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.     

Molecular evolution and domain structure of plasminogen-related growth factors (HGF/SF and HGF1/MSP).

Plasminogen-related growth factors, a new family of polypeptide growth factors with the basic domain organization and mechanism of activation of the blood proteinase plasminogen, include hepatocyte growth factor/scatter factor (HGF/SF), a potent effector of the growth, movement, and differentiation of epithelia and endothelia, and hepatocyte growth factor-like/macrophage stimulating protein (HGF1/MSP), an effector of macrophage chemotaxis and phagocytosis. Phylogeny of the serine proteinase domains and analysis of intron-exon boundaries and kringle sequences indicate that HGF/SF, HGF1/MSP, plasminogen, and apolipoprotein (a) have evolved from a common ancestral gene that consisted of an N-terminal domain corresponding to plasminogen activation peptide (PAP), 3 copies of the kringle domain, and a serine proteinase domain. Models of the N domains of HGF/SF, HGF1/MSP, and plasminogen, characterized by the presence of 4 conserved Cys residues forming a loop in a loop, have been modeled based on disulfide-bond constraints. There is a distinct pattern of charged and hydrophobic residues in the helix-strand-helix motif proposed for the PAP domain of HGF/SF; these may be important for receptor interaction. Three-dimensional structures of the 4 kringle and the serine proteinase domains of HGF/SF were constructed by comparative modeling using the suite of programs COMPOSER and were energy minimized. Docking of a lysine analogue indicates a putative lysine-binding pocket within kringle 2 (and possibly another in kringle 4). The models suggest a mechanism for the formation of a noncovalent HGF/SF homodimer that may be responsible for the activation of the Met receptor. These data provide evidence for the divergent evolution and structural similarity of plasminogen, HGF/SF, and HGF1/MSP, and highlight a new strategy for growth factor evolution, namely the adaptation of a proteolytic enzyme to a role in receptor activation.     

Hepatocyte growth factor/scatter factor and its interaction with heparan sulphate and dermatan sulphate

Hepatocyte growth factor (HGF)/scatter factor (SF) is a unique growth factor, in that it binds both heparan sulphate (HS) and dermatan sulphate (DS). The sequences in HS and DS that specifically interact with and modulate HGF/SF activity have not yet been fully identified. Ascidian DS, which uniquely possesses O-sulphation at C-6 (and not C-4) of its N -acetylgalactosamine unit, was analysed for HGF/SF-binding activity in the biosensor. The kinetic analysis revealed a strong, biologically relevant interaction with an equilibrium dissociation constant ( K (d)) of approx. 1 nM. An Erk activation assay also demonstrated stimulation of the MAP kinase pathway downstream of the Met receptor following addition of both HGF/SF and ascidian DS to the glycosaminoglycan-deficient CHO-745 mutant cell line. Furthermore, the activation of Met and the MAP kinase pathway by HGF/SF and ascidian DS leads to a cellular response in the form of migration.     

Total synthesis of biotinylated N domain of human hepatocyte growth factor

Hepatocyte growth factor/scatter factor (HGF/SF) is the high affinity ligand of MET tyrosine kinase receptor. We report here the total synthesis of a biotinylated analogue of human HGF/SF N domain. Functionally, N domain is part of the HGF/SF high affinity binding site for MET and also the main HGF/SF binding site for heparin. The 97 Aa linear chain featuring a C-terminal biotin group was assembled in high yield using an N-to-C one-pot three segments assembly strategy relying on a sequential Native Chemical Ligation (NCL)/bis(2-sulfanylethyl)amido (SEA) native peptide ligation process. The folded protein displayed the native disulfide bond pattern and showed the ability to bind heparin.     

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.     

