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Review

Mammary gland: From embryogenesis to adult life Giuseppe Musumeci a,1 , Paola Castrogiovanni a,∗,1 , Marta Anna Szychlinska a , Flavia Concetta Aiello a , Giada Maria Vecchio b , Lucia Salvatorelli b , Gaetano Magro b , Rosa Imbesi a a

Department of Biomedical and Biotechnological Sciences, Human Anatomy and Histology Section, School of Medicine, University of Catania, Catania, Italy Department of Medical and Surgical Sciences and Advanced Technologies, G.F. Ingrassia, Azienda Ospedaliero – Universitaria “Policlinico-Vittorio Emanuele”, Anatomic Pathology Section, University of Catania, Catania, Italy b

a r t i c l e

i n f o

Article history: Available online xxx Keywords: Mammary gland Embryogenesis Development Endocrine signals

a b s t r a c t The aim of this review is to focus on the molecular factors that ensure the optimal development and maintenance of the mammary gland thanks to their integration and coordination. The development of the mammary gland is supported, not only by endocrine signals, but also by regulatory molecules, which are able to integrate signals from the surrounding microenvironment. A major role is certainly played by homeotic genes, but their incorrect expression during the spatiotemporal regulation of proliferative, functional and differentiation cycles of the mammary gland, may result in the onset of neoplastic processes. Attention is directed also to the endocrine aspects and sexual dimorphism of mammary gland development, as well as the role played by ovarian steroids and their receptors in adult life. © 2015 Elsevier GmbH. All rights reserved.

Introduction In mammals, including humans, the mammary gland has a development that begins during prenatal life, but it also consists of several stages during postnatal life (neonatal, puberty up to pregnancy) (Richert et al., 2000; Castrogiovanni et al., 2014; Musumeci et al., 2013). The mammary glands are modified and highly specialized sweat glands. The whole system of mammary ducts is included

Abbreviations: AR, androgen receptor; AP1, APETALA1; BMP4, bone morphogenetic protein 4; CEBPB, CCAAT/enhancer binding protein (C/EBP), beta; Dvl1, disheveled segment polarity protein 1; EDA-R, death receptor; ER, estrogen receptor; ETS2, v-ets avian erythroblastosis virus E26 oncogene homolog 2; FGF-10, fibroblast growth factor-10; GFs, growth factors; GSK3b, glycogen synthase kinase 3b; ID2, inhibitor of DNA binding 2, dominant negative helix-loop-helix protein; IgG, immunoglobulin G; IKK-␣, inhibitor of nuclear factor kappa-B kinase-␣; JAK, Janus kinase; LEF-1, lymphocyte enhancer factor-1; LMO4, LIM-domain only protein 4; Msx, muscle segment homeobox; NF-␬B, nuclear factor kappa-light-chain-enhancer of activated B cells; PEA3, phosphatidylinositol-4-phosphate 5-kinase and related FYVE finger-containing proteins signal transduction mechanisms; PR, progesterone receptor; PRLR, prolactin receptor; PTHrP, parathyroid hormone-related protein; PTHR1, parathyroid hormone/parathyroid hormone-related protein receptor 1; RANK, receptor activator of nuclear factor kappa-B; RANKL, RANK-ligand; RTK, tyrosine kinase receptor; Sp1, specificity protein 1; SRC-3/AIB1, steroid receptor co-activator-3; STAT3, signal transducer and activator of transcription-3; TBX3, T box3; TEBs, terminal epithelial buds. ∗ Corresponding author. E-mail address: [email protected] (P. Castrogiovanni). 1 Equal contribution.

