Activation of PI3K-Akt-GSK3beta pathway mediates hepatocyte growth factor inhibition of RANTES expression in renal tubular epithelial cells

Hepatocyte growth factor (HGF) was recently reported to ameliorate renal inflammation in a rat model of chronic renal failure. HGF exerted its action through suppression of RANTES expression in renal tubules. In the present study, we utilized an in vitro model of human kidney proximal tubule epithelial cells (HKC) to elucidate the mechanisms of RANTES suppression by HGF. HGF significantly suppressed basal and TNF-alpha-induced mRNA and protein expression of RANTES in a time and dose dependent fashion. HGF elicited PI3K-Akt activation and inhibited GSK3, a downstream transducer of PI3K-Akt, by inhibitory phosphorylation at Ser-9. When the PI3K-Akt pathway was blocked by wortmannin, HGF inhibition of RANTES was abrogated, demonstrating that the PI3K-Akt pathway is necessary for HGF action. In addition, specific inhibition of GSK3 activity by lithium ion suppressed basal and TNF-alpha-induced RANTES expression, reminiscent of the action of HGF. To further investigate the role of GSK3 in modulating RANTES expression, we examined the effect of forced expression of wild type GSK3beta or an uninhibitable mutant GSK3beta, in which the regulatory Ser-9 residue is changed to alanine (S9A-GSK3beta) in HKC. Overexpression of wild type GSK3beta did not alter the inhibitory action of HGF on RANTES. In contrast, expression of S9A-GSK3beta abolished HGF inhibition of basal and TNF-alpha stimulated RANTES expression. These findings suggest that PI3K-Akt activation and subsequent inhibitory phosphorylation of GSK3beta are required for HGF-induced suppression of RANTES in HKC.     

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.     

The regulatory role of heparin on c-Met signaling in hepatocellular carcinoma cells

The role of heparin as an anticoagulant is well defined; however, its role in tumorigenesis and tumor progression is not clear yet. Some studies have shown that anticoagulant treatment in cancer patients improve overall survival, however, recent clinical trials have not shown a survival benefit in cancer patients receiving heparin treatment. In our previous studies we have shown the inhibitory effects of heparin on Hepatocyte Growth Factor (HGF)-induced invasion and migration in hepatocellular carcinoma (HCC) cells. In this study, we showed the differential effects of heparin on the behaviors of HCC cells based on the presence or absence of HGF. In the absence of HGF, heparin activated HGF/c-Met signaling and promoted motility and invasion in HCC cells. Heparin treatment led to c-Met receptor dimerization and activated c-Met signaling in an HGF independent manner. Heparin-induced c-Met activation increased migration and invasion through ERK1/2, early growth response factor 1 (EGR1) and Matrix Metalloproteinases (MMP) axis. Interestingly, heparin modestly decreased the proliferation of HCC cells by inhibiting activatory phosphorylation of Akt. The inhibition of c-Met signaling reversed heparin-induced increase in motility and invasion and, proliferation inhibition. Our study provides a new perspective into the role of heparin on c-Met signaling in HCC.     

The EGFR/ErbB3 Pathway Acts as a Compensatory Survival Mechanism upon c-Met Inhibition in Human c-Met+ Hepatocellular Carcinoma

