An Estrogen Receptor (ER){alpha} Deoxyribonucleic Acid-Binding Domain Knock-In Mutation Provides Evidence for Nonclassical ER Pathway Signaling in Vivo

Monika Jakacka, Masafumi Ito, Fred Martinson, Toshio Ishikawa, Eun Jig Lee and J. Larry Jameson

Division of Endocrinology, Metabolism, and Molecular Medicine, Northwestern University Medical School, Chicago, Illinois 60611

Address all correspondence and requests for reprints to: J. Larry Jameson, M.D., Ph.D., Department of Medicine, Northwestern University Medical School, 251 East Huron Street, Galter 3-150, Chicago, Illinois 60611. E-mail: ljameson{at}northwestern.edu.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We created a nonclassical estrogen receptor (ER) knock-in mouse model by introducing a mutation that selectively eliminates classical ER signaling through estrogen response elements, while preserving the nonclassical ER pathway. Heterozygous nonclassical ER knock-in (NERKI) females are infertile. Their ovaries contain no corpora lutea, reflecting a defect in ovulation, and the stromal cells contain lipid droplets, suggesting altered steroidogenesis. The uteri are enlarged with evidence of cystic endometrial hyperplasia, and the mammary glands are hypoplastic. These phenotypic features indicate differential ER effects on growth and development in various estrogen-responsive tissues. These findings suggest that nonclassical ER signaling pathways play an important physiological role in the development and function of the reproductive system.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ESTROGENS ARE STEROID hormones that play a central role in the regulation of reproduction. Their effects are mediated through the estrogen receptor (ER), a member of the nuclear receptor superfamily. The classical pathway for ER signaling is mediated by receptor binding to estrogen response elements (EREs). When bound to an agonist, the receptor undergoes conformational changes that induce interactions with coactivators, leading to transcriptional stimulation of target genes. This mode of ER action has been documented for many genes involved in reproduction, such as prolactin (PRL) (1), progesterone receptor (PR) (2), and uteroglobin (3), as well as genes involved in cellular growth and metabolism, such as c-fos (4), very low density lipoprotein receptor (5), and clotting factor XII (6).

In addition to this classical pathway of estrogen action, the ER can also alter gene transcription by a mechanism that does not require direct ER binding to an ERE. Instead, protein-protein interactions between the ER and other transcription factors, such as Sp1, Jun, or nuclear factor-kappa B, lead to changes in target gene transcription. Examples of genes regulated in this nonclassical pathway include heat shock protein Hsp27 (7), low density lipoprotein receptor (8), collagenase (9, 10, 11), IGF-I (12), choline acetyltransferase (13), lipoprotein lipase (14), IL-6 (15, 16), and others. In many of these cases, there is no evidence of ER binding to DNA, although sequences within the ER DNA binding domain (DBD) seem to be necessary (15, 17). In vitro studies suggest that non-ERE-dependent actions of the ER may be of physiological significance. It is also notable that many of the nonclassical pathway target genes are paradoxically stimulated by ER antagonists, raising the possibility that selective estrogen receptor modulators may act as agonists through the nonclassical pathway (9, 10).

The significance of nonclassical signaling for members of the nuclear receptor family has been elegantly demonstrated by studies of the glucocorticoid receptor (GR). Mice completely deficient in GR expression die shortly after birth due to lung atelectasis (18). Another model was created to identify GR actions that are mediated independent of DNA binding. A point mutation was introduced that abolished GR homodimerization and therefore GR binding to its response element (19). Animals homozygous for this mutation (GRdim/dim mice) survived, demonstrating that the GR activity through the classical pathway is not essential for survival. Further characterization of these animals allowed GR action to be classified as classical vs. nonclassical pathways. For example, proopiomelanocortin and PRL gene expression, as well as induction of thymocyte apoptosis, are dependent on DNA binding, whereas development and function of the adrenal medulla, and certain inflammatory responses, are independent of the classical pathway.

Knockout (KO) models of both the ER{alpha} and ERß genes have demonstrated their important role in reproduction and development. Disruption of the ERß gene impairs ovulation but males are fertile (19). Mutagenesis of the ER{alpha} gene causes phenotypic effects in multiple reproductive tissues in males and females. ER{alpha}KO males are infertile and exhibit impaired fluid resorption and obstruction of the seminiferous tubules. ER{alpha}KO females are anovulatory and their ovaries contain hemorrhagic cysts. Their uteri are hypoplastic and unresponsive to estrogen, and the development of the mammary gland is arrested. Serum levels of estradiol (E2) and LH are elevated, and the levels of PRL are diminished (summarized in Ref. 20). Studies of mice deficient in both ER{alpha} and ß (ER{alpha}ßKO) provide additional evidence for the important role of the ERs in female reproductive development and function (21). Ovarian follicles of adult ER{alpha}ßKO females differentiate into structures that resemble seminiferous tubules of the testes, a phenotype that is unlike that seen in either ER{alpha}KO or ERßKO.

