Atbf1 Regulates Pubertal Mammary Gland Development Likely by Inhibiting the Pro-Proliferative Function of Estrogen-ER Signaling

Abstract

ATBF1 is a candidate tumor suppressor that interacts with estrogen receptor (ER) to inhibit the function of estrogen-ER signaling in gene regulation and cell proliferation control in human breast cancer cells. We therefore tested whether Atbf1 and its interaction with ER modulate the development of pubertal mammary gland, where estrogen is the predominant steroid hormone. In an in vitro model of cell differentiation, i.e., MCF10A cells cultured in Matrigel, ATBF1 expression was significantly increased, and knockdown of ATBF1 inhibited acinus formation. During mouse mammary gland development, Atbf1 was expressed at varying levels at different stages, with higher levels during puberty, lower during pregnancy, and the highest during lactation. Knockout of Atbf1 at the onset of puberty enhanced ductal elongation and bifurcation and promoted cell proliferation in both ducts and terminal end buds of pubertal mammary glands. Enhanced cell proliferation primarily occurred in ER-positive cells and was accompanied by increased expression of ER target genes. Furthermore, inactivation of Atbf1 reduced the expression of basal cell markers (CK5, CK14 and CD44) but not luminal cell markers. These findings indicate that Atbf1 plays a role in the development of pubertal mammary gland likely by modulating the function of estrogen-ER signaling in luminal cells and by modulating gene expression in basal cells.

Zinc Finger Homeodomain Factor Zfhx3 Is Essential for Mammary Lactogenic Differentiation by Maintaining Prolactin Signaling Activity

“…Development-We previously measured Zfhx3 mRNA expression at different stages of mammary gland development and found that the Zfhx3 mRNA level varies at different stages, being the highest in late pregnancy and lactation (20). Here we determined Zfhx3 protein expression.…”

“…Breeding, genotyping, and preparation of mice with MMTV-Cre-mediated mammary-specific knock-out of Zfhx3 were previously described (20). All mice were fed with pathogen-free food and water and were closely monitored and humanely euthanized.…”

“…Immunoblotting-Cell lysate preparation and Western blotting were performed as previously described (20). Briefly, cells or tissues were lysed using radioimmune precipitation assay buffer supplemented with protease inhibitor (cOmplete, Roche) and phosphatase inhibitor (PhosStop, Roche).…”

“…Glands-Whole mount analysis of mouse mammary glands was performed as previously described (20) except that whole mount glands were stained with carmine alum instead of hematoxylin. Briefly, the fourth abdominal mammary gland on the left was dissected at lactation day 2, placed between two glass slides and spread by placing weights on top of the slide for 10 min, followed by fixing in 3.7% formalin overnight.…”

“…For example, ZFHX3 is necessary for neuronal and myogenic differentiation in cell culture models (11, 14 -16), and deletion of Zfhx3 in mice causes developmental defects, interrupts epithelial homeostasis, and induces neoplastic morphology in mouse prostates (17)(18)(19). In mouse mammary glands, we previously demonstrated that Zfhx3 mRNA expression varies at different stages during development, reaching the highest level at lactation (20), and that Zfhx3 regulates pubertal mammary gland development (20). In addition, both estrogen and progesterone, two hormones essential for normal mammary gland development, induce or enhance the transcription of ZFHX3 in human and mouse mammary epithelial cells (21,22), although estrogen also causes protein degradation of ZFHX3 when too much estrogen is present (21).…”