Estrogen Receptor- (ER ), But Not ER , Modulates Estrogen ...

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Endocrinology 143(11):4196 – 4202 Copyright © 2002 by The Endocrine Society doi: 10.1210/en.2002-220353

Estrogen Receptor-␣ (ER␣), But Not ER␤, Modulates Estrogen Stimulation of the ER␣-Truncated Variant, TERP-1 DEREK A. SCHREIHOFER*, DANIEL F. ROWE, EMILIE F. RISSMAN, ELKA M. SCORDALAKES, JAN-ÅKE GUSTAFSSON, AND MARGARET A. SHUPNIK Division of Endocrinology and Metabolism, Department of Internal Medicine (D.A.S., D.F.R., M.A.S.), and Department of Biology (E.F.R., E.M.S.), University of Virginia, Charlottesville, Virginia 22908; and Center for Biotechnology and Department of Medical Nutrition, Karolinska Institute, NOVUM (J.-Å.G.), S-14186 Huddinge, Sweden Estrogens regulate pituitary gene expression through two nuclear receptors (ERs), ER␣ and ER␤. Rodent pituitary also expresses high levels of the pituitary-specific ER␣ isoform, truncated ER product-1 (TERP-1), which modulates the response of both ER forms to 17␤-estradiol (E2). Under physiological conditions, E2 stimulates TERP-1 expression from an ER␣ intronic promoter containing several potential binding sites for ERs. To evaluate the role of intact ER proteins on TERP-1 expression, we measured basal expression and steroid stimulation of TERP-1 in wild-type (WT) mice and mice in which either the ER␣ (ER␣KO) or the ER␤ (ER␤KO) gene was disrupted. TERP-1 mRNA expression was assessed by semi-

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STROGENS REGULATE gene expression through nuclear estrogen receptors (ERs) that act as liganddependent transcription factors (1, 2). In pituitary tissue the ER isoforms, ER␣ and ER␤, are present in several cell types, with highest expression in lactotropes and gonadotropes (3– 6). In addition, the rodent pituitary expresses high levels of the pituitary-specific ER␣ isoform, truncated ER product-1 (TERP-1) (7–10). TERP-1 mRNA is transcribed from a novel transcriptional start site and contains a unique 31-base sequence fused to sequence encoding exons 5– 8 of the rat ER␣ gene (10). The TERP-1 promoter is located in the large intron between exons 4 and 5 in the rat ER␣ gene and appears to be transcribed primarily in the pituitary gland (10, 11). Although other TERP-1 splice variant transcripts have been detected, they are all transcribed from the same promoter, and TERP-1 is by far the predominant form (5, 10, 11). Levels of TERP-1 mRNA and protein, hereafter called TERP, are stimulated by 17␤-estradiol (E2) in both mouse and rat pituitary cells, and TERP is also dramatically regulated throughout the rat estrous cycle in a manner divergent from that of full-length ER␣ (8, 10, 12). Because E2 treatment also increases degradation of pituitary ER␣ protein and decreases ER␤ mRNA, steroid status significantly alters the ratio of TERP to the full-length ER proteins (10, 13). Less information is available on androgen regulation of TERP. TERP mRNA Abbreviations: AP-1, Activating protein-1; AR, androgen receptor; ARE, androgen response element; DHT, dihydrotestosterone; E2, 17␤estradiol; ER, estrogen receptor; ERE, estrogen response element; KO, knockout; T, testosterone; TERP-1, truncated estrogen receptor product-1; WT, wild-type.

quantitative RT-PCR, and protein expression was evaluated by immunoblots. Both TERP-1 mRNA and protein were expressed in pituitaries from castrate WT, ER␣KO, and ER␤KO male and female mice. E2 stimulated TERP-1 mRNA expression in WT and ER␤KO mice of both sexes, but had no effect on TERP-1 mRNA in either male or female ER␣KO mice. Testosterone treatment also stimulated TERP-1 in WT, ER␣KO, and ER␤KO male mice. We conclude that ER␣ is critical for E2 stimulation, but not basal expression, of the TERP promoter, and that testosterone may act through the androgen receptor to stimulate the TERP-1 promoter in males. (Endocrinology 143: 4196 – 4202, 2002)

