Purified scatter factor stimulates epithelial and vascular endothelial cell migration

Fibroblasts and smooth muscle cells release a protein activity which causes epithelial sheets to "scatter" into isolated cells. Purification of scatter factor (SF) activity from ras-transformed 3T3 cells was reported recently. We purified ras-3T3 SF by a slightly different method with essentially similar findings. Purified factor showed a single band at 77 +/- 3 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. Scatter activity was eluted from gel slices at this molecular size. Reduction with mercaptoethanol caused the loss of activity and the appearance of two bands (58 and 31 kDa). We report the amino acid composition of ras-3T3 SF and sequences of several tryptic peptides. These sequences were not similar to the known proteins in the Protein Database. We have shown previously that partially purified ras-3T3 scatter activity stimulates migration of epithelial and vascular endothelial cells in a new migration assay utilizing microcarrier beads. We now demonstrate that the same purified ras-3T3 protein scatters epithelial cells and stimulates epithelial and endothelial migration in microcarrier bead and Boyden chamber assays. Partially purified human smooth muscle scatter activity shares these activities, but the protein(s) responsible has not been isolated. Migration-stimulating activity was maximal at ras-3T3 protein concentrations less than 10 ng/ml (0.13 nM). ras-3T3 SF had no collagenolytic activity and did not stimulate DNA synthesis in fibroblast growth factor-responsive human melanocytes. ras-3T3 SF appears to be a new protein which regulates endothelial and epithelial mobility; and, therefore, it may be involved in vascular repair and wound healing.     

Interactions of multiple heparin binding growth factors with neuropilin-1 and potentiation of the activity of fibroblast growth factor-2

The hypothesis that neuropilin-1 (Npn-1) may interact with heparin-binding proteins other than vascular endothelial growth factor has been tested using an optical biosensor-based binding assay. The results show that fibroblast growth factor (FGF) 1, 2, 4, and 7, FGF receptor 1, hepatocyte growth factor/scatter factor (HGF/SF), FGF-binding protein, normal protease sensitive form of prion protein, antithrombin III, and Npn-1 itself are all able to interact with Npn-1 immobilized on the sensor surface. FGF-2, FGF-4, and HGF/SF are also shown to interact with Npn-1 in a solution assay. Moreover, these protein-protein interactions are dependent on the ionic strength of the medium and are inhibited by heparin, and the kinetics of binding of FGF-2, FGF-4 and HGF/SF to Npn-1 are characterized by fast association rate constants (270,000-1,600,000 m(-1) s(-1)). These results suggest that Npn-1 possesses a "heparin" mimetic site that is able to interact at least in part through ionic bonding with the heparin binding site on many of the proteins studied. Npn-1 was also found to potentiate the growth stimulatory activity of FGF-2 on human umbilical vein endothelial cells, indicating that Npn-1 may not just bind but also regulate the activity of heparin-binding proteins.     

Tyrosine Phosphorylation of β-Catenin and Plakoglobin Enhanced by Hepatocyte Growth Factor and Epidermal Growth Factor in Human Carcinoma Cells

The effect of hepatocyte growth factor /scatter factor (HGF/SF) and epidermal growth factor (EGF) on cadherin-mediated adhesion of human carcinoma cells was studied. HGF/SF induced scattering of colonic adenocarcinoma HT29 and gastric adenocarcinomas MKN7 and MKN74 cells. Likewise, EGF induced scattering of HT29 and MKN7 cells. These cells expressed E-cadherin, which was concentrated at cell-cell contact sites. When the scattering of these cells was induced by HGF/SF or EGF, the E-cadherin concentration at cell-cell boundaries tended to decrease. Irnmunoblotting analyses, however, demonstrated that these growth factor treatments did not alter the expression of E-cadherin and E-cadherin-associated proteins, α- and β-catenin and plakoglobin. β-Catenin, plakoglobin and an unidentified 115-kDa molecule associated with E-cadherin were found to be phosphorylated at tyrosine residues, and these phosphorylations were enhanced by the growth factor treatments. These results suggest that HGF/SF and EGF may modulate the function of the cadherin-catenin system via tyrosine phosphorylation of cadherin-associated proteins.     

