RON Receptor Tyrosine Kinase Research Articles

Central role of the threonine residue within the p+1 loop of receptor tyrosine kinase in STAT3 constitutive phosphorylation in metastatic cancer cells.

The receptor tyrosine kinases (RTKs) RET, MET, and RON all carry the Met(p+1loop)-->Thr point mutation (i.e., 2B mutation), leading to the formation of tumors with high metastatic potential. Utilizing a novel antibody array, we identified constitutive phosphorylation of STAT3 in cells expressing the 2B mutation but not wild-type RET. MET or RON with the 2B mutation also constitutively phosphorylated STAT3. Members of the EPH, the only group of wild-type RTK that carry Thr(p+1loop) residue, are often expressed unexpectedly in different types of cancers. Ectopic expression of wild-type but not Thr(p+1loop)-->Met substituted EPH family members constitutively phosphorylated STAT3. In both RTK(Metp+1loop) with 2B mutation and wild-type EPH members the Thr(p+1loop) residue is required for constitutive kinase autophosphorylation and STAT3 recruitment. In multiple endocrine neoplasia 2B (MEN-2B) patients expressing RET(M918T), nuclear enrichment of STAT3 and elevated expression of CXCR4 was detected in metastatic thyroid C-cell carcinoma in the liver. In breast adenocarcinoma cell lines expressing multiple EPH members, STAT3 constitutively bound to the promoters of MUC1, MUC4, and MUC5B genes. Inhibiting STAT3 expression resulted in reduced expression of these metastasis-related genes and inhibited mobility. These findings provide insight into Thr(p+1loop) residue in RTK autophosphorylation and constitutive activation of STAT3 in metastatic cancer cells.

Analysis of RON receptor tyrosine kinase and its splicing variant in malignant and non-malignant human colonic mucosa

The presence of RON and its variant isoform in malignant and non-malignant human colonic tissues was investigated by immunohistochemistry using paraffin-embedded sections and RT-PCR analysis followed by direct sequencing of PCR product using RNAs isolated from frozen tissues. In normal colonic mucosa of both human fetus and adult, RON was uniformly expressed in cript cells, especially in the bottom of cripta. On the other hand, the expression was distributed heterogeneously in adenomas and in colon cancer. The expression of RON was significantly related to the degree of differentiation of colon cancer. The RT-PCR analysis of RNA isolated from malignant and non-malignant colonic tissue revealed the presence of two RON mRNA isoforms. Direct sequencing of two major products was revealed to be identical to that of human wild-type RON and a splicing variant of RON transcript which has been reported in human gastric cancer cell line, KATO-III. It is indicated that both wild type RON and its valiant isoform play an important role in regulating the normal function of colonic mucosa such as differentiation and motile activity, and the expression of both wild-type RON and its valiant isoform could be considered to be reduced during malignancy of human colonic mucosa.

C-Cbl is a critical modulator of the Ron tyrosine kinase receptor.

Ron, the receptor tyrosine kinase (RTK) for the macrophage stimulating protein (MSP), activates multiple signaling pathways by recruiting several positive regulators to a multifunctional docking site. Here we show that stimulation by MSP also recruits a negative regulator, the c-Cbl ubiquitin ligase, to the multifunctional docking site as well as to a juxtamembrane tyrosine autophosphorylation site. c-Cbl recruitment to these two sites results in polyubiquitylation of Ron molecules, which are subsequently sorted for endocytosis and degradation. Both the phosphotyrosine binding domain of c-Cbl and its RING domain are essential for downregulation of Ron. Although Ron and c-Cbl are found also in physical complexes that include Grb2, these associations are insufficient for productive ubiquitylation of Ron. Our results shed light on the mechanism of receptor desensitization mediated by c-Cbl and its binding partner Grb2.

Activation of CR3-mediated phagocytosis by MSP requires the RON receptor, tyrosine kinase activity, phosphatidylinositol 3-kinase, and protein kinase C zeta.

