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. 2010 Dec;24(12):4660-7.
doi: 10.1096/fj.10-163428. Epub 2010 Jul 28.

Biological and biochemical consequences of global deletion of exon 3 from the ER alpha gene

Sylvia C Hewitt  1 , Grace E Kissling, Karen E Fieselman, Friederike L Jayes, Kevin E Gerrish, Kenneth S Korach
Affiliations

Biological and biochemical consequences of global deletion of exon 3 from the ER alpha gene

Sylvia C Hewitt et al. FASEB J. 2010 Dec.

Abstract

To address issues resulting from α estrogen receptor-knockout (αERKO) residual N-terminal truncated estrogen receptor α, and to allow tissue-selective deletion of ERα, we generated loxP-flanked exon 3 mice. Initial characterization of global sox2 cre-derived exon 3-deleted Ex3αERKO mice indicated no ERα protein in uterine tissue and recapitulation of previously described female phenotypes, confirming successful ablation of ERα. Body weights of Ex3αERKO female mice were 1.4-fold higher than wild-tupe (WT) females and comparable to WT males. Microarray indicated the Ex3αERKO uterus is free of residual estrogen responses. RT-PCR showed Nr4a1 is increased 41-fold by estrogen in WT and 7.4-fold in αERKO, and not increased in Ex3αERKO. Nr4a1, Cdkn1a, and c-fos transcripts were evaluated in WT and Ex3αERKO mice following estrogen, IGF1, or EGF injections. All 3 were increased by all treatments in WT. None were increased by estrogen in Ex3αERKO. Nr4a1 increased 24.5- and 14.7-fold, Cdkn1a increased 14.2- and 12.3-fold, and c-fos increased 20.9-fold and 16.2-fold after IGF1 and EGF treatments, respectively, in the Ex3αERKO mice, confirming that growth factor regulation is independent of ERα. Our Ex3α ERα model will be useful in studies of complete or selective ablation of ERα in target tissues.

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Figures

Figure 1.
Figure 1.
A) RT-PCR of ERα transcripts. Top panel: cDNA made from WT (lanes 1–3) or Ex3αERKO (lanes 4–6) uterine RNA was amplified using primers that spanned exon 4 to exon 8, yielding an 850-bp amplimer. Arrows show 1000- and 500-bp markers. Bottom panel: PCR from primers spanning eons 2 to 5 yielded a 560-bp amplimer in WT (lanes 1–3), and a smaller amplimer in Ex3αERKO (lanes 4–6). Arrows show 600- and 300-bp markers. B) ERα Western blot of 10 μg total uterine protein from WT (lanes 1 and 2) or Ex3αERKO (lanes 3 and 4) uteruses from a 1-min exposure (top panel) or a 10-min overexposure (bottom panel). ERα antibody sc 542 recognizes N-terminal epitope. Arrowheads show 64- and 51-kDa markers. C) ERα immunodetection in sections of WT (top and middle) or Ex3αERKO (bottom) intact uteruses. D) Ex3αERKO ovary showing hemorrhagic cystic pathology. E) Mammary gland whole mounts showing fully developed epithelial structure in adult (>10 wk) WT and rudimentary ductal structure in adult (>10 wk) Ex3αERKO (arrow) ovariectomized mice. Scale bars = 0.2 μM (C, top panel); 0.1 μM (C, middle and bottom panels); 0.5 μM (D).
Figure 2.
Figure 2.
A) Ki67 immunodetection in sections of WT (top panels) or Ex3αERKO (bottom panels) uteruses that were ovariectomized and treated for 24 h with saline (V), E2, EGF, or IGF1. B) BrdU incorporation in sections of WT (top panels) or Ex3αERKO (bottom panels) uteruses that were ovariectomized and treated for 24 h with E2, EGF, or IGF1 and for 2 h with BrdU. C) RT-PCR of ERα-independent growth factor-responsive transcripts in WT or Ex3αERKO uterine samples injected for 2 h with saline vehicle (V), E2, EGF, or IGF1 at doses described in Materials and Methods. Data were tested by 2-way ANOVA t test with a Bonferroni correction to determine significance relative to the V level. *P < 0.05. Scale bars = 0.04 μM (A); 0.10 μM (B).
Figure 3.
Figure 3.
Growth curves. Body weights were recorded weekly and compared on plots of WT vs. Ex3αERO males (A) or females (B). Males and females were also compared in plots (C, WT; D, Ex3αERKO). Ex3αERKO females resemble males in their growth pattern. Total body fat content (E) and bone mineral density (BMD; F) of adult (84–112 d old) WT and Ex3αERKO females was evaluated by Piximus imaging and compared. A) WT (n=11) and Ex3αERKO (n=14) males. B) WT (n=9) and Ex3αERKO (n=14) females. C) WT males and females. D) Ex3αERKO males and females. E, F) Fat content (E) and BMD (F) of WT (n=13) and Ex3αERKO (n=10) females.
Figure 4.
Figure 4.
Microarray analysis of uterine samples. A) Two-dimensional (2-D) cluster comparing individual uterine samples from WT or Ex3αERKO animals treated with saline vehicle (V) or for 2 or 24 h with E2. The 2-D cluster was generated using Rosetta Resolver software. It compares Z scores derived from the intensity values of individual uterine samples from WT or Ex3αERKO animals treated with saline vehicle or for 2 or 24 h with E2. B) Principal component analysis (PCA) of gene expression from triplicate WT or Ex3αERKO uterine RNA after treatments with saline vehicle, or E2 for 2 or 24 h. PCA separated the samples into 3 groups: WT V and Ex3αERKO, WT E2 2 h, and WT E2 24 h. The 2-D cluster (A) and PCA (B) were generated using Rosetta Resolver software. They compare Z scores derived from the intensity values of individual uterine samples from WT or Ex3αERKO animals. C) Two-D cluster comparing E2 2 h to vehicle treatments in ratio experiments. A total of 573 transcripts were selected from a previous data set (9) because they exhibited estrogen regulation in αERKO uteruses. These 573 transcripts were then evaluated in the WT and Ex3αERKO dataset from panel A. WT V vs. WT E2 2 h exp 1and αERKO V vs. αERKO E2 2 h show ratios of the transcript intensity of estrogen-treated to vehicle in the previous 2-color array experiment. WT V vs. WT E2 2 h exp 2 and Ex3αERKO V vs. Ex3αERKO E2 2 h show built ratios from the dataset in panel A. Red and green color indicate increased and decreased signal, respectively, as a result of estrogen; intensity signifies the fold regulation.
Figure 5.
Figure 5.
RT PCR of residual ERα E1-mediated responses. RT PCR was used to compare responses of several transcripts that have residual response to E2 in the older αERKO model uterus. For each group, n = 3 animals. Cysteine-rich protein 61 (Cyr61), FBJ osteosarcoma oncogene (c-Fos), nuclear receptor subfamily 4, group A, member 1 (Nr4a1or Nur77), and ERBB receptor feedback inhibitor 1 (Errfi1 or Mig6) lack any response in Ex3αERKO (left panels) but show residual response in the αERKO (right panels). *P < 0.05, **P < 0.01, ***P < 0.001 vs. saline vehicle treatment.
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