Repressor of estrogen receptor activity (REA) is essential for mammary gland morphogenesis and functional activities: studies in conditional knockout mice - PubMed (original) (raw)

Repressor of estrogen receptor activity (REA) is essential for mammary gland morphogenesis and functional activities: studies in conditional knockout mice

Sunghee Park et al. Endocrinology. 2011 Nov.

Abstract

Estrogen receptor (ER) is a key regulator of mammary gland development and is also implicated in breast tumorigenesis. Because ER-mediated activities depend critically on coregulator partner proteins, we have investigated the consequences of reduction or loss of function of the coregulator repressor of ER activity (REA) by conditionally deleting one allele or both alleles of the REA gene at different stages of mammary gland development. Notably, we find that heterozygosity and nullizygosity for REA result in very different mammary phenotypes and that REA has essential roles in the distinct morphogenesis and functions of the mammary gland at different stages of development, pregnancy, and lactation. During puberty, mice homozygous null for REA in the mammary gland (REAf/f PRcre/+) showed severely impaired mammary ductal elongation and morphogenesis, whereas mice heterozygous for REA (REAf/+ PRcre/+) displayed accelerated mammary ductal elongation, increased numbers of terminal end buds, and up-regulation of amphiregulin, the major paracrine mediator of estrogen-induced ductal morphogenesis. During pregnancy and lactation, mice with homozygous REA gene deletion in mammary epithelium (REAf/f whey acidic protein-Cre) showed a loss of lobuloalveolar structures and increased apoptosis of mammary alveolar epithelium, leading to impaired milk production and significant reduction in growth of their offspring, whereas body weights of the offspring nursed by females heterozygous for REA were slightly greater than those of control mice. Our findings reveal that REA is essential for mammary gland development and has a gene dosage-dependent role in the regulation of stage-specific physiological functions of the mammary gland.

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Figures

Fig. 1.

Design of the conditional targeting vector and strategy for disruption of the REA gene using PR-Cre. A, The targeting vector, described in more detail in the text, contains positive (NEO) and negative [thymidine kinase (TK)] selection markers, two frt sites that flank a neomycin resistant gene cassette (indicated as ovals), and two _lox_P sequences (triangles). The REAflox-frt-neo allele was created by homologous recombination in embryonic stem cells, and the REAflox/+ allele was derived from a REAflox-frt-neo allele through in vivo Flippase recombination enzyme (FLP)-mediated recombination. Finally, the PR-Cre mice were used to generate the conditional deletion of the REA gene by Cre-mediated excision in PR expressing cell lineages. B, Genomic DNA isolated from the indicated tissues was genotyped by PCR.

Fig. 2.

ERα, PR, and REA expression in the developing mammary gland and at L14. Immunohistochemical detection of ERα, PR, and REA in TEB and ducts of the mammary glands of virgin female mice at 6 wk of age and in alveoli of the mammary glands of L14 mice. Scale bars, 200 μm.

Fig. 3.

Mammary ductal outgrowth is accelerated in mice heterozygous for REA but severely impaired in mice homozygous null for REA. Mice heterozygous for REA show hyperstimulation of amphiregulin upon E2 treatment. A, Whole-mount staining of the number four inguinal mammary glands from REA_f/f_, REA_f_/+ PR_Cre_/+, and REA_f/f_ PR_Cre_/+ virgin mice at 6, 8, and 15 wk of age. Scale bars, 2 mm. B, Ductal length of the mammary glands at 6 wk of age. Distance from the lymph node to the end of the longest extended duct was measured. The horizontal line in box plots represents the median length from eight mammary glands per group. C, The number of TEB per mammary gland was counted in whole mounts of number four inguinal glands (n = 8 mammary glands per group) at 6 wk of age. D, REA_f/f_ and REA_f_/+ PR_Cre_/+ mice at 6 wk of age were ovariectomized, allowed to rest for 2 wk, and were then injected with oil vehicle (Veh) or E2 (0.05 μg/g of body weight/d) for 5 d. Amphiregulin mRNA was measured by qRT-PCR. n = 7 mice per group, mean ±

; #, P < 0.05 vs. wild type. N.A., Not available.

Fig. 4.

Conditional knockout of the REA gene in the mammary epithelium during pregnancy and lactation using WAP-Cre. A, Schematic diagram of the REA gene-targeting strategy. B, Genomic DNA isolated from the indicated tissues was genotyped by PCR. REA (C) mRNA and protein (D) were analyzed by qRT-PCR or immunoblotting, respectively, in the mammary gland at different stages of pregnancy and lactation. The data are mean ±

, and the REA mRNA expression is presented as relative expression normalized to wild-type P8. E, Immunohistochemical detection of REA in REA_f/f_ and REA_f/f_ WAP-Cre mammary glands at L14. WC, WAP-Cre. Scale bars, 200 μm; #, P < 0.05 vs. wild type.

