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NEUROENDOCRINOLOGY |
Women’s Health Research Institute, Wyeth-Ayerst Research (P.J.S., T.L.D., I.M.), Radnor, Pennsylvania 19087; and Molecular Genetics, Wyeth/Genetic Institute (G.R.A.), Andover, Massachusetts 01810
Address all correspondence and requests for reprints to: Dr. Paul J. Shughrue, Department of Neuroscience, Merck Research Laboratories, WP26A-3000, Sumneytown Pike and Broad Street, West Point, Pennsylvania 19486. E-mail: . paul_shughrue{at}merck.com
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Abstract |
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KO) mouse brain, suggesting a splice variant of ER or another ER. The discovery of ERß suggested that binding was due to ERß, although questions about an ER
remained. To test this hypothesis, ERßKO mice were generated and crossed with ERKO mice, and ER/ßKO animals were used for in vivo binding studies with [125I]estrogen. The results revealed nuclear binding sites in the ER/ßKO hypothalamus and amygdala. As the binding resembled the distribution of ER, we evaluated the presence of ER splicing variants. A nonphysiological splice variant of ER was identified in ER/ßKO brain and uterus, but was absent in wild-type mice. ER immunoreactivity was also detected in regions of ER/ßKO brain where residual binding was seen. To ascertain the functionality of the variant, the regulation of PR was assessed in brain. The results revealed that E2 significantly increased PR expression, an indication that the variant can regulate gene transcription. These data demonstrate the presence and functionality of an ER variant in ER/ßKO brain and suggest that the residual binding and regulation of PR in ER/ßKO brain can be accounted for by the variant. |
Introduction |
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Concurrent with these studies, a novel ER (named ERß) was isolated from rat prostate. This receptor has a high degree of sequence homology to ER, specific binding for E2, and is capable of modulating an estrogen response element reporter construct in a dose-dependent manner (4). In situ hybridization studies revealed that ERß mRNA was expressed throughout the rostral-caudal extent of the rat brain (5) as well as in regions of the ERKO (ERKO) brain (6) where residual binding was observed (3). These data suggested that the binding seen in the ERKO brain was due to the interaction of radiolabeled ligand with ERß.
Recent studies found that an estrogenic pesticide, methoxychlor, was capable of modulating lactoferrin and glucose-6-phosphate dehydrogenase mRNAs in the ERKO uterus (7). As the estrogen antagonist ICI 182,780 was unable to abate the methoxychlor-induced increase in gene expression, Ghosh et al. (7) speculated that the actions of methoxychlor were mediated through a non-ER/non-ERß mechanism. Similarly, studies with cortical explants found that ER- and ERß-selective ligands were unable to elicit phosphorylation of ERK, as seen with E2 (8). Taken together, these observations and others suggested that additional ERs may exist, perhaps an ER.
Recently, we generated and characterized an ERßKO mouse. The ERßKO and ERKO mice were then crossed to generate ER/ßKO animals. In the present study, these double ER knockout mice (ER/ßKO) were used to ascertain whether residual estrogen-binding sites are still present in brain. As binding sites were observed in the ER/ßKO brain, additional studies were conducted to determine whether the ligand was interacting with a novel nuclear ER (ER) or a splicing variant of ER and/or ERß.
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Materials and Methods |
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). In contrast, ßERKO mice (9) were generated by inserting a copy of Neo in the reverse orientation into the PstI site of exon 3. No portion of the ERß gene was removed during the target event that generated ßERKO mice (9).