The goal of this study was to distinguish the classical and nonclassical ER actions in vivo. Previous in vitro work has demonstrated that it is possible to selectively eliminate classical ER signaling and preserve nonclassical signaling by introducing a mutation into the P box of the first zinc finger of the DBD (22). This mutation (E207A/G208A in mouse ER{alpha}) abolishes ERE binding and activation of ERE-containing reporter genes, but preserves the nonclassical pathway, as demonstrated by ER regulation of reporter genes containing activator protein 1 (AP1) response elements. We hypothesized that targeted insertion (knock-in) of this mutation would contrast with the ER{alpha}KO model by preserving the physiological functions of ER that are independent of DNA binding (nonclassical pathway). Unexpectedly, heterozyous females are infertile due to anovulation and the occurrence of endometrial hyperplasia. The presence of such a strong phenotype in the heterozygous state confirms the role of nonclassical ER signaling in vivo and suggests that these features may be caused by dysregulation of classical and nonclassical signaling.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Features of the Introduced Mutation
The E207A/G208A mutation in the mouse ER{alpha} (mER{alpha}) was introduced into the first zinc finger of the DBD to eliminate ERE binding (22). The sequence of the zinc finger and the position of the mutation are depicted in Fig. 2AGo. The in vitro functional properties of this mutant in the classical and the nonclassical pathways are demonstrated in Fig. 1Go. In the classical pathway, wild-type (WT) ER{alpha} activates transcription in the presence of estrogen and represses transcription in the presence of the antiestrogen ICI 182,780 (Fig. 1AGo, ERE reporter). The E207A/G208A mutant is inactive in the classical pathway. In the nonclassical pathway, the ER ligands exhibit effects that are the opposite of those seen in the classical pathway: E2 represses transcription, and ICI 182,780 activates transcription (Fig. 1AGo, AP1 reporter). The activities of the WT and E207A/G208A ER mutants are comparable, demonstrating that the DBD mutation does not affect the nonclassical pathway.



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Figure 2. Introduction of the NERKI Mutation into Exon 3 of Mouse ER{alpha}

A, Schematic illustration of the first zinc finger of mouse ER{alpha}. Amino acids forming the proximal box are shown in bold. The introduced double mutation (E at position 207 to A and G at position 208 to A) is indicated in the figure. B, Schematic illustration of the targeting strategy used to introduce the mutation. Restriction sites are abbreviated as follows: N, NheI; B, BamHI; Sh, SphI; E, EcoRV; neo indicates the neomycin cassette flanked by lox P sites; tk indicates thymidine kinase cassette. S indicates a position of a Southern blot probe (shown in C). Introduction of the mutation introduces a novel SphI site. The neomycin cassette was excised in vivo by mating with transgenic mice expressing Cre recombinase. Resulting progeny have a remaining 34-bp lox P site in the targeted allele. C, Southern blot of total genomic DNA from ES cells, digested with EcoRV, and hybridized with a 5'-probe. The WT allele generates a 7.6-kb band, and the targeted allele generates a 5.7-kb band. D, Example of genotyping of the animals with an excised neomycin cassette using primers flanking the remaining loxP site. PCR product of a targeted allele is 234 bp in size, and the PCR product of the WT allele is 200 bp in size. E, Expression of both WT and mutant ER{alpha} in the liver, as examined by RT-PCR (PCR) and Western blot (We). The PCR product was restricted with SphI. The WT band is 400 bp, and the PCR product from the mutated allele restricts into two fragments of approximately 200 bp each.

 


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Figure 1. Mutant ERs Do Not Exert a Dominant-Negative Effect on the Classical or Nonclassical Pathways

A, Transfection studies were performed in ER-deficient TSA cells, using either ERE or AP1 reporters. Cells were transfected with WT ER{alpha} or the E207A/G208A mutant. The legend in the ERE reporter panel refers to all of the panels in Fig. 1Go. B, Transfection studies were performed in TSA cells, using ERE or AP1 reporters. Cells were transfected with either WT ER{alpha} alone (WT) or with WT ER{alpha} and E207A/G208A mutant at a 1:1 ratio (WT+MUT, 1:1), or at a 1:10 ratio (WT+MUT, 1:10). Cells were treated with ethanol vehicle, 1 nM E2, or 100 nM ICI 182,780, and luciferase activity was measured.

 
Introduction of a DBD mutation raises the possibility that the mutant receptor might exert a dominant-negative effect on the WT receptor. To address this possibility, WT and mutant receptor expression vectors were cotransfected at a 1:1 or a 1:10 ratio, and the activity of the ERE and AP1 reporters was measured (Fig. 1BGo). Addition of the mutant receptor did not affect the activity of WT ER{alpha}, either in the classical or in the nonclassical pathway, indicating that the mutant does not exert an apparent dominant-negative effect in vitro.

Knock-In Mutation of ER DBD
The aforementioned mutation (E207A/G208A) was introduced into the mER{alpha} locus by targeted mutagenesis using positive-negative selection. A {lambda}-phage clone containing exon 3, which encodes the first zinc finger (23) (Fig. 2AGo), was isolated from a mouse genomic library. The targeting construct contained a neomycin cassette flanked by lox P sites approximately 300 bp upstream of the mutated exon (Fig. 2BGo). Transfection of the targeting vector into embryonic stem (ES) cells provided three correctly targeted clones, two of which were injected into blastocysts and implanted into foster mothers. Two of the male chimeras exhibited germline transmission and served as founders for the nonclassical ER knock-in (NERKI) colony. The modified allele designation is Er(tm2jlj). However, for the remainder of this manuscript, the knock-in mouse line will be referred to as NERKI. Initial screening was performed using the 5'-probe from the region of the ER{alpha} gene external to the targeting construct (Southern blot in Fig. 2CGo; positions of the probe and restriction sites shown in Fig. 2BGo). The neomycin cassette was excised by breeding the founder males to transgenic females expressing Cre recombinase under control of the cytomegalovirus promoter (Fig. 2BGo). Genotyping was performed by PCR of sequences flanking the lox P site after excision of the neomycin cassette (Fig. 2DGo). The presence of the mutated allele was confirmed by sequencing PCR products from genomic DNA. Expression of the mutated ER allele was confirmed by RT-PCR, followed by digestion of the PCR product at a mutation-specific site (SphI, Fig. 2EGo). Expression of the mutant ER allele was comparable to the WT allele (data not shown). Western blot analyses indicated that total ER{alpha} protein levels were not altered (Fig. 2EGo).

