Regulates Hypothalamic Oxytocin (Oxt) Gene Expression

NEUROENDOCRINOLOGY

The ER␤ Ligand 5␣-androstane, 3␤,17␤-diol (3␤-diol) Regulates Hypothalamic Oxytocin (Oxt) Gene Expression Dharmendra Sharma, Robert J. Handa, and Rosalie M. Uht Department of Pharmacology and Neuroscience and Institute for Aging and Alzheimer’s Disease Research (D.S., R.M.U.), University of North Texas Health Sciences Center, Fort Worth, Texas 76107; and Department of Basic Medical Sciences (R.J.H.), University of Arizona College of Medicine, Phoenix, Arizona 85004

The endocrine component of the stress response is regulated by glucocorticoids and sex steroids. Testosterone down-regulates hypothalamic-pituitary-adrenal (HPA) axis activity; however, the mechanisms by which it does so are poorly understood. A candidate testosterone target is the oxytocin gene (Oxt), given that it too inhibits HPA activity. Within the paraventricular nucleus of the hypothalamus, oxytocinergic neurons involved in regulating the stress response do not express androgen receptors but do express estrogen receptor-␤ (ER␤), which binds the dihydrotestosterone metabolite 3␤,17␤-diol (3␤-diol). Testosterone regulation of the HPA axis thus appears to involve the conversion to the ER␤-selective ligand 5␣-androstane, 3␤-diol. To study mechanisms by which 3␤-diol could regulate Oxt expression, we used a hypothalamic neuronal cell line derived from embryonic mice that expresses Oxt constitutively and compared 3␤-diol with estradiol (E2) effects. E2 and 3␤-diol elicited a phasic response in Oxt mRNA levels. In the presence of either ligand, Oxt mRNA levels were increased for at least 60 min and returned to baseline by 2 h. ER␤ occupancy preceded an increase in Oxt mRNA levels in the presence of 3␤-diol but not E2. In tandem with ER␤ occupancy, 3␤-diol increased occupancy of the Oxt promoter by cAMP response element-binding protein and steroid receptor coactivator-1 at 30 min. At the same time, 3␤-diol led to the increased acetylation of histone H4 but not H3. Taken together, the data suggest that in the presence of 3␤-diol, ER␤ associates with cAMP response element-binding protein and steroid receptor coactivator-1 to form a functional complex that drives Oxt gene expression. (Endocrinology 153: 2353–2361, 2012)

hysiological responses to stress are mediated by extensive neurohumoral processes. Central to a concerted response is activation of the hypothalamic-pituitary-adrenal (HPA) axis. A considerable body of evidence in rodents indicates that in addition to other complexities, the response differs by gender: females have a heightened endocrine response to stress. This was described as early as 1970 by Gaskin and Kitay (1), was followed by the reports showing estradiol regulation of neuroendocrine responses (2, 3), and has been investigated increasingly since. In females, the heightened or facilitated response tracks, in part, to estradiol (E2) up-regulation of CRH gene (crh)

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expression. In males, however, the primary sex steroid, testosterone, down-regulates activation of the stress response. One mechanism for this appears to involve the conversion of testosterone to dihydrotestosterone and subsequent conversion of dihydrotestosterone to 5␣ androstane-3␤, 17␤ diol (3␤-diol; Fig. 1). 3␤-Diol, binds estrogen receptor (ER)-␤ and the resultant holo-receptor regulates gene expression, reviewed by Handa et al. (4). Oxytocin (Oxt) is a nonapeptide synthesized by magnocellular neurons in the paraventricular and supraoptic nuclei of the hypothalamus. It is best known for its systemic roles in reproduction, which arise in part from Oxt

ISSN Print 0013-7227 ISSN Online 1945-7170 Printed in U.S.A. Copyright © 2012 by The Endocrine Society doi: 10.1210/en.2011-1002 Received December 31, 2011. Accepted February 21, 2012. First Published Online March 20, 2012

Abbreviations: CBP, cAMP response element-binding protein; ChIP, chromatin immunoprecipitation; crh, corticotropin-releasing hormone gene; 3␤-diol, 5␣ androstane-3␤, 17␤ diol; E2, estradiol; ER, estrogen receptor; ERE, estrogen response element; HPA, hypothalamic-pituitary-adrenal; mHypoE-38, mouse hypothalamic neuronal; Oxt, oxytocin; qPCR, quantitative PCR; SRC, steroid receptor coactivator.

