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1  Introduction

Epithelium and mesenchyme are two distinct types of tissues that are present in virtually every organ. Lung, kidney, vascular system and most glands consist of tubular epithelial networks embedded in mesenchymal tissue. The formation of the first rudiments for these tree-like structures is specified during embryogenesis; epithelial buds invade the underlying mesenchyme and ramify to create interconnected tubules, a process known as branching morphogenesis. Lungs, salivary glands and mammary glands exhibit further developmental changes, namely alveolar morphogenesis, whereby round hollow alveoli arise from the ductal tree and differentiate into the functional units of the organ. Mesenchymal-epithelial interactions are strictly required for patterning morphogenic events (Sawer and Fallon, 1983); mesenchymal soluble factors bind to their epithelial receptors thus activating several signaling pathways which lead to local cellular responses like growth, motility, morphogenesis and differentiation.

The mammary gland is one of the most interesting models to study mesenchymal-epithelial interactions, as they play critical roles not only in embryonic mammary development but also in postnatal growth and differentiation of the gland. The mammary gland is a dynamic organ which has the unique property to undergo main developmental changes after birth. The complex mechanisms supporting mammary ductal branching and alveolar morphogenesis have been subject of extensive research, but are still far from being fully unravelled.

This work characterizes Vav2 as a novel intracellular effector of the receptor tyrosine kinase ErbB-2 that mediates lobulo-alveolar morphogenesis of the mammary gland. The first part of this Introduction reviews the developmental stages of the mammary gland and their regulation. The second part concerns the ErbB family of receptor tyrosine kinases and their role in mammary gland development. The third part describes features of the Vav family of guanine nucleotide exchange factors that may be useful for interpreting the results.

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1.1 Development of the Mammary Gland

The development of the murine mammary gland will be here outlined, since the mouse is the most thoroughly studied mammalian model (reviewed in Sakakura, 1987; Silberstein et al., 2001). During embryonic life, the mammary anlagen appear between days E10-E12 as an invagination of epidermal cells into the underlying mesenchyme, the fat pad. The mammary fat pad differentiates from deeply-placed mesenchymal cells. At day E16, primary ducts emerge from the epithelial rudiments into the fat pad and undergo an initial round of branching morphogenesis. Later, the nipples derive from epidermal invagination around the primary duct. The infant mammary gland consists of a primitive ductal tree that emanates from the nipple into the proximal fat pad. During puberty (5 to 8 weeks of age), the ducts elongate and branch further into the fat pad. The growing points for ductal growth are structures termed end buds, located at the terminal ends of the ducts. These end buds consist of two distinguishable cell populations: the cap cells, which are the progenitors for myoepithelium, and the body cells, which give rise to mammary epithelium. During the estrous cycle, alveolar buds first emerge from the lateral walls of the ducts, then regress and form again in the next cycle. Alveolar morphogenesis begins early in pregnancy with extensive budding of alveoli from the ducts; later, alveoli cluster to form lobulo-alveolar structures that are the secretory units of milk components throughout late pregnancy and lactation. Following weaning, the glands undergo a remodelling process known as involution (Lund et al., 1996; Li et al., 1997); during this phase, massive epithelial apoptosis results in destruction of lobulo-alveolar structures and regression of the gland to the pre-pregnancy state.

1.1.1 Regulation by Systemic Hormones

The development and biology of the mammary gland is controlled by a complex interplay of systemic hormones and local growth factors. Hormones are critical regulators of mammary [page 5↓]development both in embryonic and postnatal life. Once the embryonic mammary buds are formed, they stimulate expression of androgen receptors in the surrounding mesenchyme (Kratochwil, 1986). Testicular androgens from male fetuses induce regression of the epithelial mammary rudiment at E14, whereas estrogens are apparently not required for prenatal development (Korach, 1994).

The structure and functionality of the postnatal mammary tissue is orchestrated by hormonal changes occurring with age, estrous cycle and reproductive status. Pubertal growth and secretory differentiation of mammary parenchyme is dependent on ovarian steroids (estrogen, progesterone), prolactin and growth hormone. A role for estrogen and progesterone in ductal growth has been demonstrated by genetic and tissue recombination studies. Estrogen induces ductal dichotomous branching via paracrine stimulation of the stromal receptors, whereas the epithelial receptors are dispensable (Bocchinfuso and Korach, 1997; Cunha et al., 1997). Conversely, progesterone targets its epithelial receptors at puberty to promote ductal side-branching (Lydon et al., 1995; Brisken et al., 1998). Genetic ablation of prolactin or its epithelial receptor revealed that prolactin controls regression of terminal end buds and ductal side-branching during puberty (Ormandy et al., 1997; Horseman et al., 1997); these effects of prolactin are indirect and may involve regulation of ovarian production of progesterone (Brisken et al., 1999). Recent tissue recombination experiments circumvented the sterility of the abovementioned knockout mice and showed that both progesterone and prolactin act directly on their epithelial receptors to stimulate lobulo-alveolar development (Brisken et al., 1998; Brisken et al., 1999). Ectopic expression of growth hormone in transgenic mice leads to precocious mammary development and epithelial differentiation (Bchini et al., 1991), indicating that growth hormone promotes functional differentiation of the gland. This effect may be mediated by insulin-like growth factor-1 (IGF-1), as growth hormone induces synthesis of IGF-1 in stromal mammary cells (Kleinberg, 1997). Moreover, an essential role of IGF-1 in end bud formation was shown by the targeted deletion of IGF-1, which resulted in [page 6↓]retardation of ductal morphogenesis that could be rescued by exogenous IGF-1 (Ruan and Kleinberg, 1999).

