Macrophage-stimulating Protein Research Articles

HGF–MSP chimera protects kidneys from ischemia–reperfusion injury

Renal ischemia–reperfusion (I/R) injury is inevitable in transplantation and is related to long-term graft function. MF-1, a bifunctional hepatocyte growth factor (HGF)–macrophage-stimulating protein (MSP) (HGF–MSP) chimera was recently reported to prevent apoptosis. We therefore hypothesized that treatment with MF-1 would protect kidneys from I/R injury by inhibiting tubular epithelial apoptosis. MF-1 directly guarded cultured proximal tubular epithelial cells from hypoxia-induced necrosis and apoptosis in vitro. In addition, the therapeutic effects of MF-1 were evaluated using a rat I/R injury model in vivo. Saline-treated kidneys had increased creatinine and BUN, and exhibited tubular epithelial apoptosis with activated caspase 3 expression. In contrast, MF-1 treatment up-regulated Akt phosphorylation, and inhibited caspase 3 activation and tubular apoptosis, thereby ameliorating renal dysfunction. Of particular interest is that macrophage infiltration was suppressed in the MF-1-treated kidney. In conclusion, we identified a novel therapeutic approach using MF-1 to protect kidneys from I/R injury.

Apical Oxidative Hyaluronan Degradation Stimulates Airway Ciliary Beating via RHAMM and RON

Hyaluronan (HA) is synthesized in high-molecular-weight form at the apical pole of airway epithelial cells, covering the luminal surface. When human airway epithelial cells grown and redifferentiated at the air-liquid interface (ALI) were exposed to xanthine/xanthine oxidase (X/XO), ciliary beat frequency (CBF) increased. This effect was blocked by superoxide dismutase (SOD) and catalase. Inhibition of hyaluronan synthesis inhibited the CBF response to X/XO, while addition of exogenous HA amplified it. A functionally blocking antibody to the receptor for hyaluronic acid-mediated motility (RHAMM) reduced the CBF response to X/XO. Since RHAMM has no transmembrane domain and thus cannot signal on its own, the association of RHAMM with recepteur d'origine nantais (RON), a member of the hepatocyte growth factor receptor family, was explored. Immunohistochemistry of human airway epithelium showed co-localization of RHAMM and RON at the apex of ciliated cells. Physical association of RHAMM and RON was confirmed with co-immunoprecipitations. Macrophage-stimulating protein (MSP), an agonist of RON, stimulated CBF. Genistein, a nonspecific tyrosine kinase inhibitor, and MSP beta chain (beta-MSP), a specific RON inhibitor, blocked the X/XO-induced CBF increase. HA present in the apical secretions of human airway epithelial cells was shown to degrade upon exposure to X/XO, a process inhibited by SOD. Low-molecular-weight HA fragments stimulated CBF, an effect blocked by anti-RHAMM antibody and genistein. These data suggest that high molecular form HA is broken down by reactive oxygen species to form low-molecular-weight fragments that signal via RHAMM and RON to stimulate CBF.

The MET receptor tyrosine kinase in invasion and metastasis

Various cytokines and soluble growth factors upon interaction with their membrane receptors are responsible for inducing cellular proliferation, differentiation, movement, and protection from anoikis (a planned suicide activated by normal cells in absence of attachment to neighboring cells or extracellular matrix (EMC)). Among those soluble factors a major position is exerted by hepatocyte growth factor (HGF) together with its receptor MET and macrophage-stimulating protein (MSP) in cooperation with its receptor RON.

Inducing effects of macrophage stimulating protein on the expansion of early hematopoietic progenitor cells in liquid culture