NERKI+/- Females Are Infertile
Heterozygous male NERKI+/- animals successfully produced progeny when bred to WT females. Unexpectedly, however, heterozygous female NERKI+/- mice were infertile when bred to either WT or NERKI+/- males. After 4 months, none of 14 NERKI+/- females became pregnant or gave birth. In parallel matings, their WT littermates reproduced successfully. Groups of six WT and six NERKI+/- females were superovulated and mated with WT or heterozygous males known to be fertile. All WT females had vaginal plugs and became pregnant. No plugs were seen in NERKI+/- females, indicating that they did not mate, and no pregnancies occurred. Examination of vaginal smears of NERKI+/- and WT littermates demonstrated that while WT animals cycled normally, NERKI+/- animals were in constant diestrus (data not shown).

NERKI+/- Females Have Decreased Serum Progesterone Levels
Serum gonadotropin and steroid hormone levels were determined for NERKI+/- animals and their WT littermates (Table 1AGo). LH, FSH, and E2 levels were not significantly different between the two groups. Progesterone levels were reduced in the NERKI+/- mice (P < 0.01), most likely because of abnormal follicular development and anovulation (see below).


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Table 1. Serum Hormone Levels in WT and NERKI+/- Females

 
NERKI+/- females were examined for their ability to manifest estrogen negative feedback on gonadotropin expression. WT and NERKI+/- animals were ovariectomized and treated either with placebo or with estrogen, and the serum levels of LH were compared (Table 1BGo). Ovariectomy elevated LH levels in both WT and NERKI+/- animals (the lower levels in NERKI+/- were not statistically significant). Estrogen treatment reduced LH in both WT and NERKI+/- animals to the level seen in nonovarectomized females, indicating that the presence of one WT and one mutated allele of ER{alpha} is sufficient for negative regulation of LH expression by estrogen.

Differentiation of the Mammary Gland Is Inhibited in Untreated NERKI+/- Females
Mammary gland morphology was examined in young adult WT and NERKI+/- females. Ductal elongation at the borders of the mammary fat pad of NERKI+/- females was comparable to that of WT mice, indicating that mammary development was relatively normal at this stage of development (Fig. 3AGo). However, the subsequent side-branching and differentiation of ductal buds into lobuloalveolar structures, which occur after puberty, were decreased compared with WT littermates (Fig. 3BGo). Quantitative comparison of mammary gland whole mounts demonstrated a decreased number of branching sites; the number of branching points per centimeter of duct was 91.2 ± 4.5 for WT females and 44.0 ± 1.9 for NERKI+/- females (P < 0.01). Analysis of the ducts at a higher magnification revealed that alveolar bud formation was impaired in NERKI+/- females (Fig. 3BGo). We attempted to stimulate the development of the mammary glands by implanting progesterone pellets in WT and NERKI+/- females. Although some additional glandular development was observed in NERKI+/- animals, it was much less than that observed in WT animals.



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Figure 3. Mammary Gland Development Is Inhibited in NERKI+/- Females

A, Comparison of whole mounts of mammary glands from WT and NERKI+/- females. Glands were stained with carmine alum and photographed at x6.5 magnification. B, Comparison of duct ends of mammary glands from females. Glands were stained with carmine alum and photographed at x50 magnification.

 
NERKI+/- Females Are Anovulatory, but Ovulation Can Be Induced by Hormonal Treatment
The ovaries of WT and NERKI+/- females contained follicles at all stages of development (Fig. 4AGo). However, no corpora lutea were present in NERKI+/- ovaries, indicating that the animals did not ovulate spontaneously. Mature antral follicles were present but many exhibited a cystic appearance and increased size. Defective LH signaling might explain the lack of ovulation in NERKI+/- females. However, as shown above, serum LH levels are similar in NERKI+/- and WT animals. Ovarian LH receptor mRNA levels were also unchanged in NERKI+/- females (data not shown).



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Figure 4. NERKI+/- Females Are Anovulatory, but Ovulation Can Be Induced with Hormonal Treatment

A, Comparison of WT and NERKI+/- ovary at x50 magnification, H&E staining. Note the presence of corpora lutea in WT ovaries (cl). Note the presence of large cystic follicles on the periphery and lipid vesicles in the stroma of the NERKI+/- ovary (c). B, Oil red O stain of a frozen section of WT and NERKI+/- ovary. Note the presence of staining in the stroma of the NERKI+/- ovary, which is absent in the WT ovary. C, Expression of StAR, examined by a RT-PCR and Western blot in WT and NERKI+/- ovaries. Animals were untreated. H3 indicates histone 3, serving as a loading control for the Western blot.

 
Mice lacking both ER isoforms have an ovarian phenotype distinct from that seen in the individual ER{alpha}- or ERßKO models (21, 24), suggesting a combinatorial role for the ERs in ovarian function. To test the possibility that the ER{alpha} knock-in mutation could alter ERß expression, ovarian ERß expression was measured by semiquantative RT-PCR. ERß expression was similar in WT and NERKI+/- animals (data not shown).

