Abstract. We have examined the role of two mesenchymal ligands of epithelial tyrosine kinase receptors in mouse mammary gland morphogenesis. In organ cultures of mammary glands, hepatocyte growth factor (HGF, scatter factor) promoted branching of the ductal trees but inhibited the production of secretory proteins. Neuregulin (NRG, neu differentiation factor) stimulated lobulo-alveolar budding and the production of milk proteins. These functional effects are paralleled by the expression of the two factors in vivo: HGF is produced in mesenchymal cells during ductal branching in the virgin animal; NRG is expressed in the mesenchyme during lobulo-alveolar development at pregnancy. The receptors of HGF and NRG (c-met, c-erbB3, and c-erbB4), which are expressed in the epithelial cells, are not regulated. In organ culture, branching morphogenesis and lobulo-alveolar differentiation of the mammary gland could be abolished by blocking expression of endogenous HGF and NRG by the respective antisense oligonucleotides; in antisense oligonucleotide-treated glands, morphogenesis could again be induced by the addition of recombinant HGF and NRG. We thus show that two major postnatal rnorphogenic periods of mammary gland development are dependent on sequential mesenchymal-epithelial interactions mediated by HGF and NRG.ROWTH and morphogenesis of epithelia represent essential steps in the development of many eukaryotic organs. A series of developmental studies have demonstrated that growth and morphogenesis of epithelia are closely regulated by mesenchymal-epithelial interactions. These studies include transplantation and organ culture experiments, which have been carried out with various systems such as the salivary gland, the lung, the kidney, or the breast (Grobstein, 1953;Spooner and Wessells, 1970;Sax6n, 1987;Sakakura, 1991). The signaling molecules involved in mesenchymal-epithelial interactions during development are still largely unknown. Recent evidence suggests, however, that epithelial tyrosine kinase receptors and their mesenchymal ligands can take over these functions (see Birchmeier and Birchmeier, 1993; for a review).Several tyrosine kinase receptors with prevalent expression on epithelial cells have recently been characterized. The corresponding ligands are frequently synthesized by mesenchymal cells, i.e., could function in a paracrine manner (Stoker et al., 1987;Miki et al., 1991;

We have established a cell culture system that reproduces morphogenic processes in the developing mammary gland. EpH4 mouse mammary epithelial cells cultured in matrigel form branched tubules in the presence of hepatocyte growth factor/scatter factor (HGF/SF), the ligand of the c-met tyrosine kinase receptor. In contrast, alveolar structures are formed in the presence of neuregulin, a ligand of c-erbB tyrosine kinase receptors. These distinct morphogenic responses can also be observed with selected human mammary carcinoma tissue in explant culture. HGF/SF-induced branching was abrogated by the PI3 kinase inhibitors wortmannin and LY294002. In contrast, neuregulin- induced alveolar morphogenesis was inhibited by the MAPK kinase inhibitor PD98059. The c-met–mediated response could also be evoked by transfection of a c-met specific substrate, Gab1, which can activate the PI3 kinase pathway. An activated hybrid receptor that contained the intracellular domain of c-erbB2 receptor suffices to induce alveolar morphogenesis, and was observed in the presence of tyrosine residues Y1028, Y1144, Y1201, and Y1226/27 in the substrate-binding domain of c-erbB2. Our data demonstrate that c-met and c-erbB2 signaling elicit distinct morphogenic programs in mammary epithelial cells: formation of branched tubules relies on a pathway involving PI3 kinase, whereas alveolar morphogenesis requires MAPK kinase.