Macrophage-stimulating protein (MSP) promotes the phagocytosis of C3bi-coated erythrocytes by resident peritoneal macrophages, although the mechanism by which this occurs is largely unknown. We show that MSP-induced complement-mediated phagocytosis requires the RON receptor tyrosine kinase and the alphaMbeta2 integrin, as evidenced by the inability of RON-/- and alphaM-/- peritoneal macrophages to augment phagocytosis of complement-coated sheep erythrocytes in response to MSP. MSP stimulation of macrophages results in tyrosine phosphorylation and AKT activation, and inhibitor studies demonstrate a phagocytic requirement for tyrosine kinase and phosphatidylinositol 3-kinase (PI-3K) activity as well as activity of the atypical protein kinase C (PKC) isoform zeta, which localizes to MSP-induced phagosomes containing complement-coated beads. Additionally, MSP augments the ability of peritoneal macrophages to bind to intercellular adhesion molecule-1 (ICAM-1) via the alphaMbeta2 integrin. MSP-induced ICAM-1 adhesion is also dependent on tyrosine kinase activity, PI-3K, and PKC zeta, indicating that these signaling requirements are upstream of complement receptor 3 activation.

Hyaluronidase 2 negatively regulates RON receptor tyrosine kinase and mediates transformation of epithelial cells by jaagsiekte sheep retrovirus.

The candidate tumor-suppressor gene hyaluronidase 2 (HYAL2) encodes a glycosylphosphatidylinositol-anchored cell-surface protein that serves as an entry receptor for jaagsiekte sheep retrovirus, a virus that causes contagious lung cancer in sheep that is morphologically similar to human bronchioloalveolar carcinoma. The viral envelope (Env) protein alone can transform cultured cells, and we hypothesized that Env could bind and sequester the HYAL2 receptor and thus liberate a potential oncogenic factor bound and negatively controlled by HYAL2. Here we show that the HYAL2 receptor protein is associated with the RON receptor tyrosine kinase (also called MST1R or Stk in the mouse), rendering it functionally silent. In human cells expressing a jaagsiekte sheep retrovirus Env transgene, the Env protein physically associates with HYAL2. RON liberated from the association with HYAL2 becomes functionally active and consequently activates the Akt and mitogen-activated protein kinase pathways leading to oncogenic transformation of immortalized human bronchial epithelial cells. We find activated RON in a subset of human bronchioloalveolar carcinoma tumors, suggesting RON involvement in this type of human lung cancer.

Altered expression of the RON receptor tyrosine kinase in primary human colorectal adenocarcinomas: generation of different splicing RON variants and their oncogenic potential.

The RON receptor tyrosine kinase is a member of the MET proto-oncogene family that has been implicated in regulating motile-invasive phenotypes in certain types of epithelial cancers. The purpose of this study was to determine if RON expression is altered in primary human colorectal adenocarcinomas. Results from immunohistochemical staining showed that RON is highly expressed in the majority of colorectal adenocarcinomas (29/49 cases). Accumulated RON is also constitutively active with autophosphorylation in tyrosine residues. Moreover, three splicing variants of RON, namely RONdelta165, RONdelta160, and RONdelta155 were detected and cloned from two primary colon cancer samples. These RON variants were generated by deletions in different regions in extracellular domains of the RON beta chain. Functional studies showed that expression of RONdelta160 or RONdelta155 in Martin-Darby canine kidney cells resulted in increased cell dissociation (scatter-like activity). RON variants, RONdelta160 and RONdelta155, also exerted the ability to induce multiple focus formation and sustain anchorage-independent growth of transfected NIH3T3 cells. Moreover, NIH3T3 cells expressing RONdelta160 or RONdelta155 formed tumors in athymic nude mice and colonized in the lungs. These data suggest that RON expression is altered in certain primary colon cancers. Abnormal accumulation of RON variants may play a role in the progression of certain colorectal cancers in vivo.

