as 3ßHSD and 17ßHSD (15, 16). Results of amplification by real-time RT-PCR demonstrated the presence of both enzyme mRNAs in SK-N-SH cells (Fig. 5Go, A and B). Gel electrophoresis of the PCR product showed three distinct bands for 17ßHSD mRNA that correspond to the three different isoforms of 17ßHSD (Fig. 5BGo). Our results also demonstrated that ERß1 was endogenously expressed in this cell line (Fig. 5CGo). The prostate tumor-derived LNCaP cell line was used as a positive control for these experiments.


Figure 4
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  		FIG. 4. Metabolic pathway of androgens. Diagram depicts the metabolic pathway of T. Italics signify enzymes required for conversion, and bold lettering denotes the chemical name of the hormone product.

 

Figure 5
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  		FIG. 5. Characterization of enzymes and receptors in the SK-N-SH neuronal cell line. A–C, Photomicrograph showing amplified products for 3ßHSD (A), 17ßHSD (B), and ERß1 (C) mRNA after RT-PCR and 1.0% agarose gel electrophoresis. Products were stained with ethidium bromide. Total RNA was isolated from the human prostate tumor-derived cell line LNCaP (control) or the human neuroblastoma-derived cell line SK-N-SH. Presence of band indicates primary transcripts for above named products in each cell line.

 
Ligand-dependent and ligand-independent effects on full-length (5.5 kb) AVP promoter activity mediated by ERß splice variants
To determine whether DHT, or its metabolite 3ßAdiol, regulates AVP promoter activity through an ERß-mediated mechanism, we cotransfected SK-N-SH cells with the full-length (5.5 kb) AVP promoter-luciferase reporter construct and an expression vector containing either ERß1, ERß1{delta}3, or ERß2 (Fig. 6Go). Overexpression of ERß1 caused a robust ligand-independent increase in AVP promoter activity that is consistent with previous results (Fig. 6Go). In addition, treatment with DHT and 3ßAdiol, but not E2, significantly augmented the ligand-independent effect.


Figure 6
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  		FIG. 6. Effects of various ERß splice variants on full-length (5.5 kb) AVP promoter activity. Cotransfection of SK-N-SH cells with the full-length (5.5 kb) AVP promoter-luciferase reporter construct and an expression vector containing 1.0 µg/well of ERß1, ERß1{delta}3, or ERß2. After transfection, cells were treated with 0.009% EtOH (vehicle), 100 nM E2, 100 nM DHT, or 100 nM 3ßAdiol for 15 h. Data are represented as percent change ± SEM in relative light units from vehicle-treated empty vector control. *, Significant difference from empty vector controls; #, statistically significant difference among groups (P < 0.05).

 
The splice variant ERß1{delta}3 is lacking exon 3, the coding sequence for the second zinc finger of the DNA-binding domain, resulting in an inability to bind DNA efficiently (23). Cotransfection with ERß1{delta}3 had no effect on AVP promoter activity even in the presence of a ligand (Fig. 6Go), suggesting that DNA binding is required for ERß-mediated AVP promoter activity.

The splice variant ERß2 contains an 18-amino-acid sequence in the ligand-binding domain that is not present in the other ERß splice variants. Similar to ERß1, cotransfection with ERß2 significantly increased AVP promoter activity in a ligand-independent fashion, although the magnitude of the increased activity was significantly less (~50%) than that of ERß1 (P < 0.05). However, in contrast to results using ERß1, treatment of ERß2-expressing cells with E2 and 3ßAdiol, but not DHT, significantly increased AVP promoter activity (Fig. 6Go).

Determining a site of action for ERß-induced AVP promoter activity using truncated AVP promoter-luciferase reporter constructs
Previous studies suggested that ERß1 increased AVP promoter activity in a ligand-independent fashion and identified the putative site of action as located between –4.0 and –1.1 kb upstream from the transcriptional start site (27). To more precisely define a location on the proximal AVP promoter that is required for the ligand-independent and -dependent actions of ERß1 and ERß2, we deleted regions of the promoter from the 5' end at the following locations upstream from the transcriptional start site: –1370 (AVP1.3), –740 (AVP740), and –306 (AVP306). Successive deletion of the 5' end of the AVP promoter significantly increased basal activity for the AVP740 and AVP306 constructs (Fig. 7Go). The mean increase in basal activity was 195 ± 27 and 179 ± 22% for AVP740 and AVP306, respectively. There was no significant effect of the deletion on the basal activity of the AVP1.3 promoter construct (Fig. 7Go).


