Characterisation of FAP-1 expression and CD95 mediated apoptosis in the A818-6 pancreatic adenocarcinoma differentiation system

The present study investigated the expression and localisation of FAP-1 (Fas associated phosphatase-1) and CD95 in a 3D differentiation model in comparison to 2D monolayers of the pancreatic adenocarcinoma cell line A818-6. Under non-adherent growth conditions, A818-6 cells differentiate into 3D highly organised polarised epithelial hollow spheres, resembling duct-like structures. A818-6 cells showed a differentiation-dependent FAP-1 localisation. Cells grown as 2D monolayers revealed FAP-1 staining in a juxtanuclear cisternal position, as well as localisation in the nucleus. After differentiation into hollow spheres, FAP-1 was relocated towards the actin cytoskeleton beneath the outer plasma membrane of polarised cells and no further nuclear localisation was observed. CD95 surface staining was found only in a subset of A818-6 monolayer cells, while differentiated hollow spheres appeared to express CD95 in all cells of a given sphere. We rarely observed co-localisation of CD95 and FAP-1 in A818-6 monolayer cells, but strong co-localisation beneath the outer plasma membrane in polarised cells. Analysis of surface expression by flow cytometry revealed that only a subset (36%) of monolayer cells showed CD95 surface expression, and after induction of hollow spheres, CD95 presentation at the outer plasma membrane was reduced to 13% of hollow spheres. Induction of apoptosis by stimulation with agonistic anti-CD95 antibodies, resulted in increased caspase activity in both, monolayer cells and hollow spheres. Knock down of FAP-1 mRNA in A818-6 monolayer cells did not alter resposiveness to CD95 agonistic antibodies. These data suggested that CD95 signal transduction was not affected by FAP-1 expression in A818-6 monolayer cells. In differentiated 3D hollow spheres, we found a polarisation-induced co-localisation of CD95 and FAP-1. A tight control of receptor surface representation and signalling induced apoptosis ensures controlled removal of individual cells instead of a "snowball effect" of apoptotic events.     

Epimorphin Functions as a Key Morphoregulator for Mammary Epithelial Cells

Hepatocyte growth factor (HGF) and EGF have been reported to promote branching morphogenesis of mammary epithelial cells. We now show that it is epimorphin that is primarily responsible for this phenomenon. In vivo, epimorphin was detected in the stromal compartment but not in lumenal epithelial cells of the mammary gland; in culture, however, a subpopulation of mammary epithelial cells produced significant amounts of epimorphin. When epimorphin-expressing epithelial cell clones were cultured in collagen gels they displayed branching morphogenesis in the presence of HGF, EGF, keratinocyte growth factor, or fibroblast growth factor, a process that was inhibited by anti-epimorphin but not anti-HGF antibodies. The branch length, however, was roughly proportional to the ability of the factors to induce growth. Accordingly, epimorphin-negative epithelial cells simply grew in a cluster in response to the growth factors and failed to branch. When recombinant epimorphin was added to these collagen gels, epimorphin-negative cells underwent branching morphogenesis. The mode of action of epimorphin on morphogenesis of the gland, however, was dependent on how it was presented to the mammary cells. If epimorphin was overexpressed in epimorphin-negative epithelial cells under regulation of an inducible promoter or was allowed to coat the surface of each epithelial cell in a nonpolar fashion, the cells formed globular, alveoli-like structures with a large central lumen instead of branching ducts. This process was enhanced also by addition of HGF, EGF, or other growth factors and was inhibited by epimorphin antibodies. These results suggest that epimorphin is the primary morphogen in the mammary gland but that growth factors are necessary to achieve the appropriate cell numbers for the resulting morphogenesis to be visualized.     