is present at much higher levels in female than in male rats, but can be stimulated by E2 in males (7, 14). However, treatment with either E2 or the nonaromatizable androgen dihydrotestosterone (DHT) stimulated TERP mRNA in female rats in vivo and in the GH3 and RC4B clonal rat pituitary cell lines (10). The expression of TERP protein relative to ER␣ and ER␤ can alter estrogen-regulated transcriptional responses (11, 15, 16). Although TERP protein cannot bind DNA and does not bind steroid effectively, it contains the dimerization region of the ligand-binding domain and can also interact with ER coregulatory proteins (16, 17). High levels of TERP expression, resulting in ratios of TERP/ER greater than 1:1, suppress ER transcriptional activity on estrogen response element (ERE)-containing promoters (11, 15). Transcriptional suppression occurs by the formation of TERP heterodimers with either ER␣ or ER␤, which have a decreased ability to bind to the ERE (16). We have also found that lower levels of TERP and ratios of TERP/ER less than 1:1 stimulate fulllength ER activity at an ERE, possibly by titration of repressor molecules (15). Although TERP mRNA levels are stimulated by E2 treatment, the roles of specific ER isoforms in this stimulation or the importance of ER␣ gene expression for TERP expression are not clear. We examined the pituitary glands from male and female mice in which either the ER␣ (ER␣KO) or the ER␤ (ER␤KO) gene was disrupted for TERP expression and steroid regulation compared with those in their wild-type (WT) siblings. Both male and female ER␣KO mice are infertile and have distinct pituitary phenotypes, although pituitary cell development appears to be normal (18, 19). ER␣KO mice

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exhibit significant deficits in PRL transcription and secretion, elevated levels of LH ␤-subunit mRNA and serum LH in males and females, and elevated FSH␤ mRNA in females (18 –20). ER␣KO mice also have very different steroid levels from those in wild-type mice. Basal E2 serum levels in WT female mice have been reported as approximately 24 pg/ml, with E2 at proestrus near 100 pg/ml, and are lower than those in ER␣KO females (241 pg/ml) (21). E2 serum levels in WT male mice (11 pg/ml) are roughly equivalent to levels in ER␣KO males (13 pg/ml). Testosterone (T) levels are less dramatically affected in ER␣KO (0.8 ng/ml) compared with WT (0.4 ng/ml) females, but are elevated in ER␣KO (8.5 ng/ml) compared with WT (3.9 ng/ml) male mice (22, 23). In contrast to the severe reproductive phenotype in ER␣KO mice, ER␤KO mice may reproduce, although females are subfertile, apparently via impaired ovarian follicular development (18, 24, 25). ER␤KO female mice have normal estrous cycles, but lower FSH levels on estrus compared with WT littermates (Amory, E., and E. F. Rissman, unpublished data). No dramatic pituitary phenotype has been reported to date (24). Our studies with these genetic models demonstrate that the expression of ER␣ is not required for TERP expression, but is critical for E2 stimulation. Furthermore, androgen treatment can stimulate TERP expression in male mice in the absence or presence of functional ER isoforms. Materials and Methods Animals All animal procedures were performed in accordance with the guidelines established by the University of Virginia animal care and use committee. Mice were produced by mating heterozygotes carrying a single copy of either the disrupted ER␣ gene (26) or the disrupted ER␤ gene (24). Both ERs were disrupted by insertion of the neomycin resistance gene (neo). The resulting offspring were genotyped by PCR amplification of DNA extracted from tails. For the ER␣KO mice this was accomplished with three primers; one from the 5⬘ end of the ER␣ gene (5⬘-CGGTCTACGGCCAGTCGGGCATC-3⬘), another from the 5⬘ end of the neo insert present in the disrupted ER␣ gene (5⬘-CTCTTGATTCCCACTTTGTGGTTC-3⬘), and a reverse primer from the 3⬘ end of the ER␣ gene (5⬘-CGCTGGGCTCGTTCTCCAGGTAGTA-3⬘). The products yielded included a 200-bp band spanning bases 414 – 614 on the ER␣ gene amplified from the WT gene, a 300-bp band amplified from the disrupted gene, and both bands for heterozygotes. For ER␤ mice the following primers were used: one from intron 2 (5⬘-GGAGTAGAAACAAGCAATCCAGACATC-3⬘), another from the 3⬘ end of the neo insert (5⬘-GCAGCCTCTGTTCCACATACACTTC-3⬘), and a third from exon 3 (5⬘-AGAATGTTGCACTGCCCCTGCTGCT-3⬘). A 665-bp band (intron 2 and exon 3 primers) was amplified for homozygous WT mice, a 500-bp band (intron 2 and neo primers) for homozygous gene-disrupted mice, and both bands for heterozygous mice. Male and female mice used in the experiments were WT or homozygous knockout mice, lacking either the functional ER␣ (ER␣KO) or ER␤ (ER␤KO). The ER␣KO mice were of a mixed 129/SvJ and C57BL/6J background, approximately eight generations back-crossed into C57BL/6J at the time of this study. ER␤KO mice were approximately two generations backcrossed into C57BL/6J at the time of this study. After weaning (18 –20 d of age) mice were individually housed in plastic cages on a 12-h light, 12-h dark cycle (lights off at 1200 h Eastern standard time) and received food (Purina mouse chow 5001, Ralston Purina Co., St. Louis, MO) and water ad libitum. There were not significant differences in mRNA or protein expression between untreated WT groups from various litters; however, in each study WT and homozygous knockout littermates (ER␣⌲⌷ or ER␤KO) from the same genetic background were compared.

