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

Nakatsuji M, Minami M, Seno H, Yasui M, Komekado H, Higuchi S, Fujikawa R, Nakanishi Y, Fukuda A, Kawada K, Sakai Y, Kita T, Libby P, Ikeuchi H, Yokode M, Chiba T - PLoS Genet. (2015)

Bottom Line: Prostaglandin E2 plays important roles in the maintenance of colonic homeostasis.To investigate the in vivo roles of EPRAP, we examined the effects of EPRAP on colitis and colitis-associated tumorigenesis.Thus, EPRAP may be a potential therapeutic target for inflammatory bowel disease and associated intestinal tumorigenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastroenterology and Hepatology, Kyoto University Graduate School of Medicine, Kyoto, Japan.

ABSTRACT
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 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.

No MeSH data available.


Related in: MedlinePlus

EPRAP overexpression in macrophages ameliorates DSS-induced colitis and colitis-associated tumorigenesis.(A) Representative photographs of colons at the end of DSS treatment; colons were obtained from WT and CD68–mEPRAP transgenic (TG) mice. (B) Percent changes in colon length expressed relative to DSS-free water controls (n = 8 [WT]; n = 6 [TG]). (C) Histological colitis scores (n = 10 [WT]; n = 8 [TG]). (D) Representative H&E staining of rectal sections. (E) The numbers of F4/80-, Gr-1–, B220-, CD4-, and CD8-positive cells infiltrated in colonic tissues of WT and TG mice, per high-power field (400× magnification) (n = 8 each). (F) The expression levels of TNF-α, IL-1β, IL-6, CXCL1, and MCP-1 protein in colonic tissue lysates from DSS-treated WT and TG mice (n = 15 [WT]; n = 8 [TG]). (G) Survival curves during the course of AOM/DSS treatment in WT and TG mice (n = 18 [WT]; n = 11 [TG]; P < 0.05). (H) The numbers of colonic polyps per mouse, with size distribution (left) and total number (right) in colonic tissues of AOM/DSS-treated WT and TG mice (n = 9 [WT]; n = 10 [TG]). (I) Representative photographs of colons at the end of AOM/DSS treatment. (J) Ki-67–positive cells in rectal polyps of AOM/DSS-treated WT and TG mice (left). TG mice exhibited markedly fewer Ki-67–positive cells than WT mice (n = 5 each) (right). (K) Representative photographs of TUNEL assay performed on rectal polyps of AOM/DSS-treated WT and TG mice. The arrows indicate TUNEL-positive apoptotic cells: the insets show higher magnifications of selected regions (indicated by dashed boxes) (left). TG mice exhibited more TUNEL-positive cells than WT mice (right) (n = 5 each). All values represent means ± SEM. *P < 0.05, **P < 0.01 vs. WT mice. Scale bars: 100 μm.
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pgen.1005542.g005: EPRAP overexpression in macrophages ameliorates DSS-induced colitis and colitis-associated tumorigenesis.(A) Representative photographs of colons at the end of DSS treatment; colons were obtained from WT and CD68–mEPRAP transgenic (TG) mice. (B) Percent changes in colon length expressed relative to DSS-free water controls (n = 8 [WT]; n = 6 [TG]). (C) Histological colitis scores (n = 10 [WT]; n = 8 [TG]). (D) Representative H&E staining of rectal sections. (E) The numbers of F4/80-, Gr-1–, B220-, CD4-, and CD8-positive cells infiltrated in colonic tissues of WT and TG mice, per high-power field (400× magnification) (n = 8 each). (F) The expression levels of TNF-α, IL-1β, IL-6, CXCL1, and MCP-1 protein in colonic tissue lysates from DSS-treated WT and TG mice (n = 15 [WT]; n = 8 [TG]). (G) Survival curves during the course of AOM/DSS treatment in WT and TG mice (n = 18 [WT]; n = 11 [TG]; P < 0.05). (H) The numbers of colonic polyps per mouse, with size distribution (left) and total number (right) in colonic tissues of AOM/DSS-treated WT and TG mice (n = 9 [WT]; n = 10 [TG]). (I) Representative photographs of colons at the end of AOM/DSS treatment. (J) Ki-67–positive cells in rectal polyps of AOM/DSS-treated WT and TG mice (left). TG mice exhibited markedly fewer Ki-67–positive cells than WT mice (n = 5 each) (right). (K) Representative photographs of TUNEL assay performed on rectal polyps of AOM/DSS-treated WT and TG mice. The arrows indicate TUNEL-positive apoptotic cells: the insets show higher magnifications of selected regions (indicated by dashed boxes) (left). TG mice exhibited more TUNEL-positive cells than WT mice (right) (n = 5 each). All values represent means ± SEM. *P < 0.05, **P < 0.01 vs. WT mice. Scale bars: 100 μm.

Mentions: To verify the pivotal role of EPRAP in macrophages, we generated transgenic mice in which the murine CD68 promoter directed murine EPRAP expression (CD68–mEPRAP transgenic mice), leading to overexpression of EPRAP selectively in macrophages (S8 Fig). CD68–mEPRAP transgenic mice were fertile, accumulated body weight normally, and did not develop spontaneous diarrhea. During the course of DSS-induced colitis, WT and CD68–mEPRAP transgenic mice had no significant difference in mortality or body weight loss (S9A and S9B Fig); however, CD68–mEPRAP transgenic mice showed markedly reduced colon shortening (Fig 5A and 5B). Histological examination of the colon obtained from DSS-treated CD68–mEPRAP transgenic mice revealed reduced crypt loss and fewer inflammatory cells than in colons of WT mice (Fig 5C and 5D). Indeed, DSS-treated CD68–mEPRAP transgenic mice, had fewer neutrophils, B cells, CD4+ T cells, and CD8+ T cells as well as macrophages (Figs 5E and S9C), and reduced concentrations of TNF-α, IL-1β, IL-6, CXCL1, or MCP-1 (Fig 5F). As well, after 3 cycles of DSS treatment following intraperitoneal AOM injection, CD68–mEPRAP transgenic mice also exhibited less crypt loss, decreased infiltration of inflammatory cells with lower histological damage scores than WT mice (S10 Fig). In colitis-associated tumorigenesis, in contrast to WT mice, all CD68–mEPRAP transgenic mice survived the AOM/DSS treatment (Fig 5G), and CD68–mEPRAP transgenic mice developed fewer polyps (Fig 5H and 5I). In addition, polyps of CD68–mEPRAP transgenic mice contained significantly fewer Ki-67–positive cells (Fig 5J), but more TUNEL-positive cells, than those of WT mice (Fig 5K). These results indicated that forced expression of EPRAP in macrophages suppressed colitis and colitis-associated tumorigenesis.


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

Nakatsuji M, Minami M, Seno H, Yasui M, Komekado H, Higuchi S, Fujikawa R, Nakanishi Y, Fukuda A, Kawada K, Sakai Y, Kita T, Libby P, Ikeuchi H, Yokode M, Chiba T - PLoS Genet. (2015)