Conformational lability in serine protease active sites: structures of hepatocyte growth factor activator (HGFA) alone and with the inhibitory domain from HGFA inhibitor-1B

Hepatocyte growth factor activator (HGFA) is a serine protease that converts hepatocyte growth factor (HGF) into its active form. When activated HGF binds its cognate receptor Met, cellular signals lead to cell growth, differentiation, and migration, activities which promote tissue regeneration in liver, kidney and skin. Intervention in the conversion of HGF to its active form has the potential to provide therapeutic benefit where HGF/Met activity is associated with tumorigenesis. To help identify ways to moderate HGF/Met effects, we have determined the molecular structure of the protease domain of HGFA. The structure we determined, at 2.7 A resolution, with no pseudo-substrate or inhibitor bound is characterized by an unconventional conformation of key residues in the enzyme active site. In order to find whether this apparently non-enzymatically competent arrangement would persist in the presence of a strongly-interacting inhibitor, we also have determined, at 2.6 A resolution, the X-ray structure of HGFA complexed with the first Kunitz domain (KD1) from the physiological inhibitor hepatocyte growth factor activator inhibitor 1B (HAI-1B). In this complex we observe a rearranged substrate binding cleft that closely mirrors the cleft of other serine proteases, suggesting an extreme conformational dynamism. We also characterize the inhibition of 16 serine proteases by KD1, finding that the previously reported enzyme specificity of the intact extracellular region of HAI-1B resides in KD1 alone. We find that HGFA, matriptase, hepsin, plasma kallikrein and trypsin are potently inhibited, and use the complex structure to rationalize the structural basis of these results.     

A new crystal form of the NK1 splice variant of HGF/SF demonstrates extensive hinge movement and suggests that the NK1 dimer originates by domain swapping

NK1 is a splice variant of the polypeptide growth factor HGF/SF that consists of the N terminal (N) and first kringle (K) domains and retains receptor binding and signalling. While NK1 behaves as a monomer in solution, two independent crystallographic structures have previously shown an identical, tightly packed dimer. Here we report a novel orthorhombic crystal form of NK1 at 2.5 A resolution in which four NK1 protomers are packed in two distinct dimers in the asymmetric unit. Although the basic architecture of the new NK1 dimers is similar to the two described earlier, the new crystal form demonstrates extensive hinge movement between the N and K domain that leads to re-orientation of the receptor-binding sites. The hinge bending is evidence of the paucity of strong interactions between domains within the protomer, in contrast to the extensive interactions between protomers in the dimer. These observations are consistent with domain swapping in the dimer, such that the interdomain interactions of the monomer are replaced by equivalent interprotomer interactions in the dimer and offer a route for protein engineering of NK1 variants which may act as receptor antagonists.     

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.     

Dissociation of heparan sulfate and receptor binding domains of hepatocyte growth factor reveals that heparan sulfate-c-met interaction facilitates signaling.

Hepatocyte growth factor (HGF) is a secreted, heparan sulfate (HS) glycosaminoglycan-binding protein that stimulates mitogenesis, motogenesis, and morphogenesis in a wide array of cellular targets, including hepatocytes and other epithelial cells, melanocytes, endothelial cells, and hematopoietic cells. NK1 is an alternative HGF isoform that consists of the N-terminal (N) and first kringle (K1) domains of full-length HGF and stimulates all major HGF biological activities. Within NK1, the N domain retains the HS binding properties of full-length HGF and mediates HS-stimulated ligand oligomerization but lacks significant mitogenic or motogenic activity. In contrast, K1 does not bind HS, but it stimulates receptor and mitogen-activated protein kinase activation, mitogenesis, and motogenesis, demonstrating that structurally distinct and dissociable domains of HGF are the primary mediators of HS binding and receptor activation. Despite the absence of HS-K1 binding, K1 mitogenic activity in HS-negative cells is strictly dependent on added soluble heparin, whereas K1-stimulated motility is not. We also found that, like the receptors for fibroblast growth factors, the HGF receptor c-Met binds tightly to HS. These data suggest that HS can facilitate HGF signaling through interaction with c-Met that is independent of HGF-HS interaction and that the recruitment of specific intracellular effectors that mediate distinct HGF responses such as mitogenesis and motility is regulated by HS-c-Met interaction at the cell surface.     

Hepatocyte growth factor/scatter factor binds to small heparin-derived oligosaccharides and stimulates the proliferation of human HaCaT keratinocytes.