Macrophage stimulating protein (MSP) is produced by human bone marrow endothelial cells. In this study, we sought to observe its effects on inducing the expansion of early hematopoietic progenitor cells which were cultured in a liquid culture system in the presence of the combination of stem cell factor (SCF), interleukin 3 (IL-3), interleukin 6 (IL-6), granulocyte macrophage-colony stimulating factor (GM-CSF), erythropoietin (EPO) (Cys) and MSP or of Cys and bone marrow endothelial cell conditioned medium (EC-CM). Human bone marrow CD34(+) cells were separated and cultured in a liquid culture system for 6 days. Granulocyte-macrophage colony forming unit (CFU-GM) and colony forming unit-granulocyte, erythrocyte, macrophage, megakaryocyte (CFU-GEMM) were employed to assay the effects of different treatment on the proliferation of hematopoeitic stem/progenitor cells. The nitroblue tetrazolium (NBT) reductive test and hoechest 33258 staining were employed to reflect the differentiation and apoptosis of the cells respectively. MSP inhibited the proliferation of CFU-GM and CFU-GEMM in semi-solid culture and the inhibitory effect on CFU-GEMM was stronger than on CFU-GM. MSP inhibited the differentiation of early hematopoietic progenitor cells induced by hematopoietic stimulators. Bone marrow (BM) CFU-GEMM was 2.3-fold or 1.7-fold increase or significantly decreased in either Cys + EC-CM, Cys + MSP or Cys compared with 0 hour control in liquid culture system after 6 days. MSP, a hematopoietic inhibitor, inhibits the differentiation of early hematopoietic progenitor cells induced by hematopoietic stimulators and makes the early hematopoietic progenitor cells expand in a liquid culture system.

The RON Receptor Tyrosine Kinase Mediates Oncogenic Phenotypes in Pancreatic Cancer Cells and Is Increasingly Expressed during Pancreatic Cancer Progression

Pancreatic cancer is an aggressive disease characterized by rapid growth and early metastasis. The recepteur d'origine nantais (RON) receptor tyrosine kinase is overexpressed and/or constitutively active in several epithelial cancers, but its role in pancreatic cancer is unknown. In this study, we have characterized RON expression in both murine and human pancreatic cancer. Immunoblotting indicates that RON is expressed in pancreatic intraepithelial neoplasia (PanIN), primary, and metastatic cell lines both in the human and mouse. Immunostaining revealed that 93% of high-grade PanIN, 79% of primary, and 83% of metastatic lesions from human pancreatic tissue samples expressed RON, with minimal expression in normal ducts and low-grade PanIN (6% and 18%, respectively). Moreover, we show a dose-dependent effect of hepatocyte growth factor-like protein (HGFL), the RON-specific ligand, on pancreatic cancer cell migration and invasion, which was reversed by RON inhibition. Although stimulation with HGFL had no effect on proliferation, concurrent RON receptor blockade and gemcitabine treatment increased apoptosis of RON-expressing pancreatic cancer cells versus gemcitabine treatment alone. Finally, HGFL stimulation of pancreatic cancer cells resulted in increased expression of phospho-mitogen-activated protein kinase and phospho-Akt. Taken together, these findings suggest that RON receptor signaling may contribute to pancreatic carcinogenesis, and that further investigation is warranted to assess the potential of RON-directed therapies in this deadly disease.

The macrophage-stimulating protein pathway promotes metastasis in a mouse model for breast cancer and predicts poor prognosis in humans

A better understanding of tumor metastasis requires development of animal models that authentically reproduce the metastatic process. By modifying an existing mouse model of breast cancer, we discovered that macrophage-stimulating protein promoted breast tumor growth and metastasis to several organs. A special feature of our findings was the occurrence of osteolytic bone metastases, which are prominent in human breast cancer. To explore the clinical relevance of our model, we examined expression levels of three genes involved in activation of the MSP signaling pathway (MSP, MT-SP1, and MST1R) in human breast tumors. We found that overexpression of MSP, MT-SP1, and MST1R was a strong independent indicator of both metastasis and death in human breast cancer patients and significantly increased the accuracy of an existing gene expression signature for poor prognosis. These data suggest that signaling initiated by MSP is an important contributor to metastasis of breast cancer and introduce an independent biomarker for assessing the prognosis of humans with breast cancer.