Mesenchymal epimorphin is important for pancreatic duct morphogenesis

Epithelial-mesenchymal interactions are crucial for the proper development of many organs, including the pancreas. Within the pancreas, the ducts are thought to harbor stem/progenitor cells, and possibly to give rise to pancreatic ductal carcinoma. Little is known about the mechanism of formation of pancreatic ducts in the embryo. Pancreatic mesenchyme contains numerous soluble factors which help to sustain the growth and differentiation of exocrine and endocrine structures. Here, we report that one such morphoregulatory mesenchymal protein, epimorphin, plays an important role during pancreatic ductal proliferation and differentiation. We found that epimorphin is expressed in pancreatic mesenchyme during early stages of development, and at mesenchymal-epithelial interfaces surrounding the ducts at later stages. Strong upregulation of epimorphin expression was seen during in vitro pancreatic duct differentiation. Similarly, in vitro pancreatic duct formation was inhibited by a neutralizing antibody against epimorphin, whereas addition of recombinant epimorphin partially rescued duct formation. Together, our study demonstrates the role of epimorphin in pancreatic ductal morphogenesis.     

Antibodies against domain E3 of laminin-1 and integrin alpha 6 subunit perturb branching epithelial morphogenesis of submandibular gland, but by different modes

Branching epithelial morphogenesis requires interactions between the surrounding mesenchyme and the epithelium, as well as interactions between basement membrane components and the epithelium. Embryonic submandibular gland was used to study the roles of two mesenchymal proteins, epimorphin and tenascin-C, as well as the epithelial protein laminin-1 and one of its integrin receptors on branching morphogenesis. Laminin-1 is a heterotrimer composed of an alpha 1 chain and two smaller chains (beta 1 and gamma 1). Immunofluorescence revealed a transient expression of laminin alpha 1 chain in the epithelial basement membrane during early stages of branching morphogenesis. Other laminin-1 chains and alpha 6, beta 1, and beta 4 integrin subunits seemed to be expressed constitutively. Expression of epimorphin, but not tenascin-C, was seen in the mesenchyme during early developmental stages, but a mAb against epimorphin did not perturb branching morphogenesis of this early epithelium. In contrast, inhibition of branching morphogenesis was seen with a mAb against the carboxy terminus of laminin alpha 1 chain, the E3 domain. An inhibition of branching was also seen with a mAb against the integrin alpha 6 subunit. The antibodies against laminin alpha 1 chain and integrin alpha 6 subunit perturbed development in distinct fashions. Whereas treatment with the anti-E3 resulted in discontinuities of the basement membrane at the tips of the branching epithelium, treatment with the mAb against alpha 6 integrin subunit seemed to leave the basement membrane intact. We suggest that the laminin E3 domain is involved in basement membrane formation, whereas alpha 6 beta 1 integrin binding to laminin-1 may elicit differentiation signals to the epithelial cells.     

The interplay of matrix metalloproteinases, morphogens and growth factors is necessary for branching of mammary epithelial cells.

The mammary gland develops its adult form by a process referred to as branching morphogenesis. Many factors have been reported to affect this process. We have used cultured primary mammary epithelial organoids and mammary epithelial cell lines in three-dimensional collagen gels to elucidate which growth factors, matrix metalloproteinases (MMPs) and mammary morphogens interact in branching morphogenesis. Branching stimulated by stromal fibroblasts, epidermal growth factor, fibroblast growth factor 7, fibroblast growth factor 2 and hepatocyte growth factor was strongly reduced by inhibitors of MMPs, indicating the requirement of MMPs for three-dimensional growth involved in morphogenesis. Recombinant stromelysin 1/MMP3 alone was sufficient to drive branching in the absence of growth factors in the organoids. Plasmin also stimulated branching; however, plasmin-dependent branching was abolished by both inhibitors of plasmin and MMPs, suggesting that plasmin activates MMPs. To differentiate between signals for proliferation and morphogenesis, we used a cloned mammary epithelial cell line that lacks epimorphin, an essential mammary morphogen. Both epimorphin and MMPs were required for morphogenesis, but neither was required for epithelial cell proliferation. These results provide direct evidence for a crucial role of MMPs in branching in mammary epithelium and suggest that, in addition to epimorphin, MMP activity is a minimum requirement for branching morphogenesis in the mammary gland.     

