Scatter factor and hepatocyte growth factor are indistinguishable ligands for the MET receptor.

Scatter Factor (SF) is a fibroblast-secreted protein which promotes motility and matrix invasion of epithelial cells. Hepatocyte Growth Factor (HGF) is a powerful mitogen for hepatocytes and other epithelial tissues. SF and HGF, purified according to their respective biological activities, were interchangeable and equally effective in assays for cell growth, motility and invasion. Both bound with identical affinities to the same sites in target cells. The receptor for SF and HGF was identified as the product of the MET oncogene by: (i) ligand binding and coprecipitation in immunocomplexes; (ii) chemical crosslinking to the Met beta subunit; (iii) transfer of binding activity in insect cells by a baculovirus carrying the MET cDNA; (iv) ligand-induced tyrosine phosphorylation of the Met beta subunit. SF and HGF cDNA clones from human fibroblasts, placenta and liver had virtually identical sequences. We conclude that the same molecule (SF/HGF) acts as a growth or motility factor through a single receptor in different target cells.     

Targeting of the SF/HGF receptor to the basolateral domain of polarized epithelial cells

Scatter Factor, also known as Hepatocyte Growth Factor (SF/HGF), has pleiotropic functions including direct control of cell-cell and cell- substrate adhesion in epithelia. The subcellular localization of the SF/HGF receptor is controversial. In this work, the cell surface distribution of the SF/HGF receptor was studied in vivo in epithelial tissues and in vitro in polarized MDCK monolayers. A panel of monoclonal antibodies against the beta chain of the SF/HGF receptor stained the basolateral but not the apical surface of epithelia lining the lumen of human organs. Radiolabeled or fluorescent-tagged anti- receptor antibodies selectively bound the basolateral cell surface of MDCK cells, which form a polarized monolayer sealed by intercellular junctions, when grown on polycarbonate filters in a two-chamber culture system. The receptor was concentrated around the cell-cell contact zone, showing a distribution pattern overlapping with that of the cell adhesion molecule E-cadherin. The basolateral localization of the SF/HGF receptor was confirmed by immunoprecipitation after domain selective cell surface biotinylation. When cells were fully polarized the SF/HGF receptor became resistant to non-ionic detergents, indicating interaction with insoluble component(s). In pulse-chase labeling and surface biotinylation experiments, the newly synthesized receptor was found exclusively at the basolateral surface. We conclude that the SF/HGF receptor is selectively exposed at the basolateral plasma membrane domain of polarized epithelial cells and is targeted after synthesis to that surface by direct delivery from the trans-Golgi network.     

HGF/SF Induces Mesothelial Cell Migration and Proliferation by Autocrine and Paracrine Pathways

Mesothelial repair differs from that of other epithelial-like surfaces as healing does not occur solely by centripetal in-growth of cells as a sheet from the wound margins. Mesothelial cells lose their cell-cell junctions, divide, and adopt a fibroblast-like morphology while scattering across and covering the wound surface. These features are consistent with a cellular response to hepatocyte growth factor/scatter factor (HGF/SF). In this study, we examined the ability of mesothelial cells to secrete HGF/SF and investigated its possible role as an autocrine regulator of mesothelial cell motility and proliferation. We found that human primary mesothelial cells expressed HGF/SF mRNA and secreted active HGF/SF into conditioned medium as determined by ELISA and in a scattering bioassay. These cells also expressed the HGF/SF receptor, Met, as shown by RT-PCR and by Western blot analysis and immunofluorescence. Incubation of mesothelial cells with neutralizing antibodies to HGF/SF decreased cell migration to 25% of controls, whereas addition of HGF/SF disrupted cell-cell junctions and induced scattering and enhanced mesothelial cell migration. Furthermore, HGF/SF showed a small but significant mitogenic effect on all mesothelial cell lines examined. In conclusion, HGF/SF is produced by mesothelial cells and induces both motility and proliferation of these cells. These data are consistent with HGF/SF playing an autocrine role in mesothelial healing.     

HGF/SF-met signaling in the control of branching morphogenesis and invasion

Hepatocyte growth factor/Scatter factor (HGF/SF) is a multifunctional growth factor which can induce diverse biological events. In vitro, these include scattering, invasion, proliferation and branching morphogenesis. In vivo, HGF/SF is responsible for many processes during embryonic development and a variety of activities in adults, and many of these normal activities have been implicated in its role in tumorgenesis and metastasis. The c-Met receptor tyrosine kinase is the only known receptor for HGF/SF and mediates all HGF/SF induced biological activities. Upon HGF/SF stimulation, the c-Met receptor is tyrosine-phosphorylated which is followed by the recruitment of a group of signaling molecules and/or adaptor proteins to its cytoplasmic domain and its multiple docking sites. This action leads to the activation of several different signaling cascades that form a complete network of intra and extracellular responses. Different combinations of signaling pathways and signaling molecules and/or differences in magnitude of responses contribute to these diverse series of HGF/SF-Met induced activities and most certainly are influenced by cell type as well as different cellular environments. In this review, we focus on HGF/SF-induced branching morphogenesis and invasion, and bring together recent new findings which provide insight into how HGF/SF, via c-Met induces this response.     

