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Eliot M Rosen - One of the best experts on this subject based on the ideXlab platform.
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role of src signal transduction pathways in Scatter Factor mediated cellular protection
Journal of Biological Chemistry, 2009Co-Authors: Qinghui Meng, John Laterra, Eliot M RosenAbstract:Scatter Factor (SF) (hepatocyte growth Factor) is a pleiotrophic cytokine that accumulates in tumors, where it may induce invasion, angiogenesis, and chemoresistance. We have studied the mechanisms by which SF and its receptor (c-Met) protect cells against the DNA-damaging agent adriamycin (ADR) as a model for chemoresistance of SF/c-Met-overexpressing tumors. Previous studies identified a phosphatidylinositol 3-kinase/c-Akt/Pak1/NF-κB cell survival pathway in DU-145 prostate cancer and Madin-Darby canine kidney epithelial cells. Here we studied Src signaling pathways involved in SF cell protection. Src enhanced basal and SF stimulated NF-κB activity and SF protection against ADR, in a manner dependent upon its kinase and Src homology 3 domains; and endogenous Src was required for SF stimulation of NF-κB activity and cell protection. The ability of Src to enhance SF stimulation of NF-κB activity was due, in part, to its ability to stimulate Akt and IκB kinase activity; and Src-mediated stimulation of NF-κB was due, in part, to a Rac1/MKK3/6/p38 pathway and was Akt-dependent. SF caused the activation of Src and the Rac1 effector Pak1. Furthermore, SF induced activating phosphorylations of MKK3, MKK6, and p38 within the c-Met signalsome in an Src-dependent manner. The NF-κB-inducing kinase was found to act downstream of TAK1 (transforming growth Factor-β-activated kinase 1) as a mediator of SF- and Src-stimulated NF-κB activity. Finally, the Src/Rac1/MKK3/6/p38 and Src/TAK1/NF-κB-inducing kinase pathways exhibited cross-talk at the level of MKK3. These findings delineate some novel signaling pathways for SF-mediated resistance to ADR.
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role of nf κb signaling in hepatocyte growth Factor Scatter Factor mediated cell protection
Oncogene, 2005Co-Authors: Qinghui Meng, Itzhak D Goldberg, John Laterra, Marc Symons, Sal Coniglio, Richard G Pestell, Eliot M RosenAbstract:The cytokine Scatter Factor/hepatocyte growth Factor (HGF/SF) protects epithelial, carcinoma, and other cell types against cytotoxicity and apoptosis induced by DNA-damaging agents such as ionizing radiation and adriamycin (ADR, a topoisomerase IIα inhibitor). We investigated the role of nuclear Factor kappa B (NF-κB) signaling in HGF/SF-mediated protection of human prostate cancer (DU-145) and Madin–Darby canine kidney (MDCK) epithelial cells against ADR. HGF/SF caused the rapid nuclear translocation of the p65 (RelA) subunit of NF-κB associated with the transient loss of the inhibitory subunit IκB-α. Exposure to HGF/SF caused the activation of an NF-κB luciferase reporter that was blocked or attenuated by the expression of a mutant ‘super-repressor’ IκB-α. Electrophoretic mobility shift assay supershift assays revealed that HGF/SF treatment induced the transient binding of various NF-κB family proteins (p65, p50, c-Rel, and RelB) with radiolabeled NF-κB-binding oligonucleotides. The HGF/SF-mediated protection of DU-145 and MDCK cells against ADR (demonstrated using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays) was abrogated by the IκB-α super-repressor. The ability of HGF/SF to activate NF-κB signaling was dependent on c-Akt → Pak1 (p21-associated kinase-1) signaling (with Pak1 downstream of c-Akt) and was inhibited by the tumor suppressor PTEN (phosphatase and tensin homolog). Inhibitors of phosphatidylinositol-3′-kinase and Src family kinases significantly inhibited HGF/SF-mediated activation of NF-κB, while inhibitors of MEK, protein kinase C, and p70 S6 kinase had a modest effect or no effect on NF-κB activity. HGF/SF induced the expression of several known NF-κB target genes (cIAP-1 (cellular inhibitor of apoptosis-1), cIAP-2, and TRAF-2 (TNF receptor-associated Factor-2)) in an NF-κB-dependent manner; HGF/SF blocked the inhibition of expression of these genes by ADR. Experimental manipulation of expression of these genes suggests that they (particularly TRAF-2 and cIAP-2) contribute to the protection against ADR by HGF/SF. These findings suggest that HGF/SF activates NF-κB through a c-Akt → Pak1 signaling pathway that is also dependent on Src, and that NF-κB contributes to HGF/SF-mediated protection against ADR.
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the multisubstrate adapter gab1 regulates hepatocyte growth Factor Scatter Factor c met signaling for cell survival and dna repair
Molecular and Cellular Biology, 2001Co-Authors: Yong Xian, Itzhak D Goldberg, Qinghui Meng, Ren-qi Yuan, Eliot M RosenAbstract: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 IIα 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.
