Macrophage-stimulating Protein Research Articles

Plasma Level of Placenta-Derived Macrophage-Stimulating Protein -Chain in Preeclampsia before 20 Weeks of Pregnancy.

ObjectThis study aimed to investigate the diagnostic value of placenta-derived macrophage-stimulating protein α-chain (MSP-α) before the 20th week of gestation for the early diagnosis of preeclampsia (PE).Methods and MaterialsTwo parts of this nested case-control study were simultaneously executed, and 1500 pregnant women were recruited. A total of 124 pregnant women were included in the plasma analysis part of this study. The MSP-α plasma level was measured before the 20th week of gestation, and the participants were followed until delivery. A case group of 62 women with PE and a control group of 62 women matched by gestational age, maternal age, and pre-pregnancy BMI (with normotensive pregnancies) were evaluated. In the placenta analysis part of this nested case-control study, the placentas of 34 pregnant women were randomly obtained. The placental levels of MSP were measured in 17 individuals with PE (case group) and in 17 women with a normotensive pregnancy matched by gestational age and maternal age (control group).ResultsThe plasma level of MSP-α was higher in the PE group than in the control group before the 20th week of gestation (p < 0.001). In addition, compared to the women with severe features in the PE group, those without severe features had a significantly higher plasma MSP-α level before the 20th week of gestation (p < 0.001). The area under the receiver operating characteristic curve (AUC) of MSP-α before the 20th week of gestation was 0.905 (95% CI, 0.811–0.962) for the women with early-onset PE without severe features. With regard to the placenta, the PE group (accumulated optical density, IOD [SUM] = 8862.37 ± 2064.42) exhibited increased MSP staining (more intense MSP staining or more extensive staining) compared with the control group (normal pregnancies (IOD [SUM] = 447.92 ± 114.72, P < 0.001). Furthermore, increased MSP staining was detected among the women without severe features compared with those with severe features in the PE group (IOD [SUM]: 12192.65 ± 5325.56 vs. 4104.83 ± 2383.06, P = 0.021).ConclusionAccording to the findings of this study, the plasma level of MSP-α may be associated with PE, and MSP-α may be considered a candidate protein for further analysis in studies of PE. Multicenter studies with larger sample sizes must be performed in the future to obtain accurate results regarding the predictive value of MSP-α in combination with other protein factors for the early diagnosis of PE.

Abstract A26: The RON tyrosine kinase accelerates pancreatic duct neoplasia and activates a positive autocrine/paracrine signaling loop that promotes alternative macrophage activation

Abstract Introduction: The RON (receptor d'origine nantais) tyrosine kinase is overexpressed in &amp;gt;80% of human pancreatic cancers, but its effect on pancreatic carcinogenesis is largely unknown. Methods: We developed a transgenic mouse that overexpresses RON specifically in the pancreas (Pdx-1-RON). Additionally, we utilized an existing transgenic mouse with a functionally inactive RON tyrosine kinase domain (Pdx-1-RTK-/-). These strains were crossed with the established CK murine model of pancreatic cancer yielding compound strains, RCK and CK/TK-/-. We performed immunofluorescent staining, flow cytometry, and western blot analysis on the tissues from these animals to study the effects of RON overexpression and the loss of RON signaling in the setting of oncogenic Kras. Results: RON overexpression results in increased acinar-ductal metaplasia, accelerates PanIN progression to invasive cancer, and results in an accumulation of alternatively-activated (MRC1+) macrophages in the tumor microenvironment. We found that RON overexpression results in upregulation of its own ligand, macrophage-stimulating protein (MSP), activating a positive autocrine/paracrine signaling loop that effects tumor growth and the differentiation of tumor-associated macrophages. Knockdown of RON expression and/or MSP expression results in decreased tumor growth in vivo. Additionally, loss of RON signaling in CK/TK-/- mice leads to a profound decrease in alternatively-activated (MRC1+) macrophages in the tumor microenvironment. In contrast, these animals demonstrate an accumulation of classically activated (CD11c+) macrophages. RON signaling is also associated with downregulation of the chemokine, monocyte chemoattractant protein-1 (MCP-1), a known driver of CD11c+ macrophage activation. The correlation of RON and MSP and the inverse correlation with MCP-1 expression were validated in a database analysis of human pancreatic tumor specimens. Conclusion: Our studies demonstrate that murine RON overexpression accelerates the development of invasive pancreatic cancer in the setting of oncogenic Kras. Active RON signaling results in upregulation of its own ligand, MSP, initiating a positive autocrine/paracrine feedback loop that promotes tumor growth and alters the differentiation of macrophages in the tumor microenvironment. Citation Format: Michele L. Babicky, Megan M. Harper, Evangeline S. Mose, Dawn J. Jaquish, Randall P. French, Susan E. Waltz. The RON tyrosine kinase accelerates pancreatic duct neoplasia and activates a positive autocrine/paracrine signaling loop that promotes alternative macrophage activation. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr A26.