Backgroundc-Met, a high-affinity receptor for Hepatocyte Growth Factor (HGF), plays a critical role in tumor growth, invasion, and metastasis. Hepatocellular carcinoma (HCC) patients with activated HGF/c-Met signaling have a significantly worse prognosis. Targeted therapies using c-Met tyrosine kinase inhibitors are currently in clinical trials for HCC, although receptor tyrosine kinase inhibition in other cancers has demonstrated early success. Unfortunately, therapeutic effect is frequently not durable due to acquired resistance.MethodsWe utilized the human MHCC97-H c-Met positive (c-Met⁺) HCC cell line to explore the compensatory survival mechanisms that are acquired after c-Met inhibition. MHCC97-H cells with stable c-Met knockdown (MHCC97-H c-Met KD cells) were generated using a c-Met shRNA vector with puromycin selection and stably transfected scrambled shRNA as a control. Gene expression profiling was conducted, and protein expression was analyzed to characterize MHCC97-H cells after blockade of the c-Met oncogene. A high-throughput siRNA screen was performed to find putative compensatory survival proteins, which could drive HCC growth in the absence of c-Met. Findings from this screen were validated through subsequent analyses.ResultsWe have previously demonstrated that treatment of MHCC97-H cells with a c-Met inhibitor, PHA665752, results in stasis of tumor growth in vivo. MHCC97-H c-Met KD cells demonstrate slower growth kinetics, similar to c-Met inhibitor treated tumors. Using gene expression profiling and siRNA screening against 873 kinases and phosphatases, we identified ErbB3 and TGF-α as compensatory survival factors that are upregulated after c-Met inhibition. Suppressing these factors in c-Met KD MHCC97-H cells suppresses tumor growth in vitro. In addition, we found that the PI3K/Akt signaling pathway serves as a negative feedback signal responsible for the ErbB3 upregulation after c-Met inhibition. Furthermore, in vitro studies demonstrate that combination therapy with PHA665752 and Gefitinib (an EGFR inhibitor) significantly reduced cell viability and increased apoptosis compared with either PHA665752 or Gefitinib treatment alone.Conclusionc-Met inhibition monotherapy is not sufficient to eliminate c-Met⁺ HCC tumor growth. Inhibition of both c-Met and EGFR oncogenic pathways provides superior suppression of HCC tumor growth. Thus, combination of c-Met and EGFR inhibition may represent a superior therapeutic regimen for c-Met⁺ HCC.     

A new anti-c-met antibody selected by a mechanism-based dual-screening method: Therapeutic potential in cancer

c-Met, the high affinity receptor for hepatocyte growth factor (HGF), is one of the most frequently activated tyrosine kinases in many human cancers and a target for cancer therapy. However, inhibitory targeting of c-Met with antibodies has proven difficult, because most antibodies have intrinsic agonist activity. Therefore, the strategy for reducing the agonism is critical for successful development of cancer therapies based on anti-c-Met antibodies. Here we developed a mechanism-based assay method for rapid screening of anti-c-Met antibodies, involving the determination of Akt phosphorylation and c-Met degradation for agonism and efficacy, respectively. Using the method, we identified an antibody, F46, that binds to human c-Met with high affinity (Kd = 2.56 nM) and specificity, and induces the degradation of c-Met in multiple cancer cells (including MKN45, a gastric cancer cell line) with minimal activation of c-Met signaling. F46 induced c-Met internalization in both HGF-dependent and HGF-independent cells, suggesting that the degradation of c-Met results from antibody-mediated receptor internalization. Furthermore, F46 competed with HGF for binding to c-Met, resulting in the inhibition of both HGF-mediated invasion and angiogenesis. Consistently, F46 inhibited the proliferation of MKN45 cells, in which c-Met is constitutively activated in an HGF-independent manner. Xenograft analysis revealed that F46 markedly inhibits the growth of subcutaneously implanted gastric and lung tumors. These results indicate that F46, identified by a novel mechanism-based assay, induces c-Met degradation with minimal agonism, implicating a potential role of F46 in therapy of human cancers.     

Molecular imaging of c-Met tyrosine kinase activity

The receptor tyrosine kinase c-Met and its ligand, hepatocyte growth factor/scatter factor (HGF/SF), modulate signaling cascades implicated in cellular proliferation, survival, migration, invasion, and angiogenesis. Therefore, dysregulation of HGF/c-Met signaling can compromise the cellular capacity to moderate these activities and can lead to tumorigenesis, metastasis, and therapeutic resistance in various human malignancies. To facilitate studies investigating HGF/c-Met receptor coupling or c-Met signaling events in real time and in living cells and animals, here we describe a genetically engineered reporter where bioluminescence can be used as a surrogate for c-Met tyrosine kinase activity. c-Met kinase activity in cultured cells and tumor xenografts was monitored quantitatively and dynamically in response to the activation or inhibition of the HGF/c-Met signaling pathway. Treatment of tumor-bearing animals with a c-Met inhibitor and the HGF neutralizing antibody stimulated the reporter’s bioluminescence activity in a dose-dependent manner and led to a regression of U-87 MG tumor xenografts. Results obtained from these studies provide unique insights into the pharmacokinetics and pharmacodynamics of agents that modulate c-Met activity and validate c-Met as a target for human glioblastoma therapy.     