Figure 7
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  		FIG. 7. Basal activity of truncated AVP promoter-luciferase reporter constructs. SK-N-SH cells were transiently transfected with 1.0 µg of various AVP promoter-luciferase reporter constructs: AVP5.5, AVP1.3, AVP740, and AVP306. Data are expressed as a percent change ± SEM in relative light units from the full-length (5.5 kb) AVP promoter-luciferase reporter construct. *, Significant difference from the full-length promoter activity (P < 0.05).

 
AVP1.3 promoter construct.
Cotransfection of AVP1.3 with ERß1 and ERß2 resulted in similar responses to those seen with the full-length promoter. Overexpression of ERß1 induced a significant increase in AVP1.3 promoter activity in the absence of hormone treatment (Fig. 8AGo). However, contrary to results using the full-length promoter, hormone treatments did not further augment the ligand-independent increase in AVP1.3 promoter activity induced by ERß1 (Fig. 8AGo). Cotransfection of ERß2 with the AVP1.3 promoter produced the same response seen with the full-length (5.5 kb) AVP promoter (Figs. 6Go and 8AGo). A significant ligand-independent increase was observed, and the effect was further enhanced with E2 and 3ßAdiol, but not DHT, treatment (Fig. 8AGo).


Figure 8
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  		FIG. 8. Characterization of AVP promoter site of action for ERß1 and ERß2-mediated effects. A–C, Cotransfection of SK-N-SH cells with the truncated AVP promoter-luciferase reporter constructs AVP1.3 (A), AVP740 (B), or AVP306 (C) and an expression vector containing 1.0 µg/well of ERß1 or ERß2. After transfection, cells were treated with 0.009% EtOH (vehicle), 100 nM E2, 100 nM DHT, or 100 nM 3ßAdiol for 15 h. Data are represented as mean ± SEM percent change in relative light units from vehicle-treated empty vector control. *, Significant difference from empty vector controls; #, statistically significant difference among groups (P < 0.05).

 
AVP740 promoter construct.
Cotransfection with ERß1 increased ligand-independent activity of the AVP740 promoter; however, the effect was greatly reduced from that of the full-length (5.5 kb) promoter (330.9 and 1169.7%, respectively; Figs. 7Go and 9BGo). Hormone treatments had no effect on AVP740 promoter activity mediated by ERß1. The pattern of AVP740 promoter activity when cotransfected with ERß2 was similar to the AVP1.3 and AVP5.5 constructs. Overexpression of ERß2 induced a significant ligand-independent increase in AVP740 promoter activity, although, like ER-ß1, the effect was not as robust as seen with the full-length construct (228.3 and 619.5%, respectively; Figs. 6Go and 8BGo). Treatment with DHT and 3ßAdiol significantly increased AVP740 promoter activity above the ligand-independent levels to a similar degree observed with the full-length construct (Fig. 8BGo).


Figure 9
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  		FIG. 9. Site-directed mutagenesis of putative ERE located in proximal AVP promoter. Cotransfection of SK-N-SH cells with the truncated AVP promoter-luciferase reporter construct AVP1.3 containing a deletion of the putative ERE sequence AGGTCAAGGTCA located at position –995/–984 upstream from the transcription start site and an expression vector containing 1.0 µg/well of ERß1 or ERß2. After transfection, cells were treated with 0.009% EtOH (vehicle), 100 nM E2, 100 nM DHT, or 100 nM 3ßAdiol for 15 h. Data are represented as percent change in relative light units from vehicle-treated empty vector control (mean ± SEM). *, Significant difference from empty vector controls. Dissimilar symbols denote a statistically significant difference among groups (P < 0.05).

 
AVP306 promoter construct.
Deletion of the AVP promoter at position –306 resulted in greatly reduced ligand-independent effects of ERß1 and ERß2. Unlike what was observed with the previous AVP promoter constructs, both ERß1 and ERß2 increased AVP306 promoter activity equivalently (Fig. 8CGo). Furthermore, the hormone-dependent increase observed with ERß2 was completely abolished with the AVP306 promoter (Fig. 8CGo).

Effects of putative ERE deletion on ERß-induced ligand-independent AVP promoter activity
Two functional consensus ERE sequences have been identified in the most distal 1-kb region of the AVP promoter (27). These regions were shown to be important for some of the ligand-dependent regulation of the AVP promoter mediated by ER{alpha}. Here, we have identified a putative ERE in the proximal region of the promoter beginning at position –984 (Fig. 2Go). This experiment was designed to determine whether the proximal ERE is important for the ligand-independent action of ERß1 and ERß2. We used site-directed mutagenesis to destroy the putative ERE in the AVP1.3 promoter construct (AVP1.3{delta}ERE; Fig. 2Go). This construct was selected because it lacked the distal EREs, yet it functionally resembled the activity of the full-length promoter when cotransfected with ERß1 and ERß2.