HIV‑1 downregulates the expression and phosphorylation of receptor tyrosine kinase by targeting the NF‑κB pathway.

Macrophages are major targets of human immunodeficiency virus (HIV) and can act as long-term reservoirs of the virus. Chronic HIV-1 infection is associated with dysregulated inflammation. Recepteur d'origine nantais (RON) is expressed in tissue resident macrophages and functions to maintain inflammatory homeostasis. The present study aimed to compare the expression of RON on HIV-positive and -negative participants, and to investigate the mechanism by which HIV-1 influences the expression and function of RON in the JLTRG T cell line. The levels of RON and the RON ligand, macrophage-stimulating protein (MSP), in the peripheral blood of HIV-1-positive patients that were receiving (n=22) or not receiving highly active anti-retroviral therapy (HAART) (n=82) and 37 healthy control participants were determined by enzyme-linked immunosorbent assay. Expression of RON and MSP in the JLTRG T cell line was assessed by western blotting and the subcellular location was analyzed by fluorescence microscopy. JLTRG cells were co-cultured with a cell line that stably expresses HIV, H9/HTLV-IIIB, and alterations in the levels of RON and nuclear factor-κB (NF-κB) in JLTRG cells were assessed by western blotting. The expression of RON and MSP were significantly different in the serum of HIV-1- positive patients that were receiving HAART compared with those not receiving HAART (P<0.05) and healthy control patients (P<0.01). RON was detected in JLTRG cells, and was shown to be downregulated by HIV-1 infection. HIV-1 infection of JLTRG cells also reduced NF-κB phosphorylation. Thus, HIV-1 was shown to downregulate the expression and phosphorylation of RON by targeting the NF-κB pathway.

Diagnosis value and correlation of macrophage stimulating protein with immune regulatory factors in acute-on-chronic liver failure patients

Objective To observe the expression level of macrophage stimulating protein (MSP) in acute-on-chronic liver failure (ACLF) patients, and to explore the clinical significance and correlation with different immune regulatory factors. Methods The double antigen sandwich enzyme-linked immunosorbent assay method was used to detect MSP in the peripheral blood of 45 patients who were diagnosed with ACLF and 32 cases of chronic hepatitis B (CHB). The MSP levels were compared among ACLF patients with different outcomes, and the MSP level of healthy person was used as control. Meanwhile, liver function and hepatitis B virus (HBV) load were detected, and the expressions of peripheral blood CD4+ interferon (IFN)γ+ (helper T cell 1 Th1), CD4+ interleukin (IL)-4+ (helper T cell 2, Th2), CD4+ IL-17+ (helper T cell 17, Th17) and CD4+ CD25+ Foxp3+ (regular T cell, Treg) were measured by flow cytometry. The comparison of means between two samples was done by t test, and one-way ANOVA and linear correlation analysis were also used. Results The serum MSP levels in ACLF patients , CHB group and healthy control were (1.65±0.46) ng/mL, (1.43±0.32) ng/mL and (1.23±0.21) ng/mL, respectively. The serum MSP level in ACLF patients was significantly higher than both CHB patients (t=2.163, P=0.035) and healthy control (t=4.032, P=0.01). The MSP level in ACLF survival group was statistically higher compared with ACLF death group ( ng/mL vs ng/mL, t=1.973, P=0.042). Th2, Th17 cells in ACLF group, CHB group and healthy control group were (1.51±0.27)% and (1.94±1.02)%, (0.42±0.08)% and (0.55±0.36)%, (0.23±0.19) % and (0.26±0.19)%, respectively, which were all significantly different (F=7.759 and 37.229, respectively; both P 0.05). Conclusion MSP is involved in the progress of ACLF, and it may be associated with clinical outcomes and cellular immune imbalance of ACLF patients. Key words: Acute-on-chronic liver failure; Macrophage stimulating protein; Immunity, cellular