1.1.2 Local Effects of Multiple Growth Factors

The complexity of signals that control postnatal mammary growth and differentiation is intriguing. Systemic hormones may synergize with a variety of growth factors that are locally produced either in the mammary mesenchyme (like HGF/SF, FGF-4, FGF-2, KGF, HRG1α, TGF-β, insulin-like growth factors) or in the mammary epithelium (Wnts, EGF, TGF-α, amphiregulin, FGF-1). This section concerns stimulatory and inhibitory growth factors that are implicated in growth and differentiation of the mammary gland. The function of EGF-like growth factors and their receptors in mammary development will be addressed separately.

The role of Wnt signals in mammary development begins in embryonic life. Lef-1 null mice fail to form mammary anlagen (van Genderen et al., 1994). Synergism of Wnt and parathyroid hormone-related peptide (PTHrP) signals has been shown to be essential for branching morphogenesis, sexual dimorphism and nipple formation of the mammary gland during embryogenesis (Wysolmerski et al., 1998; Dunbar and Wysolmerski, 1999; Foley et al., 2001). In postnatal life, the Wnt pathway is essential for mammary growth and differentiation during pregnancy. Wnt-4 and progesterone signals trigger interconnected cascades that control ductal side-branching and alveolar morphogenesis; using genetic and tissue recombination techniques, Brisken et al. (2000) showed that progesterone impacts nearby epithelial cells to induce expression of Wnt-4, which in turn synergistically acts in a paracrine fashion to promote ductal side-branching. Further support for a role of Wnt signaling in mammary development at pregnancy comes from genetic experiments with β-catenin, a downstream effector of the Wnt pathway. Mammary glands from virgin mice overexpressing an active β-catenin mutant show pregnancy-like lobulo-alveolar development and differentiation (Imbert et al., 2001); however, ductal side-branching is normal in these transgenic mice. These findings suggest that the canonical Wnt signaling pathway contributes to alveolar [page 7↓]morphogenesis, while Wnt control of ductal side-branching during pregnancy is not mediated by β-catenin but by other as yet unknown downstream effectors. Alternative Wnt-induced effectors are reviewed elsewhere (Hülsken and Birchmeier, 2001).

Previous observations suggest a role for hepatocyte growth factor/scatter factor (HGF/SF) and its receptor c-Met in ductal growth of the virgin mammary gland. HGF/SF and its receptor c-Met are coordinately produced in mammary mesenchyme and epithelium, respectively, with highest expression levels throughout puberty and in adult life until mid-pregnancy (Pepper et al., 1995; Niranjan et al., 1995; Yang et al., 1995); HGF/SF is expressed at low levels during late pregnancy and throughout lactation, and expression again increases during involution of the glands. In support of a role in ductal growth, HGF/SF stimulates branching when human mammary organoids are cultured on collagen, resembling the ductal elongation events that are observed in the mammary gland during puberty (Niranjan et al., 1995). Similarly, exogenous HGF induces extensive ductal branching in organ culture, whereas antisense HGF oligonucleotides block ductal growth of explanted mouse mammary glands (Yang et al., 1995). Branching morphogenesis is also observed in organotypic cell culture experiments whereby primary mammary epithelial cells, or cells from the mammary epithelial cell lines NMuMG and EpH4 are grown on three-dimensional matrices in the presence of HGF/SF (Niranjan et al., 1995; Soriano et al., 1995; Niemann et al., 1998). However, direct proof of a physiological role of HGF/SF signaling in the mammary gland in vivo is still awaited.

Factors that regulate differentiation of cells from the mononuclear phagocytic lineage also appear to be involved in mammary development. The colony stimulating factor 1 gene (CSF-1) is disrupted by an inactivating mutation in the recessive osteopetrosis (op) allele; homozygous op/op mice show a lactational defect due to incomplete mammary ductal side-branching and lactational failure, despite normal lobulo-alveolar morphogenesis and expression of milk proteins (Pollard and Hennighausen., 1994). The TNF family member osteoprotegerin-ligand or osteclast differentiation factor (ODF) synergizes with CSF-1 in [page 8↓]osteoclastogenesis (Lacey et al., 1998); absence of ODF or its epithelial receptor RANK (receptor activator of NFκB) impairs formation of lobulo-alveolar structures during pregnancy due to enhanced cell death; however, and in contrast to CSF-1 ablation, it does not affect side-branching (Fata et al., 2000). These results suggest a model whereby CSF-1 regulates ductal sprouting, and ODF subsequently promotes lobulo-alveolar development and terminal differentiation.

The formation of the adult mammary gland depends not only on growth stimulation but also on active inhibition, which prevents infilling the extraglandular mesenchyme. There is ample evidence that various TGF-β factors reversibly inhibit growth of the mammary end buds (reviewed in Silberstein, 2001). Overexpression of TGF-β1 in the mammary gland leads to impaired ductal elongation (Pierce et al., 1993) and absence of alveolar outgrowth and milk secretion (Jhappan et al., 1993), indicating that TGF-β1 negatively regulates both ductal and alveolar morphogenesis. Conversely, inhibition of TGF-β1 signaling promotes excessive ductal branching in mice that overexpress a kinase-deficient TGF-β type II receptor in the mammary stroma (Gorska et al., 1998; Joseph et al., 1999); this effect may be driven by up-regulation of HGF/SF expression, thus suggesting that the chronic inhibition of ductal growth by TGF-β1 results from its down-regulatory effect on the periductal synthesis of HGF/SF. Transgenic mice and transplantation experiments identified TGFβ-3 as a local factor that is secreted by alveolar cells upon milk stasis, and initiates apoptosis during the first stage of involution (Nguyen et al., 2000); the autocrine pro-apoptotic effect of TGFβ-3 involves activation of Stat3 followed by up-regulation of IGF-binding protein-5 (IGFBP-5), which sequesters and inactivates the mitogen IGF-1 (Li et al., 1997; Tonner et al., 1997; Chapman et al., 1999; Nguyen et al., 2000).

