1. INTRODUCTION During development, differentiation and morphogenesis of epithelial organs are essential processes and lead to an astounding array of different structures. Signals for growth and differentiation of epithelia are frequently provided by neighbouring mesenchymal cells. The molecular basis for signals exchanged during mesenchymal-epithelial interaction is provided by paracrine signalling systems consisting of epithelial tyrosine kinase receptors and their mesenchymal ligands. Hepatocyte Growth Factor/ Scatter Factor (HGF/SF) and the c-met receptor, neuregulin and the members of the c-erbB receptor family, Glial cell line Derived Neurotrophic factor (GDNF) and the c-ret receptor as well as the Fibroblast Growth Factors (FGFs) and their receptors constitute such paracrine signalling systems. During recent years, the analysis of mice that carry targeted mutations or express transdominant receptors have demonstrated an important role for epithelial tyrosine kinase receptors and their mesenchymal ligands for epithelial development. We have previously identified an important function for HGF/SF and neuregulin in the development of the mammary gland by using the whole organ culture system. In this review we will concentrate on the effects HGF/SF and neuregulin exert on mammary epithelial cells cultured on matrigel. We observe that these two growth factors elicit fundamentally distinct morphogenic responses. HGF/SF induces the formation of tubular structures, whereas neuregulin evokes the formation of alveolar structures. The structures generated in cell culture by these two growth factors resemble the ones which are formed during mammary gland development in vivo. We used this cell culture system to further analyse the signalling pathways that become activated by HGF/SF or neuregulin and lead to the different morphogenic responses of mammary epithelial cells. 2. THE EFFECT OF HGF/SF AND NEUREGULIN ON MORPHOGENESIS AND DIFFERENTIATION OF
MAMMARY EPITHELIAL CELLS During mouse mammary gland development HGF/SF and neuregulin are expressed by the mesenchym that surrounds the epithelial cells which produce receptors for both ligands. Interestingly, HGF/SF is highly expressed in the mammary gland during puberty whereas neuregulin is specifically highly expressed during pregnancy of the mouse2. In experiments using whole organ cultures of mouse mammary glands HGF/SF promotes branching of ductal trees and inhibits terminal differentiation, as assessed by the expression of milk proteins. Neuregulin stimulates lobulo-alveolar differentiation in this culture system and promotes the expression of milk components such as β-casein2. These results indicate that different growth factors elicit distinct responses in organ culture of the mammary gland and raise the questions of whether different cellular populations of the mammary epithelium are affected by these factors and how these different responses are evoked on a molecular level. On one hand, the organ culture system provides a good model for the analysis of morphogenic events but on the other hand, its complex cellular composition precludes a biochemical analysis of signalling cascades activated by morphogenic factors. In the search for an appropriate way to examine signalling pathways responsible for morphogenic changes, we found that mammary epithelial cells (EpH4) grown on matrigel are affected by two growth factors, HGF/SF and neuregulin. EpH4/K6 mouse mammary epithelial cells cultured on matrigel in the presence of hormones (insulin, prolactin and hydrocortisone) form small spheroids and secrete milk protein4-6. These aggregates consist mainly of 3 to 7 single-layered epithelial cells surrounding a small lumen. When HGF/SF was added, EpH4/K6 cells started to grow faster and built long tubular structures that could reach a length of up to several millimeters. Histological analysis revealed that the tubular structures induced by HGF/SF consist of several layers of cells lining the elongated lumina. In contrast, neuregulin induced an entirely different response in EpH4/K6 cells grown on matrigel: large alveolar-like structures or lobulo-alveolar-like aggregates are formed. Histological analysis demonstrated that neuregulin-induced alveoli and lobulo-alveoli consist of single-layered cells surrounding large lumina. We also tested other growth factors for their morphogenic effect on this mammary epithelial cells grown on matrigel: epidermal growth factor (EGF) stimulated growth, whereas keratinocyte growth factor (KGF) had only a moderate effect on proliferation. However, neither growth factor, EGF or KGF, elicited any particular morphogenic response: tubular structures or large alveoli were not induced. We went on to analyse the distribution of the cell adhesion molecule E-cadherin and the production of the differentiation marker β-casein by immunofluorescence-staining (Fig. 1). In control aggregates, β-casein production was high but E-cadherin was evenly distributed along all cell surfaces, suggesting only moderate polarization of the cells (Fig 1A). HGF/SF reduced β-casein expression in the tubular structures as assessed by the reduction of immunofluorescence-staining intensity (corroborated by a decrease of mRNA expression for casein, observed by northern blotting). E-cadherin was largely distributed along the whole cell surface (Fig 1B). However, in accordance with the more pronounced polarization of cells in neuregulin-induced alveoli, E-cadherin was predominantly located at lateral membranes. β-Casein was strongly expressed in these cells as assessed by the staining intensity observed by immunofluorescence analysis (Fig 1 C). Thus, EpH4 mammary gland epithelial cells respond in a distinct manner when stimulated with HGF/SF or neuregulin. While HGF/SF induces branched multilayered tubes and inhibits the production of milk proteins, neuregulin causes the formation of alveoli and lobulo-alveolar-like aggregates which often consist of a monolayered epithelium and possess a higher degree of polarization and differentiation. Both growth factors can induce complex morphogenic programs in mammary gland epithelial cells, which resemble those observed in the mammary gland during postnatal development.
