Cigarette smoke extract-induced p120-mediated NF-κB activation in human epithelial cells is dependent on the RhoA/ROCK pathway.

Cigarette smoke exposure is a major cause of chronic obstructive pulmonary disease (COPD), but the underlying molecular inflammatory mechanisms remain poorly understood. Previous studies have found that smoke disrupts cell-cell adhesion by inducing epithelial barrier damage to the adherens junction proteins, primarily E-cadherin (E-cad) and p120-catenin (p120). Recently, the anti-inflammatory role of p120 has drawn increasing attention. In this study, we demonstrate that p120 has a role in the cigarette smoke extract-induced inflammatory response, presumably by regulating NF-κB signaling activation. Mechanistically, we show that p120-mediated NF-κB signaling activation in airway epithelial inflammation is partially RhoA dependent and is independent of E-cad. These results provide novel evidence for the role of p120 in the anti-inflammatory response.

p120-Catenin modulating nuclear factor-κB activation is partially RhoA/ROCKdependent in scratch injury.

p120-catenin (p120) is known as a cadherin-associated protein that participates in tumor metastasis and invasion, as well as an anti-inflammatory mediator. Recently, its anti-inflammatory role is drawing increasing attention, but the regulatory mechanisms are still unknown. Here, we report that p120 modulated inflammatory responses partially depends on RhoA/ROCK pathway in scratch-induced injury in human bronchial epithelial cells (BECs). For the first time, we found that p120 was significantly reduced in BECs after scratching, which could induce interleukin-8 (IL-8) production through nuclear factor-κB (NF-κB) activation accompanied with IκBα phosphorylation. Over-expression of p120 3A could inhibit NF-κB activation and IL-8 mRNA expression and protein synthesis after scratching, while p120 knockdown by small interfering RNA could promote NF-κB activation and IL-8 mRNA expression and protein synthesis after scratching. Furthermore, we found that RhoA was the binding partner of p120 in BECs. Although total RhoA and p120-binded RhoA remained unchanged, the RhoA activity was increased after scratching. Chemical blockade of RhoA/ROCK signaling (Y27632) inhibited scratch-induced nuclear translocation of NF-κB p65. Over-expression of p120 3A attenuated scratch-induced RhoA activation, whereas silence of p120 significantly elevated scratch-induced RhoA activation in BCEs. Conclusively, these results indicate an anti-inflammatory effect of p120 in bronchial epithelial cells through its modulation of NF-κB signaling depending on RhoA/ROCK pathway.

p120 modulates LPS-induced NF-κB activation partially through RhoA in bronchial epithelial cells.

p120-Catenin (p120) is an adherens junction protein recognized to regulate cell-cell adhesion. Emerging evidence indicates that p120 may also play an important role in inflammatory responses, and the regulatory mechanisms are still unknown. In the present study, we showed that p120 was associated with airway inflammation. p120 downregulation induced nuclear factor-κB (NF-κB) activation, accompanied with I κ B α degradation, p65 nuclear translocation, and increased expression of interleukin-8 (IL-8) in lipopolysaccharide (LPS)- treated C57BL mice and human bronchial epithelial cells (BECs). Moreover, we first found that p120 directly coprecipitated with RhoA in BECs. After LPS stimulation, although total RhoA and p120-bound RhoA were unchanged, RhoA activity was increased. Y27632, a ROCK inhibitor, could partially inhibit nuclear translocation of p65. Overexpression of p120 inactivated RhoA and NF-κB in BECs, whereas p120 loss significantly increased RhoA activity, p65 nuclear translocation, and IL-8 expression. Taken together, our study supports the regulatory role of p120 in airway inflammation and reveals that p120 may modulate NF-κB signaling partially through RhoA.

Epithelial-mesenchymal transition and apoptosis of renal tubular epithelial cells are associated with disease progression in patients with IgA nephropathy.

