RON-regulated innate immunity is protective in an animal model of multiple sclerosis.

The tyrosine kinase receptor RON and its ligand, macrophage stimulating protein (MSP), exert inhibitory effects on systemic innate immunity, but their CNS expression and impact on human neuroinflammatory diseases are unknown were RON and MSP present in human brain perivascular macrophages and microglia, but RON mRNA and protein abundance in the CNS were diminished in both MS patients and the MS animal model, experimental autoimmune encephalomyelitis (EAE). Treatment of differentiated human monocytoid cells with MSP resulted in significant reduction of interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha and MMP-9 mRNA levels, whereas minimal effects were observed in human astrocytes. After induction of EAE, RON knockout and heterozygote animals exhibited significantly increased CNS proinflammatory gene (TNF-alpha, MMP-12) expression compared with wild-type littermate controls, although IL-4 levels were suppressed in both RON-deficient groups. Neurological disease in RON-deficient animals showed a more rapid onset with overall worsened severity, together with exacerbated demyelination, axonal injury, and neuroinflammation after EAE induction. The proto-oncogene, c-Cbl, which modulates ubiquitylation of RON, was increased in glia in both MS brains and EAE spinal cords. Thus, the MSP-RON pathway represents a novel regulatory mechanism within the CNS by which innate immunity and its pathogenic effects could be targeted for future therapeutic interventions.

Ron receptor signaling augments mammary tumor formation and metastasis in a murine model of breast cancer.

The tyrosine kinase receptor Ron has been implicated in several types of cancer, including overexpression in human breast cancer. This is the first report describing the effect of Ron signaling on tumorigenesis and metastasis in a mouse model of breast cancer. Mice with a targeted deletion of the Ron tyrosine kinase signaling domain (TK-/-) were crossed to mice expressing the polyoma virus middle T antigen (pMT) under the control of the mouse mammary tumor virus promoter. Both pMT-expressing wild-type control (pMT+/- TK+/+) and pMT+/- TK-/- mice developed mammary tumors and lung metastases. However, a significant decrease in mammary tumor initiation and growth was found in the pMT+/- TK-/- mice compared with controls. An examination of mammary tumors showed that there was a significant decrease in microvessel density, significantly decreased cellular proliferation, and a significant increase in terminal deoxynucleotidyl transferase-mediated nick end labeling-positive staining in mammary tumor cells from the pMT+/- TK-/- mice compared with the pMT+/- TK+/+ mice. Biochemical analyses on mammary tumor lysates showed that whereas both the pMT-expressing TK+/+ and TK-/- tumors have increased Ron expression compared with normal mammary glands, the pMT-expressing TK-/- tumors have deficits in mitogen-activated protein kinase and AKT activation. These results indicate that Ron signaling synergizes with pMT signaling to induce mammary tumor formation, growth, and metastasis. This effect may be mediated in part through the regulation of angiogenesis and through proliferative and cell survival pathways regulated by mitogen-activated protein kinase and AKT.

Ron tyrosine kinase receptor regulates papilloma growth and malignant conversion in a murine model of skin carcinogenesis.

Recent studies demonstrate that the receptor tyrosine kinase (TK) Ron is tumorigenic when overexpressed and plays a role in regulating skin homeostasis. We hypothesized that Ron signaling promotes skin carcinogenesis. To test this hypothesis, mice deficient in the TK domain of Ron (TK(-/-) mice) were crossed with v-Ha-ras (Tg.AC) transgenic mice; the resulting TK(-/-) Tg.AC(+/-) mice, and their controls, were utilized in a model of chemically induced Ras-mediated skin carcinogenesis. The mice were treated with 2.5 microg of 12-O-tetradecanoylphorbol-13-acetate applied weekly to the shaved back of 36 control (TK(+/+) Tg.AC(+/-)) and 35 experimental (TK(-/-) Tg.AC(+/-)) mice. In an analysis of the resulting papillomas, a reduction in cellular proliferation and papilloma volume was found in the TK(-/-) Tg.AC(+/-) mice compared to controls. Further, Ron protein expression was upregulated during papilloma formation. Ablation of Ron signaling resulted in partial defects in MAPK and Akt signaling that may account for the decreased papilloma growth in the TK(-/-) Tg.AC(+/-) mice. The papilloma-bearing mice were monitored for the occurrence of malignant skin tumors and other malignant tumor types for a period of 48 weeks. Loss of Ron receptor signaling significantly reduced the percent of papillomas that underwent malignant conversion as well as the number of mice developing other malignant tumor types. In conclusion, these studies demonstrate that Ron signaling augments papilloma growth and malignant conversion in vivo.