Figure 1. Scanning confocal micrograph of EpH4/K6 aggregates treated with HGF/SF (B) or neuregulin (C); controls are shown in A. 3. SIGNALLING PATHWAYS ACTIVATED BY HGF/SF AND NEUREGULIN IN MAMMARY
EPITHELIAL CELLS
We used this cell culture system to further analyse components of the signalling cascades activated by HGF/SF and its receptor c-met as well as cascades regulated by neuregulin and the members of the c-erbB family of receptor tyrosine kinases. EpH4/K6 cells were transfected with the cDNA of the recently identified substrate of the c-met receptor, Gab1. When grown on matrigel, the transfected cells produced tubular structures, even in the absence of HGF/SF. These structures were not observed with cells transfected with a control plasmid only. The histology of the Gab1-induced structures was identical to those induced by HGF/SF. In order to investigate which member of the c-erbB receptor family or what receptor combination is responsible for the neuregulin-induced phenotype of mammary epithelial cells, we performed transfection experiments with hybrid receptor molecules. Neuregulin signals are mediated by direct interaction with the high affinity receptors c-erbB3 or c-erbB4. In addition, c-erbB2 acts as an important coreceptor for the transmission of neuregulin9-11. We transfected EpH4/K6 cells with cDNAs for hybrid receptors consisting of extracellular portion of trk (the receptor for nerve growth factor, NGF) and intracellular c-erbBs. Remarkably, clones of EpH4 cells which stably expressed the hybrid trK/c-erbB2 receptor produced alveolar-like structures in the presence of NGF. These structures were found to be histologically identical to the neuregulin-induced alveoli. Control transfectants expressing pSVneo, trk or an trk/KGF hybrid receptor did not show this morphogenic response in matrigel. Similarly, stable cell clones expressing a trK/c-erbB4 hybrid receptor did not form alveolar structures in the presence of NGF. We then examined EpH4/K6 cells expressing mutants of trK/c-erbB2 to identify essential docking sites in the c-erbB2 receptor that elicit this morphogenic response. Recently, five phosphorylated tyrosine residues in the COOH-terminal substrate-binding region have been identified as important docking sites for signalling substrates acting downstream of the c-erbB212,13. We found that a hybrid receptor trK/c-erbB4 containing only the first four tyrosines was sufficient to induce alveolar structures when overexpressed in EpH4 cells and activated by NGF. In contrast, a hybrid receptor of trK/c-erbB4 containing only the last one of these five tyrosines was not capable of inducing alveolar morphogenesis. These findings demonstrate that an activated trK/c-erbB2 is sufficient to elicit a specific morphogenic response in mammary epithelial cells, the formation of alveoli and lobulo-alveolar structures, even in the absence of active c-erbB3 and c-erbB4 coreceptors. Furthermore, our results indicate that only the first four (Y1028, Y1144, Y1201 and Y1226/27) but not the last tyrosine (Y1253) are important for mediating the neuregulin-induced morphogenic response. In order to examine which of the known signalling cascades mediates the morphogenic responses evoked by HGF/SF and neuregulin we used inhibitors that interfere with specific signalling pathways. Addition of the PI3 kinase inhibitors, wortmannin and LY294002, to EpH4/K6 cells reduced the formation of elongated structures, whereas the MAPK kinase inhibitor PD98059 had no effect on the stimulation of elongated aggregates. In contrast, PD98059 quenched the induction of larger alveoli, whereas wortmannin was without effect on the formation of larger alveolar structures. These results suggest that the formation of tubular structures induced by HGF/SF requires pathways involving Gab1 and PI3 kinase, whereas for the induction of alveoli and lobulo-alveolar structures by neuregulin, an activated c-erbB2 receptor is sufficient and most likely the activity of MAPK kinase is required.