Deletion of the Ron receptor tyrosine kinase domain in mice provides protection from endotoxin-induced acute liver failure

The targeted deletion of the cytoplasmic domain of the Ron receptor tyrosine kinase (TK) in mice leads to exaggerated responses to injury in several murine models of inflammation as well as increased lethality in response to endotoxin (lipopolysaccharide [LPS]). Using a well-characterized model of LPS-induced acute liver failure (ALF) in galactosamine (GalN)-sensitized mice, we show that Ron TK −/− mice display marked protection compared with control Ron TK +/+ mice. Whereas control mice have profound elevation of serum aminotransferase levels (a marker of hepatocyte injury) and hemorrhagic necrosis of the liver, in dramatic contrast, Ron TK −/− mice have mild elevation of aminotransferase levels and relatively normal liver histology. These findings are associated with a reduction in the number of liver cells undergoing apoptosis in Ron TK −/− mice. Paradoxically, treatment of Ron TK −/− mice with LPS/GalN leads to markedly elevated (3.5-fold) serum levels of tumor necrosis factor (TNF) α, a key inflammatory mediator in this liver injury model, as well as reduced amounts of interleukin (IL) 10 (a suppressor of TNF-α production) and interferon (IFN)-γ (a TNF-α sensitizer). These results show that ablation of the TK activity of the Ron receptor leads to protection from the development of hepatocellular apoptosis in response to treatment with LPS/GalN, even in the presence of excessive levels of serum TNF-α. In conclusion, our studies show that the Ron receptor TK plays a critical role in modulating the response of the liver to endotoxin. (H EPATOLOGY 2002;36:1053-1060.)

Activation of the RON Receptor Tyrosine Kinase by Macrophage-stimulating Protein Inhibits Inducible Cyclooxygenase-2 Expression in Murine Macrophages

The RON receptor tyrosine kinase is activated by macrophage-stimulating protein, which regulates macrophage migration, phagocytosis, and nitric oxide production. We report here the inhibitory effect of RON on lipopolysaccharide (LPS)-induced cyclooxygenase (Cox)-2 expression in mouse macrophages. In RON-expressing macrophages treated with macrophage stimulating protein, LPS-induced prostaglandin E(2) (PGE(2)) production was significantly reduced. The inhibition was accompanied by reduction of Cox-2 protein and mRNA expression. Transcriptional studies indicated that RON activation inhibits LPS-induced luciferase activity driven by the Cox-2 gene promoter. To determine whether RON activation affects LPS-induced NF-kappa B pathway, which is important for Cox-2 expression. Western blot analyses were performed showing that RON activation inhibits LPS-induced I kappa B alpha degradation. The decreased I kappa B alpha degradation was due to reduced I kappa B alpha phosphorylation at Ser-32 as determined by I kappa B alpha (Ser-32) phosphor-antibody. Moreover, we found that LPS-induced IKK beta activity, an enzyme responsible for phosphorylation of I kappa B alpha, was inhibited upon RON activation. Interestingly, these inhibitory effects were not regulated by RON-mediated phosphatidylinositol-3 kinase. These results suggest that RON activation inhibits LPS-induced macrophage Cox-2 expression. The inhibitory effect is mediated by impairing LPS-activated cascade enzymes that activate NF-kappa B. The inhibition of Cox-2 expression might represent a novel mechanism for the inhibitory functions of RON in vivo against LPS-induced inflammation and septic shock.

Targeted expression of the receptor tyrosine kinase RON in distal lung epithelial cells results in multiple tumor formation: oncogenic potential of RON in vivo.

