Role Of Glycogen Synthase Kinase 3 In Airway Epithelial Cell And Alveolar Type II Epithelial Cell Injury Caused By Cigarette Smoke

Part I Role of glycogen synthase kinase 3 in squamous differentiation induced by cigarette smoke in porcine airway epithelial cellSquamous differentiation of airway (tracheobronchial) epithelium induced by diverse stimuli, such as cigarette smoke, has been thought to be an adaptive response to chronic injury as well as a precancerous lesion of lung squamous carcinoma. The molecular mechanisms of squamous differentiation have not been fully elucidated. Glycogen synthase kinase 3 (GSK3) is a multifunctional protein kinase that plays important roles in metabolism, cell proliferation, differentiation, apoptosis and cell motility. There are two mammalian GSK3 isoforms, designated GSK3αand GSK3β. GSK3 is constitutively active in resting cell, and can be inactivated by phosphorylation of Ser-21 in GSK3αor Ser-9 in GSK3β. GSK3 acts as a key and negative regulator of numerous signal pathways, including Wnt/β-catenin and activator protein-1 (AP-1) signaling pathway. Recent researches indicated that GSK3 and AP-1 signaling might be implicated in squamous differentiation of airway epithelium induced by cigarette smoke. In the present study, we examined the expression of GSK3 in lung tissue of several experimental animals and cultured porcine airway epithelial cells (PAECs), and further investigated the role of GSK3 and AP-1 signaling in squamous differentiation of airway epithelium induced by cigarette smoke in vitro.Immunostaining showed that both GSK3αand GSK3βwere prominently expressed in plasm of airway epithelial cells, submucosal gland cells, smooth muscle cells and alveolar epithelial cells of human, rat, mouse or pig. However, there were unexpected high levels of GSK3αin cartilage lacuna cells when compared with those of GSK3β. No signals of phosphorylated GSK3α/βwere observed in lung tissues. Immunofluorescence demonstrated that the expression of GSK3αand GSK3βwere high but phosphorylated GSK3α/βwas low in PAECs.Cytotoxicity assay and cell morphological observation demonstrated cigarette smoke components inhibited the growth of PAECs and resulted in morphological changes, which showed delayed confluence, more widely spread and flattened appearance with widened cell-cell interspaces, compared with the classic cobblestone epithelial morphology. As expected from previous studies, it was confirmed by Western blot and RT-PCR in PAECs that cigarette smoke components enhance the expression of involucrin protein and small proline-rich protein mRNA, two markers of squamous differentiation.Moreover, it was found that in vitro cigarette smoke components notably inhibited glycogen synthase kinase 3 (GSK3) by increasing inactive phosphorylated GSK3α/βand reducing GSK3βexpression. The inactivation of GSK3 by two highly selective inhibitors, lithium and SB216763, also significantly enhanced involucrin expression in cultured porcine airway epithelial cells.In addition, Transcription factor activity assay showed that cigarette smoke components significantly promoted AP-1 binding activities to the upstream regulatory region of involucrin gene, and similar results were observed by further studies through using GSK3 inhibitors to imitate the effects of cigarette smoke components.Taken together, these data suggest: (1) cigarette smoke components can inhibit the growth and induce squamous differentiation of PAECs. (2) GSK3 is highly expressed in airway epithelium, and involved in involucrin expression induced by cigarette smoke in PAEC probably via negatively regulating AP-1 activity, implying a possible mechanism responsible for squamous differentiation induced by cigarette smoke. Part II Role of glycogen synthase kinase 3 andβ-catenin/TCF signaling in alveolar type II epithelial cell injury caused by cigarette smokeCigarette smoke is known to have various injurious effects on alveolar epithelial cells. It suppresses migration, proliferation and differentiation of the cells so that they can not cover the defects resulted from the injury, and inhibits surfactant secretion and collagen production. Thus injury of alveolar epithelial cells may contribute to the development of lung diseases induced by cigarette smoke. However, the molecular mechanisms of alveolar epithelial cell injury caused by cigarette smoke remain unclear.β-cantenin is a multifunctional protein that plays an important role in cellular development, cell adhesion, repair and injury, cell cycle regulation and tumor formation. It is not only associated with E-cadherin to maintain strong cell-cell adhesion and tissue integrity in epithelium, but also a key component in Wnt signaling to regulate the expression of a variety of genes by its translocation to the nucleus and interaction with transcription factor TCF/LEF (T cell factor/lymphoid enhancer factor). Recent researches demonstrated that nicotine and NNK, two components of cigarette smoke, increased phosphorylation of glycogen synthase kinase 3 (GSK3) in vitro. GSK3βacts as a key and negative regulator of the classical Wnt/β-catenin signaling pathway, and a primary kinase responsible for phosporylation and down-regulation ofβ-cantenin levels. Our former studies showed that the expression and location ofβ-cantenin altered in the injury and repair process of airway epithelium induced by cigarette smoke. But it is unclear whether GSK3βandβ-cantenin/TCF signaling are involved in alveolar epithelial cell injury caused by cigarette smoke. In the present study, we examined the expression of GSK3βin cultured alveolar epithelial cell line (A549), and further investigated the role of GSK3βandβ-cantenin/TCF signaling in alveolar epithelial cell injury caused by cigarette smoke in vitro. Immunofluorescence demonstrated that the expression of GSK3βwas high in A549 cells. Western blot analysis showed that cigarette smoke extract (CSE) notably inhibited GSK3βby reducing GSK3βexpression and increasing inactive phosphorylated GSK3βin a dose-dependent manner.Moreover, it was found by Western blot that CSE increased the expression ofβ-catenin protein in a dose-dependent manner and induced nuclear translocation ofβ-catenin. It was also demonstrated that CSE activatedβ-cantenin/TCF signaling by transient transfection of TCF luciferase reporter plasmids(pGL3-OT)followed by CSE treatment, using the mutant of TCF luciferase reporter plasmids (pGL3-OF) as controls.Finally, we further studied the role of GSK3βin the activation ofβ-cantenin/TCF signaling induced by CSE. We detected the expression of GSK3βandβ-catenin after transient transfection of a stable mutant of GSK3β(GSK3βS9A), which is continuously active and unable to be inhibited by the upstream kinase of GSK3β. It was observed that the GSK3βprotein level was increased dramatically and the expression ofβ-catenin was reduced significantly. These suggest that the high expression of active GSK3βpromote the degradation ofβ-catenin. Then, we examined theβ-catenin/TCF transcriptional activity after cotransfected GSK3βS9A with the TCF luciferase reporter plasmids followed by CSE treatment. We demonstrated that active GSK3βinhibitedβ-catenin/TCF transcriptional activity induced by CSE (P<0.01, compared with the group of CSE treatment). This result suggests that CSE activatesβ-cantenin/TCF signaling via inhibiting GSK3βactivity.Taken together, these data suggest: (1) CSE can inhibit GSK3βby reducing GSK3βexpression and increasing inactive phosphorylated GSK3βin A549 cells. (2) CSE can increase the expression ofβ-catenin and induce nuclear translocation ofβ-catenin in A549 cells. (3) CSE activatesβ-cantenin/TCF signaling via inhibiting GSK3βactivity, implying a possible mechanism responsible for alveolar epithelial cell injury caused by cigarette smoke.