Tyrosine kinase receptor RON functions downstream of the erythropoietin receptor to induce expansion of erythroid progenitors.

Erythropoietin (EPO) is required for cell survival during differentiation and for progenitor expansion during stress erythropoiesis. Although signaling pathways may couple directly to docking sites on the EPO receptor (EpoR), additional docking molecules expand the signaling platform of the receptor. We studied the roles of the docking molecules Grb2-associated binder-1 (Gab1) and Gab2 in EPO-induced signal transduction and erythropoiesis. Inhibitors of phosphatidylinositide 3-kinase and Src kinases suppressed EPO-dependent phosphorylation of Gab2. In contrast, Gab1 activation depends on recruitment and phosphorylation by the tyrosine kinase receptor RON, with which it is constitutively associated. RON activation induces the phosphorylation of Gab1, mitogen-activated protein kinase (MAPK), and protein kinase B (PKB) but not of signal transducer and activator of transcription 5 (Stat5). RON activation was sufficient to replace EPO in progenitor expansion but not in differentiation. In conclusion, we elucidated a novel mechanism specifically involved in the expansion of erythroblasts involving RON as a downstream target of the EpoR.

The receptor tyrosine kinase Ron is expressed in the mouse ovary and regulates inducible nitric oxide synthase levels and ovulation.

OBJECTIVE: To determine the reproductive effects in mice of the deletion of the tyrosine kinase domain of the Ron receptor. DESIGN: Controlled animal studies. SETTING: Academic research environment. ANIMAL(S): Immature mice with deletion of the tyrosine kinase domain of the Ron receptor (TK-/-) at 22-30 days of age and adult black Swiss female mice at 5-6 weeks of age. INTERVENTION(S): Hormonal stimulation of immature female TK-/- animals to induce ovulation. MAIN OUTCOME MEASURE(S): Ovulation rates measured by counting the total number of cumulus oocyte complexes in the ampullar region of the murine oviduct after hormonal stimulation. Western blot analysis measured murine ovarian protein levels of endothelial and inducible nitric oxide synthase (iNOS). Immunohistochemical analysis localized iNOS in the developing murine ovarian follicle. RESULT: Immature TK-/- mice (22-30 days) ovulate significantly fewer cumulus oocyte complexes. Western blot analyses demonstrated increased levels of iNOS before and after ovulation compared with controls. Conversely, endothelial nitric oxide synthase levels were similar and remained constant during corresponding time periods. Immunohistochemical analyses demonstrated a significant increase in iNOS staining throughout the ovary in TK-/- mice with a significant amount of iNOS in granulosa cells surrounding the oocyte when compared with controls. CONCLUSION(S): The increased level of nitric oxide in the TK-/- mice is likely due to an elevated level of iNOS, which may contribute to a decrease in the size of the ovaries and ovulation rates of immature TK-/- animals.

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) alpha, a key inflammatory mediator in this liver injury model, as well as reduced amounts of interleukin (IL) 10 (a suppressor of TNF-alpha production) and interferon (IFN)-gamma (a TNF-alpha 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-alpha. In conclusion, our studies show that the Ron receptor TK plays a critical role in modulating the response of the liver to endotoxin.

The role of the receptor tyrosine kinase Ron in nickel-induced acute lung injury.

Acute lung injury (ALI), a severe respiratory syndrome, develops in response to numerous insults and responds poorly to therapeutic intervention. Recently, cDNA microarray analyses were performed that indicated several pathogenic responses during nickel-induced ALI, including marked macrophage activation. Macrophage activation is mediated, in part, via the receptor tyrosine kinase Ron. To address the role of Ron in ALI, the response of mice deficient in the cytoplasmic domain of Ron (Ron tk-/-) were assessed in response to nickel exposure. Ron tk-/- mice succumb to nickel-induced ALI earlier, express larger, early increases in interleukin-6, monocyte chemoattractant protein-1, and macrophage inflammatory protein-2, display greater serum nitrite levels, and exhibit earlier onset of pulmonary pathology and augmented pulmonary tyrosine nitrosylation. Increases in cytokine expression and cellular nitration can lead to tissue damage and are consistent with the differences between genotypes in the early onset of pathology and mortality in Ron tk-/- mice. These analyses indicate a role for the tyrosine kinase receptor Ron in ALI.