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subsequent report, Adan et al. (18) showed that the corresponding sequence between ⫺172 and ⫺148 is responsive to retinoic acid. Koohi et al. (19) analyzed the bovine promoter and found a complex noncanonic response element in the same region that binds the estrogen-related receptor-␣. Wehrenberg et al. (20) also examined this region in the bovine promoter and found sequences similar to chicken ovalbumin upstream promoter transcription factor and steroidogenic factor 1. Because this region is conserved across species and mechanisms by which ER␤ regulates Oxt expression have not been established, we sought to elucidate ER␤-mediated effects on Oxt expression in the proximal region of the promoter, with particular attention paid to the ligand 3␤-diol.

Materials and Methods FIG. 1. Metabolic pathway for the synthesis of 3␤-diol from testosterone. HSD, Hydroxysteroid dehydrogenase.

secretion by the posterior pituitary. Oxt is not only important for female reproductive functions such as milk ejection and parturition but also plays a role in female and male reproductive behaviors as well (5, 6). In addition to expression in magnocellular neurons, hypothalamic Oxt is also expressed in parvocellular neurons that project to extrahypothalamic sites, and its expression changes in response to the nonreproductive physiological states of the animal. One of these states is stress. For example, Oxt administered centrally decreases corticosterone and ACTH responses to given stressors (7–9). E2 has long been known to regulate Oxt expression in the magnocellular neurons of the hypothalamus; however, E2 also regulates Oxt expression in other neuronal populations in the brain (10). The mechanisms by which it does so are incompletely understood. However, the regulation of Oxt expression in the brain does appear to involve ER␤ (11, 12). ER␤ regulates promoters through an estrogen response element (ERE) (13), and the Oxt promoter of all mammalian species has a functional composite response element that is thought to mediate estrogen responses (14, 15). In mouse, the noncanonic ERE that mediates E2 responses is GATGACCTTGACC (16). In rat, by using various deletion mutants of the 5⬘-flanking region of the promoter, Adan et al. (17) showed that an ERE between ⫺172 and ⫺149 plays a key role in Oxt regulation; indeed, it confers maximum estrogen responsiveness (15-fold stimulation). In distinction, sequences further upstream (up to ⫺3200) fail to confer estrogen responsiveness (17). In a

Cell culture and immunocytochemistry Mouse hypothalamic neuronal (mHypoE-38) cells (CELLutions Biosystems, Ontario, Canada) were the only cells used in the experiments. They were maintained in phenol red-free DMEM/Ham’s F12 (Hyclone Laboratories, Logan, UT) and supplemented with 10% newborn calf serum (Gemini Bioproducts, Woodland, CA); 100 U/ml penicillin/streptomycin; 0.1 mM nonessential amino acids; 1 mM sodium pyruvate; and 2 mM L-glutamine (all from Cellgro, Mediatech, Inc., Manassas, VA). For immunocytochemical analysis, cells were plated and grown in Lab-Tek II chamber slides (Nalge Nunc International, Rochester, NY) for 24 h. Cells were then fixed in 4% paraformaldehyde, permeablized with 0.1% Triton X-100, and exposed to a blocking solution (5% normal goat serum, 2% BSA in PBS) to reduce nonspecific binding of the antibody. To ensure that the cells express Oxt, ER␣, and ER␤ in our conditions, cells were incubated in primary antibody for 24 h at 4 C. Polyclonal antiOxt antibody was purchased from Sigma Aldrich Co. (St. Louis MO); monoclonal antibodies against ER␣ and ER␤ were purchased from Santa Cruz Biotechnology (1:250; Santa Cruz, CA). After washing with PBS, cells were incubated for 1 h with either Alexa Fluor 594 goat antirabbit or Alexa Fluor 488 goat antimouse IgG (1:1000; Molecular Probes, Grand Island, NY). Cells were counterstained with Hoechst for DNA. After washing, cells were mounted in FluorSave reagent (Calbiochem, San Diego, CA). Images were captured with Nuance Multispectral Imaging System (Cri, Hopkinton, MA), and digitized images were arranged using Adobe Photoshop (San Jose, CA). For experiments involving treatments, cells were incubated with media containing stripped serum for 48 h before treatment with ligands. Serum was stripped with charcoal and dextran. Briefly, 2.5 g activated charcoal (C3345; Sigma) and 0.25 g dextran (T-70; catalog no. 31390; Sigma) were mixed in 1 liter of 0.1 M Tris (pH 8.0). The solution was then stirred overnight at 4 C. The charcoal/dextran mixture was centrifuged at 1000 ⫻ g. The pellets were mixed with 250 ml of sera and incubated at 45 C for 1 h. Aliquots were subsequently centrifuged to remove charcoal. Finally, the stripped serum was sterilized by filtering through a 0.22-␮m filter (Millipore, Bedford, MA). Treatment with E2