Stimulation of DNA synthesis and cell proliferation of human mammary myoepithelial-like cells by hepatocyte growth factor/scatter factor depends on heparan sulfate proteoglycans and sustained phosphorylation of mitogen-activated protein kinases p42/44

Hepatocyte growth factor/scatter factor (HGF/SF) is a heparan/dermatan sulfate-binding growth factor produced by stromal cells that acts as a paracrine effector on neighboring epithelia. HGF/SF stimulated DNA synthesis in human mammary (Huma) 109 myoepithelial-like cells grown on collagen I and fibronectin substrata but not when grown on plastic. Dual phosphorylation of mitogen-activated protein kinases (p42/44(MAPK)) was required for this stimulation of DNA synthesis. In Huma 109 cells cultured on plastic, HGF/SF stimulated a transient phosphorylation of p42/44(MAPK), which reached a maximum at 10 min after addition of the growth factor and returned to near basal levels after 20 min. In contrast, the phosphorylation of p42/44(MAPK) stimulated by HGF/SF in cells cultured on collagen I or fibronectin was sustained over 45 min. In Huma 109 cells deficient in sulfated glycosaminoglycans, HGF/SF failed to stimulate p42/44(MAPK) phosphorylation or DNA synthesis on any substratum, even when soluble heparan sulfate proteoglycans purified from the cells or from the culture medium were added. However, HGF/SF stimulated DNA synthesis and a sustained phosphorylation of p42/44(MAPK) in sulfated glycosaminoglycan-deficient Huma 109 cells plated on a substratum of medium HSPGs but not cell HSPGs. The HGF/SF-induced proliferation is thus highly dependent on heparan sulfate proteoglycans in myoepithelial-like cells.     

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.     

Quantitation of cytokine-stimulated migration of endothelium and epithelium by a new assay using microcarrier beads

Recent studies have identified a group of cytokines which appear to be cell-specific regulators of mobility in nonleukocytic mammalian cells. One example is scatter factor (SF), a soluble protein(s) produced by cultured fibroblasts and vascular smooth muscle cells which causes spreading and separation ("scattering") of tight, cohesive colonies of epithelial cells. Studies of SF action have been limited because the degree of scattering is difficult to quantitate and because scattering assays cannot be used to study potential target cells that do not form tight, cohesive colonies. We developed a simple, quantitative assay of SF-stimulated mobility based on migration of target cells off microcarrier beads onto plastic culture surfaces in 24-well plates. We showed that crude and partially purified SF derived from ras-transformed 3T3 cells stimulates migration of both epithelial and vascular endothelial cells but not of producer or nonproducer fibroblasts. Scatter and migration-stimulating activities copurified on cation exchange chromatography; and the degree of stimulation was closely correlated with scattering titer regardless of SF purity. Migration of endothelial cells from beads, while extremely sensitive to SF, was not affected by serum concentration (1 to 10%), various purified growth factors, or fibronectin. Both scattering and migration from beads were blocked by cycloheximide (0.1 microgram/ml) during assay incubation, suggesting that these processes require protein synthesis. The microcarrier bead assay may be a useful quantitative tool to study the biochemical mechanisms of SF-stimulated cell migration.     

Endocan is a novel chondroitin sulfate/dermatan sulfate proteoglycan that promotes hepatocyte growth factor/scatter factor mitogenic activity