RON, a member of the MET proto-oncogene family, has been implicated in the progression of certain epithelial cancers. The purpose of this study was to determine the oncogenic potential of RON in vivo in lung epithelial cells. Transgenic mice were established using surfactant protein C promoter to express human RON in the distal lung epithelial cells. These mice were born normal but developed multiple lung tumors with distinct morphology and growth patterns. Tumors appeared as a single mass in the lung around 2 months of age and gradually developed into multiple nodules located mostly in the peripheral portions of the lung. A transition from early adenomas to later adenocarcinomas was observed. Morphologically, tumors were characterized as cuboidal epithelial cells with a type II cell phenotype, grew along the alveolar walls, and projected into the alveolar septa. RON was highly expressed and constitutively activated in tumors. These results indicate that overexpression of human wild-type RON causes the formation of lung tumors with unique biological characteristics in vivo. This model provides opportunities to study the role of RON in the pathogenesis of lung tumors and to elucidate the mechanisms underlying this distinct lung tumor.

Activation of the RON receptor tyrosine kinase protects murine macrophages from apoptotic death induced by bacterial lipopolysaccharide

RON is a receptor tyrosine kinase activated by macrophage-stimulating protein. We demonstrate here that RON activation inhibits LPS-induced apoptosis of mouse peritoneal macrophages and Raw264.7 cells expressing RON or a constitutively active RON mutant. The antiapoptotic effect of RON was accompanied with the inhibition of LPS-induced production of nitric oxide (NO), a molecule responsible for LPS-induced cell apoptosis. This conclusion is supported by experiments using a chemical NO donor GSNO, in which RON activation directly blocked GSNO-induced apoptotic death of Raw264.7 cells and inhibited LPS-induced p53 accumulation. Furthermore, we showed that treatment of cells with wortmannin, which inhibits phosphatidylinositol (PI)-3 kinase, prevents the inhibitory effect of RON on LPS-induced macrophage apoptosis. These results were confirmed further by expression of a dominant inhibitory PI-3 kinase p85 subunit. These data suggest that by activating PI-3 kinase and inhibiting p53 accumulation, RON protects macrophage from apoptosis induced by LPS and NO. The antiapoptotic effect of RON might represent a novel mechanism for the survival of activated macrophages during inflammation.

Macrophage-stimulating protein is a neurotrophic factor for embryonic chicken hypoglossal motoneurons.

Macrophage-stimulating protein (MSP) exerts a variety of biological actions on many cell types, but has no known functions in the brain. MSP is structurally related to hepatocyte growth factor (HGF), another pleiotropic factor whose many functions include promoting neuronal survival and growth. To investigate whether MSP is also capable of acting as a neurotrophic factor, we purified hypoglossal motoneurons from the embryonic chicken hindbrain because these neurons are known to express the MSP receptor tyrosine kinase RON. MSP promoted the in vitro survival of these neurons during the period of naturally occurring neuronal death and enhanced the growth of neurites from these neurons. MSP mRNA was detected in the developing tongue whose musculature is innervated by hypoglossal neurons. Our study demonstrates that MSP is a neurotrophic factor for a population of developing motoneurons.

Oncogenic mutants of RON and MET receptor tyrosine kinases cause activation of the beta-catenin pathway.

beta-Catenin is an oncogenic protein involved in regulation of cell-cell adhesion and gene expression. Accumulation of cellular beta-catenin occurs in many types of human cancers. Four mechanisms are known to cause increases in beta-catenin: mutations of beta-catenin, adenomatous polyposis coli, or axin genes and activation of Wnt signaling. We report a new cause of beta-catenin accumulation involving oncogenic mutants of RON and MET receptor tyrosine kinases (RTKs). Cells transfected with oncogenic RON or MET were characterized by beta-catenin tyrosine phosphorylation and accumulation; constitutive activation of a Tcf transcriptional factor; and increased levels of beta-catenin/Tcf target oncogene proteins c-myc and cyclin D1. Interference with the beta-catenin pathway reduced the transforming potential of mutated RON and MET. Activation of beta-catenin by oncogenic RON and MET constitutes a new pathway, which might lead to cell transformation by these and other mutant growth factor RTKs.

Cross-talk between RON receptor tyrosine kinase and other transmembrane receptors.