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). The construct was electroporated into R1 ES cells (50 µg DNA/1 x 107 cells in 800 µl), and G418 resistant clones were screened for homologous recombination by Southern analysis of HindIII genomic DNA digests using a 500-bp HindIII-Xba 5' outside probe. Two gene-targeted ES cell clones were identified, and each clone was expanded and used to generate germline chimeras by blast injection into C57BL/6 host blasts. The validity of the resulting ERßKO mice was then established by ribonuclease (RNase) protection assay (RPA) using a probe capable of distinguishing ERßKO from wild-type (wt) mRNA. An RPA probe was prepared by PCR cloning a 300-bp fragment of exon 1 using primers of the sequence (forward, 5'-CGCAAGACATGGAGATC-3') and (reverse, 5'-AACGCACGGGTGTTGGGTCG-3') into pGEM-T-Easy. This probe extends 100 bp beyond the insertion site in the targeting vector and therefore produces 300- and 200-bp protected fragments from the wt and ERßKO-targeted mRNA, respectively. Radiolabeled RNA probe was prepared using linearized, gel-purified plasmid template and an in vitro transcription kit following the manufacturer’s protocol (Promega Corp., Madison, WI). RNA was prepared from frozen dissected tissues using TRIzol (Life Technologies, Inc., Grand Island, NY) and the manufacturer’s suggested protocol. Probe:RNA hybrids were prepared by pre-melting the RNA in 30 µl hybridization buffer at 85 C for 5 min [80% formamide, 400 mM NaCl, 10 mM PIPES (pH 6.5), 1 mM EDTA, and 0.05% SDS], followed by overnight incubation at 42 C. Probe:RNA hybrids were digested in 350 µl T2 RNase buffer [100 mM NaCl, 50 mM NaOAc (pH 5.2), 2 mM EDTA, and 10 U T2 RNase] at 37 C for 30 min, precipitated, and separated on an 8% acrylamide gel. The gels were then dried and exposed to autoradiographic film or phosphorimaging screens overnight. As shown in Fig. 2, RPA analysis of total RNA isolated from wt (+/+) and heterozygous (+/-) mice revealed the predicted 300-bp fragment in the wt lanes and both the wt (300-bp) and predicted 200-bp fragments (the truncated transcript) in the heterozygous (+/-) mice. These data demonstrate that the targeted ERß allele synthesized the predicted transcript, including the stop codons, whereas the results from Western blot and immunocytochemistry studies (see Fig. 3), confirmed that the ERßKO mice lacked ERß.
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KO-C57BL/6Jx129 (1) and ERßKO-129 (see above) mice were genotyped using PCR analysis of tail biopsy. As adults, heterozygous ERKO females were crossed with homozygous ERßKO males to generate ER/ßKO. Double heterozygous ER/ßKO females were intercrossed with ER (+/-) / ERß(-/-) males to produce homozygous ER/ßKO mice. The studies described in this paper were reviewed and approved by the Wyeth-Ayerst animal care and use committee.
In vivo binding studies
Adult (60- to 90-d-old) wt and ER knockout mice were ovariectomized (ovx) for 7 d and then sc injected with 2 µg/kg BW 17-iodovinyl-11ß-methoxyestradiol ([125I]estrogen; SA, 2200 Ci/mM) (10) in 100 µl vehicle (50% dimethylsulfoxide and 50% saline; n = 5/group). For competition studies, additional mice (n = 2) were injected twice with 125 µg/kg BW 17ß-E2; once 30 min before [125I]estrogen and again with the radiolabeled ligand. Four to 6 h after injection of [125I]estrogen, the brains were collected from all groups and frozen, and 20-µm coronal cryostat sections were thaw-mounted onto gelatin-coated slides. The slide-mounted sections were then fixed and washed as previously described (11). The sections were apposed to x-ray film (BMR-1, Kodak, Rochester, NY) for 4 d, coated with gelatin, and then dipped in liquid emulsion (Kodak, NTB-2, diluted 1:1 with water). After 10–40 d of exposure, slides were developed, stained with cresyl violet, and coverslipped. The section-mounted slides from all animals were washed, apposed to film, exposed, and photographically processed together to minimize differences due to variations in conditions.
RT-PCR and nested RT- PCR ER cDNA
RNA was prepared from frozen dissected tissues (preoptic area of the brain and uterus from ER/ßKO mice) using TRIzol (Life Technologies, Inc.) and the manufacturer’s suggested protocol. First strand synthesis was prepared from 2–10 µl total RNA using a primer of the sequence (5'-CCTTTCTCGTTACTGC-3') with the SuperScript II RT kit (Life Technologies, Inc.). First round PCR amplification of 1 µl first strand template was performed in a 50-µl reaction using the AmpliTaq buffer and enzyme for 10 cycles with primers of the sequence (forward, 5'-ATTCCTTCCTTCCGTCTT-3') and (reverse, 5'-CCTTTCTCGTTACTGC-3'). Nested PCR amplification of 1 µl from the first amplification was performed in a 50-µl reaction for 25 cycles with primers of the sequence (forward, 5'-GCCGGTCTACGGCCAGTCGG-3') and (reverse, 5'-GCACACGGCACA GTAGCGAG-3'). Both amplification reactions were performed with the following thermal cycling conditions: 95 C for 1 min, 58 C for 2 min, and 72 C for 2 min, preceded by incubations at 95 C for 5 min and followed by an incubation at 72 C for 7 min. Products of nested PCR amplification were cloned using the PGem-T Easy cloning system (Promega Corp.) and then sequenced.