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Scatter Factor hepatocyte growth Factor sf hgf content and function in human gliomas
International Journal of Developmental Neuroscience, 1999Co-Authors: Katrin Lamszus, John Laterra, Manfred Westphal, Eliot M RosenAbstract:Scatter Factor/hepatocyte growth Factor (SF/HGF) is a pleiotrophic cytokine that stimulates motility and invasion of several cancer cell types and induces angiogenesis. Its receptor MET is a transmembrane tyrosine kinase encoded by the C-MET proto-oncogene. To assess the potential relevance of SF/HGF in gliomas we performed functional studies in vivo and in vitro, expression analyses and correlative studies. We showed that both SF/HGF and MET are expressed in gliomas in vivo and are upregulated during transition from low grade to malignant glioma. When SF/HGF cDNA was transfected into glioma cells that expressed the MET receptor the cells formed considerably larger and more vascularized intracranial tumors in vivo than SF/HGF negative control clones. In other glioma cells, which constitutively expressed both SF/HGF and MET, we abolished SF/HGF expression by antisense ribozyme-targeting, which led to a significant decrease in tumorigenicity and tumor growth. In vitro SF/HGF strongly stimulated glioma cell motility and to a lesser degree proliferation. SF/HGF also strongly increased endothelial cell motility in vitro and extracts of tumors derived from SF/HGF-transfected glioma cells were more mitogenic for endothelial cells and more angiogenic in the rat cornea angiogenesis assay than extracts from control tumors. In a three-dimensional in vitro angiogenesis assay basic fibroblast growth Factor (bFGF) was found to synergize with either SF/HGF or vascular endothelial growth Factor (VEGF) in inducing endothelial capillary-like tubes, whereas neither SF/HGF nor VEGF alone or in combination were effective. Interestingly, while both VEGF and SF/HGF levels appeared to be increased in malignant gliomas compared with low grade ones, this was not the case for bFGF of which biologically relevant levels were already present in low grade gliomas. It thus seems that bFGF alone is insufficient to induce angiogenesis in gliomas but may act synergistically with either VEGF and/or SF/HGF when these become upregulated during malignant progression. In conclusion, we showed that SF/HGF may contribute to glioma progression by stimulating tumor invasiveness, proliferation and neovascularization.
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Scatter Factor protects epithelial and carcinoma cells against apoptosis induced by DNA-damaging agents
Oncogene, 1998Co-Authors: Ji-an Wang, Itzhak D Goldberg, Ren-qi Yuan, Sara Rockwell, Janet Andres, Andrey Zlatapolskiy, Eliot M RosenAbstract:Scatter Factor (SF) (hepatocyte growth Factor) is a cytokine that may play a role in human breast cancer invasiveness and angiogenesis. We now report that SF can block the induction of apoptosis by various DNA damaging-agents, including cytotoxic agents used in breast cancer therapy. SF protected MDA-MB-453 human breast cancer cells, EMT6 mouse mammary tumor cells and MDCK renal epithelial cells against apoptosis induced by adriamycin (ADR), X-rays, ultraviolet radiation, and other agents. Protection was observed in assays of DNA fragmentation, cell viability (MTT), and clonogenic survival. Protection of MDA-MB-453 cells against ADR was dose- and time-dependent; maximal protection required pre-incubation with 75–100 ng/ml of SF for 48 h or more. Protection required functional SF receptor ( c-Met ), but was not dependent on p53. Western blotting analysis revealed that pre-treatment of MDA-MB-453 cells with SF inhibited the ADR-induced decreases in the levels of Bcl-X _ L , an anti-apoptotic protein related to Bcl-2; and the dose-response and time course characteristics for SF-mediated increases in the Bcl-X _ L protein levels of ADR-treated cells were consistent with the degrees of protection against apoptosis observed under the same conditions. Furthermore, Bcl-X _ L levels were not down-regulated by ADR in MDA-MB-231 breast cancer cells, consistent with the finding that SF failed to protect these cells against ADR, despite the fact that they contain functional c-Met receptor. In contrast to Bcl-X _ L , SF blocked ADR-induced increases in c-Myc and inhibited the expression of p21^ WAF1/CIP1 and of the BRCA1 protein in MDA-MB-453 cells. However, SF did not cause significant changes in the cell cycle distribution of ADR-treated cells. These findings suggest that SF-mediated protection of human breast cancer cells may involve inhibition of one or more pathways required for the activation of apoptosis and may particularly target the anti-apoptotic mitochondrial membrane pore-forming protein Bcl-X _ L as a component of the protective mechanism. By implication, the accumulation of SF within human breast cancers may contribute to the development of a radio- or chemoresistant phenotype.
George Vande F Woude - One of the best experts on this subject based on the ideXlab platform.