Similar to results shown with the AVP1.3 promoter construct, cotransfection of ERß1 with AVP1.3({delta}ERE) induced a robust ligand-independent increase in promoter activity (Fig. 9Go). However, deletion of the proximal ERE resulted in a significant ligand-dependent increase in promoter activity mediated by ERß1. Treatment with DHT and 3ßAdiol, but not E2, significantly augmented the ligand-independent increase in promoter activity (Figs. 8AGo and 9Go). Cotransfection with AVP1.3({delta}ERE) and ERß2 yielded similar results as those observed with the AVP1.3 promoter construct (Figs. 8AGo and 9Go). Thus, deletion of the proximal ERE had no significant effect on ligand-independent promoter activity mediated by ERß2 (Fig. 9Go). However, treatment with 3ßAdiol was significantly more effective at augmenting the ligand-independent effect than E2 (Fig. 9Go). This result is contrary to what was observed with the AVP5.5, AVP1.3, and AVP740 promoters. With those promoter constructs, E2 and 3ßAdiol equally increased promoter activity above levels achieved with the ligand-independent effect (P < 0.05; Figs. 6Go and 8Go, A and B).

The coregulatory protein GRIP1 is required for 3ßAdiol-induced stimulation of AVP promoter activity mediated by ERß1
The recruitment of coregulatory proteins is an important component of ER signaling pathways (28). To determine whether the coactivator GRIP1 is required for ERß1-induced AVP promoter activity, the full-length (5.5 kb) AVP promoter-luciferase reporter construct was cotransfected with an expression vector containing ERß1 and an expression vector containing the NR-box sequence (amino acids 629–760) from GRIP1 (GRIP-NRbox). The GRIP-NRbox expression vector serves as a dominant negative by preventing endogenous GRIP1 protein from interacting with ERß1 (29). Overexpression of ERß1 induced a robust ligand-independent increase in AVP promoter activity that was unaffected by the GRIP-NRbox construct (Fig. 10Go). However, although treatment with 3ßAdiol significantly augmented the ligand-independent effect in control cells, it had no effect in cells transfected with GRIP-NRbox (Fig. 10Go).


Figure 10
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  		FIG. 10. Effects of squelching endogenous GRIP1 protein in SK-N-SH cells on ERß1-mediated AVP promoter activity. Cotransfection of SK-N-SH cells with the full-length (5.5 kb) AVP promoter-luciferase reporter construct and an expression vector containing 1.0 µg/well of ERß1 and/or GRIP1-NRbox. After transfection, cells were treated with 0.009% EtOH (vehicle) or 100 nM 3ßAdiol for 15 h. Data are represented as percent change in relative light units from vehicle-treated empty vector control (mean ± SEM). *, Significant difference from empty vector controls; #, statistically significant difference among groups (P < 0.05).

 

   Discussion
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 Abstract
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 Materials and Methods
 Results
 Discussion
References
 
Together, these experiments revealed important information that furthers our understanding of how gonadal steroid hormones regulate AVP promoter activity. First, we have demonstrated a potential molecular mechanism for the androgenic regulation of AVP expression in some brain areas. Our results show that DHT acts through AR to inhibit AVP promoter activity but does not work through AR to increase AVP promoter activity, whereas DHT and its metabolite 3ßAdiol can stimulate AVP promoter activity through ERß1 and ERß2. Second, we have provided evidence that ERß splice variants might play an important role in differentially modulating AVP expression in neuronal cells. ERß1 and -ß2 induced a strong constitutive activation of the AVP promoter in addition to their ligand-dependent effects, suggesting that up-regulation of these receptors in AVP-containing cells is sufficient to alter AVP expression profiles in the brain. Third, ERß1 and ERß2 have differing ligand-dependent effects and act at different regions of the AVP promoter to exert their ligand-dependent effects, thereby providing an additional level of complexity for gonadal steroid hormone regulation of AVP. Finally, our results confirmed that the nuclear receptor coactivator protein GRIP is required for 3ßAdiol to induce AVP promoter activity when mediated by ERß1. This raises the possibility that ERß1 and ERß2 can differentially regulate AVP expression through the selective recruitment of coregulatory proteins.

Several lines of evidence suggested that androgens exert a stimulatory effect on AVP expression in specific brain regions (11, 30, 31). In adult male rats, castration abolished and T treatment restored AVP expression in the BST and MeA (9). Furthermore, treatment with DHT significantly increased AVP expression in the SON of hyperosmotic rats (30) and in the medial parvocellular neurons of the PVN, an important neural component regulating the hormonal response to a stressor (31). Because DHT has been thought to be a nonaromatizable metabolite of T that acts exclusively through AR, these data provided strong evidence that AR played a critical role in mediating central AVP expression. However, our results argue against this hypothesis. Instead, we have shown that DHT inhibited AVP promoter activity when mediated by low levels of AR.

