1. Volume 142, Issue 5, Pages 1183-1194.e4 (May 2012)
A Variant of Smurf2 Protects Mice Against Colitis-Associated Colon Cancer by Inducing Transforming Growth Factor β Signaling 
Heike Dornhoff, Christoph Becker, Stefan Wirtz, Dennis Strand, Stefan Tenzer, Susanne Rosfa, Clemens Neufert, Jonas Mudter, Jürgen Markl, Jürgen Siebler, Markus F. Neurath 
Gastroenterology 
Volume 142, Issue 5, Pages e4 (May 2012)
DOI: /j.gastro
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2. Figure 1
Smurf2 expression is dependent on TGF-β and up-regulated in tumor-infiltrating T cells in a murine model of colitis-associated colon cancer. (A) Real-time PCR analysis of Smurf2 mRNA expression in isolated tumors and control tissue without tumors from AOM/DSS-treated WT mice (*P < .05). (B) Assessment of Smurf2 mRNA expression in indicated cell populations from the colon of AOM/DSS-treated WT mice. CD4+ T cells, B220+ B cells, intestinal epithelial cells (IEC), CD8+ T cells, and CD11c+ dendritic cells. One representative experiment out of 4 independent experiments is shown. (C) Analysis of Smurf2 mRNA expression in CD4+ T cells on stimulation with TGF-β (*P < .05). (D) PCR analysis of Smurf2 mRNA expression in tumor-infiltrating CD4+ T cells in the AOM/DSS model. Sequence analysis of WTSmurf2 and ΔE2Smurf2 is shown. (E) (Upper panels) Analysis of WTSmurf2 and ΔE2Smurf2 mRNA expression in CD4+ T cells on stimulation with TGF-β. Data are expressed as mean values ± SD of 3 independent experiments (*P < .05). (Lower panels) Real-time PCR analysis of WTSmurf2 and ΔE2Smurf2 mRNA expression in AOM/DSS colitis and colitis-associated colon tumors (**P < .001, ***P < .05).
Gastroenterology  , e4DOI: ( /j.gastro )
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3. Figure 2
Characterization of the novel Smurf2 variant and structural changes in its C2 domain. (A) Western blot analysis of WTSmurf2 and ΔE2Smurf2 expressing GFP in Cos7 cells. (B) Schematic diagram of Smurf1 and WTSmurf2 and ΔE2Smurf2. (C) WTSmurf2 and ΔE2Smurf2 RNA expression in human CD4+ T cells. Sequence analysis of the DNA sequences of the ΔE2Smurf2 variants in mice and humans. Real-time PCR of both isoforms of Smurf2 was performed in samples from control patients and patients with IBD. WT Smurf expression levels were significantly different between both groups (*P < .05). Although there was a more than 2-fold induction of ΔE2Smurf2 expression in patients with IBD compared with controls, this effect did not reach statistical significance due to the markedly higher variability of ΔE2Smurf2 expression between individual patients with IBD. The reasons for this high variability between individual patients with IBD are currently unknown but might relate to the duration of the disease, the disease phenotype, or drug therapy. In any case, a significant decrease in the ratio between WTSmurf2 and ΔE2Smurf2 was noted in patients with IBD compared with controls. (D) NMR solution structure of the membrane-binding C2 domain of human WTSmurf2 (pdb ID 2JQZ) for hypothetical structure analysis. The 13 amino acids deleted in ΔE2Smurf2 and corresponding to exon 2 are shown in red. (E) Homology model of ΔE2Smurf2. The regions encoded by the spliced exons 1 and 3 are highlighted in gray and yellow.
Gastroenterology  , e4DOI: ( /j.gastro )
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4. Figure 3
ΔE2Smurf2 is localized in lysosomal compartments and up-regulates Smad signaling. (A) (Left panel) Cellular localization of WTSmurf2 and ΔE2Smurf2 in Cos7 cells expressing both GFP-tagged isoforms of Smurf2. Untransfected cells are shown as controls. (Right panel) Western blot analysis of both GFP-tagged Smurf2 proteins from Cos7 cells in the lipid fraction of the cell membrane. (B) Colocalization of ΔE2Smurf2 with markers for endosomes (EEA1), lysosomes, and TGFβRII by confocal imaging. (C) Analysis of the Smad signaling pathway in Cos7 cells on transfection of expression vectors for WTSmurf2 and ΔE2Smurf2. (D) Smad3 reporter assay was performed. Luciferase activity of the Smad3 promoter was measured in a luminometer. Data are representative of 3 independent experiments. Mean values ± SD are shown (*P < .05, **P < .005). (E) Western blot analysis of TGFβRII expression in transfected Cos7 cells. (F) FACS analysis of primary splenic CD4+CD25− T cells transfected with Smurf2 expression constructs for CD4-FITC and TGFβRII-PE.