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structural basis of hepatocyte growth Factor Scatter Factor and met signalling
Proceedings of the National Academy of Sciences of the United States of America, 2006Co-Authors: Ermanno Gherardi, George Vande F Woude, Mark Youles, Maxim V Petoukhov, Sara Sandin, J T Finch, Larsgoran Ofverstedt, Ricardo Nunez Miguel, T L Blundell, Ulf SkoglundAbstract:The polypeptide growth Factor, hepatocyte growth Factor/Scatter Factor (HGF/SF), shares the multidomain structure and proteolytic mechanism of activation of plasminogen and other complex serine proteinases. HGF/SF, however, has no enzymatic activity. Instead, it controls the growth, morphogenesis, or migration of epithelial, endothelial, and muscle progenitor cells through the receptor tyrosine kinase MET. Using small-angle x-ray Scattering and cryo-electron microscopy, we show that conversion of pro(single-chain)HGF/SF into the active two-chain form is associated with a major structural transition from a compact, closed conformation to an elongated, open one. We also report the structure of a complex between two-chain HGF/SF and the MET ectodomain (MET928) with 1:1 stoichiometry in which the N-terminal and first kringle domain of HGF/SF contact the face of the seven-blade β-propeller domain of MET harboring the loops connecting the β-strands b–c and d–a, whereas the C-terminal serine proteinase homology domain binds the opposite “b” face. Finally, we describe a complex with 2:2 stoichiometry between two-chain HGF/SF and a truncated form of the MET ectodomain (MET567), which is assembled around the dimerization interface seen in the crystal structure of the NK1 fragment of HGF/SF and displays the features of a functional, signaling unit. The study shows how the proteolytic mechanism of activation of the complex proteinases has been adapted to cell signaling in vertebrate organisms, offers a description of monomeric and dimeric ligand-receptor complexes, and provides a foundation to the structural basis of HGF/SF-MET signaling.
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enhanced growth of human met expressing xenografts in a new strain of immunocompromised mice transgenic for human hepatocyte growth Factor Scatter Factor
Oncogene, 2005Co-Authors: Yuwen Zhang, Yanli Su, Nathan J Lanning, Margaret Gustafson, Nariyoshi Shinomiya, Ping Zhao, Galia Tsarfaty, Lingmei Wang, George Vande F WoudeAbstract:Enhanced growth of human met-expressing xenografts in a new strain of immunocompromised mice transgenic for human hepatocyte growth Factor/Scatter Factor
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hepatocyte growth Factor Scatter Factor mediates angiogenesis through positive vegf and negative thrombospondin 1 regulation
Proceedings of the National Academy of Sciences of the United States of America, 2003Co-Authors: Yuwen Zhang, Yanli Su, Olga V Volpert, George Vande F WoudeAbstract:Hepatocyte growth Factor/Scatter Factor (HGF/SF), acting through the Met receptor, plays an important role in most human solid tumors, and inappropriate expression of this ligand–receptor pair is often associated with poor prognosis. The molecular basis for the malignant potential of the HGF/SF–Met signal in cancer cells has mostly been attributed to its mitogenic and invasive properties. However, HGF/SF also induces angiogenesis, but the signaling mechanism has not been fully explained, nor has this activity been directly associated with HGF/SF–Met-mediated tumorigenesis. It is known that HGF/SF induces in vitro expression of vascular endothelial growth Factor (VEGF), a key agonist of tumor angiogenesis; by contrast, thrombospondin 1 (TSP-1) is a negative regulator of angiogenesis. Here, we show that, in the very same tumor cells, in addition to inducing VEGF expression, HGF/SF dramatically down-regulates TSP-1 expression. We show that TSP-1 shut-off plays an important, extrinsic role in HGF/SF-mediated tumor development, because ectopic expression of TSP-1 markedly inhibits tumor formation through the suppression of angiogenesis. Interestingly, although VEGF-induced expression is sensitive to inhibitors of several pathways, including mitogen-activated protein kinase, phosphoinositide 3-kinase, and signal transducer and activator of transcription 3, TSP-1 shut-off by HGF/SF is prevented solely by inhibiting mitogen-activated protein kinase activation. These studies identify HGF/SF as a key switch for turning on angiogenesis. They suggest that TSP-1 is a useful antagonist to tumor angiogenesis and that it may have therapeutic value when used in conjunction with inhibitors of VEGF.
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normal and malignant prostate epithelial cells differ in their response to hepatocyte growth Factor Scatter Factor
American Journal of Pathology, 2001Co-Authors: Glenn A Gmyrek, George Vande F Woude, Marc Walburg, Craig P Webb, Hsiaoman Yu, Darracott E Vaughan, Beatrice S KnudsenAbstract:Hepatocyte growth Factor/Scatter Factor (HGF/SF) promotes the proliferation, differentiation, motility, and invasion of epithelial cells by binding to its cell surface receptor, the Met tyrosine kinase. In the prostate, Met is expressed predominantly by prostate epithelial cells (PrEC), whereas HGF/SF is synthesized by prostate stromal cells (PrSC). Met is also expressed in localized and metastatic prostate cancers. Our results show that PrECs in in vitro culture maintain expression of Met at a level comparable to DU145 cancer cell expression. HGF/SF secreted by PrSC stimulates tyrosine phosphorylation of the Met receptor. In normal PrEC, HGF/SF causes growth inhibition, sustained phosphorylation of mitogen-activated protein kinase, and increased CK18 expression consistent with cell differentiation. In contrast, HGF/SF significantly stimulates the proliferation of DU145 prostate cancer cells. HGF/SF in the conditioned medium of PrSC specifically induces migration of both normal and malignant prostate epithelial cells through MatriGel-coated Transwell filters. HGF/SF depletion reduces cell migration by approximately 50%. The response of PrEC is specific for HGF/SF since the other growth Factors tested do not significantly affect growth or migration of PrECs. These results support the in vivo importance of the prostate stroma and specifically of HGF/SF as a unique stromal derived Factor in the development and progression of prostate cancer.
