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Epithelial Sel1L is required for the maintenance of intestinal homeostasis.

ABSTRACT: Inflammatory bowel disease (IBD) is an incurable chronic idiopathic disease that drastically decreases quality of life. Endoplasmic reticulum (ER)-associated degradation (ERAD) is responsible for the clearance of misfolded proteins; however, its role in disease pathogenesis remains largely unexplored. Here we show that the expression of SEL1L and HRD1, the most conserved branch of mammalian ERAD, is significantly reduced in ileal Crohn's disease (CD). Consistent with this observation, laboratory mice with enterocyte-specific Sel1L deficiency (Sel1L(?IEC)) develop spontaneous enteritis and have increased susceptibility to Toxoplasma gondii-induced ileitis. This is associated with profound defects in Paneth cells and a disproportionate increase of Ruminococcus gnavus, a mucolytic bacterium with known association with CD. Surprisingly, whereas both ER stress sensor IRE1? and effector CHOP are activated in the small intestine of Sel1L(?IEC) mice, they are not solely responsible for ERAD deficiency-associated lesions seen in the small intestine. Thus our study points to a constitutive role of Sel1L-Hrd1 ERAD in epithelial cell biology and the pathogenesis of intestinal inflammation in CD.

SUBMITTER: Sun S

PROVIDER: S-EPMC4751599 | biostudies-literature | 2016 Feb

REPOSITORIES: biostudies-literature

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Epithelial Sel1L is required for the maintenance of intestinal homeostasis.

Sun Shengyi S Lourie Rohan R Cohen Sara B SB Ji Yewei Y Goodrich Julia K JK Poole Angela C AC Ley Ruth E RE Denkers Eric Y EY McGuckin Michael A MA Long Qiaoming Q Duhamel Gerald E GE Simpson Kenneth W KW Qi Ling L

Molecular biology of the cell 20151202 3

Inflammatory bowel disease (IBD) is an incurable chronic idiopathic disease that drastically decreases quality of life. Endoplasmic reticulum (ER)-associated degradation (ERAD) is responsible for the clearance of misfolded proteins; however, its role in disease pathogenesis remains largely unexplored. Here we show that the expression of SEL1L and HRD1, the most conserved branch of mammalian ERAD, is significantly reduced in ileal Crohn's disease (CD). Consistent with this observation, laboratory ...[more]

PMID: 26631554

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Project description:Background & aimsIntestinal epithelial homeostasis depends on a tightly regulated balance between intestinal epithelial cell (IEC) death and proliferation. While the disruption of several IEC death regulating factors result in intestinal inflammation, the loss of the anti-apoptotic BCL2 family members BCL2 and BCL2L1 has no effect on intestinal homeostasis in mice. We investigated the functions of the antiapoptotic protein MCL1, another member of the BCL2 family, in intestinal homeostasis in mice.MethodsWe generated mice with IEC-specific disruption of Mcl1 (Mcl1ΔIEC mice) or tamoxifen-inducible IEC-specific disruption of Mcl1 (i-Mcl1ΔIEC mice); these mice and mice with full-length Mcl1 (controls) were raised under normal or germ-free conditions. Mice were analyzed by endoscopy and for intestinal epithelial barrier permeability. Intestinal tissues were analyzed by histology, in situ hybridization, proliferation assays, and immunoblots. Levels of calprotectin, a marker of intestinal inflammation, were measured in intestinal tissues and feces.ResultsMcl1ΔIEC mice spontaneously developed apoptotic enterocolopathy, characterized by increased IEC apoptosis, hyperproliferative crypts, epithelial barrier dysfunction, and chronic inflammation. Loss of MCL1 retained intestinal crypts in a hyperproliferated state and prevented the differentiation of intestinal stem cells. Proliferation of intestinal stem cells in MCL1-deficient mice required WNT signaling and was associated with DNA damage accumulation. By 1 year of age, Mcl1ΔIEC mice developed intestinal tumors with morphologic and genetic features of human adenomas and carcinomas. Germ-free housing of Mcl1ΔIEC mice reduced markers of microbiota-induced intestinal inflammation but not tumor development.ConclusionThe antiapoptotic protein MCL1, a member of the BCL2 family, is required for maintenance of intestinal homeostasis and prevention of carcinogenesis in mice. Loss of MCL1 results in development of intestinal carcinomas, even under germ-free conditions, and therefore does not involve microbe-induced chronic inflammation. Mcl1ΔIEC mice might be used to study apoptotic enterocolopathy and inflammatory bowel diseases.

