Macrophage-stimulating Protein Research Articles

Abstract B240: Inhibition of MSP-RON signaling pathway in cancer cells by a novel soluble form of RON variant containing the complete sema sequences

Abstract The RON receptor tyrosine kinase and its ligand macrophage stimulating protein (MSP) play a role in epithelial tumorigenesis. We report here a novel RON variant that antagonizes the RON-MSP pathway in various cancer cells. The variant was an 85 kDa soluble protein from a mRNA transcript with an insertion of 49 nucleotides between exons 5 and 6. The insertion created a stop code leading to a RON variant composed of a full 35kDa α-chain and a 45 kDa partial extracellular β-chain. The protein was featured by a sema domain, a hinge and a portion of the first IPT unit (designated as RON 85). RON 85 directly bound to the MSP, formed the MSP-RON 85 complex, and inhibited RON phosphorylation. RON 85 disrupted RON or RON 160 dimerization, prevented their phosphorylation, and attenuated their downstream signaling events. The action of RON 85 was specific to RON and had no effect on MET and EGFR. In colon and pancreatic cancer cells, RON 85 inhibited spontaneous or MSP-induced Erk1/2 and AKT phosphorylation, which results in impaired cell proliferation and colony formation. RON 85 also inhibited spontaneous or MSP-induced cell migration. We concluded that RON 85 is an antagonist to the MSP-RON pathway with therapeutic potentials for controlling tumorigenic phenotypes of cancer cells. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B240.

Hepatocyte growth factor activator is a serum activator of single‐chain precursor macrophage‐stimulating protein

Macrophage-stimulating protein (MSP) is a plasma protein that circulates as a single-chain proform. It acquires biological activity after proteolytic cleavage at the Arg483-Val484 bond, a process in which serum and cell surface serine proteinases have been implicated. In this article, we report that hepatocyte growth factor activator (HGFA), a serum proteinase which activates hepatocyte growth factor in response to tissue injury, may have a critical role in the activation of pro-MSP. In vitro analysis has revealed that human HGFA efficiently cleaves human pro-MSP at the physiological activation site without further degradation, resulting in biologically active MSP, as measured by the chemotactic response and MSP-induced morphological change of peritoneal macrophages. The processing of pro-MSP by HGFA is 10-fold more efficient than processing by factor XIa. To search for a role of HGFA in pro-MSP activation, we analyzed the processing of mouse pro-MSP in sera from HGFA-knockout (HGFA(-/-)) mice. The proform of MSP was the predominant molecular form in the plasma of both wild-type and HGFA(-/-) mice. In wild-type sera, endogenous pro-MSP was progressively converted to the mature two-chain form during incubation at 37 degrees C. However, this conversion was significantly impaired in sera from HGFA(-/-) mice. The addition of recombinant HGFA to HGFA-deficient serum restored pro-MSP convertase activity in a dose-dependent manner, and a neutralizing antibody to HGFA significantly reduced the conversion of pro-MSP in wild-type serum. Moreover, initial infiltration of macrophages into the site of mechanical skin injury was delayed in HGFA(-/-) mice. We suggest that HGFA is a major serum activator of pro-MSP.

Transactivation of RON receptor tyrosine kinase by interaction with PDGF receptor β during steady-state growth of human mesangial cells

Although it is well known that platelet-derived growth factor (PDGF) causes mesangial cell proliferation (presumably contributing to progression of glomerular disease), targeted inhibition of the PDGF receptor system has shown only limited efficacy against glomerular diseases. To examine whether this discrepancy is due to the involvement of other pathways, we used phosphorylated receptor tyrosine kinase arrays and found that RON (recepteur d'origine nantais) was phosphorylated while the PDGF receptor was dephosphorylated (thus inactive) in human mesangial cells (HMCs) at the time of cell cycle entry. Further, RON remained active during steady-state growth. Activation of RON was independent of its canonical ligand, macrophage-stimulating protein, but was mediated by transactivation from the PDGF-engaged PDGF receptor. Following stimulation with PDGF we found that the two receptors physically interacted. Knockdown of RON by siRNA increased the number of apoptotic cells without affecting the rate of DNA synthesis, suggesting that RON has anti-apoptotic functions. Immunohistochemical analysis found phosphorylated RON in glomerular lesions of patients with IgA nephropathy but not those with minimal change nephrotic syndrome, a disease not associated with mesangial proliferation. These results suggest that RON is involved in mesangial cell proliferation under both physiological and pathological conditions, and may be a relevant target for therapeutic intervention.