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(Sigma) and 3␤-diol (Steraloids Inc., Providence, RI) were administered at 10⫺7 M each.

RNA isolation and RT-quantitative PCR (qPCR) Total RNA was isolated from cells at 80 –90% confluence using Trizol reagent (Invitrogen, Carlsbad, CA). For reverse transcription, 1 ␮g of total RNA was used and cDNA synthesized at 42 C for 30 min using a Verso cDNA kit (Thermo Fisher Scientific, Fair Lawn, NJ). To determine whether the cells expressed ER␣, ER␤, and Oxt mRNA, RT-PCR was performed. The primers used to amplify the cDNA were the following: forward, 5⬘-CGGTCTACGGCCAGTCGGGCATC-3⬘ and reverse, 5⬘-CGCTGGGCTCGTTCTCCAGGTAGTA-3⬘ (ER␣); forward, 5⬘-GCTGTGATGAACTACAGTGTTCCC-3⬘ and reverse, 5⬘-TGGACTAGTAACAGGGCTGGCACA-3⬘ (ER␤); forward, 5⬘-CCTACAGCGGATCTCAGACTGA-3⬘ and reverse, 5⬘-TCAGAGCCAGTAAGCCAAGCA-3⬘ (Oxt); forward, 5⬘-GCTCCGGCATGTGCAAA-3⬘ and reverse, 5⬘-AGGATCTTCATGAGGTAGT-3⬘ (actin). Amplicons were evaluated by electrophoresis using a 1.5% agarose gel and ethidium bromide staining. To study changes in the level of Oxt mRNA after treatment with E2 or 3␤-diol, RT-qPCR was performed using IQ Sybr Green Super Mix (Bio-Rad Laboratories, Hercules, CA) and 400 nM of each primer in a CFX96 qPCR detection system (Bio-Rad, Hercules, CA). The primer sequences used to amplify Oxt mRNA were forward, 5⬘-CCTACAGCGGATCTCAGACTGA-3⬘ and reverse, 5⬘-TCAGAGCCAGTAAGCCAAGCA-3⬘ (Oxt). ␤-Actin primer sequences used were forward, 5⬘-TGGCACCACACCTTCTACAATGAG-3⬘ and reverse, 5⬘-GGGTCATCTTTTCACGGTTGG-3⬘. Samples were amplified by an initial denaturation at 95 C for 3 min and then cycled (45 times) at 95 C for 30 sec, 60 C for 30 sec, and 72 C for 30 sec. The ⌬⌬cycle threshold method of calculation (21) was used with minor modifications (22) and data were normalized to ␤-actin.

Chromatin immunoprecipitation (ChIP) followed by qPCR To determine the promoter occupancy by ER and coactivators after E2 or 3␤-diol treatments, ChIP analysis was performed. Cells were grown to 80 –90% confluency and then incubated for 48 h in media containing stripped serum. After incubation in stripped serum, cells were treated with ␣-amanitin for 2 h to synchronize the cell cycle, washed twice with PBS, and then treated with ethanolic vehicle, E2 (10⫺7 M), or 3␤-diol (10⫺7 M) for 5 min, 10 min, 30 min, 60 min, 2 h, and 4 h. ChIP was performed as described (21, 22). In brief, after treatment, cells underwent 1% formaldehyde fixation for 10 min at room temperature. Fixation was stopped by addition of glycine (0.125 M). After washing twice in ice-cold PBS, cells were collected and pelleted by centrifugation at 590 ⫻ g for 1 min. Pellets were resuspended in 1 ml of cell sonication buffer [1% Triton X-100; 0.1% deoxycholate; 50 mM Tris, pH 8.1; 150 mM NaCl; 5 mM EDTA; 1 mM phenylmethylsulfonyl fluoride; and protease inhibitor cocktail, which contained 4-(2-aminoethyl)benzene sulfonyl fluoride, aprotinin, bestatin, E-64, leupeptin, and pepstatin A; Sigma]. They were then incubated on ice for 10 min and subjected to sonication using a Misonix sonicator Q700 with cup horn (QSonica, Newtown, CT). Five pulses of 30 sec each at 50% amplitude were used to shear chromatin. Samples were centrifuged at 16000 ⫻ g for 15 min at 4 C and supernatants were

