Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

The protein disulfide isomerase AGR2 is essential for the production of intestinal mucus

ABSTRACT: Protein disulfide isomerases (PDIs) aid protein folding and assembly by catalyzing formation and shuffling of cysteine disulfide bonds in the endoplasmic reticulum (ER). Many members of the PDI family are expressed in mammals but the roles of specific PDIs in vivo are poorly understood. A recent homology-based search for additional PDI family members identified anterior gradient homolog 2 (AGR2), a protein originally presumed to be secreted by intestinal epithelial cells, but the function of AGR2 has been obscure. Here we show that AGR2 is expressed in the ER of secretory cells and is essential for in vivo production of intestinal mucin, a large cysteine-rich glycoprotein that forms the protective mucus gel lining the intestine. A cysteine residue within the AGR2 thioredoxin-like domain forms mixed disulfide bonds with MUC2, consistent with a direct role for AGR2 in mucin processing. Despite a complete absence of intestinal mucin, mice lacking AGR2 appeared healthy but were highly susceptible to dextran sodium sulfate-induced experimental colitis, indicating a critical role for AGR2 in protection from environmental insults. We conclude that AGR2 is a unique member of the PDI family that has a specialized and non-redundant role in intestinal mucus production. Keywords: small intestine and colon gene expression profiles for Agr2-/- and littermate control mice DNA miocroarrays were used to analyze small intenstine and colon mRNA expression of AGR2 KO and littermate control mice. The experiment incorporated a 1 color design and used Agilent arrays that contained roughly 44,00 60mer probes that provide complete coverage of the mouse genome. 12 arrays were hybridized and represent 8 small intestine samples ( 4 each KO and WT) and 4 colon samples (2 each KO and WT)

ORGANISM(S): Mus musculus

SUBMITTER: Sung-Woo Park

PROVIDER: E-GEOD-12503 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

ACCESS DATA

Publications

The protein disulfide isomerase AGR2 is essential for production of intestinal mucus.

Park Sung-Woo SW Zhen Guohua G Verhaeghe Catherine C Nakagami Yasuhiro Y Nguyenvu Louis T LT Barczak Andrea J AJ Killeen Nigel N Erle David J DJ

Proceedings of the National Academy of Sciences of the United States of America 20090409 17

Protein disulfide isomerases (PDIs) aid protein folding and assembly by catalyzing formation and shuffling of cysteine disulfide bonds in the endoplasmic reticulum (ER). Many members of the PDI family are expressed in mammals, but the roles of specific PDIs in vivo are poorly understood. A recent homology-based search for additional PDI family members identified anterior gradient homolog 2 (AGR2), a protein originally presumed to be secreted by intestinal epithelial cells. Here, we show that AGR ...[more]

PMID: 19359471

Similar Datasets

Project description:Colorectal cancer risk is associated with diets high in red meat. Heme, the pigment of red meat, induces cytotoxicity of colonic contents and elicits epithelial damage and compensatory hyperproliferation, leading to hyperplasia. Here we explore the possible causal role of the gut microbiota in heme-induced hyperproliferation. To this end, mice were fed a purified control or heme diet (0.5 μmol/g heme) with or without broad-spectrum antibiotics for 14 d. Heme-induced hyperproliferation was shown to depend on the presence of the gut microbiota, because hyperproliferation was completely eliminated by antibiotics, although heme-induced luminal cytotoxicity was sustained in these mice. Colon mucosa transcriptomics revealed that antibiotics block heme-induced differential expression of oncogenes, tumor suppressors, and cell turnover genes, implying that antibiotic treatment prevented the heme-dependent cytotoxic micelles to reach the epithelium. Our results indicate that this occurs because antibiotics reinforce the mucus barrier by eliminating sulfide-producing bacteria and mucin-degrading bacteria (e.g., Akkermansia). Sulfide potently reduces disulfide bonds and can drive mucin denaturation and microbial access to the mucus layer. This reduction results in formation of trisulfides that can be detected in vitro and in vivo. Therefore, trisulfides can serve as a novel marker of colonic mucolysis and thus as a proxy for mucus barrier reduction. In feces, antibiotics drastically decreased trisulfides but increased mucin polymers that can be lysed by sulfide. We conclude that the gut microbiota is required for heme-induced epithelial hyperproliferation and hyperplasia because of the capacity to reduce mucus barrier function. Mice were fed a Westernized high fat control diet, or the same diet supplemented with 0.5 µmol heme/g diet. One group of control and one group of heme mice received a mixture of broad spectrum Antibiotics (Abx) (ampicilin, neomycin and metronidazole) in their drinking water. After 14 days of intervention, mice were killed and gene expression was profiled in colon.

Project description:Epithelial cells of the mammalian intestine are covered with a mucus layer that prevents direct contact with intestinal microbes but also constitutes a substrate for mucus-degrading bacteria. To study the effect of mucus degradation on the host response, germ-free mice were colonized with Akkermansia muciniphila. This anaerobic bacterium belonging to the Verrucomicrobia is specialized in the degradation of mucin, the glycoprotein present in mucus, and found in high numbers in the intestinal tract of human and other mammalian species. Efficient colonization of A. muciniphila was observed with highest numbers in the cecum, where most mucin is produced. In contrast, following colonization by Lactobacillus plantarum, a facultative anaerobe belonging to the Firmicutes that ferments carbohydrates, similar cell-numbers were found at all intestinal sites. Whereas A. muciniphila was located closely associated with the intestinal cells, L. plantarum was exclusively found in the lumen. The global transcriptional host response was determined in intestinal biopsies and revealed a consistent, site-specific, and unique modulation of about 750 genes in mice colonized by A. muciniphila and over 1500 genes after colonization by L. plantarum. Pathway reconstructions showed that colonization by A. muciniphila altered mucosal gene expression profiles toward increased expression of genes involved in immune responses and cell fate determination, while colonization by L. plantarum led to up-regulation of lipid metabolism. These indicate that the colonizers induce host responses that are specific per intestinal location. In conclusion, we propose that A. muciniphila modulates pathways involved in establishing homeostasis for basal metabolism and immune tolerance toward commensal microbiota. Keywords: Analysis of target gene regulation by using microarrays Adult germ-free female NMRI-KI mice (45 – 65 days) were used for bacterial mono-association. Two bacterial strains were used in this study, A. muciniphila MucT (ATTC BAA-835) and L. plantarum WCFS1 (NCIMB 8826). A. muciniphila was grown anaerobically in a basal mucin based medium and L. plantarum was grown anaerobically at 37°C in Man-Rogosa-Sharpe broth (MRS; Le Pont de Claix, France). After 7 days of colonization, mice were killed by cervical dislocation and terminal ileum, cecum and ascending colon specimens were sampled.