Abstract B41: HGFL-Ron signaling enhances breast cancer stem cell populations

Abstract The Ron receptor tyrosine kinase is overexpressed in a high fraction of human breast cancers (BCs). Ron and its ligand, the Hepatocyte Growth Factor-Like protein (HGFL), play important roles during BC initiation, progression, and therapeutic resistance, with overexpression of this signaling cascade associated with increased BC metastasis and poor prognosis in humans. Reports demonstrate that a small subpopulation of cancer cells, called the breast cancer stem cells (BCSCs), are essential for the development and maintenance of BC, leading to tumor formation and recurrence. Despite the role of HGFL-Ron signaling as a key regulator of BC, nothing is known about this signaling pathway in BCSC populations. To investigate the function of HGFL-Ron signaling in BCSCs, the expression levels of Ron and HGFL were analyzed in BCSCs. mRNA analyses show increased levels of Ron and HGFL in BCSC-enriched Mammospheres (MSs) compared to parental BC epithelial cells. Next, the association between Ron signaling and the BCSC population was examined. Aldefluor assays of several Ron replete and depleted human and murine BC epithelial cell lines show that Ron overexpression increases the BCSC population. In vivo, flow cytometry analyses of breast tumors for BCSC markers show that Ron signaling is associated with increases in the BCSC population. MS formation assays using several Ron replete and depleted human and murine BC cell lines also demonstrate that cells expressing high levels of Ron have increased MS formation. Ex vivo studies of sorted BCSCs further support that Ron signaling enhances the MS formation ability of BCSCs. Interestingly, our data suggest that both genetic ablation and chemical inhibition of Ron signaling is sufficient to decrease BCSC MS formation ability. Mammosphere culture over several passages also suggest that Ron signaling BCSCs have higher self-renewal capabilities than Ron deficient BCSCs, highlighting the role of HGFL-Ron signaling in regulating BCSC function. Recent preliminary studies correlate Ron signaling in BCSCs with increased β-catenin and NF-κB pathways, suggesting these as the potential molecular mechanisms through which Ron signaling promotes BCSC function in BC. Overall, our data provide (i) the first evidence demonstrating both HGFL and Ron as critical factors regulating the BCSC population and (ii) that this regulation might occur in a β-catenin and NF-κB pathway dependent manner. These studies suggest both Ron and HGFL as potential therapeutic targets to eradicate BCSCs and improve the outcome of BC patients. Citation Format: Sasha J. Ruiz-Torres, Susan E. Waltz. HGFL-Ron signaling enhances breast cancer stem cell populations. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B41.

RON Nuclear Translocation under Hypoxia Potentiates Chemoresistance to DNA Double-Strand Break-Inducing Anticancer Drugs.

Tumor hypoxia is associated with radioresistance, chemoresistance, and metastasis, which eventually lead to cancer progression and a poor patient prognosis. RON [also known as macrophage-stimulating protein receptor (MST1R)] belongs to the c-MET [also known as hepatocyte growth factor receptor (HGFR)] receptor tyrosine kinase (RTK) superfamily. To identify the interaction partners of RON nuclear translocation in response to hypoxia, the nuclear extract of TSGH8301 bladder cancer cells was immunoprecipitated for tandem mass profiling analysis. Nuclear RON interacted with adenosine triphosphate (ATP)-dependent DNA helicase 2 (Ku70) and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) to activate nonhomologous end joining (NHEJ) DNA repair. The interaction was time dependent, extending 3 to 24 hours posthypoxia or until the components had been exposed to the chemotherapeutic drugs doxorubicin and epirubicin. Stable knockdown experiments in vitro suggest the importance of RON for the chemoresistance of cancer cells under hypoxia. In addition, the tyrosine kinase domain of nuclear RON is crucial for interaction with Ku70 under hypoxia. J82 cells transfected with RON showed a survival advantage in the presence of epirubicin and hypoxia. This suggests that nuclear RON activates NHEJ repair by interacting with Ku70/DNA-PKcs and inhibiting RON activity to increase cancer cell chemosensitivity. Mol Cancer Ther; 15(2); 276-86. ©2016 AACR.

Identification of short-form RON as a novel intrinsic resistance mechanism for anti-MET therapy in MET-positive gastric cancer.

Despite the promising results from initial studies, there are significant limitations in the application of MET-targeted therapy in gastric cancer. Intrinsic resistance is one of the major obstacles. The aim of this study is to identify the responsible receptor tyrosine kinases (RTKs) that determine the unresponsiveness of MET inhibitor in MET-positive gastric cancer. through an RNA-interference-based functional screen targeting most human RTKs, we identified that activation of the fibroblast growth factor receptor 2 (FGFR2) and recepteur d'origine nantais (RON) pathways attenuated MET inhibitor-induced suppression of cell proliferation and migration. Notably, in the two forms of RON pathway activation, only upregulation of short-form RON (sf-RON), but not stimulation of full length RON with macrophage stimulating protein, conferred MET inhibitor resistance in vitro and in vivo. Furthermore, the profile of the gastric cancer samples observed that sf-RON was frequently upregulated in MET-positive gastric cancer. Our findings indicate that activation of the sf-RON signaling pathway represents a novel mechanism underlying MET inhibitor unresponsiveness. A combination strategy with drugs targeting both RON and MET pathways is believed to improve the efficacy of MET-targeted therapy.

A nutritional nonalcoholic steatohepatitis minipig model

Background and AimsThe objective of this study was to elucidate whether a Western diet was associated with nonalcoholic steatohepatitis (NASH), and the relationship between NASH, autophagy and endoplasmic reticulum (ER) stress. MethodsFour-month-old Lee–Sung minipigs were randomly assigned to two groups: control diet (C) and Western diet (W), for a 5-month experimental period. ResultsFeeding a Western diet produced a body composition with more fat, less lean and a greater liver weight. Compared with C pigs, W pigs also exhibited an elevated level of plasma insulin and free fatty acid. The W pigs displayed glucose intolerance, lower circulation antioxidant capacity and greater hepatic oxidative stress. Furthermore, pig fed the W diets had increased collagen accumulation in the liver and elevated systemic inflammation [tumor necrosis factor α and interleukin (IL)-6]. Compared with C pigs, W pigs had higher hepatic ER stress-related protein expression of GRP94, CHOP and caspase-12. The W pigs also had greater hepatic autophagy-related protein expression of p62 and LC3II. In an obesity antibody array analysis, W pigs had higher type 2 diabetes mellitus- (insulin-like growth factor 1, osteoprotegerin and resistin), atherosclerosis- (vascular endothelial growth factor, platelet-derived growth factor-AA and plasminogen activator inhibitor-I) and inflammation- [IL-1, macrophage-stimulating protein alpha, X-linked ectodermal dysplasia receptor and serum amyloid A (SAA)] related protein expressions. In addition, W pigs had greater plasma SAA concentration than C pigs and plasma SAA level was highly associated with IL-6. ConclusionsWe successfully established a NASH pig model, and our findings suggested an association of NASH with ER stress and autophagy. The SAA has potential as a novel plasma biomarker for nonalcoholic fatty liver disease pigs.

Plasma macrophage-stimulating protein and hepatocyte growth factor levels are associated with prostate cancer progression.

Hepatocyte growth factor (HGF) is a well-known multifunctional growth factor, and evidence has accumulated indicating that the HGF/MET (HGF receptor) signaling axis is involved in the progression of cancer. Macrophage-stimulating protein (MSP) is also known as a growth factor which activates not only macrophages but also cancer cells and osteoclasts through the activation of the specific Receptor d'origine nantais (RON). Pro-HGF and pro-MSP lack biological activity and, therefore, require proteolytic activation for conversion to an active two-chain form by HGF activator (HGFA). Although, there are several studies on HGF/MET signaling with castration-resistant prostate cancer (CRPC) and bone metastasis, reports on plasma protein are rare. In addition, the MSP/RON signaling axis in PC is not well understood. Here, we analyzed associations between PC progression and plasma HGF and MSP levels. We tested plasma samples from 58 patients with PC: 36 with castration-resistant (CR) PC and 22 with pretreatment for PC as control. We used enzyme-linked immunosorbent assay (ELISA) kit to determine plasma levels of HGF, MSP and HGFA, and examined correlations with clinicopathological characteristics such as Gleason grade and bone metastasis. PCR was used to evaluate HGF and MSP-related molecules in PC cell lines. Plasma levels of HGF, MSP and HGFA in the CRPC group were higher than in the control group (HGF: P < 0.001; MSP: P = 0.008; HGFA: P < 0.001). HGF and MSP levels were significantly correlated (P = 0.003). In the CRPC group, plasma HGF and MSP levels and Gleason score were not correlated; however, high plasma MSP level correlated with bone metastasis. (P = 0.016). In cell lines, PC3 expressed significantly more HGF, MET and RON than did LNCaP (P < 0.001), and both cell lines expressed MSP. Plasma concentrations of HGF, MSP and HGFA are significantly elevated in patients with CRPC. Also, as plasma MSP levels are significantly associated with bone metastasis in CRPC patients, MSP may be a candidate for serum marker of bone metastasis. Our results show the importance of the HGF/MET and MSP/RON signaling systems in CRPC.

Abstract 3922: Novel hepatocyte growth factor and macrophage stimulating protein antagonists for the treatment of castration-resistant prostate cancer

Abstract Castration-resistant prostate cancer (CRPC) is an advanced form of prostate cancer that is refractory to androgen deprivation therapy and characterized by aggressive growth, extensive metastasis formation, and poor prognosis. CRPC is temporarily manageable by the first-line chemotherapeutic docetaxel, but will eventually progress as a chemoresistant cancer, prompting a need for additional therapeutics to either replace docetaxel or to be used in combination therapies that increase the effectiveness of chemotherapy. The aberrant activation of the tyrosine kinase receptor c-Met by the active, dimeric form of hepatocyte growth factor (HGF) and the tyrosine kinase receptor RON by macrophage stimulating protein (MSP) results in the overactivation of signaling pathways that encourage cell proliferation, migration, and survival, contributing to the malignant behavior and chemoresistance of many cancers. The onset of metastatic prostate cancer is associated with both increased serum levels of HGF and expression of c-Met and RON in metastatic growths. Our laboratory has developed small molecules that inhibit HGF dimerization, c-Met activation, and malignant cell behaviors in several cancer cell types. Considering the high degree of homology between HGF and MSP, we predict that our strategy for inhibiting the activation of HGF can be extended to develop compounds that dually inhibit HGF and MSP. We hypothesize that inhibiting HGF and MSP with our antagonists will correspondingly reduce activation of c-Met and RON and suppress tumor growth and metastasis in CRPC. We further anticipate a therapy that combines our antagonists and docetaxel will increase the efficacy of chemotherapy by blunting the pro-survival properties of the HGF/c-Met and MSP/RON systems. Preliminary results indicate that our HGF dimerization antagonist Norleual [Nle-Tyr-Ile-ψ-(CH2-NH2)3-4-His-Pro-Phe] significantly inhibits the migration of CRPC cells and increases their susceptibility to docetaxel treatment in vitro. The primary objective of this study is to investigate the feasibility of HGF, MSP, and HGF/MSP dual antagonists as a therapy for CRPC either alone or in combination with docetaxel. Citation Format: Brett Vanderwerff, Kevin Church, Leen Kawas, Joseph Harding. Novel hepatocyte growth factor and macrophage stimulating protein antagonists for the treatment of castration-resistant prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3922. doi:10.1158/1538-7445.AM2015-3922

HGFL supports mammary tumorigenesis by enhancing tumor cell intrinsic survival and influencing macrophage and T-cell responses.

The Ron receptor is overexpressed in human breast cancers and is associated with heightened metastasis and poor survival. Ron overexpression in the mammary epithelium of mice is sufficient to induce aggressive mammary tumors with a high degree of metastasis. Despite the well-documented role of Ron in breast cancer, few studies have examined the necessity of the endogenous Ron ligand, hepatocyte growth factor-like protein (HGFL) in mammary tumorigenesis. Herein, mammary tumor growth and metastasis were examined in mice overexpressing Ron in the mammary epithelium with or without HGFL. HGFL ablation decreased oncogenic Ron activation and delayed mammary tumor initiation. HGFL was important for tumor cell proliferation and survival. HGFL loss resulted in increased numbers of macrophages and T-cells within the tumor. T-cell proliferation and cytotoxicity dramatically increased in HGFL deficient mice. Biochemical analysis of HGFL proficient tumors showed increased local HGFL production, with HGFL loss decreasing β-catenin expression and NF-κB activation. Re-expression of HGFL in HGFL deficient tumor cells stimulated cell migration and invasion with coordinate activation of NF-κB and reduced apoptosis. Together, these results demonstrate critical in vivo functions for HGFL in promoting breast tumorigenesis and suggest that targeting HGFL may inhibit tumor growth and reactivate anti-tumor immune responses.