EPRAP overexpression in macrophages ameliorates DSS-induced colitis and colitis-associated tumorigenesis.(A) Representative photographs of colons at the end of DSS treatment; colons were obtained from WT and CD68–mEPRAP transgenic (TG) mice. (B) Percent changes in colon length expressed relative to DSS-free water controls (n = 8 [WT]; n = 6 [TG]). (C) Histological colitis scores (n = 10 [WT]; n = 8 [TG]). (D) Representative H&E staining of rectal sections. (E) The numbers of F4/80-, Gr-1–, B220-, CD4-, and CD8-positive cells infiltrated in colonic tissues of WT and TG mice, per high-power field (400× magnification) (n = 8 each). (F) The expression levels of TNF-α, IL-1β, IL-6, CXCL1, and MCP-1 protein in colonic tissue lysates from DSS-treated WT and TG mice (n = 15 [WT]; n = 8 [TG]). (G) Survival curves during the course of AOM/DSS treatment in WT and TG mice (n = 18 [WT]; n = 11 [TG]; P < 0.05). (H) The numbers of colonic polyps per mouse, with size distribution (left) and total number (right) in colonic tissues of AOM/DSS-treated WT and TG mice (n = 9 [WT]; n = 10 [TG]). (I) Representative photographs of colons at the end of AOM/DSS treatment. (J) Ki-67–positive cells in rectal polyps of AOM/DSS-treated WT and TG mice (left). TG mice exhibited markedly fewer Ki-67–positive cells than WT mice (n = 5 each) (right). (K) Representative photographs of TUNEL assay performed on rectal polyps of AOM/DSS-treated WT and TG mice. The arrows indicate TUNEL-positive apoptotic cells: the insets show higher magnifications of selected regions (indicated by dashed boxes) (left). TG mice exhibited more TUNEL-positive cells than WT mice (right) (n = 5 each). All values represent means ± SEM. *P < 0.05, **P < 0.01 vs. WT mice. Scale bars: 100 μm.
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pgen.1005542.g005: EPRAP overexpression in macrophages ameliorates DSS-induced colitis and colitis-associated tumorigenesis.(A) Representative photographs of colons at the end of DSS treatment; colons were obtained from WT and CD68–mEPRAP transgenic (TG) mice. (B) Percent changes in colon length expressed relative to DSS-free water controls (n = 8 [WT]; n = 6 [TG]). (C) Histological colitis scores (n = 10 [WT]; n = 8 [TG]). (D) Representative H&E staining of rectal sections. (E) The numbers of F4/80-, Gr-1–, B220-, CD4-, and CD8-positive cells infiltrated in colonic tissues of WT and TG mice, per high-power field (400× magnification) (n = 8 each). (F) The expression levels of TNF-α, IL-1β, IL-6, CXCL1, and MCP-1 protein in colonic tissue lysates from DSS-treated WT and TG mice (n = 15 [WT]; n = 8 [TG]). (G) Survival curves during the course of AOM/DSS treatment in WT and TG mice (n = 18 [WT]; n = 11 [TG]; P < 0.05). (H) The numbers of colonic polyps per mouse, with size distribution (left) and total number (right) in colonic tissues of AOM/DSS-treated WT and TG mice (n = 9 [WT]; n = 10 [TG]). (I) Representative photographs of colons at the end of AOM/DSS treatment. (J) Ki-67–positive cells in rectal polyps of AOM/DSS-treated WT and TG mice (left). TG mice exhibited markedly fewer Ki-67–positive cells than WT mice (n = 5 each) (right). (K) Representative photographs of TUNEL assay performed on rectal polyps of AOM/DSS-treated WT and TG mice. The arrows indicate TUNEL-positive apoptotic cells: the insets show higher magnifications of selected regions (indicated by dashed boxes) (left). TG mice exhibited more TUNEL-positive cells than WT mice (right) (n = 5 each). All values represent means ± SEM. *P < 0.05, **P < 0.01 vs. WT mice. Scale bars: 100 μm.
Mentions: To verify the pivotal role of EPRAP in macrophages, we generated transgenic mice in which the murine CD68 promoter directed murine EPRAP expression (CD68–mEPRAP transgenic mice), leading to overexpression of EPRAP selectively in macrophages (S8 Fig). CD68–mEPRAP transgenic mice were fertile, accumulated body weight normally, and did not develop spontaneous diarrhea. During the course of DSS-induced colitis, WT and CD68–mEPRAP transgenic mice had no significant difference in mortality or body weight loss (S9A and S9B Fig); however, CD68–mEPRAP transgenic mice showed markedly reduced colon shortening (Fig 5A and 5B). Histological examination of the colon obtained from DSS-treated CD68–mEPRAP transgenic mice revealed reduced crypt loss and fewer inflammatory cells than in colons of WT mice (Fig 5C and 5D). Indeed, DSS-treated CD68–mEPRAP transgenic mice, had fewer neutrophils, B cells, CD4+ T cells, and CD8+ T cells as well as macrophages (Figs 5E and S9C), and reduced concentrations of TNF-α, IL-1β, IL-6, CXCL1, or MCP-1 (Fig 5F). As well, after 3 cycles of DSS treatment following intraperitoneal AOM injection, CD68–mEPRAP transgenic mice also exhibited less crypt loss, decreased infiltration of inflammatory cells with lower histological damage scores than WT mice (S10 Fig). In colitis-associated tumorigenesis, in contrast to WT mice, all CD68–mEPRAP transgenic mice survived the AOM/DSS treatment (Fig 5G), and CD68–mEPRAP transgenic mice developed fewer polyps (Fig 5H and 5I). In addition, polyps of CD68–mEPRAP transgenic mice contained significantly fewer Ki-67–positive cells (Fig 5J), but more TUNEL-positive cells, than those of WT mice (Fig 5K). These results indicated that forced expression of EPRAP in macrophages suppressed colitis and colitis-associated tumorigenesis.

Bottom Line: Prostaglandin E2 plays important roles in the maintenance of colonic homeostasis.To investigate the in vivo roles of EPRAP, we examined the effects of EPRAP on colitis and colitis-associated tumorigenesis.Thus, EPRAP may be a potential therapeutic target for inflammatory bowel disease and associated intestinal tumorigenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastroenterology and Hepatology, Kyoto University Graduate School of Medicine, Kyoto, Japan.

ABSTRACT
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 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.

No MeSH data available.


Related in: MedlinePlus