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Experimental protocols Mice were gonadectomized via a midline incision under general anesthesia (100 mg/kg ketamine and 10 mg/kg xylazine, ip). For steroid treatments ovariectomized female mice (⬎14 d post ovariectomy) were injected for 3 d with oil vehicle or E2 (20 or 50 ␮g) at 1000 h. On d 3 animals were anesthetized with halothane and were killed by decapitation at 1500 h. Serum E2 levels for treated animals were 150 ⫾ 32 pg/ml (20 ␮g) and 300 ⫾ 52 pg/ml (50 ␮g) at this time. Pituitaries were rapidly removed, placed on dry ice, and stored at ⫺70 C for subsequent isolation of RNA and protein. Proteins were analyzed from WT oil-treated (n ⫽ 7), WT E2-treated (n ⫽ 6), ER␣KO E2-treated (n ⫽ 8), and ER␣KO (n ⫽ 8) ovariectomized female mice. Male mice were castrated 10 d before death and implanted with either empty SILASTIC-brand implants (Dow Corning Corp., Midland, MI; inside diameter, 1.02 mm; outside diameter, 2.16 mm) or implants filled with 5 mm T. Implants were inserted sc in the dorsal midscapular region. The number of T implants inserted was varied to create different dose groups, including empty SILASTIC capsules (control) and one (low) or six (high) 5-mm T implants.

Semiquantitative RT-PCR Total RNA was extracted from pituitaries by lysis in Tri-Reagent and polyacryl carrier (Molecular Research, Inc., Cincinnati, OH) according to manufacturer’s instructions. Conditions for semiquantitative RT-PCR were determined separately for each mRNA using WT pituitary mRNA from pooled, randomly cycling female mice. One microgram of total RNA was reverse transcribed in a 20-␮l mixture consisting of 5 mm MgCl2, 1⫻ PCR Buffer II (Perkin-Elmer, Norwalk, CT), 2 mm deoxyribonucleotides, 1 U ribonuclease inhibitor, 2.5 ␮m random hexamers, and 2.5 U murine leukemia virus reverse transcriptase. RT reactions were incubated 10 min at room temperature, 15 min at 42 C, and 5 min at 99 C, then cooled to 4 C for 5 min in an Eppendorf Mastercycler (Eppendorf Scientific, Hamburg, Germany) gradient thermocycler. For PCRs, MgCl2 was adjusted to 2 mm, and buffer was adjusted to 1.2⫻. Water, 0.2 mm primer oligonucleotides (Operon Technologies, Alameda, CA), and 2.5 U/100 ␮l Platinum Taq DNA polymerase (Life Technologies, Inc., Grand Island, NY) were added to a final volume of 16 ␮l. PCR was performed in an Eppendorf Mastercycler gradient thermocycler. General PCR conditions consisted of an initial denaturation step of 2 min at 94 C, followed by additional cycles with a 30-sec denaturation at 94 C, a 30-sec annealing step, and a 30-sec extension step at 72 C. A final 10-min extension step was performed at 72 C. Optimization was performed separately for each set of primers. Annealing temperature (56 – 66 C) for each primer set was determined at 35 cycles with 100-1000 ng input RNA. The optimal cycle number was determined over a range of 15– 47 cycles. For all mRNAs 100 ng input RNA fell in the linear range for all primer sets. Cycle numbers used to measure each mRNA were as follows: ER␣ N terminus, 30 cycles; ER␤ N terminus, 38 cycles; TERP-1, 38 cycles; and ␤-actin, 26 cycles. For all mRNAs, 100 –500 ng input RNA fell in the linear range for all primer sets. TERP mRNA was amplified from an aliquot of the reverse transcriptase reaction equivalent to 200 ng input RNA, and all other mRNAs were amplified from the equivalent of 100 ng input RNA. PCR primers for ER␣ were the same as those used for genotyping WT mice and amplified a 200-bp sequence. A 370-bp sequence for TERP was amplified with primers to the 5⬘-untranslated TERP-specific sequence (5⬘-CCATTTCTTGAGCTTGTTGAACAG-3⬘) and ER␣ exon 7 (5⬘-AGTGTCTGTGATCTTGTCCAGGAC-3⬘). PCR primers for ER␤ amplified a 258-bp sequence from exons 1 (5⬘-GCTGTGATGAACTACAGTGTTCCC-3⬘) and 2 (5⬘-TGGACTAGTAACAGGGCTGGCACA-3⬘) and spanned the site of the neo insert. For semiquantitative analysis pituitaries were pooled into groups of two or three for each genotype and treatment group. All examined mRNAs were amplified three times from separate RT reactions. ␤-Actin was amplified in its linear range with exon 2 (5⬘-ATGGGTCAGAAGGACTCCTACGTG-3⬘) and exon 3 (5⬘GGAGTCCATCACAATGCCAGTGGT-3⬘) primers and used as a control for mRNA concentration and quality. After PCR amplification, reactions were separated on 1% agarose gels containing ethidium bromide (0.5 ␮g/ml). Gels were photographed and evaluated by fluoroimaging with a Fluorimager 515 (Molecular Dynamics, Inc., Sunnyvale, CA). Data were analyzed with ImageQuant software (Molecular Dynamics, Inc.).