Neutralization of Macrophage-Stimulating Protein Ameliorates Renal Injury in Anti–Thy 1 Glomerulonephritis

Macrophage-stimulating protein (MSP) is a scatter factor that causes cell proliferation and migration, and receptor origin nantaise (RON) is its receptor. RON is expressed in macrophages and mesangial cells, and MSP is produced by renal tubular cells. This study investigated whether MSP/RON participate in the pathogenesis of anti-Thy 1 nephritis, a glomerular disease that is characterized by invasion of circulating monocytes into glomeruli and migration and proliferation of mesangial cells. In vivo, renal function and histopathology were studied in rats that had anti-Thy 1 disease and were untreated and treated with a neutralizing anti-MSP antibody. In vitro, whether monocytes express RON and whether MSP has a chemotactic effect on monocytes were studied. In vivo, in anti-Thy 1 disease, MSP was expressed de novo in glomeruli, and neutralization of MSP attenuated the rise in serum creatinine and proteinuria, stopped glomerular neutrophil and monocyte influx, protected from glomerular injury, and lessened mesangial cell overgrowth. In vitro, unstimulated monocytes did not express RON, but the stimulation with LPS induced de novo RON expression. LPS-stimulated monocytes were attracted by MSP. These results demonstrate a pathogenic role of the MSP/RON system in anti-Thy 1 nephritis.

Tyrosine kinase receptor RON in human pancreatic cancer

Specific tyrosine kinase receptors such as c-MET mediate epithelial-mesenchymal (EMT) transition, leading to phenotypic alterations associated with increased cell motility. It was hypothesized that RON, a tyrosine kinase receptor related to c-MET, would be expressed in human pancreatic cancer cells, induce EMT, and would thus serve as a target for therapy in a preclinical model. RON expression in human pancreatic cancer specimens was assessed by immunohistochemistry. In pancreatic cancer cell lines, RON expression was assessed by reverse-transcriptase polymerase chain reaction (PCR) and Western blot analysis. The human pancreatic cancer cell line L3.6pl, with high RON expression, was exposed to macrophage stimulating protein (MSP), the RON ligand, and assessed for cell migration, invasion, and changes associated with EMT. Western blot analysis and immunofluorescent staining were used to assess alterations in protein expression and cellular location, respectively. A RON monoclonal antibody (MoAb) was used to block ligand-induced activation of RON. Immunohistochemical staining revealed RON overexpression in 93% of human pancreatic cancer specimens relative to nonmalignant ductal tissue. RON mRNA and protein was expressed in 9 of 9 human pancreatic cancer cell lines. Treatment of L3.6pl cells with MSP increased Erk phosphorylation, cell migration, and invasion (P < .001). RON activation led to a decrease in membrane-bound E-cadherin in association with nuclear translocation of beta-catenin. RON MoAb inhibited downstream signaling as well as cell migration and invasion. In nude mice, RON MoAb inhibited subcutaneous and orthotopic tumor growth by about 60%. RON activation induced molecular and cellular alterations consistent with EMT. Inhibition of RON activation inhibited tumor growth in vivo. Novel antineoplastic therapies designed to inhibit RON activity may hinder mechanisms critical for pancreatic tumor progression.

MET and RON Receptor Tyrosine Kinases: Novel Therapeutic Targets in Squamous Cell Carcinoma of the Head and Neck

MET (hepatocyte growth factor receptor) and RON (recepteur dorigine Nantaise) are members of the MET proto-oncogene family of receptor tyrosine kinases (RTKs). Signalling from MET or RON activates multiple signalling pathways and ultimately promotes tumorigenesis and the formation of metastases. Mutations in MET have been detected in abundance in squamous cell carcinoma of the head and neck (SCCHN) metastases relative to the primary tumour, suggesting that this is a critical oncogene regulating dissemination. The biological significance of RON in SCCHN is still relatively unexplored. As survival has plateaued for patients with SCCHN, novel therapies with effects on the primary tumour and metastatic disease are urgently required. Small molecule inhibition of MET has been achieved in the pre-clinical setting and future clinical development is an exciting prospect. In this review, we summarise the biology of MET and RON RTKs and their contribution to an invasive tumour phenotype. We highlight their potential as therapeutic targets and address putative roles for MET and RON in resistance to conventional therapy, with particular reference to SCCHN. Keywords: MET, RON, tyrosine kinase, squamous cell carcinoma of the head and neck, hepatocyte growth factor, macrophage stimulating protein, metastasis, small molecule tyrosine kinase inhibitors

Changes In Gene Expression Induced by Tienilic Acid and Sulfamethoxazole: Testing the Danger Hypothesis