RON is a transmembrane receptor tyrosine kinase that mediates biological activities of Macrophage Stimulating Protein (MSP). MSP is a multifunctional factor regulating cell adhesion, motility, growth and survival. MSP binding to RON causes receptor tyrosine phosphorylation leading to up-regulation of RON catalytic activity and subsequent activation of downstream signaling molecules. Recent studies show that RON is spatially and functionally associated with other transmembrane molecules including adhesion receptors integrins and cadherins, and cytokine and growth factor receptors IL-3 betac, EPOR and MET. For example, MSP-induced cell shape change is mediated via RON-activated IL-3 betac receptor. Activation of integrins causes MSP-independent RON phosphorylation, and the integrin/RON collaboration regulates cell survival. Thus, RON can be activated without MSP by ligand stimulation of RON-associated receptors, and MSP-activated RON can cause ligand-independent activation of RON-associated receptors. As a result of the receptor cross-activation RON-specific pathways become a part of a signal transduction network of other receptors, and conversely signaling pathways activated by other receptors can be used by RON. This receptor collaboration extends the spectrum of cellular responses generated by MSP and by putative ligands of RON-associated receptors. However signaling pathways involved in the receptor cross-talk and underlying activation mechanisms remain to be investigated. The purpose of this review is to summarize data and to discuss a role of cross-talk between RON and other transmembrane receptors.

The role of the RON receptor tyrosine kinase in a murine model of fulminant hepatic failure

The role of the RON receptor tyrosine kinase in a murine model of fulminant hepatic failure

The role of the RON receptor tyrosine kinase in a murine model of fulminant hepatic failure

as well as colorimetric immunoassays. Results: 1. In LR, maximal Itih4 expression was observed at 30 minutes and 12 hours, predominantly centrizonal in distribution. 2. Itih-4 expression was prominent in early liver development at day 9 and reached a second peak at day 16, being restricted to hepatoblasts, immature hepatncytes and differentiated hepatocytes. 3. A marked increase in Itih-4 labeling was noted in proliferating hepatocytes, but not bile duct cells in liver explant cultures treated with I L-6 (100 units/ml). Itih-4 expression was not altered in explants cultured with TNF alpha. 4. The fluorescence emission spectrum of metal free ITIH-4 showed a maximum at 332 nm with no change on addition of 5 mM Ca. The GST-fusion protein was recovered in the void fraction with no protein binding to the column. Similarly covalent binding of Itih-4 to hyaiuronic acid was not detected. Conclusions: In LR, Itih-4 expression corresponds to that of immediate early genes, such as c-myc, c-los, c-jun, IGFBP-2 and may contribute to the entry of normally quiescent hepetocytes into the early stages of the cell cycle. The markedly high expression of Itih-4 in early liver development and in explants treated with IL-6 suggest a prominent role for Itih-4 in the tissue remodeling that occurs during regenerative and developmental aspects of liver formation.

Immunohistochemical analysis of distribution of RON receptor tyrosine kinase in human digestive organs.

The immunohistochemical distribution of RON receptor tyrosine kinase in digestive organs of both human fetus and adult, including the esophagus, stomach, duodenum, small intestine, colon, rectum, liver, gallbladder, pancreas, and spleen, was investigated semiquantitively using an affinity-purified rabbit polyclonal antibody. RON was observed to be widely distributed throughout various digestive organs and cell types in humans. The immunoreactivity for RON was observed in the epithelium of the esophagus, small intestine, colon, hepatocytes, Kupffer cells, and splenic macrophages both in the adult and the fetus, suggesting that the MSP/RON signaling pathway possesses the proper biological properties to possibly be involved in morphogenesis or differentiation of cells in these organs and cell types. Several organs differed in immunoreactivity between adult and fetus. No immunoreactive cells were found in the pancreas of adults; however, immunoreactivity was observed in acinar cells and in some of the duct or ductular cells and endocrine cells of the islet of the fetus. Similarly, immunoreactivity was not observed in gastric mucosa except in the intestinal metaplastic cells in adults; however, immunoreactivity was found in the foveolar epithelium of the stomach of the fetus. Although the biological significance of RON in malignancy is unclear, the presence of RON immunoreactivity in the fetus and it lack in the adult may indicate that RON is a oncofetal substance in human pancreas and stomach.