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Immunoblot analysis

Statistical analysis

Total pituitary or uterine protein was extracted using Tri-Reagent according to the manufacturer’s instructions. The protein concentration was determined using a bicinchoninic acid kit (Pierce Chemical Co., Rockford, IL). Approximately 20 ␮g (uterine) or 100 ␮g (pituitary) total protein were separated on 12% polyacrylamide-SDS gels, and ER␣ and TERP protein expression was determined by immunoblot analysis using a rabbit polyclonal antibody as previously described (12). The antibody (C1355) was generated against C-terminal amino acids 586– 600 of the rat ER␣ and also detects mouse ER␣, but does not cross-react with ER␤ (10). High sensitivity detection was achieved using 6 ⫻ 8-cm gels for protein resolution and the SuperSignal Pico West chemiluminescence detection system (Pierce Chemical Co.), which results in very low background even with high levels of protein loading. This permitted us to perform Western analysis with the C1355 primary antibody at 1:7,500 dilution for 1 h at room temperature, followed by a 1-h incubation with a horseradish peroxidase-conjugated donkey antirabbit IgG secondary antibody (Amersham Pharmacia Biotech, Arlington Heights, IL) at 1:5,000 dilution compared with dilutions of 1:5,000 for C1355 and 1:800 for secondary antibody in previous studies. The C1355 antibody did not detect any immunopositive band in any ER-negative cell system or in serum, and preabsorption of the antibody with the antigenic peptide resulted in a loss of the immunopositive full-length ER␣ and TERP protein bands. In some blots two additional faint bands were observed above the TERP protein band, as observed by others (9, 11), and these were not eliminated with preabsorbed serum. Full-length ER␣ protein, but not TERP protein, could also be detected with antibodies to the N terminus (N21, a gift from Dr. Geoffrey Greene, University of Chicago, Chicago, IL) and hinge (ER715, a gift from Dr. Jack Gorski, University of Wisconsin, Madison, WI) regions, whereas both proteins were detected with additional C-terminal antibodies (7, 12). ER blots were stripped in 50 mm Tris buffer containing 100 mm ␤-mercaptoethanol at 50 C for 30 min. After washing, protein loading was normalized to ␤-actin on the same blots using a monoclonal primary antibody at 1:5,000 dilution (Sigma, St. Louis, MO) and a horseradish peroxidase-conjugated goat antimouse IgG secondary antibody at 1:40,000 dilution (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). Enhanced chemiluminescence (Amersham Pharmacia Biotech) was used to detect ␤-actin. In no case did secondary antibodies alone result in visualization of any immunopositive bands. In some cases, in vitro translated proteins or proteins from transfected COS cells were included on blots as markers and positive controls for experimental conditions. To calculate ER␣ and TERP protein levels, the intensities of immunopositive bands were measured by densitometry and normalized for the intensity of the immunopositive ␤-actin band measured in the same lane on the same blot for each sample. Densitometry was performed with a Personal Densitometer SI and analyzed with ImageQuant software (Molecular Dynamics, Inc.).