Activated c-Met signals through PI3K with dramatic effects on cytoskeletal functions in small cell lung cancer

Small cell lung cancer (SCLC) is an aggressive illness with early metastases. There are several receptor tyrosine kinases (RTKs) overexpressed in SCLC, including c-Met. c-Met contains an external semaphorin-like domain, a cytoplasmic juxtamembrane domain, tyrosine kinase domain and multiple tyrosines that bind to adapter molecules. We have previously reported that c-Met is abundantly expressed in the NCI-H69 SCLC cell line and now have determined the downstream effects of stimulating c-Met via its ligand hepatocyte growth factor (HGF). Utilizing unique phospho-specific antibodies generated against various tyrosines of c-Met, we show that Y1003 (binding site for c-Cb1 and a negative regulatory site), Y1313 (binding site for PI3K), Y1230/Y1234/Y1235 (autophosphorylation site), Y1349 (binding site for Grb2), Y1365 (important in cell morphogenesis) are phosphorylated in response to HGF (40 ng/ml, 7.5 min) in H69 cells. Since multiple biological and biochemical effects are transduced through the PI3K pathway, we determine the role of PI3K in the c-Met/HGF stimulation pathway. We initially determined that by inhibiting PI3K with LY294002 (50μM over 72 hours), there was at least a 55% decrease in viability of H69 cells. Since H69 SCLC cells form clusters in cell culture, we determined the effects of HGF and LY294002 on cell motility of the clusters by time-lapse video microscopy. In response to HGF, SCLC moved much faster and formed more clusters, and this was inhibited by LY294002. Finally, we determined the downstream signal transduction of HGF stimulation of c-Met with and without inhibition of c-Met (with geldanamycin, an anisamycin antibiotic that inhibits c-Met in SCLC) or PI3K (with LY294002). We show that association of c-Met with PI3K and GAB2 is diminished by inhibiting c-Met. In summary, activation of the c-Met pathway targets the PI3K pathway in SCLC and this may be an important therapeutic target.     

Reciprocal Activating Crosstalk between c-Met and Caveolin 1 Promotes Invasive Phenotype in Hepatocellular Carcinoma

c-Met, the receptor for Hepatocyte Growth Factor (HGF), overexpressed and deregulated in Hepatocellular Carcinoma (HCC). Caveolin 1 (CAV1), a plasma membrane protein that modulates signal transduction molecules, is also overexpressed in HCC. The aim of this study was to investigate biological and clinical significance of co-expression and activation of c-Met and CAV1 in HCC. We showed that c-Met and CAV1 were co-localized in HCC cells and HGF treatment increased this association. HGF-triggered c-Met activation caused a concurrent rise in both phosphorylation and expression of CAV1. Ectopic expression of CAV1 accelerated c-Met signaling, resulted in enhanced migration, invasion, and branching-morphogenesis. Silencing of CAV1 downregulated c-Met signaling, and decreased migratory/invasive capability of cells and attenuated branching morphogenesis. In addition, activation and co-localization of c-Met and CAV1 were elevated during hepatocarcinogenesis. In conclusion reciprocal activating crosstalk between c-Met and CAV1 promoted oncogenic signaling of c-Met contributed to the initiation and progression of HCC.     

Hepatocyte growth factor and c-Met promote dendritic maturation during hippocampal neuron differentiation via the Akt pathway

During central nervous system development, growth factors and their associated receptor protein tyrosine kinases regulate many neuronal functions such as neurite extension and dendrite maturation. Hepatocyte growth factor (HGF) and its receptor, c-Met, can promote formation of neurites and enhance elaboration of dendrites in mature neurons, but their effects on the early stages of dendrite maturation in hippocampal neurons and the signaling pathways by which they promote dendrite formation have not been studied. Exogenous HGF treatment effectively enhanced the phosphorylation and activation of c-Met in cultured hippocampal neurons at 4 days in vitro. HGF treatment increased the number of dendrites and promoted dendrite elongation in these neurons. Consistent with these results, HGF activated Akt, which phosphorylates glycogen synthase kinase-3beta (GSK-3beta) to inactivate it, and reduced phosphorylation of microtubule-associated protein 2 (MAP2), which can promote microtubule polymerization and dendrite elongation when dephosphorylated. Conversely, pharmacological inhibition of c-Met with its specific inhibitor, PHA-665752, or genetic knock-down of c-Met with short hairpin RNAs (shRNAs) suppressed HGF-induced phosphorylation of Akt and GSK-3beta, increased phosphorylation of MAP2, and reduced dendrite number and length in cultured hippocampal neurons. Moreover, suppressing c-Met with PHA-665752 or by shRNA decreased MAP2 expression. Inhibiting Akt activity with the phosphoinositide-3-kinase inhibitor LY294002 or Akt inhibitor X suppressed HGF-induced phosphorylation of GSK-3beta, increased MAP2 phosphorylation, and blocked the ability of HGF to enhance dendritic length. These observations indicate that HGF and c-Met can regulate the early stages of dendrite maturation via activation of the Akt/GSK-3beta pathway.     