in the context of an adipose mesenchyme that exerts considerable influence on its growth and evolution (Giordano et al., 2014). These influences are the result of a complex multifactorial process that progresses through both prenatal and postnatal stages (Richert et al., 2000; Musumeci et al., 2014, 2015). The histogenesis of the mammary gland begins early in the embryonic period with the development, in females, of a draft consisting of a small-branched channels system located in the mammary adipose tissue. Although its development proceeds with the isometric growth until the neonatal period, a greater impulse is in the prepubertal period; it continues in the peripubertal phase with elongation and branching of the ducts; finally, it culminates in the pubertal stage characterized by full sexual maturity, period in which the branching of the ducts increases and on their tips, the alveolar buds will form. Pregnancy represents the last developmental stage of the mammary gland during which the functional differentiation of the glandular parenchyma takes place in order to prepare the lobule–alveolar structure to lactation (Richert et al., 2000). This structure undergoes involution in response to the interruption of pregnancy and the cessation of menstrual cycles (Fig. 1). The regulation of all the stages of mammary gland development, characterized by repeated cycles of morphological growth and functional differentiation, is given by a series of systemic endocrine signals (Neville et al., 2002; Need et al., 2014; Musumeci et al., 2014, 2015). Nevertheless, it seems also necessary to consider the existence of other regulatory molecules capable of integrating all the involved signals, both the endocrine ones and those

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originating from the surrounding microenvironment (mesenchymal cells, cellular matrix, growth factors, hormones, paracrine/autocrine factors, cytokines, etc.). Only the integration and coordination of all these factors ensures the optimal development of the mammary gland, the maintenance of its own evolutionary destiny and tissue identity. The coordination of this complex process is largely organized and supported by the Homeobox genes (Chen and Sukumar, 2003; Satoh et al., 2004; Pagani et al., 2010). However, even if the spatiotemporal regulation of the proliferative, differentiative and functional cycles of the mammary gland by the homeotic genes may be effective, it is always possible that their wrong expression in mammary cells may lead to a defective or insufficient cell differentiation or, on the contrary, to an uncontrolled proliferation, contributing to the onset of a neoplastic process (Ligresti et al., 2008; Douglas and Papaioannou, 2013; Howard and Lu, 2014).

Embryogenesis of the human mammary gland In humans, as well as in other mammals, the tegumentary ectodermal epithelium, after the interaction with the mesenchymal cells of the underlying dermis, originates the epidermis and activates the differentiation of various specialized dermal–epidermal structures such as hair, nails, teeth and several exocrine glands such as sebaceous, sweat and mammary glands. These latter arise, since the end of week 4 of embryonic development, from bilateral thickenings that extend from the axillary to the inguinal region, called mammary ridges or “milk lines” (Moore et al., 2011; Macias and Hinck, 2012). These regress, except for those in a small region of the chest, where they form the mammary placodes (Fig. 2) (Moore et al., 2011; Macias and Hinck, 2012). These areas, characterized by a lenticular form, subsequently expand in the underlying mesenchyme and constitute the primordium of the mammary gland (Cowin and Wysolmerski, 2010; Moore et al., 2011). The development of the milk lines continues until week 6. Between weeks 7 and 8, the mammary parenchyma begins to invade the underlying stroma forming a primitive mammary disk. A further proliferative surge of mammary parenchyma starts at week 9, and a simultaneous discrete rarefaction of the epithelial layers of the overlying skin occurs. Between weeks 10 and 12 some epithelial buds originate from the mammary proliferation. These buds branch out and extend to the epithelial–mesenchymal boundaries (Macias and Hinck, 2012; Moore et al., 2011). The additional ramification between weeks 13 to 20 leads to the formation of about 15–20 solid epithelial cords. These give rise to other epithelial cords (lactiferous) that converge in the nipples. During the next ramification processes that continue to week 32, the solid epithelial cords undergo apoptosis of the internal epithelial cells. Between weeks 32 to 40 of gestation, starting from the ends of the epithelial gems, a tubule-alveolar proliferation is established (Cowin and Wysolmerski, 2010; Moore et al., 2011). The alveoli are covered by a monolayer of epithelial cells and circumscribed by a mesenchymeconnective stroma. Before birth, the mammary glands are equally developed in the male and female and each of them consists of about 20 lactiferous ducts that open into a dimple. In a few weeks, the proliferation of the underlying mesoderm transforms the dimple into an everted nipple and the skin surrounding it proliferates forming the areola (Moore et al., 2011). The spatiotemporal sequence of these phases in the mammary gland development is subjected to control and regulation by different genetic and transcriptional factors such as nuclear regulatory proteins (transcription factors) that play a key role in the development of the mammary gland, and if deregulated can contribute to breast cancer (Ligresti et al., 2008; Douglas and Papaioannou, 2013; Howard and Lu, 2014; Musumeci et al., 2014, 2015).

