Yoshiaki Arimura

A genome-wide association study identifies three new susceptibility loci for ulcerative colitis in the Japanese population

Ulcerative colitis is one of the principal forms of inflammatory bowel disease with complex manifestations. Although previous studies have indicated that there is a genetic contribution to the pathogenesis of ulcerative colitis, the genes influencing susceptibility to the disease have not been fully determined. To identify genetic factors conferring risk of ulcerative colitis, here we conducted a two-stage genome-wide association study and subsequent replication study using 1,384 Japanese individuals with ulcerative colitis and 3,057 control subjects. In addition to the expected strong association with the major histocompatibility complex (MHC) region, we identified three new susceptibility loci: the immunoglobulin receptor gene FCGR2A (rs1801274, P = 1.56 × 10−12), a locus on chromosome 13q12 (rs17085007, P = 6.64 × 10−8) and the glycoprotein gene SLC26A3 (rs2108225, P = 9.50 × 10−8). rs1801274 is a nonsynonymous SNP of FCGR2A that is reported to have a critical effect on receptor binding affinity for IgG and to be associated with other autoimmune diseases. Our findings provide insight into the molecular pathogenesis of ulcerative colitis.

Enhancing epithelial engraftment of rat mesenchymal stem cells restores epithelial barrier integrity.

The cellular origin, in vivo function and fate of donor bone marrow‐derived cells residing in the recipient intestinal epithelial cells, pericryptal myofibroblasts or endothelial cells remain obscure. Although ‘immunoprivileged’ mesenchymal stem cells (MSCs) are prime candidates for cell‐ and gene‐based therapy, their precise role in colitis remains largely undetermined. Using a dextran sulphate sodium (DSS) colitis with busulphan (BU)‐induced hypoplastic marrow model, we examined the therapeutic effects of MSC transplantation, focusing on the role of MSCs as both cell providers and immunomodulators. Donor‐derived MSCs were detected by eGFP immunofluorescence and fluorescence in situ hybridization for Y‐chromosome (Y‐FISH) analysis. Western blot analysis of apical‐most tight junction proteins was performed with antibodies against claudin‐2, ‐7, ‐8, ‐12, ‐13, ‐15 and ZO‐1. Cytokine and cell cycle profiles were analysed by semi‐quantitative RT‐PCR and flow cytometry. Susceptibility to DSS colitis was significantly increased by co‐existing BU‐induced bone marrow hypoplasia and this increase was significantly reduced by enhancing epithelial engraftment of MSCs, an effect depending on restoring epithelial barrier integrity rather than inhibiting host immune responses. We provide evidence that implicates MSCs in maintaining epithelial barrier function by reassembling apical‐most tight junction proteins, claudins. The therapeutic efficacy of extrinsic MSCs depends on enhancing epithelial engraftment in damaged crypts by busulphan conditioning. Such a role for the MSC‐derived intestinal cells in colitis therapy merits further examination and may offer a promising new treatment for inflammatory bowel disease (IBD). Copyright

Myogenic lineage differentiated mesenchymal stem cells enhance recovery from dextran sulfate sodium-induced colitis in the rat

BackgroundAlthough mounting evidence implicates mesenchymal stem cells (MSCs) in intestinal tissue repair, uncertainty remains concerning the distribution, function, and fate of repopulating MSCs in recipient colonic tissues. Therefore, we investigated the role of transplanted MSCs in the repair phase of DSS colitis.MethodsLacZ-labeled rat MSCs were transplanted into rats with colitis induced by 4% DSS on day 2. Regular water replaced the DSS solution on day 6. Therapeutic effect was evaluated on day 9 by clinicopathologic and growth factor/cytokine expression profiles. We analyzed the Notch signaling pathway by Western blotting and characterized immunofluorescence of lacZ-labeled MSCs with confocal laser microscopy. In vivo differentiation of MSC was confirmed by transmission electron microscopy (TEM).ResultsRecovery of colitis was modestly but significantly promoted by MSC transplantation due to proceeding cell cycle and inhibiting apoptosis in the epithelia. Tgfa mRNA expression increased significantly, while Notch signaling was inhibited in the colonic tissues with MSC transplantation. β-Galactosidase-positive cells, which expressed α-SMA, desmin, and vimentin, were infrequently detected in the lamina propria stroma. DSS exposure in vitro proved to be the most potent inducer for α-SMA in MSCs where TEM demonstrated myogenic lineage differentiation.ConclusionsWe found that MSCs transplantation modestly promoted the repair of DSS colitis. The donor-derived MSCs were likely reprogrammed to differentiate to myogenic lineage cells by cues from the micro milieu. Further characterization of these cells is warranted as a basis for applying cell-based therapy for inflammatory bowel disease.