Proteoglycans that modulate the activities of growth factors, chemokines, and coagulation factors regulate in turn the vascular endothelium with respect to processes such as inflammation, hemostasis, and angiogenesis. Endothelial cell-specific molecule-1 is mainly expressed by endothelial cells and regulated by pro-inflammatory cytokines (Lassalle, P., Molet, S., Janin, A., Heyden, J. V., Tavernier, J., Fiers, W., Devos, R., and Tonnel, A. B. (1996) J. Biol. Chem. 271, 20458-20464). We demonstrate that this molecule is secreted as a soluble dermatan sulfate (DS) proteoglycan. This proteoglycan represents the major form either secreted by cell lines or circulating in the human bloodstream. Because this proteoglycan is specifically secreted by endothelial cells, we propose to name it endocan. The glycosaminoglycan component of endocan consists of a single DS chain covalently attached to serine 137. Endocan dose-dependently increased the hepatocyte growth factor/scatter factor (HGF/SF)-mediated proliferation of human embryonic kidney cells, whereas the nonglycanated form of endocan did not. Moreover, DS chains purified from endocan mimicked the endocan-mediated increase of cell proliferation in the presence of HGF/SF. Overall, our results demonstrate that endocan is a novel soluble dermatan sulfate proteoglycan produced by endothelial cells. Endocan regulates HGF/SF-mediated mitogenic activity and may support the function of HGF/SF not only in embryogenesis and tissue repair after injury but also in tumor progression.     

Growth Factor–dependent Activation of αvβ3 Integrin in Normal Epithelial Cells: Implications for Tumor Invasion

Integrin activation is a multifaceted phenomenon leading to increased affinity and avidity for matrix ligands. To investigate whether cytokines produced during stromal infiltration of carcinoma cells activate nonfunctional epithelial integrins, a cellular system of human thyroid clones derived from normal glands (HTU-5) and papillary carcinomas (HTU-34) was employed. In HTU-5 cells, αvβ3 integrin was diffused all over the membrane, disconnected from the cytoskeleton, and unable to mediate adhesion. Conversely, in HTU-34 cells, αvβ3 was clustered at focal contacts (FCs) and mediated firm attachment and spreading. αvβ3 recruitment at FCs and ligand-binding activity, essentially identical to those of HTU-34, occurred in HTU-5 cells upon treatment with hepatocyte growth factor/scatter factor (HGF/SF). The HTU-34 clone secreted HGF/SF and its receptor was constitutively tyrosine phosphorylated suggesting an autocrine loop responsible for αvβ3 activated state. Antibody-mediated inhibition of HGF/SF function in HTU-34 cells disrupted αvβ3 enrichment at FCs and impaired adhesion. Accordingly, activation of αvβ3 in normal cells was produced by HTU-34 conditioned medium on the basis of its content of HGF/SF. These results provide the first example of a growth factor–driven integrin activation mechanism in normal epithelial cells and uncover the importance of cytokine-based autocrine loops for the physiological control of integrin activation.     

Antagonistic effect of NK4 on HGF/SF induced changes in the transendothelial resistance (TER) and paracellular permeability of human vascular endothelial cells

Hepatocyte growth factor/scatter factor (HGF/SF) is a multi-function cytokine that has been shown to regulate the expression of cell adhesion molecules in human endothelial cells. It is also a key cytokine in the development and progression of cancer, particularly during metastasis. NK4 is a variant of HGF/SF that has already been shown to be antagonistic to HGF/SF. This study shows that HGF/SF decreased transendothelial resistance (TER) and increased paracellular permeability in human vascular endothelial cells can that such effects can be inhibited by addition of the NK4 variant. In addition, HGF/SF-stimulated invasion of endothelium by breast cancer cells was inhibited by the addition of NK4. Western blotting revealed that HGF/SF decreased the protein level, and increased tyrosine phosphorylation of ZO-1, but did not cause a change in level of occludin or claudin-1, both molecules involved in tight junction function. RT-PCR revealed that addition of HGF/SF caused no change in signal for claudin-5 or junctional adhesion molecule (JAM), but there was a decrease in the signal for claudin-1. NK4 was able to prevent the decrease in levels of ZO-1 protein by HGF/SF.     