The Met receptor tyrosine kinase transduces motility, proliferation, and morphogenic signals of scatter factor/hepatocyte growth factor in epithelial cells

Depending on the target cells and culture conditions, scatter factor/hepatocyte growth factor (SF/HGF) mediates several distinct activities, i.e., cell motility, proliferation, invasiveness, tubular morphogenesis, angiogenesis, or cytotoxicity. A small isoform of SF/HGF encoded by a natural splice variant, which consists of the NH2-terminal hairpin structure and the first two kringle domains but not the protease homology region, induces cell motility but not mitogenesis. Two types of SF/HGF receptors have recently been discovered in epithelial cells, the high affinity c-Met receptor tyrosine kinase, and low affinity/high capacity binding sites, which are probably located on heparan sulfate proteoglycans. In the present study, we have addressed the question whether the various biological activities of SF/HGF are transduced into cells by a single type of receptor. We have here examined MDCK epithelial cells transfected with a hybrid cDNA encoding the ligand binding domain of the nerve growth factor (NGF) receptor and the membrane-spanning and tyrosine kinase domains of the Met receptor. We demonstrate that all biological effects of SF/HGF upon epithelial cells such as the induction of cell motility, proliferation, invasiveness, and tubular morphogenesis can now be triggered by the addition of NGF. Thus, it is likely that all known biological signals of SF/HGF are transduced through the receptor tyrosine kinase encoded by the c-Met protooncogene.     

Hepatocyte growth factor/scatter factor modulates cell motility, proliferation, and proteoglycan synthesis of chondrocytes

Hepatocyte growth factor/scatter factor (HGF/SF) is a multifunctional growth factor that promotes proliferation, motility, and morphogenesis in epithelial cells. Recently the HGF receptor, c-met protooncogene product, has been shown to be expressed in developing limb buds (Sonnenberg, E., D. Meyer, M. Weidner, and C. Birchmeiyer, 1993. J. Cell Biol. 123: 223-235), suggesting that some populations of mesenchymal cells in limb buds respond to HGF/SF. To test the possibility that HGF/SF is involved in regulation of cartilage development, we isolated chondrocytes from knee joints and costal cartilages of 23-d embryonic and 4-wk-old rabbits, and analyzed the effects of HGF/SF on migration and proliferation of these cells. We found that HGF/SF stimulated migration of cultured articular chondrocytes but did not scatter limb mesenchymal fibroblasts or synovial fibroblasts in culture. HGF/SF also stimulated proliferation of chondrocytes; a maximum three-fold stimulation in DNA synthesis was observed at the concentration of 3 ng/ml of HGF/SF. Moreover, HGF/SF had the ability to enhance proteoglycan synthesis in chondrocytes. The responsiveness of chondrocytes to HGF/SF was also supported by the observation that they expressed the HGF/SF receptor. Addition of the neutralizing antibody to rat HGF/SF affected neither DNA synthesis nor proteoglycan synthesis in rat chondrocytes, suggesting a paracine mechanism of action of HGF/SF on these cells. In situ hybridization analysis showed that HGF/SF mRNA was restrictively expressed in the areas of future joint regions in developing limb buds and in the intercostal spaces of developing costal cartilages. These findings suggest that HGF/SF plays important roles in cartilage development through its multiple activities.     

Hepatocyte growth factor enhances MMP activity in human endothelial cells

Scatter factor (SF) or hepatocyte growth factor (HGF) has been identified as an angiogenic factor. Angiogenesis requires not only tube formation but also invasion of pericytes and extracellular matrix (ECM) remodeling to promote new vessel stabilization. In the current study, the effect of SF/HGF on endothelial cell (EC) production of matrix metalloproteinases (MMPs) was explored. We showed that SF/HGF enhanced MT1-MMP synthesis and induced MMP-2 activation in two human EC lines: dermal microvessel EC and coronary arterial EC. Furthermore, SF/HGF accelerated EC invasion into matrix, an activity that could be inhibited by a MMP inhibitor. We also demonstrated that the MAP kinase cascade is critical in signal transduction pathway from SF/HGF stimulation to MT1-MMP up-regulation. The current study indicates that MMP activation is a novel effect of SF/HGF on ECs.     

