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. 2008 Jun;149(6):2970-9.
doi: 10.1210/en.2007-1526. Epub 2008 Mar 13.

An estrogen receptor-alpha knock-in mutation provides evidence of ligand-independent signaling and allows modulation of ligand-induced pathways in vivo

Kerstin W Sinkevicius  1 , Joanna E Burdette, Karolina Woloszyn, Sylvia C Hewitt, Katherine Hamilton, Sonia L Sugg, Karla A Temple, Fredric E Wondisford, Kenneth S Korach, Teresa K Woodruff, Geoffrey L Greene
Affiliations

An estrogen receptor-alpha knock-in mutation provides evidence of ligand-independent signaling and allows modulation of ligand-induced pathways in vivo

Kerstin W Sinkevicius et al. Endocrinology. 2008 Jun.

Abstract

Estrogen-nonresponsive estrogen receptor-alpha (ERalpha) knock-in (ENERKI) mice were generated to distinguish between ligand-induced and ligand-independent ER-alpha actions in vivo. These mice have a mutation [glycine 525 to leucine (G525L)] in the ligand-binding domain of ERalpha, which significantly reduces ERalpha interaction with and response to endogenous estrogens, whereas not affecting growth factor activation of ligand-independent pathways. ENERKI mice had hypoplastic uterine tissues and rudimentary mammary gland ductal trees. Females were infertile due to anovulation, and their ovaries contained hemorrhagic cystic follicles because of chronically elevated levels of LH. The ENERKI phenotype confirmed that ligand-induced activation of ERalpha is crucial in the female reproductive tract and mammary gland development. Growth factor treatments induced uterine epithelial proliferation in ovariectomized ENERKI females, directly demonstrating that ERalpha ligand-independent pathways were active. In addition, the synthetic ERalpha selective agonist propyl pyrazole triol (PPT) and ER agonist diethylstilbestrol (DES) were still able to activate ligand-induced G525L ERalpha pathways in vitro. PPT treatments initiated at puberty stimulated ENERKI uterine development, whereas neonatal treatments were needed to restore mammary gland ductal elongation, indicating that neonatal ligand-induced ERalpha activation may prime mammary ducts to become more responsive to estrogens in adult tissues. This is a useful model for in vivo evaluation of ligand-induced ERalpha pathways and temporal patterns of response. DES did not stimulate an ENERKI uterotrophic response. Because ERbeta may modulate ERalpha activation and have an antiproliferative function in the uterus, we hypothesize that ENERKI animals were particularly sensitive to DES-induced inhibition of ERalpha due to up-regulated uterine ERbeta levels.