Regulation of scatter factor production via a soluble inducing factor

Scatter factor (SF) (also known as hepatocyte growth factor [HGF]) is a fibroblast-derived cytokine that stimulates motility, proliferation, and morphogenesis of epithelia. SF may play major roles in development, repair, and carcinogenesis. However, the physiologic signals that regulate its production are not well delineated. We found that various human tumor cell lines that do not produce SF secrete factors that stimulate SF production by fibroblasts, suggesting a paracrine mechanism for regulation of SF production. Conditioned medium from these cell lines contained two distinct scatter factor-inducing factor SF-IF activities: a high molecular weight (> 30 kD), heat sensitive activity and a low molecular weight (< 30 kD) heat stable activity. Further studies revealed that SF-producing fibroblasts also secrete factors that stimulate their own SF production. We characterized the < 30-kD SF-IF activity from ras-3T3 (clone D4), a mouse cell line that overproduces both SF and SF-IF. The < 30-kD filtrate from ras-3T3 conditioned medium induced four- to sixfold increases in expression of SF biologic activity, immunoreactive protein, and mRNA by multiple SF- producing fibroblast lines. Ras-3T3 SF-IF activity was stable to boiling, extremes of pH, and reductive alkylation, but was destroyed by proteases. We purified ras-3T3 SF-IF about 10,000-fold from serum-free conditioned medium by a combination of ultrafiltration, cation exchange chromatography, and reverse phase chromatography. The purified protein exhibited electrophoretic mobility of about 12 kD (reduced) and 14 kD (nonreduced) by SDS-PAGE. The identity of the protein was verified by elution of biologic activity from gel slices. Purified SF-IF stimulated SF production in a physiologic concentration range (about 20-400 pM). Its properties and activities were distinct from those of IL-1 and TNF, two known inducers of SF production. We suggest that SF-IF is a physiologic regulator of SF production.     

Phosphorylation and sulfation of arylsulfatase A accompanies biosynthesis of the enzyme in normal and carcinoma cell lines

Arylsulfatase A (arylsulfate sulfohydrolase, EC 3.1.6.1), a mammalian lysosomal enzyme, is initially synthesized as a 69, 67 and 64 kDa precursor polypeptide in a prostate carcinoma cell line PC-3SF12, in HeLa cells and in a normal human embryonic lung cell line WI-38, respectively. These precursor polypeptides are secreted into the medium or processed to mature enzymes of apparent molecular mass 66, 64 or 62 kDa in PC-3SF12, HeLa or WI-38 cells, respectively. The precursor and mature polypeptides in WI-38 cells are phosphorylated, and the phosphate is lost upon treatment with endo-beta-hexosaminidase H. Arylsulfatase A is also shown to be sulfated in WI-38 cells. The presence of castanospermine, an inhibitor of sulfation of the second N-acetylglucosamine residue of the chitobiose core, does not reduce the extent of sulfation of arylsulfatase A, suggesting that either terminal sugars or the protein is sulfated. Sulfation may have a protective function similar to that of terminal sialic acid residues in glycoproteins. Although the subcellular location of arylsulfatase A is identical in PC-3SF12 and in WI-38 cells, pulse-chase experiments indicate that arylsulfatase A protein has a slower turnover in the prostate carcinoma cell line than it does in the normal human lung cell line. The differences in the apparent molecular weights of arylsulfatase A in the normal and carcinoma cell lines are shown to be due to variations in the carbohydrate content of the enzyme. The apparent molecular mass of the polypeptide chain obtained after endo-beta-hexosaminidase H treatment is 59 kDa, a value which is identical for all three cell lines studied here. These results suggest the possibility of an enhanced activity of terminal glucosyltransferase enzymes in carcinoma cell lines and in tumor tissues. Arylsulfatase A may be a useful marker for studying transformation-related processes in human cell lines.     

Inhibition of the MET Kinase Activity and Cell Growth in MET-Addicted Cancer Cells by Bi-Paratopic Linking

MET, the product of the c-MET proto-oncogene, and its ligand hepatocyte growth factor/scatter factor (HGF/SF) control survival, proliferation and migration during development and tissue regeneration. HGF/SF-MET signaling is equally crucial for growth and metastasis of a variety of human tumors, but resistance to small-molecule inhibitors of MET kinase develops rapidly and therapeutic antibody targeting remains challenging. We made use of the designed ankyrin repeat protein (DARPin) technology to develop an alternative approach for inhibiting MET. We generated a collection of MET-binding DARPins covering epitopes in the extracellular MET domains and created comprehensive sets of bi-paratopic fusion proteins. This new class of molecules efficiently inhibited MET kinase activity and downstream signaling, caused receptor downregulation and strongly inhibited the proliferation of MET-dependent gastric carcinoma cells carrying MET locus amplifications. MET-specific bi-paratopic DARPins may represent a novel and potent strategy for therapeutic targeting of MET and other receptors, and this study has elucidated their mode of action.     

