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Intestinal gene expression in ENU mutagenesis mouse strains with missense mutations in Muc2 mucin and ER stress

ABSTRACT: 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, Crohn’s 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. Keywords: Single gene, multiple mutant comparison

ORGANISM(S): Mus musculus

PROVIDER: GSE9913 | GEO | 2008/03/01

SECONDARY ACCESSION(S): PRJNA103905

REPOSITORIES: GEO

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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.

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Intestinal gene expression in ENU mutagenesis mouse strains with missense mutations in Muc2 mucin and ER stress

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