Proteolytic cleavage and activation of pro-macrophage-stimulating protein by hepatocyte growth factor activator and human airway trypsin-like protease

Background and Objectives: Macrophage-stimulating protein (MSP) is a plasma protein homologous to hepatocyte growth factor (HGF). MSP circulates as inactive precursor (pro-MSP) in the blood and requires proteolytic activation to transduce signals via its specific cell surface receptor tyrosine kinase, RON (recepteur d’origine nantais). Serine proteinases on the cell surface and also in serum have been implicated in this activation. The aim of this study was to examine the effects of serum serine proteinase, HGF activator (HGFA) and cell surface serine proteinase of stratified squamous epithelium such as human airway trypsin-like proteinase (HAT) and its family protein, differentially expressed in squamous cell carcinoma gene 1 (DESC1), on the activation of pro-MSP.

Recepteur d'Origine Nantais Tyrosine Kinase Is a Direct Target of Hypoxia-inducible Factor-1α-mediated Invasion of Breast Carcinoma Cells

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.

RON receptor tyrosine kinase in human gliomas: expression, function, and identification of a novel soluble splice variant

Malignant gliomas are incurable because of their diffuse infiltration of the surrounding brain. The recepteur d'origine nantais (RON) receptor tyrosine kinase is highly expressed in several epithelial cancer types and mediates tumorigenic, pro-invasive as well as metastatic effects. Analyzing RON expression in human gliomas, we found that different splice variants with known oncogenic activity are expressed in glioblastomas (GBM). In addition, the RON ligand macrophage-stimulating protein (MSP) is secreted by cultured GBM cells. MSP showed no mitogenic effect on GBM cells but displayed significant chemotactic activity for several GBM cell lines. We identified a novel splice variant, RONDelta90, which is generated by a transcript missing exon 6. As a result of a frameshift, translation is terminated in exon 7, resulting in a truncated soluble protein. RONDelta90 transcripts are expressed in normal human brain as well as in low grade astrocytomas but only in approximately 50% of highly malignant astrocytomas. In addition, RONDelta90 is detectable in supernatants of GBM cell lines. We cloned the RONDelta90 cDNA, and purified the recombinant protein from transfected cells. RONDelta90 inhibited MSP-induced phosphorylation of cellular RON and also attenuated basal activation levels. In addition, RONDelta90 inhibited MSP-induced glioma cell migration as well as random motility. To conclude, RONDelta90 is a novel soluble receptor variant with antagonistic activity that may act as a physiological modulator of RON signaling. The expression of several oncogenic RON splice variants in malignant gliomas suggests that these could represent candidate targets for treatment with agents inhibiting RON activity.

Macrophage stimulating protein is a neurotrophic factor for a sub-population of adult nociceptive sensory neurons

Macrophage stimulating protein (MSP) is a pleiotropic growth factor that signals via the RON receptor tyrosine kinase. Here we demonstrate that MSP increases the proportion of cultured adult mouse DRG neurons displaying discernable neuritic processes and promotes the elongation and branching of these processes in a dose dependent manner. RON expression in adult DRG is largely restricted to nerve growth factor (NGF)-responsive nociceptive neurons, and MSP mimics the effects of NGF by increasing the expression of several mRNAs that encode functionally important proteins that are characteristically expressed by this neuronal sub-population. MSP mRNA is expressed at high levels in the peripheral target fields of DRG somatic afferents, but is undetectable in DRG, spinal cord or freshly dissected sciatic nerve. These results suggest that MSP is a peripheral target-derived neurotrophic factor for NGF-responsive adult DRG neurons.