Treating animals with a super-physiological concentration of gonadotropins (superovulation) can sometimes bypass ovulatory defects (25). This technique was partially successful for inducing ovulation in NERKI+/- animals (data not shown). In WT animals, superovulation increased both the size of the ovary and the number of corpora lutea present. In NERKI+/- animals, superovulation also increased ovarian size, but not all mature follicles formed corpora lutea. Many of the follicles were arrested at the antral stage, and others formed hemorrhagic cysts, resembling those seen in untreated ER{alpha}KO mice (26). Some follicles were successfully released, however, as demonstrated by the presence of corpora lutea in the ovaries and ova in the oviducts (data not shown).

The ovarian stroma in NERKI+/- animals was increased, and the stromal cells contained cytoplasmic vacuoles (Fig. 4AGo, NERKI). Staining of frozen sections with Oil Red O demonstrated increased lipid in these vesicles (Fig. 4BGo). Similar lipid-filled cells have been observed in mice lacking steroidogenic acute regulatory protein (StAR) (27). Because of this similarity, levels of StAR expression were measured by RT-PCR and Western blot (Fig. 4CGo). Levels of both StAR mRNA and protein were decreased in the ovaries of untreated NERKI+/- animals (Fig. 4CGo). Quantitation of the results demonstrated that StAR expression was reduced by about 50% in the NERKI+/- ovary compared with WT. In the WT ovary, StAR is expressed at the highest levels in the corpus luteum, followed by the thecal cells (reviewed in Ref. 28), suggesting that the lack of ovulation may account for part of decreased StAR expression. No significant differences were seen in the expression of other genes involved in steroidogenesis, such as P450scc, P450c17, or aromatase (data not shown).

Uteri of NERKI+/- Mice Are Enlarged and Exhibit Cystic Endometrial Hyperplasia
NERKI+/- uteri are significantly elongated and heavier when compared with WT littermates (Fig. 5AGo, insets). The average wet uterine weight in an 8- to 12-wk-old animal was 104 ± 10 mg in WT compared with 265 ± 20 mg in NERKI+/- mice (P < 0.01). The difference in weight became even more pronounced in older animals (data not shown). Each of the structural compartments of the uterus—the myometrium, stroma, and endometrium—are present in both WT and NERKI+/- uteri. However, NERKI+/- animals have grossly enlarged endometrial glands, filled with secretory material (Fig. 5AGo), a phenotype that also progressed with age (data not shown).



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Figure 5. The Uterus of NERKI+/- Heterozygotes Is Hypersensitive to Estrogen in Untreated Animals, but Responds Normally to Hormone Replacement

A, Comparison of longitudinal sections of uteri from WT and NERKI+/- females at x50 magnification, H&E stain. Insets are photographs of a whole uterus with ovaries next to the cm scale. B, Comparison of endometrium from ovariectomized WT and NERKI+/- females treated with placebo, E2, or TMX. Pictures were taken at x200 magnification and stained with H&E. C, Comparison of longitudinal sections of uteri from WT and NERKI+/- females at x100 magnification, stained with an antibody to PR.

 
The animals were ovariectomized and treated with E2 or TMX to test the responsiveness of the uteri to exogenous hormonal treatment. Uterine weights in WT animals from the control group (placebo pellet) decreased by approximately 5-fold (average wet uterine weight was 20 ± 10 mg). In the NERKI+/- placebo-treated group, uterine weight also decreased, but only by approximately 2-fold (average wet uterine weight was 155 ± 40 mg). However, ovariectomy did not diminish the number of cysts. TMX, which is an ER antagonist in some tissues (such as the mammary gland), acts as an agonist in the uterus (29) and is known to stimulate the nonclassical ER pathway (9, 10, 11). The uteri of WT E2- and TMX-treated groups enlarged in response to treatment (average wet uterine weight was 155 ± 60 mg in the E2 group and 105 ± 20 mg in the TMX group), and exhibited hyperemia and proliferation of the columnar epithelium (Fig. 5BGo). The uteri of treated NERKI+/- animals also increased in size, but the difference in weight between the treatment and placebo groups was less pronounced than in the WT (average uterine weight was 185 ± 80 mg for the E2 group and 195 ± 90 mg for the TMX group). E2 and TMX both caused hyperemia and proliferation of the endometrial epithelium (Fig. 5BGo). The vaginal smears of both WT and NERKI+/- animals treated with E2 or TMX contained abundant epithelial cells, characteristic of estrogen-exposed animals (data not shown). The proliferative response of the endometrial epithelium and vaginal cornification indicate that the uterus and vagina of NERKI+/- animals are responsive to estrogen.

PR is one of the uterine estrogen-responsive genes that is regulated by the classical pathway (2). Its expression in intact animals was analyzed by immunohistochemistry and found to be similar in WT and NERKI+/- animals (Fig. 5CGo). When animals were ovariectomized and estrogen was replaced, PR expression increased comparably in WT and NERKI+/- animals (data not shown). These data indicate that one active ER{alpha} allele is sufficient for PR regulation in the uterus.

The Mutant ER Allele Functions in Vivo Similar to in Vitro
The activity of the classical and nonclassical pathways was examined in the uteri of NERKI+/- and WT animals. The classical pathway was assessed by measuring the expression of an ERE-regulated target gene PRL (1). Pituitary PRL levels were measured by RT-PCR using total RNA from females that were ovariectomized and treated either with placebo or estrogen (Fig. 6AGo). Basal levels of PRL were decreased in NERKI+/- females, but the degree of PRL induction by estrogen was comparable in both groups, indicating that the remaining allele of WT ER{alpha} was fully functional.