Immunocytochemistry
Our standard immunocytochemistry method (12) was used to assess the localization of ER immunoreactivity in the wt and ER/ßKO brain and ERß immunoreactivity in the wt and ERßKO brain and ovary. Ovx mice were transcardially perfused with 4% paraformaldehyde/3.75% acrolien (pH 7.4), and the brains were postfixed in 4% paraformaldehyde overnight at 4 C. Vibratome sections (35 µm) were treated with 0.2% Triton X-100 and 0.1 M glycine and then incubated in blocking solution (20% normal donkey serum/1% BSA/1% H2O2 in PBS). The sections were incubated for 48–72 h at 4 C with a rabbit polyclonal antiserum raised against the C terminus of ER (FMS-ER7, diluted 1:10,000) (13) or ERß (Z8P, Zymed Laboratories, Inc., South San Franscisco, CA; 50 ng/ml) (12). The sections were washed and incubated with a biotinylated donkey antirabbit serum (The Jackson Laboratory, Bar Harbor, ME; 1:1000), and the immunoreactivity was visualized with a standard avidin-biotin peroxidase complex method.
In situ hybridization
PR gene expression was evaluated in the preoptic area and uterus of wt, ERKO ERßKO, and ER/ßKO mice with in situ hybridization as described previously (3, 14). Briefly, adult female mice (2–4 month old) were ovx for 10 d and then treated with 10 µg/kg E2 dissolved in 50% dimethylsulfoxide/50% saline or vehicle alone (n = 6/group). Six hours after E2 injection, animals were killed, their brains and uteri were frozen, and 16-µm coronal cryostat sections were collected on gelatin-coated slides. [35S]UTP-labeled cRNA probe was then generated using our PR-815 plasmid (14) linearized with HindIII (sense; control) or BamHI (antisense). Processed section-mounted slides were hybridized with 200 µl antisense or sense (control) riboprobe (6 x 106 dpm/slide)-50% formamide hybridization mix and incubated overnight at 55 C. The slides were washed at 67 C, dehydrated, and apposed to BioMax (BMR-1, Kodak) x-ray film for 4 d. The slides from all animals were hybridized, washed, exposed, and photographically processed together to eliminate differences due to interassay variations in conditions.
Data analysis
PR hybridization signal was analyzed in the medial preoptic area because this area has the highest level of residual binding in the ER/ßKO brain, and the regulation of PR mRNA by estrogens has been well characterized (3, 14). Changes in PR expression were also evaluated in wt and ER/ßKO uteri. The relative OD measurements obtained from film autoradiograms were obtained with a computer-based image analysis system (C-Imaging, Inc., Pittsburgh, PA). The results from two sequential sections per animal were averaged, and two-way ANOVA was used to test for differences in the level of PR mRNA. The numerical values are reported as the mean ± SE, and all statements of difference imply that P < 0.05.
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Results |
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), Western blot analysis, and immunocytochemistry (Fig. 3). The results from each of these methods confirmed the absence of ERß in the ERßKO mice. An example of these results is shown in Fig. 3. Through the use of immunocytochemistry, nuclear ERß staining was detected in the paraventricular nucleus of wt mouse brain and in the ovarian follicles (Fig. 3, A and C), tissues known to express high levels of ERß mRNA (4, 6). In contrast, no ERß immunoreactivity was seen in the ERßKO brain, including the paraventricular nucleus (Fig. 3B), or the ovary (Fig. 3D).
