Germline and somatic mutations in the Apc (Adenomatous polyposis coli ) gene are thought to be seminal genetic events in the etiology of human and murine colorectal cancer. ApcMin mice carry a germline mutation in the Apc gene and experience reduced lifespan due to adenocarcinoma burden. Wild type, but not mutated, APC binds to and regulates beta-catenin, the mammalian homolog of armadillo required for cadherin-mediated cell adhesion. Beta-catenin released from its binding to membrane E-cadherin forms a heterodimer with Tcf/LEF and functions as a transcriptional factor. To study the regulation of this pathway, we used two conditional immortal murine intestinal epithelial cell lines contrasting in Apc genotype(""""""""Immortomouse""""""""/Min Colonic Epithelia, Apc +/-; Young Adult Mouse Colon epithelia, Apc +/+). We have demonstrated that IMCE cells which have defective degradation of beta-catenin, have higher levels of beta-catenin/LEF-1 transcriptional factor by EMSA and higher expression of COX-2 than YAMC cells in response to lipopolysaccharide (LPS) and interferon-gamma (IFN-g). The critical role of nitric oxide (NO) in this response was shown by the abrogation of the LPS, IFN-g effect by inhibitors of nitric oxide synthase II. Additionally, NO-donors increased beta-catenin/LEF-1 formation by EMSA as well as the expression of COX-2. That the effect was mediated by the availability of beta-catenin was supported by the differential response in IMCE and YAMC cells and by the direct demonstration of free, cytoplasmic beta-catenin in response to NO treatment. Our current work is focused on the mechanism by which NO increases free, unbound beta-catenin. Preliminary findings suggest that NO stimulates the degradation of membrane bound E-cadherin with the concomitant release of beta-catenin from the cytoplasmic E-cadherin binding site. Using an antibody recognizing the extracellular domain of E-cadherin, we found that treatment with NO donors markedly increased E-cadherin degradation products accumulating in the medium. Since metalloproteinases mediate the degradation of E-cadherin, we tested several inhibitors of metalloproteinases and found that they not only blocked the effect of NO on E-cadherin degradation but also abrogated its effect on the formation of beta-catenin/LEF-1 transcriptional complexes. These findings suggest that the activation of metalloproteinases by NO releases free beta-catenin from E-cadherin to form beta-catenin/LEF-1 transcriptional complexes. We are identifying the specific metalloproteinase activated by NO and elucidating the mechanism of this activation.