HGF and c-Met Interaction Promotes Migration in Human Chondrosarcoma Cells

Chondrosarcoma is a type of highly malignant tumor with a potent capacity for local invasion and causing distant metastasis. Chondrosarcoma shows a predilection for metastasis to the lungs. Hepatocyte growth factor (HGF) has been demonstrated to stimulate cancer proliferation, migration, and metastasis. However, the effect of HGF on migration activity of human chondrosarcoma cells is not well known. Here, we found that human chondrosarcoma tissues demonstrated significant expression of HGF, which was higher than that in normal cartilage. We also found that HGF increased the migration and expression of matrix metalloproteinase (MMP)-2 in human chondrosarcoma cells. c-Met inhibitor and siRNA reduced HGF-increased cell migration and MMP-2 expression. HGF treatment resulted in activation of the phosphatidylinositol 3′-kinase (PI3K)/Akt/PKCδ/NF-κB pathway, and HGF-induced expression of MMP-2 and cell migration was inhibited by specific inhibitors or siRNA-knockdown of PI3K, Akt, PKCδ, and NF-κB cascades. Taken together, our results indicated that HGF enhances migration of chondrosarcoma cells by increasing MMP-2 expression through the c-Met receptor/PI3K/Akt/PKCδ/NF-κB signal transduction pathway.     

Benzyl isothiocyanate inhibits basal and hepatocyte growth factor-stimulated migration of breast cancer cells

Benzyl isothiocyanate (BITC), which is found in cruciferous vegetables, has been shown to have anti-carcinogenic properties. Hepatocyte growth factor (HGF) has the ability to stimulate dissociation, migration, and invasion in various tumor cells, and abnormally increased expressions of HGF and its transmembrane tyrosine kinase receptor, c-Met, have previously been detected in human breast cancer, and are associated with high tumor grade and poor prognosis. In this study, in order to assess the mechanisms relevant to the BITC-induced regulation of breast cancer cell migration and invasion, MDA-MB-231 human breast cancer cells and 4T1 murine mammary carcinoma cells were cultured in the presence of 0-4?μmol/l BITC with or without 10?μg/l of HGF. BITC inhibited both the basal and HGF-induced migration of MDA-MB-231 and 4T1 cells in a dose-dependent manner. In MDA-MB-231 cells, BITC reduced both basal and HGF-induced secretion and activity of urokinase-type plasminogen activator (uPA). In addition, BITC increased the protein levels of plasminogen activator inhibitor-1. HGF stimulated c-Met and Akt phosphorylation, but did not affect the phosphorylation of extracellular signal-regulated kinase-1/2 or stress-activated protein/c-jun N-terminal kinase. BITC suppressed NF-κB activity and reduced the HGF-induced phosphorylation of c-Met and Akt in a dose-dependent manner. LY294002, a specific Akt inhibitor, reduced both basal and HGF-induced uPA secretion and migration of MDA-MB-231 cells. In this study, we demonstrated that BITC profoundly inhibits the migration and invasion of MDA-MB-231 cells, which is associated with reduced uPA activity, and also that these phenomena are accompanied by the suppression of Akt signaling.     

Biochemical and pharmacological characterization of human c-Met neutralizing monoclonal antibody CE-355621