The Efficacy of IGF-I Receptor Monoclonal Antibody against Human Gastrointestinal Carcinomas is Independent of k-ras Mutation Status

Purpose: Insulin-like growth factor (IGF)-I receptor (IGF-IR) signaling is required for carcinogenicity and proliferation of gastrointestinal cancers. We have previously shown successful targeting therapy for colorectal, pancreatic, gastric, and esophageal carcinomas using recombinant adenoviruses expressing dominant negative IGF-IR. Mutation in k-ras is one of key factors in gastrointestinal cancers. In this study, we sought to evaluate the effect of a new monoclonal antibody for IGF-IR, figitumumab (CP-751,871), on the progression of human gastrointestinal carcinomas with/without k-ras mutation. Experimental Design: We assessed the effect of figitumumab on signal transduction, proliferation, and survival in six gastrointestinal cancer cell lines with/without k-ras mutation, including colorectal and pancreatic adenocarcinoma, esophageal squamous cell carcinoma, and hepatoma. Combination effects of figitumumab and chemotherapy were also studied. Then figitumumab was evaluated in the treatment of xenografts in nude mice. Results: Figitumumab blocked autophosphorylation of IGF-IR and its downstream signals. The antibody suppressed proliferation and tumorigenicity in all cell lines. Figitumumab inhibited survival by itself and up-regulated chemotherapy (5-FU and gemcitabine) induced apoptosis. Moreover, the combination of this agent and chemotherapy was effective against tumors in mice. The effect of figitumumab was not influenced by the mutation status of k-ras. Figitumumab reduced expression of IGF-IR but not insulin receptor in these xenografted tumors. The drug did not affect murine body weight or blood concentrations of glucose, insulin, IGF binding protein 3, and growth hormone. Conclusions: IGF-IR might be a good molecular therapeutic target and figitumumab may thus have therapeutic value in human gastrointestinal malignancies even in the presence of k-ras mutations. Clin Cancer Res; 17(15); 5048–59. ©2011 AACR.

Genetic Variants in Surfactant, Pulmonary associated Protein D (SFTPD) and Japanese Susceptibility to Ulcerative Colitis

Background: Identifying culprit genes for a complex trait in a homogeneous population such as the Japanese remains challenging. We aimed to use previous achievements of genome‐wide association studies to identify precise susceptibility loci for inflammatory bowel disease in Japanese people and to simultaneously investigate the replication of recently identified susceptibility loci. Methods: An unrelated Japanese population of 174 Crohns disease patients, 296 ulcerative colitis (UC) patients, and 394 healthy controls was consecutively enrolled in this study. Genotype and haplotype analyses focusing on susceptibility to inflammatory bowel disease were performed using 7 single nucleotide polymorphisms (SNPs) including 5 HapMap tag SNPs within surfactant, pulmonary‐associated protein D (SFTPD) along with the 2 Caucasian susceptibility loci. We performed fine‐scale mapping of trait‐associated loci with the extension of a shattered coalescent process in a Bayesian framework. Epistasis on disease phenotypes was statistically explored with the interaction dendrogram. Results: A minor allele G of rs911887 reached statistical significance for susceptibility to UC. The 2‐allele haplotype GG comprising rs911887 and rs2243639 (Ala160Thr) within SFTPD was significantly associated with susceptibility to UC. A posterior density plot shows that trait‐associated variants in the vicinity of rs911887 are likely to exist. An association between NKX2‐3 and UC susceptibility was replicated and diverse evidence of epistasis in Japan was suggested. Conclusions: This preliminary study suggests that SFTPD is both a susceptibility gene and a disease‐modifying gene for UC in Japanese. Replication of the causality and functional analyses of SFTPD is urgently warranted. (Inflamm Bowel Dis 2009;)

Targeting for insulin-like growth factor-I receptor with short hairpin RNA for human digestive/gastrointestinal cancers