In vitro cytodifferentiation of perinatal rat islet cells within a tridimensional matrix of collagen

Summary Cell suspensions prepared by collagenase digestion of pancreases obtained from rat fetuses (21.5 d old) and newborns (2.5 d old) were mixed with a collagen solution and inoculated on a collagen base layer. At the onset of the culture, most acinar cells became necrotic, whereas other epithelial cells proliferated. Most of the cell clusters arranged themselves into simple polarozed structures composed of epithelial cells forming hollow spheres, and from these budded neoformed endocrine islets. Scarce fibroblasts were located close to these structures. Immunocytochemical localization of insulin and glucagon, as well as ultrastructural characteristics of the cell types revealed an intrainsular distribution similar to the in vivo localization. Tridimensional matrix of collagen offers, to perinatal pancreatic cells in culture, an environment close to the in vivo conditions: cells reorganize themselves in tissuelike structures and cell interactions concerned in the cytodifferentiation of pancreatic islets occur. This system allows for the study of undifferentiated epithelial cells—the presumed stem cells—differentiating and differentiated endocrine cells in the same preparation. B.A. is supported by a doctoral scholarship from the Institut pour l'Encouragement de la Recherche Scientifique dans l'Industrie et l'Agriculture, Brussels. This work was supported by grants from the Fonds National de la Recherche Scientifique, Brussels, and from Petrofina S.A., Brussels.     

In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

The pancreas is an essential organ that regulates glucose homeostasis and secretes digestive enzymes. Research on pancreas embryogenesis has led to the development of protocols to produce pancreatic cells from stem cells ¹. The whole embryonic organ can be cultured at multiple stages of development ^2-4. These culture methods have been useful to test drugs and to image developmental processes. However the expansion of the organ is very limited and morphogenesis is not faithfully recapitulated since the organ flattens. We propose three-dimensional (3D) culture conditions that enable the efficient expansion of dissociated mouse embryonic pancreatic progenitors. By manipulating the composition of the culture medium it is possible to generate either hollow spheres, mainly composed of pancreatic progenitors expanding in their initial state, or, complex organoids which progress to more mature expanding progenitors and differentiate into endocrine, acinar and ductal cells and which spontaneously self-organize to resemble the embryonic pancreas. We show here that the in vitro process recapitulates many aspects of natural pancreas development. This culture system is suitable to investigate how cells cooperate to form an organ by reducing its initial complexity to few progenitors. It is a model that reproduces the 3D architecture of the pancreas and that is therefore useful to study morphogenesis, including polarization of epithelial structures and branching. It is also appropriate to assess the response to mechanical cues of the niche such as stiffness and the effects on cell´s tensegrity.     

Morphogenic protein epimorphin protects intestinal epithelial cells from oxidative stress by the activation of EGF receptor and MEK/ERK, PI3 kinase/Akt signals

Epimorphin is a mesenchymal protein that regulates morphogenesis of epithelial cells. Our preliminary study suggested a novel function of epimorphin in enhancing survival of intestinal epithelial cells (IEC). Oxidative stress leads to cell injury and death and is suggested to be a key contributor to pathogenesis of inflammatory bowel disease. This study was conducted to determine whether epimorphin protects IEC from oxidative stress. Rat intestinal epithelial cell line IEC-6 was cultured with epimorphin (10 and 20 mug/ml), and the life span of IEC was assessed. The mean life span of IEC-6 cells was prolonged 1.9-fold (P < 0.0006) by treatment with epimorphin. We then examined the epimorphin signaling pathways. Epimorphin phosphorylated epidermal growth factor (EGF) receptor, activated the MEK/extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase and phosphatidylinositol 3 (PI3) kinase/Akt pathways, phosphorylated Bad, and induced Bcl-X(L) and survivin. Hydrogen peroxide (1 mM) induced cell death in 92% of IEC-6 cells, but epimorphin dramatically diminished (88.7%) cell death induced by hydrogen peroxide (P < 0.0001). This protective effect of epimorphin was significantly attenuated by inhibitors of MEK and PI3 kinase (P < 0.0001) or EGF receptor-neutralizing antibody (P = 0.0007). In wound assays, the number of migrated cells in the wound area decreased (72.5%) by treatment with 30 muM hydrogen peroxide, but epimorphin increased the number of migrated cells 3.18-fold (P < 0.0001). These results support a novel function of epimorphin in protecting IEC from oxidative stress. This anti-oxidative function of epimorphin is dramatic and is likely mediated by the activation of EGF receptors and the MEK/extracellular signal-regulated kinase and PI3 kinase/Akt signaling pathways and through the induction of anti-apoptotic factors.     