3ßAdiol, a metabolite of DHT, is a functional ligand for ERß1 and ERß2 in neuronal cells (18), raising the possibility that DHT-induced AVP expression in the brain is mediated by ERß. Previously, we demonstrated that in vivo, 3ßAdiol activated ERß-expressing neurons in the PVN of adult rats (32). Here, we show that 3ßAdiol significantly potentiated the ligand-independent effect of ERß1 and ERß2 on AVP promoter activity in vitro. DHT has a very low binding affinity for ER (19); therefore, it is reasonable to assume that the stimulatory effects of DHT on AVP promoter activity mediated by ERß1 were accomplished through its intracellular conversion to 3ßAdiol. Moreover, DHT cannot be converted to T, ruling out the possibility that the observed DHT effect is through back conversion to T and then E2. Also, we have confirmed that the enzymes necessary to convert DHT to 3ßAdiol are expressed in the SK-N-SH cell line. The failure of DHT to stimulate ERß2-mediated AVP promoter activity might be due to the considerably lower binding affinity 3ßAdiol has for ERß2 than ERß1 (Ki = 58.1 and 1.7 nM, respectively) (18).

Over the last decade several splice variants have been identified for both ER{alpha} and ERß mRNAs (22, 24, 33, 34, 35). In humans, there are five isoforms of ERß (ERß1–ERß5), all of which diverge at similar positions in helix 10 (36). In the rat brain, four splice variants of ERß1 are expressed: ERß1{delta}3, E-ß1{delta}4, ERß2, and ERß2{delta}3 (24, 25). Although these splice variants have been identified in some regions of the rat brain that express AVP (SON, PVN, and MeA), it remains to be determined which splice variants are coexpressed with AVP in individual cells. These variants arise from the alternative splicing of heterogeneous nuclear RNA and thus have distinct structural and functional characteristics. For example, ERß2 has an 18-amino-acid insert in the ligand-binding domain that is absent from ERß1, thus allowing for differential ligand selectivity. In humans, the isoform termed ERß2 (also called ERßcx) is not homologous to the rat ERß2 but rather contains a novel carboxy terminal end. To date, a human equivalent of the rat ERß2 (i.e. one containing an 18-amino-acid insert in the ligand-binding domain) has not been identified. The {delta}3 variants are missing exon 3, which codes for the second zinc finger of the DNA-binding domain (23), whereas the {delta}4 variant is missing exon 4, which codes for the portion of the hinge region that contains the nuclear localization signal (24). In this study, we used the different splice variants of ERß to better understand the dynamics of ERß-mediated promoter activity. For instance, our results using ERß1{delta}3 suggest that direct DNA binding of the receptor is required for both the ligand-independent and ligand-dependent effects of ERß1 on AVP promoter activity.

Perhaps the most intriguing finding in this study is the observation that ERß2 activated AVP promoter activity differently from ERß1. Previous studies examining the function of ERß2 have indicated that it activates transcription similar to that of ERß1 (20, 21). Here we showed that unlike ERß1, both E2 and 3ßAdiol significantly augmented the ligand-independent increase in AVP promoter when mediated by ERß2. Furthermore, the site of action for 3ßAdiol/ERß2-induced activation of the AVP promoter lies at –306 to –740 bp upstream from the transcription start site, whereas the functional site of 3ßAdiol/ERß1-mediated AVP promoter activity lies more than 1.3 kb upstream. These data demonstrate that the regulation of the AVP promoter varies depending on which ER variant is present in a given cell. However, determining precisely which splice variants are coexpressed with AVP in differing brain areas is severely limited by current techniques. Given that the number of newly described nuclear receptor splice variants has grown at a rapid pace and is constantly changing, they represent potential alternative mechanisms to unlock the complexities of gonadal steroid hormone-regulated gene transcription.