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the geldanamycins are potent inhibitors of the hepatocyte growth Factor Scatter Factor met urokinase plasminogen activator plasmin proteolytic network
Cancer Research, 2000Co-Authors: Craig P Webb, Michael Jeffers, Shahriar Koochekpour, Curtis Hose, Marianne Oskarsson, Edward A Sausville, Anne Monks, George Vande F WoudeAbstract:The Met receptor tyrosine kinase and its ligand, hepatocyte growth Factor/Scatter Factor (HGF/SF), have been implicated in human tumor development and metastasis. HGF/SF induces the expression of urokinase plasminogen activator (uPA) and the uPA receptor (uPAR), important mediators of cell invasion and metastasis. We have developed a cell-based assay to screen for inhibitors of this signaling system using the induction of endogenous uPA and uPAR and the subsequent conversion of plasminogen to plasmin as the biological end point. Assay validation was established using a neutralizing antiserum to HGF/SF and a uPA inhibitor (B428), as well as inhibitors of the MKK-MAPK1/2 pathway, shown previously to be important in the induction of uPA and uPAR. Using this assay, we found several classes of molecules that exhibited inhibition of HGF/SF-dependent plasmin activation. However, we discovered that certain members of the geldanamycin family of anisamycin antibiotics are potent inhibitors of HGF/SF-mediated plasmin activation, displaying inhibitory properties at femtomolar concentrations and nine orders of magnitude below their growth inhibitory concentrations. At nanomolar concentrations, the geldanamycins down-regulate Met protein expression, inhibit HGF/SF-mediated cell motility and invasion, and also revert the phenotype of both autocrine HGF/SF-Met transformed cells as well as those transformed by Met proteins with activating mutations. Thus, the geldanamycins may have important therapeutic potential for the treatment of cancers in which Met activity contributes to the invasive/metastatic phenotype.
Paolo M Comoglio - One of the best experts on this subject based on the ideXlab platform.
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an uncleavable form of pro Scatter Factor suppresses tumor growth and dissemination in mice
Journal of Clinical Investigation, 2004Co-Authors: Massimiliano Mazzone, Paolo M Comoglio, Silvia Cavassa, Luigi Naldini, Cristina Basilico, Selma Pennacchietti, Mauro Risio, Paolo MichieliAbstract:Scatter Factor (SF), also known as hepatocyte growth Factor, is ubiquitously present in the extracellular matrix of tissues in the form of an inactive precursor (pro-SF). In order to acquire biological activity, pro-SF must be cleaved by specific proteases present on the cell surface. The mature form of SF controls invasive cues in both physiological and pathological processes through activation of its receptor, the Met tyrosine kinase. By substituting a single amino acid in the proteolytic site, we engineered an unprocessable form of pro-SF (uncleavable SF). Using lentivirus vector technology, we achieved local or systemic delivery of uncleavable SF in mice. We provide evidence that (a) uncleavable SF inhibits both protease-mediated pro-SF conversion and active SF–induced Met activation; (b) local expression of uncleavable SF in tumors suppresses tumor growth, impairs tumor angiogenesis, and prevents metastatic dissemination; and (c) systemic expression of uncleavable SF dramatically inhibits the growth of transplanted tumors and abolishes the formation of spontaneous metastases without perturbing vital physiological functions. These data show that proteolytic activation of pro-SF is a limiting step in tumor progression, thus suggesting a new strategy for the treatment or prevention of the malignant conversion of neoplastic lesions.
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interplay between Scatter Factor receptors and b plexins controls invasive growth
Oncogene, 2004Co-Authors: Paolo Conrotto, Simona Corso, Sara Gamberini, Paolo M Comoglio, Silvia GiordanoAbstract:Met and Ron tyrosine kinases are members of the Scatter Factor Receptor family. Met is the receptor for hepatocyte growth Factor while Ron is that for macrophage stimulating protein. On ligand stimulation, activation of these receptors induces ‘invasive growth’, a complex biological response involved in tissue morphogenesis and, when deregulated, in tumor progression and metastasis. Scatter Factor Receptors share structural homology with Plexins, transmembrane receptors for Semaphorins, a family of ligands originally identified as axon guidance molecules. A physical and functional association between Met and Plexin B1, the prototype of class B Plexin subfamily, has been previously demonstrated. Here, we show that both Met and Ron receptors can interact with each of the three members of class B Plexins, even in the absence of their ligands and that Plexin B1 ligand, Sema 4D, can induce activation of Met and Ron receptors, promoting an invasive response. Furthermore, in some human neoplastic cell lines Plexin B1 is overexpressed, constitutively tyrosine phosphorylated, and associated with Scatter Factor Receptors. These data extend the crosstalk previously described between Met and Plexin B1 to the entire families of Scatter Factor Receptors and class B Plexins and show that interaction with multiple upstream activators can finely tune the invasive growth process both in physiological conditions and in tumor growth and metastatization.