Tienilic acid (TA) was withdrawn due to idiosyncratic hepatotoxicity. Two hypotheses for the mechanisms of idiosyncratic reactions are the hapten and danger hypotheses, which are not mutually exclusive. Both human CYP 2C9 and rat CYP 2C11 metabolize TA to a reactive metabolite that was reported to bind exclusively to these enzymes. TA-Induced liver toxicity is associated with antibodies against CYP 2C9, thus TA appears to act as a hapten. However, if the binding were limited to CYP 2C, it is unlikely that this would lead to significant cell stress. If TA does not cause cell stress it would suggest that acting as a hapten is sufficient to induce an idiosyncratic reaction. To test whether TA can cause cell stress rats were dosed with TA and hepatic gene expression was profiled at 6 and 24 hr after drug administration. TA induced changes in genes involved in oxidative stress (aldo-keto reductase, glutathione-S-transferase, thioredoxin reductase, epoxide hydrolase), inflammation (IL-1β, interferon regulatory factor 1, macrophage stimulating protein 1), cytotoxicity (caspase-12), and liver regeneration (p27Kip1, DUSP6, serine dehyratase, spectrin β II, inhibin βA). These data support the hypothesis that danger signals in the form of cell-stress may be involved in initiating the immune response observed in TA-induced toxicity. In separate experiments, we examined the changes in gene expression induced in mice by sulfamethoxazole, which also causes idiosyncratic reactions. Sulfamethoxazole is an aromatic amine, and aromatic amines in general are associated with idiosyncratic drug reactions. They form reactive metabolites that both act as electrophiles and can redox cycle; therefore, it was assumed that sulfamethoxazole would cause some type of cell stress, the only question was what changes in mRNA expression would occur. In contrast to expectations, no changes induced by sulfamethoxazole could easily be interpreted as a danger signal. These data are presented together because they are the opposite of the expected results and convey a complex story.

Ubiquitylation, trafficking and degradation of the Ron tyrosine kinase receptor

Ron, the tyrosine kinase receptor for Macrophage Stimulating Protein (MSP) activates multiple intracellular signaling pathways promoting cell proliferation, motility, invasiveness and morphogenesis. Decoration of Receptor Tyrosine Kinases (RTKs) by ubiquitin is a signal for receptor internalization and routing to degradative compartments. Here we report the existence of a dual mechanism for Ron receptor downregulation. Ron is ubiquitylated in ligand-independent manner by the U-box E3 ligase CHIP (C-terminal Hsc70 interacting protein), recruited via chaperone intermediates Hsp90 and Hsc70. CHIP controls receptor turnover, as demonstrated by gene silencing experiments. Following ligand-induced activation, Ron recruits the negative regulator c-Cbl, which mediates receptor polyubiquitylation and degradation mostly via proteasome, in contrast with lysosomal-dependent degradation of multi-ubiquitilyated receptors. The molecular mechanisms regulating Ron trafficking to degradative compartments was assessed by function-impaired mutants or by gene silencing of chlatrin- or caveolin-dependent endocytosis. While chlatrin is not involved in receptor degradation, Ron can directly interact with caveolin-1 and interference with caveolae/raft-dependent endocytosis affects Ron sorting to degradative compartments. This research was supported by PRIN Italian Public Funding Agency.

Macrophage-Stimulating Protein Stimulates Erythropoietin Receptor Phosphorylation To Promote Enhanced Erk1/2 and Akt Activation in a Gab2-Dependent Pathway.