The c-Met proto-oncogene is a multifunctional receptor tyrosine kinase that is stimulated by its ligand, hepatocyte growth factor (HGF), to induce cell growth, motility and morphogenesis. Dysregulation of c-Met function, through mutational activation or overexpression, has been observed in many types of cancer and is thought to contribute to tumor growth and metastasis by affecting mitogenesis, invasion, and angiogenesis. We identified human monoclonal antibodies that bind to the extracellular domain of c-Met and inhibit tumor growth by interfering with ligand-dependent c-Met activation. We identified antibodies representing four independent epitope classes that inhibited both ligand binding and ligand-dependent activation of c-Met in A549 cells. In cells, the antibodies antagonized c-Met function by blocking receptor activation and by subsequently inducing downregulation of the receptor, translating to phenotypic effects in soft agar growth and tubular morphogenesis assays. Further characterization of the antibodies in vivo revealed significant inhibition of c-Met activity (≥ 80% lasting for 72–96 h) in excised tumors corresponded to tumor growth inhibition in multiple xenograft tumor models. Several of the antibodies identified inhibited the growth of tumors engineered to overexpress human HGF and human c-Met (S114 NIH 3T3) when grown subcutaneously in athymic mice. Furthermore, lead candidate antibody CE-355621 inhibited the growth of U87MG human glioblastoma and GTL-16 gastric xenografts by up to 98%. The findings support published pre-clinical and clinical data indicating that targeting c-Met with human monoclonal antibodies is a promising therapeutic approach for the treatment of cancer.     

Contact inhibition of hepatocyte growth regulated by functional association of the c-Met/hepatocyte growth factor receptor and LAR protein-tyrosine phosphatase

Contact inhibition, the inhibition of cell proliferation by tight cell-cell contact is a fundamental characteristic of normal cells. Using primary cultured hepatocytes, we investigated the mechanisms of contact inhibition that decrease the mitogenic activity of hepatocyte growth factor (HGF), focusing on the regulation of c-Met/HGF-receptor activation. In hepatocytes cultured at a sparse cell density, HGF stimulation induced prolonged c-Met tyrosine phosphorylation for over 5 h and a marked mitogenic response. In contrast, HGF stimulation induced transient c-Met tyrosine phosphorylation in <3 h and failed to induce mitogenic response in hepatocytes cultured at a confluent cell density. Treatment of the confluent cells with HGF plus orthovanadate, a broad spectrum protein-tyrosine phosphatase inhibitor, however, prolonged c-Met tyrosine phosphorylation for over 5 h and permitted the subsequent mitogenic response. The mitogenic response to HGF was associated with the duration of c-Met tyrosine phosphorylation even in the sparse cells. We found that the activity and expression of the protein-tyrosine phosphatase LAR increased following HGF stimulation specifically in confluent hepatocytes and not in sparse hepatocytes. LAR and c-Met were associated, and purified LAR dephosphorylated tyrosine-phosphorylated c-Met in in vitro phosphatase reactions. Furthermore, antisense oligonucleotides specific for LAR mRNA suppressed the expression of LAR, allowed prolonged c-Met tyrosine phosphorylation, and led to acquisition of a mitogenic response in hepatocytes even under the confluent condition. Thus functional association of LAR and c-Met underlies the inhibition of c-Met-mediated mitogenic signaling through the dephosphorylation of c-Met, which specifically occurs under the confluent condition.     

The Multisubstrate Adapter Gab1 Regulates Hepatocyte Growth Factor (Scatter Factor)–c-Met Signaling for Cell Survival and DNA Repair

Hepatocyte growth factor (scatter factor) (HGF/SF) is a pleiotrophic mediator of epithelial cell motility, morphogenesis, angiogenesis, and tumorigenesis. HGF/SF protects cells against DNA damage by a pathway from its receptor c-Met to phosphatidylinositol 3-kinase (PI3K) to c-Akt, resulting in enhanced DNA repair and decreased apoptosis. We now show that protection against the DNA-damaging agent adriamycin (ADR; topoisomerase IIalpha inhibitor) requires the Grb2-binding site of c-Met, and overexpression of the Grb2-associated binder Gab1 (a multisubstrate adapter required for epithelial morphogenesis) inhibits the ability of HGF/SF to protect MDCK epithelial cells against ADR. In contrast to Gab1 and its homolog Gab2, overexpression of c-Cb1, another multisubstrate adapter that associates with c-Met, did not affect protection. Gab1 blocked the ability of HGF/SF to cause the sustained activation of c-Akt and c-Akt signaling (FKHR phosphorylation). The Gab1 inhibition of sustained c-Akt activation and of cell protection did not require the Gab1 pleckstrin homology or SHP2 phosphatase-binding domain but did require the PI3K-binding domain. HGF/SF protection of parental MDCK cells was blocked by wortmannin, expression of PTEN, and dominant negative mutants of p85 (regulatory subunit of PI3K), Akt, and Pak1; the protection of cells overexpressing Gab1 was restored by wild-type or activated mutants of p85, Akt, and Pak1. These findings suggest that the adapter Gab1 may redirect c-Met signaling through PI3K away from a c-Akt/Pak1 cell survival pathway.     