The aim of the present study was to examine the effects of epithelial-mesenchymal transition (EMT) and apoptosis of renal tubular epithelial cells on the prognosis of immunoglobulin A (IgA) nephropathy. Renal biopsy tissues from 74 cases of IgA nephropathy were divided into a mild mesangial proliferation group (27 cases), a focal hyperplasia group (28 cases) and a proliferative sclerosis group (19 cases). The blood pressure, serum creatinine and 24 h urinary protein excretion of all patients were detected. To define EMT, α-smooth muscle actin (α-SMA), vimentin and collagen fibers were assessed. Apoptosis was determined by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). The blood pressure, serum creatinine and 24 h urinary protein excretion of patients with IgA nephropathy altered with increasing pathological grade. All clinical indices of patients in the proliferative sclerosis group were higher than those of the other two groups, and the 24 h urinary protein excretion of the focal hyperplasia group was statistically higher than that of the mild mesangial proliferation group. The expression of tubular interstitial α-SMA, vimentin and collagen fibers increased with the pathological grade and was closely correlated with clinical indices, including collagen fibers and 24 h urinary protein excretion. TUNEL-positive cells increased with the exacerbation of pathological changes. The EMT and apoptosis of renal tubular epithelial cells reflected the clinical severity of IgA nephropathy. α-SMA, vimentin and the apoptotic index may be used as important markers for evaluating the prognosis of IgA nephropathy.

Involvement of p120 in LPS-induced NF-kappaB activation and IL-8 production in human bronchial epithelial cells.

P120-catenin (p120), a prototypic member of a subfamily of Armadillo repeat domain (Arm domain) proteins, not only participates in cell-cell adhesion, but also mediates inflammatory responses in the skin. In the present study, we demonstrated the effect of p120 on lipopolysaccharide (LPS)-induced inflammatory responses in human bronchial epithelial cells (BECs). We first confirmed that p120 expression was significantly reduced after LPS stimulation in BECs, the p65 subunit of nuclear factor-kappaB (NF-kappaB) nuclear translocation was promoted and NF-kappaB activity was rapidly induced. Moreover, the expression level of interleukin-8 (IL-8) increased after LPS treatment. Over-expression of p120 attenuated LPS-stimulated NF-kappaB reporter gene expression and IL-8 mRNA expression and protein synthesis. On the contrary, transfection with p120 small interfering RNA (siRNA) significantly elevated LPS-stimulated NF-kappaB transcriptional activity, p65 nuclear translocation and IL-8 expression. Collectively, these results indicate an anti-inflammatory effect of p120 in BECs, through its modulation of NF-kappaB signaling.

Cigarette smoke extract induces activation of beta-catenin/TCF signaling through inhibiting GSK3beta in human alveolar epithelial cell line.

Cigarette smoke is known to have various injurious and cytotoxic effects on alveolar epithelial cells. However, the mechanism about the effects caused by cigarette smoke on alveolar epithelial cells remains unclear. In the present study, we first validated that cigarette smoke extract (CSE) impaired the viability of alveolar epithelial cells (A549 cells) and resulted in some morphological changes. Next, we found that glycogen synthase kinase 3beta (GSK3beta) was highly expressed in A549 cells, and CSE significantly inhibited GSK3beta by reducing GSK3beta expression and increasing inactive phosphorylated GSK3beta. It was also observed that CSE promoted beta-catenin accumulation and nuclear translocation, and further activated beta-catenin/TCF signaling. Finally, we demonstrated that GSK3beta over-expression promoted the degradation of beta-catenin and abolished beta-catenin/TCF transcriptional activity that was induced by CSE in alveolar epithelial cells. These results suggest that CSE induces the activation of beta-catenin/TCF signaling through inhibiting GSK3beta, implying a possible mechanism responsible for the injurious and cytotoxic effects on alveolar epithelial cell caused by cigarette smoke.

Bleomycin-induced nuclear factor-kappaB activation in human bronchial epithelial cells involves the phosphorylation of glycogen synthase kinase 3beta.