Data for each ER and animal experiment were analyzed separately. After one-way ANOVA, a priori pairwise comparisons were made between treatment groups and controls using t tests. P ⬍ 0.05 was considered significant.

Results Basal TERP expression in wild-type, ER␣KO, and ER␤KO female mice

Immunoblotting studies were performed with total pituitary protein (100 ␮g) from WT (n ⫽ 6) and ER␣KO (n ⫽11) or WT (n⫽6) and ER␤KO (n ⫽ 7) female mice and an antibody specific for the C-terminal 15 amino acids of ER␣. In WT and ER␤KO female mouse pituitaries, immunoreactive fulllength ER␣ protein of approximately 64 – 66 kDa was clearly observed, but was lacking in pituitaries from ER␣KO mice (Fig. 1, left panel). In contrast, TERP protein migrating at approximately 22–26 kDa was clearly observed in pituitaries from all female mice in Fig. 1 regardless of genotype. ER␣ protein was easily detected in total uterine protein (20 ␮g) from WT and ER␤KO females, but not ER␣KO females (Fig. 1, right panel). The immunopositive TERP band was not seen in uterine protein from WT, ER␣KO, or ER␤KO mice, indicating that TERP expression is restricted to the pituitary, as in the rat. We also tested 50 –100 ␮g uterine protein from several animals of each genotype, with identical results. Overall, significant levels of TERP protein were detected in mouse pituitaries, and basal expression did not require both full-length ER proteins. Estrogen stimulation of TERP-1 mRNA and protein in male and female mice requires ER␣

TERP mRNA, quantitated by RT-PCR with a TERP-specific primer, could easily be detected in both male and female mice regardless of genotype (Fig. 2). Three days of E2 treatment significantly increased TERP-1 mRNA levels in pituitaries from both male and female WT and ER␤KO mice (Fig. 2). However,

FIG. 1. Expression of ER␣ and TERP-1 protein in pituitaries and uteri from ovariectomized WT, ER␣KO, and ER␤KO female mice. Left panel, One hundred micrograms of total pituitary protein from one ovariectomized WT and two ER␣KO female mice or 50 ␮g protein from one WT and one ER␤KO female mice were separated on 12% denaturing polyacrylamide gels and transferred to membranes. Immunopositive proteins were detected with an ER␣-specific antibody, C1355. Right panel, Twenty micrograms of total uterine protein from one WT and one ER␣KO or one WT and one ER␤KO mouse were separated on 12% SDS-polyacrylamide gels, and immunopositive proteins were detected as described above. The migrations of the 64- to 66-kDa ER␣ and the 22- to 26-kDa TERP proteins are designated by arrowheads. Migration positions of molecular mass markers (MW) in kilodaltons are shown on the right.



FIG. 2. Estrogen regulation of TERP-1 mRNA expression in mouse pituitary. Relative TERP mRNA expression (TERP/␤-actin) detected by semiquantitative RT-PCR in gonadectomized WT (⫹/⫹) and knockout (⫺/⫺) male (M) and female (F) mice treated with oil (䡺) or estrogen (E2; f). Results for ER␣KO (A) and ER␤KO (B) mice are shown with those for WT littermates from each genetic background. Data represent the mean ⫾ SEM from 7–10 mice/group, and significant differences (P ⬍ 0.05) between oil and E2 groups are denoted with asterisks. The right panels depict PCR products from individual samples in each treatment group.

FIG. 3. Estrogen regulation of ER␣ and TERP protein in female mouse pituitary. Fifty micrograms of total pituitary protein from ovariectomized WT and ER␣KO mice treated for 3 d with oil or E2. Proteins were separated on 12% denaturing polyacrylamide gels and detected with an ER␣-specific antibody. In vitro translated (IVT) ER␣ and TERP are included for comparison, and ␤-actin is shown as a loading control. Two immunopositive bands migrating above the authentic TERP protein band are nonspecific, as determined by immunoabsorption of antibody. Migration of relevant molecular mass (MW) markers are shown on the right of individual panels. Similar results were observed in six to eight animals per group, and the experiment was performed twice.

