Role of ERK and JNK pathways in regulating cell motility and matrix metalloproteinase 9 production in growth factor-stimulated human epidermal keratinocytes

Invasion is an essential cellular response that plays an important role in a number of physiological and pathological processes. Matrix metalloproteinase (MMP) production and cell movement are diverse cellular responses integral to the process of invasion. The complexity of the invasive process suggests the necessity of coordinate activation of more than one signaling pathway in order to activate specific factors responsible for regulating these cellular responses. In this report, we demonstrate that cell movement and MMP-9 production are both directly dependent on the activation of endogenous ERK signaling in hepatocyte growth factor (HGF)-or epidermal growth factor (EGF)-stimulated human epidermal keratinocytes. The kinetic profiles of endogenous MEK and ERK activity suggest that prolonged activation of these signal transducers is an underlying mechanism involved in stimulating cell motility and MMP-9 production. In support of this finding, a transient MEK/ERK signal elicited by keratinocyte growth factor (KGF) or insulin-like growth factor-1 (IGF-1) fails to stimulate these invasion-related responses. Specific inhibition of MEK leads to suppression of ERK activation, marked reduction in steady-state levels of c-Fos, and inhibition of cell movement and MMP-9 production. This occurs despite continued activation of JNK and c-Jun signaling in the presence of MEK-specific inhibition. In contrast, when JNK activity is specifically inhibited in HGF-stimulated cells, AP-1 activity is suppressed but cell motility is not affected. This evidence suggests that while ERK and JNK activity are necessary for AP-1 activation, ERK but not JNK is sufficient in stimulating cell motility. J. Cell. Physiol. 180:271–284, 1999. © 1999 Wiley-Liss, Inc.     

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.     

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.     

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.     

Receptor-type protein tyrosine phosphatase beta (RPTP-beta) directly dephosphorylates and regulates hepatocyte growth factor receptor (HGFR/Met) function

Protein tyrosine phosphorylation is a ubiquitous, fundamental biochemical mechanism that regulates essential eukaryotic cellular functions. The level of tyrosine phosphorylation of specific proteins is finely tuned by the dynamic balance between protein tyrosine kinase and protein tyrosine phosphatase activities. Hepatocyte growth factor receptor (also known as Met), a receptor protein tyrosine kinase, is a major regulator of proliferation, migration, and survival for many epithelial cell types. We report here that receptor-type protein tyrosine phosphatase β (RPTP-β) specifically dephosphorylates Met and thereby regulates its function. Expression of RPTP-β, but not other RPTP family members or catalytically inactive forms of RPTP-β, reduces hepatocyte growth factor (HGF)-stimulated Met tyrosine phosphorylation in HEK293 cells. Expression of RPTP-β in primary human keratinocytes reduces both basal and HGF-induced Met phosphorylation at tyrosine 1356 and inhibits downstream MEK1/2 and Erk activation. Furthermore, shRNA-mediated knockdown of endogenous RPTP-β increases basal and HGF-stimulated Met phosphorylation at tyrosine 1356 in primary human keratinocytes. Purified RPTP-β intracellular domain preferentially dephosphorylates purified Met at tyrosine 1356 in vitro. In addition, the substrate-trapping mutant of RPTP-β specifically interacts with Met in intact cells. Expression of RPTP-β in human primary keratinocytes reduces HGF induction of VEGF expression, proliferation, and motility. Taken together, the above data indicate that RPTP-β is a key regulator of Met function.     

Heparan sulfate-modified CD44 promotes hepatocyte growth factor/scatter factor-induced signal transduction through the receptor tyrosine kinase c-Met