Novel serum biomarkers for detection of excessive alcohol use.

Construct interview that correctly identifies those with alcohol use disorder have limitation, especially when the subjects are motivated to minimize the magnitude of drinking behavior. Current laboratory tests to detect excessive alcohol consumption are limited by marginal sensitivity/specificity. Excessive drinking has been shown to affect several organ systems, which may be reflected in changes in quantity of plasma proteins. Our aim was to employ novel proteomic analyses to identify potential markers for excessive alcohol use. A prospective case-control study included 49 controls and 54 excessive drinkers (discovery cohort). The serum proteomic analyses in these subjects were performed, and the results were tested in the verification cohort (40 controls and 40 excessive drinkers). Using the appropriate cutoff and confirmation with ELISA, we identified 4 proteins which were significantly elevated in the serum of excessive drinkers: AT-rich interactive domain-containing protein 4B (ARID4B), phosphatidylcholine-sterol acyltransferase (LCAT), hepatocyte growth factor-like protein (MST1), and ADP-ribosylation factor 6 (ARL6). The performance of the conventional markers (aspartate aminotransferase [AST], alanine aminotransferase [ALT], gamma-glutamyl transpeptidase [GGT], percentage of carbohydrate-deficient transferrin [%CDT], and mean corpuscular volume [MCV]) discriminating between excessive alcohol use and controls had an area under the curve (AUC) ranging from 0.21 (ALT) to 0.67 (MCV). The AUC of these novel proteins showed the improvement in the detection of excessive drinkers compared to conventional laboratory tests, ranging from 0.73 (for ARID4B) to 0.86 (for ARL6). We have identified 4 novel proteins that can discern subjects with excessive alcohol use. Further studies are needed to determine the clinical implications of these markers to detect excessive alcohol use and confirm abstinence.

Expression of Hepatocyte Growth Factor-Like Protein in Human Wound Tissue and Its Biological Functionality in Human Keratinocytes.

Hepatocyte growth factor-like protein (HGFl) and its receptor, Recepteur d'Origine Nantais (RON), have been implicated in the development of wound chronicity. HGFl and RON expression was detected in acute wound tissue, chronic wound tissue and in normal skin using quantitative polymerase chain reaction (Q-PCR). HGFl and RON expression was also assessed in chronic healing and chronic non-healing wound tissues using Q-PCR and immunohistochemical staining. Expression was similarly detected in the HaCaT immortalized human keratinocyte cell line using reverse transcription polymerase chain reaction (RT-PCR). rhHGFl was used to assess the impact of this molecule on HaCaT cell functionality using in vitro growth assays and electric cell-substrate impendence sensing (ECIS) migration assays. HGFl and RON transcript expression were significantly increased in acute wound tissue compared to chronic wound tissue and were also elevated, though non-significantly, in comparison to normal skin. Minimal expression was seen in both healing and non-healing chronic wounds. Treatment of HaCaT cells with rhHGFl had no effect on growth rates but did enhance cell migration. This effect was abolished by the addition of a phospholipase C gamma (PLCγ) small molecule inhibitor. The increased expression of HGFl and RON in acute, healing wounds and the pro-migratory effect of HGFl in an in vitro human keratinocyte model, may indicate a role for HGFl in active wound healing.

The Effects of Macrophage-Stimulating Protein on the Migration, Proliferation, and Collagen Synthesis of Skin FibroblastsIn VitroandIn Vivo