Delivering the message: epimorphin and mammary epithelial morphogenesis

The mammary gland consists of a highly branched tubular epithelium surrounded by a complex mesenchymal stroma. Epimorphin is an extracellular protein that is expressed by mammary mesenchymal cells that directs epithelial morphogenesis. Depending upon the context of presentation--polar versus apolar--epimorphin can selectively direct two key processes of tubulogenesis: branching morphogenesis (processes involved in tubule initiation and extension) and luminal morphogenesis (required for enlargement of tubule caliber). Here, we outline the fundamentals of mammary gland development and describe the function of epimorphin in these processes. We conclude with a review of recent studies that suggest similar morphogenic roles for epimorphin in other glandular organs.     

Changes in the distribution of the 34-kdalton tyrosine kinase substrate during differentiation and maturation of chicken tissues

We examined the distribution of the 34-kilodalton (34-kD) tyrosine kinase substrate in tissues of adult and embryonic chicken using both a mouse monoclonal antibody and a rabbit polyclonal antibody raised against the affinity purified 34 kD protein. We analyzed the localization by immunoblotting of tissue extracts, by immunofluorescence staining of frozen tissue sections, and by staining sections of paraffin-embedded organs by the peroxidase antiperoxidase method. The 34-kD protein was present in a variety of cells, including epithelial cells of the skin, gastrointestinal, and respiratory tracts, as well as in fibroblasts and chondrocytes of connective tissue and mature cartilage, and endothelial cells of blood vessels. The 34-kD protein was also found in subpopulations of cells in thymus, spleen, bone marrow, and bursa. The protein was not detected in cardiac, skeletal, or smooth muscle cells, nor in epithelial cells of liver, kidney, pancreas, and several other glands. Although most neuronal cells did not contain the 34-kD protein, some localized brain regions did contain detectable amounts of this protein. The 34-kD protein was not detected in actively dividing cells of a number of tissues. Changes in the distribution of the 34-kD protein were observed during the differentiation or maturation of cells in several tissues including epithelial cells of the skin and gastrointestinal tract, fibroblasts of connective tissue, and chondroblasts.     

Factors affecting morphogenesis of rabbit gallbladder epithelial cells cultured in collagen gels

Although peptide growth factors play an important role in the morphogenesis of gallbladder, little is known about how they effect the morphogenesis of gallbladder epithelial cells. Rabbit gallbladder epithelial cells (RGEC) were isolated and cultured in monolayer or collagen gels. Epidermal growth factor (EGF), hepatocyte growth factor (HGF), epimorphin, transforming growth factor-beta 1 (TGF-beta 1), and fibroblast-conditioned medium (FCM) were added to the cultured cells to clarify the effects of these peptides and FCM on morphogenesis of RGEC. RGEC suspended in collagen gels form spherical cysts with morphologic polarity. EGF, HGF, epimorphin, and FCM promoted cyst maturation by accelerating the proliferation and aggregation of clear, polarized vesicles. In contrast, TGF-beta 1 markedly inhibited DNA synthesis in both monolayer and collagen gel cultures and promoted formation of branching structures in collagen gels. Furthermore, in the presence of EGF, TGF-beta 1 induced a drastic change in morphogenesis, with the formation of branching networks that showed cell-cell contact only at sites where branches touched. RGEC-forming multicellular cysts did not express vimentin but expressed significant amounts of cytokeratin and regained junctional complexes. In contrast, TGF-beta 1-treated cells strongly expressed vimentin along with branching structures and showed decreases in cytokeratin expression and junctional complexes. Thus, TGF-beta 1 induces a mesenchyme-like cell shape accompanied by cytoskeletal molecular changes, with loss of both epithelial polarization and junctional complexes. These results suggest that the morphogenetic program of RGEC is likely to be determined by the interaction of these peptides and the timing of their presence.     

