PI3K-Dependent GSK3β(Ser9)-Phosphorylation Is Implicated in the Intestinal Epithelial Cell Wound-Healing Response

Introduction: The ability of the intestinal epithelial barrier to respond to various injurious insults is an essential component of intestinal homeostasis. However, the molecular mechanisms responsible for wound-healing and repair in the intestine are poorly understood. The glycogen synthase kinase 3 beta (GSK3 beta) has been implicated in various biological processes such as cellular motility, cell spreading and recently inflammation. Aim: To investigate the role of GSK3 beta in intestinal epithelial cell restitution. Methods: Rat intestinal epithelial IEC18 cells were serum-starved for 16 to 24h and wounded by multiple scraping. Akt(Ser473)-, GSK3 beta(Ser9)-and RelA(Ser536)-phosphorylation were determined by Western blot using specific phospho-antibodies. The inhibitors AG1478 (1 mu M) and Ly294002 (25 mM) were used to block EGF-R autophosphorylation and PI3K-activation, respectively. beta-catenin/LEF/TCF dependent transcription was determined by reporter gene assay (TOP/FOP system). C-myc gene expression was evaluated by real-time RT-PCR. GSK3 beta(-/-) mouse embryonic fibroblasts were used to characterize the role of GSK3 beta in wounding-induced cell migration. Results: Wounding induced GSK3 beta(Ser9) phosphorylation in IEC-18 cells, which led to beta-catenin accumulation as well as nuclear translocation of beta-catenin. beta-catenin stabilization/nuclear translocation led to enhanced LEF-TCF transcriptional activity and subsequent c-myc mRNA accumulation in wounded cell monolayers. Blocking PI3K/Akt signaling with Ly294002 prevented wound-induced GSK3 beta(Ser9) phosphorylation as well as beta-catenin nuclear translocation and significantly attenuated restitution. Additionally, wounding induced rapid NF-kB(Ser536) phosphorylation, which was inhibited by AG1478, but not by Ly294002. GSK3 beta(-/-) cells demonstrated significantly attenuated wound-induced restitution compared to wild-type cells. Conclusion: We conclude that PI3K-mediated GSK3 beta phosphorylation is involved in the intestinal epithelial wound-healing response. Phosphorylation of GSK3 beta may be important for intestinal restitution by promoting cell motility in response to wounding.