The role of fibroblast growth factor (FGF) signaling in the mammary gland is still controversial; whereas the different members of the FGF family show a temporally and spatially regulated expression in mammary tissue, genetic experiments suggest that they may [page 9↓]have redundant roles in the development of the gland. Expression of endogenous FGF4 and its receptor, FGFR1, is detected in virgin females, but not during pregnancy and lactation (Coleman-Knarcik et al., 1994; Chodosh et al., 2000). However, transgenic mice for FGF4 exhibit hyperplastic lobulo-alveolar structures that persist longer after weaning due to impaired apoptosis, and mice expressing an ectopic dominant-negative form of FGFR1 in mammary epithelium surprisingly lack a discernible phenotype (Morini et al., 2000). It is possible that FGF4, like TGFβ factors, plays a role in the control of apoptotic remodelling during ductal development and involution. In contrast to the transgenics for inactive FGFR1, mice overexpressing a dominant-negative FGFR2 transgene show an impairment in lobulo-alveolar development by mid-pregnancy (Jackson et al., 1997); knockout mice for FGF-7, its putative mammary ligand, show normal mammary development (Guo et al., 1996). Future research employing combined FGF-factor knockout mice may help to understand the function of this family in mammary tissue.

To conclude, the mammary development is regulated by multiple growth and differentiation factors and their intracellular signaling cascades. Loss of a single factor is often not compensated by the others, indicating that these factors trigger essential interacting or intersecting signaling cascades. Therefore, integration of these signals and the identification of new mammogenic factors still represent a major challenge.

1.1.3 The Role of Extracellular Matrix

The inducing effects of embryonic mesenchyme or its postnatal counterpart, termed stroma, are partially mediated by components of the extracellular matrix (ECM). In the adult gland, epithelial cells are in direct contact with basal myoepithelial cells and with the ECM structure known as basement membrane. The basement membrane is a complex and organized three-dimensional array of laminin, type IV collagen, heparan sulphate, proteoglycans and other proteins (Timpl, 1996). It is known that cell-ECM interactions influence tissue architecture through modulation of signaling pathways that affect cell growth, differentiation, survival and [page 10↓]morphogenesis (reviewed in Adams and Watt, 1993). The use of reconstituted basement membranes provides an excellent system to study the essential role of ECM for lactogenic differentiation in vitro. In organotypic cell culture, synthesis and secretion of milk components by mammary epithelial cells results from the interrelated effects of hormones, cell-cell and cell-ECM contacts (reviewed in Lin and Bissell, 1993). Primary mammary epithelial cells fail to differentiate to a secretory phenotype when cultured on plastic or onto a thin collagen type I layer (Emerman and Pitelka, 1977; Berdichevsky et al., 1992); however, these cells undergo alveolar morphogenesis and secrete milk proteins vectorially when cultured on a reconstituted, laminin-rich matrix from Engelbreth-Holm-Swarm (EHS) tumours (termed Matrigel; Kleinman et al., 1986; Barcellos-Hoff et al., 1989). Indeed, adhesion of mammary epithelial cells to laminin is critical for β-casein gene expression and for activation of Stat5, an essential regulator of milk gene expression (Streuli et al., 1995a; Streuli et al., 1995b; see also section 2.5 of this Introduction). However, the extent of morphogenic events in vitro is limited, indicating that additional signals from living stromal cells are required to support formation of fully-developed ducts or alveoli.

Integrins are cellular receptors for laminin, a major ECM component (Sonnenberg et al., 1990). Integrins are expressed at the basal membrane of epithelial cells both in mammary alveolar tissue and in EHS-cultured alveoli (Streuli et al., 1991). A role of integrins as physiological receptors for ECM was suggested by cell culture studies in which integrin function was blocked by pan-antibodies (Streuli et al., 1991); such blocking antibodies prevented the expression of milk proteins by mammary epithelial cells embedded in EHS matrix, indicating that the ECM induces lactogenic differentiation via integrin signaling. Genetic studies confirmed that integrins are cellular laminin receptors that control mammary functional differentiation; targeted expression of a transgene coding for an inactive β1-integrin subunit impairs lobulo-alveolar development and secretory differentiation during pregnancy (Faraldo et al., 1998). Moreover, laminin accumulates at the lateral surface of [page 11↓]luminal cells in the transgenic glands, indicating that integrin-ECM interactions determine baso-apical polarity of alveolar cells.

Overall, it is evident that direct cell-ECM contacts contribute to the induction of morphogenic and lactogenic events in mammary development. Future work will help to fully understand the cooperative effects of the ECM and other mitogenic factors in growth, morphogenesis and functional differentiation of the mammary epithelium.

