Functional Consequences of the Macrophage Stimulating Protein 689C Inflammatory Bowel Disease Risk Allele

<div><p>Background</p><p>Macrophage stimulating protein (MSP) is a serum growth factor that binds to and activates the receptor tyrosine kinase, Recepteur d'Origine Nantais (RON). A non-synonymous coding variant in MSP (689C) has been associated with genetic susceptibility to both Crohn's disease and ulcerative colitis, two major types of inflammatory bowel disease (IBD) characterized by chronic inflammation of the digestive tract. We investigated the consequences of this polymorphism for MSP-RON pathway activity and IBD pathogenesis.</p><p>Methods</p><p>RON expression patterns were examined on mouse and human cells and tissues under normal and disease conditions to identify cell types regulated by MSP-RON. Recombinant MSP variants were tested for their ability to bind and stimulate RON and undergo proteolytic activation. MSP concentrations were quantified in the serum of individuals carrying the MSP 689R and 689C alleles.</p><p>Results</p><p>In intestinal tissue, RON was primarily expressed by epithelial cells under normal and disease conditions. The 689C polymorphism had no impact on the ability of MSP to bind to or signal through RON. In a cohort of normal individuals and IBD patients, carriers of the 689C polymorphism had lower concentrations of MSP in their serum.</p><p>Conclusions</p><p>By reducing the quantities of circulating MSP, the 689C polymorphism, or a variant in linkage disequilibrium with this polymorphism, may impact RON ligand availability and thus receptor activity. Given the known functions of RON in regulating wound healing and our analysis of RON expression patterns in human intestinal tissue, these data suggest that decreased RON activity may impact the efficiency of epithelial repair and thus underlie the increased IBD susceptibility associated with the MSP 689C allele.</p></div

Binding kinetics and affinities of MSP 689R and 689C proteins to RON.

<p>^aData are the average ± standard deviations of three or more independent determinations in general.</p><p>SPR capturing RON-Fc at 25°C,</p><p>SPR using immobilized RON Sema/PSI at 25°C,</p><p>BLI using RON-Fc at 30°C,</p><p>Radioligand binding to 3T3-hRON cells for 2 h at room temperature,</p><p>Radioligand binding to 3T3-hRON cells for 4 h on ice.</p

The 689C polymorphism in MSP does not affect binding to RON.

<p>(A) Overview of recombinant human MSP proteins used in this study. MSP α- and β-chains, PAN domain (N), kringle domains (K), serine protease-like domain (SPL) are indicated. scMSP is inactive due to the R483E mutation at the proteolytic cleavage site (arrow). 689R and 689C versions of all MSP proteins were generated. (B) Cell-free binding assay consisting of plate bound RON-Fc and soluble MSP. Means of three replicates per group are shown. Lines represent dose-response curves fit to a 4 parameter equation, which yielded EC₅₀ values of 0.1, 0.2, 0.05 and 0.1 nM for MSP 689R, 689C, MSP β 689R and MSP β 689C, respectively. (C) SPR analysis of MSP proteins binding to immobilized RON Sema/PSI, showing relative response in response units. Data are representative of three independent experiments. (D) Radioligand binding assay of MSP binding to RON. Competition binding to 3T3-hRON cells (large graphs) used to generate affinities and Scatchard plots (inset graphs) are shown. Mean +/− SD of three independent experiments is shown. (E) Homology model of the structure of RON Sema/PSI (PDB code 4FWW) in beige bound to MSP β (PDB code 2ASU) in blue. RON and MSP β were globally aligned to the Met/HGF β complex (PDB code 1SHY). Predicted locations of the MSP β contact residues within 4 Å of RON are highlighted in magenta. MSP β residue arginine 689 was mutated to a cysteine and is shown in yellow.</p

The 689C polymorphism does not affect MSP signaling through RON.

<p>(A) Western blot analysis of total Akt and pAkt in A2780-hRON and BxPC3 cells treated as indicated. Blot was performed in triplicate and mean of the pAkt/total ratios is shown. (B) Quantitation of pAkt by MSD analysis in 3T3-hRON cells treated with scMSP or MSP variants. Mean +/− SD of triplicate wells is shown. Data are representative of three independent experiments. (C) Quantification of pAkt by MSD analysis in HCT15 cells treated with MSP variants. Mean +/− SD of triplicate wells is shown. Data are representative of two independent experiments. (D) Quantitation of pAkt by MSD analysis in human primary colon cells treated with MSP variants. Mean +/− SD of triplicate wells is shown. Data are representative of two independent experiments. (E) Images from scratch wound assay of 3T3-hRON cells treated with scMSP or MSP variants. Images are from the same cell culture at the indicated times after scratch wounding. Dashed lines represent position of initial scratch. (F) Quantitation of scratch wound assay from 3T3-hRON cells treated with medium-alone, scMSP, or MSP variants. Mean +/− SD of three treatments is shown. Data are representative of three independent experiments. ^#not significant, **p≤0.0003 for comparisons between medium and MSP variants.</p

Carriers of the 689C polymorphism have lower quantities of serum MSP.