4. DISCUSSION
Morphogenic activities of various growth factors on mammary gland development have been examined previously; for instance it has been shown that EGF (or TGF-α) and TGF-β can influence ductal as well as alveolar deveIopment. These factors are predominantly expressed in an autocrine fashion by epithelial cells and are produced throughout mammary gland postnatal development. Mesenchymal-epithelial interactions are essential for pre- and postnatal development of the mammary gland, so we have focused on the functional analysis of mesenchymal ligands, HGF/SF and neuregulin, and their epithelial tyrosine kinase, c-met and the c-erbBs. Interestingly, both growth factors are highly expressed within the mammary gland during specific developmental phases: HGF/SF is high expressed during puberty, whereas neuregulin is strongly expressed only during pregnancy. It has already been shown that epithelial cells isolated from kidney, breast and other organs respond to HGF/SF by morphological differentiation, the formation ofbranched tubules when grown in a collagen matrix. Here we used matrigel as a substrate to investigate effects of HGF/SF and neuregulin on functional differentiation, that is, expression of milk proteins. HGF/SF inhibits the expression of β-casein, indicating that a morphogenic program without concomitant functional differentiation is activated. This is consistent with developmental processes in vivo: milk production is blocked during branching of the ductal tree during puberty. In the whole organ culture system of the mouse mammary gland, HGF/SF inhibits the expression of milk components whereas neuregulin stimulates the production of milk components. We did not observe an increase of β-casein expression by neuregulin in our cell culture system. It seems possible that neuregulin promotes an already programmed differentiation process leading to alveolar morphogenesis and concomitant production of milk components in the whole organ cultures ofthe mammary gland whereas such a complex differentiation program does not exist in our EpH4 cell system. Interestingly, neuregulin induced a redistribution of the cell adhesion molecule E-cadherin to the lateral membranes of EpH4/K6 cells. Recently, this redistribution of cadherins has also been reported for other epithelial cell lines. In addition, it has been demonstrated that neuregulin can promote the formation of ring-shaped multicellular structures in various cancer cell lines cultured on plastic. Several tyrosine kinase receptors have been reported to affect epithelial cells (e.g. trk, c-ros, KGFR) but only c-met was found to induce tubulogenesis. Although a variety of substrates were found to bind to tyrosine phosphorylation sites in the COOH-terminus of met, a substrate which can mediate the signal responsible for branching morphogenesis, Gab1, has only recently been identified. It has previously demonstrated that Gab 1 is the predominant protein phosphorylated following activation of the c-met receptor in epithelial cells. The c-met receptor can signal through ras and PI3 kinase Signalling through ras, resulting in cellular growth, requires the substrate Grb2, which binds to tyrosine residue Y1254 of c-met (i.e. the second tyrosine residue of the bidentate docking site. Motility and morphogenic responses evoked by an active c-met receptor require Gab1, which binds strongly to Y1347 (the first residue of the bidentate docking site. Gab1 can also bind to Y1354, but in the presence of Grb2 binding to Y1347 is preferred and stabilised . Furthermore, it has recently been shown that the pleckstrin homology domain of Gab1 is crucial for its subcellular localization to cell-cell contacts as well as for the morphological response in MDCK cells38. Gab1 harbours three potential PI3 kinase-binding sites. Indeed, we found that inhibitors of PI3 kinase, wortmannin and LY294002, specifically reduce HGF/SF-induced formation of elongated structures. Recently it has been shown that LY294002 could inhibit the localization of Gab1 to areas of cell-cell contacts. These data indicate that tubular and branching morphogenesis depends on pathways involving PI3 kinase. In contrast, the neuregulin-induced formation of larger alveoli could be inhibited by a MAPK kinase inhibitor, PD98059. Thus, components of the ras/MAPK kinase pathway are important for alveolar development of mammary epithelial cells. It will now be important to identify which substrates or substrate combinations elicit the alveolar morphogenesis through the activated c-erbB2 receptor.
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Hepatocyte & Neuregulin