In vivo binding studies
[125I]Estrogen binding studies were conducted with wt, ERKO, ERßKO, and ER/ßKO mice to investigate the presence and distribution of ER-containing neurons in the brain and to ascertain how this pattern changes in ERKO mice (Figs. 4, 5). In wt animals, cells with a nuclear concentration of radiolabeled ligand were seen throughout the forebrain, including the medial preoptic area (Fig. 4); bed nucleus of the stria terminalis (Fig. 4); paraventricular, ventromedial, and arcuate nuclei of the hypothalamus; and medial amygdala. Similarly, [125I]estrogen binding was seen throughout the ERßKO mouse forebrain (Figs. 4 and 5B). However, a reduction in labeled cell number was seen in regions where both ERs are expressed, and only a few labeled cells were detected in the paraventricular nucleus, an area where ERß is the predominant ER. In contrast, there was no apparent attenuation in labeled cell number in the ERßKO ventromedial and arcuate nuclei, brain regions where ERß mRNA is sparse. In the ERKO mouse brain (Fig. 4), the degree of radiolabeled ligand binding was markedly reduced in most forebrain regions, including the preoptic area and bed nucleus of the stria terminalis, with the exception of the paraventricular nucleus. Interestingly, some weak binding was still seen in the ventromedial and arcuate nuclei, despite the presumed lack of functional ER in these brain regions. Finally, when we looked at the results of binding studies conducted with ER/ßKO mice, we were surprised to find weakly labeled neurons in the preoptic area (Figs. 4 and 5A), bed nucleus of the stria terminalis (Fig. 4 and 5A), ventromedial nucleus, arcuate nucleus, and medial amygdala. Moreover, analysis of the subcellular distribution of [125I]estrogen revealed that the ligand was concentrated in the nucleus of cells, suggesting the presence of a third nuclear ER or an active splice variant of ER or ERß.
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splice variants/ßKO mouse brain resembled the known localization of ER, RT-PCR cloning was used to assess the presence of ER wt and splice variant mRNA in the uterus and brain of wt and ER/ßKO mice. Using PCR primers that spanned exons 2 and 3 of ER, a 184-nucleotide variant of ER, was amplified from the ER/ßKO uterus and brain. The splice variant was not detected in wt uterus or brain, which only exhibit the 355-nucleotide product of wt ER mRNA (Fig. 6). Sequence analysis of the cloned splice variant product revealed that the transcript was the same as E1, a splice variant of ER (15) that arises from a cryptic splice site and contains the first 20 bp of the Neo cassette and the 3'-portion of exon 2. Thus, the E1 variant lacks half of exon 2, the sequence that encodes part of the AB domain of the receptor, but still contains both the DNA-binding and ligand-binding domains.
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splice variants, C and D) with an antiserum raised against the C terminus of ER (13), we investigated the distribution of the E1 variant in the ER/ßKO brain. The results demonstrate the presence of nuclear immunoreactivity (the ER splice variant) in the ER/ßKO brain (Fig. 5C, inset), including the medial preoptic area (Fig. 5C), bed nucleus of the stria terminalis (Fig. 5C), arcuate nucleus, ventromedial nucleus, and medial amygdala. Moreover, the localization of immunoreactivity seen in the ER/ßKO brain matched both the number and the distribution of [125I]estrogen-binding sites seen with in vivo autoradiography (Fig. 5, A vs. C).
Functional capacity of the E1 ER splice variant
To evaluate the functional capacity of the ER splice variant in ER/ßKO mice, the regulation of PR mRNA by E2 was assessed in the preoptic nucleus of the hypothalamus and uterus. When ovx wt mice were injected with E2, a significant increase in the level of PR mRNA was observed in the preoptic nucleus compared with that in vehicle-treated ovx animals (Fig. 7). Similarly, E2 treatment of ovx ERKO, ERßKO, and ER/ßKO mice significantly augmented the level of PR mRNA in the preoptic nucleus, although to a different extent, compared with that in vehicle-treated ovx littermates (Fig. 7). Interestingly, treatment of ovx ER/ßKO mice with E2 did not significantly increase the expression of PR in the uterus, whereas E2 dramatically augmented the levels of PR mRNA in ovx wt females.