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Figure 6. Effects of the NERKI Mutation on Gene Expression in Vivo

A, Induction of PRL expression by estrogen in pituitaries of WT and NERKI+/- females. Animals were ovariectomized and treated with either placebo or E2. Levels of PRL mRNA in total RNA from the pituitary were measured by semiquantitive RT-PCR and normalized to tubulin levels (P < 0.01 for the WT and P < 0.05 for the NERKI). The ratio of PRL to tubulin is shown below the PCR results. B, Induction of IGF-I expression in uteri of WT and NERKI+/- females. Sexually immature females were treated with either placebo or E2. Levels of IGF-I mRNA in total RNA from the uterus were measured by semiquantitive RT-PCR and normalized to actin levels (P < 0.05 for the WT and P < 0.001 for the NERKI+/-). The ratio of IGF-I to actin is shown below the PCR results.

 
Regulation of nonclassical genes is more difficult to characterize because ER-mediated nonclassical effects have previously been studied only in vitro. One possible nonclassical candidate gene is IGF-I, as its expression requires ER{alpha} (30) and its promoter contains a functional AP1 site (12). The expression of IGF-I was examined in uteri of immature females treated with estrogen and compared with untreated controls (Fig. 6BGo). Estrogen up-regulated the IGF-I expression in both WT and NERKI+/- females, indicating that the nonclassical activity of the mutant was not altered.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Interpretation of the Heterozygous Phenotype
The severe phenotype observed in the NERKI+/- females was unexpected, as one intact ER{alpha} allele in the ER{alpha}KO mouse is sufficient to sustain essentially normal ER action (20). Several potential mechanisms might account for how the heterozygous NERKI mutation could exert its phenotypic effects. One possibility is that the NERKI+/- mutant exerts a dominant-negative effect on the WT receptor. We addressed this issue in cell culture studies by examining the activity of the classical and nonclassical pathways after coexpression of WT and mutant ER{alpha}. No apparent dominant-negative effect was detected in either pathway. This conclusion was supported by analysis of PRL, an ERE-regulated gene. PRL expression was slightly decreased in NERKI+/- females, consistent with partial loss-of-function of the classical pathway. Estrogen responsiveness was retained when ovariectomized animals were treated with exogenous estrogen. Although it is still possible that the mutation exerts a dominant-negative effect in certain tissues (e.g. ovary), this property does not appear to be a prominent feature of the DBD mutation.

A second possibility might involve an imbalance in the relative activities of the classical and nonclassical pathways. For example, the lack of ERE binding may selectively reduce the expression of genes in the classical pathway. In contrast, genes in the nonclassical pathway may not be altered. To the extent that some estrogen-responsive genes might control cell growth whereas others control differentiation, it is plausible that phenotypic effects might result from selective changes in the composition of expressed genes. This hypothesis can ultimately be tested by performing gene expression array studies that compare heterozygous ER{alpha}KO and NERKI gene expression in specific tissues.

A third possibility could be that the phenotype in some of the tissues (e.g. ovary) is due to an inhibitory effect of the mutant ER on another signaling pathway. This hypothesis will ultimately be tested by generating a compound heterozygote (ER{alpha} null/NERKI mice).

The NERKI+/- Phenotype Confirms Activity of the Nonclassical ER Pathway in Vivo
The ovaries of NERKI+/- animals contained cytoplasmic lipid deposits in the stromal cells; this feature is not seen in the ovaries of ER{alpha}KO mice, suggesting that this phenotype is specific to the introduced mutation. Because of the similarity to the lipid deposits seen in StAR KO mice (27), the levels of StAR were examined and found to be decreased in untreated NERKI+/- ovaries. The StAR promoter is not known to contain an ERE, suggesting that the altered expression level is not the result of defective signaling through the classical pathway. Enhancer elements important for StAR expression include an SF1 binding site and a cAMP response element (31), the latter of which has been shown to participate in cross-talk with the ER (summarized in Ref. 32). Thus, it is possible that the lipid deposits are caused by inhibition through nonclassical ER activity, but this will require further studies of the StAR promoter. Interestingly, despite the decreased StAR levels, the serum levels of estrogen in NERKI+/- are normal, similar to the situation seen in StAR KO females (27).

The importance of ER{alpha} for uterine growth is consistent with its high expression in the female reproductive tract during development, puberty, and adulthood (reviewed in Ref. 33). In the absence of ER{alpha}, the uterus is hypoplastic, resulting in a uterine weight that is only half that of WT littermates (26). The increased size and weight of the uterus in NERKI+/- females is strikingly different from the appearance of the uterus of ER{alpha}KO mice, suggesting a specific contribution of the nonclassical activity of ER{alpha} to uterine growth and development. Cross-talk between the ER and growth factors in the uterus is well established (34, 35, 36). It is known that estrogen can regulate levels of growth factors and their receptors in the uterus (37, 38, 39), possibly through the nonclassical pathway. For example, the epidermal growth factor promoter does not contain an ERE, but it has Sp1 sites that have been implicated in the regulation of epidermal growth factor expression (40). Given that impaired growth factor signaling causes uterine hypoplasia (41), and growth factor overexpression can increase uterine weight (42), altered growth factor expression could contribute to the increased uterine size in NERKI+/- animals.

Examination of IGF-I expression demonstrated that estrogen induces IGF-I in uteri of NERKI+/- animals to a similar or greater extent than in WT mice. More investigation is needed to establish the effects of the introduced mutation on growth factor expression in NERKI+/- animals. However, these initial data suggest that the mutation preserves the ER{alpha} activity in the nonclassical pathway in vivo.

