Identification of a fibroblast-derived epithelial morphogen as hepatocyte growth factor.

We have previously shown that Madin-Darby canine kidney (MDCK) epithelial cells grown in collagen gels in the presence of fibroblasts or fibroblast-conditioned medium (CM) form branching tubules, instead of the spherical cysts that develop under control conditions. We now report that the fibroblast-derived molecule responsible for epithelial tubulogenesis is hepatocyte growth factor (HGF). First, addition of exogenous HGF to cultures of MDCK cells induces formation of epithelial tubules. Second, the tubulogenic activity of fibroblast CM is completely abrogated by antibodies to HGF. These results demonstrate that HGF, a polypeptide that was identified as a mitogen for cultured hepatocytes, has the properties of a paracrine mediator of epithelial morphogenesis, and suggest that it may play important roles in the formation of parenchymal organs during embryonic development.     

Hepatocyte growth factor increases urokinase-type plasminogen activator (u-PA) and u-PA receptor expression in Madin-Darby canine kidney epithelial cells.

We have recently demonstrated that fibroblast-conditioned medium induces Madin-Darby canine kidney (MDCK) epithelial cells to form branching tubules when grown in three-dimensional collagen or fibrin gels (Montesano, R., Schaller, G., and Orci, L. (1991) Cell 66, 697-711), and that this morphogenetic effect is mediated by hepatocyte growth factor (HGF), also known as scatter factor (Montesano, R., Matsumoto, K., Nakamura, T., and Orci, L. (1991) Cell 67, 901-908). In fibrin gels, this effect is inhibited by addition of exogenous serine protease inhibitors, which suggests a role for plasminogen activators (PAs) in the matrix remodeling required for tubulogenesis. In the studies reported in this paper, we have investigated the effect of fibroblast-conditioned medium (CM) and HGF on the production of PAs by MDCK cells. We have found that urokinase-type PA (u-PA) activity and mRNA are increased 4.9-fold by CM from human MRC-5 fibroblasts, which has tubulogenic activity, but not by CM from human Detroit-550 fibroblasts, which lacks tubulogenic activity. The u-PA inductive property of MRC-5 CM was completely inhibited by preincubation with antibodies to recombinant human HGF (rhHGF). Exogenously added rhHGF also increased u-PA activity and mRNA 5.9-fold in MDCK cells, with an optimal effect at approximately 10 ng/ml. MRC-5 CM also increased u-PA receptor mRNA 34.9-fold in MDCK cells, an effect which was inhibited by 71% by preincubating the CM with antibodies to rhHGF, and which was mimicked by exogenously added rhHGF (31.3-fold increase). These results demonstrate that HGF, which induces tubulogenesis by MDCK cells in vitro, also increases u-PA and u-PA receptor expression in these cells. Taken together with our previous observations, this suggests that the resulting increase in extracellular proteolysis, appropriately localized to the cell surface, is required for epithelial morphogenesis.     

Mammary gland morphogenesis in vitro: Formation of branched tubules in collagen gels by a cloned rat mammary cell line

The three-dimensional growth in vitro of cloned rat mammary cell lines on floating type I collagen gels has been investigated. Multicellular outgrowths formed by the various cell types show morphological differences on serial histological sectioning and electron microscopy. One cell line, Rama 25, an epithelial cell line derived from a dimethylbenz(a)anthracene (DMBA)-induced mammary adenocarcinoma can form branched tubules within the matrix. The amount of collagen in the matrix modified the structure of the predominant outgrowths formed by this cell line. High-concentration (0.6% w/v) collagen gels support the growth of tubules up to 0.5 mm in length which have an extensive lumen surrounded by rings of up to 26 cells. Absence of differentiated myoepithelial elements around the ring suggests a resemblence to primitive ducts found in the mammary glands of neonatal rats. The spectrum of cellular polarity toward the lumen seen throughout the tubules and the occasional irregular arrangement of epithelial cells are features of adenocarcinoma. Lumen formation occurs by central cell necrosis and separation of the external layers of initially solid cords. The tubules branch either dichotomously, by bifurcation at the distal ends or monopodially, by budding at the sides of the outgrowths. Rama 25 grown on gels containing lower concentrations of collagen (0.1 or 0.3% w/v) produce narrow branching structures with incomplete lumina and spikes of elongated cells. Tubular structures are not formed by Rama 25 grown on nonfloating gels. At the light microscopic level the layer of spindle cells formed beneath the surface monolayer on nonfloated gels resembles the sarcomatous regions of tumors, however at the ultrastructural level the spindle cells show some evidence of being myoepithelial-like rather than fibroblast-like. Sandwiching the epithelial cell sheet between two layers of collagen gel results in loss of contact with the media and the formation of spindle cells. The myoepithelial-like cell lines Rama 29 and Rama 401 form spiked branches of elongated cells and solid branching cords of cells, respectively. However, no lumen formation is observed. The fibroblast-like cell line Rama 27 shows extensive migration of either single cells or chains of cells into the gel. Thus only one cell type (Rama 25) is necessary to form branched tubules in vitro and the structure of the tubules can be modified by collagen, a component of the extracellular matrix.     