E2 did not increase TERP mRNA expression in male or female ER␣KO mice, demonstrating a primary role for ER␣ in mediating estrogen regulation of TERP expression (Fig. 2). No Nterminal ER␣ mRNA was detected in ER␣KO mice using primers spanning the neo insert, confirming the loss of full-length ER␣ expression. Similarly, no ER␤ mRNA was detected in ER␤KO mice. E2 stimulation of TERP mRNA was specific for this transcript, as there was no effect of E2 on ER␣ mRNA in WT or ER␤KO mice, and E2 slightly suppressed ER␤ mRNA in WT and ER␣KO mice (not shown). Immunoblots from ovariectomized female WT mice treated with either oil (n ⫽ 7) or E2 (n⫽ 6) showed a similar pattern of TERP-1 protein regulation. E2 treatment reduced ER␣ protein levels by approximately 70% (oil, 1.39 ⫾ 0.09; E2, 0.41 ⫾ 0.03 ER␣/␤-actin; P ⬍ 0.01) and increased TERP

expression (oil, 0.06 ⫾ 0.01; E2, 0.13 ⫾ 0.02 TERP/␤-actin; P ⬍ 0.05; Fig. 3). In ER␣KO ovariectomized female mice (oil, n ⫽ 8; E2, n ⫽ 8), no ER␣ protein was seen, but mice expressed detectable TERP protein (Fig. 3). TERP protein expression, when normalized for ␤-actin expression on the same blot, was not stimulated by E2 treatment (oil, 0.05 ⫾ 0.025; E2, 0.07 ⫾ 0.02 TERP/␤-actin). Thus, at both mRNA and protein levels, stimulation of TERP by E2 requires ER␣. TERP-1 mRNA stimulation by T in WT, ER␣KO, and ER␤KO males

Because TERP was expressed in male mice, we examined the ability of T to regulate ER mRNAs in castrate and Treplaced male mice with intact or disrupted ERs. Intact WT

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and ER␤KO mice have plasma T levels of approximately 2 ng/ml, whereas ER␣KO mice have plasma levels of approximately 6 ng/ml (22) (Rissman, E., unpublished data). The castrate WT and knockout mice in the present study had plasma T levels of 0.01– 0.15 ng/ml. Low T implants raised T levels to 2.22–2.24 ng/ml, and high T implants further raised T levels to 12.99 –13.61 ng/ml. T had no effect on ER␣ mRNA levels in WT or ER␤KO mice (not shown). As with E2 treatment, TERP mRNA expression in WT and ER␤KO male mice was stimulated by T (Fig. 4). In contrast to E2, however, T also increased TERP mRNA in ER␣KO mice. In each case the highest T dose led to a significant increase in TERP mRNA, and the low dose was intermediate between the castrate and high T conditions. In contrast, ER␤ mRNA levels were suppressed by T in WT, ER␣KO, and ER␤KO animals (Fig. 4), similar to results with DHT in clonal mouse pituitary cells and suggesting that T is acting through the androgen receptor (AR) to mediate these transcriptional effects.

FIG. 4. T regulation of TERP-1 and ER␤ mRNA expression in mouse pituitary. Relative TERP-1 (A; TERP/␤-actin) and ER␤ (B; ER␤/␤actin) mRNA expression was detected by semiquantitative RT-PCR in castrate WT (⫹/⫹) and knockout (⫺/⫺) male (M) mice treated with oil (䡺), low T (f), or high T (p). Results from ER␣KO and ER␤KO mice are shown with those from WT littermates for each genetic background. Data represent the mean ⫾ SEM from 7–10 mice/group, and significant differences (P ⬍ 0.05) between oil and T groups are denoted with asterisks.