The tyrosine-phosphorylated hepatocyte growth factor/scatter factor receptor associates with phosphatidylinositol 3-kinase.

The receptor for hepatocyte growth factor, also known as scatter factor (HGF/SF), has recently been identified as the 190-kDa heterodimeric tyrosine kinase encoded by the MET proto-oncogene (p190MET). The signaling pathway(s) triggered by HGF/SF are unknown. In A549 cells, a lung epithelial cell line, nanomolar concentrations of HGF/SF induced tyrosine phosphorylation of the p190MET receptor. The autophosphorylated receptor coprecipitated with phosphatidylinositol 3-kinase (PI 3-kinase) activity. In GTL16 cells, a cell line derived from a gastric carcinoma, the p190MET receptor, overexpressed and constitutively phosphorylated on tyrosine, coprecipitated with PI 3-kinase activity and with the 85-kDa PI 3-kinase subunit. In these cells activation of protein kinase C or the increase of intracellular [Ca2+] inhibits tyrosine phosphorylation of the p190MET receptor as well as the association with both PI 3-kinase activity and the 85-kDa subunit of the enzyme. In an in vitro assay, tyrosine phosphorylation of the immobilized p190MET receptor was required for binding of PI 3-kinase from cell lysates. These data strongly suggest that the signaling pathway activated by the HGF/SF receptor includes generation of D-3-phosphorylated inositol phospholipids.     

Hereditary papillary renal carcinoma type I

Germline missense mutations in the tyrosine kinase domain of the hepatocyte growth factor/scatter factor (HGF/SF) receptor, c-Met, are thought to be responsible for hereditary papillary renal carcinoma (HPRC) type 1, a form of human kidney cancer. In addition to extensive linkage analysis of HPRC families localizing the HPRC type 1 gene within chromosome 7, the demonstration that individual c-Met mutations reconstituted in cultured cells display enhanced and dysregulated kinase activity, and confer cell transformation and tumorigenicity in mice, solidifies this conclusion. Our prior knowledge of HGF/SF biology and c-Met signaling enabled rapid progress in unraveling the molecular pathogenesis of HPRC type 1, and in laying the framework for the development of novel therapeutics for the treatment of this cancer. At the same time, the study of HPRC type 1 has refined our appreciation of the oncogenic potential of c-Met signaling, and challenges our current understanding of HGF/SF and c-Met function in health and disease.     

Smooth muscle releases an epithelial cell scatter factor which binds to heparin

Summary We report that culture bovine calf aorta and human adult iliac artery smooth muscle cells release a soluble factor which causes spreading and separation of cells in normally tight, cohesive epithelial colonies, similar to the morphologic changes induced by the fibroblast-derived scatter factor (SF). Smooth muscle-derived SF was heat sensitive, trypsin labile, and nondialyzable, consistent with a protein (or proteins). Its effects on epithelium were not mimicked by a variety of proteolytic enzymes, growth factors, or hormones, and were not blocked by antiproteases or by antibodies to fibronectin and basic fibroblast growth factor. Epithelial cell proliferation was unaffected or only mildly stimulated by partially purified SF at concentrations that produced cell scattering. Both smooth muscle-and MRC5 human embryo fibroblast-derived SFs could be partially purified with similar elution patterns on a number of different chromatographic columns, including DEAE-agarose, heparin-sepharose, Bio-Rex 70, concanavalin A-sepharose, and MonoQ. SF from both sources bound tightly to heparin-sepharose, requiring 1.3 to 1.4M NaCl for elution. The morphologically obvious cell scattering effect was markedly inhibited by soluble heparin at concentrations down to 5 μg/ml, and this inhibition was prevented by protamine. These data suggest that vascular smooth muscle cells produce an epithelial cell scattering factor with properties similar to the fibroblast-produced factor, including a high affinity for heparin. Such factors are potentially important because they may represent a new class of proteins that primarily regulate cell mobility rather than growth and differentiation. Supported by American Cancer Society grant ACS IN-31-28-5, an Argail L. and Anna G. Hull Cancer Research Award, and grants-in-aid from the American Heart Association (#880981) and the American Lung Association of Connecticut. Dr. Goldberg was supported by the LIJ-Harvard Research Consortium and the Finkelstein Foundation.     