Hepatocyte growth factor/scatter factor (HGF/SF) acts via a dual receptor system consisting of the MET tyrosine kinase receptor and heparan sulfate or dermatan sulfate proteoglycans. In optical biosensor binding assays, competition by oligosaccharides for binding of HGF/SF to immobilized heparin showed that disaccharides failed to compete, whereas tetrasaccharides inhibited HGF/SF binding (IC(50) 8 microg/ml). The inhibitory potency of the oligosaccharides increased as their length increased by successive disaccharide units, to reach a maximum (IC(50) 1 microg/ml) at degree of polymerization (dp) 10. In binding assays, HGF/SF was found to bind directly to oligosaccharides as small as dp 4, and the binding parameters were similar for oligosaccharides of dp 4-14 (k(a) 2.2-45.3 x 10(6) m(-1) s(-1), k(d) 0.033-0.039 s(-1), and K(d) 9-16 nm). In human keratinocytes, HGF/SF stimulated DNA synthesis, and this was dependent on a sustained phosphorylation of p42/44(MAPK). In chlorate-treated and hence sulfated glycosaminoglycan-deficient HaCaT cells, the stimulation of DNA synthesis by HGF/SF was almost abolished. Heparin-derived oligosaccharides from dp 2 to dp 24 were added together with HGF/SF to chlorate-treated cells to determine the minimum size of oligosaccharides able to restore HGF/SF activity. At restricted concentrations of oligosaccharides (4 ng/ml), HGF/SF required decasaccharides, whereas at higher concentrations (100 ng/ml) even tetrasaccharides were able to partly restore DNA synthesis. The results suggest that HGF/SF binds to a tetrasaccharide and that although this is sufficient to enable the stimulation of DNA synthesis, longer oligosaccharides are more efficient, perhaps by virtue of their ability to bind more easily other molecules.     

Inhibition by copper(II) binding of hepatocyte growth factor (HGF) interaction with its receptor Met and blockade of HGF/Met function

Overexpression of hepatocyte growth factor (HGF) and its receptor Met often occurs in carcinoma cells, leading to establishment of an HGF/Met autocrine loop. Therefore, disruption of the HGF/Met autocrine loop may lead to down-regulation of tumorigenesis. To study the HGF/Met interaction, we have developed a cell-free system to detect HGF binding to a Met fusion protein, Met-IgG, using a modified enzyme-linked immunosorbent assay methodology. Since we previously showed that HGF can be purified by copper(II) affinity chromatography, we further explored the effect of copper(II) on the HGF/Met interaction. The divalent metal cations copper(II) and zinc(II) significantly inhibited HGF binding to immobilized Met-IgG with IC(50) values of 230-270 microM, respectively, whereas manganese(II) and magnesium(II) were less inhibitory with 20-60-fold higher IC(50) values. Incubation of 1 mM copper(II) with HGF resulted in nondenaturing and denaturing gel-mobility shifts, indicating that copper(II) binds directly to HGF. This interaction occurs at the N terminus of HGF, as incubation of 1 mM copper(II) with both HGF and the HGF derivative NK1 yielded similar results on SDS-PAGE. HGF-induced activation of Met and cell scattering were inhibited upon addition of HGF in the presence of 1 mM and 500 microM copper(II), respectively. Chemical protonation with diethyl pyrocarbonate of HGF histidine residues impeded the ability of 500 microM copper(II) to inhibit the binding of HGF to immobilized Met-IgG. Based on the NK1 domain structure, we propose that copper(II) may interact with HGF via the histidine residues in either N-terminal or kringle domains. The inhibition of HGF/Met interaction and subsequent downstream cellular functions may be through direct interference by copper(II), such as a change in charge or an induced local conformational change. This putative copper(II) binding domain may be the basis for developing potential inhibitors of HGF/Met binding and downstream functions and could lead to novel strategies for anti-cancer treatment.     

Functional characterization of human hepatocyte growth factor mutants obtained by deletion of structural domains.