Friend-virus mediated erythroleukemia is a well-established model of murine oncogenesis. Several host factors have been shown to be necessary for Friend-mediated infection including a productive interaction between the Erythropoietin Receptor (EPO-R) and the 55 kDa glycoprotein product of the retroviral envelope gene (gp55). The Fv2 locus has been mapped and shown to be a truncated form of the STK tyrosine kinase (Sf-STK). Sf-STK expression and kinase activity is required for Friend virus infection of sensitive mouse strains. While the EPO-R and Sf-STK are required for Friend disease progression, it is unknown whether Sf-STK modulates EPO-R mediated signaling. EPO-R co-immunoprecipitated Sf-STK and STK specifically in COS-7 and Ba/F3 cells expressing both receptors. While the association between EPO-R and STK was shown to be constitutive, intriguingly, we discovered that co-stimulation with the STK ligand, Macrophage-Stimulating Protein (MSP) and EPO resulted in enhanced tyrosine phosphorylation of the EPO-R, compared to stimulation with EPO alone. Interestingly, STK kinase activity was required for EPO-dependent STK tyrosine phosphorylation, but was not required for MSP dependent EPO-R phosphorylation. Analysis of downstream signaling revealed STK and EPO-R co-operated in ligand induced dose-dependent phosphorylation of Erk1/2 and Akt. Interestingly, MSP and EPO also co-operated to stimulate tyrosine phosphorylation of the adaptor protein Gab2, leading to the recruitment of Shp2 and the p85 subunit of Phosphatidylinositol 3 kinase. These data suggest that Gab2 can couple to enhanced phosphorylation of Erk1/2 and Akt. However, no synergy was observed in MSP co-stimulation of JAK2, STAT1, STAT5, Jnk or p38 phosphorylation, demonstrating that STK activates specific EPO-dependent signaling pathways. EPO-dependent regulation of Socs family members was examined in Ba/F3-EPO-R cell lines expressing various STK constructs. The presence of STK induced Cis and Socs3, but not Socs1, transcript levels by 2.0-fold upon EPO stimulation. STK multi-functional docking sites (Y1330/Y1337) were required to observe optimal induction of Socs3. In contrast, Cis induction required STK tyrosine kinase activity. Our data demonstrate that EPO-R and STK interact at a biochemical level leading to augmentation of downstream signaling. These data suggest that Sf-STK contributes to Friend disease through delivery of mitogenic and/or survival signals to target erythroblast progenitor cells.

Therapeutic Implications of a Human Neutralizing Antibody to the Macrophage-Stimulating Protein Receptor Tyrosine Kinase (RON), a c-MET Family Member

RON is a member of the c-MET receptor tyrosine kinase family. Like c-MET, RON is expressed by a variety of epithelial-derived tumors and cancer cell lines and it is thought to play a functional role in tumorigenesis. To date, antagonists of RON activity have not been tested in vivo to validate RON as a potential cancer target. In this report, we used an antibody phage display library to generate IMC-41A10, a human immunoglobulin G1 (IgG1) antibody that binds with high affinity (ED50 = 0.15 nmol/L) to RON and effectively blocks interaction with its ligand, macrophage-stimulating protein (MSP; IC50 = 2 nmol/L). We found IMC-41A10 to be a potent inhibitor of receptor and downstream signaling, cell migration, and tumorigenesis. It antagonized MSP-induced phosphorylation of RON, mitogen-activated protein kinase (MAPK), and AKT in several cancer cell lines. In HT-29 colon, NCI-H292 lung, and BXPC-3 pancreatic cancer xenograft tumor models, IMC-41A10 inhibited tumor growth by 50% to 60% as a single agent, and in BXPC-3 xenografts, it led to tumor regressions when combined with Erbitux. Western blot analyses of HT-29 and NCI-H292 xenograft tumors treated with IMC-41A10 revealed a decrease in MAPK phosphorylation compared with control IgG-treated tumors, suggesting that inhibition of MAPK activity may be required for the antitumor activity of IMC-41A10. To our knowledge, this is the first demonstration that a RON antagonist and specifically an inhibitory antibody of RON negatively affects tumorigenesis. Another major contribution of this report is an extensive analysis of RON expression in approximately 100 cancer cell lines and approximately 300 patient tumor samples representing 10 major cancer types. Taken together, our results highlight the potential therapeutic usefulness of RON activity inhibition in human cancers.