HGF-Induced Tubulogenesis and Branching of Epithelial Cells Is Modulated by Extracellular Matrix and TGF-β

Beginning with the observation that hepatocyte growth factor (HGF) induces the formation of branching tubular structures in Madin-Darby canine kidney (MDCK) cells cultured in Type I collagen gels but not in basement membrane Matrigel, we examined the individual components within this complex basement membrane extract to determine the effect of these proteins on the morphogenetic changes mediated by HGF. After extraction of several growth factors from Matrigel, HGF was still unable to induce process formation, an early event in tubulogenesis, indicating that one or more of the remaining extracellular matrix (ECM) proteins or growth factors were exerting the inhibitory effect. By individually adding back these components to MDCK cells grown in Type I collagen gels in the presence of HGF, we were able to establish that: (1) certain ECM proteins, such as laminin, entactin, and fibronectln, actually facilitated the formation of branching tubular structures and increased their complexity; (2) other ECM proteins, such as Type IV collagen, heparan sulfate proteoglycan, and vitronectin, caused marked inhibition of HGF-induced morphogenesis; and (3) not only did transforming growth factor-β (TGF-β) inhibit the formation of tubular structures, but those which did form exhibited little branching, thereby suggesting that TGF-β modulates tubulogenesis as well as branching. These results suggest that a tubulogenic morphogen such as HGF and a tubulogenesis-inhibitory morphogen such as TGF-β can, in the context of the dynamic matrix known to exist during epithelial tissue development, modulate the degree of tubule (or ductal) formation, the length of these tubules, and the extent of their arborization. The relevance of these findings to tubulogenesis and branching during kidney development is discussed.     

Nucleotide exchange factor ECT2 regulates epithelial cell polarity

Cell polarity regulates diverse biological events such as localization of embryonic determinants and establishment of tissue and organ architecture. Epithelial cell polarity is regulated by the polarity complex Par6/Par3/atypical protein kinase C (aPKC). We previously found that the nucleotide exchange factor ECT2 associates with this polarity complex and regulates aPKC activity, but the role of ECT2 in cell polarity is still unclear. Here we show that expression of a dominant negative (ECT2-N2) or constitutively active (ECT2-DeltaN5) form of ECT2 inhibits normal cyst formation of MDCK cells in 3-dimensional collagen gels. Central lumens were not observed in cysts formed by cells expressing either ECT2-DeltaN5 or ECT2-N2. Apical localization of ZO-1 and basolateral localization of beta-catenin were no longer observed in these cells. Interestingly, cells expressing ECT2-N2 did form normal cysts when cultured in the basement membrane matrix Matrigel instead of collagen gels. Addition of a major Matrigel component, laminin, partially rescued the normal cyst formation inhibited by ECT2-N2 in 3-dimensional collagen gels. Thus, signaling through laminin might override the defects of signaling through collagen and ECT2. Whereas ECT2-N2 inhibited the lumen formation of MDCK cysts, caspase-3, which is reportedly involved in lumen formation through apoptosis, was activated at various locations of cells in the cysts. It is likely that perturbation of ECT2 signaling inhibits the establishment of epithelial cell polarity leading to the inhibition of selected elimination of cells at the center of cysts. Thus, ECT2 appears to play a critical role in epithelial cell polarity.     