Macrophage-stimulating protein (MSP), an important cytokine with multiple functions, is highly expressed in adipose-derived stem cells-conditioned medium (ASC-CM). ASCs can effectively promote wound healing through paracrine mechanism, suggesting that MSP may play a critical role in wound healing. Through binding to its receptor, RON (Receptuerd'OrigineNantaise, also called macrophage stimulation 1 receptor; MST1R), it can activate epithelial cells and work as an inflammatory mediator. In this study, we found RON was also expressed on dermal fibroblasts and investigated the effects of MSP on proliferation, migration, and collagen synthesis of fibroblasts. With the treatment of different concentrations of MSP (0, 1, 10, 20, 50, and 100 ng/mL) on fibroblasts, proliferation, migration, and collagen synthesis were analyzed by Cell Counting Kit-8 (CCK-8), transwell and real-time polymerase chain reaction. Under the treatment of MSP, the migration, Collagen I, III synthesis, and matrix metalloproteinase-1 (MMP-1) mRNA expression of fibroblasts were upregulated significantly, although there was no effect on fibroblasts proliferation, and the optimal concentration of MSP for migration and collagen synthesis was 10 ng/mL. In the in vivo study, 10 ng/mL MSP was applied to full-thickness skin wound with bacterial cellulose membranes, and this treatment could accelerate the wound healing rate and increased the collagen synthesis of wound sites. This study suggested that MSP appears to promote the migration of fibroblasts, enhances collagen synthesis and remodeling, and effectively improves wound healing.

Variant RONΔ160 of the RON receptor tyrosine kinase promotes the growth and invasion in vitro and in vivo in gastric cancer cell lines.

BackgroundRecepteur d’origine nantais (RON) is a receptor tyrosine kinase whose overexpression has been observed in human gastric cancers. This study aimed to determine whether overexpression of the variant RONΔ160 could induce tumorigenicity of gastric cancer cells in vitro or in vivo, and whether its specific small molecule inhibitor (Compound I) could inhibit the effect of RONΔ160.MethodsWe constructed human gastric cancer cell line MGC-803 that was stably transfected with a recombinant plasmid expressing RONΔ160, and the effect of RONΔ160 overexpression and macrophage-stimulating protein (MSP) activation on proliferation, migration and invasion abilities of MGC-803 cells were evaluated. Tumor-bearing mice with gastric cancer cells were used to analyze the effects of RONΔ160 overexpression and Compound I on implanted tumor growth.ResultsIn vitro, overexpression of RONΔ160 in MGC-803 cells resulted changes to their cell morphology, and promoted cell proliferation, migration and invasion. In addition, overexpression of RONΔ160 increased the proportion of cells in the S phase. The effect of RONΔ160 was significantly enhanced by induction of MSP inducing (p < 0.05). In vivo, RONΔ160 promoted the growth of MGC-803 cells in nude mice, including increased tumor size and weight, and lower tumor incubation period. The Compound I inhibited the tumorigenic abilities of RONΔ160 (p <0.05).ConclusionsThe results indicate that overexpression of the variant RONΔ160 altered the phenotype and tumorigenicity of MGC-803 cells. Its specific small molecule inhibitor could inhibit the effect of RONΔ160. Therefore, the variant RONΔ160 may become a potential therapeutic target for gastric cancer.

Deficiency in Macrophage-Stimulating Protein Results in Spontaneous Intestinal Inflammation and Increased Susceptibility Toward Epithelial Damage in Zebrafish

Several genome-wide association studies have identified the genes encoding for macrophage-stimulating protein (MSP) and its receptor RON (Recepteur d'Origine Nantais) as possible susceptibility factors in inflammatory bowel disease. While it has been shown that the MSP-RON signaling pathway is involved in tissue injury responses, current mouse models for MSP and RON deficiency have not clearly demonstrated a role of MSP-RON signaling in the context of intestinal inflammation. In this study, we report that the recently identified zebrafish Msp mutant (msp(t34230)) develops spontaneous intestinal inflammation over time. From 14 to 28 weeks postfertilization Msp-deficient zebrafish show intestinal eosinophilia, increased intestinal expression of inflammatory marker mmp9, and activation of intestinal goblet cells. Moreover, these Msp mutant zebrafish are more susceptible toward ethanol-induced epithelial damage, which resulted in increased infiltration and proliferation of immune cells within the lamina propria and prolonged intestinal proinflammatory cytokine responses in some mutant fish. In light of the recent development of many tools to visualize, monitor, and genetically modify zebrafish, these Msp-deficient zebrafish will enable in-depth in vivo analysis of epithelial and macrophage-specific MSP-RON signaling in the context of intestinal inflammation.

