Macrophage stimulating protein (MSP) evokes superoxide anion production by human macrophages of different origin.
ABSTRACT: 1. Macrophage Stimulating Protein (MSP), a serum factor related to Hepatocyte Growth Factor, was originally discovered to stimulate chemotaxis of murine resident peritoneal macrophages. MSP is the ligand for Ron, a member of the Met subfamily of tyrosine kinase receptors. The effects of MSP on human macrophages and the role played in human pathophysiology have long been elusive. 2. We show here that human recombinant MSP (hrMSP) evokes a dose-dependent superoxide anion production in human alveolar and peritoneal macrophages as well as in monocyte-derived macrophages, but not in circulating human monocytes. Consistently, the mature Ron protein is expressed by the MSP responsive cells but not by the unresponsive monocytes. The respiratory burst evoked by hrMSP is quantitatively higher than the one induced by N-formylmethionyl-leucyl-phenylalanine and similar to phorbol myristate acetate-evoked one. 3. To investigate the mechanisms involved in NADPH oxidase activation, leading to superoxide anion production, different signal transduction inhibitors were used. By using the non selective tyrosine kinase inhibitor genistein, the selective c-Src inhibitor PP1, the tyrosine phosphatase inhibitor sodium orthovanadate, the phosphatidylinositol 3-kinase inhibitor wortmannin, the p38 inhibitor SB203580, the MEK inhibitor PD098059, we demonstrate that hrMSP-evoked superoxide production is mediated by tyrosine kinase activity, requires the activation of Src but not of PI 3-kinase. We also show that MAP kinase and p38 signalling pathways are involved. 4. These results clearly indicate that hrMSP induces the respiratory burst in human macrophages but not in monocytes, suggesting for the MSP/Ron complex a role of activator as well as of possible marker for human mature macrophages.
Project description:The RON receptor tyrosine kinase regulates the balance between classical (M1) and alternative (M2) macrophage activation. In primary macrophages, the ligand for Ron, macrophage-stimulating protein (MSP), inhibits the expression of inducible NO synthase, a marker of classically activated macrophages, whereas promoting the expression of arginase I, a marker of alternative activation. Ron(-/-) mice express increased levels of IL-12, a product of classically activated macrophages, after endotoxin administration, resulting in increased serum IFN-? levels and enhanced susceptibility to septic shock. In this study, we demonstrate that MSP inhibits LPS-induced IL-12p40 expression, and this inhibition is dependent on the docking site tyrosines in Ron. To further define this inhibition, we examined the effect of Ron on signaling pathways downstream of Ron. We found that MSP does not inhibit the MyD88-independent activation of IFN regulatory factor 3 and production of IFN-? in response to LPS, nor does it inhibit MyD88-dependent TGF-?-activated kinase phosphorylation or MAPK activation in primary macrophages. However, the induction of I?B kinase activity, I?B degradation, and DNA binding of NF-?B after LPS stimulation is delayed in the presence of MSP. In addition, Ron inhibits serine phosphorylation of p65 and NF-?B transcriptional activity induced by LPS stimulation of TLR4. Finally, MSP inhibits the NF-?B-dependent upregulation of the nuclear I?B family member, I?B?, a positive regulator of secondary response genes including IL-12p40. LPS also induces expression of Ron and an N-terminally truncated form of Ron, Sf-Ron, in primary macrophages, suggesting that the upregulation of Ron by LPS could provide classical feedback regulation of TLR signaling.
Project description:A multidisciplinary method combining transcriptional data, specificity profiling, and biological characterization of an enzyme may be used to predict novel substrates. By integrating protease substrate profiling with microarray gene coexpression data from nearly 2,000 human normal and cancerous tissue samples, three fundamental components of a protease-activated signaling pathway were identified. We find that MT-SP1 mediates extracellular signaling by regulating the local activation of the prometastatic growth factor MSP-1. We demonstrate MT-SP1 expression in peritoneal macrophages, and biochemical methods confirm the ability of MT-SP1 to cleave and activate pro-MSP-1 in vitro and in a cellular context. MT-SP1 induced the ability of MSP-1 to inhibit nitric oxide production in bone marrow macrophages. Addition of HAI-1 or an MT-SP1-specific antibody inhibitor blocked the proteolytic activation of MSP-1 at the cell surface of peritoneal macrophages. Taken together, our work indicates that MT-SP1 is sufficient for MSP-1 activation and that MT-SP1, MSP-1, and the previously shown MSP-1 tyrosine kinase receptor RON are required for peritoneal macrophage activation. This work shows that this triad of growth factor, growth factor activator protease, and growth factor receptor is a protease-activated signaling pathway. Individually, MT-SP1 and RON overexpression have been implicated in cancer progression and metastasis. Transcriptional coexpression of these genes suggests that this signaling pathway may be involved in several human cancers.