Protein levels of CC chemokine ligand (CCL)15, CCL16 and macrophage stimulating protein in patients with sarcoidosis

The objective of this study was to assess protein levels for candidate cytokines, chemokines, growth factors, matrix metalloproteinases and their inhibitors in bronchoalveolar lavage fluid (BALF) in patients with polar forms of pulmonary sarcoidosis, i.e. Löfgren's syndrome (LS) and more advanced chest X-ray (CXR) stage III disease. Twenty-four inflammatory molecules were analysed in unconcentrated BALF samples from 10 sarcoidosis patients with CXR stage III and 10 patients with LS by semiquantitative protein array. Four novel molecules [CC chemokine ligand (CCL)15, CCL16, macrophage migration inhibitory factor (MIF) and macrophage stimulating protein (MSP)], detected for the first time in association with sarcoidosis, were then quantified by enzyme-linked immunosorbent assay in a second cohort of 68 sarcoidosis patients and 17 control subjects. The protein levels of CCL15, CCL16, CCL24, CXCL8, CXCL9, CXCL10, interleukin-16, MIF, MSP and matrix metallopeptidase 1 were increased in CXR stage III patients when compared with patients with LS. CCL15 and MSP up-regulation in CXR stage III patients in comparison with LS patients and controls was confirmed by enzyme-linked immunosorbent assay. Moreover, MSP was associated with treatment requirement (P = 0.001) and CCL15 was elevated in patients with disease progression at 2-year follow-up (P = 0.016). CCL16 levels were increased in sarcoidosis versus controls (P < 0.05), but no difference was observed between patient subgroups. MIF up-regulation was not confirmed in a larger patient group. In conclusion, chemokines CCL15, CCL16 and MSP were found elevated for the first time in BALF from sarcoidosis patients; our results showed that CCL15 and MSP may affect disease course.

Expression of the RON receptor tyrosine kinase and its association with gastric carcinoma versus normal gastric tissues

BackgroundRecepteur d'origine nantais (RON) is a receptor tyrosine kinase that is activated by a serum-derived, macrophage stimulating protein (MSP) growth factor and is expressed in many malignant tumors. The aim of the present study was to reveal the protein expression profile of RON and its relationship with clinicopathological characteristics of gastric carcinoma and prognosis.MethodsGastric carcinoma tissue from 98 patients, along with 29 specimens of paraneoplastic tissue and 10 specimens of normal gastric mucosa, were examined by immunohistochemistry (IHC). Western blot analysis of 19 samples of gastric carcinoma tissue and corresponding paraneoplastic tissue, 8 specimens of normal gastric mucosa, and 2 specimens of normal lymph node samples also detected expression of a splice variant of RON, RONΔ165. All samples obtained were accompanied by patient follow-up data that ranged from 3 to 89 months (median time: 22 months).ResultsThe rate of positive RON expression differed significantly between gastric carcinoma tissues [56.1%, (55/98)] and paraneoplastic tissues [25.6%, (8/29)] (p = 0.007). In contrast, RON expression was absent in normal gastric mucosa samples. RON expression positively correlated with the invasive depth of the tumor (p = 0.019), perigastric lymph nodes metastasis (p = 0.019), and TNM stage (p = 0.001). However, RON expression was independent of tumor growth pattern according to Bormann criteria (p = 0.209), histopathological grade (p = 0.196), and incidence of distant metastasis (p = 0.400). RON expression was not related to a patient's survival rate (p = 0.195). RONΔ165 was strongly expressed in fresh gastric carcinoma tissue, corresponding paraneoplastic tissue, and perigastric lymph nodes with metastatic carcinoma. In contrast, expression of RONΔ165 was not observed in normal gastric mucosa and normal lymph node tissue samples.ConclusionRON expression is significant in gastric carcinoma tissue and corresponding paraneoplastic tissue, but is not expressed in normal gastric mucosa. Expression of RONΔ165 was similarly observed in gastric carcinoma tissue and in metastases present in lymph node tissues. We hypothesize that RON and its splice variant play an important role in the occurrence, progression, and metastasis of gastric carcinoma, and therefore may represent a useful marker to evaluate the biological activity of gastric carcinoma.

