Disruption of the intestinal epithelial barrier occurs in many intestinal diseases, but neither the mechanisms nor the contribution of barrier dysfunction to disease pathogenesis have been defined. We utilized a murine model of T cell-mediated acute diarrhea to investigate the role of the epithelial barrier in diarrheal disease. We show that epithelial barrier dysfunction is required for the development of diarrhea. This diarrhea is characterized by reversal of net water flux, from absorption to secretion; increased leak of serum protein into the intestinal lumen; and altered tight junction structure. Phosphorylation of epithelial myosin II regulatory light chain (MLC), which has been correlated with tight junction regulation in vitro, increased abruptly after T cell activation and coincided with the development of diarrhea. Genetic knockout of long myosin light chain kinase (MLCK) or treatment of wild-type mice with a highly specific peptide MLCK inhibitor prevented epithelial MLC phosphorylation, tight junction disruption, protein leak, and diarrhea following T cell activation. These data show that epithelial MLCK is essential for intestinal barrier dysfunction and that this barrier dysfunction is critical to pathogenesis of diarrheal disease. The data also indicate that inhibition of epithelial MLCK may be an effective non-immunosuppressive therapy for treatment of immune-mediated intestinal disease.

Intestinal polyposis, a precancerous neoplasia, results primarily from an abnormal increase in the number of crypts, which contain intestinal stem cells (ISCs). In mice, widespread deletion of the tumor suppressor Phosphatase and tensin homolog (PTEN) generates hamartomatous intestinal polyps with epithelial and stromal involvement. Using this model, we have established the relationship between stem cells and polyp and tumor formation. PTEN helps govern the proliferation rate and number of ISCs and loss of PTEN results in an excess of ISCs. In PTENdeficient mice, excess ISCs initiate de novo crypt formation and crypt fission, recapitulating crypt production in fetal and neonatal intestine. The PTEN-Akt pathway probably governs stem cell activation by helping control nuclear localization of the Wnt pathway effector β-catenin. Akt phosphorylates β-catenin at Ser552, resulting in a nuclear-localized form in ISCs. Our observations show that intestinal polyposis is initiated by PTEN-deficient ISCs that undergo excessive proliferation driven by Akt activation and nuclear localization of β-catenin. Accession codes. Gene Expression Omnibus (GEO): GSE6078.URLs. GEO: http://www.ncbi.nlm.nih.gov/projects/geo/.Supplementary information is available on the Nature Genetics website. COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests. HHS Public AccessAuthor manuscript Nat Genet. Author manuscript; available in PMC 2015 December 16. Published in final edited form as:Nat Genet. 2007 February ; 39(2): 189-198. doi:10.1038/ng1928. Author Manuscript Author ManuscriptAuthor Manuscript Author ManuscriptThe failure of most current therapies to cure cancer has led to the hypothesis that treatments targeted at malignant proliferation spare a more slowly cycling 'cancer stem cell' population that has the ability to regenerate the tumor 1 . Recently, cancer stem cells have been identified and shown to seed tumors upon transplantation into a secondary host [2][3][4] . However, little is known about the process by which mutation(s) in a stem cell result in primary tumor initiation.Although there are many 'causes' of intestinal cancer, it is well established that almost all cases begin with the development of benign polyps, mainly involving benign neoplastic proliferation of epithelium. The epithelium of the small intestine is composed of a proliferation compartment (crypt) and a differentiation compartment in the villus (Fig. 1a). ISCs, located near the crypt base and above Paneth cells 5,6 , give rise to enterocytes, goblet cells, enteroendocrine cells and Paneth cells [6][7][8] . Intestinal polyposis features a substantial increase in the number of crypts (crypt expansion) and a reduction in epithelial cell differentiation 6,7,9,10 . A key question 7,9,11 is whether stem cells are involved in the abnormal crypt expansion during polyp initiation.Studies of human hereditary intestinal polyposis syndromes (which typically, but not uniformly, predispose affected individuals to gastrointes...

No abstract

Background & Aims Mechanisms responsible for crypt architectural distortion in chronic ulcerative colitis (CUC) are not well understood. Data indicate that Akt signaling cooperates with Wnt to activate β-catenin in intestinal stem and progenitor cells through phosphorylation at Ser552 (P-β-catenin552). We investigated whether phosphoinositide 3- kinase (PI3K) is required for Akt-mediated activation of β-catenin during intestinal inflammation. Methods The class IA subunit of PI3K was conditionally deleted from intestinal epithelial cells in mice. Acute inflammation was induced in these mice (I-pik3r1KO) and their intestines were analyzed by biochemical and histological methods. The effects of chemically blocking PI3K in colitic IL-10−/− mice were examined. Biopsy samples from patients were examined. Results Compared to wild type mice, I-pik3r1KO mice had reduced T-cell–mediated Akt and β-catenin signaling in intestinal stem and progenitor cells and limited crypt epithelial proliferation. Biochemical analyses indicated that PI3K–Akt signaling increased nuclear total β-catenin and P-β-catenin552 levels and reduced phosphorylation of N-terminal β-catenin, which is associated with degradation. PI3K inhibition in IL-10−/− mice impaired colitis-induced epithelial Akt and β-catenin activation, reduced progenitor cell expansion, and prevented dysplasia. Human samples had increased numbers of progenitor cells with P-β-catenin552 throughout expanded crypts and increased mRNA expression of β-catenin target genes in CUC, colitis-associated cancer, tubular adenomas, and sporadic colorectal cancer, compared with control samples. Conclusions PI3K–Akt signaling cooperates with Wnt to increase β-catenin signaling during inflammation. PI3K-induced and Akt-mediated β-catenin signaling are required for progenitor cell activation during the progression from CUC to CAC; these factors might be used as biomarkers of dysplastic transformation in the colon.

No abstract