Overexpression and Activation of the RON Receptor Tyrosine Kinase in a Panel of Human Colorectal Carcinoma Cell Lines

RON is a receptor tyrosine kinase belonging to the MET proto-oncogene family. The purposes of this study are to determine the expression and activation of RON in a panel of human colon carcinoma cell lines. Western blotting showed that RON is barely detectable in normal and SV-40-transformed colon epithelial cells, but highly expressed and constitutively activated in several colon carcinoma cell lines including Colo201, HT-29, HCT116, and SW837. Moreover, a novel RON variant with a molecular mass of 160 kDa (RONΔ160) was identified from HT-29 cells. The cDNA encoding RONΔ160 has an in-frame deletion of 109 amino acids in the extracellular domain of the RON β chain, which is caused by splicing out of two exons in the RON mRNA. No mutations were found in the kinase domain of the RON gene in five carcinoma cell lines screened. By expressing RON in colon epithelial cells, we found that RON activation increases cell motile-invasive activities and protects cells against apoptotic death. These data suggest that RON expression and activation are deregulated in colon carcinoma cell lines. By abnormal activation of RON, this receptor and its variant may regulate motile-invasive phenotypes of certain colon carcinoma cells in vivo.

Gene structure of the human receptor tyrosine kinase RON and mutation analysis in lung cancer samples

The human RON gene (MST1R) maps to 3p21.3, a region frequently altered in lung cancer and other malignancies. It encodes a receptor tyrosine kinase (RTK) closely related to MET, whose mutations are associated with neoplasia. We investigated whether RON might be involved in the development or progression of lung cancer. We first determined the exon-intron structure of the gene by direct sequencing of RON cosmid DNA and PCR products containing intronic sequences, and then developed primers suitable for mutation analysis by the single-strand conformation polymorphism (SSCP) method. Twenty coding exons were characterized, all but the first one small (average size: 170 bp), a feature shared with other RTK genes. We performed SSCP analysis of RON in small and non-small cell lung cancer samples, upon detection of its expression in a sample of lung cancer cell lines. A mutation (T915C: L296P) was found in an adenocarcinoma specimen. Several single nucleotide polymorphisms were also found. The panel of intron-anchored primers developed in this work will be useful for mutation analysis of the RON gene in different types of human tumors. © 2000 Wiley-Liss, Inc.

Identification of a novel splicing product of the RON receptor tyrosine kinase in human colorectal carcinoma cells

The RON receptor tyrosine kinase is a 180 kDa heterodimeric protein composed of a 40 kDa alpha chain and a 145 kDa beta chain with intrinsic tyrosine kinase activity. Activation of RON causes cell dissociation, motility and invasion of extracellular matrices, suggesting that RON might be involved in tumor metastasis. We report here the cloning of a novel splice variant of RON in human colorectal carcinoma cell line HT-29. This RON variant is first produced as a single chain precursor with a molecular mass of 160 kDa. Proteolytic cleavage results in a 40 kDa alpha chain and a short form of the beta chain with a molecular mass of 125 kDa. The altered receptor is synthesized from a transcript differing from the full-length RON mRNA by an in-frame deletion of 109 amino acids in the extracellular domain of the RON beta chain. The consequence of the deletion is constitutive activation of the protein with autophosphorylation. Expression of the RON variant in colon epithelial CoTr cells results in increased cell migration and invasion of extracellular matrices. These data suggest that generation of the activated splice variant of RON may contribute to the invasive phenotype of human colorectal carcinomas in vivo.

Identification of a novel splicing product of the RON receptor tyrosine kinase in human colorectal carcinoma cells

Identification of a novel splicing product of the RON receptor tyrosine kinase in human colorectal carcinoma cells