Fig. 1. The diagram shows the different stages of mammary development starting from the embryonic mammary primordium, followed by the various postnatal developmental stages, up to the menopause. The mammary placode differentiates into mammary buds that penetrate the underlying surrounding mesenchyme, sprout and develop a lumen. In the neonatal period the arborized gland invaded the developing fat pad. At puberty morphogenesis begins under control of estrogen and progesterone that regulate side branching. In pregnancy, estrogen, progesterone and prolactin play roles in alveolar expansion. In the late stages of pregnancy and during lactation, prolactin plays a key role in establishing the secretory state. After lactation, the gland involutes.

Endocrine aspects of mammary development and sexual dimorphism The development of the mammary gland is determined by endocrine interactions that begin during the early stages of

Fig. 2. Development of mammary glands. (A) Diagram of the embryo at about 28 days, which shows the mammary ridges (milk lines). (B) Diagram of embryo at week 6, showing what remains of the mammary ridges.

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embryonic development and continue throughout the period of postnatal life. The entity of these endocrine interactions depends on changes in the plasma levels of specific hormones and on the expression and the location of the receptors for which they have high binding affinity (Neville et al., 2002; Moise et al., 2013; Need et al., 2014). In mouse and rat, the sexual dimorphism occurs already in the uterus (Cardy, 1991). The transcription of the androgen (ARs) and estrogen receptors (ERs) is induced by epithelium on the mesenchymal cells simultaneously to the expression of the parathyroid hormone-related protein (PTHrP) (Hiremath and Wysolmerski, 2013; Moise et al., 2013). At the same time, the epithelium induces the mammary mesenchymal stroma to express receptors for this protein. In this way a paracrine epithelial–mesenchymal interaction takes place, and, through the interaction with the Wnt signaling pathway, it induces two processes: (1) the migration of mammary epithelial cells to the mammary adipose layer; (2) the morphological differentiation of overlying epidermal epithelial cells into the nipple (Boras-Granic and Hamel, 2013). All this happens in the human embryo at week 16 of gestation. In female mice, on day 14 of gestation, the epithelium of the mammary draft responds to estrogen, thanks to the presence of ERs, and evolves in the nipple (Sampayo et al., 2013). In male mice, the testis precociously produces testosterone that enters the bloodstream and binds the ARs expressed by mesenchymal cells of the mammary stroma, whose response results in the proliferation and the condensation of the stroma around the epithelial extroversion of the nipple, thus inducing its regression and then necrosis. If this process does not happen, the epithelial rudiment of the nipple remains dormant for many years during pre- and postnatal life (Sampayo et al., 2013). Therefore, it is sensitive to any exogenous or endogenous estrogen, as occurs during male puberty. This results in stimulation of growth of mammary ducts and primitive mammary alveoli and it is one of the most frequent causes of unilateral or bilateral gynecomastia in the male. In the male, the sexual dimorphic development of the mammary draft occurs after the onset of puberty (Cardy, 1991). In the late gestation stages, the distal portions of the mammary ducts evolve into alveolar structures, whose epithelia appear to have secretory activities. In fact, at birth, the mammary ducts may contain milk-like secreted material, known as “witch’s milk”, secreted by some infants during the first 6 weeks after birth (Jain et al., 2013). Between birth and puberty, however, except for a limited extension of the ductal system in the mammary adipose layer, the mammary draft remains relatively quiescent. The morphogenetic changes, concerning the mammary gland during puberty, pregnancy and lactation, are basically determined by steroids (estrogen and progesterone) (Need et al., 2014) and peptidic hormones (prolactin) (Oakes et al., 2008). Since puberty is a complex phenomenon dominated by estrogen and progesterone, in this phase the ductal system is stimulated to grow and further ramify (secondary ducts) (Need et al., 2014). At the end of puberty, a simple system of primary and secondary ducts are formed, and they emanate epithelial sprouts as terminal epithelial buds (TEBs), which constitute specialized mammary structures as they contain the precursors of luminal epithelial cells and myoepithelial cells (Moore et al., 2011). These ducts have an internal coating consisting of a single layer of epithelium, circumscribed by a layer of myoepithelial cells which is discontinuous in the small ducts. With the succession of menstrual cycles, the preexisting ducts continue to branch, forming more right-angle ductal sprouts (“collaterals”) and making the mammary gland more and more complex (Stute et al., 2004). During pregnancy, the gland becomes hypertrophic thanks to the new lobule–alveolar formations. This development phase is mainly driven by progesterone and other hormones induced by

