Tumor Suppressor PTEN Inhibits Human Airway Smooth Muscle Cells Migration

BackgroundBronchial asthma is considered to be a chronic airway inflammatory disease, characterized by airway wall remodeling,airway eosinophilic inflammation,and airway hyperresponsiveness(AHR).Airway remodeling in asthma is a complex process that involves structural changes in virtually all tissues of the airway wall.The histologic changes to the airways consist of epithelial proliferation and goblet cell differentiation,subepithelial fibrosis,airway smooth muscle(ASM) growth, angiogenesis,matrix protein deposition,gland hyperplasia and hypertrophy. Cytokines,chemokines,and growth factors from inflammatory cells and structural cells contribute to remodeling.Smooth muscle cells,which are seen in grate numbers and size,play an important role in airway remodeling,which are considered as the most important cells in the procedure of airway remodeling.The changes of ASMCs like contraction,proliferation,hypertrophy and excreting ECM,which also promote the airway remodeling and AHR.In the past several years,some researches found that,like the migration of vascular smooth muscle cells(VSMCs) in atherosclerosis,ASMCs also possesses the capability of migrating.ASMCs migrates to the tunica intima of airway and proliferate,which resulting in the airway thicken and narrow.Although there is,as yet, no direct evidence showing that ASMCs migrate in vivi in the adult airway,it is possible that ASMCs migration is an important aspect of the airway remodeling. Moreover,it is also possible that the migration of ASMCs toword the lumen of the airway plays a role in the appearance of myofibroblasts in the lamina reticularis of the asthmatic airway.The myfibroblast has an intermediate betwen differentiated smooth muscle cells and fibroblasts.By electron microscopy,myfibroblasts of the airway are elongate cells and located in the lamina reticularis,a layer that lies between the true epithelial basement membrane and the airway smooth muscle layer.Thus,the myofibroblasts may comes from the migtated ASMCs.PTEN is a dual-specificity phosphatase with both protein phosphatase and lipid phosphatase activity.PTEN is the first phosphatase identified as a tumor suppressor. Not since the discovery of p53 has a tumor suppressor generated such interest.Initial studies performed on cancer cell lines suggested that PTEN may be responsible for almost all types of cancer,both solid tumors and hematological malignancies.The deletion of PTEN has been associated with advanced stage tumors or metastatic disease.PTEN has been shown to play a pivotal role in apoptosis,cell cycle arrest, and possibly cell migration.The first report about that PTEN plays a pivotal role between airway inflammation and airway hyperresponsiveness was shown in 2003, the activity and protein levels of PTEN were decreased significantly after ovalbumin inhalation,Administration of adenoviruses carrying PTEN cDNA reduced the symptoms of asthma and decreased the increased levels of plasma extravasation and IL-4,IL-5and eosinophil cationic protein(ECP) in allergen-induced asthmatic lungs. A lot of studies have confirmed that PTEN can inhibit the proliferation and migration of tumour cell,myocardium and vascular smooth muscle through inhibit Akt,ERK_1/2 and FAK signal pathways.And these signal pathways also takes part in migration of ASMCs,we want to confirm that they have the same effect.This research will concentrate on two aspects: First,To construct a recombinant adenovirus vector containing PTEN through the pAdxsi system.Second,to study the role of PTEN in the migration of ASMCs.Methords1,The bronchus samples were got from the Pulmonary lobectomy,chest surgery department of nanfang hospital,after obtaining the permission of the lung cancer patients. 2,ASMCs was primarily cultured by explant method from the trachea of human,and identified by light inverted microscope and cell immunohistochemistry.3,Recombinant shuttle plasmid and recombinant adenovirus vector were confirmed by restrictive digestion,and we got a 1.3 kb small fragment and digestion of strap. The recombinant adenovirus which has the capability of transfection was packaged and propagated in HEK293 cell.The viral titer was examined by plaque assay and the mRNA of PTEN in cell were determined by polymerase chain reaction(PCR).The PTEN recombinant adenovirus is constructed successfully.3,In vitro wound healing assay,as shown in our results,with the approximate same wound area at 0 h,and the of the cell number of cell-free areas of wounds in Ad-PTEN(38.40±7.73)-treated were significantly larger than that treated with the Ad-GFP(94.40±12.05)- and uninfected(83.20±7.59)-groups at 48h.The decrease did not appear to be due to the effects of the adenovirus since it showed no inhibitory effect on cell migration,because there was no significant differences were observed in the in vitro cell migration between the uninfected-and Ad-GFP-treated groups.And PDGF treatment significantly increased the migration of ASM cells into the wounds. These results indicated that the migration of ASM cells was down-regulated by expression of PTEN.4,We performed in vitro transwell chamber analysis.As shown in the results,the migration ability of ASMCs in the uninfected(658.80±61.81)-and Ad-GFP(658.00±46.58)-treated groups was obviously higher than that in the Ad-PTEN(206.40±20.48)-treated groups but not significantly difference with that in the two control groups.And PDGF induced significant increases in migration in uninfected and Ad-GFP-infected ASM cells.These results also demonstrated that PTEN overexpression inhibited both basal and PDGF-induced ASM cells migration.5,There is a little of stress fiber and no filopodia in the Ad-GFP,DMEM control groups,while ASMC were pretreated with Ad-PTEN,the changes of actin cytoskeleton and stress fibers were inhibited.When three groups were stimulated with PDGF-BB(10nmol/L),ASMCs induced reorganization of actin cytoskeleton and formation of stress fibers in 60 minues.6,Western blot:(1)The ratio of p-Akt/Akt in ASMCs after were infected with Ad-PTEN,Ad-GFP and DMEM was as follows:0.059±0.001,0.503±0.001,0.502±0.001.But when the three groups were stimulated with 10 ng/ml of PDGF significantly decrease the p-Akt expression in Ad-PTEN group(P＜0.00),compared with control group,there is no significant difference(P＞0.054).So we can comfirm that PTEN inhibit migration of ASMCs by block Akt.(2) The ratio of p-ERK_1/2/ ERK_1/2 in ASMCs after were infected with Ad-PTEN,Ad-GFP and DMEM was no significant difference(P＞0.15).(3) The ratio of p-FAK/FAK in ASMCs after were infected with Ad-PTEN,Ad-GFP and DMEM was as follows:0.099±0.002,0.647±0.001,0.649±0.001.But when the three groups were stimulated with 10 ng/ml of PDGF significantly decrease the p-FAK expression in Ad-PTEN group(P＜0.00), compared with control group,there is no significant difference(P＞0.767).So we can comfirm that PTEN inhibit migration of ASMCs by block FAK.7,All statistical tests were operated with the program SPSS13.0,all the data was expressed by(?)±S,Factorial exprimengt was applied in the comparison of more samples.We accepted statistical significant values of P＜0.05.