Human hepatocyte growth factor (hHGF) consists of characteristic structural domains. In this study, we prepared mutant proteins lacking each of these domains and examined their biological activities for stimulation of hepatocyte DNA synthesis, inhibition of Meth A cell growth, and induction of MDCK cell dissociation. We also examined their interactions with the c-met/HGF receptor by competition analysis and by analysis of levels of tyrosine phosphorylation. The mutant proteins lacking the N-terminal, the first kringle, or the second kringle domain were not biologically effective and could not compete with hHGF bound to the c-met/HGF receptor. The results indicate that these domains are necessary for the biological activities of hHGF mediated by binding to the c-met/HGF receptor. The mutant proteins lacking the third or fourth kringle domain moderately retained biological activities and the receptor binding. The relative levels of the tyrosine phosphorylation of the c-met/HGF receptor by these mutant proteins correlated well with the relative potencies of the biological activities when compared with that of the wild-type hHGF. The mutant protein lacking the light chain was not effective in the biological activities and tyrosine phosphorylation of the c-met/HGF receptor, but competed with hHGF bound to the c-met/HGF receptor. These results suggest that the heavy chain plays an important role in the interaction of hHGF with the c-met/HGF receptor and that the light chain is further required for the tyrosine phosphorylation of the c-met/HGF receptor.     

肝细胞生长因子及受体传递的调节与内吞作用

肝细胞生长因子(HGF)是一种具有多重功能的细胞调控因子。HGF与其受体Met酪氨酸激酶(c-Met)的结合可激发多种生物学反应,从而调节细胞的增殖、分化、形态发生和侵袭运动等。有多种因素参与了HGF/c-Met信号传导的调控,从而防止信号的过度放大,其中Cbl1、Rab、泛素化激酶和HGF/c-Met的内吞等发挥了重要的作用。因此,对HGF/c-Met内吞过程的研究,了解内吞对于HGF/c-Met的信号传导及其调控的影响,探讨HGF/c-Met信号传导通路的调控机理和相互作用模式,可进一步阐明HGF/c-Met信号传导的调控机制,从而验证肝细胞中内吞作用直接调节HGF/c-Met信号通路的作用机制。     

Radioimmunoscintigraphy of tumors autocrine for human met and hepatocyte growth factor/scatter factor

Inappropriate expression of the c-met-protooncogene product (Met) and/or of its ligand, hepatocyte growth factor/scatter factor (HGF/SF), has been correlated with poor prognosis in a variety of human solid tumors. We are developing animal models for nuclear imaging of Met and HGF/SF expression in tumors in vivo. We radioiodinated a mixture of monoclonal antibodies (MAbs) that bind to human HGF/SF and to the external ligand-binding domain of human Met, and then injected the I-125-MAb mixture intravenously into mice bearing tumors either autocrine for human HGF/SF and human Met or autocrine-paracrine for murine HGF/SF and murine Met. Serial total body gamma camera images were obtained, and regional activity was determined by quantitative region-of-interest (ROI) analysis. Tumors autocrine for human HGF/SF and Met demonstrated significantly more rapid uptake and more rapid clearance of the I-125-MAb mixture than tumors expressing one or both murine homologues, reaching a mean tumor to total body activity ratio of > 0.3 by 1 day postinjection. We conclude that radioimmunodetection of tumors autocrine for human HGF/SF and Met is feasible with an I-125-MAb mixture reactive against the ligand-receptor pair.     

Structure of the human receptor tyrosine kinase met in complex with the Listeria invasion protein InlB

The tyrosine kinase Met, the product of the c-met proto-oncogene and the receptor for hepatocyte growth factor/scatter factor (HGF/SF), mediates signals critical for cell survival and migration. The human pathogen Listeria monocytogenes exploits Met signaling for invasion of host cells via its surface protein InlB. We present the crystal structure of the complex between a large fragment of the human Met ectodomain and the Met-binding domain of InlB. The concave face of the InlB leucine-rich repeat region interacts tightly with the first immunoglobulin-like domain of the Met stalk, a domain which does not bind HGF/SF. A second contact between InlB and the Met Sema domain locks the otherwise flexible receptor in a rigid, signaling competent conformation. Full Met activation requires the additional C-terminal domains of InlB which induce heparin-mediated receptor clustering and potent signaling. Thus, although it elicits a similar cellular response, InlB is not a structural mimic of HGF/SF.     