Although sporadic cystic endometrial hyperplasia has been observed in WT mice aged 1 yr or older (43), NERKI+/- animals exhibited this phenotype as young adults (8–12 wk of age). The extent of hyperplasia worsens with age, as observed in the 6-month-old NERKI+/- animals, whereas the WT littermates appear normal (data not shown). Endometrial hyperplasia can also be induced by treatment with certain ER ligands, such as TMX (44), or triarylalkenes similar to TMX (45). These agents act as agonists in the nonclassical pathway (9, 10), consistent with the possibility that activation of nonclassical signaling may contribute to the endometrial hyperplasia seen in NERKI mice. Mice lacking PR also exhibit uterine hyperplasia and inflammation (46). Although the PR levels in the uterus appear to be normal, it is possible that lower levels of progesterone, or defective PR signaling, may contribute to endometrial phenotype in NERKI+/- females.

ER{alpha} is necessary for the uterine response to E2 or TMX after ovariectomy, as demonstrated by diminished hormonal responses in the ER{alpha}KO mouse (47, 48). On the other hand, a single functional ER{alpha} allele is sufficient for these responses (20). Similarly, the uteri of NERKI+/- mice are estrogen responsive, indicating that the mutation does not abolish the signaling pathways necessary for this response. This result provides additional evidence that the mutant does not exert a dominant-negative effect on estrogen signaling. Instead, it is more consistent with the provocative possibility that the phenotype results from an altered balance between the classical and the nonclassical pathways. For example, the WT allele may provide the necessary background for uterine development, as demonstrated by the active estrogen response. Subsequently, the nonclassical pathway can be fully engaged or even exaggerated due to reduced expression of genes in the classical pathway.

ER{alpha} Loss-of-Function May Contribute to the NERKI+/- Phenotype
Analysis of the ovarian phenotype reveals similarities between NERKI+/- and homozygous ER{alpha}KO females. Females of both models are anovulatory. However, ER{alpha}KO ovaries contain hemorrhagic cysts as a result of elevated gonadotropins (49). The cysts in NERKI+/- ovaries were not hemorrhagic, unless animals were treated with superphysiological levels of gonadotropins. This finding is consistent with the role of gonadotropins in the establishment of the hemorrhagic ovarian phenotype in both models.

Treatment with gonadotropins induced ovulation in NERKI+/- females. However, none of the superovulated NERKI+/- animals became pregnant when mated to fertile males (data not shown). It is likely that the endometrial hyperplasia, which has been implicated in infertility in several species (50, 51, 52), contributes to infertility in the NERKI+/- model, independent of the ovarian phenotype.

Mouse mammary glands develop through two major stages: ductal elongation, occurring during puberty, and postpubertal side-branching and alveolar development (53). Mammary glands of ER{alpha}KO mice are growth inhibited at the prepubertal stage (54). Interestingly, mammary glands of NERKI+/- animals undergo ductal elongation normally, but subsequent branching and lobuloalveolar development are arrested. As a similar phenotype was seen in animal models that had defective progesterone or PRL signaling (55, 56, 57, 58), it is possible that there is a related defect in NERKI+/- animals. The regulation of both progesterone and PRL signaling pathways is largely dependent on classical ER{alpha} activity, and an ERE has been identified in both the PR and PRL promoters (1, 2). However, PR levels are comparable in the mammary glands of WT and NERKI+/- females, and treatment of NERKI+/- females with progesterone did not induce mammary gland development. It is still possible that the mammary phenotype in NERKI+/- animals is caused by decreased activity of the classical pathway (59), but the phenotype is not a direct effect of decreased levels of progesterone.

The NERKI Model Provides an Opportunity to Examine the Molecular Basis of ER{alpha} Signaling in Vivo
The phenotype of NERKI+/- females illustrates the complexity of estrogen signaling in whole-animal physiology. It is notable that the effect of the NERKI mutant is distinctly different in various estrogen target tissues, i.e. it causes hyperplasia in the uterus and hypoplasia in the mammary gland. This apparent discrepancy resembles the effects of the selective estrogen receptor modulators, which are used for clinical therapy. For example, TMX acts as an antagonist in the breast and results in decreased growth, but it acts as an agonist in the uterus, where it induces hyperplasia (reviewed in Ref. 60). Such differences could arise from varied contributions of the classical vs. the nonclassical pathways in each estrogen target tissue. Understanding the molecular mechanisms by which the ER mediates its effects opens new possibilities for tissue-specific therapies. It will be especially interesting to study the NERKI mutation in the homozygous state. Efforts are currently underway to create homozygous NERKI animals using in vitro fertilization techniques and foster mothers to bypass the ovulatory and uterine defects.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Gene Targeting
NERKI mice were generated by homologous recombination in HM-1 embryonic stem (ES) cells (61) using dominant-negative selection. {lambda}-Phage clones containing exon 3 of mER{alpha} were isolated from the {lambda}PIXII-129SvJ genomic library (Stratagene, La Jolla, CA). A targeting vector was constructed using approximately 10 kb of genomic sequence, a thymidine kinase cassette, and a neomycin cassette flanked by loxP sites (62). The E207A/G208A mutation was introduced into exon 3 by overlapping PCR (codons GAA GGC were changed to GCT GCA). ES targeting was performed at the Children’s Memorial Institute for Education and Research (Chicago, IL). The linearized targeting construct (30 µg) was electroporated into approximately 4 x 106 HM-1 ES cells. Cells were cultured in the presence of 200 µg/ml G418 (Life Technologies, Inc., Gaithersburg, MD) and 2 µM gancyclovir (Roche Laboratories, Nutley, NJ). DNA was purified using the Puregene DNA Isolation Kit (Gentra Systems, Inc., Minneapolis, MN) according to the manufacturer’s instructions. Clones confirmed for homologous recombination were selected using Southern blot analysis. Five clones of 118 contained a targeted ER{alpha} allele. Three of those clones contained the mutation in the correct position. Those clones were karyotyped, and two of them were used to produce chimeric animals by blastocyst injection. Male chimeras were bred to WT 129SvJ females to achieve germline transmission. Resulting progeny contained the neomycin cassette flanked by loxP sites. Preliminary experiments did not reveal detectable effects of the neomycin cassette. Heterozygous males were then bred to transgenic 129SvJ females expressing Cre recombinase under the control of cytomegalovirus promoter (62). Animals were genotyped by PCR using primers flanking one remaining loxP site: sense 5'-GTGTTCTGAAATCCCACCCAGCTGC and antisense: 5'-GAAATGGACTGCATACCCCAGAACAG. Each PCR contained: 0.15 M Tris-HCl (pH 8.8), 0.1 M MgCl2, 0.1 M (NH4)2SO4, 0.1% ß-mercaptoethanol, 10% dimethylsulfoxide, 0.8 mM deoxynucleotide triphosphates, 10 pmol of each primer, and 2.5 U Taq polymerase (Promega Corp., Madison, WI). PCRs consisted of the following thermal cycles: 94 C for 4 min, followed by 35 cycles of 94 C for 1 min, 55 C for 1 min, and 72 C for 1 min 30 sec. The WT allele yields a PCR 200-bp product, and the mutant allele with the loxP site gives a 235-bp product.