MSP: An emerging player in metabolic syndrome

MSP (Macrophage Stimulating Protein; also known as Hepatocyte Growth Factor-like protein (HGFL) and MST1) is a secreted protein and the ligand for transmembrane receptor tyrosine kinase Recepteur d’Origine Nantais (RON; also known as MST1R). Since its discovery, MSP has been demonstrated to play a key role in regulating inflammation in the peripheral tissues of multiple disease models. Recent evidences also point toward a beneficial role of MSP in the regulation of hepatic lipid and glucose metabolism, thereby implicating MSP as a crucial regulator in maintaining metabolic homeostasis while simultaneously suppressing inflammatory processes. In this review, we discuss the recent advances that demonstrate the significance of MSP in metabolic syndrome and build a strong case supporting its therapeutic potential.

Regulation of Macrophage Polarization by RON Receptor Tyrosine Kinase Signaling.

The M1 and M2 states of macrophage polarization are the two extremes of a physiologic/phenotypic continuum that is dynamically influenced by environmental signals. The M1/M2 paradigm is an excellent framework to understand and appreciate some of the diverse functions that macrophages perform. Molecular analysis of mouse and human macrophages indicated that they gain M1 and M2-related functions after encountering specific ligands in the tissue environment. In this perspective, I discuss the function of recepteur d’origine nantais (RON) receptor tyrosine kinase in regulating the M2-like state of macrophage activation Besides decreasing pro-inflammatory cytokine production in response to toll-like receptor-4 activation, macrophage-stimulating protein strongly suppresses nitric oxide synthase and at the same time upregulates arginase, which is the rate limiting enzyme in the ornithine biosynthesis pathway. Interestingly, RON signaling preserved some of the characteristics of the M1 state, while still promoting the hallmarks of M2 polarization. Therefore, therapeutic modulation of RON activity can shift the activation state of macrophages between acute and chronic inflammatory states.

Hepatocyte growth factor activator inhibitor type-1 in cancer: Advances and perspectives (Review)

Cancer is one of the most common diseases, with high morbidity and mortality rates. Large‑scale efforts have been made to understand the pathogenesis of the disease, particularly in the advanced stages, in order to develop effective therapeutic approaches. Hepatocyte growth factor activator inhibitor type-1 (HAI-1), also known as serine protease inhibitor Kunitz type 1, inhibits the activity of several trypsin-like serine proteases. In particular, HAI-1 suppresses hepatocyte growth factor (HGF) activator and matriptase, resulting in subsequent inhibition of HGF/scatter factor and macrophage‑stimulating protein (MSP). HGF and MSP are involved in cancer development and progression, via the receptors Met receptor tyrosine kinase (RTK) and Ron RTK, respectively. Therefore, HAI-1-mediated downregulation of HGF and MSP signaling may suppress tumorigenesis and progression in certain types of cancers. Abnormal HAI-1 expression levels have been observed in various types of human cancer. The exact function of HAI-1 in cancer pathogenesis, however, has not been fully elucidated. In this review, the focus is on the potential impact of aberrant HAI-1 expression levels on tumorigenesis and progression, the underlying mechanisms, and areas that require further investigation to clarify the precise role of HAI-1 in cancer.

Inhibitors of HGFA, Matriptase, and Hepsin Serine Proteases: A Nonkinase Strategy to Block Cell Signaling in Cancer

Hepatocyte growth factor activators (HGFA), matriptase, and hepsin are S1 family trypsin-like serine proteases. These proteases proteolytically cleave the single-chain zymogen precursors, pro-HGF (hepatocyte growth factor), and pro-MSP (macrophage stimulating protein) into active heterodimeric forms. HGF and MSP are activating ligands for the oncogenic receptor tyrosine kinases (RTKs), c-MET and RON, respectively. We have discovered the first substrate-based ketothiazole inhibitors of HGFA, matriptase and hepsin. The compounds were synthesized using a combination of solution and solid-phase peptide synthesis (SPPS). Compounds were tested for protease inhibition using a kinetic enzyme assay employing fluorogenic peptide substrates. Highlighted HGFA inhibitors are Ac-KRLR-kt (5g), Ac-SKFR-kt (6c), and Ac-SWLR-kt (6g) with K is = 12, 57, and 63 nM, respectively. We demonstrated that inhibitors block the conversion of native pro-HGF and pro-MSP by HGFA with equivalent potency. Finally, we show that inhibition causes a dose-dependent decrease of c-MET signaling in MDA-MB-231 breast cancer cells. This preliminary investigation provides evidence that HGFA is a promising therapeutic target in breast cancer and other tumor types driven by c-MET and RON.