Nuclear factor-kappaB (NF-kappaB) plays a central role in the development of bleomycin (BLM) lung toxicity, but the regulatory mechanisms are still unknown. In the present study, we investigated the cytotoxic effect of BLM on cultured human bronchial epithelial cells (BECs) and first confirmed that BLM induced the transcriptional activation of NF-kappaB signaling in BECs. We also found that BLM activated Akt (protein kinase B, PKB) and increased the phosphorylation level of glycogen synthase kinase 3beta (GSK3beta). GSK3beta is known to be a key downstream target of Akt, and LY294002, the PI3K (phosphatidylinositol 3-kinase)/Akt inhibitor, which promoted the dephosphorylation of GSK3beta, significantly attenuated BLM-induced NF-kappaB activation. Next, we further observed that constitutively active GSK3beta stabilized the inhibitor of NF-kappaB (IkappaBalpha), inhibited p65 nuclear translocation and partially blocked BLM-induced NF-kappaB activation. Importantly, a co-immunoprecipitation assay revealed that GSK3beta formed a complex with IkappaBalpha, while GSK3beta phosphorylation caused by BLM led to their dissociation. These results suggest that BLM can induce the activation of NF-kappaB signaling in BECs and this process is tightly associated with the phosphorylation status of GSK3beta, implying a possible regulatory mechanism of NF-kappaB signaling in BECs during the toxic lung injury induced by BLM.

Glycogen synthase kinase 3beta induces apoptosis in cancer cells through increase of survivin nuclear localization.

Glycogen synthase kinase 3beta (GSK3beta) regulates numerous signaling pathways that control a wide range of cellular processes, including cell proliferation, differentiation, apoptosis and metabolism. We report a novel function of GSK3beta: It interacts with the inhibitor-of-apoptosis protein (IAP) survivin to modulate its expression, thus regulating apoptosis in human lung cancer cells. A co-immunoprecipitation assay revealed that GSK3beta can bind survivin. Activation of GSK3beta induced translocation of survivin from the cytoplasm to the nucleus, resulting in G1 cell-cycle arrest and apoptosis, as well as sensitization to the chemotherapeutic drug doxorubicin. In contrast, inactivation of GSK3beta, either by transfection of a dominant-negative mutant inhibitor DN-GSK3beta or with selective inhibitor LiCl, increased cytoplasmic survivin expression, leading to cell-cycle progression and resistance to apoptosis. These results identify a pro-apoptotic role for GSK3beta in cancer cells, through its modulation of survivin in subcellular redistribution. This new role suggests that there is a potential for pharmacologic activation of GSK3beta to enhance treatment of cancer patients, including those with resistance.

IQGAP1 activates Tcf signal independent of Rac1 and Cdc42 in injury and repair of bronchial epithelial cells.

The process of injury and repair involves spreading, migration and cell proliferation. The functions of Rho GTPases and their effector IQGAP1 are poor known in this process of airway epithelium. In the present study, we employed a widely used in vitro model by scratching a monolayer of BECs. We found that scratching induced decreasing of the GTP-bound Rac1 and Cdc42, but increasing the amounts of IQGAP1 at different time points. Next, we confirmed that IQGAP1 interacted with the constitutively active Rac1 (Rac1(V12)) and Cdc42 (Cdc42(V12)) rather than the dominant negative Rac1 (Rac1(N17)) and Cdc42 (Cdc42(N17)). Over-expressions of wild type (WT) IQGAP1 and its mutant (T1050AX2), which was defective to interact with Rho GTPases, induced translocation of beta-catenin from the cytoplasm into the nucleus. These results activated Tcf/Lef and increased the expression levels of its target genes of c-myc and cyclin D1. Likewise, the amounts of c-myc and cyclin D1 increased after scratching. Our results suggested that IQGAP1 mediated cell proliferation through activating Tcf in a manner independent of Rac1 and Cdc42 in wound repair of BECs.

IQ domain GTPase-activating protein 1 mediates the process of injury and repair in bronchial epithelial cells.

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 ß-catenin from the cytoplasm into the nucleus and activated the Tcf/Lef signal. Scratching altered the associations of IQGAP1 with ß-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.

IQGAP1 promotes cell proliferation and is involved in a phosphorylation-dependent manner in wound closure of bronchial epithelial cells.