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pregnancy (Need et al., 2014). Development continues further, but especially during lactation. During the weaning phase, with the cessation of lactation, the mammary gland undergoes remodeling, until it returns to the virginal phenotype (Fig. 1) (Need et al., 2014). Prolactin, milk secretion and mammary carcinogenesis Milk secretion is controlled by prolactin. On the mammary gland, this hormone exerts a synergistic action with estrogen and progesterone increasing growth, differentiation and development of the gland (Neville et al., 2002). In recent years, these hormones have been the subject of pilot studies in epidemiology in order to determine the endocrine interactions in the onset and progression of breast cancer (Ligresti et al., 2008). The results of these studies are highly controversial, also because they were mainly based on the effects of endocrine circulating prolactin (hormone of adenohypophyseal origin) on the mammary gland, but the prolactin synthesized by the cells of the mammary gland themselves was little considered. In the mammary gland, prolactin exerts mainly an autocrine/paracrine action (Fernandez et al., 2010). Further studies on prolactin synthesized by the cells of the mammary gland are necessary, especially because the dual aspect of this hormone seems more evident: (1) as a circulating hormone and (2) as a true cytokine. Prolactin and cytokines show many common properties. Prolactin, as well as cytokines, is synthesized in several other extrapituitary sites of the organism; prolactin receptors (PRLRs) have a nearly ubiquitous distribution and their structure is homologous to the cytokine receptors, in addition to a similar system of signal transduction (Binart et al., 2000). Furthermore, prolactin has a chameleon-like behavior: post-translational modifications, such as glycosylation, phosphorylation, segmentation and polymerization generate a relevant molecular heterogeneity. The glycosylation of prolactin, for example, induces a reduced binding affinity to its receptor (Pellegrini et al., 1988), while the phosphorylation increases the binding affinity, but it is functionally turned into an antagonist (Ueda et al., 2009); a segmented form of prolactin has antiangiogenic properties, while in the presence of other cytokines, it can exert angiogenic effects (Clapp et al., 2012; Reuwer et al., 2012). The polymerization of the molecule, or its conjugation to immunoglobulin G (IgG), results in large molecular complexes called “big” and “macro” prolactin, respectively 50–60 kDa and 150–170 kDa, found in serum of patients with hyperprolactinemia (Malaguarnera et al., 2004, 2005; Shimatsu and Hattori, 2012; Fahie-Wilson and Smith, 2013). It is also known that prolactin “regulates” the response to ovarian hormones in various tissues of the female reproductive system including the ovaries, uterus and the mammary gland (Veldhuis and Hammond, 1980). This action is certainly limited to paracrine prolactin, not endocrine. In fact, it is secreted in extrapituitary locations as the decidua, the myometrium, the mammary gland, the immune system and also the prostate. In these areas, the synthesis and/or the local accumulation of autocrine/paracrine prolactin may play an important tumorigenic role (Muthuswamy, 2012). Lymphocyte enhancer factor-1 (LEF-1), homeotic genes and ˇ-catenin in the development and carcinogenesis of the mammary gland In the early stages of human embryonic development, the LEF1 and homeotic genes muscle segment homeobox 1 (Msx1) and 2 (Msx2), play a very important role, not only in the development of the mammary gland placodes, but also in several other ectodermal epithelial drafts (Chen and Sukumar, 2003; Satoh et al., 2004; Pagani et al., 2010). In the early phases of development, Msx1, Msx2 and LEF-1 are coexpressed in the epithelium of the mammary gland draft,

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Fig. 3. Diagram of transverse section at the level of a placode showing breast and signaling pathways involved in its development (see text). BMP4: bone morphogenetic protein 4; FGF-10: fibroblast growth factor-10; TBX3: T box3.