Type I collagen gel induces Madin-Darby canine kidney cells to become fusiform in shape and lose apical-basal polarity

In the embryo, epithelia give rise to mesenchyme at specific times and places. Recently, it has been reported (Greenburg, G., and E. D. Hay. 1986. Dev. Biol. 115:363-379; Greenberg, G., and E. D. Hay. 1988. Development (Camb.). 102:605-622) that definitive epithelia can give rise to fibroblast-like cells when suspended within type I collagen gels. We wanted to know whether Madin-Darby canine kidney (MDCK) cells, an epithelial line, can form mesenchyme under similar conditions. Small explants of MDCK cells on basement membrane were suspended within or placed on top of extracellular matrix gels. MDCK cells on basement membrane gel are tall, columnar in shape, and ultrastructurally resemble epithelia transporting fluid and ions. MDCK explants cultured on type I collagen gel give rise to isolated fusiform-shaped cells that migrate over the gel surface. The fusiform cells extend pseudopodia and filopodia, lose cell membrane specializations, and develop an actin cortex around the entire cell. Unlike true mesenchymal cells, which express vimentin and type I collagen, fusiform cells produce both keratin and vimentin, continue to express laminin, and do not turn on type I collagen. Fusiform cells are not apically-basally polarized, but show mesenchymal cell polarity. Influenza hemagglutinin and virus budding localize to the front end or entire cell surface. Na,K-ATPase occurs intracellularly and also symmetrically distributes on the cell surface. Fodrin becomes diffusely distributed along the plasma membrane, ZO-1 cannot be detected, and desmoplakins distribute randomly in the cytoplasm. The loss of epithelial polarity and acquisition of mesenchymal cell polarity and shape by fusiform MDCK cells on type I collagen gel was previously unsuspected. The phenomenon may offer new opportunities for studying cytoplasmic and nuclear mechanisms regulating cell shape and polarity.     

Galectin-3 and polarized growth within collagen gels of wild-type and ricin-resistant MDCK renal epithelial cells

Previous studies (Q. Bao and R. C. Hughes (1995) J. Cell Sci., 108, 2791-2800) showed that the beta-galactoside-binding protein, galectin-3, is secreted onto the basolateral surface domains of Madin-Darby canine kidney MDCK cells growing as polarized cysts within a collagen gel. The growth and enlargement of such cysts were shown to be increased significantly when cultured in the presence of antibodies directed against the lectin and were slowed down by addition of exogenous galectin-3. These results suggested a role for galectin-3, interacting with appropriately glycosylated surface receptors, as a negative growth regulator in the development of MDCK cysts, a well-known model for renal epithelial morphogenesis. In the present report we have tested this proposal by use of a ricin-resistant mutant of MDCK cells that is unable to transfer galactose residues during biosynthesis of cellular glycoconjugates and hence lacks extracellular receptors for galectin-3. We find that when grown within collagen gels, the mutant cell cysts grow significantly faster than wild-type cell cysts. Furthermore, they form nonspherical and tubular cysts that are induced in wild-type cell cysts only under the influence of the morphogen, hepatocyte growth factor (HGF).     

Dissecting tumor cell invasion: epithelial cells acquire invasive properties after the loss of uvomorulin-mediated cell-cell adhesion

The generation of invasiveness in transformed cells represents an essential step of tumor progression. We show here, first, that nontransformed Madin-Darby canine kidney (MDCK) epithelial cells acquire invasive properties when intercellular adhesion is specifically inhibited by the addition of antibodies against the cell adhesion molecule uvomorulin; the separated cells then invade collagen gels and embryonal heart tissue. Second, MDCK cells transformed with Harvey and Moloney sarcoma viruses are constitutively invasive, and they were found not to express uvomorulin at their cell surface. These data suggest that the loss of adhesive function of uvomorulin (which is identical to E-cadherin and homologous to L-CAM) is a critical step in the promotion of epithelial cells to a more malignant, i.e., invasive, phenotype. Similar modulation of intercellular adhesion might also occur during invasion of carcinoma cells in vivo.     

HH2A,an immortalized bovine mammary epithelial cell line,expresses the gene encoding mammary derived growth inhibitor (MDGI)

Summary We have established and partially characterized a spontaneously immortalized bovine mammary epithelial cell line, designated HH2a. The cells express the gene encoding for mammary derived growth inhibitor (MDGI) when grown on released collagen gels in the presence of lactogenic hormones. This is the first report of a cell line that expresses MDGI. Immunohistochemical studies showed that HH2a cells contain keratin intermediate filaments and desmosomes. When plated on confluent monolayer of live fibroblasts, HH2a cells extensively contacted with fibroblasts. When embedded in the collagen gels, they rearranged themselves to produce three-dimensional duct-like outgrowths extending into the matrix. The HH2a cell line should be useful in investigations of the roles of cell-cell and cell-extracellular interactions in regulation of breast epithelial cell proliferation, and of the hormonal regulation of MDGI gene expression.     