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Figures

Figure 1
Figure 1
Ligand-induced transcriptional activity of ERα and G525L ERα. A, Ishikawa cells were transfected with mouse ERα or G525L ERα and 3xERE-luc plasmids, and treated with E2, DES, or PPT (top panel). For E2 treatments, G525L ERα transcriptional activity is significantly different from ERα activity (P < 0.001). Western blots confirmed that there were equal levels of ERα and G525L ERα in these experiments, indicating that the G525L mutation does not affect ERα protein stability (bottom panel). B, A dose curve shows that G525L ERα transcription was only stimulated by low concentrations of DES or PPT, and only a very high concentration of E2.
Figure 2
Figure 2
Targeting strategy for G525L ERα knock-in mutation. A, Schematic illustration of the targeting strategy used to introduce the mutation. Diagrams show the WT ERα locus, targeting construct, ES-targeted mutant allele, and F1 mutant allele after ACN cassette self-excision. The targeting construct contained ERα exons 8 (gray box) and 9 (white box), the G525L mutation (black bar in exon 9), an extra XbaI site, and an 18 bp 6xHis-tag epitope. The ACN cassette was flanked at the 5′ and 3′ ends by loxP sites (black arrowheads). Restriction enzyme sites shown include XmnI (Xm), XhoI (Xh), HindIII (H), and XbaI (Xb). B, Screening for target insertion by homologous recombination. ES cell DNA was digested with XmnI and Southern blotting was performed with a 5′ external probe (black rectangle in panel A), resulting in WT and ES allele products of 7.3 and 9.8 kb, respectively (left panel). PCR amplification with primers in the targeting construct and external to the 3′ end of the targeting construct (black arrowheads in A) resulted in WT and ES allele products of 3.5 and 7.0 kb, respectively (right panel). There was less 7.5-kb ES mutant targeted product than 3.8-kb WT product because it is very difficult to amplify long genomic sequences. The integrity of both products was confirmed by sequencing. C, DNA isolated from tail snips was analyzed by PCR using primers surrounding the remaining loxP site in the F1 mutant allele (black arrows in Fig. 2A). The WT and F1 mutant allele generate a 497- and 544-bp PCR product, respectively. HET, Heterozygous. D, Uterine ERα levels from 12-wk-old representative WT and ENERKI (EN) mice. Uterine extracts were analyzed by Western blotting using ERα or actin antibodies. Both full-length ERα (66 kDa) and a splicing variant (46 kDa) lacking the first 173 amino acids were present (14). ERα levels were equal among all genotypes.
Figure 3
Figure 3
Reproductive tract histology of 12-wk-old representative mice. Uterine (top) and ovarian (bottom) tissue H&E staining from WT (left) and ENERKI (right) mice. WT uteri (A) developed normally, but ENERKI tissues (B) did not exhibit an increase in uterine wet weight, and were immature and hypoplastic. Insets depict reproductive tract morphology. WT ovaries (C) contained many corpora lutea and healthy follicles, whereas ENERKI ovaries (D) had no corpora lutea (CL), and the majority contained large hemorrhagic cysts. Bars, 100 μm.
Figure 4
Figure 4
Mammary gland whole mounts from 6-wk-old representative mice. WT (left) ductal trees extended to the lymph node (white asterisk) and had enlarged terminal end buds (white arrowhead), whereas ENERKI (right) mammary glands only had a rudimentary epithelial ductal tree (white arrow). Bars, 400 μm.
Figure 5
Figure 5
Uterine Ki67 immunohistochemistry after vehicle, E2, or IGF-I treatments of representative mice. Uterine tissues were removed 16–24 h after vehicle, E2, or IGF-I treatments in 12-wk-old ovariectomized WT (left) or ENERKI (right) females and analyzed for proliferation via Ki67 immunohistochemistry. WT and ENERKI animals treated with vehicle (A and B) had no uterine Ki67 staining. WT, but not ENERKI, animals treated with E2 (C and D) exhibited uterine glandular and luminal epithelial Ki67 staining. Both WT and ENERKI animals treated with IGF-I (E and F) exhibited epithelial uterine Ki67 staining. WT proliferation was robust (E), whereas ENERKI proliferation was patchy, with a marginal response in some regions (black arrow) and a rare strong response in a few areas (black arrowhead) (F). Bars, 100 μm.
Figure 6
Figure 6
Uterotrophic activity of E2 and PPT. A and B, Immature WT or ENERKI female mice were sc injected with the indicated doses of vehicle, E2 (A), or PPT (B) for 3 consecutive days. Uterine wet weight was measured on the fourth day. Values represent mean ± sem with three to five animals per group. WT values are significantly different from ENERKI values at all E2 concentrations (P < 0.001) and at 1000–100,000 μg/kg PPT (P < 0.04). C, Adult ovariectomized WT or αERKO female mice were ip injected with vehicle or 10,000 μg/kg PPT. Uterine wet weight was measured 24 h later. Values represent mean ± sem with three to five animals per group. Vehicle and PPT WT values are significantly different (P < 0.01).
Figure 7
Figure 7
Uterotrophic activity of DES. A and B, Immature WT or ENERKI female mice were sc injected with the indicated doses of vehicle, DES (A), or PPT and DPN (B) for 3 consecutive days. Uterine wet weight was measured on the fourth day. Values represent mean ± sem with four to five animals per group. WT values are significantly different from ENERKI values at all DES concentrations (P < 0.03). The WT 10,000 μg/kg PPT value is significantly different from both the 10,000 μg/kg PPT and 10,000 μg/kg DPN, and 10,000 μg/kg PPT and 30,000 μg/kg DPN values (P < 0.02). The ENERKI 10,000 μg/kg PPT value is significantly different from the 10,000 μg/kg PPT and 30,000 μg/kg DPN value (P < 0.01). C, Relative expression levels of WT and ENERKI uterine ERβ transcript levels. ERβ expression levels were normalized to RPL13A expression, and the relative expression was determined by normalizing to the WT control. The reported results represent the average ± sem of triplicate samples. G525L ERβ transcript levels are significantly different from WT levels (P < 0.01).
Figure 8
Figure 8
Effect of long-term PPT treatments on ENERKI uterine and mammary gland tissues. Groups of four to five ENERKI females were injected sc with vehicle from 4 d of age (A and B), 10,000 μg/kg PPT from 3.5 wk of age (C and D), or 10,000 μg/kg PPT from 4 d of age (E and F) every fourth day until 8 wk of age. Uterine tissue H&E staining (A, C, and E) and mammary gland whole mounts (B, D, and F) were then performed. Bars, 100 μm (A, C, and E) and 400 μm (B, D, and F). Although uterine growth was stimulated with either PPT treatment schedule (C and E), mammary gland ductal development was only stimulated with neonatal treatments (F). In ENERKI animals treated with 10,000 μg/kg PPT from 4 d to 8 wk of age, mammary ductal trees extended past the lymph node (white asterisk) and had enlarged terminal end buds (white arrowhead) (F). Animals treated with vehicle or with PPT from 3.5 wk had a rudimentary ductal tree (white arrow) (B and D).
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References

    1. Klinge CM 2000 Estrogen receptor interaction with co-activators and co-repressors. Steroids 65:227–251 - PubMed
    1. Manolagas SC, Kousteni S 2001 Perspective: nonreproductive sites of action of reproductive hormones. Endocrinology 142:2200–2204 - PubMed
    1. Coleman KM, Smith CL 2001 Intracellular signaling pathways: nongenomic actions of estrogens and ligand-independent activation of estrogen receptors. Front Biosci 6:D1379–D1391 - PubMed
    1. Hewitt SC, Kenneth KS 2002 Estrogen receptors: structure, mechanisms and function. Rev Endocr Metab Disord 3:193–200 - PubMed
    1. Driggers PH, Segars JH 2002 Estrogen action and cytoplasmic signaling pathways. Part II: the role of growth factors and phosphorylation in estrogen signaling. Trends Endocrinol Metab 13:422–427 - PMC - PubMed

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