Identification of a Novel Recepteur d'Origine Nantais/c-Met Small-Molecule Kinase Inhibitor with Antitumor Activity In vivo

Recepteur d'origine nantais (RON) is a receptor tyrosine kinase closely related to c-Met. Both receptors are involved in cell proliferation, migration, and invasion, and there is evidence that both are deregulated in cancer. Receptor overexpression has been most frequently described, but other mechanisms can lead to the oncogenic activation of RON and c-Met. They include activating mutations or gene amplification for c-Met and constitutively active splicing variants for RON. We identified a novel inhibitor of RON and c-Met, compound I, and characterized its in vitro and in vivo activities. Compound I selectively and potently inhibited the kinase activity of RON and c-Met with IC(50)s of 9 and 4 nmol/L, respectively. Compound I inhibited hepatocyte growth factor-mediated and macrophage-stimulating protein-mediated signaling and cell migration in a dose-dependent manner. Compound I was tested in vivo in xenograft models that either were dependent on c-Met or expressed a constitutively active form of RON (RONDelta160 in HT-29). Compound I caused complete tumor growth inhibition in NIH3T3 TPR-Met and U-87 MG xenografts but showed only partial inhibition in HT-29 xenografts. The effect of compound I in HT-29 xenografts is consistent with the expression of the activating b-Raf V600E mutation, which activates the mitogen-activated protein kinase pathway downstream of RON. Importantly, tumor growth inhibition correlated with the inhibition of c-Met-dependent and RON-dependent signaling in tumors. Taken together, our results suggest that a small-molecule dual inhibitor of RON/c-Met has the potential to inhibit tumor growth and could therefore be useful for the treatment of patients with cancers where RON and/or c-Met are activated.

The RON Receptor Tyrosine Kinase Regulates IFN-γ Production and Responses in Innate Immunity

Receptor tyrosine kinases are emerging as a class of key regulators of innate immune responses. We have shown previously that the RON receptor tyrosine kinases (murine Stk), expressed on tissue-resident macrophages, inhibit classical macrophage activation while promoting hallmarks of alternative activation, thus regulating the critical balance between the inflammatory and wound-healing properties of activated macrophages. We have also shown previously that RON(-/-) mice are more susceptible to in vivo endotoxin challenge than wild-type mice, suggesting that the expression of this receptor confers a degree of endotoxin resistance to these animals. Here we demonstrate that, in response to in vivo LPS challenge, RON(-/-) mice harbor significantly increased systemic levels of IFN-gamma and IL-12p70 and increased levels of IL-12p40 transcript in their spleen. This elevation of IFN-gamma can be attributed to splenic NK cells responding to the elevated levels of IL-12. Analysis of RON and IFN-gamma receptor double-knockout mice indicates that the enhanced susceptibility of RON(-/-) mice to endotoxin challenge is dependent on IFN-gamma-mediated signals. In vitro studies demonstrate that stimulation of primary peritoneal macrophages with macrophage-stimulating protein, the ligand for RON, inhibits IFN-gamma-induced STAT1 phosphorylation and CIITA expression, resulting in reduced surface levels of MHC class II. Further studies demonstrating the induction of suppressor of cytokine signaling 1 via macrophage-stimulating protein/RON signaling provide a potential mechanistic insight into this regulatory pathway. These results indicate that the RON receptor regulates both the production of and response to IFN-gamma, resulting in enhanced susceptibility to endotoxin challenge.

Macrophage Stimulating Protein May Promote Tubular Regeneration after Acute Injury

Macrophage-stimulating protein (MSP) exerts proliferative and antiapoptotic effects, suggesting that it may play a role in tubular regeneration after acute kidney injury. In this study, elevated plasma levels of MSP were found both in critically ill patients with acute renal failure and in recipients of renal allografts during the first week after transplantation. In addition, MSP and its receptor, RON, were markedly upregulated in the regenerative phase after glycerol-induced tubular injury in mice. In vitro, MSP stimulated tubular epithelial cell proliferation and conferred resistance to cisplatin-induced apoptosis by inhibiting caspase activation and modulating Fas, mitochondrial proteins, Akt, and extracellular signal-regulated kinase. MSP also enhanced migration, scattering, branching morphogenesis, tubulogenesis, and mesenchymal de-differentiation of surviving tubular cells. In addition, MSP induced an embryonic phenotype characterized by Pax-2 expression. In conclusion, MSP is upregulated during the regeneration of injured tubular cells, and it exerts multiple biologic effects that may aid recovery from acute kidney injury.