Using the splice variant ERß1{delta}3, we have shown that DNA binding is required for both the ligand-independent and ligand-dependent actions of ERß1 and ERß2. However, we found only one candidate sequence for a putative ERE in the proximal 1.3-kb region of the AVP promoter. This proximal ERE consists of two consecutive canonical half sites at position –995/–984 upstream from the transcription start site. We used site-directed mutagenesis to abolish this region to determine whether it was required for ERß-mediated AVP promoter activity. Our results showed that neither the ligand-independent nor ligand-dependent effects of ERß1 and -ß2 on AVP promoter activity was altered by destruction of the putative ERE. Moreover, in this region, there is no evidence of other known sites where steroid receptors can interact, such as an activator protein-1 or Sp-1 site. The absence of a canonical ERE at the ERß site of action in the AVP promoter raises the possibility that ERß1 and ERß2 can bind and activate novel DNA sequences. In support of this hypothesis, we have previously shown that ERß1 significantly increased GnRH promoter activity in a ligand-independent fashion and that the site of action lacked a known hormone response element (37).

The constitutive activation of gene promoters by ERß1 and its splice variants demonstrate a unique property of ERß that is not shared by its counterpart, the originally described estrogen receptor ER{alpha}. Of the gene promoters investigated to date, all are regulated in a ligand-independent fashion by ERß, including CRH (38), GnRH (37), and as shown herein and by others, AVP (27). Collectively, these data suggest that ER{alpha} and ERß serve different physiological functions and potentially regulate gene networks through very different intracellular mechanisms. Consistent with our findings, Shapiro and colleagues (27) showed a concentration-dependent, ERß-induced increase in AVP promoter activity that occurred in the absence of hormone treatment. In comparison, we observed significant ligand-independent increases in AVP promoter activity at concentrations of ERß1 that were one tenth of that used by Shapiro and colleagues (27) to see an effect and one half of that which elicited no effect (0.5 µg/well; data not shown). Moreover, Shapiro et al. (27) found that the effect of E2 treatment on ERß-mediated AVP promoter activity was dependent on the concentration of ERß. E2 increased AVP promoter activity at low concentrations of ERß, whereas it failed to increase AVP promoter activity at higher concentrations. However, in our hands, E2 failed to further augment AVP promoter activity above the ligand-independent effect at concentrations of ERß similar to those tested by Shapiro et al. (27). One possible explanation for these discrepancies is that the ERß construct used in these studies was 45 amino acids longer than that used by Shapiro et al. (27). Shortly after the originally reported sequence of 485 amino acids for ERß (19), it was discovered that the true transcription start site reflected a length of 530 amino acids (39). The higher basal activity of ERß1 observed in our study might be explained by these additional 45 amino acids.

Nuclear receptor signaling is propagated by the recruitment of various coregulatory proteins. Coregulators are integral components of the transcriptional machinery participating in everything from chromatin remodeling to the stabilization of RNA polymerases (40, 41). The nuclear receptor coactivator protein GRIP1 (also called SRC-2, TIF2, and NCoA-2) has been shown to be an important regulator of ER signaling (42, 43). Moreover, ERß differentially recruits coactivators in response to different ligands (28). In this study, we used a dominant-negative GRIP1 expression vector (29) to begin to elucidate some of the downstream molecular participants involved in ERß-mediated regulation of the AVP promoter. The expression vector we used contained the NR-box sequence (amino acids 629–760) from GRIP1 subcloned into the pM plasmid vector. The NR-box sequence will bind to the LXXLL motif of the transfected ERß1 construct thereby preventing any endogenous GRIP1 protein from interacting with ERß1. Using this strategy, we found that GRIP1 was not required for the ligand-independent activation of AVP promoter activity by ERß1. By contrast, by squelching endogenous GRIP1 activity we completely abolished the 3ßAdiol-induced activation of the AVP promoter, suggesting that the GRIP1 (NR-box) interfered with the function of the endogenously expressed GRIP1 protein. These results indicate that GRIP1 is an important player in ERß1’s ligand-dependent regulation of AVP by 3ßAdiol and also suggest that the downstream pathways regulating ligand-dependent and ligand-independent activation of ERß1 are distinct.

The role of central AVP release has been at the forefront of research examining the intricacies of social and intimate behaviors. For males in particular, activation of central AVP receptors has been shown to influence the pair-bonding preferences and paternal care behaviors in voles (44, 45). AVP also mediates scent-marking behaviors in hamsters (46). Furthermore, AVP expression in brain regions that mediate these male-typical behaviors has been shown to be androgen dependent (9). The data presented herein demonstrate that ERß is a likely candidate for mediating the DHT effects on AVP expression, suggesting that ERß might be required for the proper execution of male social behaviors. This is particularly significant given the differential distribution of ER{alpha}, ERß, and its splice variants in specific brain nuclei. Moreover, we have shown specific regions of the AVP promoter that are important for ERß regulation despite the lack of any known hormone response elements. These data raise important new questions about novel DNA-binding sites and/or interacting proteins and their significance for ERß-mediated gene transcription. Finally, these data underscore the importance of complex promoters and the necessity for multiple levels of regulation. In the case of AVP, it allows for differential regulation of gene expression in discrete neuronal populations, under varying homeostatic conditions (i.e. hypernatremia, circulating hormone levels, etc.), and with varying expression of ER splice variants within a given cell.