Epimorphin expression in interstitial pneumonia

Epimorphin modulates epithelial morphogenesis in embryonic mouse organs. We previously suggested that epimorphin contributes to repair of bleomycin-induced pulmonary fibrosis in mice via epithelium-mesenchyme interactions. To clarify the role of epimorphin in human lungs, we evaluated epimorphin expression and localization in normal lungs, lungs with nonspecific interstitial pneumonia (NSIP), and lungs with usual interstitial pneumonia (UIP); we also studied the effect of recombinant epimorphin on cultured human alveolar epithelial cells in vitro. Northern and Western blotting analyses revealed that epimorphin expression in NSIP samples were significantly higher than those in control lungs and lungs with UIP. Immunohistochemistry showed strong epimorphin expression in mesenchymal cells of early fibrotic lesions and localization of epimorphin protein on mesenchymal cells and extracellular matrix of early fibrotic lesions in the nonspecific interstitial pneumonia group. Double-labeled fluorescent images revealed expression of matrix metalloproteinase 2 in re-epithelialized cells overlying epimorphin-positive early fibrotic lesions. Immunohistochemistry and metalloproteinase activity assay demonstrated augmented expression of metalloproteinase induced by recombinant epimorphin in human alveolar epithelial cells. These findings suggest that epimorphin contributes to repair of pulmonary fibrosis in nonspecific interstitial pneumonia, perhaps partly by inducing expression of matrix metalloproteinase 2, which is an important proteolytic factor in lung remodeling.     

Epimorphin: a mesenchymal protein essential for epithelial morphogenesis.

A novel 150 kd protein expressed on the surface of mesenchymal cells of mouse embryonic tissues was identified. A monoclonal antibody to this molecule inhibited various processes of epithelial morphogenesis, such as hair follicle growth and lung epithelial tubular formation, in organ cultures of these tissues. Sequence analysis of cDNA encoding this protein revealed that it had 289 amino acids with a hydrophobic stretch at the C-terminus. NIH 3T3 cells transfected with the cDNA of this protein expressed the exogenous 150 kd protein on their surface. When lung epithelial cells were cocultured with these transfected cells, they showed normal tubular morphogenesis, but not with untransfected NIH 3T3 cells. These results indicate that this protein, termed epimorphin, plays a central role in epithelial-mesenchymal interactions.     

Identification and characterization of a sphere organelle protein

Sphere organelles are nuclear structures in amphibian oocytes that are easily visible by light microscopy. These structures are up to 10 microns in diameter and have been described morphologically for decades, yet their function remains obscure. The present study defines a protein component of the sphere organelle, named SPH-1, which is recognized by a mAb raised against purified Xenopus laevis oocyte nucleoplasm. SPH-1 is an 80-kD protein which is localized specifically to spheres and is undetectable elsewhere on lampbrush chromosomes or in nucleoli. We show using confocal microscopy that SPH-1 is localized to the cortex of sphere organelles. Furthermore, we have isolated a cDNA that can encode SPH-1. When epitope-tagged forms of SPH-1 are expressed in X. laevis oocytes the protein specifically localizes to spheres, demonstrating that the cloned cDNA encodes the sphere antigen. Comparison of the predicted amino acid sequence with sequence databases shows SPH-1 is related to p80-coilin, a protein associated with coiled bodies; coiled bodies are nuclear structures found in plant and animal cells. The sphere-specific mAb stains X. laevis tissue culture cells in a punctate nuclear pattern, showing that spheres or sphere antigens are present in somatic cells as well as germ cells and suggesting a general and essential function for spheres in all nuclei.     

Colocalization of F-actin and 34-kilodalton actin bundling protein in Dictyostelium amoebae and cultured fibroblasts

The Ca2+-sensitive actin-binding protein isolated from Dictyostelium discoideum, 30,000-D protein (Fechheimer and Taylor: J. Biol. Chem. 259:4514-4520, 1984;) has recently been localized in filipodia of substrate-adhered amoebae (Fechheimer: J. Cell Biol. 104:1539-1551, 1987). We have determined that this protein has a Mr of 34,000 daltons and is strictly colocalized with actin filaments in both substrate-attached Dictyostelium amoebae and cultured fibroblasts. 3T3 fibroblasts, as well as normal and virally transformed rat kidney fibroblasts (NRK) contain a 34-kilodalton (kD) protein that cross-reacts specifically with antibody to the Dictyostelium bundling protein. Mammalian 34-kD protein is colocalized with F-actin in stress fibers and the cortical cytoskeleton in substrate-adhered fibroblasts. In substrate-adhered vegetative Dictyostelium, F-actin and 34-kD protein are concentrated in regions of the cell cortex exhibiting filipodia and membrane ridges. Multiple filipodia formed after exposure to the chemoattractant folic acid stain intensely for 34-kD protein, implying participation in the assembly of actin bundles during filipod formation. The cortex of pseudopodia also contained high concentrations of bundling protein, but pseudopod interiors did not. In contrast to vegetative Dictyostelium, F-actin and 34-kD protein were not colocalized in cells that had progressed through the developmental cycle. In fruiting bodies, 34-kD protein was detected by immunofluorescence microscopy only in prespore cells, while F-actin appeared in stalk cells and spores.     