<p>(A) Analysis of rs3197999 alleles in DNA samples from normal, UC, and CD patients. The C allele encodes MSP 689R and T allele encodes MSP 689C. Allele frequency and number are indicated for each group. (B) MSP concentrations in matched serum samples of EMBARK subjects determined by ELISA, with results grouped by rs3197999 genotype. *p<0.005, **p<0.0005. (C) Serum MSP concentrations grouped by genotype and disease status. *p<0.05, **p<0.005, ***p<0.0005.</p

RON is preferentially expressed by epithelial cells in human tissues under steady-state and disease conditions.

<p>(A) Representative images from IHC analysis of RON protein expression (brown) on tissue sections from normal, UC, and CD colons. Scale bar  =  100 µm. (B) Representative images from ISH analysis of RON transcript expression (red) on tissue sections from normal, UC, and CD colons. Scale bar  =  20 µm. (C) Quantitative RT-PCR analysis of RON expression in human intestinal biopsies samples taken from normal individuals and uninflamed and inflamed regions of UC and CD patients. (D) Analysis of RON expression in single cell suspensions of resected intestinal tissues from an ulcerative colitis patient by flow cytometry using a monoclonal antibody specific for human RON (blue histogram) or an isotype control antibody (shaded histogram). Macrophages were gated as CD45⁺CD14⁺HLADR⁺ and epithelial cells were gated as CD45⁻EpCAM⁺ cells. (E) Quantification of RON expression by flow cytometry on macrophages and epithelial cells from resected intestinal tissue of multiple donors (macrophages: 1 non-IBD (N), 3 UC, 2 CD; epithelial cells: 2 UC, 1 CD). Patient numbers are indicated (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083958#pone.0083958.s007" target="_blank">Table S1</a>). Data represents the mean fluorescence intensity (MFI) ratio of RON staining to isotype control staining. Dashed line indicates a ratio of 1∶1, <i>i.e.</i> lack of RON expression. N, non-IBD.</p

RON is expressed by myeloid and epithelial cell populations in mice.

<p>(A) Single cell suspensions of the indicated cell populations or tissues were analyzed by flow cytometry for RON expression using a monoclonal antibody specific for murine RON (blue histogram) or an isotype control antibody (shaded histogram). Macrophage populations were gated as CD45⁺F4/80⁺CD11b⁺MHC class II⁺ for <i>ex vivo</i>-derived cells and F4/80⁺CD11b⁺ for bone marrow cultured cells. Colonic epithelial cells were gated as CD45⁻ECadherin⁺. B) Representative image from IHC analysis of RON expression (brown) on tissue sections from normal mouse colon. Scale bars  =  100 µm. Data are representative of at least three similar experiments.</p

The 689C polymorphism does not affect proteolytic activation of MSP by hepsin.

<p>Purified MSP 689R (upper panel) and MSP 689C (lower panel) consisting of both pro-MSP and MSP were mixed with 100 nM to 97 pM hepsin in a 2-fold dilution series and analyzed by gel electrophoresis under reducing conditions. Relative mobilities corresponding to pro-MSP, MSP α, and MSP β are indicated. Data are representative of three independent experiments.</p

RON is preferentially expressed by human epithelial cells.

<p>(A) Quantitative RT-PCR survey of RON expression in human cell lines and primary cells. Columns 1: HEK293 control, columns 2–4: epithelial cell lines, columns 5–6: monocytic cell lines, column 7: human CD14⁺ monocytes (monos), columns 8–11: monocyte-derived macrophages (MDMs) left untreated or treated with the indicated stimuli. Data in columns 1-7 are the mean +/− SD of three independent samples and columns 8–11 are the mean of two donors. (B) Analysis of single-cell suspensions for RON expression by flow cytometry. Cells were stained with a monoclonal antibody specific for human RON (blue histogram) or an isotype control antibody (shaded histogram). MDMs were gated as CD14⁺CD33⁺.</p