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Discussion |
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/ßKO mouse brain with nuclear uptake and retention of radiolabeled estrogen despite the expected lack of functional ERs. Characterization of nuclear binding sites with RT-PCR revealed a splicing variant of ER at exon 2, thus producing a truncated ER protein that retained both the DNA- and ligand-binding domains. Using an antiserum raised against the C terminus of ER and immunocytochemistry, we demonstrated that the ER variant was localized in the same regions of the ER/ßKO brain where [125I]estrogen was observed. These observations and the finding that E2 regulates the expression of PR mRNA in the ER/ßKO preoptic area suggest that an ER splice variant is present in the ERKO brain, a receptor capable of binding ligand and modulating gene transcription.
Previous studies in mice found nuclear estrogen-binding sites throughout the forebrain, including the cerebral cortex; preoptic area; bed nucleus of the stria terminalis; paraventricular, arcuate, and ventromedial nuclei of the hypothalamus; and amygdala (11, 16, 17, 18, 19, 20, 21). Recent studies from our laboratory also detected nuclear estrogen-binding sites in similar regions of the ERKO forebrain and showed that these sites were specific to estrogen (3, 11). The localization of ERß mRNA in regions of the ERKO forebrain (6) where residual binding was seen further suggested that the binding was due to the interaction with ERß. Moreover, these observations provided evidence that the binding seen in brain was due to ER and/or ERß. Surprisingly, the present autoradiography studies with ER/ßKO mice still detected nuclear binding sites in the preoptic area, bed nucleus of the stria terminalis, arcuate nucleus, ventromedial nucleus, and amygdala, although both the number of labeled cells and the intensity of signal were dramatically attenuated. The similar distribution of binding seen in the ER/ßKO brain and the known distribution of ER suggested the presence of a variant form of ER, although the existence of another ER, perhaps ER, could not be ruled out.
In an attempt to characterize the nuclear binding site(s) detected in the ER/ßKO brain, RT-PCR was used to assess the presence of ER splice variants in the preoptic area and uterus. PCR primers that spanned exons 2 and 3 of ER amplified a single 184-bp product from both tissues in ER/ßKO mice, whereas the same primer sets amplified a 355-bp product from wt tissues. Sequence analysis of the PCR products revealed that a splicing variant of ER was present in ER/ßKO uterus and brain. The variant arises from a splicing event that uses sequence in the Neo cassette as a donor site and the exon 3 acceptor site and retains the ER reading frame. The resulting ER splice variant, seen only in ERKO mice, contains exon 1 and the 5' half of exon 2, the first 20 bp of the Neo cassette, and exons 3–9. Although the ER splice variant protein is missing the 3' half of the A/B domain, it retains the remainder of the receptor, including both the DNA- and ligand-binding domains. Interestingly, the splice variant that we sequenced from the ER/ßKO uterus and brain was previously found in the ERKO uterus and called E1 (15). In vitro studies with E1 showed that the transcript generated a 60-kDa protein, capable of binding E2 and activating the transcription of an estrogen response element reporter gene construct (15). However, as E2 binding in the ERKO uterus was markedly attenuated (
10% of wt), and estrogen failed to significantly increase a number of uterine genes, including PR (15), the importance of this variant was discounted. In the present report, we also failed to detect a significant increase in PR mRNA in the ER/ßKO uterus after treatment of ovx mice with E2. As the levels of E1 in the uterus appear to be similar to or greater than those in the preoptic area of the ER/ßKO brain, it is unlikely that the differential modulation of PR mRNA is due to gross differences in tissue distribution. Instead, the regulation of PR in the brain, but not the uterus, of ER/ßKO mice, may reflect differences in the cellular environment (i.e. cofactors and/or comodulators). That is, the presence or absence of critical cofactors in the brain vs. uterus may determine the activity of the E1 variant and thereby determine the ability of E1 to regulate genes.