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collected. One hundred-microgram aliquots of the supernatant were diluted in 1 ml of cell sonication buffer and then incubated overnight with antibody. Polyclonal antibodies against ER␣, ER␤, cAMP response element-binding protein (CBP) and steroid receptor coactivator (SRC)-1 were obtained from Santa Cruz Biotechnology. Polyclonal antibody against acetylated H3 and H4 were obtained from Active Motif (Carlsbad, CA). Three micrograms of each antibody were used to precipitate the chromatin. Further processing of the chromatin samples were performed as described (22). Lastly, DNA was eluted in 100 ␮l of 10 mM Tris (pH 7.5). For qPCR, 5 ␮l of template DNA was used and amplified using SYBR green super mix (Bio-Rad) and 400 nM of each primer. The primer sequences were designed to target the region of the mouse Oxt promoter that contains the composite estrogen response element centered at approximately ⫺160. Primer sequences are forward, 5⬘-TCCAACCCCTCCCAAGTCTCTC-3⬘ (⫺264 to ⫺242), and reverse, 5⬘-AGAGGCAGGCCCTTCATTTGC-3⬘ (⫺145 to ⫺124). They yield a 140-bp product. In a preliminary experiment to validate the primers, we used an untreated chromatin sample to perform a melting curve analysis. Omission of either the chromatin sample or primers was used as controls. We evaluated the amplified product by 1.5% agarose gel electrophoresis. The amplicons migrated at the predicted molecular weight (140 bp), and no products were detected for the controls. In addition, we monitored the melting temperature to ensure that there was one peak. After the initial characterization of the amplified product, we monitored the melting temperature only. We did so for all products in all experiments to ensure that there was a single peak for each set of amplicons. qPCR values were calculated by the ⌬⌬cycle threshold method (19) and expressed as fold differences of vehicle.

Western blot analysis Whole-cell extracts were prepared from a single, 10-cm plate of confluent cells by using radioimmunoprecipitation assay buffer. Extracts were from cells that had been treated with vehicle. Twenty micrograms of cell extracts were subjected to Western blot analysis after electrophoresis on a 12% sodium dodecyl sulfate-polyacrylamide gel using either polyclonal anti-ER␣ (1: 1000; Santa Cruz Biotechnology) or anti-ER␤ (1:1000; Santa Cruz Biotechnology) antibody.

Statistical analysis To determine significant differences between treatments, statistical analyses were performed using either Student’s t test or by a two-way ANOVA (treatment and time), followed by Bonferroni multiple comparison test for post hoc analysis using the GraphPad Prism 5 program (GraphPad Inc., San Diego, CA). For ER␣ and ER␤ profile at different times, a two-way ANOVA was performed (treatment and time). However, to test significant difference from the control for individual treatment at a particular time, a Student’s t test was performed as indicated in the figure legends. Data are presented as the average ⫾ SEM of the number of observations indicated in the figure legends. A P ⬍ 0.05 was considered significant.

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Results mHypoE-38 cell characterization To extend the molecular analysis of 3␤-diol-regulated Oxt expression required identification of a suitable cell line. Belsham et al. (23) had generated and characterized neuronal cell lines from embryonic mouse hypothalami. One of these, the mHypoE-38 (previously referred to as N38) was shown to express Oxt, ER␣, and ER␤ (24). We chose this line to elucidate 3␤-diol mechanisms of Oxt regulation. To further assess the line’s suitability, we measured levels of ER␣, ER␤, and Oxt mRNA by RT-PCR analysis. To rule out the possibility of spurious amplifications, amplicons were visualized using agarose gel electrophoresis. The primers used resulted in amplicons of the expected size (ER␣, 200bp; ER␤, 258 bp; Oxt, 82 bp; and actin, 541bp, Fig. 2A). The cell line was further evaluated for expression of ER␣, ER␤, and Oxt peptides by Western blot analysis (Fig. 2B). In the case of ER␤, two proteins were observed. The more prominent band was taken to be ER␤1. The band that migrated more quickly was taken to be one of several ER␤ splice variants, most likely ER␤1␦3, which in the rat has a predicted weight of 49 kDa. The molecular

