Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2007 Mar 27;104(13):5455-60.
doi: 10.1073/pnas.0611647104. Epub 2007 Mar 16.

Amphiregulin is an essential mediator of estrogen receptor alpha function in mammary gland development

Laura Ciarloni  1 , Sonia Mallepell, Cathrin Brisken
Affiliations

Amphiregulin is an essential mediator of estrogen receptor alpha function in mammary gland development

Laura Ciarloni et al. Proc Natl Acad Sci U S A. .

Abstract

Most mammary gland development occurs after birth under the control of systemic hormones. Estrogens induce mammary epithelial cell proliferation during puberty via epithelial estrogen receptor alpha (ERalpha) by a paracrine mechanism. Epidermal growth factor receptor (EGFR) signaling has long been implicated downstream of ERalpha signaling, and several EGFR ligands have been described as estrogen-target genes in tumor cell lines. Here, we show that amphiregulin is the unique EGF family member to be transcriptionally induced by estrogen in the mammary glands of puberal mice at a time of exponential expansion of the ductal system. In fact, we find that estrogens induce amphiregulin through the ERalpha and require amphiregulin to induce proliferation of the mammary epithelium. Like ERalpha, amphiregulin is required in the epithelium of puberal mice for epithelial proliferation, terminal end buds formation, and ductal elongation. Subsequent stages, such as side-branching and alveologenesis, are not affected. When amphiregulin(-/-) mammary epithelial cells are in close vicinity to wild-type cells, they proliferate and contribute to all cell compartments of the ductal outgrowth. Thus, amphiregulin is an important paracrine mediator of estrogen function specifically required for puberty-induced ductal elongation, but not for any earlier or later developmental stages.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Regulation of EGFR ligands' expression by estrogens. Quantitative RT-PCR analysis of mammary gland mRNA for amphiregulin and other EGFR ligands normalized to keratin 18. (A and B) Mice ovariectomized before puberty were injected with either vehicle (open bars) or 17-β-estradiol (filled bars) and analyzed either for EGFR ligand expression 8 h later (A) or for amphiregulin over 48 h (B). (C and D) Mammary glands of 14- to 28-day-old mice were analyzed for amphiregulin expression (C) or for EGFR ligand expression (14- and 21-day-old females, open and filled bars, respectively) (D). Bars report the mean values obtained from three different mice. Error bars indicate standard deviation. Relative increase refers to control treated (A and B) or to the 14-day-old mice (C and D). (E) RT-PCR analysis of amphiregulin and keratin 18 expression in glands from ovariectomized WT and ERα−/− mice 6 h after administration of 17-β-estradiol (+) or vehicle (−).
Fig. 2.
Fig. 2.
Developmental analysis of amphiregulin−/− mammary glands. (A–F) Whole-mount micrographs of inguinal glands from amphiregulin−/− (A, C, and E) and WT (B, D, and F) females were analyzed at the following developmental stages: day 24 (A and B), 6 weeks (C and D), 3 months (E and F). (Scale bars: A–F, 5 mm; A′–F′, 1 mm.) Micrographs are representative of glands from 12 mice analyzed per time point. (G) Number of branching points in inguinal mammary glands of 24-day-old amphiregulin−/− (filled bar, n = 6) and WT (open bar, n = 7) females. (H and I) Histological sections of mammary glands from 24-day-old amphiregulin−/− and WT mice stained by immunohistochemistry with an anti-SMAα antibody to highlight the myoepithelial cells (brown) counterstained with hematoxylin. H&E (J and K) staining of paraffin sections from 6-week-old amphiregulin−/− (J) and WT (K) glands. (Scale bar: 50 μm.) (L and M) Whole-mount micrographs of amphiregulin−/− (L) and WT (M) glands at 16.5 days of pregnancy. (N and O) Histological sections of amphiregulin−/− (N) and WT (O) mammary glands at 18.5 days of pregnancy. (P) Whole-mount micrograph of gland from an amphiregulin−/− female after 12 pregnancies.
Fig. 3.
Fig. 3.
Estrogen-induced proliferation in amphiregulin−/− and WT mammary glands. Immunohistochemistry was performed on sections of amphiregulin−/− (A and C) or WT (B and D) glands either from 3-week-old ovariectomized females 48 h after 17-β-estradiol stimulation (A and B) or 26-day-old mice (C and D) with an anti-BrdU antibody. Positively stained lymph nodes from the same glands are shown as positive control for BrdU incorporation (A and C Insets). (Scale bars: 50 μm.)
Fig. 4.
Fig. 4.
Amphiregulin−/− epithelial transplants. Representative outgrowth of amphiregulin−/− (A) or WT (B) GFP+ mammary epithelium transplanted into WT cleared fat pads. The glands were derived from virgin recipients and observed directly under the fluorescence stereomicroscope (B). (Scale bar: 5 mm.)
Fig. 5.
Fig. 5.
Amphiregulin−/− and WT chimeric mammary epithelia. Whole-mount preparations of a cleared fat pad showing representative mammary reconstitution on injection with a mixture of amphiregulin−/− GFP+ (green), and WT ROSA26+ (blue) epithelial cells in a 1:1 ratio. (A and B) GFP was visualized directly under the fluorescence stereomicroscope (A); β-galactosidase activity revealed after X-Gal staining (B). (C and D) Histological sections of glands reconstituted with amphiregulin−/− LacZ+ and WT cells. Amphiregulin−/− LacZ+ cells (blue) were found both among the body (C, arrow) and the cap cells (D, arrow). (E and F) Glands reconstituted with amphiregulin−/− GFP+ and WT cells were sectioned and stained with anti-GFP antibody. Both luminal (F, arrow) and myoepithelial cells (E, arrow) show GFP staining (brown). (G) Glands reconstituted with amphiregulin−/− GFP+ and WT cells were sectioned and costained with anti-BrdU (Left), and anti-GFP antibodies (Center) merge (Right) reveals double-positive cells. (Scale bars: A and B, 500 μm; C–G, 50 μm.)
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Lyons WR, Li CH, Johnson RE. Recent Prog Horm Res. 1958;14:219–248. - PubMed
    1. Nandi S. J Natl Cancer Inst. 1958;21:1039–1063. - PubMed
    1. Hinck L, Silberstein GB. Breast Cancer Res. 2005;7:245–251. - PMC - PubMed
    1. Shyamala G. J Mammary Gland Biol Neoplasia. 1999;4:89–104. - PubMed
    1. Brisken C. J Mammary Gland Biol Neoplasia. 2002;7:39–48. - PubMed

Publication types

MeSH terms