Epithelial morphogenesis.

The identification of protein factors, such as epimorphin, scatter factor, and activin, that induce epithelial branching and convergent extension-like movements in embryonic tissues are important breakthroughs in our understanding of the role of mesenchyme in epithelial morphogenesis. Moreover, the development of simple in vitro epithelial cell systems that undergo morphogenesis in response to these factors should provide a means to investigate the cellular and molecular bases of the morphogenetic movements themselves. Although many different cellular processes are involved in such morphogenetic behaviors, cell rearrangement is a particularly intriguing one that will be important to study further. Several considerations lead to the prediction that a dynamic regulation of cell-cell adhesion is likely to play a central role in cell rearrangements and epithelial morphogenesis. Ultimately, a greater issue to be addressed is how the different cellular mechanisms participating in epithelial morphogenesis are coordinated and regulated, so as to generate the diverse patterns found in various epithelia.     

α1- and α5-containing Laminins Regulate the Development of Bile Ducts via β1 Integrin Signals

Signals derived from basal lamina components are important for developing three-dimensional architecture of epithelial tissues. Laminins consisting of α, β, and γ subunits in basal lamina play pivotal roles in the formation and maintenance of epithelial tissue structures. However, it remains unclear which laminin isoforms transmit signals and how epithelial cells receive them to regulate multiple developmental processes. In three-dimensional culture of a liver progenitor cell line, Hepatic Progenitor Cells Proliferating on Laminin (HPPL), the cells establish apicobasal polarity and form cysts with a central lumen. Neutralizing antibody against β1 integrin blocked the formation and maintenance of the cyst structure, indicating that β1 integrin signaling was necessary throughout the morphogenesis. Although the addition of α1-containing laminin, a ligand of β1 integrin, induced cyst formation, it was dispensable for the maintenance of the cyst, suggesting that HPPL produces another ligand for β1 integrin to maintain the structure. Indeed, we found that HPPL produced α5-containing laminin, and siRNA against laminin α5 partially inhibited the lumen formation. In fetal liver, p75NTR(+) periportal fibroblasts and bile duct epithelial cells, known as cholangiocytes, expressed α1- and α5-containing laminins, respectively. In laminin α5 KO liver, cholangiocytes normally emerged, but the number of bile ducts was decreased. These results suggest that α1-containing laminin is sufficient as a component of the basal lamina for the commitment of bipotential liver progenitors to cholangiocytes and the apicobasal polarization, whereas α5-containing laminin is necessary for the formation of mature duct structures. Thus, α1- and α5-containing laminins differentially regulate the sequential events to form epithelial tissues via β1 integrin signals.     

Development of Foreign Mammary Epithelial Morphology in the Stroma of Immunodeficient Mice