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weight of the Oxt species is consistent with the size of the Oxt prohormone, that is, the Oxt peptide along with its neurophysin before processing. Immunoreactivity for ER␣, ER␤, and Oxt was also evaluated by immunocytochemistry (Fig. 2C). Experiments to determine the specificity of the ER antibodies have been reported in an earlier paper (22). Briefly, we reported that preadsorption of the antibody with corresponding immunizing peptides blocked immunoreactive labeling. Cross-adsorption with immunogen for the opposite ER did not alter the staining profile. For the negative control, the primary antiserum was omitted. In summary, mHypoE-38 cells express Oxt, ER␣, and ER␤ mRNA and immunoreactivity and are therefore suitable for studying ER-mediated Oxt gene expression. E2 and 3␤-diol increase Oxt mRNA levels in a phasic manner To compare the effect of E2 with 3␤-diol on mRNA levels, cells were treated for 5 min, 10 min, 30 min, 60 min, 2 h, and 4 h with either ligand. Total RNA was isolated and mRNA levels were measured by RT-qPCR. 3␤-Diol and E2 elicited an increase in the expression of Oxt mRNA

FIG. 2. mHypoE-38 cells display features necessary for studying E2 and 3␤-diol regulation of Oxt gene expression. A, The cells express ER␣, ER␤, and Oxt mRNA. Amplicons are shown. B, Western blots were performed to evaluate the expression of ER␣, ER␤, and Oxt. Twenty micrograms of protein were separated using a 12% SDS-PAGE gel. Blots were probed with Oxt, ER␣, or ER␤ antibodies (n ⫽ 3 for all assays, representative data are shown). C, Immunocytochemical localization of ER␣, ER␤, and Oxt. Cells were stained with anti-ER␣ (upper panel), ER␤ (middle panel), or antiOxt (lower panel) and counterstained with Hoechst to visualize nuclei. Primary antisera were omitted for the negative control. For a size reference, the Hoecht’s stained ER␣ nuclei are 10 –15 ␮m in diameter. ir, Immunoreactivity.

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up through 60 min, which then decreased to vehicle levels by 2 h (Fig. 3). The only differences between treated and untreated levels occurred at 30 and 60 min, for both E2 and 3␤-diol, when a significant increase in the mRNA levels was observed. Thus, in the case of mRNA levels, E2 and 3␤-diol elicit similar patterns of response. 3␤-Diol leads to different levels of Oxt occupancy by ER␣ and ER␤ To compare the pattern of ER␣ and ER␤ occupancy in the region of the Oxt promoter in response to E2 or 3␤diol, ChIP analyses were performed. The region targeted for amplification contains the functional ERE sequence found at 160 to ⫺180 bp (16). By qPCR analysis, there was no significant increase in ER␣ occupancy after treatment with E2 at any time point; however, E2 repressed ER␣ occupancy at 60 min (Fig. 4A). In distinction, there was a peak of ER␤ occupancy associated with 3␤-diol treatment at 30 min and, similar to ER␣, there was repression at 60 min (Fig. 4B). Compared with vehicle, 3␤diol led to increased occupancy of ER␣ at 10 min. 3␤-Diol induces recruitment of CBP and SRC-1 to the Oxt promoter Given that 3␤-diol regulated ER␤ occupancy of the Oxt promoter, we next explored promoter occupancy by the coactivators CBP and SRC-1 at 30 and 60 min after treatment with either ligand. These times were chosen because they corresponded to significant changes in ER␣ and ER␤ occupancy (Fig. 4). ChIP analyses of the Oxt promoter revealed a pattern of CBP and SRC-1 occupancy that was strikingly similar to the pattern of ER␤ occupancy. At 30 min, 3␤-diol significantly increased promoter occupancy by both CBP and SRC-1, whereas E2 had no effect (Fig. 5,