Discussion

TERP-1 is a highly regulated, pituitary-specific protein that can modulate the transcriptional activity of full-length ER isoforms. Previous results demonstrate that TERP-1 expression is regulated throughout the rat estrous cycle differently from ER␣ and ER␤ (7). In vivo studies in rats and in rat and mouse pituitary cells lines showed that E2 is a potent stimulator of TERP mRNA and protein expression (7–12, 14). The present study sought to determine whether the expression of TERP-1, which has recently been shown to be transcribed from an intronic promoter in the rat ER␣ gene (11), is dependent upon the expression of full-length ERs in the mouse pituitary. Our results demonstrate that in the mouse, ER␣ is critical for the E2 stimulation of TERP expression in the pituitary, and that in both species TERP mRNA and protein levels are stimulated by E2 (7, 12, 14). In mouse pituitaries basal TERP expression was maintained in the absence of either ER␣ or ER␤, suggesting that TERP expression may not be fully dependent on ERs. Furthermore, in male mice T appears to stimulate TERP expression independently of full-length ERs, consistent with the results of DHT treatment in female rats and rat pituitary cell lines (10). The rat TERP promoter has recently been cloned (11), and our preliminary results suggest that the mouse TERP promoter has a similar location and structure. In this ER␣KO model, the gene is disrupted in the second exon, whereas the TERP promoter in the rat and in the mouse (our unpublished data) is in an intron downstream of this site (intron between exons 4 and 5 in the rat). Thus, initiation and completion of transcription would occur appropriately for TERP mRNA, which contains the equivalent of exons 5– 8 in the rat gene, if ER␣ was not required for basal expression. No full-length ER␣ mRNA or neo splice variant mRNA is detected in the ER␣KO mouse pituitary (2, 28), and we have not observed this at the mRNA or protein level in our studies. The TERP promoter in intron 4 of the rat ER␣ gene contains several regulatory elements that are consistent with its localization in the pituitary and its regulation by E2. Notably, the first 2 kb upstream of the transcriptional start site contains one putative palindromic ERE, four half-EREs, and several AP-1 sites (11). Because both ligand-activated ER␣ and ER␤ can stimulate transcription from ERE-containing promoters (17, 27), E2 could stimulate TERP expression in ER␣KO via ER␤. One possible explanation for our results is that ER␤ may not play a significant role in either basal or E2-stimulated TERP expression. ER␤ levels in the mouse pituitary are very low and may not be able to support E2-dependent transcriptional activity. We have been unable to detect ER␤ in the adult mouse pituitary by immunoblotting using several different antibodies. In addition, although ER␤ is detectable by RTPCR, less sensitive techniques, such as ribonuclease protection assay, failed to detect ER␤ mRNA in either the WT or ER␣KO mouse pituitary (28). However, others have noted the presence of ER␤ mRNA using in situ hybridization in both adult and neonatal rats as well as RT-PCR (5, 6, 10, 29, 30). ER␤ protein has also been detected by immunocytochemistry in rat pituitary by some investigators, but this expression is generally much lower than ER␣ and is much greater during early development than in the adult (31). In


addition, fewer pituitary cells express ER␤ than ER␣ protein in the adult rat pituitary (6, 29, 30). ER␤ mRNA and protein were detected in both human pituitaries and pituitary tumors, but generally at lower levels than ER␣ (32, 33). A second possible explanation for the importance of ER␣, but not ER␤, in E2-dependent TERP regulation is that ER␣, TERP, and ER␤ are differentially expressed in different cell types, with the highest TERP expression in cells expressing full-length ER␣. Although some investigators have reported widespread colocalization of ERs in many pituitary cell types, others have seen more distinct cell expression patterns (5, 29, 31–33). In the human pituitary and clonal pituitary tumors, ER␣ is expressed at its highest levels in lactotropes and gonadotropes, whereas ER␤ is higher in gonadotropes than in lactotropes and is the only ER expressed in TSH-, GH-, and ACTH-expressing tumors (32, 33). In the rat, ER␤ and ER␣ appear to colocalize in about 20% of pituitary cells, but several studies suggest that lactotropes express primarily ER␣, with only 11% of the cells expressing ER␤, whereas 67% of gonadotropes express ER␤ alone or in addition to ER␣ (5, 31). ER␤ may be expressed in other cell types and is expressed at its highest levels in the rat during early development, before the expression of the gonadotropins; the physiological function of ER␤ during this time period is unknown (31, 34). The distribution of TERP in the pituitary seems to follow the pattern of ER␣ expression, as it is more highly expressed in lactotropes than in other cell types (6). Finally, it is possible, although less likely, that E2 regulation of the TERP promoter is different in the presence of different ER isoforms, as occurs for the vasopressin gene or on activating protein-1 (AP-1)-containing promoters (35, 36). The TERP promoter contains both ERE and AP-1 elements, and this possibility requires further study. However, all data obtained to date suggest that the expression of active ER␣ is an important prerequisite to effective stimulation of TERP by E2, and ER␤ cannot compensate for the loss of ER␣. Overall, these results are in keeping with the physiological function of ER␣ and ER␤ in adult pituitary. Studies of reproductive function in ER␣KO and ER␤KO mice show that ER␤KO mice have relatively normal pituitary morphology and function, whereas ER␣KO mice show severe abnormalities in gonadotropin and PRL secretion and steroid feedback on the hypothalamic-pituitary axis (18 –21). TERP mRNA was stimulated by T in male mice with either intact or disrupted ER genes and in female rats (10). This was somewhat unexpected, as pituitary TERP expression in male rats is less than that in females and is stimulated in both males and females by E2 (3, 7, 14). In our studies the effect of T cannot be solely due to aromatization to E2, because T stimulates TERP expression in ER␣KO mice, and E2 cannot. Furthermore, the nonaromatizable androgen DHT stimulates TERP expression in ovariectomized female rats and in pituitary cell lines (10). Thus, T is probably acting at least partly through ARs, which are present in numerous pituitary cells (36, 37) and can directly modulate gonadotropin secretion in rodent pituitary cells (38). Although there is no canonical androgen response element (ARE) sequence in the TERP promoter, androgens can act to modulate gene expression through the AR via protein-protein interactions. For example, the TERP promoter contains putative binding sites