Curcumin Blocks RON Tyrosine Kinase–Mediated Invasion of Breast Carcinoma Cells

We have recently shown that macrophage-stimulating protein (MSP) promotes the invasion of recepteur d'origine nantais (RON), a tyrosine kinase receptor-positive MDA-MB-231, MDA-MB-468 breast cancer cells, and also identified the regulatory elements required for RON gene expression. In this report, we have analyzed the efficacy of a chemopreventive agent, curcumin, in blocking RON tyrosine kinase-mediated invasion of breast cancer cells. Reverse transcription-PCR and Western analysis indicated the down-regulation of the RON message and protein, respectively, in MDA-MB-231 and MDA-MB-468 cells. Significantly, curcumin-mediated inhibition of RON expression resulted in the blockade of RON ligand, MSP-induced invasion of breast cancer cells. We have identified two putative nuclear factor-kappaB p65 subunit binding sites on the RON promoter. Using chromatin immunoprecipitation analysis and site-directed mutagenesis of the RON promoter, we have confirmed the binding of p65 to the RON promoter. Our data show that curcumin reduces RON expression by affecting p65 protein expression and transcriptional activity. Treatment of MDA-MB-231 cells with pyrrolidine dithiocarbamate, an inhibitor of p65, or small interfering RNA knockdown of p65, blocked RON gene expression and MSP-mediated invasion of MDA-MB-231 cells. This is the first report showing the regulation of human RON gene expression by nuclear factor-kappaB and suggests a potential therapeutic role for curcumin in blocking RON tyrosine kinase-mediated invasion of carcinoma cells.

Mechanisms of hepatocyte growth factor–mediated and epidermal growth factor–mediated signaling in transdifferentiation of rat hepatocytes to biliary epithelium

Previous studies from our laboratory have demonstrated that hepatocytes can transdifferentiate into biliary epithelium (BE) both in vivo and in vitro; however, the mechanisms are unclear. The current study was designed to investigate the mechanisms of hepatocyte transdifferentiation in vitro. Rat hepatocytes were cultured in roller bottles to obtain hepatocyte organoid cultures, which were stimulated with various growth factors (GFs) including hepatocyte growth factor (HGF), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), stem cell factor (SCF), macrophage-stimulating protein (MSP), fibroblast growth factor-a (FGF-a), fibroblast growth factor-b (FGF-b), and fibroblast growth factor-8b (FGF-8b). Only the cultures treated with HGF, EGF, and their combination exhibited formation of hepatocyte-derived biliary epithelium (BE) despite the presence and activation of all the pertinent cognate membrane receptors of the rest of the GFs. Microarray analysis of the organoid cultures identified specific up-regulation of approximately 500 target genes induced by HGF and EGF, including members of the extracellular matrix (ECM) protein family, Wnt/beta-catenin pathway, transforming growth factor beta (TGF-beta)/bone morphogenetic protein (BMP) pathway, and CXC (cysteine-any amino acid-cysteine) chemokines. To investigate the downstream signaling involved in hepatocyte to biliary epithelial cell (BEC) transdifferentiation, we investigated expression and activities of mitogen-activated protein (MAP) kinases [extracellular signal-regulated kinase (ERK)1/2, p38, and c-Jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK)] as well as serine/threonine kinase AKT. The analysis indicated that AKT phosphorylation was particularly increased in cultures treated with HGF, EGF, and their combination. Whereas phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 completely inhibited biliary epithelium formation, AKT inhibitor could only moderately reduce formation of BE in the organoid cultures treated with HGF+EGF. Most of the HGF+EGF target genes were altered by LY294002. Taken together, these data indicate that hepatocyte to BE transdifferentiation is regulated by HGF and EGF receptors and that PI3 kinase-mediated signaling independent of AKT is a crucial component of the transdifferentiation process.