The re-epithelialization process of the airway involves spreading and migration followed by cell proliferation. Scaffold IQ domain GTPase-activating protein (IQGAP1), an effector of Rho GTPases, is a key component in a series of cell processes, although its exact mechanism in injury and repair of the airway is still unclear. In this study, we utilized a widely used model in vitro by scratching bronchial epithelial cells (BECs). At different time points after scratching, the amounts of IQGAP1 in mRNA and protein were greater than that in the control. PKCepsilon-mediated phosphorylation of IQGAP1 was involved in the process of injury and repair. The overexpression of PKCepsilon or treatment with phorbol-12-myristate-13-acetate (the PKC activator) promoted wound closure. On the contrary, the group treated with GF109203X (the PKC inhibitor) had the opposite effect. Scratching or overexpression of IQGAP1 induced increasing amounts of total beta-catenin and the transposition of beta-catenin from the cytoplasm into the nucleus. These results activated the T cell factor/lymphoid enhanced factor and induced expression levels of its target genes of c-myc and cyclin D1. The reduction of IQGAP1 by the transfection of small interference RNA of IQGAP1 attenuated these effects and directly impaired the scratching-induced wound closure. Taken together, our results suggest that IQGAP1 promotes cell proliferation and phosphorylation of IQGAP1 is involved in the process of wound closure in BECs.

beta-catenin/Tcf signaling in squamous differentiation of porcine airway epithelial cells.

For a preliminary study of the role of beta-catenin/Tcf signaling in squamous differentiation of airway (tracheobronchial) epithelial cells, a stable mutant of beta-catenin was transfected into primarily cultured porcine airway epithelial cells. Western blotting revealed that exogenous protein was observed in large quantity in cytoplasm and nucleus. When co-transfected with Tcf luciferase reporter plasmids, beta-catenin mutant increased the reporter's transcriptional activities. However, mRNA expression of a squamous differentiation marker, small proline-rich protein (SPRP), was not elevated, as shown by reverse transcription-polymerase chain reaction. These findings suggest that beta-catenin/Tcf signaling may not be directly involved in the squamous differentiation of porcine airway epithelial cells.

[Mutations of p53 gene in 41 cases of human brain gliomas].

BACKGROUND & OBJECTIVE: p53 gene plays an important role in regulating cell cycle, maintaining completeness of cellular genomes, inducing cell differentiation and apoptosis. p53 gene mutation occurs usually in gliomas, especially astrocytomas. This study was to investigate p53 gene mutation and its correlation to the development of glioma. METHODS: p53 gene mutation in 41 specimens of human gliomas was analyzed by polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) and DNA sequencing. RESULTS: p53 gene mutations were found in 17 (41.5%) of the 41 specimens of gliomas. The p53 mutations located on exons 5-8: 7 cases (41.2%) on exon 5; 1 (5.9%) on exon 6; 4 (23.5%) on exon 7; 5 (29.4%) on exon 8. The mutation rate of p53 gene was significantly higher in grade III-IV gliomas than in grade I-II gliomas (P<0.01). DNA sequencing indicated that the p53 gene mutations were point mutations and deletions of exons in the 17 positive cases, including 13 cases (76.5%) of missense mutation, 2 cases (11.8%) of samesense mutation, and 2 cases (11.8%) of frame shift mutation. G_A and A-->G were major base mutations (55.6%). CONCLUSIONS: p53 mutations always locate on exons 5 and 8. Missense mutation is major mutant type of p53 gene. p53 mutation plays an important role in the development and malignant transformation of human gliomas.

Stem cell factor/c-kit signaling mediated cardiac stem cell migration via activation of p38 MAPK.

OBJECTIVE: It was reported that there are cardiac stem cells (CSCs) in the rat heart, and they could reconstitute well-differentiated myocardium that are formed by blood-carrying new vessels and myocytes. However, how do the CSCs migrate into the peri-infarcted areas after myocardial infarction (MI)? It remains entirely unknown about the signal transduction involved in the migration of CSCs. METHODS AND RESULTS: Rat heart MI was induced by left coronary artery ligation. Both immunohistochemical staining and Western blotting analysis was performed to detect the expression of SCF protein, and RT-PCR was conducted for the expression of SCF mRNA. Cardiac stem cells were isolated from rat hearts, and a cardiac stem cell migration assay was performed using a 48-well chemotaxis chamber system. On day 5 after MI in rats, the expression of stem cell factor (SCF) mRNA and protein was significantly increased in the peri-infarcted area, which was matched with more accumulation of CSCs in the region and improvement of cardiac function, which was blocked by p38 MAPK selective inhibitor SB203580. In in vitro experiments, SCF induced CSC migration in a concentration-dependent manner, and the antibody against SCF receptor (c-kit) blocked the SCF-induced CSC migration. Western blot analysis showed that the phosphorylated p38 MAPK (Phospho-p38 MAPK) was highly increased in the SCF-treated CSCs, and the inhibition of p38 MAPK activity significantly attenuated SCF-induced the migration of CSCs. CONCLUSION: It demonstrated that SCF/c-kit signaling may mediate the migration of CSCs via activation of p38 MAPK.

Lysosomal acid lipase over-expression disrupts lamellar body genesis and alveolar structure in the lung.

The functional role of neutral lipids in the lung is poorly understood. Lysosomal acid lipase (LAL) is a critical enzyme in hydrolysis of cholesteryl esters and triglycerides to generate free fatty acids and cholesterol in lysosomes. Human LAL was over-expressed in a doxycycline-controlled system in mouse respiratory epithelial cells to accelerate intracellular neutral lipid degradation and perturb the surfactant homeostasis in the lung. In this animal system, neutral lipid concentrations of pulmonary surfactant were reduced in bronchoalveolar lavage fluid (BALF) in association with decrease of surfactant protein C (SP-C) gene expression. The size and the number of lamellar bodies in alveolar type II epithelial cells (AT II cells) were significantly reduced accordingly. The number of macrophages required for surfactant recycling in BALF was also significantly reduced. As a result of these combinatory effects, emphysema of the alveolar structure was observed. Taken together, neutral lipid homeostasis is essential for maintenance of lamellar body genesis and the alveolar structure in the lung.

[Activation and prognostic significance of AKT, NF-kappaB and STAT3 in breast cancer with lymph node metastasis and estrogen receptor expression].

BACKGROUND & OBJECTIVE: Being downstream targets of the phosphatase and tensin homolog deleted on chromosome ten (PTEN) and epidermal growth factor receptor (EGFR) pathways, serine/threonine kinase AKT, nuclear factor-kappaB (NF-kappaB) and signal transducer and activator of transcription-3 (STAT3), play important roles in cell proliferation, apoptosis and oncogenesis. This study was to investigate the activation and prognostic values of AKT, NF-kappaB and STAT3 in breast cancer with lymph node metastasis and estrogen receptor (ER) expression. METHODS: The expression of phosphatized AKT (p-AKT), NF-kappaB (p-NF-kappaB) and STAT3 (p-STAT3), and the expression of EGFR, PTEN, HER-2, and Ki67 in tissue samples from 130 breast cancer patients with lymph node metastasis and ER expression were detected by immunohistochemistry. RESULTS: The activity of AKT (p-AKT) and NF-kappaB (p-NF-kappaB) were correlated to HER-2 overexpression (P=0.023 and P=0.017) and histological grade of breast cancer (P=0.035 and P=0.004). The expression of p-AKT, p-NF-kappaB and p-STAT3 had no correlation to tumor size, EGFR overexpression, and proliferation index assessed by Ki67. The expression of p-AKT was positively correlated to that of p-NF-kappaB (r=0.43, P<0.001) and negatively correlated to that of p-PTEN (r=-0.20, P=0.002). The overexpression of p-AKT and p-NF-kappaB were significantly related to shorter survival (P=0.002 and P=0.003). The up-regulation of p-NF-kappaB expression was also related to enhanced risk of recurrence and metastasis (P=0.006). Cox multivariate analysis showed that the expression of p-AKT and p-NF-kappaB were correlated to shorter overall survival (OS) (P=0.017 and P=0.008) and disease-free survival (DFS) (P=0.005 and P=0.012), while the expression of p-STAT3 had no correlation to OS (P=0.332) and DFS (P=0.237). CONCLUSIONS: The activation of AKT and NF-kappaB, but not STAT3, significantly contributes to the progression of breast cancer. Activated AKT and NF-kappaB may indicate poor prognosis of breast cancer with lymph node metastasis and ER expression.