The presence of [125I]estrogen-binding sites in the ER/ßKO brain and amplification of E1, an ER splice variant, from the preoptic area suggest that the binding in brain is due to the interaction with E1. Alternatively, the radiolabeled estrogen may be binding to a novel nuclear ER (ER), a receptor with a distribution similar to that of ER. To resolve this issue we used an antiserum raised against the C terminus of ER (FMS-ER7) to investigate the distribution of ER immunoreactivity in the ER/ßKO brain. The results demonstrated the presence of nuclear ER immunoreactivity in cells of the preoptic area, bed nucleus of the stria terminalis, arcuate nucleus, ventromedial nucleus, and amygdala. Moreover, the apparent number of immunoreactive nuclei and their distribution within subregions of the brain matched the topography of [125I]estrogen-binding sites. These observations are in good agreement with a previous study that looked at the distribution of ER immunoreactivity in the ERKO brain using a different antiserum (C1355) raised against the C terminus of ER (22). Using C1355, nuclear ER immunoreactivity was found in the ERKO preoptic area, bed nucleus of the stria terminalis, arcuate nucleus, ventromedial nucleus, and amygdala, with the number of labeled cells being dramatically attenuated compared with those in wt animals (22). These observations support the present findings and indicate that the E1 variant is translated into protein in certain regions of the ERKO and ER/ßKO brain.
The localization of an ER splice variant in brain, a receptor that contains both DNA- and ligand-binding domains, suggested that estrogen might still regulate genes in the ER/ßKO mouse brain. Using in situ hybridization, we found that estrogen significantly increased the level of PR mRNA in the preoptic area of the ovx ER/ßKO brain. However, despite the significant induction of PR in the ER/ßKO brain, estrogen treatment was only capable of inducing PR to a level equivalent to that in vehicle-treated ovx wt animals. Similarly, estrogen modulated PR expression in wt, ERKO, and ERßKO mice, but the level of hybridization signal was attenuated in all KO brains compared with that in wt animals. Previous in situ hybridization studies with ERKO mice showed that 17ß-E2, diethylstilbestrol, 4-hydroxyestradiol, and 17-E2 induced PR mRNA in the preoptic area (4), indicating that a variety of steroidal and nonsteroidal estrogens can modulate PR. Using immunocytochemistry, Moffatt and colleagues (22) found that E2 up-regulated PR in the preoptic area of ovx ERKO mice. Interestingly, the number of PR-immunoreactive cells was also augmented in the ERKO ventromedial and arcuate nuclei (22), brain regions where ERß mRNA is sparse (6). As the present study detected [125I]estrogen-binding sites and ER immunoreactivity in these same regions of the ER/ßKO brain, it is likely that estrogen, acting through the ER splice variant, also regulates PR in the ventromedial and arcuate nuclei.
The present studies demonstrate the existence and functionality of an ER splice variant in the ER/ßKO brain and provide the opportunity to look for additional nuclear ERs in the brain. Data from several recent studies have suggested that additional ERs may exist in the rodent uterus and brain, perhaps an ER (7, 8). This conclusion is based on the findings that some, but not all, estrogenic compounds are capable of eliciting a response in ERKO tissues that is not blocked by the antiestrogen ICI 182,780 (7, 8). Although these results may be due to the presence of a novel ICI-insensitive ER, it is also possible that the deletion of sequence in the A/B domain of the ER splice variant alters the binding affinity and/or activity of certain estrogens, such as ICI 182,780 (23). This hypothesis is supported by in vitro studies with mutant ER constructs that show both mutant and/or ligand-dependent differences in receptor activation and function (24, 25, 26, 27).
Although the present studies provide evidence that the ER splice variant is present and functionally active in selected brain regions, the physiological relevance of the receptor in ERKO mice remains unknown. Based on behavioral observations, it is clear that the low number of [125I]estrogen-binding sites and the weak induction of PR by estrogen are insufficient to maintain normal female reproductive behaviors in ERKO mice (28, 29). However, despite these observations, we cannot assume that the presence and functionality of the ER splice variant are irrelevant in all neuronal systems. Future studies with the ERKO and ER/ßKO mice are clearly needed to ascertain the importance of the ER splice variant and to determine whether this variant may preclude the use of these animals in studying certain endocrine systems. Alternatively, the generation and characterization of new transgenic mice lacking ER using traditional KO strategies and/or conditional KO technologies will help us to ascertain the importance of ER and its splice variants in the organization and function of the brain. More importantly, the use of transgenic animals that lack both ERs and their associated variants will enable us to confirm the present findings and clearly demonstrate that there are only two nuclear ERs, ER and ERß.
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Footnotes |
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Received October 2, 2001.
Accepted for publication January 9, 2002.
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References |
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-[125I]iodoestradiol binding: neuronal localization of estrogen binding sites in the developing rat brain. Steroids 60:726–737[CrossRef][Medline]
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