but they do not develop. Furthermore, in humans, the mutation of PTHR1 causes Blomstrand type chondrodysplasia, a syndrome characterized by defects in ossification and lack of development of teats and udder (Ogata, 2010). Although in different ways, other homeotic genes are expressed in the epithelium and mesenchymal stroma of the mammary gland, such as Hoxc6, Hoxc8, Hoxd8, Hoxd9 and Hoxd10, whose main role seems to be the control and the regulation of the epithelial–mesenchymal interaction system (Friedmann et al., 1994; Friedmann and Daniel, 1996; Chen and Capecchi, 1999). Previous evidence, however, indicates that in adult life, the expression of these genes could undergo deregulation, which, in turn, is likely to be connected with the process of mammary carcinogenesis (Friedmann et al., 1994; Friedmann and Daniel, 1996; Chen and Capecchi, 1999). Ovarian steroids, their receptors and mammary gland in adult life

afterwards they are also expressed in surrounding mesenchymal cells. In particular Msx2 is mainly expressed in periepithelial mesenchymal cells where it plays a key role in the ductal branching process (Satoh et al., 2004). In postnatal life, puberty and early pregnancy, Msx1 and Msx2 continue to be expressed, respectively, in the epithelium and in mesenchymal stroma. This gene expression, however, is drastically reduced in the late stages of pregnancy and during lactation (Chen and Sukumar, 2003; Satoh et al., 2004; Pagani et al., 2010). The reduced or abolished expression of LEF-1 in knockout mice, induces a deficiency or even cessation in the growth of mammary placodes. In adult life, LEF-1 is less expressed compared to the embryonic period. This transcription factor plays an “architectural” monitoring role in the glandular development. The LEF-1 recognizes an interaction domain in ␤-catenin and it becomes one of the transcription factors of the Wnt signaling pathway (BorasGranic and Hamel, 2013). In consequence of the accumulation of ␤-catenin in the cytosol and its subsequent translocation into the nucleus, ␤-catenin binds LEF-1 acting as a coactivator (Boras-Granic and Hamel, 2013). The crucial role of ␤-catenin/LEF signaling in the development of several cancers in man is also known, though it is not clear if this could happen also in the breast (Table 1) (Gebeshuber et al., 2007). The expression of the transcription factor T box3 (TBX3) in the mammary ridges is critical for the activation of the signaling pathway Wnt/␤-catenin and EDA-R (death receptor), both necessary for the differentiation of mammary placodes. In mice lacking the TBX3-gene mice none of the placodes of the 5 pairs of breasts are formed, while heterozygous mice and women with TBX3 haploinsufficiency (lack of one of the two alleles of a gene) show a severe breast hypoplasia together with a deficiency of the development of upper limbs (ulnar–mammary syndrome) (Douglas and Papaioannou, 2013). In this regard it may be possible that in circumscribed areas of the mammary line, the increase of TBX3 depends on the integration of fibroblast growth factor-10 (FGF10) and Wnt signals. FGF-10, secreted by the cells of the extension of the ventral hipoassial somites, provides a vertical point signal that is integrated with the lateral signal of Wnt, produced from the ectoderm and the dorso-lateral mesoderm (Fig. 3). Bone morphogenetic protein 4 (BMP4), produced by the ectoderm and the ventral mesenchyme, inhibits the expression of TBX3, preventing the excessive ventralization of placodes (Cho et al., 2006) (Fig. 3). The further development of the mammary buds depends on the production of PTHrP by the ectoderm. The PTHrP stimulates the underlying mesenchyme, which expresses the parathyroid hormone/parathyroid hormone-related peptide receptor 1 (PTHR1), to produce factors that promote the further proliferation of the epithelium (Hiremath and Wysolmerski, 2013). In mice mutated for PTHrP or for its receptor PTHR1, mammary buds are formed,