   Footnotes
 
This work was supported by United States Public Health Service Grants RO1 NS039951 (R.J.H.) and F32 HD046301 (T.R.P.).

Disclosure Statement: T.R.P, W.C.J.C., L.R.H., and R.J.H. have nothing to declare.

First Published Online April 5, 2007

Abbreviations: 3ßAdiol, 5{alpha}-Androstane-3ß,17ß-diol; AR, androgen receptor; AVP, arginine vasopressin; BST, bed nucleus of the stria terminalis; DHT, 5{alpha}-dihydrotestosterone; E2, 17ß-estradiol; ER, estrogen receptor; ERE, estrogen response element; EtOH, ethanol; GRIP1, glucocorticoid receptor interacting protein-1; 3{alpha}HSD, 3{alpha}-hydroxysteroid oxidoreductase; 3ßHSD, 3ß-hydroxysteroid oxidoreductase; 17ßHSD, 17ß-hydroxysteroid dehydrogenase; MeA, medial amygdala; PVN, paraventricular nucleus; SCN, suprachiasmatic nucleus; SON, supraoptic nucleus; T, testosterone.

Received January 22, 2007.

Accepted for publication March 27, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Blanchard RJ, Griebel G, Farrokhi C, Markham C, Yang M, Blanchard, DC 2005 AVP V1b selective antagonist SSR149415 blocks aggressive behaviors in hamsters. Pharmacol Biochem Behav 80:189–194[CrossRef][Medline]
  2. Scordalakes EM, Rissman EF 2004 Aggression and arginine vasopressin immunoreactivity regulation by androgen receptor and estrogen receptor {alpha}. Genes Brain Behav 3:20–26[CrossRef][Medline]
  3. Wang Z, Aragona BJ 2004 Neurochemical regulation of pair bonding in male prairie voles. Physiol Behav 83:319–328[CrossRef][Medline]
  4. Isobe Y, Isobe M 1998 Circadian rhythm of Arg-vasopressin contents in the suprachiasmatic nucleus in relation to corticosterone. Brain Res 800:78–85[CrossRef][Medline]
  5. Isobe Y, Nishino H 2004 Signal transmission from the suprachiasmatic nucleus to the pineal gland via the paraventricular nucleus: analysed from arg-vasopressin peptide, rPer2 mRNA and AVP mRNA changes and pineal AA-NAT mRNA after the melatonin injection during light and dark periods. Brain Res 1013:204–211[CrossRef][Medline]
  6. Albeck DS, McKittrick CR, Blanchard DC, Blanchard RJ, Nikulina J, McEwen BS, Sakai RR 1997 Chronic social stress alters levels of corticotropin-releasing factor and arginine vasopressin mRNA in rat brain. J Neurosci 17:4895–4903[Abstract/Free Full Text]
  7. Ebner K, Wotjak CT, Landgraf R, Engelmann M 2002 Forced swimming triggers vasopressin release within the amygdala to modulate stress-coping strategies in rats. Eur J Neurosci 15:384–388[CrossRef][Medline]
  8. Erkut ZA, Pool C, Swaab DF 1998 Glucocorticoids suppress corticotropin-releasing hormone and vasopressin expression in human hypothalamic neurons. J Clin Endocrinol Metab 83:2066–2073[Abstract/Free Full Text]
  9. DeVries GJ, Buijs RM, Van Leeuwen FW, Caffe AR, Swaab DF 1985 The vasopressinergic innervation of the brain in normal and castrated rats. J Comp Neurol 233:236–254[CrossRef][Medline]
  10. Miller MA, DeVries GJ, al-Shamma HA, Dorsa DM 1992 Decline of vasopressin immunoreactivity and mRNA levels in the bed nucleus of the stria terminalis following castration. J Neurosci 12:2881–2887[Abstract]
  11. De Vries, GJ, Wang, Z, Bullock, NA, Numan, S 1994 Sex differences in the effects of testosterone and its metabolites on vasopressin messenger RNA levels in the bed nucleus of the stria terminalis of rats. J Neurosci 14:1789–1794[Abstract]
  12. Giguere V, Yang N, Segui P, Evans RM 1988 Identification of a new class of steroid hormone receptors. Nature 331:91–94[CrossRef][Medline]
  13. Jirikowski GF, Herbert Z, Petrusz P, Sendemir E, Caldwell JD 2005 Co-expression of vasopressin and androgen-binding protein in the rat hypothalamus. J Chem Neuroanat 29:233–237[CrossRef][Medline]
  14. Zhou, L, Blaustein, JD, De Vries, GJ 1994 Distribution of androgen receptor immunoreactivity in vasopressin- and oxytocin-immunoreactive neurons in the male rat brain. Endocrinology 134:2622–2627[Abstract]