Extracellular proteolytic cleavage by urokinase is required for activation of hepatocyte growth factor/scatter factor.

The extracellular protease urokinase is known to be crucially involved in morphogenesis, tissue repair and tumor invasion by mediating matrix degradation and cell migration. Hepatocyte growth factor/scatter factor (HGF/SF) is a secretory product of stromal fibroblasts, sharing structural motifs with enzymes of the blood clotting cascade, including a zymogen cleavage site. HGF/SF promotes motility, invasion and growth of epithelial and endothelial cells. Here we show that HGF/SF is secreted as a single-chain biologically inactive precursor (pro-HGF/SF), mostly found in a matrix-associated form. Maturation of the precursor into the active alpha beta heterodimer takes place in the extracellular environment and results from a serum-dependent proteolytic cleavage. In vitro, pro-HGF/SF was cleaved at a single site by nanomolar concentrations of pure urokinase, generating the active mature HGF/SF heterodimer. This cleavage was prevented by specific urokinase inhibitors, such as plasminogen activator inhibitor type-1 and protease nexin-1, and by antibodies directed against the urokinase catalytic domain. Addition of these inhibitors to HGF/SF responsive cells prevented activation of the HGF/SF precursor. These data show that urokinase acts as a pro-HGF/SF convertase, and suggest that some of the growth and invasive cellular responses mediated by this enzyme may involve activation of HGF/SF.     

Stimulation of DNA synthesis in skin fibroblasts by human hepatocyte growth factor/scatter factor.

The effect of hepatocyte growth factor/scatter factor (HGF/SF) on the proliferation of human skin fibroblasts was examined. At concentrations above 1.0 ng/ml, both native and recombinant human HGF/SF stimulated the DNA synthesis determined by [³H]thymidine incorporation, which was completely inhibited by an anti-human HGF/SF monoclonal antibody. The maximal DNA synthesis in the treated cells was nearly twice that in untreated cells. HGF/SF also caused an increase in the labelling index, DNA content and cell number. The effect of HGF/SF was more than additive to the maximal effect of insulin and epidermal growth factor, other mitogens for the fibroblasts. These results indicate that human skin fibroblasts are sensitive to the mitogenic action of HGF/SF.     

How to make tubes: signaling by the Met receptor tyrosine kinase

Hepatocyte growth factor/scatter factor (HGF/SF), acting through the receptor tyrosine kinase Met, stimulates cells derived from a variety of different organs to form elongated hollow tubules when grown in three-dimensional gels. In vivo data also indicate a role for HGF/SF and Met in tubule formation during liver and kidney regeneration and mammary gland formation. Activation of Met results in the recruitment of a myriad of signal transducers that regulate dissociation of adherens junctions and the stimulation of cellular motility, survival, proliferation and morphogenesis during tubule formation. Among these many signal transducers, the Gab1 adaptor protein and its effector, the SHP2 tyrosine phosphatase, have been found to be crucial for tubulogenesis and for the sustained stimulation of the ERK/MAP kinase pathway. Here, we discuss the contribution of these and other signaling pathways and the role of HGF/SF and Met in the formation of epithelial cell tubules both in vitro in branching-morphogenesis assays and in vivo during organogenesis.     

Hepatocyte growth factor induces delayed STAT3 phosphorylation through interleukin-6 expression

Met receptor tyrosine kinase mediates pleiotropic cellular responses following its activation by hepatocyte growth factor or scatter factor (HGF/SF). STAT3 was reported to be one of direct downstream molecules in HGF/SF-Met signaling. In the present study, however, we observed that Tyr705 of STAT3 was phosphorylated from 2 h or 6 h in NIH3T3 and Chang liver cells, respectively, after HGF/SF treatment. Blocking of the phosphorylation by cycloheximide or actinomycin D and the rapid STAT3 phosphorylation with the conditioned medium from HGF/SF-treated NIH3T3 cells suggested that a newly synthesized secretory protein was responsible for the delayed STAT3 phosphorylation. Among the known mediators to induce STAT3 phosphorylation, interleukin-6 (IL-6) mRNA and protein were induced by HGF/SF, and the released IL-6 was accumulated in the conditioned medium after HGF/SF treatment. Furthermore, the neutralizing IL-6 antibody abolished the STAT3 phosphorylation. Treatment with LY294002, a PI3 kinase inhibitor, but not with other signal inhibitors, resulted in the loss of delayed STAT3 phosphorylation by HGF/SF, showing the involvement of PI3 kinase pathway. Collectively, these results demonstrate that HGF/SF-Met signal cascade stimulates IL-6 production via PI3 kinase pathway, leading to STAT3 phosphorylation as a secondary effect.     