Project description:ObjectiveRecent studies using whole-body clock-disrupted animals identified a disruption in the circadian rhythm of the intestinal L-cell incretin hormone, glucagon-like peptide-1 (GLP-1). Although GLP-1 plays an essential role in metabolism through enhancement of both glucose-stimulated insulin secretion and satiety, recent evidence has also demonstrated its importance in regulating intestinal and microbial homeostasis. Therefore, using in vivo and in vitro models, this study assessed the role of the core circadian clock gene Arntl in the regulation of time-dependent GLP-1 secretion and its impact on the intestinal environment.MethodsOral glucose tolerance tests were conducted at zeitgeber time 2 and 14 in control and inducible Gcg-Arntl knockout (KO) mice. Colonic intraepithelial lymphocytes were isolated, mucosal gene expression analysis was conducted, and 16S rRNA gene sequencing of colonic feces as well as analysis of microbial metabolites were performed. Time-dependent GLP-1 secretion and transcriptomic analysis were conducted in murine (m) GLUTag L-cells following siRNA-mediated knockdown of Arntl.ResultsGcg-Arntl KO mice displayed disrupted rhythmic release of GLP-1 associated with reduced secretion at the established peak time point. Analysis of the intestinal environment in KO mice revealed a decreased proportion of CD4+ intraepithelial lymphocytes in association with increased proinflammatory cytokine gene expression and increased colonic weight. Moreover, increased Actinobacteria within the colonic microbiome was found following L-cell Arntl disruption, as well as reductions in the microbial products, short chain fatty acids, and bile acids. Finally, siRNA-mediated knockdown of Arntl in mGLUTag L-cells resulted in both impaired time-dependent GLP-1 secretion and the disruption of pathways related to key cellular processes.ConclusionsThese data establish, for the first time, the essential role of Arntl in the intestinal L-cell in regulating time-dependent GLP-1 secretion. Furthermore, this study revealed the integral role of L-cell Arntl in mediating the intestinal environment, which ultimately may provide novel insight into the development of therapeutics for the treatment of intestinal and metabolic disorders.

Project description:Although genome-wide association studies have identified the gene RNF186 encoding an E3 ubiquitin-protein ligase as conferring susceptibility to ulcerative colitis, the exact function of this protein remains unclear. In the present study, we demonstrate an important role for RNF186 in macroautophagy/autophagy activation in colonic epithelial cells and intestinal homeostasis. Mechanistically, RNF186 acts as an E3 ubiquitin-protein ligase for EPHB2 and regulates the ubiquitination of EPHB2. Upon stimulation by ligand EFNB1 (ephrin B1), EPHB2 is ubiquitinated by RNF186 at Lys892, and further recruits MAP1LC3B for autophagy. Compared to control mice, rnf186-/- and ephb2-/- mice have a more severe phenotype in the DSS-induced colitis model, which is due to a defect in autophagy in colon epithelial cells. More importantly, treatment with ephrin-B1-Fc recombinant protein effectively relieves DSS-induced mouse colitis, which suggests that ephrin-B1-Fc may be a potential therapy for human inflammatory bowel diseases.Abbreviations: ACTB: actin beta; ATG5: autophagy related 5; ATG16L1: autophagy related 16 like 1; ATP: adenosine triphosphate; Cas9: CRISPR associated protein 9; CD: Crohn disease; CQ: chloroquine; Csf2: colony stimulating factor 2; Cxcl1: c-x-c motif chemokine ligand 1; DMSO: dimethyl sulfoxide; DSS: dextran sodium sulfate; EFNB1: ephrin B1; EPHB2: EPH receptor B2; EPHB3: EPH receptor B3; EPHB2K788R: lysine 788 mutated to arginine in EPHB2; EPHB2K892R: lysine 892 mutated to arginine in EPHB2; ER: endoplasmic reticulum; FITC: fluorescein isothiocyanate; GFP: green fluorescent protein; GWAS: genome-wide association studies; HRP: horseradish peroxidase; HSPA5/BiP: heat shock protein family A (Hsp70) member 5; IBD: inflammatory bowel diseases; Il1b: interleukin 1 beta; Il6: interleukin 6; IRGM:immunity related GTPase M; i.p.: intraperitoneally; IPP: inorganic pyrophosphatase; KD: knockdown; KO: knockout; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MTOR: mechanistic target of rapamycin kinase; NOD2: nucleotide binding oligomerization domain containing 2; PI3K: phosphoinositide 3-kinase; PtdIns3K: class III phosphatidylinositol 3-kinase; RNF186: ring finger protein 186; RNF186A64T: alanine 64 mutated to threonine in RNF186; RNF186R179X: arginine 179 mutated to X in RNF186; RPS6: ribosomal protein S6; Tnf: tumor necrosis factor; SQSTM1: sequestosome 1; Ub: ubiquitin; UBE2D2: ubiquitin conjugating enzyme E2 D2; UBE2H: ubiquitin conjugating enzyme E2 H; UBE2K: ubiquitin conjugating enzyme E2 K; UBE2N: ubiquitin conjugating enzyme E2 N; UC: ulcerative colitis; ULK1:unc-51 like autophagy activating kinase 1; WT: wild type.

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Epithelial Sel1L is required for the maintenance of intestinal homeostasis.

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Epithelial Sel1L is required for the maintenance of intestinal homeostasis.

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