CD44 has been implicated in tumor progression and metastasis, but the mechanism(s) involved is as yet poorly understood. Recent studies have shown that CD44 isoforms containing the alternatively spliced exon v3 carry heparan sulfate side chains and are able to bind heparin-binding growth factors. In the present study, we have explored the possibility of a physical and functional interaction between CD44 and hepatocyte growth factor/scatter factor (HGF/SF), the ligand of the receptor tyrosine kinase c-Met. The HGF/SF-c-Met pathway mediates cell growth and motility and has been implicated in tumor invasion and metastasis. We demonstrate that a CD44v3 splice variant efficiently binds HGF/SF via its heparan sulfate side chain. To address the functional relevance of this interaction, Namalwa Burkitt's lymphoma cells were stably co-transfected with c-Met and either CD44v3 or the isoform CD44s, which lacks heparan sulfate. We show that, as compared with CD44s, CD44v3 promotes: (i) HGF/SF-induced phosphorylation of c-Met, (ii) phosphorylation of several downstream proteins, and (iii) activation of the MAP kinases ERK1 and -2. By heparitinase treatment and the use of a mutant HGF/SF with greatly decreased affinity for heparan sulfate, we show that the enhancement of c-Met signal transduction induced by CD44v3 was critically dependent on heparan sulfate moieties. Our results identify heparan sulfate-modified CD44 (CD44-HS) as a functional co-receptor for HGF/SF which promotes signaling through the receptor tyrosine kinase c-Met, presumably by concentrating and presenting HGF/SF. As both CD44-HS and c-Met are overexpressed on several types of tumors, we propose that the observed functional collaboration might be instrumental in promoting tumor growth and metastasis.     

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.     

Hepatocyte Growth Factor (HGF)-Induced Cell Migration Is Negatively Modulated by Epidermal Growth Factor through Tyrosine Phosphorylation of the HGF Receptor

Hepatocyte growth factor (HGF) stimulated cell migration of human gastric carcinoma cell lines MKN1, MKN7, and MKN28. Epidermal growth factor (EGF) also stimulated the cell migration of these three cell lines. In MKN7 cells, HGF-stimulated cell migration was rather reduced in the presence of EGF, whereas such an observation was not made with MKN1 and MKN28 cells. Therefore, we compared the effect of EGF on HGF-stimulated HGF receptor phosphorylation in these cell lines. HGF induced a rapid tyrosine phosphorylation of the HGF receptor in all these cell lines. In MKN7 cells, the increased phosphorylation was further enhanced by EGF, although EGF alone did not affect tyrosine phosphorylation of the HGF receptor. In MKN1 and MKN28 cells, EGF did not influence tyrosine phosphorylation of the HGF receptor, whether HGF was present or not. The data presented here suggest that EGF negatively modulates the cellular response to HGF by increasing tyrosine phosphorylation of the HGF receptor in certain types of epithelial cells, e.g., MKN7 cells.     

Pericellular activation of hepatocyte growth factor/scatter factor (HGF/SF) in colorectal carcinomas: roles of HGF activator (HGFA) and HGFA inhibitor type 1 (HAI-1).

Activation of hepatocyte growth factor/scatter factor (HGF/SF) is a critical limiting step in the HGF/SF-induced signaling pathway mediated by MET receptor tyrosine kinase. Although HGF/SF-MET signaling could have potentially important roles in the invasive growth of tumors and tumor angiogenesis, little is known about the regulation of HGF/SF activation in the tumor tissues. This activation occurs in the extracellular milieu caused by proteolytic cleavage at the bond between Arg194-Val195 in the single-chain HGF precursor to generate the active two-chain heterodimeric form. Here we show that activation of HGF/SF is significantly enhanced in colorectal carcinoma tissues compared with normal colorectal mucosa, and HGF activator (HGFA), a recently identified factor XII-like serine proteinase, is critically involved in this process. Furthermore, we also show that HGF activator inhibitor type 1 (HAI-1) should have an important regulatory role in the pericellular activation of HGF/SF having diverse roles acting as a cell surface specific inhibitor of active HGFA and a reservoir of this enzyme on the cell surface. The latter property might paradoxically ensure the concentrated pericellular HGFA activity in certain cellular conditions in which shedding of HAI-1/HGFA complex from the plasma membrane is upregulated.     

Regulation of scatter factor/hepatocyte growth factor responses by Ras, Rac, and Rho in MDCK cells.