ROCK-generated contractility regulates breast epithelial cell differentiation in response to the physical properties of a three-dimensional collagen matrix

Breast epithelial cells differentiate into tubules when cultured in floating three-dimensional (3D) collagen gels, but not when the cells are cultured in the same collagen matrix that is attached to the culture dish. These observations suggest that the biophysical properties of collagenous matrices regulate epithelial differentiation, but the mechanism by which this occurs is unknown. Tubulogenesis required the contraction of floating collagen gels through Rho and ROCK-mediated contractility. ROCK-mediated contractility diminished Rho activity in a floating 3D collagen gel, and corresponded to a loss of FAK phosphorylated at Y397 localized to 3D matrix adhesions. Increasing the density of floating 3D collagen gels also disrupted tubulogenesis, promoted FAK phosphorylation, and sustained high Rho activity. These data demonstrate the novel finding that breast epithelial cells sense the rigidity or density of their environment via ROCK-mediated contractility and a subsequent down-regulation of Rho and FAK function, which is necessary for breast epithelial tubulogenesis to occur.     

Mammary gland morphogenesis in vitro: Extracellular requirements for the formation of tubules in collagen gels by a cloned rat mammary epithelial cell line

Summary The mechanism of induction of tubular outgrowths in vitro on floating collagen gels and the influence of extracellular factors on this process have been investigated using the clonal rat mammary epithelial cell line, Rama 25. Growth of Rama 25 on such floating gels causes their contraction. Contraction of the gel is accompanied by a 10-fold increase in the number of cells per unit area, a change in cell shape, and a convolution of the epithelial cell sheet. Gels folded over manually show an 11-times higher incidence of tubules along the folds than on the flat surface. Tubular formation begins when cords of cells develop from local proliferations of the cell sheet and become canalized. Tubules follow wrinkles in the gel and branch to yield monopodial, dichotomous, or lobular architecture. Hydrocortisone and insulin, in the presence of serum, stimulate both narrow and thick tubular structures on folded gels, whereas extra additions of 1 ng/ml cholera toxin or 100 ng/ml epidermal growth factor preferentially stimulate thick tubular structures. Floating glutaraldehyde-fixed gels, very thick collagen gels, and collagen gels prepared on the top of rigid steel grids fail to support the formation of tubules, suggesting that flexibility and access of the medium to basal surfaces are important to their genesis. Incorporation of hyaluronic acid into the gel matrix preferentially inhibits the thick tubular outgrowths. Thus, the branching tubular structures generated by Rama 25 can be influenced in different ways by various extracellular factors in the medium and in the gel. During the course of this work E. J. Ormerod was in receipt of a Ludwig Research Studentship.     

Low concentrations of transforming growth factor-beta-1 induce tubulogenesis in cultured mammary epithelial cells

Background  

Formation of branching tubes is a fundamental step in the development of glandular organs. To identify extracellular cues that orchestrate epithelial tubulogenesis, we employed an in vitro assay in which EpH4-J3B1A mammary epithelial cells form spheroidal cysts when grown in collagen gels under serum-free conditions, but form branching tubules in the presence of fetal calf serum (FCS).     

Differential Sensitivity of Epithelial Cells to Extracellular Matrix in Polarity Establishment

Establishment of apical-basal polarity is crucial for epithelial sheets that form a compartment in the body, which function to maintain the environment in the compartment. Effects of impaired polarization are easily observed in three-dimensional (3-D) culture systems rather than in two-dimensional (2-D) culture systems. Although the mechanisms for establishing the polarity are not completely understood, signals from the extracellular matrix (ECM) are considered to be essential for determining the basal side and eventually generating polarity in the epithelial cells. To elucidate the common features and differences in polarity establishment among various epithelial cells, we analyzed the formation of epithelial apical-basal polarity using three cell lines of different origin: MDCK II cells (dog renal tubules), EpH4 cells (mouse mammary gland), and R2/7 cells (human colon) expressing wild-type α-catenin (R2/7 α-Cate cells). These cells showed clear apical-basal polarity in 2-D cultures. In 3-D cultures, however, each cell line displayed different responses to the same ECM. In MDCK II cells, spheroids with a single lumen formed in both Matrigel and collagen gel. In R2/7 α-Cate cells, spheroids showed similar apical-basal polarity as that seen in MDCK II cells, but had multiple lumens. In EpH4 cells, the spheroids displayed an apical-basal polarity that was opposite to that seen in the other two cell types in both ECM gels, at least during the culture period. On the other hand, the three cell lines showed the same apical-basal polarity both in 2-D cultures and in 3-D cultures using the hanging drop method. The three lines also had similar cellular responses to ECM secreted by the cells themselves. Therefore, appropriate culture conditions should be carefully determined in advance when using various epithelial cells to analyze cell polarity or 3-D morphogenesis.     