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A and B). At 60 min, however, both ligands significantly decreased CBP occupancy compared with the vehicle (Fig. 5A). In contrast, there was no change in SRC-1 occupancy (Fig. 5B). In the case of CBP, this decrease was below baseline, as was the case for both ER␣ and ER␤ (Fig. 4). 3␤-Diol and E2 induce different patterns of H3 and H4 acetylation To determine whether E2 or 3␤-diol elicited a change in histone acetylation, the levels of H3 and H4 acetylation were assessed using ChIP analysis. The E2 profile was the same for both H3 and H4. It failed to alter the level of acetylation at 30 min but led to decreased acetylation of both H3 and H4 at 60 min (Fig. 6, A and B). In contrast, the 3␤-diol profile differed between the two histones. 3␤Diol had no effect on H3 acetylation at 30 or 60 min but tripled H4 acetylation at 30 min, an effect that was gone by 60 min (Fig. 6B).

Discussion The present study indicates that 3␤-diol, an androgen metabolite and ER␤ agonist, plays a role in the regulation of Oxt expression. The mHypoE-38 hypothalamic cell line, which expresses Oxt and ER␣ and ER␤, permitted kinetic analyses of Oxt mRNA levels and ER␣ and ER␤ occupancy of the rat Oxt promoter. Both ligands elicited a phasic increase in Oxt mRNA levels. The increase was preceded by ER␣ and ER␤ occupancy in the presence of 3␤-diol but not E2. Levels of CBP and SRC-1 occupancy of the Oxt promoter were assessed at the peak and trough of ER occupancy. Lastly, levels of H3 and H4 acetylation

FIG. 3. 3␤-Diol and E2 alter Oxt mRNA levels. Cells were incubated in media containing stripped serum for 48 h and then treated with E2 or 3␤diol (10⫺7 M each) for the times indicated. Bars represent the mean ⫾ SEM of the data obtained from three to four independent experiments and are presented as the fold difference of vehicle (Veh). **, P ⬍ 0.01 (two way ANOVA followed by post hoc analysis).

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FIG. 4. 3␤-Diol and E2 lead to different patterns of promoter occupancy by ER␣ and ER␤. Cells were treated as in Fig. 3. For both profiles, bars represent the mean ⫾ SEM of the data obtained from three independent experiments and are presented as the fold difference of vehicle (Veh). ***, P ⬍ 0.001 (two way ANOVA followed by post hoc analysis); #, P ⬍ 0.05; ##, P ⬍ 0.01 (significance determined by t test).

were monitored to provide an indirect measure of CBP and/or SRC-1 histone acetyltransferase activity. Taken in isolation, the profile of mRNA responses to E2 and 3␤-diol are sufficiently similar that they could be interpreted as resulting from identical mechanisms. The differences in the promoter occupancy and degree of histone acetylation, however, indicate clearly that this is not the case. This is particularly so for events surrounding the Oxt mRNA peak at 30 – 60 min. Events leading up to the peak are seen only in the presence of 3␤-diol. They include a combination of increased promoter occupancy by ER␣ and ER␤, CBP, and SRC-1 and an increase in the level of H4 acetylation. Taken together, the data suggest that by 30 min 3␤-diol leads to formation of a functional complex of ER␤, CBP, and SRC-1 in the region of the Oxt promoter. That such a complex could form is supported by the literature. Specifically, ER␤ binds SRC-1 (25) and SRC-1 binds the CBP homolog, p300 (26). Given that CBP is a prototypic histone acetyltransferase and acetyltransferase activity has been ascribed to SRC-1 (27), recruitment to the promoter of either could explain the observed increase in H4 acetylation. That this putative complex forms only

in the presence of 3␤-diol is striking, particularly in comparison with events that occur by 60 min. After 60 min, the levels of Oxt mRNA begin to decrease, reaching control levels by 2 h. The decline in mRNA coincides with all other measures being at or below control levels. Specifically, there is a reduction from 30 to 60 min in all cases except for SRC-1 and Ac-H3. In the case of SRC-1, there is no effect of E2 at either time, and in the case of Ac-H3, there is no effect of 3␤-diol at either time. The concerted response of all other parameters suggests two opposing possibilities: either that there is redundancy in mechanism and/or that the mechanism requires precise synchronization of a number of events. Experiments that involve reduction of individual components of the putative complex are needed to determine the extent to which either or both are the case. Performing kinetic analyses of ER and coregulator occupancy to generate models of complex assembly at a given promoter has been reported in several different contexts. Prime examples of this approach are the comprehensive analyses of ER␣ and associated factors in the breast cancer cell line, MCF-7 (28, 29). Another example