Scatter factor/hepatocyte growth factor (SF/HGF) stimulates the motility of epithelial cells, initially inducing centrifugal spreading of cell colonies followed by disruption of cell-cell junctions and subsequent cell scattering. These responses are accompanied by changes in the actin cytoskeleton, including increased membrane ruffling and lamellipodium extension, disappearance of peripheral actin bundles at the edges of colonies, and an overall decrease in stress fibers. The roles of the small GTP-binding proteins Ras, Rac, and Rho in regulating responses to SF/HGF were investigated by microinjection. Inhibition of endogenous Ras proteins prevented SF/HGF-induced actin reorganization, spreading, and scattering, whereas microinjection of activated H-Ras protein stimulated spreading and actin reorganization but not scattering. When a dominant inhibitor of Rac was injected, SF/HGF- and Ras-induced spreading and actin reorganization were prevented, although activated Rac alone did not stimulate either response. Microinjection of activated Rho inhibited spreading and scattering, while inhibition of Rho function led to the disappearance of stress fibers and peripheral bundles but did not prevent SF/HGF-induced motility. We conclude that Ras and Rac act downstream of the SF/HGF receptor p190Met to mediate cell spreading but that an additional signal is required to induce scattering.     

Role of the hepatocyte growth factor receptor, c-Met, in oncogenesis and potential for therapeutic inhibition.

Receptor tyrosine kinases have become important therapeutic targets for anti-neoplastic molecularly targeted therapies. c-Met is a receptor tyrosine kinase shown to be over-expressed and mutated in a variety of malignancies. Stimulation of c-Met via its ligand hepatocyte growth factor also known as scatter factor (HGF/SF), leads to a plethora of biological and biochemical effects in the cell. There has been considerable knowledge gained on the role of c-Met-HGF/SF axis in normal and malignant cells. This review summarizes the structure of c-Met and HGF/SF and their family members. Since there are known mutations of c-Met in solid tumors, particularly in papillary renal cell carcinoma, we have summarized the various mutations and over-expression of c-Met known thus far. Stimulation of c-Met can lead to scattering, angiogenesis, proliferation, enhanced cell motility, invasion, and eventual metastasis. The biological functions altered by c-Met are quite unique and described in detail. Along with biological functions, various signal transduction pathways, including the cytoskeleton are altered with the activation of c-Met-HGF/SF loop. We have recently shown the phosphorylation of focal adhesion proteins, such as paxillin and p125FAK in response to c-Met stimulation in lung cancer cells, and this is detailed here. Finally, c-Met when mutated or over-expressed in malignant cells serves as an important therapeutic target and the most recent data in terms of inhibition of c-Met and downstream signal transduction pathways is summarized.     

Engineering the NK1 fragment of hepatocyte growth factor/scatter factor as a MET receptor antagonist

The growth and motility factor hepatocyte growth factor/scatter factor (HGF/SF) and its receptor MET, the tyrosine kinase encoded by the c-MET proto-oncogene, exert major roles in cancer invasion and metastasis and are key targets for therapy. NK1 is an alternative spliced variant of HGF/SF that consists of the N-terminal (N) and first kringle (K1) domains and has partial agonistic activity. NK1 crystallises as a head-to-tail dimer with an extensive inter-protomeric interface resulting from contacts between the two short interdomain linkers and reciprocal contacts between the N and K1 domains. Here we show that a subset of mutants at the NK1 dimer interface, such as the linker mutants Y124A or N127A or the kringle mutant V140A:I142A, bind the MET receptor with affinities comparable to wild-type NK1 but fail to assemble a dimeric, signalling competent NK1-MET complex. These NK1 variants have no detectable agonistic activity on, behave as bona fide receptor antagonists by blocking cell migration and DNA synthesis in target cells and have strong prospects as therapeutics for human cancer.     

