Epithelial to mesenchymal transition (EMT) is a process occurring during embryonic development and cancer progression. Using recepteur d'origine nantais (RON)-expressing epithelial cells as a model, we showed that RON activation causes spindle-shaped morphology with increased cell motilities. These activities resemble those observed in EMT induced by transforming growth factor (TGF)-β1 or by Ras-Raf signaling. By immunofluorescent and Western blot analyses, we found that constitutive RON expression results in diminished expression of Ecadherin, redistribution of b-catenin, reorganization of actin cytoskeleton, and increased expression of vimentin, a mesenchymal filament. RON expression is also essential for TGF-β1-induced expression of a-smooth muscle actin (a-SMA), a specialized mesenchymal marker. In the study of signaling pathways responsible for RON-mediated EMT, it was found that PD98059, a MAP kinase inhibitor, blocks the collaborative activities of RON and TGF-β1 in induction of a-SMA expression and restores epithelial cells to their original morphology. Moreover, we showed that RON expression increases Smad2 gene promoter activities and protein expression, which significantly lowers TGF-β1 threshold for EMT induction. These results suggest that persistent RON expression and activation cause the loss of epithelial phenotypes. These changes, collaborating with TGF-β1 signaling, could play a critical role in epithelial transdifferentiation towards invasiveness and metastasis of certain cancers.

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 RON△165, RON△160, and RON△155 were detected and cloned from two primary colon cancer samples.The se RON variants were generated by deletions in different regions in extracellular domains of the RON β chain. Functional studies showed that expression of RON△160 or RON△155 in Martin-Darby canine kidney cells resulted in increased cell dissociation (scatter-like activity). RON variants, RON△160 and RON△155, also exerted the ability to induce multiple focus formation and sustain anchorageindependent growth of transfected NIH3T3 cells. Moreover, NIH3T3 cells expressing RON△160 or RON△155 formed tumors in athymic nude mice and colonized in the lungs. These data suggest that RON expression is altered in certain primary colon cancers. Abnorma l accumulation of RON variants may play a role in the progression of certain colorectal cancers in vivo.

The receptor tyrosine kinase RON (recepteur d'origine nantais), a member of the MET proto-oncogene family, has been implicated in the pathogenesis of certain epithelial cancers including lung adenocarcinomas. To determine the oncogenic potential of RON, transgenic mice were generated using the surfactant protein C promoter to express human wild-type RON in the distal lung epithelial cells. The mice were born normal without morphological defects in the lung, however, multiple lung adenomas with distinct morphology and growth pattern were observed. Tumors appeared as a single mass in the lung around 2 months of age and gradually developed into multiple nodules throughout the lung. Most of the tumors were characterized as cuboidal epithelial cells with type Ⅱ cell phenotypes. They grew along the alveolar walls and projected into the alveolar septa. A transition from premalignant adenomas to adenocarcinomas was observed. The RON transgene is highly expressed and constitutively activated in the tumors as evident by immunohistochemical staining and western blot analyses. Moreover, we found that Ras expression was dramatically increased in the majority of tumors. However, no mutation in the 'hot spots' of the K-Ras or p53 gene was observed, although limited genomic instability occurs in individual tumors. Taken together, this is a mouse lung tumor model with unique biological characteristics. The model may provide an opportunity to study the role of RON in lung tumors and to elucidate the mechanisms underlying this distinct lung tumor.

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 E2 (PGE2) 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-κB pathway, which is important for Cox-2 expression. Western blot analyses were performed showing that RON activation inhibits LPS-induced IκBα degradation. The decreased IκBα degradation was due to reduced IκBα phosphorylation at Ser-32 as determined by IκBα (Ser-32) phosphor-antibody. Moreover, we found that LPS-induced IKKβ activity, an enzyme responsible for phosphorylation of IκBα, 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-κB. 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.

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 uncogenic 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 mdules located mostly in the peripheral portions of the lung. A transition from early adenomas to later adenocareinomas was observed. Morphologically, tumors were characterized as enboidal epithelial cells with a lype 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 formatiml of lung tumors with nnique biological characteristics in virtu. This model provides opportunities to stndy the role of RON in the pathogenesis of lung tumors and to elucidate the mechanisms underlying this distinct hmg tumor.

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.

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 160kDa (ROND160) was identified from HT-29 cells. The cDNA encoding ROND160 has an in-frame deletion of 109 amino acids in the extracellular domain of the RON b 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.

The RON receptor tyrosine kinase is a 180kDa heterodimeric protein composed of a 40kDa α chain and a 145kDa β 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 160kDa. Proteolytic cleavage results in a 40kDa α chain and a short form of the β chain with a molecular mass of 125kDa. 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 β chain. The consequence of the deletion is constitutive activation of the protein with auto-phosphorylation. 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.

Previous studies have shown that activation of the RON receptor tyrosine kinase inhibits inducible NO production in murine peritoneal macrophages. The purpose of this study is to determine whether inflammatory mediators such as LPS, IFN-γ, and TNF-α regulate RON expression. Western blot analysis showed that RON expression is reduced in peritoneal macrophages collected from mice injected with a low dose of LPS. The inhibition was seen as early as 8 h after LPS challenge. Experiments in vitro also demonstrated that the levels of the RON mRNA and protein are diminished in cultured peritoneal macrophages following LPS stimulation. TNF-α plus IFN-γ abrogated macrophage RON expression, although individual cytokines had no significant effect. Because LPS and TNF-a plus IFN-g induce NO production, we reasoned that NO might be involved in the RON inhibition. Two NO donors, S-nitroglutathione (GSNO) and (6)-S-nitroso-N-acetylpenicillamine (SNAP), directly inhibited macrophage RON expression when added to the cell cultures. Blocking NO production by NO inhibitors like TGF-b prevented the LPS-mediated inhibitory effect. In Raw264.7 cells transiently transfected with a report vector, GSNO or SNAP inhibited the luciferase activities driven by the RON gene promoter. Moreover, GSNO or SNAP inhibited the macrophage-stimulating proteininduced RON phosphorylation and macrophage migration. We concluded from these data that RON expression in macrophages is regulated during inflammation. LPS and TNF-α plus IFN-γ are capable of down-regulating RON expression through induction of NO production. The inhibitory effect of NO is mediated by suppression of the RON gene promoter activities.