Id-1, ITF-2, and Id-2 Comprise a Network of Helix-Loop-Helix Proteins That Regulate Mammary Epithelial Cell Proliferation, Differentiation, and Apoptosis

Abstract

Mammary epithelial cells proliferate, invade the stroma, differentiate, and die in adult mammals by mechanisms that are poorly understood. We found that Id-1, an inhibitor of basic helix-loop-helix transcription factors, regulates mammary epithelial cell growth, differentiation, and invasion in culture. Here, we show that Id-1 is expressed highly during mammary development in virgin mice and during early pregnancy, when proliferation and invasion are high. During mid-pregnancy, Id-1 expression declined to undetectable levels as the epithelium differentiated fully. Surprisingly, Id-1 increased during involution, when the epithelium undergoes extensive apoptosis. To determine whether Id-1 regulates both proliferation and apoptosis, we constitutively expressed Id-1 in mammary epithelial cell cultures. Id-1 stimulated proliferation in sparse cultures but induced apoptosis in dense cultures, which reflect epithelial cell density during early pregnancy and involution, respectively. To understand how Id-1 acts, we screened a yeast two-hybrid library from differentiating mammary epithelial cells and identified ITF-2, a basic helix-loop-helix transcription factor, as an Id-1-interacting protein. Overexpression of ITF-2 significantly reduced Id-1-stimulated proliferation and apoptosis. We show further that, in contrast to Id-1, Id-2 was expressed highly in differentiated mammary epithelial cells in vivo and in culture. In culture, Id-2 antisense transcripts blocked differentiation. Our results suggest that Id-1, ITF-2, and Id-2 comprise a network of interacting molecular switches that govern mammary epithelial cell phenotypes.The mammary gland undergoes striking changes in morphology and function during development and puberty and adult life. During each menstrual cycle, and particularly during pregnancy, mammary epithelial cells undergo cycles of proliferation, invasion, differentiation, and apoptotic cell death. During pregnancy, mammary epithelial cells proliferate, invade the surrounding stromal extracellular matrix (ECM), 1 and form lobulo-alveolar structures that prepare the gland for lactation (1-3). At late pregnancy, prior to parturition, breast epithelial cells cease proliferation and invasion and functionally differentiate into cells that express and secrete milk proteins. Throughout lactation, epithelial cells remain quiescent but continue to express milk proteins (4). After weaning, the mammary gland undergoes involution, a phase of extensive remodeling characterized by degradation of ECM and epithelial cell death by apoptosis.The molecular mechanisms that regulate and coordinate the changes in mammary epithelial cell phenotypes are poorly understood. Genes that are up-or down-regulated at different developmental stages have been identified, but little is known about how they are regulated and coordinated. The identification of tissue-specific transcriptional regulators in mammary epithelial cells is crucial, not only for deciphering the regulatory mechanisms of normal growth and differentiation but also for ...

Regulation of clusterin expression in mammary epithelial cells

“…2) as we previously determined for the expression of Zfp289 (19) and for the expression of ß-casein and Id-1 (20). Consistent with previous observations (14), we detected a strong and transient up-regulation of clusterin mRNA at the beginning of involution (between day 20 of lactation and day 1 of involution).…”

“…Moreover, these changes correlate inversely with Id-1 expression. We previously determined that Id-1 was down-regulated during the second part of pregnancy (when clusterin expression increases) and was up-regulated at day 3 of involution (when clusterin expression decreases) (20).…”

Id-1 and Id-2 are markers for metastasis and prognosis in oesophageal squamous cell carcinoma

“…In vitro experiments are being undertaken to investigate the functional role of Id-1 in metastatic progression of ESCC. Id-2 has been suggested to promote cellular differentiation in normal murine mammary epithelial cells (Parrinello et al, 2001). In breast cancer, Id-2 expressed at high levels maintains the cancer cells in a non-aggressive phenotype ).…”

Smad3 mediates immediate early induction of Id1 by TGF-β

“…They mostly act as negative transcriptional regulators in many biological processes such as cell differentiation, cell senescence, neurogenesis, apoptosis and angiogenesis [6][7][8][9][10][11][12][13]. As all of them lack a DNA-binding domain, they heterodimerize with and inhibit the function of basic HLH (bHLH) transcription factors [6,14], for examples, myogenic regulatory factors, such as MyoD, myogenin, Myf5 and MRF4/Myf6 [15,16], or class A of E proteins, such as E12, E47, E2-2 and ITF-2 [15,[17][18][19]. Inhibitor of differentiation (Id) proteins also interact with non-HLH proteins such as Ets2 [20] and MIDA1 [21] to regulate their activities.…”