Gastroenterology  , e4DOI: ( /j.gastro )
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5. Figure 4
The ΔE2Smurf2 variant controls Smurf2 expression via ubiquitination and Smad7. (A) TGF-β–dependent degradation of WTSmurf2-GFP by ΔE2Smurf2. Western blot analysis of WTSmurf2-GFP levels is dependent on ΔE2Smurf2 expression levels. (B) The degradation of Smurf2 is dependent on Smad7. Western blot analysis revealed degradation of the GFP-tagged WTSmurf2 and ΔE2Smurf2 proteins after expression of corresponding isoforms. This effect was markedly augmented by cotransfection of a Smad7 expression construct. (C) Coimmunoprecipitation of WTSmurf2 and ΔE2Smurf2. Cells were transfected with GFP-tagged ΔE2Smurf2 in the presence (right lane) or absence (left lane) of the WTSmurf2-Flag vector. Immunoprecipitation of GFP-tagged ΔE2Smurf2 is shown in the lower panel, and Western blot analysis for coimmunoprecipitated Flag-tagged WTSmurf2 is shown in the upper panel. (D) Coimmunoprecipitation in EL-4 cells. The ΔE2Smurf2 variant induces Smad7-dependent monoubiquitination of WTSmurf2. In the absence of Smad7, polyubiquitination was noted.
Gastroenterology  , e4DOI: ( /j.gastro )
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6. Figure 5
Generation of a T cell–specific ΔE2Smurf2 transgenic mouse. (A) Schematic diagram showing the transgenic ΔE2Smurf2 mouse construct. (B) T cell–specific overexpression of ΔE2Smurf2 mRNA in transgenic mice. In contrast to B220+ splenic B cells and CD11c+ antigen-presenting cells, splenic and thymic CD4+ and CD8+ T cells as well as intraepithelial lymphocytes (IEL) expressed ΔE2Smurf2 transgenic mRNA. (C) Western blot of total Smurf2 levels in splenic CD4+ T cells from WT and transgenic (TG) mice. (D) Analysis of WTSmurf2 and ΔE2Smurf2 mRNA expression in CD4+ T cells from WT and transgenic mice. Data are expressed as the mean ± SD of 3 independent experiments (*P < .05, **P < .01). (E) Western blot analyses of total Smad2/3, pSmad2/3, and TGFβRII levels in WT and transgenic mice. (F) Carboxyfluorescein succinimidyl ester proliferation assay of CD4+CD25− T cells from WT and transgenic mice.
Gastroenterology  , e4DOI: ( /j.gastro )
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7. Figure 6
ΔE2Smurf2 protects mice from tumor development in the AOM/DSS model of colitis-associated colon cancer. (A) Analysis of ΔE2Smurf2 transgenic and WT control mice in the AOM/DSS model of colitis-associated colon cancer. Representative endoscopic images at day 63 in both groups are shown. Data are representative of 5 independent experiments with 5 animals per group (*P < .05). (B) Colonic cryosections from AOM/DSS-treated WT and transgenic mice and immunohistochemistry for CD4, CD11c, myeloperoxidase, TGFβRII, and IL-6. (C) Proinflammatory cytokine expression pattern of AOM/DSS-treated WT and TG mice (*P < .05). Cells were stimulated as indicated.
Gastroenterology  , e4DOI: ( /j.gastro )
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8. Figure 7
Model of ΔE2Smurf2 function in TGF-β signaling. Proposed mechanism of action of the ΔE2Smurf2 variant in TGF-β signaling. (A) TGF-β induces activation of Smad2/Smad3 on binding to the TGF-β receptor. (B) WTSmurf2 induces degradation of the TGFβRII through interaction with Smad7 via ubiquitination and proteasomal degradation. Thus, WTSmurf2 leads to inhibition of TGF-β signaling. (C) The novel ΔE2Smurf2 splice variant can bind to the Smad7/WTSmurf2 complex, resulting in lysosomal degradation of the complex. By using this mechanism, ΔE2Smurf2 blocks the inhibitory Smad–dependent degradation of TGFβRII, increases TGFβRII levels, and augments TGF-β–mediated signaling in T lymphocytes.
Gastroenterology  , e4DOI: ( /j.gastro )
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9. Supplementary Figure 1
Schematic diagram of the AOM/DSS model. Mini-colonoscopy and chromoendoscopy at indicated time points are shown: Endoscopic analysis (E).
Gastroenterology  , e4DOI: ( /j.gastro )
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10. Supplementary Figure 2
(A) Left panel: EL-4 cells were transfected with WTSmurf2 and ΔE2Smurf2 expression constructs followed by FACS analysis for TGFβRII. Mean values +/− SEM are reported (*p < 0,05). Right panel: A representative FACS staining is shown. (B) Left panel: FACS analysis of transfected EL-4 cells. Cells were transfected with WTSmurf2-Flag and increasing amounts of ΔE2Smurf2 constructs followed by Flag analysis (*p < 0,05). (C) Confocal microscopy of transfected EL-4 cells with ΔE2Smurf2 (right panel) and costaining with Lysotracker (left panel).
Gastroenterology  , e4DOI: ( /j.gastro )
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11. Supplementary Figure 3
(A) Co-immunoprecipitation in Cos7 cells: the ΔE2Smurf2 variant induces Smad7-dependent mono-ubiquitination of wild-type Smurf2. (B) Analysis of the Smad signalling pathway in EL-4 cells upon transfection of expression vectors for wild-type Smurf2 and ΔE2Smurf2. (C) 2D gel electrophoresis and analysis of Smurf2 expression in splenic CD4+ T cells from WT and transgenic mice. While the left spot represents WTSmurf2, the right spot represents ΔE2Smurf2 protein. pH values for WTSmurf2 and ΔE2Smurf2 were 8.18 and 7.6, respectively.
Gastroenterology  , e4DOI: ( /j.gastro )
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