Project description:Background; MUC2 mucin produced by intestinal goblet cells is the major component of the intestinal mucus barrier. MUC2 homo-oligomerizes intracellularly into large secreted polymers which give mucus its viscous properties. The inflammatory bowel disease (IBD) ulcerative colitis is characterized by depleted goblet cells and a reduced mucus layer, whereas goblet cells and the mucus layer are increased in the other major inflammatory bowel disease, Crohnâs disease. Methods and Findings; By murine N-ethyl-N-nitrosourea-mutagenesis we identified two distinct non-complementing missense mutations in Muc2 exons encoding N- and C-terminal homo-oligomerization domains causing an ulcerative colitis-like phenotype. Both strains developed mild spontaneous distal intestinal inflammation, chronic diarrhea, rectal bleeding and prolapse, increased susceptibility to acute and chronic colitis induced by a luminal toxin, aberrant Muc2 biosynthesis, smaller goblet cell thecae (less stored mucin) and a diminished mucus barrier. Enhanced local production of IL-1beta, TNF-alpha and IFN-gamma was seen in the distal colon. The number of leukocytes within mesenteric lymph nodes was increased five-fold and leukocytes cultured in vitro produced both Th1 and Th2 cytokines (IFN-gamma, TNF-alpha and IL-13). Intestinal permeability was increased and the luminal bacterial flora were more heavily coated with immunoglobulin as occurs in IBD. This pathology was accompanied by accumulation of the Muc2 precursor and ultrastructural and biochemical evidence of endoplasmic reticulum (ER) stress in goblet cells, activation of the unfolded protein response, and altered intestinal expression of genes involved in ER stress, inflammation, apoptosis and wound repair. Expression of mutated Muc2 oligomerization domains in vitro demonstrated that aberrant Muc2 oligomerization underlies the ER stress. These models show that mutations in Muc2 oligomerization domains can lead to aberrant assembly of the Muc2 complex leading to ER stress, a depleted mucus barrier and intestinal inflammation. In ulcerative colitis we demonstrate similar accumulation of non-glycosylated MUC2 precursor in goblet cells together with ultrastructural and biochemical evidence of ER stress even in non-inflamed intestinal tissue. Conclusions; The observations that mucin misfolding and ER stress lead directly to intestinal inflammation and that ER stress and goblet cell pathology occur in ulcerative colitis suggest that ER stress-related mucin depletion could be a fundamental component of the pathogenesis of colitis. Experiment Overall Design: 3 individual mice from the Eeyore, Winnie or Wild-type strains were compared as groups. An Affymetrix ID was compared between groups if the ID was Present within two of the three mice within each grouping. IDs were compared by calculating the log2 of Group One average signal divided by Group 2 average signal.

Project description:In this study we compared the intestinal mucus phenotype of Clca1-deficient (Clca1-/-) mice and wild-type (WT) controls. The human CLCA1 (chloride channel regulator, calcium-activated) and its murine ortholog mouse CLCA1 (formerly known as CLCA3) are secreted proteins which have been linked to human respiratory diseases such as asthma, cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) as well as to the corresponding murine models of these diseases. Common, disease severity-related features are goblet cell hyperplasia, mucus overproduction and disturbed clearance. Neutralization of mouse CLCA1 ameliorates the symptoms in the asthmatic mouse model and restoration of the decreased mouse CLCA1 protein level in a CF mouse model ameliorates the intestinal CF lesions, pointing toward these proteins being potential therapeutic targets. The expression CLCA1 can be induced in airways but this protein is normally expressed and secreted by goblet cells of the intestinal tract in both human and mouse together with the intestinal MUC2 mucin. This makes CLCA1 a major constituent of the extracellular intestinal mucus in which the MUC2 mucin is the main structural component. To study the possible further effects of murine CLCA1 deficiency on Muc2 and on other important components of the mucus, such as Agr2, Fcgbp, Klk and Zg16, a shotgun label-free proteomics study was performed which compared their relative abundance in the mucus layer, showing a tight and significant correlation between WT and Clca1-/- mucus proteins (Pearson r = 0.931, r2 = 0.868, p < 0.0001, based on 809 valid pairs). No significant differences in known mucus protein expression were observed. However, the abundance of CLCA1 in the mucus together with the domain structure suggests a structural role and other methods to reveal the function of these domains might shed light on the overall function in mucus. Because of its abundance in the mucus and the well preserved structure thought a long evolution together with mucus we still speculate that CLCA1 was developed to protect the expressing organism, although the threat is elusive.

Dataset Information

The protein disulfide isomerase AGR2 is essential for the production of intestinal mucus

Publications

The protein disulfide isomerase AGR2 is essential for production of intestinal mucus.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure