Chitinase 3-Like 1 Promotes Macrophage Recruitment and Angiogenesis in Colorectal Cancer

Chitinase 3-like 1 (CHI3L1), one of mammalian members of the chitinase family, is expressed in several types of human cancer, and elevated serum level of CHI3L1 is suggested to be a biomarker of poor prognosis in advanced cancer patients. However, the overall biological function of CHI3L1 in human cancers still remains unknown. Studies were performed to characterize the role of CHI3L1 in cancer pathophysiology utilizing human colorectal cancer samples and human cell lines. Plasma protein and tissue mRNA expression levels of CHI3L1 in colorectal cancer were strongly upregulated. Immunohistochemical analysis showed that CHI3L1 was expressed in cancer cells and CHI3L1 expression had a significant association with the number of infiltrated macrophages and microvessel density. By utilizing trans-well migration and tube formation assays, overexpression of CHI3L1 in SW480 cells (human colon cancer cells) enhanced the migration of THP-1 cells (human macrophage cells) and HUVECs (human endothelial cells), and the tube formation of HUVECs. The knockdown of CHI3L1 by RNA interference or the neutralization of CHI3L1 by anti-CHI3L1 antibody displayed strong suppression of CHI3L1-induced migration and tube formation. Cell proliferation assay showed that CHI3L1 overexpression significantly enhanced the proliferation of SW480 cells. ELISA analysis showed that CHI3L1 increased the secretion of inflammatory chemokines, IL-8 and MCP-1, from SW480 cells through mitogen-activated protein kinase (MAPK) signaling pathway. Both neutralization of IL-8 or MCP-1 and inhibition or knockdown of MAPK in SW480 cells significantly inhibited CHI3L1-induced migration and tube formation. In a xenograft mouse model, overexpression of CHI3L1 in HCT116 cells (human colon cancer cells) enhanced the tumor growth as well as macrophage infiltration and microvessel density. In conclusion, CHI3L1 expressed in colon cancer cells promotes cancer cell proliferation, macrophage recruitment and angiogenesis. Thus, the inhibition of CHI3L1 activity may be a novel therapeutic strategy for human colorectal cancer

Wnt-3a タンパクシツ ノ トウサ シュウショク ト パルミチンサン シュウショク ガ キョウヤクスル コト ニ ヨリ カッセイガタ ノ Wnt-3a タンパクシツ ガ ゴウセイサレル

京都大学0048新制・課程博士博士(医学)甲第12960号医博第3120号新制||医||952(附属図書館)UT51-2007-H233京都大学大学院医学研究科内科系専攻(主査)教授 武藤 誠, 教授 松田 道行, 教授 永田 和宏学位規則第4条第1項該当Doctor of Medical ScienceKyoto UniversityDA

Deletion of nardilysin prevents the development of steatohepatitis and liver fibrotic changes.

Nonalcoholic steatohepatitis (NASH) is an inflammatory form of nonalcoholic fatty liver disease that progresses to liver cirrhosis. It is still unknown how only limited patients with fatty liver develop NASH. Tumor necrosis factor (TNF)-α is one of the key molecules in initiating the vicious circle of inflammations. Nardilysin (N-arginine dibasic convertase; Nrd1), a zinc metalloendopeptidase of the M16 family, enhances ectodomain shedding of TNF-α, resulting in the activation of inflammatory responses. In this study, we aimed to examine the role of Nrd1 in the development of NASH. Nrd1+/+ and Nrd1-/- mice were fed a control choline-supplemented amino acid-defined (CSAA) diet or a choline-deficient amino acid-defined (CDAA) diet. Fatty deposits were accumulated in the livers of both Nrd1+/+ and Nrd1-/- mice by the administration of the CSAA or CDAA diets, although the amount of liver triglyceride in Nrd1-/- mice was lower than that in Nrd1+/+ mice. Serum alanine aminotransferase levels were increased in Nrd1+/+ mice but not in Nrd1-/- mice fed the CDAA diet. mRNA expression of inflammatory cytokines were decreased in Nrd1-/- mice than in Nrd1+/+ mice fed the CDAA diet. While TNF-α protein was detected in both Nrd1+/+ and Nrd1-/- mouse livers fed the CDAA diet, secretion of TNF-α in Nrd1-/- mice was significantly less than that in Nrd1+/+ mice, indicating the decreased TNF-α shedding in Nrd1-/- mouse liver. Notably, fibrotic changes of the liver, accompanied by the increase of fibrogenic markers, were observed in Nrd1+/+ mice but not in Nrd1-/- mice fed the CDAA diet. Similar to the CDAA diet, fibrotic changes were not observed in Nrd1-/- mice fed a high-fat diet. Thus, deletion of nardilysin prevents the development of diet-induced steatohepatitis and liver fibrogenesis. Nardilysin could be an attractive target for anti-inflammatory therapy against NASH

EP4 Receptor–Associated Protein in Macrophages Ameliorates Colitis and Colitis-Associated Tumorigenesis

Prostaglandin E2 plays important roles in the maintenance of colonic homeostasis. The recently identified prostaglandin E receptor (EP) 4–associated protein (EPRAP) is essential for an anti-inflammatory function of EP4 signaling in macrophages in vitro. To investigate the in vivo roles of EPRAP, we examined the effects of EPRAP on colitis and colitis-associated tumorigenesis. In mice, EPRAP deficiency exacerbated colitis induced by dextran sodium sulfate (DSS) treatment. Wild-type (WT) or EPRAP-deficient recipients transplanted with EPRAP-deficient bone marrow developed more severe DSS-induced colitis than WT or EPRAP-deficient recipients of WT bone marrow. In the context of colitis-associated tumorigenesis, both systemic EPRAP null mutation and EPRAP-deficiency in the bone marrow enhanced intestinal polyp formation induced by azoxymethane (AOM)/DSS treatment. Administration of an EP4-selective agonist, ONO-AE1-329, ameliorated DSS-induced colitis in WT, but not in EPRAP-deficient mice. EPRAP deficiency increased the levels of the phosphorylated forms of p105, MEK, and ERK, resulting in activation of stromal macrophages in DSS-induced colitis. Macrophages of DSS-treated EPRAP-deficient mice exhibited a marked increase in the expression of pro-inflammatory genes, relative to WT mice. By contrast, forced expression of EPRAP in macrophages ameliorated DSS-induced colitis and AOM/DSS-induced intestinal polyp formation. These data suggest that EPRAP in macrophages functions crucially in suppressing colonic inflammation. Consistently, EPRAP-positive macrophages were also accumulated in the colonic stroma of ulcerative colitis patients. Thus, EPRAP may be a potential therapeutic target for inflammatory bowel disease and associated intestinal tumorigenesis

TNF-α was not sufficiently secreted from in <i>Nrd1^−/−</i> mice fed the CDAA diet.

<p>A. A liver fragment was divided into two pieces. One was directly subjected to protein extraction directly and measurement of TNF-α (produced TNF-α). The other piece was cultured in serum-free medium for 12 hours, and the supernatant was subjected to measurement of TNF-α (secreted TNF-α). The relative optical density (O.D.) to that of <i>Nrd1^+/+</i> fed the CSAA diet was determined by ELISA for TNF-α. Production of TNF-α from liver specimens were not significantly different between <i>Nrd1^+/+</i> and <i>Nrd1^−/−</i> mice fed the CDAA diet for 20 weeks (“produced TNF-α”). In contrast, TNF-α secreted from liver specimens was significantly increased in <i>Nrd1^+/+</i> mice fed the CDAA diet for 20 weeks compared with those fed the CSAA diet; however, the elevation was not observed in <i>Nrd1^−/−</i> mice under the same condition (“secreted TNF-α”). *<i>P</i><0.05. B. Production of IL6 and IL1-β proteins were significantly increased only in <i>Nrd1^+/+</i> mice fed the CDAA diet for 20 weeks compared with those in <i>Nrd1^−/−</i> mice fed the CSAA diet for 20 weeks. *<i>P</i><0.05. C. mRNA (upper, ‘produced’) and protein (lower, ‘secreted’) production of IL6 and IL1-β were significantly increased after LPS treatment in <i>Nrd1^+/+</i> mouse peritoneal macrophages. However, administration of anti-TNF-α neutralizing antibodies significantly suppressed the production of IL6 and IL1-β in the presence of LPS. *<i>P</i><0.05.</p

Liver fibrotic area was not observed in <i>Nrd1^−/−</i> mice fed the CDAA diet.

<p>A. Liver fibrosis was determined by Sirius red staining (red) in <i>Nrd1^+/+</i> and <i>Nrd1^−/−</i> mice at 4 (upper), 12 (middle), and 20 (lower) weeks in the livers of <i>Nrd1^+/+</i> and <i>Nrd1^−/−</i> mice fed the CSAA or CDAA diet. Fibrotic changes were not observed in <i>Nrd1^+/+</i> or <i>Nrd1^−/−</i> mice fed the CSAA diet (left). Fibrotic changes were prominent in <i>Nrd1^+/+</i> mice, but not in <i>Nrd1^−/−</i> mice fed the CDAA diet (right). Bars indicate 100 µm. B. Quantification of fibrotic areas. Fibrotic areas was observed and increased in a time-dependent manner only in the livers of <i>Nrd1^+/+</i> mice fed the CDAA diet. n = 5–8, each. *<i>P</i><0.05.</p

TNF-α was expressed in <i>Nrd1^+/+</i> and <i>Nrd1^−/−</i> mice fed the CDAA diet.

<p>A. Immunohistochemistry showed that TNF-α protein (red, arrowheads) was expressed in F4/80-positive Kupffer cells or macrophages (green, arrowheads) in the livers of both <i>Nrd1^+/+</i> and <i>Nrd1^−/−</i> mouse fed the CDAA diet for 20 weeks (right), but not in mice fed the CSAA diet for 20 weeks (left). A blue color indicates DAPI-positive nuclei. Bars indicate 50 µm. B. The number of F4/80-positive cells/×100 high-power field (HPF) in livers slightly increased (approximately 1.2 times) only in <i>Nrd1^+/+</i> mice fed the CDAA diet (right). C. Relative expression of mRNA are shown as relative values compared to those at 0 w. The mRNA expression level of CCR2 was increased in <i>Nrd1^+/+</i> mice fed the CDAA diet, and the levels were significantly higher than respective values in <i>Nrd1^−/−</i> mice fed the CDAA diet. *<i>P</i><0.05.</p

Liver fibrogenesis was not observed in <i>Nrd1^−/−</i> mice fed the HFD.

<p>A. Steatosis was observed in both <i>Nrd1^+/+</i> and <i>Nrd1^–/–</i> mice after 20-week HFD administration (right), but not in those fed a normal control diet (left). Bars indicate 100 µm. B. Quantification of triglyceride in the liver. Triglyceride was elevated in the livers of both <i>Nrd1^+/+</i> and <i>Nrd1^–/–</i> mice after 20-week HFD administration, although it was significantly higher in <i>Nrd1^+/+</i> mice. n = 4, each. *<i>P</i><0.05. C. Serum ALT levels were significantly elevated in <i>Nrd1^+/+</i> mice upon administration of the HFD, but were not elevated in other mouse groups. *<i>P</i><0.05. D. Fibrotic area was less prominent in <i>Nrd1^−/−</i> mice than in <i>Nrd1^+/+</i> mice (right). Bars indicate 100 µm. E. Fibrotic area was observed only in the livers of <i>Nrd1^+/+</i> mice fed the HFD (right). n = 5, each. *<i>P</i><0.05.</p

CDAA diet caused hepatic steatosis in <i>Nrd1^+/+</i> and <i>Nrd1^−/−</i> mice.

<p>A. Histology of the livers of <i>Nrd1^+/+</i> and <i>Nrd1^−/−</i> mice fed the CSAA (left) or CDAA (right) diets. Representative changes of the liver with regard to fat deposition at 4 (upper), 12 (middle), and 20 (lower) weeks during the experiments are depicted. Fat deposits were confirmed by oil red O staining shows (orange, inset). CV indicates central vein, and PT marks portal triad. Bars indicate 100 µm. B. Quantification of triglyceride in the liver. Triglyceride in the liver was increased in the livers of both <i>Nrd1^+/+</i> and <i>Nrd1^−/−</i> mice during administration of the CDAA or CSAA diets, although it was significantly more prominent in <i>Nrd1^+/+</i> mice. n = 4–5, each. *<i>P</i><0.05. C. There was no significant difference in the liver/body weight ratio between <i>Nrd1^+/+</i> and <i>Nrd1^−/−</i> mice during the experiments.</p