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hgf Scatter Factor selectively promotes cell invasion by increasing integrin avidity
The FASEB Journal, 2000Co-Authors: Livio Trusolino, Paolo M Comoglio, Silvia Cavassa, Paola Angelini, Margherita Ando, Andrea Bertotti, Carla BoccaccioAbstract:Hepatocyte growth Factor/Scatter Factor (HGF/SF) controls a genetic program known as ‘invasive growth’, which involves as critical steps cell adhesion, migration, and trespassing of basement membranes. We show here that in MDA-MB-231 carcinoma cells, these steps are elicited by HGF/SF but not by epidermal growth Factor (EGF). Neither Factor substantially alters the production or activity of extracellular matrix proteases. HGF/SF, but not EGF, selectively promotes cell adhesion on laminins 1 and 5, fibronectin, and vitronectin through a PI3-K-dependent mechanism. Increased adhesion is followed by enhanced invasiveness through isolated matrix proteins as well as through reconstituted basement membranes. Inhibition assays using function-blocking antibodies show that this phenomenon is mediated by multiple integrins including β1, β3, β4, and β5. HGF/SF triggers clustering of all these integrins at actin-rich adhesive sites and lamellipodia but does not quantitatively modify their membrane expression. These da...
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regulation of Scatter Factor hepatocyte growth Factor responses by ras rac and rho in mdck cells
Molecular and Cellular Biology, 1995Co-Authors: Anne J Ridley, Paolo M Comoglio, Anita C HallAbstract:: 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.
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a multifunctional docking site mediates signaling and transformation by the hepatocyte growth Factor Scatter Factor receptor family
Cell, 1994Co-Authors: Carola Ponzetto, Silvia Giordano, Alberto Bardelli, Andrea Graziani, Z Zhen, Flavio Maina, Paolo Dalla Zonca, George Panayotou, Paolo M ComoglioAbstract:Abstract Signaling by tyrosine kinase receptors is mediated by selective interactions between individual Src homology 2 (SH2) domains of cytoplasmic effectors and specific phosphotyrosine residues in the activated receptor. Here, we report the existence in the hepatocyte growth Factor/Scatter Factor (HGFSF) receptor of a multifunctional docking site made of the tandemly arranged degenerate sequence YVHNV. Phosphorylation of this site mediates intermediate- to high-affinity interactions with multiple SH2-containing signal transducers, including phosphatidylinositol 3-kinase, phospholipase Cγ, pp60 c-src , and the GRB-2-Sos complex. Mutation of the two tyrosines results in loss of biological function, as shown by abrogation of the transforming activity in the oncogenic counterpart of the receptor. The same bidentate motif is conserved in the evolutionarily related receptors Sea and Ron, suggesting that in all members of the HGFSF receptor family, signal transduction is channeled through a multifunctional binding site.
Ermanno Gherardi - One of the best experts on this subject based on the ideXlab platform.
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engineering the nk1 fragment of hepatocyte growth Factor Scatter Factor as a met receptor antagonist
Journal of Molecular Biology, 2008Co-Authors: Mark Youles, Oliver Holmes, Maxim V Petoukhov, Merel A Nessen, Simona Stivala, Dmitri I Svergun, Ermanno GherardiAbstract: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.
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insights into the structure function of hepatocyte growth Factor Scatter Factor from studies with individual domains
Journal of Molecular Biology, 2007Co-Authors: O Holmes, Malcolm Lyon, Jon A Deakin, S Pillozzi, F Carafoli, L Kemp, P J G Butler, Ermanno GherardiAbstract:Abstract Hepatocyte growth Factor/Scatter Factor (HGF/SF), the ligand for the receptor tyrosine kinase encoded by the c-Met proto-oncogene, is a multidomain protein structurally related to the pro-enzyme plasminogen and with major roles in development, tissue regeneration and cancer. 1 We have expressed the N-terminal (N) domain, the four kringle domains (K1 to K4) and the serine proteinase homology domain (SP) of HGF/SF individually in yeast or mammalian cells and studied their ability to: (i) bind the Met receptor as well as heparan sulphate and dermatan sulphate co-receptors, (ii) activate Met in target cells and, (iii) map their binding sites onto the β-propeller domain of Met. The N, K1 and SP domains bound Met directly with comparable affinities (Kd = 2.4, 3.3 and 1.4 μM). The same domains also bound heparin with decreasing affinities (N > K1 >> SP) but only the N domain bound dermatan sulphate. Three kringle domains (K1, K2 and K4) displayed agonistic activity on target cells. In contrast, the N and SP domains, although capable of Met binding, displayed no or little activity. Further, cross-linking experiments demonstrated that both the N domain and kringles 1-2 bind the β-chain moiety (amino acid residues 308–514) of the Met β-propeller. In summary, the K1, K2 and K4 domains of HGF/SF are sufficient for Met activation, whereas the N and SP domains are not, although the latter domains contribute additional binding sites necessary for receptor activation by full length HGF/SF. The results provide new insights into the structure/function of HGF/SF and a basis for engineering the N and K1 domains as receptor antagonists for cancer therapy.