1.2 The ErbB Family of Receptor Tyrosine Kinases

ErbB proteins belong to subclass I of the superfamily of receptor tyrosine kinases. This family has evolved from a single ligand-receptor pair in Caenorhabditis elegans, lin-3/let-23 (Aroian et al., 1990). Drosophila melanogaster alsoexpress one ErbB receptor, but three activating ligands and one inhibitor (Freeman, 1998). In vertebrates, the ErbB family comprises four membrane receptors: the epidermal growth factor receptor (EGF receptor; also termed ErbB-1, HER1), ErbB-2 (c-Neu, HER2), ErbB-3 (HER3) and ErbB-4 (HER4) (reviewed in Olayioye et al., 2000). These receptors share a glycosylated extracellular ligand-binding region with two cysteine-rich domains, a transmembrane stretch, and an intracellular region that encompasses a tyrosine kinase domain and a C-terminal tail containing the autophosphorylation sites. ErbB-1, ErbB-2 and ErbB-4 encode ligand-activated tyrosine kinases, whereas the corresponding ErbB-3 sequence is apparently devoid of kinase activity (Guy et al., 1994). ErbB receptors show different patterns of expression: ErbB-1 is expressed by liver parenchymal cells, fibroblasts, keratinocytes and several epithelial tissues, like the basal layer of the skin (Adamson, 1990; Partanen, 1990); ErbB-2 is expressed in a variety of tissues including nervous system, connective tissue and secretory epithelium (Kokai et al., 1987); ErbB-3 is expressed primarily in epithelium of various organs, in peripheral nervous system and in oligodendrocytes, while ErbB-4 is mostly restricted to central nervous system, cardiac muscle and glial cells (Pinkas-Kramarski et al., 1997). These differential expression [page 12↓]patterns are consistent with specific biological activities during embryonic life (discussed in Section 2.4 of this Introduction).

The ErbB receptors are activated upon ligand binding, a general mechanism that is shared by various receptor tyrosine kinases. Ligand binding induces formation of receptor dimers; this key step allows each receptor subunit to cross-phosphorylate tyrosine residues in the activation loop of the kinase domain of its partner, thus enhancing the catalytic activity (Hubbard et al., 1998). Following activation of the kinases, specific tyrosine residues on the C-terminal tail of the receptors become autophosphorylated; these phosphotyrosine residues are docking sites for intracellular signaling molecules that couple the receptors to signal transduction cascades, thus ultimately resulting in specific cellular responses. The hallmark of the ErbB family is the formation of both homo- and heterodimers following ligand binding. Moreover, each ligand induces the formation of preferential dimers in tissues where more than two ErbB receptors are expressed, leading to signal diversification (see below). The cellular routing of each receptor after ligand binding also differs for each family member: ErbB-1 undergoes rapid internalization and targets EGF for lysosomal degradation, whereas the other ErbBs are slowly internalized and are recycled back to the cell surface without significant degradation of the endocytosed ligand (Baulida et al., 1996; Pinkas-Kramarski et al., 1996; Lenferink et al., 1998).

1.2.1 ErbB Ligands Contain an EGF-like Domain

ErbB receptors are activated upon binding ligands that are known as EGF-related growth factors. These factors are mostly produced as transmembrane precursors, which can be proteolytically cleaved, thus releasing the extracellular, biologically active region. ErbB ligands are characterized by the presence of a conserved EGF-like domain of 35-50 amino acids that is essential for receptor binding. Six cystein residues form three disulfide bonds that hold together the characteristic three-loop structure of this motif. EGF-like ligands can be grouped according to their receptor-binding affinity: EGF, amphiregulin and transforming [page 13↓]growth factor-α (TGF-α) specifically bind to ErbB-1; betacellulin, heparin-binding EGF (HB-EGF) and epiregulin bind both ErbB-1 and ErbB-4; neuregulins are a complex family of proteins that include NRG-1 and NRG-2, which bind to ErbB-3 and ErbB-4, and the recently described NRG-3 and NRG-4, which bind to ErbB-4. So far, no direct ligand for ErbB-2 has been described (Peles et al., 1993; Tzahar et al., 1994).

NRG-1 (also termed Neu differentiation factor) was first isolated from medium of Ras-transformed rat fibroblasts, and the human counterpart Heregulin was detected in medium of breast cancer cells (reviewed in Pelesand Yarden, 1993). Two neuronal factors, termed glial growth factor (GGF) and acetylcholine receptor-inducing activity (ARIA) are alternatively spliced variants of NRG-1. Four different nrg genes code for the neuregulin isoforms and their related variants (Holmes et al., 1992; Wen et al., 1992; Falls et al., 1993; Marchionni et al., 1993; Carraway et al., 1997; Chang et al., 1997; Harari et al., 1999). NRG-1 shows a wide expression pattern during embryogenesis, being detectable in the central nervous system and in ventricular endothelium (Meyer et al., 1995). NRG-2 is also expressed in embryonic neural tissue and heart but otherwise is largely non-overlapping with NRG-1 expression (Carraway et al., 1997; Chang et al., 1997). NRG-3 expression is restricted to developing and adult nervous tissue (Zhang et al., 1997); NRG-4 is highly expressed in adult pancreatic tissue and weakly in muscle, but no data exist about embryonic expression (Harari et al., 1999).