Southern Blots
A 444-bp probe from the 5'-region outside of the targeting construct was prepared by PCR using a cloned genomic fragment of mER{alpha} as a template. The same probe was used for screening ES cells and for screening of the progeny of chimeric mice. Total genomic DNA purified from tail fragments was restricted with EcoRV, separated on an 0.8% agarose gel at 20 V in 0.5x Tris-borate-EDTA and DNA and transferred onto a Nytran membrane using the Turbo Blotter rapid downward transfer system (Schleicher \|[amp ]\| Schuell, Inc., Dassel, Germany) according to the manufacturer’s protocol. DNA was attached to the membrane by cross-linking. The membrane was blocked in hybridization buffer [1 M NaCl, 50 mM Tris-HCl (pH 7.5), 40% formamide, 10% dextran sulfate, 1% sodium dodecyl sulfate (SDS)] in the presence of 200 µg/ml denatured herring testes DNA at 42 C overnight. The denatured probe was added at 106 cpm/ml hybridization buffer and incubated overnight at 42 C. After incubation with the probe, the blots were washed at room temperature twice with 2x standard sodium citrate, 0.1% SDS prewarmed to 37 C, followed by one wash with 2x standard sodium citrate, 0.1% SDS prewarmed to approximately 65 C. Blots were then exposed to film.

RT-PCR
RNA was purified from tissues using TRIzol (Life Technologies, Inc.) according to the manufacturer’s protocol. RNA was subsequently treated with DNase I (Promega Corp., Madison, WI) for 15 min at 37 C followed by a 15-min incubation at 75 C. cDNA was prepared using 30 U of reverse transcriptase (Promega Corp.) per reaction in the presence of reverse transcriptase buffer (Promega Corp.), 0.8 mM deoxynucleotide triphosphates, 80 U of RNasin (Promega Corp.) and 1 µg random hexamers per reaction. The reaction mixture was incubated at 25 C for 10 min, followed by 42 C for 30 min and 99 C for 10 min. The primers used for ER{alpha} PCR were sense: 5'-AGAACGAGCCCAGCGCCTACG and antisense: 5'-GACGCTTGTGCTTCAACATTC. The PCR reaction was performed according to the previous protocol for 30–33 cycles. The PCR product was approximately 400 bp in size. The PCR product was purified using QIAGEN (Chatsworth, CA) PCR Purification kit and restricted using SphI restriction enzyme (Promega Corp.), generating two bands of approximately 200 bp. PCR primers used for semiquantitive PCR reactions were: tubulin sense, 5'-CCGGTGTCTGCTTCTATC; antisense, 5'-GAGCCGCTCCATCAGCAG; GAPDH sense, 5'-CCCTTCATTGAGACCTCAACCTA; antisense, 5'-CCAAAGTTGTCATGGATGAC; ß-actin sense, 5'-GAAGCTGTGCTATGTTGCTC; antisense, 5'-CCAGAGCAGTAATCTCCTTC; PRL sense, 5'-CTGCCAATCTGTTCCGCTGGTGAC; antisense, 5'-CCTACTGCAGTTATTAGTTGAAAC (63); IGF-I sense, 5'-CACATCTCTTCTACCTGGCG; antisense, 5'-TGAGTCTTGGGGCATGTCAGT. 32P-{alpha}-dCTP was added to the reaction, products were separated on a 6% polyacrylamide gel, and results were quantitated using a phosphor imager.

Animal Housing
Animals were housed in a barrier facility with a 14-h light,10-h dark cycle. Unless stated for the purpose of the experiments, animals of the same sex were housed five per cage. For continuous matings, one male and one female were placed in a cage. Superovulation was accomplish by ip injection of pregnant mare’s serum gonadotropin (10 IU/mouse; Sigma, St. Louis, MO) at the end of the light phase followed by ip injection of 5 IU/mouse of human chorionic gonadotropin (hCG; Sigma) 48 h later (25).