Cell swelling stimulates cytosol to membrane transposition of ICln

ICln is a multifunctional protein that is essential for cell volume regulation. It can be found in the cytosol and is associated with the cell membrane. Besides its role in the splicing process, ICln is critically involved in the generation of ion currents activated during regulatory volume decrease after cell swelling (RVDC). If reconstituted in artificial bilayers, ICln can form ion channels with biophysical properties related to RVDC. We investigated (i) the cytosol versus cell membrane distribution of ICln in rat kidney tubules, NIH 3T3 fibroblasts, Madin-Darby canine kidney (MDCK) cells, and LLC-PK1 epithelial cells, (ii) fluorescence resonance energy transfer (FRET) in living fibroblasts between fluorescently tagged ICln and fluorochromes in the cell membrane, and (iii) possible functional consequences of an enhanced ICln presence at the cell membrane. We demonstrate that ICln distribution in rat kidneys depends on the parenchymal localization and functional state of the tubules and that cell swelling causes ICln redistribution from the cytosol to the cell membrane in NIH 3T3 fibroblasts and LLC-PK1 cells. The addition of purified ICln protein to the extracellular solution or overexpression of farnesylated ICln leads to an increased anion permeability in NIH 3T3 fibroblasts. The swelling-induced redistribution of ICln correlates to altered kinetics of RVDC in NIH 3T3 fibroblasts, LLC-PK1 cells, and MDCK cells. In these cells, RVDC develops more rapidly, and in MDCK cells the rate of swelling-induced depolarization is accelerated if cells are swollen for a second time. This coincides with an enhanced ICln association with the cell membrane.     

Scatter factor: molecular characteristics and effect on the invasiveness of epithelial cells

The generation of invasiveness in transformed cells represents an essential step of tumor progression. We have previously shown that MDCK epithelial cells, which are deprived of intracellular adhesion by the addition of anti-Arc-1/uvomorulin antibodies, become invasive for collagen gels and embryonal heart tissue (Behrens, J., M. M. Mareel, F. M. Van Roy, and W. Birchmeier. 1989. J. Cell Biol. 108: 2435-2447.). Here we examined whether invasiveness is also induced by scatter factor, which is known to dissociate epithelial cells (Stoker, M., E. Gherardi, M. Perryman, and J. Gray. 1987. Nature (Lond.). 327:239- 242.). Scatter factor was purified to homogeneity from conditioned medium of human fibroblasts by heparin-Sepharose chromatography, followed by cation exchange chromatography, gel filtration, or preparative SDS gel electrophoresis. We found that scatter factor represents a 92,000 mol wt glycoprotein which, apparently, is converted by limited proteolysis into disulfide-linked 62,000 and 34/32,000 mol wt subunits. Reversed phase HPLC and sequence analysis of tryptic peptides confirmed the suggested molecular structure, and revealed further that scatter factor exhibits sequence similarities to hepatocyte growth factor and to plasminogen. Purified scatter factor in fact induces the invasiveness into collagen matrices of MDCK epithelial cells, and induces or promotes the invasiveness of a number of human carcinoma cell lines. Apparently, the effect on the human cells depends on their respective degree of differentiation, i.e., cell lines with a less pronounced epithelial phenotype were more susceptible to the factor. Scatter factor does not seem to influence synthesis, steady- state level, and phosphorylation of the cell adhesion molecule Arc- 1/uvomorulin. Thus, scatter factor represents a clearly defined molecular species which induces, in vitro, the progression of epithelial cells to a more motile, i.e., invasive phenotype.     