1.2.2 Generation of Signal Diversity

It has been proposed that EGF-like ligands are bivalent; in case of NRG-1, an N-terminally located high affinity site with narrow specificity first binds a direct receptor (ErbB-3 or ErbB-4), and then a second C-terminal site recruits a co-receptor with lower affinity but broader specificity, ErbB-2 usually being the preferred one (Tzahar et al., 1997). Such a mechanism may account for the diversity of receptor dimers that are observed with a single ligand, as well as for the activation of the orphan receptor ErbB-2 in response to different EGF-like growth factors (Karunagaran et al., 1996; Graus-Porta et al., 1997). Evidence for the existence of all [page 14↓]ten possible homo- and heterodimers of ErbB proteins has been reported, including the ErbB-2 homodimer that is stabilized by oncogenic mutation or overexpression (Riese et al., 1995; Tzahar et al., 1996). This network of inter-receptor interactions displays a strict hierarchy rather than a random pattern (Tzahar et al., 1996). In fact, ErbB-2 is the preferred heterodimerization partner for all other ErbB family members. A driving force for the preferential binding to ErbB-2 could be that heterodimers containing ErbB-2 have a very low rate of ligand dissociation compared to other receptor pairs; this property of ErbB-2 can significantly prolong cell stimulation by every ErbB ligand (Graus-Porta et al., 1995; Karunagaran et al., 1996). In the absence of ErbB-2, NRG-1 induces formation of other heterodimers like ErbB-1/ErbB-3 and ErbB-1/ErbB-4 heterodimers, which explains the inhibition in trans of EGF binding to ErbB-1 when NRG-1 is present (Karunagaran et al., 1995). The existence of several ligands, together with their distinct ability to stabilize preferential homo- and heterodimeric receptor pairs, points to the existence of a hierarchical network of ligand-stimulated receptor dimerization events within the ErbB family (Pinkas-Kramarski et al., 1996).

Autophosphorylation of tyrosine residues may also be influenced by the heterodimer combination (Olayioye et al., 1998); in this way, each receptor has the ability to interact with distinct sets of intracellular signaling proteins thus increasing the functional versatility of the ErbB family. There are four potential mechanisms which may account for ligand-induced differential phosphorylation of the receptors (reviewed in Sweeney and Carraway, 2000). One ligand may induce receptor dimerization and phosphorylation of a particular subset of tyrosine residues. Binding to a different ligand could influence site usage for phosphorylation by promoting the association of the dimeric receptor complex with other cellular proteins like kinases, phosphatases or even cell surface molecules, which may mediate phosphorylation or dephosphorylation of specific sites. Alternatively, this second ligand could stimulate the assembly of oligomeric receptor complexes, or induce a different conformation of the receptor pair. Taken together, combinatorial dimerization and ligand-induced diversification of [page 15↓]signaling appear to confer ErbB receptors the potential to give rise to a broad range of cellular responses; moreover, because each receptor is unique in terms of catalytic activity, cellular routing and transmodulation, the resulting network allows fine tuning and stringent control of cellular functions.

1.2.3 Intracellular Effectors of ErbB Receptors

As for other receptor tyrosine kinases, ligand-induced autophosphorylation of ErbB receptors on specific tyrosine residues creates docking sites for cytoplasmic signaling proteins containing Src-homology2 (SH2) or phosphotyrosine-binding (PTB) domains (reviewed in Pawson, 1995). The binding specificity of these proteins is determined by the amino acid sequences adjacent to the phosphorylated tyrosines; amino acids located N-terminally determine the binding of specific PTB domains, and amino acids that are C-terminally located select SH2 domains. All ErbB family members, including the C. elegans and D. melanogaster homologs Let23 and DER, couple via Shc and/or Grb-2 to the mitogen-activated protein kinase (MAPK) pathway (Pinkas-Kramarski et al., 1996). However, certain intracellular proteins are preferential substrates of specific ErbB receptors. ErbB-3 is a preferred activator of p85 subunit of phosphatidylinositol-3-kinase (PI-3-K) due to the multiple specific binding motifs present in the ErbB-3 C-terminal tail, which are virtually absent in case of ErbB-2 (Fedi et al., 1994; Prigent et al., 1994). Similarly, the negative regulator c-Cbl and phospholipase Cγ1 (PLCγ1) couple to both ErbB-1 and ErbB-2 but not to ErbB-3 or ErbB-4 (Fazioli et al., 1991; Fedi et al., 1994; Cohen et al., 1996; Levkowitz et al., 1998; Klapper et al., 2000; Levkowitz et al., 2000). Olayioye et al. (2000) offers an excellent review of the present knowledge about the intracellular mediators of ErbB signals. As this work aimed to find novel substrates for the ErbB-2 receptor, its known downstream effectors will be described in detail.

ErbB-2 contains 5 putative autophosphorylation sites in its C-terminal tail, termed here Y1 (the most N-terminal tyrosine residue) to Y5 (the most C-terminal one; Hazan et al., 1989; [page 16↓]Segatto et al., 1990; Akiyama et al., 1991). It has been described that Shc binds to Y4 through its PTB domain (Segatto et al., 1993; Ricci et al., 1995; Dankort et al., 1997). Grb-2 directly binds to Y2 via its SH2 domain, and indirectly via Shc (Ricci et al., 1995; Dankort et al., 1997). Chk binds to Y5 (Zrihan-Licht et al., 1998). Grb-7, c-Src, Ras-GTPase activating protein (Ras-GAP) and the abovementioned PLCγ1 also interact with ErbB-2, though the binding sites are unclear (Fazioli et al., 1991; Jallal et al., 1992; Stein et al., 1994; Muthuswamy and Muller, 1995b).