Systemic growth and branching stimuli, and appropriate interactions with the host stroma are essential for the development of foreign epithelia in the mammary gland of immunodeficient mice. These factors were manipulated to promote and investigate the generation of representative bovine epithelial morphology in the transplanted mouse mammary stroma. The bovine mammary epithelium is unique in its commitment to rapid proliferation and high rate of differentiation. Its morphological organization within a fibrotic stroma resembles that of the human breast, and differs significantly from the rudimentary ductal network that penetrates a fatty stroma in mice. Transplantation of bovine mammary epithelial cells into the cleared mammary fat pad of NOD-SCID mice led to continuous growth of epithelial structures. Multilayered hollow spheres developed within fibrotic areas, but in contrast to mice, no epithelial organization was formed between adipocytes. The multilayered spheres shared characteristics with the heifer gland’s epithelium, including lumen size, cell proliferation, cytokeratin orientation, estrogen/progesterone receptor expression and localization, and milk protein synthesis. However, they did not extend into the mouse fat pad via ductal morphology. Pre-transplantation of fibroblasts increased the number of spheres, but did not promote extension of bovine morphology. The bovine cells preserved their fate and rarely participated in chimeric mouse–bovine outgrowths. Nevertheless, a single case of terminal ductal lobuloalveolar unit (TDLU) development was recorded in mice treated with estrogen and progesterone, implying the feasibility of this representative bovine morphology’s development. In vitro extension of these studies revealed paracrine inhibition of bovine epithelial mammosphere development by adipocytes, which was also generalized to breast epithelial mammosphere formation. The rescue of mammosphere development by fibroblast growth factor administration evidences an active equilibrium between inhibitory and supportive effects exerted by the adipose and fibrotic regions of the stroma, respectively, which determines the development of foreign epithelium.     

Characterization of palladin, a novel protein localized to stress fibers and cell adhesions

Here, we describe the identification of a novel phosphoprotein named palladin, which colocalizes with alpha-actinin in the stress fibers, focal adhesions, cell-cell junctions, and embryonic Z-lines. Palladin is expressed as a 90-92-kD doublet in fibroblasts and coimmunoprecipitates in a complex with alpha-actinin in fibroblast lysates. A cDNA encoding palladin was isolated by screening a mouse embryo library with mAbs. Palladin has a proline-rich region in the NH(2)-terminal half of the molecule and three tandem Ig C2 domains in the COOH-terminal half. In Northern and Western blots of chick and mouse tissues, multiple isoforms of palladin were detected. Palladin expression is ubiquitous in embryonic tissues, and is downregulated in certain adult tissues in the mouse. To probe the function of palladin in cultured cells, the Rcho-1 trophoblast model was used. Palladin expression was observed to increase in Rcho-1 cells when they began to assemble stress fibers. Antisense constructs were used to attenuate expression of palladin in Rcho-1 cells and fibroblasts, and disruption of the cytoskeleton was observed in both cell types. At longer times after antisense treatment, fibroblasts became fully rounded. These results suggest that palladin is required for the normal organization of the actin cytoskeleton and focal adhesions.     

Hepatocyte growth factor/scatter factor induces a variety of tissue- specific morphogenic programs in epithelial cells

Hepatocyte growth factor/scatter factor (HGF/SF) is the mesenchymal ligand of the epithelial tyrosine kinase receptor c-Met. In vitro, HGF/SF has morphogenic properties, e.g., induces kidney epithelial cells to form branching ducts in collagen gels. Mutation of the HGF/SF gene in mice results in embryonic lethality due to severe liver and placenta defects. Here, we have evaluated the morphogenic activity of HGF/SF with a large variety of epithelial cells grown in three- dimensional collagen matrices. We found that HGF/SF induces SW 1222 colon carcinoma cells to form crypt-like structures. In these organoids, cells exhibit apical/basolateral polarity and build a well- developed brush border towards the lumen. Capan 2 pancreas carcinoma cells, upon addition of HGF/SF, develop large hollow spheroids lined with a tight layer of polarized cells. Collagen inside the cysts is digested and the cells show features of pancreatic ducts. HGF/SF induces EpH4 mammary epithelial cells to form long branches with end- buds that resemble developing mammary ducts. pRNS-1-1 prostate epithelial cells in the presence of HGF/SF develop long ducts with distal branching as found in the prostate. Finally, HGF/SF simulates alveolar differentiation in LX-1 lung carcinoma cells. Expression of transfected HGF/SF cDNA in LX-1 lung carcinoma and EpH4 mammary epithelial cells induce morphogenesis in an autocrine manner. In the cell lines tested, HGF/SF activated the Met receptor by phosphorylation of tyrosine residues. These data show that HGF/SF induces intrinsic, tissue-specific morphogenic activities in a wide variety of epithelial cells. Apparently, HGF/SF triggers respective endogenous programs and is thus an inductive, not an instructive, mesenchymal effector for epithelial morphogenesis.