for AP-1, specificity protein 1, cAMP response element-binding protein, and other transcription factors (11), and AR could interact with these factors, as has recently been demonstrated for other genes (39, 40). Alternatively, AR could bind directly to DNA via a nonconsensus ARE. The physiological role of T stimulation of TERP is unknown. Because only the high T dose was consistently stimulatory, and plasma concentrations of T in these animals exceed those in intact males, direct T stimulation of TERP may not play a major regulatory role physiologically. Alternatively, because some investigators have observed that high concentrations of TERP suppress the expression of ARE-containing reporter genes via the AR, we cannot rule out a role for TERP in modulating AR activity when T levels are high (11). Basal expression of TERP-1 in mice does not require the expression of either ER␣ or ER␤ and is probably dependent on other transcription factors. The TERP promoter contains potential DNA-binding sites for several factors that direct pituitary cell development and pituitary-specific gene expression, including Pitx-1, Pit-1, steroidogenic factor-1, and neural zinc finger factor 1 (11, 41– 44), although specific functions for these sites have not been demonstrated. The presence of multiple potential tissue-specific elements and E2 regulatory elements in the TERP promoter contrast with many of the multiple upstream promoters for the rat and human ER␣ genes, which have some tissue selectivity, but, in general, are expressed in multiple tissues and are not responsive to E2 (45). The ability of TERP to both enhance and inhibit E2dependent transcription suggests that it may play an important physiological role in the regulation of E2regulated genes (9 –11, 15, 16). This could occur at low TERP ratios observed in late diestrus or early proestrus by titrating suppressive molecules such as repressor of ER activity and islet-1 to stimulate ER activity (46); as TERP protein levels increase in late proestrus, TERP-ER heterodimers with reduced DNA-binding ability are formed (11, 16). Thus, TERP could participate in waves of sensitization and desensitization of E2 actions in the pituitary. Other investigators have suggested that higher levels of TERP could have independent effects on other promoters, including AREs, suggesting that TERP could have other roles in modulating pituitary gene transcription distinct from that of ER (11). These questions will require the development of animals in which TERP expression is genetically disrupted distinct from or in addition to ER␣. The ER␣KO animals have been extensively characterized and do have disrupted pituitary gene expression compared with WT animals. The studies in this report demonstrate the expression of TERP mRNA and protein in mice, E2 stimulation of TERP with dependence on ER␣ expression, T stimulation of TERP expression in the absence of ER, and persistence of TERP expression in the absence of ER␣. These facts will form an important basis for interpretation of future studies of the physiological role of TERP in vivo. Acknowledgments The authors thank the Markey Center for Cell Signaling at University of Virginia for use of the fluoroimager, and Denis Curtin for help with image analysis.

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Received March 27, 2002. Accepted July 5, 2002. Address all correspondence and requests for reprints to: Margaret A. Shupnik, Ph.D., Box 800578 HSC, University of Virginia, Charlottesville, Virginia 22908. E-mail: [email protected]. This work was supported by the core laboratories of Center for the Study of Reproduction at University of Virginia under NICHD/NIH Cooperative Agreement U54-HD-28934 as part of the Specialized Cooperative Centers Program in Reproductive Research, and NIH Grant RO1-DK-57082 (to M.A.S.) and Grants R01-MH-57759 and K02-MH01349 (to E.F.R.). * Current address: CL2130, Department of Physiology, Medical College of Georgia, 1120 15th Street, Augusta, Georgia 30912-3000.

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