The functional role of the ErbB-2 autophosphorylation sites in receptor-mediated transformation has been assessed by mutational analysis of the rodent constitutively active ErbB-2, termed Neu (Dankort et al., 1997). Absence of all major autophosphorylation sites of Neu dramatically decreases transforming activity upon overexpression in fibroblasts. The C-terminal tyrosine residues Y2 to Y5 can independently mediate transformation, since they fully restore transforming activity when individually added back to the inactive receptor. In contrast, the first tyrosine residue Y1 may not be involved in receptor-mediated transformation, as the resulting add-back mutant lacks transforming potential; moreover, Y1 may represent a negative regulatory site, since it supresses transforming activity when restored in combination with any other single tyrosine residue. Recent studies show that the functionally-redundant add-back mutants containing tyrosines Y2, Y4 and Y5 activate Ras to induce transformation, whereas the add-back mutant containing tyrosine Y3 operates independently of Ras to activate MAPK (Dankort et al., 2001).

1.2.4 ErbB Signaling in Embryonic Development

The importance of ErbB signaling in development was revealed by genetic studies in mice. Null mutations of individual ErbB receptor loci are embryonic lethal. Loss of erbB-1 leads to embryonic or perinatal lethality depending on the genetic background of the host (Miettinen et al., 1995; Sibilia et al., 1995; Threadgill et al., 1995; Sibilia et al., 1998); the mice display abnormalities in multiple organs including brain, skin, lung and gastrointestinal tract. Mice [page 17↓]that show spontaneous or targeted mutation of TGFa, one of the various ErbB-1 ligands, show only part of the phenotype observed in erbB-1 null mice, like impaired development of the eyes and hair follicles (Luetteke et al., 1993; Mann et al., 1993); this partial overlap suggests that each ErbB-1 ligand may play a distinct developmental role. The information gained by targeted mutation of erbB-2, erbB-3 and erbB-4 receptors and their ligand NRG-1 clearly demonstrates that distinct receptor heterodimers are essential for different developmental events. ErbB-2 -/- mice die at midgestation (E10.5) due to malformation of myocardial trabeculae in the heart ventricle (Lee et al., 1995), a phenotype that is shared by the NRG-1 (Meyer et al., 1995) and the erbB-4 null mice (Gassmann et al., 1995). These results are consistent with the view that NRG-1, which is expressed in endothelial cells of the endocardium (Meyer and Birchmeier, 1994), is required for activation of myocardial ErbB-2/ErbB-4 heterodimers to promote trabecular formation in the developing heart. In contrast to ErbB-2 and ErbB-4, ErbB-3 is not expressed in myocardium but in mesenchyme of the pre-valvular endocardial cushions. Accordingly, erbB-3 null mice die at day E13.5 displaying normal heart trabeculation but defective valve formation (Erickson et al., 1997; Riethmacher et al., 1997).

In addition to cardiac abnormalities, erbB-3 -/- mice show a generalized neural crest defect that affects both central and peripheral nervous structures. ErbB-3 mutant mice fail to form cranial sensory ganglia due to impaired migration of neurons from the hindbrain neural crest (Erickson et al., 1997; Riethmacher et al., 1997). This phenotype is also observed in mice lacking erbB-2 or NRG-1 (Lee et al., 95; Meyer et al., 1995); in contrast, erbB-4 deficient mice do not exhibit deficient cellularity of cranial ganglia but rather the innervation of these ganglia is disrupted, thus suggesting a unique role for ErbB-4 (Gassmann et al., 1995). In the peripheral nervous system, erbB-3 -/- mice completely lack Schwann cell precursors and Schwann cells that normally accompany nerves (Erickson et al., 1997; Riethmacher et al., 1997). In addition, degenerative motor and sensory neurons are found in the dorsal root ganglia (Erickson et al., 1997; Riethmacher et al., 1997), and migration of sympathogenic [page 18↓]neural crest cells is also impaired in erbB-3 mutants (Britsch et al., 1998); similar defects have been observed in NRG-1 and erbB-2 deficient mice (Meyer et al., 1995; Kramer et al., 1996; Britsch et al., 1998). It is evident from these studies that ErbB-2/ErbB-3 heterodimers transmit NRG-1 signals for neural crest cells to migrate. Recently, the early mortality of erbB-2 null mice has been rescued by myocardial expression of an erbB-2 transgene (Morris et al., 1999; Woldeyesus et al., 1999); erbB-2 rescued mice show striking similarities with the erbB-3 null mice, thus confirming the role of ErbB-2/ErbB-3 complexes in the development of the peripheral nervous system. Data on genetic ablation of the other NRG genes are largely missing. Mice carrying a targeted mutation of the NRG-3 gene, however, do not show an overt phenotype, but may deserve a more detailed analysis (T. Müller and C. Birchmeier, unpublished data). So far, the differential embryonic expression of the various neuregulin proteins and their distinct biological properties in vitro suggest different physiological functions (Carraway et al., 1997; Chang et al., 1997; Crovello et al., 1998). Together, these observations show the critical function of ErbB receptors and their ligands during embryogenesis. Moreover, the above genetic studies define distinct developmental roles for certain receptor combinations and are therefore strong support for the occurrence of signal diversification in vivo.

1.2.5 ErbB Receptors in Mammary Gland Development

There is considerable evidence that ErbB signaling has important roles in both normal and neoplastic growth of the mammary gland. All four ErbB receptors are found in mammary tissue (Schroeder et al., 1998). In prepubescent mammary gland, ErbB-1 and ErbB-2 are widely expressed in epithelium, stroma and mesenchymal fat, with ErbB-1 levels being particularly high in stromal cells. In the mature gland, ErbB-3 and ErbB-4 are also detected; just at this stage, ErbB-1 and ErbB-2 are differentially located: ErbB-1 is present in stroma and adipose compartments, while ErbB-2 is prominent in epithelium. During pregnancy, the four ErbB receptors are coordinately expressed in mammary epithelium; ErbB-1 and ErbB-2 are found at high levels in the alveolar epithelium throughout pregnancy, whereas ErbB-3 and [page 19↓]ErbB-4 levels increase preferentially in the ductal epithelium later in pregnancy. During lactation, receptor levels are low. ErbB-1 and ErbB-2 expression markedly increases during involution, while ErbB-3 expression declines and ErbB-4 expression is not detectable.