Aptamers Binding to c-Met Inhibiting Tumor Cell Migration

The human receptor tyrosine kinase c-Met plays an important role in the control of critical cellular processes. Since c-Met is frequently over expressed or deregulated in human malignancies, blocking its activation is of special interest for therapy. In normal conditions, the c-Met receptor is activated by its bivalent ligand hepatocyte growth factor (HGF). Also bivalent antibodies can activate the receptor by cross linking, limiting therapeutic applications. We report the generation of the RNA aptamer CLN64 containing 2’-fluoro pyrimidine modifications by systematic evolution of ligands by exponential enrichment (SELEX). CLN64 and a previously described single-stranded DNA (ssDNA) aptamer CLN3 exhibited high specificities and affinities to recombinant and cellular expressed c-Met. Both aptamers effectively inhibited HGF-dependent c-Met activation, signaling and cell migration. We showed that these aptamers did not induce c-Met activation, revealing an advantage over bivalent therapeutic molecules. Both aptamers were shown to bind overlapping epitopes but only CLN3 competed with HGF binding to cMet. In addition to their therapeutic and diagnostic potential, CLN3 and CLN64 aptamers exhibit valuable tools to further understand the structural and functional basis for c-Met activation or inhibition by synthetic ligands and their interplay with HGF binding.     

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.     

1alpha,25(OH)2-vitamin D3 inhibits HGF synthesis and secretion from MG-63 human osteosarcoma cells

Several mesenchymally derived cells, including osteoblasts, secrete hepatocyte growth factor (HGF). 1alpha,25(OH)(2)-vitamin D(3) [1,25(OH)(2)D(3)] inhibits proliferation and induces differentiation of MG-63 osteoblastic cells. Here we show that MG-63 cells secrete copious amounts of HGF and that 1,25(OH)(2)D(3) inhibits HGF production. MG-63 cells also express HGF receptor (c-Met) mRNA, suggesting an autocrine action of HGF. Indeed, although exogenous HGF failed to stimulate cellular proliferation, neutralizing endogenous HGF with a neutralizing antibody inhibited MG-63 cell proliferation; moreover, inhibiting HGF synthesis with 1,25(OH)(2)D(3) followed by addition of HGF rescued hormone-induced inhibition of proliferation. Nonneutralized cells displayed constitutive phosphorylation of c-Met and the mitogen-activated protein kinases mitogen/extracellular signal-regulated kinase (MEK) 1 and extracellular signal-regulated kinase (Erk) 1/2, which were inhibited by anti-HGF antibody. Constitutive phosphorylation of Erk1/2 was also abolished by 1,25(OH)(2)D(3). Addition of HGF to MG-63 cells treated with neutralizing HGF antibody induced rapid phosphorylation of c-Met, MEK1, and Erk1/2. Thus endogenous HGF induces a constitutively active, autocrine mitogenic loop in MG-63 cells. The known antiproliferative effect of 1,25(OH)(2)D(3) on MG-63 cells can be accounted for by the concomitant 1,25(OH)(2)D(3)-induced inhibition of HGF production.     

Immunolocalization of hepatocyte growth factor and its receptor (c-Met) during mouse liver development

Although hepatocyte growth factor (HGF) was discovered as a potent hepatotrophic factor responsible for liver regeneration and may involve some organ development in embryogenesis, it remains to be revealed what roles HGF plays in liver development. The present study was undertaken to determine which cells express HGF and its receptor c-Met and when c-Met is activated in mouse liver development by using immunoblotting and immunohistochemical techniques. HGF was detected in hepatocytes and non-parenchymal cells, including biliary epithelial cells, periportal connective tissue cells, megakaryocytes, endothelial cells, and sinusoidal cells, throughout liver development. Positive HGF immunostaining in hepatocytes increased during postnatal development, and reached the maximal level in the adult stage. c-Met protein was also expressed in hepatocytes throughout liver development, but maximal staining was obtained in 1- or 2-week-old livers. Phosphorylation of tyrosine residues in the c-Met beta chain also occurred in these stages. These results suggest that HGF signaling is implicated in hepatocyte growth during postnatal liver development, and its action could be in a paracrine mode; HGF produced by non-parenchymal cells such as sinusoidal cells acts on hepatocytes expressing c-Met receptors. Positive immunostaining in adult and postnatal hepatocytes may be derived from their blood clearance of HGF.