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. 2012 Jul 20;287(30):25631-9.
doi: 10.1074/jbc.M112.364786. Epub 2012 May 2.

Loss of TMF/ARA160 protein renders colonic mucus refractory to bacterial colonization and diminishes intestinal susceptibility to acute colitis

Shai Bel  1 , Yoav Elkis, Tali Lerer-Goldstein, Abraham Nyska, Sally Shpungin, Uri Nir
Affiliations

Loss of TMF/ARA160 protein renders colonic mucus refractory to bacterial colonization and diminishes intestinal susceptibility to acute colitis

Shai Bel et al. J Biol Chem. .

Abstract

TMF/ARA160 is a Golgi-associated protein with several cellular functions, among them direction of the NF-κB subunit, p65 RelA, to ubiquitination and proteasomal degradation in stressed cells. We sought to investigate the role of TMF/ARA160 under imposed stress conditions in vivo. TMF(-/-) and wild-type (WT) mice were treated with the ulcerative agent dextran sulfate sodium (DSS), and the severity of the inflicted acute colitis was determined. TMF(-/-) mice were found to be significantly less susceptible to DSS-induced colitis, with profoundly less bacterial penetration into the colonic epithelia. Surprisingly, unlike in WT mice, no bacterial colonies were visualized in colons of healthy untreated TMF(-/-) mice, indicating the constitutive resistance of TMF(-/-) colonic mucus to bacterial retention and penetration. Gene expression analysis of colon tissues from unchallenged TMF(-/-) mice revealed 5-fold elevated transcription of the muc2 gene, which encodes the major component of the colonic mucus gel, the MUC2 mucin. Accordingly, the morphology of the colonic mucus in TMF(-/-) mice was found to differ from the mucus structure in WT colons. The NF-κB subunit, p65, a well known transcription inducer of muc2, was up-regulated significantly in TMF(-/-) intestinal epithelial cells. However, this did not cause spontaneous inflammation or increased colonic crypt cell proliferation. Collectively, our findings demonstrate that absence of TMF/ARA160 renders the colonic mucus refractory to bacterial colonization and the large intestine less susceptible to the onset of colitis.

