| Smoking is an important risk factor in the disease of respiratory system, such as chronic obstructive pulmonary disease (COPD), which can injure the airway epithelial cells and destroy the intact epithelium structure. After the injury of epithelium, epithelial cells need to migration and proliferation in order to close wounds in the epithelial layer; these processes depend on the interaction between microtubule and microtubule-associated proteins (MAPs). In the cell, microtubule polymers are the building materials formany important shapes, a number of factors can affect plus end behavior and play an important role in the assembly of microtubule-based structures and microtubules dynamics.Truncation mutations in the adenomatous polyposis coli (APC) protein are responsible for familial and sporadic colonic tumours. APC protein is involved in a variety of life activities and acts as a regulator of numerous signaling pathways. Otherwise, recent studies indicate that APC protein has emerged as a multifunctional protein that can affect a variety of fundamental cellular processes, in particular at the aspect of microtubule regulation. The microtubule binding domain has been mapped to a highly positively charged region in the C-terminal third of APC protein. It binds to microtubules directly or indirectly and is termed"plus-end-tracking proteins". Binding of APC protein to microtubule can stabilizes microtubules in vitro and in vivo. The length of microtubules remains higher in the presence of APC protein. Glycogen synthase kinase3β(GSK3β) is a conserved, multifunctional serine/threonine kinase found in all eukaryotes. The enzyme is a key regulator of numerous signalling pathways, and is involved in a wide range of cellular processes like metabolism, cell proliferation, differentiation, apoptosis and cell motility, ranging from glycogen metabolism to cell cycle regulation and proliferation. Binding of the APC protein to microtubules increases microtubule stability and is regulated by GSK3βphosphorylation. The interaction of APC protein with microtubules is decreased by phosphorylation of APC protein by GSK3β. In addition, GSK3βis also able to phosphorylate other a number of microtubule-associated proteins. Phosphorylation of these proteins by GSK3βdecreases their ability to stabilise microtubules.To investigate the roles of APC protein and GSK3βof smoking murine model in the repair of the injured airway epithelial cells (AEC) 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 on 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 GSK3βin the AEC. (3) Western blotting was used to detect the levels of APC protein, GSK3βand phosphoric GSK3β(P-GSK3β) in pulmonary tissue. (4) Observing the localizations of APC protein and GSK3βby immunofluorescence technique.The results are as the followings: (1) Morphology of airway epithelial cells in smoking mice showed predominant injury (smoking after 1 week and 4 weeks), repair (smoking after 8 weeks) and reinjury (smoking after 12 weeks).That in non-smoking mice with intact structure, regularly arranged cilia, tight intercellular junction and had no inflammatory cells around. 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 AEC increased after 1 w smoking (0.458±0.062 vs 0.399±0.060, P<0.05 vs control), but was significantly decreased at the end of 4 w (0.339±0.056, P<0.01 vs control) and increased at the end of 8 w and 12 w (0.387±0.041, 0.378±0.037, P<0.05 vs 4 w). The expressions of GSK3βin the AEC of smoking mice obviously decreased (P<0.01 or P<0.05 vs control). (3) Western blotting showed that the expressions of APC protein and GSK3βin lung tissue were consistent with the results of immunohistochemistry; the levels of P-GSK3βin all smoking model were higher than that in control. (4)The results of immunofluorescence showed that APC protein distributes uniformly in the AEC of the control. APC protein was localized mainly near the regions of epithelial cell membrane at the end of 1 w and 8 w after smoking, which is dissimilar with the localization in control, and this change was not seen in the locations of GSK3β. The distribution of GSK3βhas no change in the control group and the smoking group, and distributes uniformly in the AEC.Taken together, these results demonstrate that the expressions and localizations of APC protein, GSK3βand the activity of GSK3βare dynamically changed in the AEC with experimental smoking injury at different phases, suggesting that APC protein and GSK3βmay be involved in the regulation of migration and proliferation of airway epithelial cell, and play important roles in the process of repair of airway epithelium injury.
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