IQ domain GTPase-activating protein 1 mediates the process of injury and repair in bronchial epithelial cells / 生理学报

The process of injury and repair in airway epithelium involves cell spreading and migration followed by cell proliferation. IQ domain GTPase-activating protein 1 (IQGAP1) acts in a series of cell processes, but has not been clarified in lung epithelial cells. In this study, a widely used model of injury and repair in vitro by scratching bronchial epithelial cells (BECs) was utilized to investigate the function of IQGAP1. The results showed that IQGAP1 was abundant in BECs of mouse, rat, pig and human. IQGAP1 was colocalized with tubulin cytoskeleton, but was destroyed by nocodazole, a microtubule disassembly reagent. IQGAP1 mRNA and protein expressions increased at 6-9 h after scratching. In addition, overexpression of IQGAP1 translocated β-catenin from the cytoplasm into the nucleus and activated the Tcf/Lef signal. Scratching altered the associations of IQGAP1 with β-catenin, adenomatous polyposis coli (APC) and cytoplasmic linker protein-170 (CLIP-170). Silencing IQGAP1 expression by small interference RNA (siRNA) blocked the wound closure. It is concluded that IQGAP1 signal is involved in the wound closure of BECs induced by scratching.

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.

Dynamic changes of adenomatous polyposis coli protein and glycogen synthase kinase 3beta in the repair of the injured airway epithelial cells in smoking mice / 生理学报

To investigate the roles of adenomatous polyposis coli (APC) protein and glycogen synthase kinase 3beta (GSK3beta) of smoking murine model in the repair of the injured airway epithelial cells (AECs) in different stages, 30 male Kun-Ming mice were randomly divided into two groups, the control group and the smoking group. There were 24 mice in smoking group, and 6 animals were separately killed at the end of the 1st, 4th, 8th and 12th week after smoking. Then the following tests were undertaken: (1) HE staining of lung section to observe the morphological changes of the bronchi in the smoking mice. (2) Immunohistochemical staining of APC protein and GSK3beta in the AECs. (3) Western blot was used to detect the levels of APC protein, GSK3beta and phosphorated GSK3beta (p-GSK3beta) in pulmonary tissue. (4) Observing the localizations of APC protein and GSK3beta in the AECs by immunofluorescence technique. The results showed: (1) AECs showed changes of predominant injury (1-, 4-week), repair (8-week) and reinjury (12-week) along with smoking time prolonged. The experimental results indicated that the model of smoking mice was duplicated successfully. (2) Immunohistochemical results showed that the expression of APC protein in the AECs increased after 1-week smoking (0.458 +/- 0.062 vs 0.399 +/- 0.060, P< 0.05 vs control), but was significantly decreased at the end of the 4th week (0.339+/- 0.056, P<0.01 vs control) and increased at the end of the 8th and 12th week (0.387 +/- 0.041, 0.378 +/- 0.037, P<0.05 vs 4-week). The expression of GSK3beta in the AECs of smoking mice obviously decreased (P<0.01 or P<0.05 vs control). (3) Western blot showed that the expressions of APC protein and GSK3beta in lung tissue were consistent with the results of immunohistochemistry; and the levels of p-GSK3beta in all smoking models were higher than that in control. (4) The results of immunofluorescence showed that APC protein was localized mainly near the regions of epithelial cell membrane at the end of the 1st and 8th week after smoking, which were dissimilar with the localization in control, and this change was not seen in the location of GSK3beta. Taken together, these results demonstrate that the expressions and localizations of APC protein, GSK3beta and the activity of GSK3beta are dynamically changed in the AECs with experimental smoking injury at different phases, suggesting that APC protein and GSK3beta may be involved in the regulation of migration and proliferation of AECs, and play an important role in the process of repair of airway epithelium injury.

Regulatory effects on apoptotic activities of gastric cancer cell line by over-expression of Smac gene / 中华病理学杂志

<p><b>OBJECTIVE</b>To investigate the effects of overexpression of second mitochondria-derived activator of caspases (Smac) gene on apoptosis of gastric cancer cells.</p><p><b>METHODS</b>Under the induction of liposome, MKN-45 cells were transfected by Smac gene and incubated with G418 for subclone selection. Reverse transcriptase-polymerase chain reaction and Western blot were used to determine cellular Smac gene expression. After induction of apoptosis by mitomycin (MMC), cell viabilities were analyzed using trypan blue stain. Apoptosis was measured by electronic microscopy, acridine orange-ethidium bromide fluorescent staining and in situ terminally labelled transferase technique (TUNEL). Western blot and colorimetry were used to assess cellular caspase-3 expression and its activity.</p><p><b>RESULTS</b>The Smac mRNA and protein levels in MKN-45/Smac subclone cells (subclone consistently expressing Smac gene) were significantly higher than those in MKN-45 (P < 0.01). When compared with those in MKN-45, cell viabilities of MKN-45/Smac were reduced by 10.0% to 30.8% (P < 0.01), after treatment with 10 microg/ml MMC for 6 to 24 hours. Some of the MKN-45/Smac cells showed characteristic morphologic changes of apoptosis, their apoptotic rate being increased by 21.2% (P < 0.01). After treatment with MMC, caspase-3 expression and its activity in MKN-45/Smac cells were significantly higher than those in MKN-45 (P < 0.01).</p><p><b>CONCLUSIONS</b>Overexpression of Smac in gastric cancer cell line significantly improves expression and activity levels of caspase-3 after induction by MMC. Such apoptosis-inducing effect establishes a novel strategy for regulating the apoptosis activity of gastric cancer.</p>

Role of glycogen synthase kinase 3 in squamous differentiation of pig airway epithelial cells: A primary study / 生理学报

To investigate if glycogen synthase kinase 3 (GSK3) is involved in squamous differentiation of airway (tracheobronchial) epithelial cells, primary pig airway epithelial cells were treated with lithium chloride, a highly selective inhibitor of GSK3. Change in morphology of cells was monitored under microscopy, and expression of beta-catenin, phosphorylated GSK3 and involucrin, a squamous differentiation marker, were dectected by Western blotting, while expression of mRNA of another squamous differentiation marker, small proline-rich protein, was detected by RT-PCR. Further, luciferase reporter assay was used to assess the activation of beta-catenin/Tcf signaling. The results demonstrated that lithium was able to induce a squamous morphology of the cells, and to enhance the expression of involucrin and small proline-rich protein mRNA. Moreover, lithium increased inhibitory phosphorylation of GSK3, augmented nuclear translocation of beta-catenin in a dose- and time-dependent manner. Activation of beta-catenin/Tcf signaling was observed after the elevation of squamous differentiation markers. Taken together, these data suggest that GSK3 is possibly involved in squamous differentiation of pig airway epithelial cells.