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structural basis of hepatocyte growth Factor Scatter Factor and met signalling
Proceedings of the National Academy of Sciences of the United States of America, 2006Co-Authors: Ermanno Gherardi, George Vande F Woude, Mark Youles, Maxim V Petoukhov, Sara Sandin, J T Finch, Larsgoran Ofverstedt, Ricardo Nunez Miguel, T L Blundell, Ulf SkoglundAbstract:The polypeptide growth Factor, hepatocyte growth Factor/Scatter Factor (HGF/SF), shares the multidomain structure and proteolytic mechanism of activation of plasminogen and other complex serine proteinases. HGF/SF, however, has no enzymatic activity. Instead, it controls the growth, morphogenesis, or migration of epithelial, endothelial, and muscle progenitor cells through the receptor tyrosine kinase MET. Using small-angle x-ray Scattering and cryo-electron microscopy, we show that conversion of pro(single-chain)HGF/SF into the active two-chain form is associated with a major structural transition from a compact, closed conformation to an elongated, open one. We also report the structure of a complex between two-chain HGF/SF and the MET ectodomain (MET928) with 1:1 stoichiometry in which the N-terminal and first kringle domain of HGF/SF contact the face of the seven-blade β-propeller domain of MET harboring the loops connecting the β-strands b–c and d–a, whereas the C-terminal serine proteinase homology domain binds the opposite “b” face. Finally, we describe a complex with 2:2 stoichiometry between two-chain HGF/SF and a truncated form of the MET ectodomain (MET567), which is assembled around the dimerization interface seen in the crystal structure of the NK1 fragment of HGF/SF and displays the features of a functional, signaling unit. The study shows how the proteolytic mechanism of activation of the complex proteinases has been adapted to cell signaling in vertebrate organisms, offers a description of monomeric and dimeric ligand-receptor complexes, and provides a foundation to the structural basis of HGF/SF-MET signaling.
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Nitric oxide modulates hepatocyte growth Factor/Scatter Factor-induced angiogenesis
Angiogenesis, 2004Co-Authors: Shiladitya Sengupta, Ermanno Gherardi, Lynda A. Sellers, Ram SasisekharanAbstract:Objective : There is limited knowledge about potential therapeutic targets in Hepatocyte growth Factor/Scatter Factor (HGF)-induced pathophysiological angiogenesis. Recent candidates have included phosphatidylinositol-3-kinase, which is an upstream activator for endothelial nitric oxide (NO) synthase (NOS III). The current study is the first to evaluate the possible involvement of NOS-NO cascade in HGF-induced angiogenesis. Methods and results: NOS III inhibitors blocked the HGF-induced functional neovascularization in vivo , as quantified using vessel counts, ^133Xe-clearance, and immunohistology. This was reversed by L -arginine. Western blot analysis of HGF-treated cells also revealed a temporal increase in HGF-induced phosphorylation. In a deconstructional approach, HGF induced the proliferation and chemokinesis of human endothelial cells. These phenotypic effects were inhibited by NOS inhibitors, L -NAME and L -NIO, and the NO scavenger, carboxy PTIO, but unaltered by 1400W, a NOS II inhibitor. This inhibition was reversed by spermine NONOate, a NO donor, which independently exerted a biphasic effect on endothelial cell proliferation. The modulation of NO did not alter HGF-induced chemoinvasion of endothelial cells, while spermine-NONOate destabilized HGF-induced tubulogenesis, suggesting that a single assay is not sufficient for predicting the final phenotypic outcome on angiogenesis. Conclusions: The study is the first to demonstrate that the NOS III nitric oxide is a key signal cascade in HGF-induced angiogenesis, and represents a promising target for the clinical management of pathological conditions characterized by overt HGF signaling.
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the interactions of hepatocyte growth Factor Scatter Factor and its nk1 and nk2 variants with glycosaminoglycans using a modified gel mobility shift assay elucidation of the minimal size of binding and activatory oligosaccharides
Journal of Biological Chemistry, 2004Co-Authors: Malcolm Lyon, Ermanno Gherardi, Jon A Deakin, Daniel Lietha, John T GallagherAbstract:Full-length hepatocyte growth Factor/Scatter Factor interacts with both heparan and dermatan sulfates and is critically dependent upon them as coFactors for activation of the tyrosine kinase receptor Met. Two C-terminally truncated variants (NK1 and NK2) of this growth Factor also occur naturally. Their glycosaminoglycan binding properties are not clear. We have undertaken a comparative study of the heparan/dermatan sulfate binding characteristics of all three proteins. This has entailed the development of a modified gel mobility shift assay, utilizing fluorescence end-tagged oligosaccharides, that is also widely applicable to the analysis of many glycosaminoglycan-protein interactions. Using this we have shown that all three hepatocyte growth Factor/Scatter Factor variants share identical heparan/dermatan sulfate binding properties and that both glycosaminoglycans occupy the same binding site. The minimal size of the oligosaccharide that binds with high affinity in all cases is a tetrasaccharide from heparan sulfate but a hexasaccharide from dermatan sulfate. These findings demonstrate that functional glycosaminoglycan binding is restricted to a binding site situated solely within the small N-terminal domain. The same minimal size fractions are also able to promote hepatocyte growth Factor/Scatter Factor-mediated activation of Met and consequent downstream signaling in the glycosaminoglycan-deficient Chinese hamster ovary pgsA-745 cells. A covalent complex of heparan sulfate tetrasaccharide with monovalent growth Factor is also active. The binding and activity of tetrasaccharides put constraints upon the possible interactions and molecular geometry within the ternary signaling complex.
