We here demonstrate a novel role of the adhesion G-protein-coupled receptor CD97 in regulating intestinal epithelial cell function. By comparing CD97 Tg mice that constitutively overexpress CD97 in intestinal epithelial cells to WT animals and to mice that lack a functional Cd97 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 
. Unexpectedly, we found a reduction of tumor numbers in Tg mice that was caused by 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 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 β-catenin to the plasma membrane and its tight association with E-cadherin 
. We found evidence that CD97 can regulate the localization and stability of β-catenin in enterocytes. Firstly, CD97 expression levels correlated with the amount of non-phosphorylated, stable β-catenin. Secondly, the amount of p-Akt (Ser473) and p-GSK-3β was regulated consistently and reversely in Ko and Tg compared to WT mice, indicating that Akt/GSK-3β signaling is involved in the stabilization of β-catenin through CD97. Akt phosphorylates the N-terminus of GSK-3β thereby inhibiting the ability of GSK-3β to phosphorylate β-catenin 
. Because phosphorylation of β-catenin initiates its ubiquitination and degradation in the proteasome, phosphorylation of GSK-3β may explain the accumulation of membranous non-phosphorylated β-catenin in Tg enterocytes.
In tumor cells, a different distribution of β-catenin is found. Here, accumulation of β-catenin caused by mutations in the adenomatous polyposis coli (Apc
) gene, involved in the intracellular transport of β-catenin 
, or in the genes encoding axin and β-catenin leads to translocation of β-catenin into the nucleus, where it induces genes critical for cell transformation and cancer 
. It becomes increasingly clear that the junctional, cytoplasmic and nuclear pools of β-catenin are closely connected 
. In our study we identified CD97 to regulate the membrane-associated pool of β-catenin.
The observations that CD97 overexpression enhances the strength of enterocytic adherens junctions and increases random tumor cell migration 
seem to be contradictory. We have evidence that Akt signaling is involved in both the CD97-induced strengthening of adherens junctions and in the migration of tumor cells: specific inhibitors of the phosphoinositide-3 kinase (PIK3)/Akt pathway stopped CD97-induced random tumor migration 
. The possibility of a dual role of CD97 is supported by a recent study demonstrating Akt signaling in E-cadherin-based adherens junction formation as well as in tumor cell migration 
. Of note is the fact that CD97 is not restricted to adherens junctions. Peripheral blood leukocytes bear the molecule evenly distributed at their surface 
. In addition, carcinomas also show intracellular cytoplasmic staining for CD97 
. In migratory, CD97-overexpressing HT1080 cells, the molecule was located in membrane ruffles at the leading edge, co-localized with cortical F-actin 
. Accordingly, membrane ruffles of migrating tumor fibroblasts were β-catenin positive 
. All these data indicate that CD97 is found in different cellular pools where it might engage with additional molecular processes.
Adherens junctions crucially contribute to the organization and stabilization of a polarized intestinal epithelium and their dysregulation has been linked causally to inflammatory bowel disease 
–. To our knowledge, the CD97 Tg mouse is the first genetic model, in which adherens junctions are strengthened and intestinal barrier function is augmented. Interestingly, a susceptibility locus for inflammatory bowel disease has been found in close proximity to the EGF-TM7 gene cluster on chromosome 19p13 
. In addition, association of a single nucleotide polymorphism in the CD97 promotor (T64C) was associated with ulcerative colitis 
. Although these findings need further confirmation, they may indicate a role of EGF-TM7 receptors in inflammatory bowel disease. By analyzing colonic biopsies of ulcerative colitis patients, we observed a loss of CD97 from enterocytic adherens junctions in 4 out of 5 patients. Whether this was due to the ongoing disintegration of the tissue or indicates the involvement of CD97 in the disease process remains to be shown.
In summary, by establishing CD97 Tg mice and examining them in tumor- and colitis-associated models we identified a novel, yet unknown function of the adhesion G-protein-coupled receptor CD97. CD97 regulated plaque core proteins of adherens junctions in colonic enterocytes. CD97 overexpression strengthened these cell contacts, which results in an increased epithelial integrity and decreased experimental colitis sensitivity. Moreover, for the first time key molecules of intracellular signaling via an EGF-TM7 receptor were identified. Our data suggest that CD97 induces Akt/GSK-3β signaling that results in a decrease of proteolytic digestion of β-catenin and its stabilization in adherens junctions. Our data are confirmed by the reverse results obtained in Ko mice.