The N-terminal domain of hepatocyte growth factor inhibits the angiogenic behavior of endothelial cells independently from binding to the c-met receptor

Hepatocyte growth factor (HGF) is a pleiotropic factor that plays an important role in complex biological processes such as embryogenesis, tissue regeneration, cancerogenesis, and angiogenesis. HGF promotes cell proliferation, survival, motility, and morphogenesis through binding to its receptor, a transmembrane tyrosine kinase encoded by the MET proto-oncogene (c-met). Structurally speaking, HGF is a polypeptide related to the enzymes of the blood coagulation cascade. Thus, it comprises kringle domains that in some other proteins have been shown to be responsible for the anti-angiogenic activity. To check whether the isolated kringles of HGF were able to inhibit angiogenesis, we produced them as recombinant proteins and compared their biological activity with that of the recombinant HGF N-terminal domain (N). We showed that (i) none of the isolated HGF kringle exhibits an anti-angiogenic activity; (ii) N is a new anti-angiogenic polypeptide; (iii) the inhibitory action of N is not specific toward HGF, because it antagonized the angiogenic activity of other growth factors, such as fibroblast growth factor-2 and vascular endothelial growth factor; and (iv) in contrast with full-length HGF, N does not bind to the c-met receptor in vitro, but fully retains its heparin-binding capacity. Our results suggest that N inhibits angiogenesis not by disrupting the HGF/c-met interaction but rather by interfering with the endothelial glycosaminoglycans, which are the secondary binding sites of HGF.     

Differential mitogenic effects of single chain hepatocyte growth factor (HGF)/scatter factor and HGF/NK1 following cleavage by factor Xa

Hepatocyte growth factor/scatter factor (HGF/SF) is a multifunctional cytokine that is involved in many normal as well as pathological conditions. HGF/NK1, a splice variant of HGF/SF, has been reported to have either antagonistic or agonistic effects with regard to c-Met signaling depending on the cell type. In these experiments, we have determined that HGF/NK1 is a potent mitogen for rat hepatocytes in culture. Furthermore, we have found that coagulation factor Xa (fXa) is capable of cleaving HGF/NK1 and single chain HGF/SF (scHGF/SF). The products resulting from cleavage of HGF/NK1 or scHGF/SF by fXa appear as single bands under non-reducing conditions. The reaction products from the digestion of HGF/NK1 by fXa were separated under reducing conditions, and the cleavage site, as determined by N-terminal sequencing, was located C-terminal to arginine 134. Previous work established that the heparin-binding domain for HGF/SF is located in the N domain of HGF/SF. Additionally, the dimerization of the HGF/SF receptor (c-Met) by the ligand HGF/NK1 is facilitated by heparin and related sulfonated sugars on the cell surface, whereas heparin is not required for HGF/SF-mediated dimerization. Cleavage of single chain HGF/SF or HGF/NK1 by factor Xa does not alter the affinity of the respective molecules for heparin, but it did variably affect the associated mitogenic activity of these factors. The associated mitogenic activity of HGF/NK1 was reduced by more than 90%, whereas the mitogenic activity of scHGF/SF was unaffected. This suggests mandatory maintenance of a steric interaction of the N domain and the first kringle domain for HGF/NK1 to act as an agonist for rat hepatocyte growth but is not required by full-length HGF/SF.     

Crystal structure of the beta-chain of human hepatocyte growth factor-like/macrophage stimulating protein

Hepatocyte growth factor like/macrophage stimulating protein (HGFl/MSP) and hepatocyte growth factor/scatter factor (HGF/SF) define a distinct family of vertebrate-specific growth factors structurally related to the blood proteinase precursor plasminogen and with important roles in development and cancer. Although the two proteins share a similar domain structure and mechanism of activation, there are differences between HGFl/MSP and HGF/SF in terms of the contribution of individual domains to receptor binding. Here we present a crystal structure of the 30 kDa beta-chain of human HGFl/MSP, a serine proteinase homology domain containing the high-affinity binding site for the RON receptor. The structure describes at 1.85 Angstrom resolution the region of the domain corresponding to the receptor binding site recently defined in the HGF/SF beta-chain, namely the central cleft harboring the three residues corresponding to the catalytic ones of active proteinases (numbers in brackets define the sequence position according to the standard chymotrypsinogen numbering system) [Gln522 (c57), Gln568 (c102) and Tyr661 (c195)] and an adjacent loop flanking the S1 specificity pocket and containing residues Asn682 (c217) and Arg683 (c218) previously shown to be essential for binding of HGFl/MSP to the RON receptor. The study confirms the concept that the serine proteinase homology domains of HGFl/MSP and HGF/SF bind their receptors in an 'enzyme-substrate' mode, reflecting the common evolutionary origin of the plasminogen-related growth factors and the proteinases of the clotting and fibrinolytic pathways. However, analysis of the intermolecular interactions in the crystal lattice of beta-chain HGFl/MSP fails to show the same contacts seen in the HGF/SF structures and does not support a conserved mode of dimerization of the serine proteinase homology domains of HGFl/MSP and HGF/SF responsible for receptor activation.     