  15. Steckelbroeck S, Jin Y, Gopishetty S, Oyesanmi B, Penning TM 2004 Human cytosolic 3{alpha}-hydroxysteroid dehydrogenases of the aldo-keto reductase superfamily display significant 3ß-hydroxysteroid dehydrogenase activity: implications for steroid hormone metabolism and action. J Biol Chem 279:10784–10795[Abstract/Free Full Text]
  16. Torn, S, Nokelainen, P, Kurkela, R, Pulkka, A, Menjivar, M, Ghosh, S, Coca-Prados, M, Peltoketo, H, Isomaa, V, Vihko, P 2003 Production, purification, and functional analysis of recombinant human and mouse 17ß-hydroxysteroid dehydrogenase type 7. Biochem Biophys Res Commun 305:37–45[CrossRef][Medline]
  17. Weihua Z, Lathe R, Warner M, Gustafsson JA 2002 An endocrine pathway in the prostate, ERß, AR, 5{alpha}-androstane-3ß,17ß-diol, and CYP7B1, regulates prostate growth. Proc Natl Acad Sci USA 99:13589–13594[Abstract/Free Full Text]
  18. Pak TR, Chung WC, Lund TD, Hinds LR, Clay CM, Handa RJ 2005 The androgen metabolite, 5{alpha}-androstane-3ß,17ß-diol, is a potent modulator of estrogen receptor-ß1-mediated gene transcription in neuronal cells. Endocrinology 146:147–155[Abstract/Free Full Text]
  19. Kuiper GG, Carlsson B, Grandien K, Enmark E, Haggblad J, Nilsson S, Gustafsson JA 1997 Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptors {alpha} and ß. Endocrinology 138:863–870[Abstract/Free Full Text]
  20. Mosselman S, Polman J, Dijkema R 1996 ERß: identification and characterization of a novel human estrogen receptor. FEBS Lett 392:49–53[CrossRef][Medline]
  21. Hanstein, B, Liu, H, Yancisin, MC, Brown, M 1999 Functional analysis of a novel estrogen receptor-ß isoform. Mol Endocrinol 13:129–137[Abstract/Free Full Text]
  22. Petersen DN, Tkalcevic GT, Koza-Taylor PH, Turi TG, Brown TA 1998 Identification of estrogen receptor ß2, a functional variant of estrogen receptor ß expressed in normal rat tissues. Endocrinology 139:1082–1092[Abstract/Free Full Text]
  23. Price Jr RH, Butler CA, Webb P, Uht R, Kushner P, Handa RJ 2001 A splice variant of estrogen receptor ß missing exon 3 displays altered subnuclear localization and capacity for transcriptional activation. Endocrinology 142:2039–2049[Abstract/Free Full Text]
  24. Price Jr RH, Lorenzon N, Handa RJ 2000 Differential expression of estrogen receptor ß splice variants in rat brain: identification and characterization of a novel variant missing exon 4. Brain Res Mol Brain Res 80:260–268[Medline]
  25. Price Jr RH, Handa RJ 2000 Expression of estrogen receptor-ß protein and mRNA in the cerebellum of the rat. Neurosci Lett 288:115–118[CrossRef][Medline]
  26. Chomczynski P, Sacchi N 1987 Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159[Medline]
  27. Shapiro RA, Xu C, Dorsa DM 2000 Differential transcriptional regulation of rat vasopressin gene expression by estrogen receptor {alpha} and ß. Endocrinology 141:4056–4064[Abstract/Free Full Text]
  28. Kraichely DM, Sun J, Katzenellenbogen JA, Katzenellenbogen BS 2000 Conformational changes and coactivator recruitment by novel ligands for estrogen receptor-{alpha} and estrogen receptor-ß: correlations with biological character and distinct differences among SRC coactivator family members. Endocrinology 141:3534–3545[Abstract/Free Full Text]
  29. Chang C, Norris JD, Gron H, Paige LA, Hamilton PT, Kenan DJ, Fowlkes D, McDonnell DP 1999 Dissection of the LXXLL nuclear receptor-coactivator interaction motif using combinatorial peptide libraries: discovery of peptide antagonists of estrogen receptors alpha and beta. Mol Cell Biol 19:8226–8239[Abstract/Free Full Text]
  30. O’Keefe JA, Crowley RS, Hrivnak P, Kim NB, Amico JA 1995 Effect of testosterone and its metabolites upon the level of vasopressin messenger ribonucleic acid in the hypothalamus of the hyperosmotically stimulated male rat. Neuroendocrinology 61:405–411[Medline]
