It becomes increasingly clear that the junctional, cytoplasmic and nuclear pools of -catenin are closely connected [2]

It becomes increasingly clear that the junctional, cytoplasmic and nuclear pools of -catenin are closely connected [2]. by a PR52 junctional complex comprising tight and adherens junctions and desmosomes, providing different functions in cell-cell adhesion. In adherens junctions (or mice and stimulated single cell motility in an isoform-specific manner [11]. simulations suggested that CD97 can increase the invasive capacity of tumors and cause the appearance of scattered tumor cells at the invasion front [11]. To explore directly the hypothesis that CD97 expression affects colorectal carcinogenesis, we generated transgenic (Tg) mice that constitutively overexpress CD97 in intestinal epithelial cells. These CD97 Tg mice were examined in the azoxymethane (AOM)/dextran sodium sulfate (DSS) model for colitis-associated tumorigenesis. Unexpectedly, depending on the CD97 cDNA copy number integrated, carcinogenesis in Tg mice was reduced due to impaired DSS-induced injury. Ultrastructural analysis of colonic enterocytes revealed that lateral cell-cell contacts were strengthened in CD97 Tg mice and weakened in CD97 knockout (Ko) mice. We demonstrate that CD97 is located in E-cadherin-based adherens junctions and that it regulates membrane-associated -catenin associated with alterations in Akt/glycogen synthase kinase-3 (GSK-3) signaling. Materials and Methods Ethics Statement This research complied with the ethics guidelines of the University of Leipzig. For the generation of transgenic mice and for animal experiments we obtained ethics approval from the Landesdirektion 1-Azakenpaullone Leipzig (TVV01/06, TVV23/08). We obtained ethics approval from the Ethics Committee of the Medical Faculty of the University of Leipzig (No111-2009) to analyze human colonic samples and written consent from all participants involved in this study. Reagents Primers and antibodies used in this study are specified in table 1 and ?and22. Table 1 List of primers. gene, we obtained evidence that CD97 controls the structure of enterocytic adherens junctions and thereby the integrity of the intestinal barrier. We first made these observations when subjecting CD97 Tg mice to AOM treatment combined with DSS in order to study the effect of CD97 on tumorigenesis. DSS causes destruction of the epithelial cells in the basal crypts and induce an inflammatory reaction in the colonic mucosa that acts as a promoter of colorectal carcinogenesis [24], [25]. Unexpectedly, we found a reduction of tumor numbers in Tg mice that was caused by 1-Azakenpaullone amelioration of DSS-induced injury. Protection of CD97 Tg mice from DSS colitis was reproduced by treatment with DSS alone and involved lower clinical disease activity, less histological crypt damage, and reduced local and systemic immune reactivity. The finding that CD97 overexpression attenuated colitis and the fact that this amelioration correlated with the CD97 cDNA copy number integrated in the Tg mice indicated that CD97 can regulate epithelial cell function. Integrity and stability of the intestinal epithelium is maintained by different types of specialized cell contacts. Immunohistochemistry revealed co-localization of CD97 with proteins of E-cadherin-based adherens junctions in murine as well as human colonic enterocytes and colorectal 1-Azakenpaullone cell lines. In addition, electron microscopy studies showed that these basally localized cell-cell junctions were strengthened in CD97 Tg mice and weakened in CD97 Ko mice. The formation and stabilization of adherens junctions requires the recruitment of cytosolic -catenin to the plasma membrane and its tight association with E-cadherin [8]. We found evidence that CD97 can regulate the localization and stability of -catenin in enterocytes. Firstly, CD97 expression levels correlated with the amount of non-phosphorylated, stable -catenin. Secondly, the amount of p-Akt (Ser473) and p-GSK-3 was regulated consistently and reversely in Ko and Tg compared to WT mice, indicating that Akt/GSK-3 signaling is involved in the stabilization of -catenin through CD97. Akt phosphorylates the N-terminus of GSK-3 thereby inhibiting the ability of GSK-3 to phosphorylate -catenin [8]. Because phosphorylation of -catenin initiates its ubiquitination and degradation in the proteasome, phosphorylation of 1-Azakenpaullone GSK-3 may explain the accumulation of membranous non-phosphorylated -catenin in Tg enterocytes. In tumor cells, a different distribution of -catenin is found. Here, accumulation of -catenin caused by 1-Azakenpaullone mutations in the adenomatous polyposis coli ( em Apc /em ) gene, involved in the intracellular transport of -catenin [26], or in the genes encoding axin and -catenin leads to translocation of -catenin into the nucleus, where it induces genes critical.

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