Glenn Merlino - One of the best experts on this subject based on the ideXlab platform.
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hepatocyte growth Factor Scatter Factor induces feedback up regulation of cd44v6 in melanoma cells through egr 1
Cancer Research, 2003Co-Authors: Juan A Recio, Glenn MerlinoAbstract:The hepatocyte growth Factor/Scatter Factor (HGF/SF) receptor c-Met and variants of the CD44 family of surface adhesion molecules, including CD44v6, have been implicated in cancer progression and metastasis. CD44 isoforms bearing heparin sulfate chains can bind to HGF/SF and facilitate its presentation to c-Met. Here, we demonstrate that HGF/SF-Met binding up-regulates the expression of CD44v6 in murine melanoma cells, serving to compensate for loss by internalization. c-Met-mediated CD44v6 up-regulation was achieved through transcriptional activation of the immediate early gene egr-1 . Enhanced egr-1 expression was apparent at the level of RNA 40 min after exposure to HGF/SF, and Egr-1 protein was detectable between 1 and 2 h post-treatment. CD44v6 RNA levels were correspondingly elevated 2 h after HGF/SF exposure. HGF/SF induced egr-1 activation via the Ras>Erk1/2 pathway but not through either phosphatidylinositol 3′-kinase or protein kinase C. Binding of NK2, a naturally occurring splice variant of HGF/SF, to c-Met failed to induce either Egr-1 or CD44v6, accounting at least in part for its antagonistic behavior. We also identified an Egr-1-binding site in the mouse CD44 gene promoter that accounts for its responsiveness to HGF/SF in melanoma cells. The compensatory up-regulation of both c-Met and CD44v6 in response to HGF/SF has important implications with respect to strategies used by cancer cells to sustain stimulation of growth- and metastasis-promoting pathways associated with tumor progression.
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Ulcerative Proctitis, Rectal Prolapse, and Intestinal Pseudo-Obstruction in Transgenic Mice Overexpressing Hepatocyte Growth Factor/Scatter Factor
Laboratory Investigation, 2001Co-Authors: Hisashi Takayama, William J Larochelle, Hitoshi Takagi, Raj P Kapur, Glenn MerlinoAbstract:Hepatocyte growth Factor/Scatter Factor (HGF/SF) can stimulate growth of gastrointestinal epithelial cells in vitro ; however, the physiological role of HGF/SF in the digestive tract is poorly understood. To elucidate this in vivo function, mice were analyzed in which an HGF/SF transgene was overexpressed throughout the digestive tract. Nearly a third of all HGF/SF transgenic mice in this study (28 of 87) died by 6 months of age as a result of sporadic intestinal obstruction of unknown etiology. Enteric ganglia were not overtly affected, indicating that the pathogenesis of this intestinal lesion was different from that operating in Hirschsprung's disease. Transgenic mice also exhibited a rectal inflammatory bowel disease (IBD) with a high incidence of anorectal prolapse. Expression of interleukin-2 was decreased in the transgenic colon, indicating that HGF/SF may influence regulation of the local intestinal immune system within the colon. These results suggest that HGF/SF plays an important role in the development of gastrointestinal paresis and chronic intestinal inflammation. HGF/SF transgenic mice may represent a useful model for the study of molecular mechanisms associated with a subset of IBD and intestinal pseudo-obstruction. Moreover, our data identify previously unappreciated side effects that may be encountered when using HGF/SF as a therapeutic agent.