Animal Procedures
All procedures were performed according to the Master Protocol approved by the Northwestern University Animal Care and Use Committee. For tissue collection, animals were anesthetized by injection with nembutal, sodium solution (Abbott Laboratories, Abbott Park, IL; 200 µl/mouse), and blood was collected by retroorbital bleed. All results presented in this publication were obtained using females between 8 and 12 wk of age, except animals used for measuring the expression of IGF-I. For this experiment, 4-wk-old sexually immature females were used. Three animals were used for each group. At time 0, animals from the treatment group were injected with 5 µg 17ß-E2 (Sigma) per mouse. Animals were euthanized after 6 h. Ovariectomy was performed under anesthesia induced by an ip injection of 50 µl per mouse of cocktail containing 25 mg/ml ketamine (80 mg/kg; Ketaset; American Home Product Corp., Madison, NJ), 1.27 mg/ml xylazine (10 mg/kg; Rompun; Bayer Corp., Pittsburgh, PA), and 0.25 mg/ml acetopromazine (American Home Product Corp.). For ovariectomy, eight animals of each genotype were used. Animals were allowed to recover for 2 wk before implantation of pellets above the scapulae. For the implantation, animals were anesthetized using Metofane (Schering-Plough Corp., Union, NJ). Pellets contained 0.25 mg/pellet 17ß-E2 (21-d release), 0.5 mg/pellet TMX, free base (21-d release), or placebo (all pellets from Innovative Research of America, Sarasota, FL). There were four animals of each genotype in the placebo group, and two animals of each genotype in the E2 and TMX groups. Animals were euthanized 3 d after pellet implantation.

Serum Hormone Measurements
Serum E2 RIA was performed using a double antibody RIA kit according to manufacturer’s protocol (ICN Biomedicals, Inc., Costa Mesa, CA). LH and FSH RIAs were performed using antibodies and reference preparations from the National Hormone Pituitary Program (provided by Dr. A. F. Parlow). Serum progesterone was measured using an immunochemical coated tube kit (ICN Pharmaceuticals, Inc., Costa Mesa, CA). E2 levels were measured in a group of 10 NERKI+/- and 10 WT mice. Progesterone was measured in groups of 11 mice. LH and FSH were measured in groups of 19 NERKI+/- and WT mice.

Histology
Excised tissues were fixed in 4% paraformaldehyde at 4 C overnight and subsequently stored in 70% ethanol at 4 C. Tissues were embedded in paraffin at the Pathology Core Facility (Northwestern University, Chicago, IL) and sectioned at thickness 3–5 µm. Standard hematoxylin-eosin (H&E) stain was performed and cover slips mounted using ACCU.MOUNT 60 (Baxter Scientific Products, Riverdale, NJ). For PR immunohistochemistry, primary antibody sc-538 was used (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Frozen sections were stained with Oil red O according to Ref. 64 and counterstained with hematoxylin (Vector Laboratories, Inc., Burlingame, CA). Uteri and ovaries were collected from 30 animals from each group (NERKI+/- and WT). Whenever possible, comparisons were made between littermates.

Whole Mounts of Mammary Glands
Whole mounts of mammary glands were prepared as described previously (59). Glands from 15 animals from each group (NERKI+/- and WT) were collected. Glands were excised and fixed overnight on a slide at room temperature in 25% glacial acetic acid, 75% ethanol. The next morning, slides were washed with 70% ethanol for 15 min, followed by a 5-min wash in water. Slides were incubated in carmine alum solution overnight at room temperature. The following morning the tissue was rinsed with water and washed with 70% ethanol for 15 min, 95% ethanol for 15 min, 100% ethanol for 15 min, and xylene for 15 min. Coverslips were mounted using ACCU.MOUNT 60 (Baxter Scientific Products).

Cell Culture
TSA cells were cultured and transfected as described previously (22).

Western Blots
Whole protein extracts were prepared by sonicating tissues in water containing protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN), 1 mM EDTA, and 1 mM EGTA. The extracts were fractionated using 10% SDS-PAGE gels and transferred onto Hybond-P transfer membranes (Amersham Pharmacia Biotech, Piscataway, NJ). Antibody D-12 was used to detect ER (Santa Cruz Biotechnology, Inc.). StAR antibody was a gift from Dr. J. F. Strauss III (University of Pennsylvania Medical Center, Philadelphia, PA). Proteins were visualized using an ECL+Plus kit (Amersham Pharmacia Biotech) according to the manufacturer’s protocols.


    ACKNOWLEDGMENTS
 
We are grateful to Drs. Teresa Woodruff, Patricia Keh, and Michael Pins for assistance with histology. We would like to thank Jeffrey Weiss, Suresh Pillai, Richard Yu, Rachel Duan, Barry D. Gehm, and John Achermann for helpful discussions and advice. We also thank Tom Kotlar, Joshua Meeks, Jennifer Cheng, and Henry Pitzele for assistance with manuscript preparation. We appreciate excellent technical help from Lisa Hurley and Ted Russell.


    FOOTNOTES
 
This work was supported in part by NIH SPORE Grant P50 CA-89018 P50.

Abbreviations: AP1, Activator protein 1; DBD, DNA binding domain; E2, Estradiol; ER, estrogen receptor; ERE, estrogen response element; ES, embryonic stem; GR, glucocorticoid receptor; H&E, hematoxylin-eosin; KO, knockout; mER{alpha}, mouse ER{alpha}; NERKI, nonclassical ER knock-in; PR, progesterone receptor; PRL, prolactin; SDS, sodium dodecyl sulfate; StAR, steroidogenic acute regulatory protein; TMX, tamoxifen; WT, wild-type.

Received for publication August 8, 2001. Accepted for publication July 11, 2002.


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