Abstract. Mammary gland development is controlled by systemic hormones and by growth factors that might complement or mediate hormonal action. Peptides that locally signal growth cessation and stimulate differentiation of the developing epithelium have not been described. Here, we report that recombinant and wild-type forms of mammary-derived growth inhibitor (MDGI) and heart-fatty acid binding protein (FABP), which belong to the FABP family, specifically inhibit growth of normal mouse mammary epithelial cells (MEC), while growth of stromal cells is not suppressed. In mammary gland organ culture, inhibition of ductal growth is associated with the appearance of bulbous alveolar end buds and formation of fully developed lobuloalveolar structures. In parallel, MDGI stimulates its own expression and promotes milk protein synthesis. Selective inhibition of endogenous MDGI expression in MEC by antisense phosphorothioate oligonucleotides suppresses appearance of alveolar end buds and lowers the ~-casein level in organ cultures. Furthermore, MDGI suppresses the mitogenic effects of epidermal growth factor, and epidermal growth factor antagonizes the activities of MDGI. Finally, the regulatory properties of MDGI can be fully mimicked by an l 1-amino acid sequence, represented in the COOH terminus of MDGI and a subfamily of structurally related FABPs. This peptide does not bind fatty acids. To our knowledge, this is the first report about a growth inhibitor promoting mammary gland differentiation.ROWTH development, and differentiation of epithelial tissues are multistage processes that are driven by a combination of paracrine and autocrine signaling factors and interactions of a cell with its extracellular matrix (reviewed in 51, 60, 62). In the mammary gland, these complex interactions are regulated by various steroid and peptide hormones (29,32,46,60). Development of the mouse mammary gland at puberty is characterized by sparsely branching ducts which invade the stroma, followed by the development of lobuloalveolar structures and functional differentiation, i.e., synthesis of milk constituents at pregnancy (1,17,32). By use of endocrine ablation (44), organ culture systems (1,35,54,65) and mammary cells (MEC) ~ growing on a

A method for the cultivation of organ explants from abdominal mammary glands of virgin mice has been established. In a serum-free medium containing aldosterone, prolactin, insulin, and cortisol (APIH medium) mammary gland development was documented by lobuloalveolar morphogenesis. The hormonal requirements for in vitro expression of beta-casein and of the mammary-derived growth inhibitor (MDGI) were tested. To this end, a full length cDNA coding for mouse MDGI was prepared displaying strong homologies to a mouse heart fatty acid binding protein, which is also expressed in the mammary gland. MDGI and beta-casein transcripts were found to be absent in the mammary tissue from primed virgin mice, and were induced upon culture of mammary explants in the APIH medium. An immunohistochemical analysis with specific antibodies against MDGI and casein revealed a different pattern of expression for the two proteins. In the APIH medium, MDGI was expressed mainly in differentiating alveolar cells of the lobuloalveolar structures, whereas beta-casein was present in both ductules and alveoli. The relationship between functional differentiation and MDGI expression was further studied in explants from glands of late-pregnant mice. At this stage of development, MDGI is found both in ducts and in alveoli. If explants were cultured with epidermal growth factor (EGF) and insulin, the lobuloalveolar structure was still present, whereas MDGI disappeared. Reinduction of MDGI expression was achieved by subsequent PIH treatment. Independent on developmental stage, EGF strongly inhibits MDGI mRNA expression. It is concluded that MDGI-expression is associated with functional differentiation in the normal gland.

A b s tra c t C ellular fatty acid-binding proteins (FABP) are a highly conserved fam ily of proteins consisting o f several subtypes» am ong them the m am m ary-derived growth inhibitor (M D G I) w hich is quite hom ologous to or even identical with the heart-type FABP (H-FABP). The FABPs and M D G I have been suggested to be in volved in intracellular fatty acid m etabolism and traffick ing. Recently, evidence for grow th and differentiation regulating properties of M D G I and H -FA B P was provid ed. U sing four affinity-purified polyclonal antibodies against bovine and hum an antigen preparations, the cel lular localization of M D G I/H -FA B P in hum an and mouse tissues and organs was studied. T he antibodies were weakly cross-reactive w ith adipose tissue extracts known to lack H-FABP, but failed to react by W estern blot analysis with liver-type FABP (L-FABP) and intesti nal-type FA B P (I-FABP). M D G I/H -FA B P protein was mainly detected in m yocardium , skeletal and sm ooth m uscle fibres, lipid and/or steroid synthesising cells (ad renals, Leydig cells, sebaceous glands, lactating m am mary gland) and term inally differentiated epithelia of the respiratory, intestinal and urogenital tracts. The results provide evidence that expression o f H -FA B P is associat ed with an irreversibly postm itotic and term inally differ entiated status of cells. Since all the antisera em ployed show ed spatially identical and qualitatively equal im m unostaining, it is suggested that hum an, bovine and m ouse M D G I/H -FA B P proteins share highly hom ologous epi topes.