Since the nineteenth century, the involvement of the endocrine system in reproduction, development and disease was considered, but only in the first decade of the twentieth century, when the steroid hormones were first isolated, was their crucial role in reproduction and feeding recognized. However, the molecular mechanisms that underlie their physiological (as well as pathological) effects on different tissues remained unknown until the 1970s, when ERs were identified and characterized also as members of the superfamily of nuclear receptors, whose effective task is to regulate transcription (Mueller, 1971; Means and O’Malley, 1972; Gorski et al., 1973; Korolkovas, 1973; Baulieu et al., 1975; Clark and Hardin, 1977). ERs and progesterone receptors (PRs) were identified for the first time in the 1970s in the mammary gland through biochemical methodology. The evidence of the crucial role played by ERs and PRs in mammary gland gene transcription is also documented by extensive literature over the years (Lydon et al., 2000; Bigsby et al., 2004; Cheng et al., 2005; Ozawa, 2005; Zhao et al., 2010; Moise et al., 2013; Sampayo et al., 2013). Some observations clearly indicate that when nuclear receptors, such as ERs and PRs, remain unsaturated by their ligand, they tend to form, in the nucleus, latent complexes with other proteins such as heat-shock proteins (Weigel and Zhang, 1998). Therefore, it seems clear that estrogen, progesterone and their nuclear receptors can exert a strong impact on mammary carcinogenesis (Moise et al., 2013). During puberty, in the peripubertal period and during the reproductive phase, the development of the mammary gland depends on these hormones and their receptors. Two genes were identified to encode for the ERs: ER␣ and ER␤ (Zhao et al., 2010). At the same time, two isoforms of PRs (PRA and PRB) were shown to be expressed from the same gene (Sampayo et al., 2013). These receptor proteins show considerable similarity with the superfamily of nuclear receptors, in fact, their functional domains are highly homologous to those of the nuclear receptors. This structure consists of a central and well conserved DNA-binding domain and a carboxyl/ligand-binding domain that mediates dimerization and transcriptional activation. ER␣ and PRs are expressed, not only in the adult mammary gland but, in higher concentrations, also in the reproductive organs, in the central nervous system (especially the hypothalamus) and in the pituitary gland (Ozawa, 2005). ER␤ is expressed in the mammary gland, but only at very low concentrations (Cheng et al., 2005). In the mammary gland, however, only a small population of epithelial cells express ERs and PRs. Generally, these epithelial cells lack a proliferative activity, but they perform a paracrine proliferative-regulatory action on the adjacent epithelial cells (Lydon et al., 2000). ERs and PRs are also present in mesenchymal cells surrounding the mammary gland (Bigsby et al., 2004).

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Table 1 Transcription factors involved in the control of both mammary gland development and cancer. Transcription factors and cofactors

Type of transcription factor

Mammary defects in knockout mouse

Deregulation in cancer

LEF-1 ␤-Catenin Msx1 and Msx2 Hoxa9, Hoxb9, Hoxd9 Hoxc6 Hoxd10 H6 ER␣ PR SRC-1 SRC-3/AIB1 STAT3 STAT5a STAT5b STAT5a/STAT5b CEBPB ID2 c-myc ETS2 PEA3 p53 LMO4

Group of high mobility Cofactor Homeobox Homeobox Homeobox Homeobox Homeobox Nuclear hormone receptor Nuclear hormone receptor Co-activator Co-activator ␤-Barrel ␤-Barrel ␤-Barrel ␤-Barrel Leucine zipper Helix-loop-helix (HLH) BHLH leucine zipper Winged helix-turn-helix Winged helix-turn-helix Zinc finger Cofactor zinc finger

Mammary sketch not developed Not described Mammary sketch not developed Defect in the alveolar development Reduced ducts ramification No lactation Not described Development of the ductal growth Defect in the alveolar development Reduced ducts ramification Reduced ducts ramification Delayed involution Defective differentiation Not described Defects in the differentiation Defect in the ductal-alveolar development Defect in the alveolar development Not described Delay in mammary carcinogenesis Reduced ducts ramification Delayed involution, mammary cancer Not described

Not described Oncogene Not described Hoxd9 not expressed Not described No gene expression Gene overexpression Gene overexpression Gene overexpression Not described Gene overexpression Substantially active Uncertain Not described Not described Not described Not described Overexpression Uncertain Overexpression or cancer No gene expression Overexpression