Despite expression of all ErbB receptors during puberty, only ErbB-1 and ErbB-2 are tyrosine-phosphorylated while ductal growth occurs (Sebastian et al., 1998). ErbB-3 and ErbB-4 seem to be present in a non-phosphorylated, inactive state at this stage, suggesting that none of them is relevant for ductal morphogenesis. Endogenous phosphorylation of all four receptors is detected during late pregnancy and lactation, with increasing levels of tyrosine-phosphorylated ErbB-1, ErbB-2 and ErbB-4 later at lactation. Information of the phosphorylation state of the ErbB receptors during involution is missing.

Like the receptors, the six EGFR ligands and NRG-1 are differentially expressed in mammary tissue (Schroeder et al., 1998). TGFα, betacellulin and heparin-binding EGF (HB-EGF) transcripts are found in prepubescent mammary gland and through mid-pregnancy, drop markedly during late pregnancy and lactation and again increase at involution. Amphiregulin and epiregulin transcripts appear in mature virgin glands and early in pregnancy, respectively; transcript levels of both factors decline later in pregnancy, remain low throughout lactation and involution, and reappear as the gland again resembles the mature virgin state. EGF transcripts are present at low levels in the virgin gland, and they dramatically increase during late pregnancy and lactation and return to low levels during involution. The NRG-1α isoform is found in mammary mesenchyme and shows a strongly regulated pattern of expression (Yang et al., 1995). NRG-1 is present at low levels in the virgin gland. At mid-pregnancy, NRG-1 exhibits a sudden concentration peak and then rapidly decreases to basal levels, which are constant later in pregnancy, throughout lactation and involution.

Several lines of evidence support a role of ErbB signaling in the development of the mammary gland. In early studies using mice, pellets containing EGF-like factors were [page 20↓]surgically implanted into the mammary glands to allow the slow local release of these factors. Implants of EGF, TGFα and NRG-1α stimulate ductal side-branching and lobulo-alveolar morphogenesis in virgin glands (Vonderhaar et al., 1987; Jones et al., 1996). EGF- or TGFα-induced alveoli lack secretory activity; in contrast, alveoli that derive from NRG-1α treatment are differentiated into secretory structures, which accumulate secreted milk proteins in their luminal compartment. These observations indicate that all factors can promote formation of alveoli but only NRG-1α stimulates their terminal differentiation. In other experiments, EGF-like growth factors were directly injected into the mammary glands to study their ability to induce tyrosine phosphorylation of ErbB receptors. Treatment of prepubescent glands with EGF stimulates tyrosine phosphorylation of stromal ErbB-1 (the EGF receptor) and ErbB-2 (Schroeder et al., 1998; Sebastian et al., 1998); as above mentioned, endogenous phosphorylation of ErbB-1 and ErbB-2 is observed in mammary tissue during puberty (Sebastian et al., 1998). Therefore, it is likely that locally-produced EGF induces at puberty the formation of active ErbB-1/ErbB-2 heterodimers, which may be essential for mammary ductal growth. Exogenous EGF induces phosphorylation of ErbB-1 and ErbB-2 at pregnancy, despite all ErbB receptors being present; in contrast, administration of NRG-1β results in trans-phosphorylation of all ErbB receptors, clearly indicating that neuregulin can induce the formation of combinatorial receptor complexes at pregnancy. Together with the pregnancy-restricted expression of neuregulin in mammary mesenchyme (Yang et al., 1995), these findings strongly suggest that NRG-1 may play a major role in the mammary gland during pregnancy to promote alveolar morphogenesis via trans-activation of ErbB heterodimeric receptors.

Convincing evidence of the physiological role of ErbB signals in mammary development has been supplied by genetic studies in mice; moreover, the phenotypes of knockout or transgenic mice support the differential roles of the ErbB receptors and their ligands that could be expected from the abovementioned stimulation studies, and from expression and activation patterns in vivo. Mammary glands from mice carrying a targeted mutation of the amphiregulin [page 21↓]gene show impaired ductal growth, whereas TGFa/EGF double null mice show normal mammary development at this stage (Luetteke et al., 1999). Amphiregulin null mammary glands are competent for lobuloalveolar differentiation; however, additional loss of TGFa and/or EGF severely compromises lactogenesis. Coherent with these findings, expression of a TGFa transgene in mammary tissue induces precocious alveolar development in virgin females, alveolar hyperplasia during pregnancy and delayed involution (Matsui et al., 1990; Sandgren et al., 1995). Together, these results suggest distinct functions of the various ErbB-1 ligands in the mammary gland: amphiregulin may be critical for ductal growth during puberty, while TGFα or EGF may be involved in alveolar differentiation during pregnancy. ErbB-1 is activated in virgin tissue; mammary expression of a transgene encoding a dominant-negative ErbB-1 receptor (the EGF receptor) inhibits ductal branching in the glands of virgin mice thus showing a role for ErbB-1 signaling in pubertal mammary development (Xie et al., 1997); similarly, female waved-2 mice, which carry a spontaneous inactivating mutation of the erbB-1 gene, display impaired glandular development (Fowler et al., 1995). Transplantation and tissue recombination experiments further support a role for ErbB-1 in ductal morphogenesis. Mammary gland grafts from neonatal erbB-1 -/- mice fail to undergo ductal growth (Sebastian et al., 1998); however, they develop lobulo-alveolar structures when stimulated by prolactin, indicating that the EGF receptor is essential for ductal branching but not for alveolar morphogenesis (Wiesen et al., 1999). Moreover, tissue recombinants revealed that wild-type fat pad supports outgrowth of erbB-1 -/- epithelium whereas the -/- fat pad does not, thus clearly showing the relevant role of stromal ErbB-1 in mammary ductal growth.