Geldanamycins Trigger a Novel Ron Degradative Pathway, Hampering Oncogenic Signaling

Ron, the tyrosine kinase receptor for macrophage-stimulating protein is responsible for proliferation and migration of cells from different tissues. Ron can acquire oncogenic potential by single point mutations in the kinase domain, and dysregulated Ron signaling has been involved in the development of different human cancers. We have previously shown that ligand-activated Ron recruits the negative regulator c-Cbl, which mediates its ubiquitylation and degradation. Here we report that Ron is ubiquitylated also by the U-box E3 ligase C-terminal Hsc70-interacting protein (CHIP), recruited via chaperone intermediates Hsp90 and Hsc70. Gene silencing shows that CHIP activity is necessary to mediate Ron degradation upon cell treatment with Hsp90 inhibitors geldanamycins. The oncogenic Ron(M1254T) receptor escapes from c-Cbl negative regulation but retains a strong association with CHIP. This constitutively active mutant of Ron displays increased sensitivity to geldanamycins, enhanced physical interaction with Hsp90, and more rapid degradation rate. Cell growth and migration, as well as the transforming potential evoked by Ron(M1254T), are abrogated upon Hsp90 inhibition. These data highlight a novel mechanism for Ron degradation and propose Hsp90 antagonists like geldanamycins as suitable pharmacological agents for therapy of cancers where altered Ron signaling is involved.

Macrophage‐stimulating protein differently affects human alveolar macrophages from smoker and non‐smoker patients: evaluation of respiratory burst, cytokine release and NF‐κB pathway

Macrophage activation is a key feature of inflammatory reactions occurring during bacterial infections, immune responses and tissue injury. We previously demonstrated that human macrophages of different origin express the tyrosine kinase receptor recepteur d'origine nantaise, the human receptor for MSP (RON) and produce superoxide anion (O(2)(-)) when challenged with macrophage-stimulating protein (MSP), the endogenous ligand for RON. This study was aimed to evaluate the role of MSP in alveolar macrophages (AM) isolated from healthy volunteers and patients with interstitial lung diseases (sarcoidosis, idiopathic pulmonary fibrosis), either smokers or non-smokers, by evaluating the respiratory burst, cytokine release and nuclear factor-kappa B (NF-kappaB) activation. MSP effects were compared with those induced by known AM stimuli, for example, phorbol myristate acetate, N-formyl-methionyl-leucyl-phenylalanine, lipopolysaccharide.MSP evokes O(2)(-) production, cytokine release and NF-kappaB activation in a concentration-dependent manner. By evaluating the respiratory burst, we demonstrate a significantly increased O(2)(-) production in AM from healthy smokers or smokers with pulmonary fibrosis, as compared to non-smokers, thus suggesting MSP as an enhancer of cigarette smoke toxicity. Besides inducing interleukin-1 beta (IL-1beta) and interleukin-10 (IL-10) production, MSP triggers an enhanced tumor necrosis factor-alpha release, especially in healthy and pulmonary fibrosis smokers. On the contrary, MSP-induced IL-10 release is higher in AM from healthy non-smokers. MSP activates the transcription factor NF-kappaB; this effect is more potent in healthy and fibrosis smokers (2.5-fold increase in p50 subunit translocation). This effect is receptor-mediated, as it is prevented by a monoclonal anti-human MSP antibody. The higher effectiveness of MSP in AM from healthy smokers and patients with pulmonary fibrosis is suggestive of its role in these clinical conditions.

Oncogenesis of RON receptor tyrosine kinase: a molecular target for malignant epithelial cancers1

Recepteur d'origine nantais (RON) belongs to a subfamily of receptor tyrosine kinases (RTK) with unique expression patterns and biological activities. RON is activated by a serum-derived growth factor macrophage stimulating protein (MSP). The RON gene transcription is essential for embryonic development and critical in regulating certain physiological processes. Recent studies have indicated that altered RON expression contributes significantly to cancer progression and malignancy. In primary tumors, such as colon and breast cancers, overexpression of RON exists in large numbers and is often accompanied by the generation of different splicing variants. These RON variants direct a unique program that controls cell transformation, growth, migration, and invasion, indicating that altered RON expression has the ability to regulate motile/invasive phenotypes. These activities were also seen in transgenic mice, in which targeted expression of RON in lung epithelial cells resulted in numerous tumors with pathological features of human bronchioloalveolar carcinoma. Thus, abnormal RON activation is a pathogenic factor that transduces oncogenic signals leading to uncontrolled cell growth and subsequent malignant transformation. Considering these facts, RON and its variants can be considered as potential targets for therapeutic intervention. Experiments using small interfering RNA and neutralizing monoclonal antibodies demonstrated that suppressing RON expression and activation decreases cancer cell proliferation, increases apoptotic death, prevents tumor formation in nude mice, and reduces malignant phenotypes. Thus, blocking RON expression and activation has clinical significance in reversing malignant phenotypes and controlling tumor growth.