  31. Viau V, Soriano L, Dallman MF 2001 Androgens alter corticotropin releasing hormone and arginine vasopressin mRNA within forebrain sites known to regulate activity in the hypothalamic-pituitary-adrenal axis. J Neuroendocrinol 13:442–452[CrossRef][Medline]
  32. Lund TD, Hinds LR, Handa RJ 2006 The androgen 5{alpha}-dihydrotestosterone and its metabolite 5{alpha}-androstan-3ß,17ß-diol inhibit the hypothalamo-pituitary-adrenal response to stress by acting through estrogen receptor ß-expressing neurons in the hypothalamus. J Neurosci 26:1448–1456[Abstract/Free Full Text]
  33. Poola I, Koduri S, Chatra S, Clarke, R 2000 Identification of twenty alternatively spliced estrogen receptor {alpha} mRNAs in breast cancer cell lines and tumors using splice targeted primer approach. J Steroid Biochem Mol Biol 72:249–258[CrossRef][Medline]
  34. Skipper JK, Young LJ, Bergeron JM, Tetzlaff MT, Osborn CT, Crews D 1993 Identification of an isoform of the estrogen receptor messenger RNA lacking exon four and present in the brain. Proc Natl Acad Sci USA 90:7172–7175[Abstract/Free Full Text]
  35. Chu S, Fuller PJ 1997 Identification of a splice variant of the rat estrogen receptor ß gene. Mol Cell Endocrinol 132:195–199[CrossRef][Medline]
  36. Moore JT, McKee DD, Slentz-Kesler K, Moore LB, Jones SA, Horne EL, Su JL, Kliewer SA, Lehmann JM, Willson TM 1998 Cloning and characterization of human estrogen receptor ß isoforms. Biochem Biophys Res Commun 247:75–78[CrossRef][Medline]
  37. Pak TR, Chung WC, Roberts JL, Handa RJ 2006 Ligand-independent effects of estrogen receptor ß on mouse gonadotropin-releasing hormone promoter activity. Endocrinology 147:1924–1931[Abstract/Free Full Text]
  38. Miller WJ, Suzuki S, Miller LK, Handa R, Uht RM 2004 Estrogen receptor (ER)ß isoforms rather than ER{alpha} regulate corticotropin-releasing hormone promoter activity through an alternate pathway. J Neurosci 24:10628–10635[Abstract/Free Full Text]
  39. Leygue E, Dotzlaw H, Lu B, Glor C, Watson PH, Murphy LC 1998 Estrogen receptor ß: mine is longer than yours? J Clin Endocrinol Metab 83:3754–3755[Free Full Text]
  40. Hall JM, McDonnell DP 2005 Coregulators in nuclear estrogen receptor action: from concept to therapeutic targeting. Mol Interv 5:343–357[Abstract/Free Full Text]
  41. Xu J, Li Q 2003 Review of the in vivo functions of the p160 steroid receptor coactivator family. Mol Endocrinol 17:1681–1692[Abstract/Free Full Text]
  42. Hong H, Kohli K, Trivedi A, Johnson DL, Stallcup MR 1996 GRIP1, a novel mouse protein that serves as a transcriptional coactivator in yeast for the hormone binding domains of steroid receptors. Proc Natl Acad Sci USA 93:4948–4952[Abstract/Free Full Text]
  43. Norris JD, Fan D, Stallcup MR, McDonnell DP 1998 Enhancement of estrogen receptor transcriptional activity by the coactivator GRIP-1 highlights the role of activation function 2 in determining estrogen receptor pharmacology. J Biol Chem 273:6679–6688[Abstract/Free Full Text]
  44. Wang Z, Ferris CF, De Vries GJ 1994 Role of septal vasopressin innervation in paternal behavior in prairie voles (Microtus ochrogaster). Proc Natl Acad Sci USA 91:400–404[Abstract/Free Full Text]
  45. Cho MM, DeVries AC, Williams JR, Carter CS 1999 The effects of oxytocin and vasopressin on partner preferences in male and female prairie voles (Microtus ochrogaster). Behav Neurosci 113:1071–1079[CrossRef][Medline]
  46. Delville Y, De Vries GJ, Schwartz WJ, Ferris CF 1998 Flank-marking behavior and the neural distribution of vasopressin innervation in golden hamsters with suprachiasmatic lesions. Behav Neurosci 112:1486–1501[CrossRef][Medline]



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