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FIG. 5. 3␤-Diol treatment leads to increased CBP and SRC-1 occupancy of the Oxt promoter. Cells were treated as indicated in Fig. 3. Bars represent the mean ⫾ SEM of the data obtained from three independent experiments and are presented as fold difference of vehicle (Veh). *, P ⬍ 0.05; ***, P ⬍ 0.001 (two way ANOVA followed by post hoc analysis); ##, P ⬍ 0.01 (significance determined by t test).

is the comparative analysis of the ER␣ and ER␤ occupancy at a major stress-responsive gene, crh, in the neuronal line, AR-5 (22). In the latter case, the temporal patterns of occupancy suggested that ER␣ and ER␤ could form two different complexes and do so in as little as 1 and 3 min, respectively. The current report focused on Oxt occupancy by ER and their coactivators and markers of activation. As is always the case, an increase in the level of gene expression may arise either from increased expression or decreased repression. Thus, the increased levels of Oxt mRNA could result from ER-induced CBP/SRC-1 recruitment and/or a coactivator/corepressor exchange mechanism. An unexpected finding reported recently is that the glucocorticoid receptor appears at the crh promoter at the same time as histone deacetylase 1 in the presence of dexamethasone (30). Thus, it is conceivable that an ER could appear in the company of a repressive entity. Given the number of reports of ER␤ opposing the effects of ER␣, it may be that the recruitment of ER␤ leads to recruitment of corepressors and their associated enzymes. This could also explain

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FIG. 6. 3␤-Diol increases H4 acetylation. Cells were treated as indicated for Fig. 3. Data are presented as fold difference of vehicle (Veh). Bars represent the mean ⫾ SEM of the data obtained from three independent experiments and are presented as fold difference of vehicle. *, P ⬍ 0.05; ***, P ⬍ 0.001 (two way ANOVA followed by post hoc analysis).

the kinetics of CRH mRNA previously reported (22). In that case too, the increased occupancy by ER␤ occurred just before the decrease in mRNA. The investigations described above were begun in an effort to shed light on molecular mechanisms by which testosterone might inhibit the HPA axis when it acts in the central nervous system. Testosterone down-regulates HPA axis activity by acting centrally via its metabolite, 3␤-diol (8, 31, 32). This occurs through an action at the level of the paraventricular nucleus, presumably through oxytocinergic neurons that express ER␤, as supported by the finding that implants of 3␤-diol to the paraventricular nucleus can inhibit HPA reactivity to stress (33). That ER␤ plays a physiological role in the 3␤-diol regulation of the HPA axis is further supported by recent findings in ER␤ knockout mice. In these mice, the ability of the ER␤-selective ligand 2,3-bis(4-hydroxyphenyl)-propionitrile to inhibit the stress-induced rise in corticosterone is eliminated. Furthermore, this is accompanied by a loss of the behavioral actions of ER␤ ligands (34).

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Taken together, the data presented indicate that in addition to E2, the dihydrotestosterone metabolite 3␤-diol plays a role in Oxt expression. That there is specificity to the 3␤-diol modulation of Oxt expression is seen in the profiles of ER␣, ER␤, coactivator occupancy, and H4 acetylation. These data provide a glimpse of the intricacies of Oxt regulation. One layer of complexity that bears further investigation is the role that ER␤ splice variants may play in Oxt expression. There is likely at least one variant in these cells as suggested by the ER␤ immunoblot data. An additional layer to investigate is at the level of ER interactions with other transcription factors known to influence the manner by which the Oxt is expressed (19, 20). All of these questions may be addressed using cell-based systems. The answers could lead to further elucidation of ER molecular mechanisms of Oxt regulation, which in turn could lead to future therapeutic targets for the control of pathologies associated with a dysregulation of HPA axis function.

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Acknowledgments

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Address all correspondence and requests for reprints to: Rosalie Marie Uht, M.D., Ph.D., University of North Texas Health Science Center, Fort Worth, Texas 76107. E-mail: [email protected]. This work was supported by National Institutes of Health Grants R01-MH082900 (to R.M.U.) and R01-NS039951 (to R.J.H.). Disclosure Summary: D.S., R.J.H., and R.M.U. have nothing to declare.

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