Inhibitory effect of luteolin on hepatocyte growth factor/scatter factor-induced HepG2 cell invasion involving both MAPK/ERKs and PI3K-Akt pathways

Hepatocyte growth factor (HGF), also known as scatter factor (SF), and its receptor, the c-Met tyrosine kinase, play roles in cancer invasion and metastasis in a wide variety of tumor cells. Clinical observations suggest that HGF can promote metastasis of hepatoma cells while stimulating tumor invasiveness. We use HGF as an invasive inducer of human hepatoma HepG2 cells to investigate the effect of flavonoids on anti-invasion. In our preliminary study, we investigated the effect of flavonoids including luteolin, quercetin, baicalein, genistein, taxifolin and catechin on HGF-mediated migration and invasion of HepG2 cells. We found that luteolin presented the most potent potential on anti-migration and anti-invasion by Boyden chamber assay. Furthermore, luteolin inhibited HGF-induced cell scattering and cytoskeleton change such as filopodia and lamellipodia was determined by both phase-contrast and fluorescence microscopy studies. In addition, Western blotting and immunoprecipitation were performed to confirm luteolin suppressed the phosphorylation of c-Met, the membrane receptor of HGF, as well as ERK1/2 and Akt, but not JNK1/2, which is activated by HGF. Our investigation demonstrated that luteolin similar to PD98059, which acts as a specific inhibitor of MEK, an up stream kinase regulating ERK1/2, and wortmannin, a PI3K inhibitor, inhibited the invasiveness induced by HGF. In conclusion, the luteolin inhibited HGF-induced HepG2 cell invasion involving both MAPK/ERKs and PI3K-Akt pathways.     

NK1, a Natural Splice Variant of Hepatocyte Growth Factor/Scatter Factor, Is a Partial Agonist In Vivo

Hepatocyte growth factor/scatter factor (HGF/SF) is a potent mitogen, motogen, and morphogen for epithelial cells expressing its tyrosine kinase receptor, the c-met proto-oncogene product, and is required for normal development in the mouse. Inappropriate stimulation of Met signal transduction induces aberrant morphogenesis and oncogenesis in mice and has been implicated in human cancer. NK1 is a naturally occurring HGF/SF splice variant composed of only the amino terminus and first kringle domain. While the biological activities of NK1 have been controversial, in vitro data suggest that it may have therapeutic value as an HGF/SF antagonist. Here, we directly test this hypothesis in vivo by expressing mouse NK1 in transgenic mice and comparing the consequent effects with those observed for mice carrying an HGF/SF transgene. Despite robust expression, NK1 did not behave as an HGF/SF antagonist in vivo. Instead, NK1-transgenic mice displayed most of the phenotypic characteristics associated with HGF/SF-transgenic mice, including enlarged livers, ectopic skeletal-muscle formation, progressive renal disease, aberrant pigment cell localization, precocious mammary lobuloalveolar development, and the appearance of mammary, hepatocellular, and melanocytic tumors. And like HGF/SF-transgenic livers, NK1 livers had higher levels of tyrosine-phosphorylated complexes associated with Met, suggesting that the mechanistic basis for the effects of NK1 overexpression in vivo was autocrine activation of Met. We conclude that NK1 acts in vivo as a partial agonist. As such, the efficacy of NK1 as a therapeutic HGF/SF antagonist must be seriously questioned.