Project description:Bone destruction occurs in aging and numerous diseases, including osteoporosis and cancer. Many cancer patients have bone osteolysis that is refractory to state-of-the-art treatments, which block osteoclast activity with bisphosphonates or by inhibiting the receptor activator of nuclear factor ?B ligand (RANKL) pathway. We previously showed that macrophage-stimulating protein (MSP) signaling, which is elevated in about 40% of breast cancers, promotes osteolytic bone metastasis by activation of the MSP signaling pathway in tumor cells or in the bone microenvironment. We show that MSP signals through its receptor, RON tyrosine kinase, expressed on host cells, to activate osteoclasts directly by a previously undescribed pathway that is complementary to RANKL signaling and converges on proto-oncogene, non-receptor tyrosine kinase SRC (SRC). Genetic or pharmacologic inhibition of RON kinase blocked cancer-mediated bone destruction and osteoporosis in several mouse models. Furthermore, the RON kinase inhibitor BMS-777607/ASLAN002 altered markers of bone turnover in a first-in-human clinical cancer study, indicating the inhibitor's potential for normalizing bone loss in patients. These findings uncover a new therapeutic target for pathogenic bone loss and provide a rationale for treatment of bone destruction in various diseases with RON inhibitors.
Project description:Human RON (Recepteur d'Origine Nantais) receptor tyrosine kinase is a cell surface receptor for Macrophage Stimulating Protein (MSP). RON mediates signal transduction pathways that regulate cell adhesion, invasion, motility and apoptosis processes. Elevated levels of RON and its alternatively spliced variants are implicated in the progression and metastasis of tumor cells. The binding of MSP α/β heterodimer to the extracellular region of RON receptor induces receptor dimerization and activation by autophosphorylation of the intracellular kinase domains. The ectodomain of RON, containing the ligand recognition and dimerization domains, is composed of a semaphorin (Sema), Plexins-Semaphorins-Integrins domain (PSI), and four Immunoglobulins-Plexins-Transcription factor (IPT) domains. High affinity association between MSP and RON is mediated by the interaction between MSP β-chain and RON Sema, although RON activation requires intact RON and MSP proteins. Here, we report the structure of RON Sema-PSI domains at 1.85 Å resolution. RON Sema domain adopts a seven-bladed β-propeller fold, followed by disulfide bond rich, cysteine-knot PSI motif. Comparison with the homologous Met receptor tyrosine kinase reveals that RON Sema-PSI contains distinguishing secondary structural features. These define the receptors' exclusive selectivity towards their respective ligands, RON for MSP and Met for HGF. The RON Sema-PSI crystal packing generates a homodimer with interface formed by the Sema domain. Mapping of the dimer interface using the RON homology to Met, MSP homology to Hepatocyte Growth Factor (HGF), and the structure of the Met/HGF complex shows the dimer interface overlapping with the putative MSPβ binding site. The crystallographically determined RON Sema-PSI homodimer may represent the dimer assembly that occurs during ligand-independent receptor activation and/or the inhibition of the constitutive activity of RONΔ160 splice variant by the soluble RON splice variant, RONΔ85.
Project description:Toll-like receptors (TLRs) enable metazoans to mount effective innate immune responses to microbial and viral pathogens, as well as to endogenous host-derived ligands. It is understood that genetic background of the host can influence TLR responsiveness, altering susceptibility to pathogen infection, autoimmunity and cancer. Macrophage stimulatory protein (MSP), which activates the receptor tyrosine kinase recepteur d'origine nantais (RON), promotes key macrophage functions such as motility and phagocytic activity. MSP also acts via RON to modulate signaling by TLR4, which recognizes a range of pathogen or endogenous host-derived molecules. Here, we show that RON exerts divergent control over TLR4 activity in macrophages from different mouse genetic backgrounds. RON potently modulated the TLR4 response in macrophages from M2-prone FVB mice, as compared with M1-skewed C57Bl6 mice. Moreover, global expression analysis revealed that RON suppresses the TLR4-dependent type-I interferon gene signature only in FVB macrophages. This leads to attenuated production of the potent inflammatory mediator, tumor necrosis factor-?. Eliminating RON kinase activity markedly decreased carcinogen-mediated tumorigenesis in M2/Th2-biased FVB mice. We propose that host genetic background influences RON function, thereby contributing to the variability in TLR4 responsiveness in rodents and, potentially, in humans. These findings provide novel insight into the complex interplay between genetic context and immune function.
Project description:Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer with poor prognosis and high rates of relapse. The lack of actionable targets for TNBC has contributed to the high mortality rates of this disease, and new candidate molecules for potential manipulation are urgently required. Here, we show that macrophage-stimulating protein (MSP) and its tyrosine kinase receptor, Recepteur d'origine nantais (RON), are potent drivers of cancer cell growth and tumor progression in a mouse model of TNBC driven by the loss of Trp53 and Brca1. After comparison of two genetically engineered mouse models of TNBC, we found that mammary tumors from K14-Cre;Brca1F/F ;Trp53F/F (KB1P) mice exhibit high endogenous levels of MSP and RON expression. We show that MSP stimulates serine/threonine kinase 1 and extracellular regulated MAPK activation as well as cancer cell growth in cell lines derived from the two mouse models, while genetic and pharmacological inhibition of RON prevents these effects. Similarly, KB1P tumor progression in mice was robustly attenuated by treatment with a RON inhibitor with accompanied reduction in the proliferation marker, Ki-67. Analysis of human gene expression data confirmed that the genes encoding MSP and RON are robustly expressed in human TNBC as well as other subsets of breast cancer. Our findings uncover a mouse model where MSP expression and RON expression are naturally increased, and they provide evidence that this receptor and its ligand are viable candidate molecules for targeted treatment of breast cancer.
