Germline and somatic mutations in the Apc (Adenomatous polyposis coli) gene are thought to be one of the seminal genetic events in the etiology of colorectal cancer. Min mice carry a germline mutation in the Apc gene and experience reduced lifespan due to adenocarcinoma burden. Germline mutations in the secretory phospholipase A2 (pla2s) gene may modify the incidence of spontaneous adenomas in mice bearing mutations in the Apc gene. This finding prompted us to investigate the hypothesis that lysophosphatidic acid (LPA), a putative product of lumenal pla2s activity, elicits specific effects on adherens junction-associated proteins in an Apc genotype-specific manner. Wild type, but not truncated, APC binds to and regulates beta-catenin, the mammalian homolog of armadillo required for cadherin-mediated cell adhesion. Using two conditionally immortal murine intestinal epithelial cell lines contrasting in Apc genotype (""""""""Immortomouse""""""""/Min Colonic Epithelia, Apc +/- ; Young Adult Mouse Colon epithelia, Apc +/+), we have demonstrated differential LPA-induced effects on beta-catenin accumulation in confluent, quiescent IMCE and YAMC cells. Immunoprecipitation experiments revealed that LPA induced a greater concentration-dependent accumulation of beta-catenin in IMCE than in YAMC cells. The level of tyrosine phosphorylated beta-catenin and pp120 increased in response to LPA treatment to a greater extent in IMCE than YAMC cells. Immunoprecipitation of beta-catenin and E-cadherin demonstrated that LPA treatment caused a time- and concentration-dependent increase in the amount of beta-catenin associated with E-cadherin in IMCE cells. In contrast to its effects on adherens junction-associated proteins, LPA induced the accumulation of similar levels of tyrosine phosphorylated focal adhesion kinase (FAK) and FAK-associated paxillin in FAK immunoprecipitates from IMCE and YAMC cells. These data indicate that LPA-induced signaling events can modulate the phosphotyrosine content of beta-catenin and its association with E-cadherin in a manner consistent with an Apc genotype-specific mechanism. These results suggest LPA initiates novel signaling events which may result in increased avidity of cell-cell adhesion in the IMCE cells. Development of a cell-cell adhesion assay and the employment of immunofluorescence microscopy are being carried out to confirm protein-protein interaction data and assess cell-cell adhesion. Furthermore, the specificity of signaling through a putative Rho-linked LPA receptor will be tested using tyrosine kinase and G-protein inhibitors.