PI3kinase mediates HGF and EGF induced hepatocyte to biliary epithelial cell transdifferentiation in organoid cultures

We have previously demonstrated that HGF and EGF can stimulate hepatocyte transdifferentiation into biliary epithelium (BE) in hepatocyte organoid cultures. To elucidate the mechanisms underlying this process rat hepatocyte organoid cultures were exposed to various growth factors (GF) including VEGF, PDGF, stem cell factor (SCF), macrophage stimulating protein (MSP), FGF‐a, FGF‐b, and FGF‐8b. None of the growth factors tested other than HGF and EGF were able to induce hepatocyte transdifferentiation despite the presence and activation of their cognate receptors. These findings indicate that HGF and EGF regulate a distinct set of genes required for transdifferentiation, which other GFs fail to activate. Microarray analysis of the organoid cultures identified upregulation of key HGF and EGF target genes such as osteopontin, CD44, CXC chemokine LIX, cyclin D1, and β‐catenin, which was confirmed by Western blot. Analysis of the signaling kinases downstream to HGF and EGF indicated increased activation of AKT. However, addition of AKT inhibitor to organoid cultures failed to prevent hepatocyte transdifferentiation. Interestingly inhibition of PI3kinase by LY294002 completely prevented development of biliary epithelium. These data indicate that the mechanism of hepatocyte to biliary transdifferentiation in hepatic organoid cultures induced by HGF and EGF is AKT‐independent PI3kinase activation.

Gene-centric association mapping of chromosome 3p implicates MST1 in IBD pathogenesis

Association mapping and candidate gene studies within inflammatory bowel diseases (IBD) linkage regions, as well as genome-wide association studies in Crohn's disease (CD) have led to the discovery of multiple risk genes, but these explain only a fraction of the genetic susceptibility observed in IBD. We have thus been pursuing a region on chromosome 3p21–22 showing linkage to CD and ulcerative colitis (UC) using a gene-centric association mapping approach. We identified 12 functional candidate genes by searching for literature cocitations with relevant keywords and for gene expression patterns consistent with immune/intestinal function. We then performed an association study composed of a screening phase, where tagging single nucleotide polymorphisms (SNPs) were evaluated in 1,020 IBD patients, and an independent replication phase in 745 IBD patients. These analyses identified and replicated significant association with IBD for four SNPs within a 1.2 Mb linkage disequilibrium region. We then identified a non-synonymous coding variant (rs3197999, R689C) in the macrophage-stimulating 1 (MST1) gene (P-value 3.62 × 10–6) that accounts for the association signal, and shows association with both CD and UC. MST1 encodes macrophage-stimulating protein (MSP), a protein regulating the innate immune responses to bacterial ligands. R689C is predicted to interfere with MSP binding to its receptor, suggesting a role for this gene in the pathogenesis of IBD.

Molecular targets in cancer therapy: the Ron approach

The receptor tyrosine kinase Ron and its ligand, Macrophage Stimulating Protein (MSP), mediate multiple processes involved in the control of cell proliferation, migration and protection from apoptosis. Dysregulated signaling of Ron, due to hyperactivation or loss of negative regulation, is involved in tumor progression and metastasis. Growing evidence indicates that Ron is abnormally expressed and activated in certain types of primary epithelial cancers (i.e. breast, colon, lung, pancreas, bladder and thyroid), where it critically contributes to the maintenance of tumorigenic and invasive phenotype. Furthermore, a positive association between aberrant Ron expression and aggressive biological indicators as well as a worse clinical outcome have been reported in breast, bladder and thyroid carcinomas. Different approaches have proved effective in targeting receptor activation/expression both in vitro and in animal models, leading to reversion of the tumorigenic phenotype. Altogether these results show that Ron is an attractive molecular target for clinical intervention.