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Figures

FIGURE 1.
FIGURE 1.
TMF/ARA160 is highly expressed in colonic epithelial columnar cells which face the intestinal lumen. Shown is an immunofluorescence staining of colon sections from healthy WT mice visualized using fluorescent (A, B, and D) and confocal (C) microscopy. Anti-TMF antibody (red), Golgi marker (green), and Hoechst staining (blue) for nuclear visualization show apical localization of TMF/ARA160 exclusively in IECs, including goblet cells (*). White arrows in C indicate TMF expression in lamina propria cells (scale bars are indicated in each image).
FIGURE 2.
FIGURE 2.
Reduced histopathological and clinical parameters severity of DSS-induced colitis in TMF−/− (KO) mice. Colon morphology of untreated WT (A) and TMF−/− (B) mice is shown. Degree of ulceration and loss of crypt architecture in DSS-treated WT (C) and TMF−/− (D) colons are shown. Images are representative examples of the different groups (n = 7 per group). Scale bars, 100 μm. Weight loss was presented as mean percent of initial weight ± S.E. (E). Histopathological semi-quantitative scores were presented as the mean score (obtained as described under “Materials and Methods”) ± S.E. (F). Presence of blood in excreta was shown as the mean (determined as described under “Materials and Methods”) ± S.E. (G). Myeloperoxidase activity was shown as percent change of activity compared with control untreated groups (n = 7 per group), reflecting neutrophil infiltrate (H). Length of colons from WT DSS-treated (left) and KO DSS-treated (right) mice are shown (I). Colon shortening is represented as mean percent change in colon length of DSS-treated mice compared with untreated mice (J) ± S.E. (n = 7 per group). *, p < 0.05; **, p < 0.07; ***, p < 0.005.
FIGURE 3.
FIGURE 3.
Reduced expression of cytokines and proinflammatory mediators in control and DSS-treated TMF−/− (KO) mice. Shown is a real-time qRT-PCR analysis of mRNAs encoding cytokines and proinflammatory mediators in untreated (control) and DSS-treated (DSS) WT and KO mice. Obtained levels were normalized to GAPDH mRNA and are presented as mean percent change in mRNA levels as compared with WT control mice ± S.E. (n = 5 per group). *, p < 0.05; **, p < 0.01.
FIGURE 4.
FIGURE 4.
Reduced bacterial infiltration in colonic tissue of DSS-treated TMF−/− (KO) mice. Fluorescent in situ hybridization with a specific probe directed toward the 16 S bacterial RNA was used to visualize bacterial penetration into colonic tissue from DSS-treated WT (A) and KO (B) mice (n = 5 per group). Bacteria (green) are indicated by arrows shown against nuclear (blue) counter stain (scale bars, 200 μm). Shown is a semi-quantitative PCR analysis of the bacterial 16 S rRNA gene using 100 ng of DNA isolated from DSS-treated WT and KO colons (C), and a density plot histogram presentation (D) of the gel in C ± S.E. PCR amplification of GAPDH is shown to ensure equal loading (n = 5 per group). *, p < 0.005.
FIGURE 5.
FIGURE 5.
Bacterial colonies are not detected in colonic lumen and mucosa of untreated TMF−/− (KO) mice. Colons of untreated WT (A and C) and KO (B and D) mice were fixed with Carnoy's fixative and stained with Giemsa. Bacterial colonies in WT colons are indicated by arrows. No bacterial colonies are visible in colons from untreated KO mice. Images are representative examples of the different groups (n = 5 per group). Scale bars, 20 μm. Shown is a semi-quantitative PCR analysis of the bacterial 16 S rRNA gene using 100 ng DNA isolated from untreated colons (E), and density plot histogram presentation (F) of the gel in E ± S.E. PCR amplification of GAPDH is shown to ensure equal loading (n = 5 per group). *, p < 0.0005.
FIGURE 6.
FIGURE 6.
Increased muc2 mRNA levels in colons of TMF−/− (KO) mice. Shown is a real-time qRT-PCR analysis of muc2 mRNA encoding the MUC2 mucin from untreated (control) and DSS-treated (DSS) WT and KO mice (A). Levels of tff3 mRNA encoding the intestinal epithelial repair factor TFF3 (B), of the mbd3 mRNA encoding the antimicrobial protein mBD3 (C), and of muc5ac mRNA encoding the MUC5AC mucin (D) were determined in colons of untreated WT and KO mice. Expression levels were normalized to GAPDH and are presented as mean change in mRNA levels as compared with WT control mice ± S.E. (n = 5 per group). *. p < 0.05.
FIGURE 7.
FIGURE 7.
Distinct mucus morphology in colons of untreated TMF−/− (KO) mice. Shown is an immunofluorescent staining of MUC2 in colon sections from untreated WT (A and C) and KO (B and D) mice (n = 5 per group). Anti-MUC2 antibody (green), combined with Hoechst staining (blue) for nuclear visualization was used to visualize the morphology of the narrow firm (filled arrowhead) and wide loose (empty arrowhead) mucus layers in WT mice in contrast to the uniform thick dense mucus layer in KO mice. Scale bars, 50 μm.
FIGURE 8.
FIGURE 8.
Up-regulation of the NF-κB subunit p65-RelA in untreated colonic epithelium of TMF−/− (KO) mice. Shown is an immunohistochemical staining of NF-κB p65 in colon sections from untreated WT (A) and KO (B) mice. PCNA-positive cells indicate the proliferation levels of IEC in colons of untreated WT (C) and KO (D) mice. Images are representative of the different groups (n = 6 per group). Scale bars, 200 μm. Nuclear extracts from colons of untreated WT and KO mice were analyzed for NF-κB p65 transcriptional activity via DNA binding by ELISA (E). Results are represented as percent change from WT mean level ± S.E. (n = 5). A real-time qRT-PCR analysis of bxl-xl mRNA encoding the Bcl-xL anti-apoptotic protein is shown (F). Expression levels were normalized to GAPDH and are presented as mean change in mRNA levels as compared with WT control mice ± S.E. (n = 5 per group). *, p < 0.0001; **, p < 0.001; ***, p < 0.05.
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