Classification of the transcription factors (Column 1) according to the type of DNA-binding or the interacting domain with protein (Column 2). The mammary gland phenotypes are identified by genetic deletion of the transcription factors, using conventional methodologies or tissue-specific strategies (Column 3). The role, certain or potential, of these factors as oncogenes in the breast tissue (Column 4). CEBPB: CCAAT/enhancer binding protein (C/EBP), beta; ER: estrogen receptor; ETS2: v-ets avian erythroblastosis virus E26 oncogene homolog 2; ID2: inhibitor of DNA binding 2, dominant negative helix-loop-helix protein; LEF-1: lymphocyte enhancer factor-1; LMO4: LIM-domain only protein 4; Msx: muscle segment homeobox; PEA3: phosphatidylinositol-4-phosphate 5-kinase and related FYVE finger-containing proteins signal transduction mechanisms; PR: progesterone receptor; SRC-3/AIB1: steroid receptor co-activator-3; STAT3: signal transducer and activator of transcription-3; STAT5: signal transducer and activator of transcription-5.

The ER␣-knockout mouse is infertile and it presents a rudimentary development of the mammary ductal system, because it lacks the sketch terminals (Hamilton et al., 2014). Despite the high plasma levels of estrogen, these terminals do not grow even in puberty (Curtis et al., 2000). It seems that the growth of ducts depends on the presence of ER␣ in the stroma, and that the availability of epithelial cells expressing ER␣ is insufficient to evoke a mammary proliferation induced by estrogen. In analogous experiments, the levels of PRs were shown to be very low, avoiding growth of glandular alveoli (Sampayo et al., 2013). Very recent evidence indicates that activation of ER␣ transcription factor can evoke, in the postnatal mammary development, a typical estrogen response, but it can also interact with other transcription factors such as specificity protein 1 (Sp1), APETALA1 (AP1) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-␬B) (Zhou et al., 2014). NF-␬B is one of the best known transcription factors as a key-regulator of inflammation and the innate immune response (Oh and Ghosh, 2013; Fan et al., 2013). As a result of recent studies, NF-␬B, as well as other transcription factors in the same group, appear to be essential for organogenesis of different epithelial tissues, including the mammary gland (Lindfors et al., 2013). The transcription factor ER␣ can also interact with other ontogenetic signaling pathways activated by cyclin D1 or other “non-classical” transcription factors such as c-Myc (Fig. 4) (Butt et al., 2005). Conclusion We can conclude that transcription factors play an essential role in development of the mammary gland, though we should emphasize that most of them are also widely expressed in different extra-mammary tissues. The specific function of these regulatory proteins in the morphogenesis of the mammary gland is their ability to interact with other cellular cofactors, which are also proteins. Several recent studies indicate that many of these transcription factors are important in determining the lobule–alveolar structure expansion, and also the differentiation of the mammary gland. Data

published in the literature also suggest that these transcription factors act as nuclear effectors in different cellular signaling pathways (Wnt, progesterone, prolactin and estrogen signaling pathways) involved in the development of mammary alveoli. Furthermore,

Fig. 4. In mammary epithelial cells, the cyclin D1 gene can be activated through several pathways. One of these begins with the binding of the receptor activator of nuclear factor kappa-B (RANK) to RANK-ligand (RANKL), which leads to the activation of NF-␬B through the inhibitor of nuclear factor kappa-B kinase-␣ (IKK-␣). This pathway is specifically activated by hormones during gestation. The second way, which leads to the activation of AP1, starts when growth factors (GFs) bind the tyrosine kinase receptor (RTK). The third way is when Wnt binds the Frizzled receptor in order to activate the disheveled segment polarity protein 1 (Dvl1) that inhibits the activity of glycogen synthase kinase 3b (GSK3b). The inactivation of GSK3b also inhibits the phosphorylation of ␤-catenin, which can no longer be degraded. As a result ␤-catenin translocates into the nucleus to activate the cyclin D1. Another way of activation of cyclin D1, consists of the prolactin/JAK/STAT receptor signaling pathway. AP1: APETALA1; Dvl1: disheveled segment polarity protein 1; GFs: growth factors; GSK3b: glycogen synthase kinase 3b; IKK-˛: inhibitor of nuclear factor kappa-B kinase-␣; JAK: Janus kinase; NF-B: nuclear factor kappa-light-chainenhancer of activated B cells; PRLR: prolactin receptor; RANK: receptor activator of nuclear factor kappa-B; RANKL: RANK-ligand; RTK: tyrosine kinase receptor; STAT-5: signal transducer and activator of transcription-5.

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