A role for NRG-1 in mammary gland development during pregnancy is substantiated by its restricted expression and pan-activating effect on ErbB receptors at this stage (see above). Indeed, mammary glands from mice that lack NRG-1a fail to undergo lobulo-alveolar morphogenesis at pregnancy (Li et al., submitted); NRG-1α is the isoform that is normally expressed in the glands. In contrast, NRG-1b null mice die during embryogenesis; the NRG-1β isoform accounts for the cardiac and neural crest phenotypes that were described in section [page 22↓]2.4 of this Introduction (C. Birchmeier, unpublished data). These results definitely demonstrate that NRG-1α is the naturally occurring isoform in mammary tissue, where it functions during pregnancy as a critical growth factor that promotes alveolar morphogenesis and secretory differentiation. Further insights into the mechanisms of neuregulin-induced morphogenesis have been gained from genetic studies with ErbB receptors. Expression of dominant-negative forms of erbB-2 and erbB-4 in the mammary gland of transgenic mice revealed a physiological role for both receptors in lobulo-alveolar development at late pregnancy and lactation (Jones et al., 1999; Jones and Stern, 1999). Moreover, there is genetic and biochemical evidence that Stat5a mediates morphogenic signals of ErbB-4 in the mammary gland during lactation (Liu et al., 1997; Jones et al., 1999). According to this data, it can be speculated that NRG-1α induces alveolar morphogenesis via signaling pathways that involve trans-activation of ErbB-2 and ErbB-4 and transcriptional regulation by Stat5a.

As already mentioned in Section 1.3 of this Introduction, the physiological processes that prepare the mammary gland for lactogenesis can be mimicked in vitro using organ culture and organotypic assays. By these means, our group has extensively contributed to understand the function of neuregulin in the mammary gland. The pioneering work of Yang et al. (1995) has shown that NRG promotes lobulo-alveolar differentiation of mammary gland explants in organ culture. The same morphogenic effect of neuregulin has been observed by Niemann et al. (1998) in organotypic cell culture. Treatment with neuregulin induces EpH4 mammary epithelial cells to form alveoli-like structures when cultured on a Matrigel reconstituted matrix; moreover, alveolar cells functionally differentiate and secrete milk components into a luminal compartment, thus reproducing in vitro the physiological responses of mammary epithelium to neuregulin. In addition, these studies show that activation of exogenous ErbB-2 receptor tyrosine kinase is sufficient for EpH4 cells to undergo alveolar morphogenesis in the Matrigel system. The present work contributes to understand the intracellular mechanisms underlying the morphogenic events of neuregulin-stimulated EpH4 cells in organotypic [page 23↓]culture, for it identifies Vav2 as an essential mediator of ErbB-2 specific signals leading to alveolar morphogenesis.

1.3 Vav Family of Guanine Nucleotide Exchange Factors

Vav proteins constitute a family of structurally related guanine nucleotide exchange factors that are involved in signaling pathways leading to cytoskeletal rearrangements and changes in gene expression (reviewed in Bustelo, 2000). Vav, the first member of the family, was identified as an oncogene encoding a constitutively active protein which lacks 67 amino acids at the N-terminus and induces transformation of NIH 3T3 fibroblasts(Katzav et al., 1989; Katzav et al., 1991). Vav expression and function is restricted to the hematopoietic system, and T and B lymphocytes derived from vav -/- mice reveal defects in antigen-receptor induced proliferation(Fischer et al., 1995; Tarakhovsky et al., 1995; Zhang et al., 1995; Fischer et al., 1998). The recently discovered vav2 and vav3 are also expressed in tissues of non-hematopoietic origin, among them epithelia (Schuebel et al., 1996; Movilla et al., 1999). Transient expression of N-terminally truncated Vav2 and Vav3 proteins in fibroblasts results in morphological changes such as membrane ruffling and formation of lamellipodia, and N-terminally truncated Vav2 is transforming in fibroblasts (Schuebel et al., 1996; Schuebel et al., 1998; Movilla et al., 1999). Vav2 has been implicated in cell-mediated killing by cytotoxic lymphocytes and in cellular responses following activation of B cell receptor and CD19 (Billadeau et al., 2000; Doody et al., 2000; Doody et al., 2001; Tedford et al., 2001). Vav and Vav3 can enhance NFκB-dependent transcription after TCR engagement (Moores et al., 2000). No biological function has as yet been assigned to Vav2 and Vav3 in epithelial tissues. Vav proteins share many different structural domains, among them a Dbl-homology domain that is responsible for their GDP/GTP exchange activity, and an SH2 domain that may link them to receptor tyrosine kinases(Bustelo, 2000). Indeed, tyrosine phosphorylation is required for the activation of full-size but not of N-terminally truncated forms of Vav proteins(Bustelo, 2000). Recently, crystallographic analysis revealed that the N-terminus of Vav acts [page 24↓]as an autoinhibitory site, and that phosphorylation of one specific tyrosine residue within this region relieves autoinhibition (Aghazadeh et al., 2000).

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