Survival and Differentiation of Mammary Epithelial Cells in Mammary Gland Development Require Nuclear Retention of Id2 Due to RANK Signaling

Abstract

RANKL plays an essential role in mammary gland development during pregnancy. However, the molecular mechanism by which RANK signaling leads to mammary gland development is largely unknown. We report here that RANKL stimulation induces phosphorylation of Id2 at serine 5, which leads to nuclear retention of Id2. In lactating Id2Tg; RANKL ؊/؊ mice, Id2 was not phosphorylated and was localized in the cytoplasm. In addition, in lactating Id2 S5A Tg mice, Id2 S5A (with serine 5 mutated to alanine) was exclusively localized in the cytoplasm of mammary epithelial cells (MECs), while endogenous Id2 was localized in the nucleus. Intriguingly, nuclear expression of Id2 S5A rescued increased apoptosis and defective differentiation of MECs in RANKL ؊/؊ mice. Our results demonstrate that nuclear retention of Id2 due to RANK signaling plays a decisive role in the survival and differentiation of MECs during mammary gland development.The mammary gland is a complex organ that proliferates and differentiates during puberty, pregnancy, and lactation under the influence of various hormones, including estrogen, progesterone, and prolactin. At birth, the mammary anlage consists of a few rudimentary ducts that occupy a small portion of the mammary fat pad. Pronounced ductal elongation and side branching commence at puberty. During pregnancy, the complexity of the ductal system increases through the addition of side branches, the formation of lobuloalveolar structures, and the differentiation of secretory epithelia (5,13,14). These proliferation, survival, and lactogenic differentiation steps are essential to form a functional lactating mammary gland during pregnancy.RANKL, a key regulator of osteoclast differentiation and/or activation (1,16,20,22), is essential for the development of a lactating mammary gland during pregnancy (10) and for the expansion of adult mammary stem cells (MaSC) in normal development (2, 17) and breast cancer (11, 36). Mice lacking RANKL or its receptor, RANK, showed impaired lobuloalveolar development during pregnancy, owing to intrinsic defects in the proliferation, survival, and lactogenic differentiation of mammary epithelial cells (MECs). Recent studies have reported that both progesterone (6, 31) and prolactin (40) signaling induce the expression of RANKL, and progesterone induces the expansion of MaSC by a paracrine effecter, RANKL, suggesting that these hormones regulate mammary gland development via the RANKL-RANK signaling pathway (2,11,17,36). However, how RANKL-RANK signaling regulates various aspects of the proliferation, survival, and differentiation of MECs in lactating mammary gland development needs to be elucidated. Cao et al. suggested that activation of a linear RANK-IB kinase ␣ (IKK␣)-NF-B-cyclin D1 signaling cascade could lead to the cellular proliferation of MECs (7). Although we suggested that RANK signaling induces ␤-casein gene expression via CCAAT/enhancer-binding protein ␤ (C/EBP␤) (18), the molecular mechanisms of RANK signaling resulting in the survival and lactogenic differentiat...

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

“…5F), both of which have established roles in mammary gland development. Id2 mediates the function of RANKL in the survival and proliferation of mammary epithelial cells (72,73), and deletion of Id2 leads to lactation defects in mice (74). The Elf5 transcription factor has a similar effect on mammary gland development as Zfhx3, and deletion of Elf5 leads to failure of Stat5 activation and defects in alveologenesis (75).…”

The Stat6-regulated KRAB domain zinc finger protein Zfp157 regulates the balance of lineages in mammary glands and compensates for loss of Gata-3

“…Id2 has recently been suggested to be a direct target of Stat5 in memory T cells (Yang et al 2011), suggesting that it is the pStat5 + cells that express Id2. Furthermore, RANKL has been reported to induce phosphorylation of Id2 at Ser 5, leading to nuclear retention, which is of itself sufficient to rescue the defective differentiation and cell death that occur in mammary glands from RANKL À/À mice (Kim et al 2011). Setdb1, which is a histone H3 Lys 9 (H3K9)-specific methyltransferase, binds the promoter of Id2 and suppresses its expression through installing H3K9 methylation ).…”

ID4 controls mammary stem cells and marks breast cancers with a stem cell-like phenotype

“…The role of the Id proteins has been studied to a limited extent in mammary gland development. Id1 is unnecessary for mammary gland development 21 , whereas Id2 is necessary for normal RANK signalling within the mammary gland 22 . Id4 loss leads to a delay in pubertal mammary gland development, associated with an increase in p38MAPK phosphorylation; however, this study did not identify a direct molecular link between Id4 as a transcriptional regulator and changes in p38MAPK phosphorylation nor did it address the role of Id4 in mammary epithelial fate decisions 23 .…”