Protein factors which regulate cell motility

Summary Cell motility (i.e., movement) is an essential component of normal development, inflammation, tissue repair, angiogenesis, and tumor invasion. Various molecules can affect the motility and positioning of mammalian cells, including peptide growth factors, (e.g., EGF, PDGF, TGF-beta), substrate-adhesion molecules (e.g., fibronectin, laminin), cell adhesion molecules (CAMs), and metalloproteinases. Recent studies have demonstrated a group of motility-stimulating proteins which do not appear to fit into any of the above categories. Examples include: 1)scatter factor (SF), a mesenchymal cell-derived protein which causes contiguous sheets of epithelium to separate into individual cells and stimulates the migration of epithelial as well as vascular endothelial cells; 2)autocrine motility factor (AMF), a tumor cell-derived protein which stimulates migration of the producer cells; and 3)migration-stimulating factor (MSF), a protein produced by fetal and cancer patient fibroblasts which stimulates penetration of three-dimensional collagen gels by non-producing adult fibroblasts. SF, AMF, and MSF are soluble and heat labile proteins with Mr of 77, 55, and 70 kd by SDS-PAGE, respectively, and may be members of a new class of cell-specific regulators of motility. Their physiologic functions have not been established, but available data suggest that they may be involved in fetal development and/or tissue repair.     

Scatter factor and hepatocyte growth factor: activities, properties, and mechanism.

Scatter factor (SF) was first identified as a fibroblast-derived protein which disperses (i.e., "scatters") cohesive colonies of epithelium. SF-like proteins were found in human smooth muscle cell conditioned medium, amniotic fluid, and placental tissue. SFs markedly stimulate migration of epithelial, carcinoma, and vascular endothelial cell types at picomolar concentrations. Hepatocyte growth factors (HGFs) were originally described as platelet- and serum-derived proteins which stimulate hepatocyte DNA synthesis. Partial amino acid sequence data for mouse and human SFs indicate significant homology with HGFs. We used biological, biochemical, and immunological assays to evaluate and compare the activities, properties, and mechanisms of action of mouse SF, human SF (fibroblast or placenta derived), and recombinant human HGF (hrHGF). We report the following findings: (a) mouse SF exhibits species-related differences in biological activities relative to the human factors; (b) human SF and hrHGF show significant overlap in biological activities (i.e., hrHGF stimulates motility of multiple normal and carcinoma cell types, whereas human SF stimulates DNA synthesis in several normal cell types); (c) the three factors contain common antigenic determinants; and (d) all three proteins stimulate rapid phosphorylation of tyrosine residues on the c-met protooncogene protein product (the putative receptor for HGF) and on another protein with Mr 110,000. A few biological and immunological differences between human SFs and hrHGF were observed. These may reflect minor variations in amino acid sequence or posttranslational modification related to the sources of the factors. Taken as a whole, our findings suggest that by structural, functional, immunological, and mechanistic criteria, human SF and human HGF are essentially identical.     

Expression of a human hepatocyte growth factor/scatter factor cDNA in MDCK epithelial cells influences cell morphology, motility, and anchorage-independent growth

The addition of exogenous hepatocyte growth factor (HGF)/scatter factor (SF) to MDCK epithelial cells results in fibroblastic morphology and cell motility. We generated HGF/SF producing MDCK cells by transfection with an expression plasmid containing human HGF/SF cDNA. Production of HGF/SF by these cells induced a change from an epithelial to a fibroblastic morphology and increased cell motility. In addition, the HGF/SF producing cells acquired efficient anchorage-independent growth in soft agar but did not form tumors in nude mice. The morphological change and the stimulation of the anchorage-independent growth were prevented by anti-HGF/SF antibody, suggesting that the factor is secreted and then exerts its effects through cell surface receptors.