Project description:Hypoxia-inducible factor-1alpha (HIF-1alpha) overexpression was shown to be associated with invasion and metastasis of tumors and tumor cell lines. The identification of molecular targets that contribute to HIF-1alpha-mediated invasion is under intensive investigation. We have analyzed the role of recepteur d'origine nantais (RON), a tyrosine kinase receptor for macrophage-stimulating protein (MSP) that plays a role in breast cancer cell invasion as one of the molecular targets of HIF-1alpha. Analysis of a panel of breast cancer cell lines indicated a correlation between HIF-1alpha and RON expression. Treatment of HIF-1alpha- and RON-positive breast cancer cells with HIF-1alpha inhibitor, echinomycin, led to the inhibition of HIF-1alpha activity and RON expression. We have identified HIF-1alpha binding site on the RON promoter. Chromatin immunoprecipitation analysis and site-directed mutagenesis of the RON promoter confirmed the binding of HIF-1alpha to RON promoter. HIF-1alpha inhibitor-, echinomycin-, or short hairpin RNA-mediated selective knockdown of HIF-1alpha or HIF-1alpha target RON tyrosine kinase abrogated RON gene expression, and the RON ligand macrophage-stimulating protein mediated invasion of breast cancer cells. Consequently, the data presented herein demonstrated RON as a novel molecular target of HIF-1alpha and suggest a potential therapeutic role for HIF-1alpha or RON tyrosine kinase inhibitors in the blockade of RON tyrosine kinase-mediated invasion of carcinoma cells.
Project description:The tyrosine kinase Ron, receptor for MSP (macrophage-stimulating protein), displays several serine residues of unknown functions. Using [(32)P]H(3)PO(4) metabolic labelling, we found that Ron is serine-phosphorylated and dephosphorylated in vitro by PP1 (protein phosphatase 1). PP1 associates with Ron obtained from cells of different origins. The association is stimulated by MSP or serum and is prevented by wortmannin, an inhibitor of the Akt/PKB (protein serine/threonine kinase B) pathway. Akt/PKB phosphorylates Ron Ser-1394, thus providing a docking site for 14-3-3 (scaffold proteins binding to phosphoserine/phosphothreonine-containing sequences). In living cells, PP1 binds to the Ron mutant S1394A, but the association is no longer regulated by serum, MSP or wortmannin. The role of PP1 association with Ron is highlighted by (1) Ser-1394 dephosphorylation by PP1 in vitro and in living cells, (2) loss of 14-3-3 association with Ron after Ser-1394 dephosphorylation by PP1 in vitro and (3) an increase in 14-3-3 association after PP1 inactivation in living cells. These results suggest that PP1 can modulate the downstream Ron signalling generated by MSP via Akt/PKB and 14-3-3 binding. This is the first report on ligand-regulated association of PP1 with a growth factor receptor.
Project description:The recepteur d'origine nantais (RON) is a receptor tyrosine kinase (RTK) in the scatter factor family, which includes the c-Met receptor. RON exhibits increased expression in a significant number of human breast cancer tissues as well as in many established breast cancer cell lines. Recent studies have indicated that in addition to ligand-dependent signaling events, RON also promotes signals in the absence of its only known ligand, MSP, when expressed in epithelial cells. In this study, we found that when expressed in MCF-10A breast epithelial cells, RON exhibits both MSP-dependent and MSP-independent signaling, which lead to distinct biological outcomes. In the absence of MSP, RON signaling promotes cell survival, increased cell spreading and enhanced migration in response to other growth factors. However, both RON-mediated proliferation and migration require the addition of MSP in MCF-10A cells. Both MSP-dependent and MSP-independent signaling by RON are mediated in part by Src family kinases. These data suggest that RON has two alternative modes of signaling that can contribute to oncogenic behavior in normal breast epithelial cells.
Project description:Metastasis is the major cause of death in cancer patients, yet the genetic and epigenetic programs that drive metastasis are poorly understood. Here, we report an epigenetic reprogramming pathway that is required for breast cancer metastasis. Concerted differential DNA methylation is initiated by the activation of the RON receptor tyrosine kinase by its ligand, macrophage stimulating protein (MSP). Through PI3K signaling, RON/MSP promotes expression of the G:T mismatch-specific thymine glycosylase MBD4. RON/MSP and MBD4-dependent aberrant DNA methylation results in the misregulation of a specific set of genes. Knockdown of MBD4 reverses methylation at these specific loci and blocks metastasis. We also show that the MBD4 glycosylase catalytic residue is required for RON/MSP-driven metastasis. Analysis of human breast cancers revealed that this epigenetic program is significantly associated with poor clinical outcome. Furthermore, inhibition of Ron kinase activity with a pharmacological agent blocks metastasis of patient-derived breast tumor grafts in vivo.