Fig. 5.

REA is required for the maintenance of differentiated lobuloalveolar structure. A, Representative photograph of pups nursed for 20 d by REA_f/f_ or REA_f/f_ WAP-Cre (WC) mothers. B, The body weights of pups nursed by REA_f/f_ or REA_f/f_ WAP-Cre mice during the first 20 d of postnatal development. The dam's genotype has a significant effect on the body weights of pups, with reduced weight gain of the pups nursed by REA_f/f_ WAP-Cre dams (GLM, F1,358 = 2280; P < 0.0001). GLM with repeated measures ANOVA. The data are mean ±

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(n = 20 per group). C, Representative hematoxylin- and eosin-stained mammary gland sections from REA_f/f_ and REA_f/f_ WAP-Cre at different stages of pregnancy, lactation, and involution. Magnification, ×40. D, qRT-PCR analysis of genes involved in milk protein expression (casein-α, casein-β, casein-κ, and WAP), lactose synthesis (α-lactalbumin), and milk lipid secretion (butyrophilin) at different stages of pregnancy (d 8 and 18) and lactation (d 2, 14, and 19). The data are mean ±

(n = 10 per group), and mRNA levels are illustrated as relative expression normalized to 36B4 by wild-type P8. E, Mammary gland lysates were analyzed by immunoblotting for REA and phospho-Stat5. ERK2 served as a loading control. #, P < 0.05 vs. wild type.

Fig. 6.

Increased body weight gain of pups nursed by REA_f_/+WAP-Cre (WC) females and its correlation with increased milk-related gene expression. A, The body weights of pups nursed by REA_f/f_ or REA_f_/+WAP-Cre mice during postnatal development. The dam's genotype has a significant effect on the body weights of pups, with increased weight gain of the pups nursed by REA_f_/+ WAP-Cre dams (GLM, F1,472 = 156; P < 0.0001). GLM with repeated measures ANOVA. The data are mean ±

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(n = 39 or 40 per group). B, mRNA levels of REA, casein-β, casein-κ, α-lactalbumin, butyrophilin, and Elf-5 were examined by qRT-PCR at different stages of pregnancy (d 18) and lactation (d 2, 5, 9, and 14). The data are mean ±

(n = 8 per group), and the expression was illustrated as relative expression normalized to 36B4 by wild-type P18. #, P < 0.05 vs. wild type.

Fig. 7.

REA_f/f_ WAP-Cre mice display increased apoptosis of mammary epithelial cells. A, The cell cycle inhibitor p21 levels are elevated in the mammary gland of REA_f/f_ WAP-Cre (WC) mice, as measured by qRT-PCR. The data are mean ±

and are illustrated as relative expression normalized to 36B4 by wild-type P8. B, Representative fluorescence images of TUNEL staining in mammary gland sections from REA_f/f_ and REA_f/f_ WAP-Cre mammary glands of L14 mice. C, Immunohistochemical detection of active caspase-3 expression in the REA_f/f_ and REA_f/f_ WAP-Cre mammary glands of L14 mice. #, P < 0.05 vs. wild type.

References

1. Asselin-Labat ML, Vaillant F, Sheridan JM, Pal B, Wu D, Simpson ER, Yasuda H, Smyth GK, Martin TJ, Lindeman GJ, Visvader JE. 2010. Control of mammary stem cell function by steroid hormone signalling. Nature 465:798–802 - PubMed
1. Richert MM, Schwertfeger KL, Ryder JW, Anderson SM. 2000. An atlas of mouse mammary gland development. J Mammary Gland Biol Neoplasia 5:227–241 - PubMed
1. LaMarca HL, Rosen JM. 2007. Estrogen regulation of mammary gland development and breast cancer: amphiregulin takes center stage. Breast Cancer Res 9:304. - PMC - PubMed
1. Hennighausen L, Robinson GW. 2005. Information networks in the mammary gland. Nat Rev Mol Cell Biol 6:715–725 - PubMed
1. Hennighausen L, Robinson GW. 2001. Signaling pathways in mammary gland development. Dev Cell 1:467–475 - PubMed

Repressor of estrogen receptor activity (REA) is essential for mammary gland morphogenesis and functional activities: studies in conditional knockout mice - PubMed (original) (raw)