Spatial-temporal distribution of glycogen synthase kinase 3beta and adenomatous polyposis coli protein are involved in the injury and repair of airway epithelial cells induced by scratching / 生理学报

To investigate the roles of glycogen synthase kinase 3beta (GSK3beta) and adenomatous polyposis coli (APC) protein in wound repair of airway epithelial cells (AECs), we established a wound model of airway epithelium in vitro. Then the following tests were undertaken: (1) Western blot was used to detect the levels of total GSK3beta and phosphorylated GSK3beta in human bronchial epithelial (16HBE) cells; (2) The localizations of APC protein was observed by using immunofluorescence technique; (3) Immunoprecipitation was used to investigate the relationship between APC protein and GSK3beta during the repair of 16HBE cells. The results were as follows: (1) The level of phosphorylated GSK3beta increased 0.5 h after scratching (P<0.05), reached a maximum at 6 h (P<0.05), and maintained until 12 h, while the total level of GSK3beta remained constant; (2) Results of immunofluorescence study showed that APC protein clustered with tubulin in the region of the migrating leading cells 6 h after scratching, which was dissimilar with that in the cells 0 h after scratching; (3) GSK3beta and APC protein were immunoprecipitated and analysed on SDS-PAGE. We found that GSK3beta and APC protein were precipitated, indicating that the two proteins existed in a complex. After scratching, dissociation of the two proteins occurred. Taken together, we conclude that scratching caused a decrease in phosphorylation of GSK3beta, and that reduced phosphorylation of GSK3beta promoted APC protein to bind to the plus ends of microtubules and stabilize the growing ends. These observations suggest that APC protein and GSK3beta may synergistically play an important role in the repair of airway epithelium.

Glycogen synthase kinase 3beta induces cell cycle arrest in a cyclin D1-dependent manner in human lung adenocarcinoma cell line A549 / 生理学报

The effect of glycogen synthase kinase 3beta (GSK3beta) has been repeatedly implicated in cell proliferation, but studies on the effect of GSK3beta in different cell lines with different stimuli have drawn different conclusions. To investigate the direct effect of GSK3beta on cell growth in human lung adenocarcinoma cell line A549, we changed its activity by transient transfection with two kinds of GSK3beta mutant plasmids, constitutively active form S9A-GSK3beta and dominant negative form KM-GSK3beta. Twenty-four hours later, cell counting, flow cytometry and Western blot detection were made respectively. The results showed that enhancing GSK3beta activity caused a decrease in cell number, as well as a higher percentage of cells at G(1) phase. Further, the expression of cyclin D1 was down-regulated by GSK3beta. Taken together, our observations suggest that GSK3beta may induce G(1) cell cycle arrest in a cyclin D1-dependent fashion and therefore possibly plays a growth-inhibitory role in A549 cells.