Estrogen receptors ER alpha and ER beta in proliferation in the rodent mammary gland - PubMed (original) (raw)

Estrogen receptors ER alpha and ER beta in proliferation in the rodent mammary gland

Guojun Cheng et al. Proc Natl Acad Sci U S A. 2004.

Erratum in

Abstract

Most evidence supports the view that ER alpha is responsible for estrogen (ovarian estradiol, E(2))-induced proliferation in the epithelial cells of the mammary gland, but despite this, proliferating epithelial cells do not express ER alpha. We have examined this apparent paradox by studying the role of ER alpha and ER beta in E(2)-induced proliferation in mammary glands (measured by BrdUrd incorporation into DNA) in mice with intact ER beta (WT mice) and those in which the ER beta gene has been inactivated (ER beta(-/-) mice). On treatment of ER beta(-/-) mice with E(2) or ovariectomized WT mice with E(2), tamoxifen, or a specific ER beta agonist (BAG), the number of BrdUrd-labeled cells in mammary glands increased from 3.4% in controls to 28-38% in the treated mice. This indicates that both ER alpha and ER beta can mediate E(2)-induced proliferation independently of each other. With specific antibodies, ER beta was found in both epithelial and stromal cells, whereas ER alpha was strictly epithelial. Within 4 h of a single dose of E(2), ER alpha was lost from the nuclei of epithelial cells. In WT mice, ER alpha reappeared by 24 h, but in ER beta(-/-) mice, return to the nucleus was delayed by 24 h. At 4 h after E(2), neither ER alpha nor progesterone receptor was detectable in BrdUrd-labeled nuclei but by 48 h after E(2), 29% of the BrdUrd-labeled cells expressed ER alpha, and 21-38% expressed progesterone receptor. During 3 weeks of continuous E(2) treatment, ER beta remained in the nucleus, but there was no detectable ER alpha. With tamoxifen treatment, ER alpha remained in the nucleus, but ER beta was lost. From these results, we conclude that ER alpha receives the proliferation signal from E(2), initiates DNA synthesis, and is then lost from cells. The subsequent steps in proliferation can proceed in the absence of either ER alpha or ER beta. ER beta facilitates the return of ER alpha to the nucleus and restores responsiveness to E(2). By down-regulating ER beta, tamoxifen may prolong refractoriness to E(2) in mammary epithelium.

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Figures

Fig. 1.

Fig. 1.

Proliferation in response to E2, tamoxifen, or BAG. (A) Four-month-old ovariectomized WT mice were treated with E2 (20 μg/kg), tamoxifen (Tam) (0.4 mg/kg), or BAG (1 mg/kg). BrdUrd was injected for 48 h before death. The BrdUrd labeling (brown) indicates cells whose DNA was synthesized during the treatment. (B) In the mammary gland, 38%, 28%, and 32% of the epithelial cells were labeled with BrdUrd in mice receiving E2, BAG, or Tam treatment, respectively, but only 3.4% in mice receiving vehicle (Con). In the uterus, BrdUrd-labeled epithelial, stromal, and myometrial cells were seen in vehicle-treated mice (Con). In mice receiving E2 or Tam, there was a striking increase in the number of BrdUrd-labeled epithelial cells, whereas no significant changes in BrdUrd-labeled cells occurred in mice receiving BAG. The proliferation amount in mammary glands of mice receiving E2, tamoxifen, or BAG was significantly higher than that in the control group (**, P < 0.01).

Fig. 2.

Fig. 2.

ERα and PR expression in mice receiving E2 or tamoxifen treatment for 3 weeks. (A) Ovariectomized adult WT or ERβ-/- mice received a pump releasing estradiol or tamoxifen (Tam) for 3 weeks. ERα and PR were expressed in most of the epithelial cells in the vehicle-treated mice (Con). In neither WT nor ERβ-/- mice was any nuclear ERα staining found after E2 treatment, whereas some cells showed cytoplasmic staining. There was still nuclear ERα staining in mice receiving tamoxifen. Nuclear PR staining was found in both WT and ERβ-/- mice receiving either E2 or tamoxifen. ERβ expression was detected by immunofluorescence. (B) More than 90% epithelial cells and 40% stromal cells in vehicle-treated WT mice expressed ERβ. This pattern was not changed on E2 treatment. In tamoxifen-treated mice, clear down-regulation of nuclear ERβ expression was detected. Some epithelial cells showed cytoplasmic staining of ERβ. In ERβ-/- mice, no ERβ staining was found. The down-regulation of ERα by E2 treatment, but not by tamoxifen, was confirmed by Western blot. (C) ERβ was expressed in WT mice and was up-regulated by E2, but not by tamoxifen treatment. No ERβ was detected in ERβ-/- mice.

Fig. 3.

Fig. 3.

Expression of ERα, PR, and cyclin D1 in mice receiving a single dose of E2. Mammary gland tissues were collected at different times from ovariectomized adult WT or ERβ-/- mice that had received a single dose of estradiol (20 μg/kg). In both WT and ERβ-/- mice, ERα expression was down-regulated at 4, 8, and 24 h postinjection, whereas nuclear ERα staining reappeared at 48 h. There were no significant changes in PR expression in either WT or ERβ-/- mice. An increase in the cyclin D1-positive cell population was evident after E2 treatment in both WT and ERβ-/- mice.

Fig. 4.

Fig. 4.

ERα and ERβ expression was detected by Western blotting in WT mice at various times after a single dose of E2. ERα and ERβ were expressed in the control group. There was no detectable ERα band at 4 and 8 h after E2 treatment, but it reappeared at 24 h and was the same as control at 48 and 72 h after E2 treatment. ERβ was up-regulated after E2 treatment.

Fig. 5.

Fig. 5.

Colocalization of BrdUrd with ERα or PR in mice receiving E2 treatment. In both WT and ERβ-/- mice 8 h after a single dose of E2 and 2 h after BrdUrd (100 mg/kg i.p.), there was no ERα, or PR (red) was colocalized with BrdUrd (green) in the mammary gland. However, 48 h after treatment, 29% BrdUrd-labeled cells expressed nuclear ERα in WT mice (yellow indicates colocalization). In the ERβ-/- mice, ERα remained in the cytoplasm at 48 h, and there was no nuclear staining. Twenty-one percent of BrdUrd-labeled cells in WT mice and 38% in ERβ-/- mice expressed PR, but most of the PR-expressing cells were not labeled with BrdUrd.

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