Maintenance and induction of morphological differentiation in dissociated mammary epithelium on floating collagen membranes

Dissociated normal mammary epithelial cells from prelactating mice were plated on different substrates in various medium-serum-hormone combinations to find conditions that would permit maintenance of morphological differentiation. Cells cultured on floating collagen membranes in medium containing insulin, hydrocortisone and prolactin maintain differentiation through 1 month in culture. The surface cells form a continous epithelial pavement. Some epithelial cells below the surface layer rearrange themselves to form alveolus-like structures. Cells at both sites display surface polarization; microvilli and tight junctions are present at their medium-facing of luminal surface and a basal lamina separates the epithelial components from the gel and stromal cells. Occasional myoepithelial cells, characterized by myofilaments and plasmalemmmal vesicles, are identified at the basal surface of the secretory epithelium. In contrast, cells cultured on plastic, glass or collagen gels attached to Petri dishes form a confluent epithelial sheet showing surface polarization, but lose secretory and myoepithelial specializations. If these dedifferentiated cells are subsequently maintained on floating collagen membranes, they redifferentiate. There is little DNA synthesis in cells on collagen gels, in contrast to Petri-dish controls. Protein synthesis in cells on floating collagen membranes increases over TO values and remains constant through 7 days in culture whereas it decreases on attached gels; however, if the gels are freed to float, protein synthesis increases sharply and parallels that seen on floating membranes.     

Maintenance and induction of morphological differentiation in dissociated mammary epithelium on floating collagen membranes

Summary Dissociated normal mammary epithelial cells from prelactating mice were plated on different substrates in various medium-serum-hormone combinations to find conditions that would permit maintenance of morphological differentiation. Cells cultured on floating collagen membranes in medium containing insulin, hydrocortisone and prolactin maintain differentiation through 1 month in culture. The surface cells form a continuous epithelial pavement. Some epithelial cells below the surface layer rearrange themselves to form alveolus-like structures. Cells at both sites display surface polarization; microvilli and tight junctions are present at their medium-facing or luminal surface and a basal lamina separates the epithelial components from the gel and stromal cells. Occasinal myoepithelial cells, characterized by myofilaments and plasmalemmal vesicles, are identified at the basal surface of the secretory epithelium. In contrast, cells cultured on plastic, glass or collagen gels attached to Petri dishes form a confluent epithelial sheet showing surface polarization, but lose secretory and myoepithelial specializations. If these dedifferentiated cells are subsequently maintained on floating collagen membranes, they redifferentiate. There is little DNA synthesis in cells on collagen gels, in contrast to Petri-dish controls. Protein synthesis in cells on floating collagen membranes increases over T₀ values and remains constant through 7 days in culture whereas it decreases on attached gels; however, if the gels are freed to float, protein synthesis increases sharply and parallels that seen on floating membranes. The work was supported by USPHS Grants CA-05388 and CA-05045 from the National Cancer Institute, DHEW.     

Cross-talk between mesenchyme and epithelium increases H19 gene expression during scattering and morphogenesis of epithelial cells

The H19 gene is an imprinted gene expressed from the maternal allele. It is known to function as an RNA molecule. We previously reported that in breast adenocarcinoma, H19 is often overexpressed in stromal cells and preferentially located at the epithelium/stroma boundary, suggesting that epithelial/mesenchymal interactions can control H19 RNA expression. In some cases of breast adenocarcinoma with poor prognosis, H19 is overexpressed in epithelial cells. Therefore we examined whether mesenchymal factors can induce H19 expression in epithelial cells. Using quantitative RT-PCR and in situ hybridization, we found that when mammary epithelial cells were cultured in collagen gels, H19 expression was strongly up-regulated compared to when cells were cultured on plastic. Collagen gels allow three-dimensional growth of epithelial cells and morphogenetic responses to soluble factors. A conditioned medium from MRC-5 fibroblasts caused branching morphogenesis of HBL-100 cells and invasive growth of MDA-MB-231 cells, whereas MCF-7 cells were unresponsive. Induction of H19 expression correlated with morphological changes in HBL-100 and in MDA-MB-231 cells, whereas H19 expression was not induced in MCF-7 cells. Using a blocking antibody, HGF/SF was identified as the fibroblast-derived growth factor capable of inducing H19 expression and cell morphogenesis. We further demonstrated that H19 promoter activity was stimulated by various growth factors using transient transfection in MDCK epithelial cells. HGF/SF was more efficient than EGF or FGF-2 in transactivating the H19 promoter, whereas IGF-2, TGFbeta-1, and TNF-alpha were ineffective. This activation by HGF/SF was prevented by pharmacological inhibition of MAP kinase or of phospholipase C. We conclude that H19 is a target gene for HGF/SF, a known regulator of epithelial/mesenchymal interactions, and suggest that the up-regulation of H19 may be implicated in morphogenesis and/or migration of epithelial cells.     