Background and aimsInsulin-like growth factor (IGF)-I receptor (IGF-IR) signaling plays important parts in both the tumorigenicity and progression of digestive/gastrointestinal malignancies. In this study, we sought to test the effectiveness of a practical approach to blocking IGF-IR signaling using RNA interference delivered by recombinant adenoviruses.MethodsWe constructed a recombinant adenovirus expressing short hairpin RNA targeting IGF-IR (shIGF-IR) and assessed its effect on signal transduction, proliferation, and survival in digestive/gastrointestinal cancer cell lines representing colorectal, gastric, and pancreatic adenocarcinoma, esophageal squamous cell carcinoma, and hepatoma. We analyzed the effects of shIGF-IR alone and with chemotherapy in vitro and in nude mouse xenografts, as well as on insulin signaling and hybrid receptor formation between IGF-IR and insulin receptor.ResultsshIGF-IR blocked expression and autophosphorylation of IGF-IR and downstream signaling by the IGFs, but not by insulin. shIGF-IR suppressed proliferation and carcinogenicity in vitro and up-regulated apoptosis in a dose-dependent fashion. shIGF-IR augmented the effects of chemotherapy on in vitro growth and apoptosis induction. Moreover, the combination of shIGF-IR and chemotherapy was highly effective against tumors in mice. shIGF-IR reduced hybrid receptor formation without effect on expression of insulin receptor.ConclusionsshIGF-IR may have therapeutic utility in human digestive/gastrointestinal cancers, both alone and in combination with chemotherapy.

Insulin-like growth factor receptor expression is associated with aggressive phenotypes and has therapeutic activity in biliary tract cancers.

Insulin‐like growth factor (IGF)‐I receptor (IGF‐IR) signaling is required for carcinogenicity and progression of several cancers but the function of this pathway and its utility as a therapeutic target have not been studied comprehensively in biliary tract carcinomas (BTC). We investigated the immunohistochemical expression of elements of the IGF axis, matrilysin, overexpression of p53 and the methylation status of the IGFBP‐3 promoter in 80 surgically resected BTC. We also assessed the effect of IGF‐IR blockade on signal transduction, proliferation and survival in three BTC cell lines using a new tyrosine kinase inhibitor, BMS‐536924, and dominant negative IGF‐IR (IGF‐IR/dn). The effects of IGF‐IR blockade was also studied in nude mouse xenograft models. IGF‐I was expressed in 60% and IGF‐II in 50% of tumors. High expression was associated with tumor size. IGF‐IR was expressed in 69% of the cases and was associated with advanced stage and matrilysin expression. Hypermethylation of the IGFBP‐3 promoter was detected in 41% of BTC and was inversely correlated with p53 expression. BMS‐536924 blocked autophosphorylation of IGF‐IR and both Akt and ERK activation by both IGF‐I and insulin. BMS‐536924 suppressed proliferation and tumorigenicity in vitro in a dose‐dependent fashion. This inhibitor upregulated chemotherapy‐induced apoptosis in a dose‐dependent fashion. Moreover, IGF‐IR blockade was effective against tumors in mice. IGF‐IR might identify a subset of BTC with a particularly aggressive phenotype and is a candidate therapeutic target in this disease. BMS‐536924 might have significant therapeutic utility. (Cancer Sci 2012; 103: 252–261)

Dynamics of claudins expression in colitis and colitis-associated cancer in rat.

Claudins comprise a multigene family of 24 species and have been shown to constitute the backbone of tight junction strands in simple epithelial cells and to be directly involved in their barrier functions. Apical-most tight junction protein complexes (TJs) are implicated in inflammatory bowel disease (IBD) pathophysiology. Except for claudin-8, TJs explored in this study (including ZO-1, claudin-1, -2, -3, -7, -12, and -15) were found to be expressed in rat colonic tissues. ZO-1 and claudin-7 were ubiquitously expressed in all study groups. As depicted in Fig. 1b, expressions of claudin-2, -12, and -15 significantly diminished after combined treatment with dextran sulfate sodium (DSS) and busulfan (BU) (lane 5), compared with either agent alone (lanes 2 and 4). Despite the lack of significance, there appeared to be a subtle dose-dependent decrease with DSS treatment (lanes 2 and 3). In contrast to these results obtained from DSS colitis, expression of claudin-1 was significantly downregulated, while expression of claudin-15 was upregulated in colitis-associated cancer tissues in the azoxymethane (AOM)/DSS model (Fig. 2b). It is very intriguing that claudins expression dynamics were mutually exclusive between colitis and colitis-associated cancer in rats. However, the biological significance of disease-specific claudin expression profiles will remain elusive until the specific expression and function of each claudin in a tissue- and cell-type relationship are comprehensively clarified. Currently, the physiologic consequences of the diversity of TJ barrier function resulting from multiple combinations of claudins are only beginning to be recognized. Full unraveling of these complexities could inspire a new paradigm of inflammation and cancer, and eventually translate to clinical practice on IBD.