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accelerated ultraviolet radiation induced carcinogenesis in hepatocyte growth Factor Scatter Factor transgenic mice
Cancer Research, 2000Co-Authors: Frances P Noonan, Miriam R Anver, Toshiyuki Otsuka, Stacey Bang, Glenn MerlinoAbstract:The dramatic rise in incidence of malignant melanoma experienced by populations both within the United States and throughout the world over the last several decades has been attributed to enhanced exposure to the UV spectrum of sunlight radiation. This hypothesis can now be tested using genetically engineered mouse models predisposed to malignant melanoma. Here we use melanoma-prone transgenic mice inappropriately expressing hepatocyte growth Factor/Scatter Factor (HGF/SF) in the skin as an experimental model system to ascertain the consequences of a chronic regimen of suberythemal UV radiation on melanoma genesis. HGF/SF is a multifunctional regulator capable of stimulating growth, motility, invasiveness, and morphogenetic transformation in cells, including melanocytes, expressing its receptor tyrosine kinase Met. HGF/SF transgenic mice demonstrate ectopic interfollicular localization and accumulation of melanocytes within the truncal dermis, epidermis, and junction and if untreated develop primary cutaneous melanoma with a mean onset age of ∼21 months. Transgenic mice and their wild-type littermates subjected to UV radiation three times weekly using FS40 sunlamps (60% UVB and 40% UVA), with daily UV doses graded from 2.25 to 6.0 kJ/m 2 , developed skin tumors with a mean onset age of 26 and 37 weeks, respectively ( P < 0.001, Kaplan-Meier log rank test). However, the repeated doses of suberythemal UV radiation used in this study failed to accelerate melanoma genesis, instead inducing the development of nonmelanoma tumors that included squamous cell carcinomas, squamous papillomas, and sarcomas. The conspicuous absence of melanocytic tumors occurred despite the immunohistochemical detection of a significant stimulation ( P < 0.001) in melanocyte-specific bromodeoxyuridine incorporation in response to only 2 weeks of UV irradiation (total UV dose of 13.5 kJ/m 2 ), resulting in 2.6- and 4.6-fold increases in the number of melanocytes in the dermis and epidermis, respectively. These data indicate that chronic suberythemal UV radiation preferentially favors the development of nonmelanocytic over melanocytic neoplasms in this transgenic animal, consistent with the pathogenesis proposed for sun exposure-associated skin cancer based on retrospective studies in the human population. Our findings suggest that the HGF/SF transgenic mouse will be useful as an experimental model for determining the consequences of exposure to various regimens of UV radiation and for elucidating the mechanisms by which such consequences are realized.
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diverse tumorigenesis associated with aberrant development in mice overexpressing hepatocyte growth Factor Scatter Factor
Proceedings of the National Academy of Sciences of the United States of America, 1997Co-Authors: Hisashi Takayama, Miriam R Anver, William J Larochelle, Richard R Sharp, Toshiyuki Otsuka, Paul W Kriebel, Stuart A Aaronson, Glenn MerlinoAbstract:Hepatocyte growth Factor/Scatter Factor (HGF/SF) is a mesenchymally derived, multifunctional paracrine regulator possessing mitogenic, motogenic, and morphogenetic activities in cultured epithelial cells containing its tyrosine kinase receptor, Met. c-met has been implicated in oncogenesis through correlation of expression with malignant phenotype in specific cell lines and tumors. Paradoxically, however, HGF/SF can also inhibit the growth of some tumor cells. To elucidate the oncogenic role of HGF/SF in vivo, transgenic mice were created such that HGF/SF was inappropriately targeted to a variety of tissues. HGF/SF transgenic mice developed a remarkably broad array of histologically distinct tumors of both mesenchymal and epithelial origin. Many neoplasms arose from tissues exhibiting abnormal development, including the mammary gland, skeletal muscle, and melanocytes, suggesting a functional link between mechanisms regulating morphogenesis and those promoting tumorigenesis. Most neoplasms, especially melanomas, demonstrated overexpression of both the HGF/SF transgene and endogenous c-met, and had enhanced Met kinase activity, strongly suggesting that autocrine signaling broadly promotes tumorigenesis. Thus, subversion of normal mesenchymal–epithelial paracrine regulation through the forced misdirection of HGF/SF expression induces aberrant morphogenesis and subsequent malignant transformation of cells of diverse origin.
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hepatocyte growth Factor Scatter Factor overexpression induces growth abnormal development and tumor formation in transgenic mouse livers
Cell Growth & Differentiation, 1996Co-Authors: Hiromi Sakata, Hisashi Takayama, Glenn Merlino, Richard R Sharp, Jeffrey S Rubin, William J LarochelleAbstract:To investigate the in vivo role of hepatocyte growth Factor/Scatter Factor (HGF/SF) in liver function, we generated transgenic mice using a mouse HGF/SF cDNA under the control of the mouse metallothionein gene promoter and 5’13’ flanking sequences. In aduft HGF/SF transgenic mice, liver weight as a percentage of total body weight was at least twice that of wildtype mice. Comparison of transgenic and control liver morphology revealed dramatic heterogeneity in the size and appearance of hepatocytes as a distinctive feature of HGF/SF overexpression. Transgenic livers exhibited a significant increase in the number of small hepatocytes with a 2N DNA content, accounting for the observed increase in liver mass. The DNA labeling index of hepatocytes increased I 1-fold at 4 weeks of age, when liver enlargement first became apparent, and was still elevated about 5-fold in adult HGF/SF transgenic mice. Moreover, hepatocytes isolated by perfusion of transgenic livers doubled every 2 days in culture, whereas little or no growth was observed with isolated control hepatocytes. The mechanistic basis of hepatocyte proliferation was elucidated as the chronic activation of the c-met proto-oncogene product. Met and substrates such as phosphatidylinositol 3-kinase, Src homology and collagen-like, pp6O��C, focal adhesion kinase pl25�, and paxillin were associated