Constitutively active mitogen-activated protein kinase kinase MEK1 disrupts morphogenesis and induces an invasive phenotype in Madin-Darby canine kidney epithelial cells.

During certain developmental processes, as well as during tumor progression, polarized epithelial cells integrated within multicellular structures convert into scattered, freely migrating fibroblast-like cells. Despite the biological and clinical importance of this phenomenon, the intracellular biochemical cascades that control the switch between the epithelial and mesenchymal phenotypes have not been elucidated. Using Madin-Darby canine kidney (MDCK) cells (clone C7) as a model system, we have assessed the potential role of the mitogen-activated protein kinase (MAPK)/ extracellular signal-regulated kinase (ERK) cascade in the modulation of epithelial plasticity. When grown in three-dimensional collagen gels, MDCK-C7 cells form spherical cysts composed of polarized epithelial cells circumscribing a central lumen. This morphogenetic behavior is profoundly subverted in MDCK-C7 cells expressing a constitutively active MAPK/ERK kinase 1 (caMEK1) mutant (C7-caMEK1 cells). When suspended in collagen gels, C7-caMEK1 cells assume an elongated fibroblastoid shape and are unable to generate multicellular cysts. In addition, when seeded onto the surface of a collagen gel, C7-caMEK1 cells penetrate extensively into the underlying matrix, unlike wild-type and mock-transfected MDCK-C7 cells, which remain confined to the surface of the gel. Similar changes in morphogenetic and invasive properties are observed in MDCK-C7F cells, a nontransfected, stably dedifferentiated derivative of MDCK-C7 cells that expresses substantially increased ERK2 activity. Both C7-caMEK1 and MDCK-C7F cells but not wild-type or mock-transfected MDCK-C7 cells express activated M(r) 72,000 gelatinase A [matrix metalloproteinase (MMP)-2] as well as elevated levels of membrane type-1 MMP. Synthetic MMP inhibitors as well as recombinant tissue inhibitor of metalloproteinases 2 and 3 suppress the invasion of collagen gels and restore the capacity of C7-caMEK1 cells to form cysts, thereby implicating the membrane type-1 MMP/MMP-2 proteolytic system in epithelial cell invasiveness and loss of multicellular organization. Taken together, our data demonstrate that increased activity of the MEK1-ERK2 signaling module in MDCK-C7 cells is associated with failure of morphogenesis and expression of a highly invasive phenotype. Sustained activation of the MAPK cascade therefore results in the destabilization of the three-dimensional architecture and the conversion of polarized epithelial cells into migrating mesenchymal-like cells.     

In vivo growth stimulation of collagen gel embedded normal human and mouse primary mammary epithelial cells

A new system for studying growth of normal human mammary epithelial cells in an in vivo environment using athymic nude mice is described. Human mammary epithelial cells dissociated from reduction mammoplasty specimens were embedded within collagen gels and subsequently transplanted subcutaneously into nude mice. Histological sections of recovered collagen gels showed epithelial cells arranged as short tubules with some branching. Proliferation of mammary epithelial cells was quantitated in vivo by 3 days' continuous infusion with 5 bromo-2′-deoxy-uridine followed by immunostaining of sections from recovered gels. Ovarian steroids administered to the host animals, resulting in blood serum levels normally found in the human female, had little or no effect on the proliferation of human mammary epithelial cells. Collagen gel embedded mouse mammary epithelial cells, mouse mammary explants, and host mammary glands all responded similarly to ovarian steroids, suggesting that the unresponsiveness of the human mammary epithelial cells under these conditions was not due to dissociation per se. However, an increased dose of 17β-estradiol or a growth factor combination containing epidermal growth factor, cholera toxin, and cortisol significantly stimulated the proliferation of human outgrowths. The growth factor response was dependent on the location of the cells, with the greatest response seen in the part of the gel proximal to the osmotic pump delivering the growth factors and the effect gradually waning in area more distal to the pump. The effect was especially striking since the mitotic figures could be easily identified and the labeling index was as high as 75%. The host mouse mammary gland also responded to growth factors, resulting in ductal hyperplasia. The proliferative and morphogenetic effects of various agents on normal human mammary epithelial cells embedded in collagen gel can be studied in vivo in nude mice. © 1995 Wiley-Liss, Inc.