Regulation of RON Tyrosine Kinase-mediated Invasion of Breast Cancer Cells

RON (recepteur d'origine nantais), a tyrosine kinase receptor for macrophage-stimulating protein (MSP) was implicated in tumor progression. However, it was not investigated how this important oncogene is regulated. We show that MSP promotes invasion of MDA MB 231 and MDA MB 468 but not MCF-7 breast cancer cells. Reverse transcription-PCR and Western analysis indicated the expression of RON message and protein, respectively, in MDA MB 231 and MDA MB 468 cells but not in MCF-7 cells. RON expression correlated with Sp1 expression. Initial analysis of a 1.2-kb and 400-bp RON promoter in MDA MB 231 and MDA MB 468 cells suggested the presence of all the necessary regulatory elements within 400 bp from the transcription start site. Site-directed mutagenesis of the 400-bp RON promoter revealed that the overlapping Sp1 sites at-94 (Sp1-3/4) and Sp1 site at -113 (Sp1-5) are essential for RON gene transcription. Electrophoretic mobility shift assays and chromatin immunoprecipitation analysis indicated that Sp1 binding to these sites is required for RON promoter activity. Ectopic Sp1 expression in Sp1 null SL2 cells confirmed the involvement of these Sp1 sites in the regulation of oncogenic RON tyrosine kinase. Treatment of MDA MB 231 cells with mithramycin A, an inhibitor of Sp1 binding, or siRNA knock-down of Sp1 blocked RON gene expression and MSP-mediated invasion of MDA MB 231 cells. This is the first report demonstrating a clear link between Sp1-dependent RON tyrosine kinase expression and invasion of breast carcinoma cells.

Regulation of cell migration and cytokine production by HGF-like protein (HLP) / macrophage stimulating protein (MSP) in primary microglia

HGF-like protein (HLP)/macrophage stimulating protein (MSP) is the only structural relative of hepatocyte growth factor (HGF), and is involved in the regulation of peripheral macrophage activation. However, the actions of HLP in microglia, a species of macrophage in the nervous system, which is closely involved in the neural degeneration and regeneration, is not yet understood. This study found that Ron, the receptor for HLP, is expressed in primary microglia using RT-PCR, immunocytochemical staining and Western blotting, and, thus, sought to elucidate the function of HLP on the primary microglia. HLP promoted microglial migration without affecting cell survival and proliferation. Furthermore, real-time quantitative RT-PCR analysis revealed that HLP greatly increased the mRNA of inflammatory cytokines, including IL-6 and GM-CSF, and iNOS. These findings provide the first evidence that HLP has the potential to modulate inflammatory actions of microglia, which proposes novel aspects for the process of degeneration and/or regeneration of the brain.

Metron factor-1 prevents liver injury without promoting tumor growth and metastasis

Hepatocyte growth factor (HGF) is the most powerful hepatotrophic factor identified so far. However, the ability of HGF to promote tumor cell "scattering" and invasion raises some concern about its therapeutic safety. We compared the therapeutic efficacy of HGF with that of Metron Factor-1 (MF-1), an engineered cytokine derived from HGF and the HGF-like factor macrophage stimulating protein (MSP), in mouse models of acute and chronic liver injury. At the same time, we tested the ability of HGF and MF-1 to promote tumor growth, angiogenesis, and invasion in several mouse models of cancer. We show that (1) MF-1 and HGF stimulate hepatocyte proliferation in vitro; (2) MF-1 and HGF protect primary hepatocytes against Fas-induced and drug-induced apoptosis; (3) HGF but not MF-1 induces scattering and matrigel invasion of carcinoma cell lines in vitro; (4) HGF but not MF-1 promotes migration and extracellular matrix invasion of endothelial cells in vitro; (5) MF-1 and HGF prevent CCl(4)-induced acute liver injury as measured by alanine aminotransferase (ALT) levels, histology, terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) analysis, and phospho-histone-3 immunostaining; (6) MF-1 and HGF attenuate liver fibrosis caused by chronic CCl(4) intoxication and promote regeneration as measured by Sirius red staining, alpha-smooth muscle actin immunostaining, and Ki-67 analysis; (7) HGF but not MF-1 promotes tumor growth, angiogenesis, and metastasis in a variety of xenograft models; (8) HGF but not MF-1 promotes intrahepatic dissemination of hepatocarcinoma cells injected orthotopically. These data suggest that MF-1 is as effective as HGF at preventing liver injury and at promoting hepatocyte regeneration, but therapeutically safer than HGF because it lacks proangiogenic and prometastatic activity.