Synergistic anti-tumor effect of recombinant chicken fibroblast growth factor receptor-1-mediated anti-angiogenesis and low-dose gemcitabine in a mouse colon adenocarcinoma model.

AIM: To evaluate whether the combination of recombinant chicken fibroblast growth factor receptor-1 (FGFR-1) protein vaccine (cFR-1) combined with low-dose gemcitabine would improve anti-tumor efficacy in a mouse CT26 colon adenocarcinoma (CT26) model. METHODS: The CT26 model was established in BABL/c mice. Seven days after tumor cell injection, mice were randomly divided into four groups: combination therapy, cFR-1 alone, gemcitabine alone, and normal saline groups. Tumor growth, survival rate of tumor-bearing mice, and systemic toxicity were observed. The presence of anti-tumor auto-antibodies was detected by Western blot analysis and enzyme-linked immunospot assay, microvessel density (MVD) of the tumors and tumor cell proliferation were detected by Immunohistochemistry staining, and tumor cell apoptosis was detected by TdT-mediated biotinylated-dUTP nick end label staining. RESULTS: The combination therapy results in apparent decreases in tumor volume, microvessel density and tumor cell proliferation, and an increase in apoptosis without obvious side-effects as compared with either therapy alone or normal control groups. Also, both auto-antibodies and the antibody-producing B cells against mouse FGFR-1 were detected in mice immunized with cFR-1 vaccine alone or with combination therapy, but not in non-immunized mice. In addition, the deposition of auto-antibodies on endothelial cells from mice immunized with cFR-1 was observed by immunofluorescent stain-ing, but not on endothelial cells from control groups. Synergistic indexes of tumor volume, MVD, cell apoptosis and proliferation in the combination therapy group were 1.71 vs 1.15 vs 1.11 and 1.04, respectively, 31 d after tumor cell injection. CONCLUSION: The combination of cFR-1-mediated anti-angiogenesis and low-dose gemcitabine synergistically enhances the anti-tumor activity without overt toxicity in mice.

Anti-angiogeneic target therapy for cancer with vaccine based on the recombinant chicken FGFR-1 in tumor-bearing mice.

To explore the anti-tumor effect of immunotherapy with recombinant protein vaccine based on FGFR-1 of chicken (cFR-1) in a mouse Meth A fibrosarcoma model, tumor volume and survival rate of the mice were observed at a 3-day interval. Microvessel density (MVD) was detected by immunohistochemistry. Auto-antibodies against self-FGFR-1 were detected by Western blotting and ELISA, respectively. The anti-FGFR-1 antibody-producing B cells (APBCs) were detected by enzyme-linked immunospot (ELISPOT) assay. Eighteen days after inoculation of tumor cells, the tumor volume was significantly smaller in cFR-1-immunized group than in mouse FGFR-1 (mFR-1) immunized group and normal saline (NS) control group (P<0.05), and the survival time was significantly longer in cFR-1-immunized group than in the control groups (P<0.01). MVD was significantly lower in cFR-1-immunized group than in mFR-1-immunized group and NS group (16.8+/-5.6 vs 64.6+/-1.8 and 59.6+/-8.7, P<0.01). Antibodies against self-FGFR-1 were found in mFR-1-immunized group, the major antibody subclasses were IgG1 and IgG2b. Compared with the two control groups, the numbers of APBCs in cFR-1-immunized group were significantly increased (P<0.01) These results demonstrated that the cFR-1-related anti-angiogenesis protein vaccine could induce the production of auto-antibodies against self-FGFR-1, which futher inhibit angiogenesis and growth of solid tumor.

[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].

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.

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.