Mast Cell and Neutrophil Peptidases Attack an Inactivation Segment in Hepatocyte Growth Factor to Generate NK4-like Antagonists

Hepatocyte growth factor (HGF) is a plasminogen-like protein with an alpha chain linked to a trypsin-like beta chain without peptidase activity. The interaction of HGF with c-met, a receptor tyrosine kinase expressed by many cells, is important in cell growth, migration, and formation of endothelial and epithelial tubes. Stimulation of c-met requires two-chain, disulfide-linked HGF. Portions of an alpha chain containing an N-terminal segment and four kringle domains (NK4) antagonize HGF activity. Until now, no physiological pathway for generating NK4 was known. Here we show that chymases, which are chymotryptic peptidases secreted by mast cells, hydrolyze HGF, thereby abolishing scatter factor activity while generating an NK4-like antagonist of HGF scatter factor activity. Thus, chymase interferes with HGF directly by destroying active protein and indirectly by generating an antagonist. The site of hydrolysis, Leu480, lies in the alpha chain on the N-terminal side of the cysteine linking the alpha and beta chains. This site appears to be specific for HGF because chymase does not hydrolyze other plasminogen-like proteins, such as macrophage-stimulating protein and plasminogen itself. Mast cell/neutrophil cathepsin G and neutrophil elastase generate similar fragments of HGF by cleaving near the chymase site. Mast cell and neutrophil peptidases are secreted during tissue injury, infection, ischemia, and allergic inflammation, where they may oppose HGF effects on epithelial repair. Thus, HGF possesses an "inactivation segment" that serves as an Achilles' heel attacked by inflammatory proteases. This work reveals a potential physiological pathway for inactivation of HGF and generation of NK4-like antagonists.

Hepatocyte Growth Factor/Scatter Factor and MET Are Involved in Arterial Repair and Atherogenesis

Several studies have shown that in the arterial wall hepatocyte growth factor/scatter factor (HGF/SF) is expressed by smooth muscle cells (SMCs) but acts on endothelial cells, not SMCs. Other studies, however, have indicated that SMCs can respond to HGF/SF. We have reinvestigated expression and activity of HGF/SF and its receptor MET in arterial SMC and endothelial cell cultures and in whole arteries after superficial or deep injury or atherogenesis. High-density cultures of SMCs produced HGF/SF but did not express MET, whereas SMCs, at the leading edge of injured cultures, expressed both ligand and receptor and showed a dramatic motility and growth response to HGF/SF. In line with these results, HGF/SF and MET expression was undetectable in the media of uninjured carotid arteries but was induced after deep arterial injury in areas of SMC migration in the neointima. Strong MET expression was also observed in the SMCs of the atherosclerotic lesions of homozygous apoE(-/-) mice, whereas HGF/SF was expressed by macrophage-derived foam cells. These results demonstrate that MET is induced in migrating and proliferating SMCs and that HGF/SF and MET are key mediators of the SMC response in atherogenesis.

Cell Motility Is Controlled by SF2/ASF through Alternative Splicing of the Ron Protooncogene

Ron, the tyrosine kinase receptor for the Macrophage-stimulating protein, is involved in cell dissociation, motility, and matrix invasion. DeltaRon, a constitutively active isoform that confers increased motility to expressing cells, is generated through the skipping of exon 11. We show that abnormal accumulation of DeltaRon mRNA occurs in breast and colon tumors. Skipping of exon 11 is controlled by a silencer and an enhancer of splicing located in the constitutive exon 12. The strength of the enhancer parallels the relative abundance of DeltaRon mRNA and depends on a sequence directly bound by splicing factor SF2/ASF. Overexpression and RNAi experiments demonstrate that SF2/ASF, by controlling the production of DeltaRon, activates epithelial to mesenchymal transition leading to cell locomotion. The effect of SF2/ASF overexpression is reverted by specific knockdown of DeltaRon mRNA. This demonstrates a direct link between SF2/ASF-regulated splicing and cell motility, an activity important for embryogenesis, tissue formation, and tumor metastasis.

Crystal structure of the β‐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.