Signal Transducer and Activator of Transcription 5a Mediates Mammary Ductal Branching and Proliferation in the Nulliparous Mouse
ABSTRACT Signal transducer and activator of transcription (Stat)5a is a critical regulator of mammary gland development. Previous studies have focused on Stat5a's role in the late pregnant and lactating gland, and although active Stat5a is detectable in mammary epithelial cells in virgin mice, little is known about its role during early mammary gland development. In this report, we compare mammary gland morphology in pubertal and adult nulliparous wild-type and Stat5a-/- mice. The Stat5a-null mammary glands exhibited defects in secondary and side branching, providing evidence that Stat5a regulates these processes. In addition, Stat5a-/- mammary glands displayed an attenuated proliferative response to pregnancy levels of estrogen plus progesterone (E+P), suggesting that it plays an important role in early pregnancy. Finally, we examined one potential mediator of Stat5a's effects, receptor activator of nuclear factor-kappaB ligand (RANKL). Stat5a-/- mammary glands were defective in inducing RANKL in response to E+P treatment. In addition, regulation of several reported RANKL targets, including inhibitor of DNA binding 2 (Id2), cyclin D1, and the cyclin-dependent kinase inhibitor p21(Waf1/Cip1), was altered in Stat5a-/- mammary cells, suggesting that one or more of these proteins mediate the effects of Stat5a in E+P-treated mammary epithelial cells.
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ABSTRACT: Menopausal hormone therapies vary widely in their effects on breast cancer risk, and the mechanisms underlying these differences are unclear. The primary goals of this study were to characterize the mammary gland transcriptional profile of estrogen+progestin therapy in comparison with estrogen-alone or tibolone and investigate pathways of cell proliferation in a postmenopausal primate model. Ovariectomized female cynomolgus macaque monkeys were randomized into the following groups: placebo (Con), oral conjugated equine estrogens (CEE), CEE with medroxyprogesterone acetate (MPA) (CEE+MPA), and tibolone given at a low or high dose (Lo or Hi Tib). All study treatment doses represented human clinical dose equivalents and were administered in the diet over a period of 2 years. Treatment with CEE+MPA had the greatest effect on global mRNA profiles and markers of mammary gland proliferation compared to CEE or tibolone treatment. Changes in the transcriptional patterns resulting from the addition of MPA to CEE were related to increased growth factors and decreased estrogen receptor (ER) signaling. Specific genes induced by CEE+MPA treatment included key members of prolactin receptor (PRLR)/signal transducer and activator of transcription 5 (STAT5), epidermal growth factor receptor (EGFR), and receptor activator of nuclear factor kappa B (RANK)/receptor activator of nuclear factor kappa B ligand (RANKL) pathways that were highly associated with breast tissue proliferation. In contrast, tibolone did not affect breast tissue proliferation but did elicit a mixed pattern of ER agonist activity. Our findings indicate that estrogen+progestin therapy results in a distinct molecular profile compared to estrogen-alone or tibolone therapy, including upregulation of key growth factor targets associated with mammary carcinogenesis in mouse models. These changes may contribute to the promotional effects of estrogen+progestin therapy on breast cancer risk.Breast cancer research: BCR 08/2013; 15(4):R62. DOI:10.1186/bcr3456 · 5.88 Impact Factor
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ABSTRACT: Progesterone receptors (PR) are transcription factors relevant to breast cancer biology. Herein, we describe an N-terminal common docking (CD) domain in PR-B, a motif first described in mitogen-activated protein kinases. Binding studies revealed PR-B interacts with dual-specificity phosphatase 6 (DUSP6) via the CD domain. Mutation of the PR-B CD domain (mCD) attenuated cell cycle progression and expression of PR-B target genes (including STAT5A and Wnt1); mCD PR-B failed to undergo phosphorylation on Ser81, a ck2-dependent site required for expression of these genes. PR-B Ser81 phosphorylation was dependent on binding with DUSP6 and required for recruitment of a transcriptional complex consisting of PR-B, DUSP6 and ck2 to an enhancer region upstream of the Wnt1 promoter. STAT5 was present at this site in the absence or presence of progestin. Furthermore, phospho-Ser81 PR-B was recruited to the STAT5A gene upon progestin treatment, suggestive of a feed-forward mechanism. Inhibition of JAK/STAT-signaling blocked progestin-induced STAT5A and Wnt1 expression. Our studies show that DUSP6 serves as a scaffold for ck2-dependent PR-B Ser81 phosphorylation and subsequent PR-B-specific gene selection in coordination with STAT5. Coregulation of select target genes by PR-B and STAT5 is likely a global mechanism required for growth promoting programs relevant to mammary stem cell biology and cancer.Nucleic Acids Research 08/2013; 41(19). DOI:10.1093/nar/gkt706 · 8.81 Impact Factor
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ABSTRACT: Progesterone (P4) stimulates proliferation of the mammary epithelium by a mechanism that involves paracrine signaling mediated from progesterone receptor (PR) positive to neighboring PR negative cells. Here we used a primary mouse mammary epithelial cell (MEC) culture system to define the molecular mechanism by which P4 regulates the expression of target gene effectors of proliferation including the paracrine factor RANKL. MECs from adult virgin mice grown and embedded in 3D basement-membrane medium resemble mammary ducts in vivo structurally and with respect to other properties including a heterogeneous pattern of PR expression, P4 induction of RANKL and other target genes in a PR-dependent manner, and a proliferative response to progestin. RANKL was demonstrated to have multiple functional P4 responsive enhancers that bind PR in a hormone dependent manner as detected by chromatin immunoprecipitation (ChIP) assay. P4 also stimulated recruitment of Stat5a to RANKL enhancers through an apparent tethering with PR. Analysis of primary MECs from Stat5a knockout mice revealed that P4 induction of RANKL and a broad range of other PR target genes required Stat5a, as did P4 stimulated cell proliferation. In the absence of Stat5a, PR binding was lost at selective RANKL enhancers, but was retained with others suggesting that Stat5a acts to facilitate PR DNA binding at selective sites and to function as a co-activator with DNA bound PR at others. These results show that RANKL is a direct PR target gene and that Stat5a has a novel role as a co-factor in PR-mediated transcriptional signaling in the mammary gland.Molecular Endocrinology 09/2013; 27(11). DOI:10.1210/me.2013-1077 · 4.20 Impact Factor