EPRAP inhibits p105 phosphorylation and MEK–ERK activation in stromal macrophages with DSS treatment.

<p><b>(A)</b> The expression levels of Akt1, and phosphorylated forms of either Akt1, MEK1, p38MAPK, Stat3 or NF-κB p65 in colonic tissue extracts from DSS-treated KO mice compared to WT mice (n = 9 [WT]; n = 7 [KO]). <b>(B)</b> Double-color immunofluorescence analyses of rectal sections from DSS-treated WT, EPRAP-deficient (KO), and CD68–mEPRAP transgenic (TG) mice. Samples were immunostained with anti-F4/80 (green) and anti–phospho-p105 (red, top), anti–phospho-MEK (red, middle), or anti–phospho-ERK (red, bottom) antibodies. Scale bars: 20 μm. <b>(C)</b> The percentages of stained cells in F4/80 positive cells. (n = 5 each). <b>(D)</b> Lamina propria macrophages were isolated from the colonic tissues of WT, KO, and TG mice with DSS treatment. Cells were analyzed by flow cytometry, with detection of phospho-p105 (top), phospho-MEK (middle), or phospho-ERK (bottom) (open histograms). Cells were also stained with isotype control antibodies (solid histograms). Figures are representative histograms performed in quintuplicate (left). The levels of phosphorylated forms of p105 (top), MEK (middle), and ERK (bottom) in viable cells from WT, KO, and TG mice were determined as the geometric mean fluorescence intensity (MFI) of the target antibody minus the MFI of the isotype control (right) (n = 5 each). All values represent means ± SEM. **<i>P</i> < 0.01 vs. WT mice.</p

The colons of ulcerative colitis (UC) showed accumulation of EPRAP-positive macrophages.

<p><b>(A)</b> Immunohistochemical staining for EPRAP in the sections of normal human and UC colons: the insets show higher magnifications of selected regions (indicated by dash boxes). Scale bars: 100 μm. <b>(B)</b> Double-color immunofluorescence analyses using sections of normal and UC colons were performed with anti-CD68 (green) and anti–EPRAP (red) antibodies (left). Scale bars: 20 μm. The percentage of EPRAP-positive cells in CD68-positive cells (right) (n = 20 [normal]; n = 20 [UC]). **<i>P</i> < 0.01 vs. normal colons. <b>(C)</b> Relationship between the percentage of EPRAP positive cells in CD68-positive macrophages and the clinical severity of UC patients (mild to moderate vs. severe) (n = 22 [mild to moderate]; n = 16 [severe]). *<i>P</i> < 0.05.</p

EPRAP in bone marrow–derived cells attenuates colitis and colitis-induced tumorigenesis.

<p>Four kinds of chimeric mice were generated by adoptive bone marrow transplantation: <b>(A)</b> Representative photographs of colons with adoptive bone marrow transplantation. <b>(B)</b> Percent changes in colon length expressed relative to DSS-free water controls in each group. (n = 4–8 each). <b>(C)</b> Histological colitis scores (n = 4–8 each). <b>(D)</b> Representative H&E staining of rectal sections. Scale bars: 100 μm. <b>(E)</b> Representative photographs of colons at the end of AOM/DSS treatment. <b>(F)</b> The number of colonic tumors per mouse, with the size distribution (left) and the total number (right) in each group (n = 4–9 each). <b>(G)</b> EPRAP did not directly affect cell proliferation. DLD-1 cell proliferation was determined by MTS assay (over expression of EPRAP, left; knockdown of EPRAP, right). Data represent fold induction of absorbance compared with mock or negative control. All values represent means ± SEM. *<i>P</i> < 0.05, **<i>P</i> < 0.01.</p

EPRAP deficiency impairs the anti-inflammatory effect of PGE/EP4.

<p><b>(A)</b> Representative photographs of colons; “Veh” indicates vehicle and “EP4” indicates EP4 agonist. <b>(B)</b> Percent changes in colon length expressed relative to DSS-free water controls (n = 5–8 each; **P < 0.01 vs. WT + Veh). <b>(C)</b> Histological colitis scores (n = 5–8 each; *<i>P</i> < 0.05 vs. WT + Veh). <b>(D)</b> Representative H&E staining. Scale bars: 100 μm. <b>(E, F)</b> Peritoneal macrophages <b>(E)</b> and colonic epithelial cells <b>(F)</b> isolated from WT and KO mice were incubated with EP4 agonist. Intracellular cAMP levels were measured as described in Supplemental Methods (*<i>P</i> < 0.05 and **<i>P</i> < 0.01 vs. 0 μM). Figures are representative of five independent experiments performed in triplicate. <b>(G, H)</b> EP4 mRNA levels in peritoneal macrophages <b>(G)</b> and colonic epithelial cells <b>(H)</b> from WT and KO mice (n = 6 each). Data represent fold induction of mRNA expression compared with WT. All values represent means ± SEM.</p

EPRAP overexpression in macrophages ameliorates DSS-induced colitis and colitis-associated tumorigenesis.

<p><b>(A)</b> Representative photographs of colons at the end of DSS treatment; colons were obtained from WT and CD68–mEPRAP transgenic (TG) mice. <b>(B)</b> Percent changes in colon length expressed relative to DSS-free water controls (n = 8 [WT]; n = 6 [TG]). <b>(C)</b> Histological colitis scores (n = 10 [WT]; n = 8 [TG]). <b>(D)</b> Representative H&E staining of rectal sections. <b>(E)</b> 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). <b>(F)</b> 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]). <b>(G)</b> Survival curves during the course of AOM/DSS treatment in WT and TG mice (n = 18 [WT]; n = 11 [TG]; <i>P</i> < 0.05). <b>(H)</b> 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]). <b>(I)</b> Representative photographs of colons at the end of AOM/DSS treatment. <b>(J)</b> 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). <b>(K)</b> 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. *<i>P</i> < 0.05, **<i>P</i> < 0.01 vs. WT mice. Scale bars: 100 μm.</p