Met-HGF/SF mediates growth arrest and differentiation in T47D breast cancer cells.

Hepatocyte growth factor/scatter factor (HGF/SF) is a pluripotent growth factor that exerts mitogenic, motogenic, and morphogenic effects. To elucidate the cellular mechanisms underlying the pluripotent function of this growth factor, T47D human breast cancer cells were transfected with human hgf/sf. The hgf/sf-positive clones exhibited different levels of biologically functional HGF/SF expression and up-regulation of endogenous Met (HGF/SF receptor) expression. In addition, a constitutive phosphorylation of the receptor on tyrosine residues was detected, establishing a Met-HGF/SF autocrine loop. The autocrine activation of Met caused marked inhibition in cell growth accompanied by cell accumulation at G0/G1. These cells underwent terminal cell differentiation as determined by morphological changes, synthesis of milk proteins such as beta-casein and alpha-lactalbumin, and production of lipid vesicles. Our results demonstrate that Met-HGF/SF, an oncogenic signal transduction pathway, is capable of inducing growth arrest and differentiation in certain breast cancer cells and, thus, may have potential as therapeutic and/or prognostic tools in breast cancer treatment.     

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.     

Suppression of adriamycin-induced apoptosis by sustained activation of the phosphatidylinositol-3'-OH kinase-Akt pathway

The mechanisms by which growth factors trigger signal transduction pathways leading to protection against apoptosis are of great interest. In this study, we investigated the effect of hepatocyte growth factor (HGF/SF) and epidermal growth factor (EGF) on adriamycin (ADR)-induced apoptosis. Treatment of human epithelial MKN74 cells with ADR, a DNA topoisomerase IIalpha inhibitor, caused apoptosis. However, cells pretreated with HGF/SF, but not those pretreated with EGF, were resistant to this apoptosis. The protective effect of HGF/SF against the ADR-induced apoptosis was abolished in the presence of either LY294002, an inhibitor of phosphatidylinositol-3'-OH kinase (PI3-K) or 1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate, an inhibitor of Akt, thus implicating the activation of PI3-K-Akt signaling in the antiapoptotic action of HGF/SF. Immunoblotting analysis revealed that HGF/SF stimulated the sustained phosphorylation of Akt for several hours but that EGF stimulated the phosphorylation only transiently. Furthermore, ADR-induced activation of caspase-9, a downstream molecule of Akt, was inhibited for at least 24 h after HGF/SF stimulation, but it was not affected by EGF stimulation. Cell-surface biotin-labeling analysis showed that the HGF/SF receptor remained on the cell surface until at least 30 min after HGF/SF addition but that the EGF receptor level on the cell surface was attenuated at an earlier time after EGF addition. These results indicate that HGF/SF, but not EGF, transmitted protective signals against ADR-induced apoptosis by causing sustained activation of the PI3-K-Akt signaling pathway. Furthermore, the difference in antiapoptotic capacity between HGF/SF and EGF is explained, at least in part, by the delayed down-regulation of the HGF/SF receptor.     

CD44 stimulation by fragmented hyaluronic acid induces upregulation and tyrosine phosphorylation of c-Met receptor protein in human chondrosarcoma cells

Hepatocyte growth factor/scatter factor (HGF/SF) can induce proliferation and motility and promote invasion of tumor cells. Since HGF/SF receptor, c-Met, is expressed by tumor cells, and since stimulation of CD44, a transmembrane glycoprotein known to bind hyaluronic acid (HA) in its extracellular domain, is involved in activation of c-Met, we have studied the effects of CD44 stimulation by ligation with HA upon the expression and tyrosine phosphorylation of c-Met on human chondrosarcoma cell line HCS-2/8. The current study indicates that (a) CD44 stimulation by fragmented HA upregulates expression of c-Met proteins; (b) fragmented HA also induces tyrosine phosphorylation of c-Met protein within 30 min, an early event in this pathway as shown by the early time course of stimulation; (c) the effects of HA fragments are critically HA size-dependent. High molecular weight HA is inactive, but lower molecular weight fragments (M(r) 3.5 kDa) are active with maximal effect in the microg/ml range; (d) the standard form of CD44 (CD44s) is critical for the response because the effect on c-Met, both in terms of upregulation and phosphorylation, is inhibited by preincubation with an anti-CD44 monoclonal antibody; and (e) phosphorylation of c-Met induced by CD44 stimulation is inhibited by protein tyrosine kinase inhibitor, tyrphostin. Therefore, our study represents the first report that CD44 stimulation induced by fragmented HA enhances c-Met expression and tyrosine phosphorylation in human chondrosarcoma cells. Taken together, these studies establish a signal transduction cascade or cross-talk emanating from CD44 to c-Met.     

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.