Green tea (-)-epigallocatechin-3-gallate inhibits HGF-induced progression in oral cavity cancer through suppression of HGF/c-Met

Hepatocyte growth factor (HGF) and c-Met have recently attracted a great deal of attention as prognostic indicators of patient outcome, and they are important in the control of tumor growth and invasion. Epigallocatechin-3-gallate (EGCG) has been shown to modulate multiple signal pathways in a manner that controls the unwanted proliferation and invasion of cells, thereby imparting cancer chemopreventive and therapeutic effects. In this study, we investigated the effects of EGCG in inhibiting HGF-induced tumor growth and invasion of oral cancer in vitro and in vivo. We examined the effects of EGCG on HGF-induced cell proliferation, migration, invasion, induction of apoptosis and modulation of HGF/c-Met signaling pathway in the KB oral cancer cell line. We investigated the antitumor effect and inhibition of c-Met expression by EGCG in a syngeneic mouse model (C3H/HeJ mice, SCC VII/SF cell line). HGF promoted cell proliferation, migration, invasion and induction of MMP (matrix metalloproteinase)-2 and MMP-9 in KB cells. EGCG significantly inhibited HGF-induced phosphorylation of Met and cell growth, invasion and expression of MMP-2 and MMP-9. EGCG blocked HGF-induced phosphorylation of c-Met and that of the downstream kinases AKT and ERK, and inhibition of p-AKT and p-ERK by EGCG was associated with marked increases in the phosphorylation of p38, JNK, cleaved caspase-3 and poly-ADP-ribose polymerase. In C3H/HeJ syngeneic mice, as an in vivo model, tumor growth was suppressed and apoptosis was increased by EGCG. Our results suggest that EGCG may be a potential therapeutic agent to inhibit HGF-induced tumor growth and invasion in oral cancer.     

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.     

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.     

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.     

Effect of hepatocyte growth factor/scatter factor and other growth factors on motility and morphology of non-tumorigenic and tumor cells

Summary Using an automated cell analyzer system, the effect of hepatocyte growth factor/scatter factor (HGF/SF), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), endothelial acidic fibroblast growth factor (a-FGF), platelet derived growth factor (PDGF), and recombinant human insulinlike growth factor (IGF) on the motility and morphology of Madin-Darby canine kidney (MDCK), rat hepatomas, C₂, and H_5–6 and murine mammary carcinoma (EMT-6) cells was investigated. Treatment of MDCK cells with HGF/SF, bFGF, EGF, and a-FGF resulted in an increase in average cell velocity and in the fraction of moving cells. Cells treated with the PDGF and IGF did not show significant alterations in velocity. MDCK cells treated with each growth factor were classified into groups of “fast” and “slow” moving cells based on their average velocities, and the average morphologic features of the two groups were quantitated. Fast-moving cells had larger average area, circularity, and flatness as compared to slow-moving cells. Factors that stimulated cell movement also induced alterations in cell morphologic parameters including spreading, flatness, area, and circularity. HGF/SF also scattered and stimulated motility of C₂ and H_5–6 hepatoma cells. In contrast to MDCK cells, there was no significant difference between the morphology of the fast moving and slow moving C₂ and H_5–6 cells. These studies suggest that growth factor cytokines have specific effects on motility of normal and tumor cells.     

The structural biology of growth factor receptor activation

Stimulation of cells by growth factors triggers cascades of signalling that result in cellular responses such as growth, differentiation, migration and survival. Many growth factors signal through receptor tyrosine kinases, leading to dimerization, trans-phosphorylation and activation of tyrosine kinases that phosphorylate components further downstream of the signal transduction cascade. Using insulin-like growth factor, nerve growth factor, hepatocyte growth factor and fibroblast growth factor as examples, we show that the globular architecture of the growth factors is essential for receptor binding. We describe how nerve growth factor (NGF) is a symmetrical dimer that binds four storage proteins (two -NGF and two γ-NGF) to give a symmetrical hetero-hexameric 7SNGF organised around the β-NGF dimer. It binds the extracellular domains of two receptor molecules in a similar way, so dimerising the receptor. Hepatocyte growth factor/scatter factor (HGF/SF) probably binds its receptor as a dimer stabilised by interactions with heparan sulfate, and fibroblast growth factor (FGF) binds its receptor as a dimer cross-linked by heparan sulfate. Surprisingly, insulin and insulin-like growth factor (IGF) bind in the monomeric form to receptors that are already covalent dimers. We propose that, in general, weak binary interactions between growth factor and individual domains of receptors are enhanced by cooperative interactions with further receptor domains, and sometimes other components like heparan, to give rise to specific multi-protein/domain complexes.