The Effects Of Cigarette Smoke Extract On MUCIN 5AC Expression And Its Signal Transduction Mechanism

Objective To explore the effects of cigarette smoke extract on mucin(MUC) 5AC expression in the airway epithelial cells and its potential signal transduction pathway.Methods (1) The well cultured airway epithelial cells of the line A549 were exposed to serial dilutions of cigarette smoke extract(CSE) for 24 hours,the effect of CSE on epithelial proliferation was assayed using methyl thiazolyl tetrazoliu(MTT) method. the levels of mRNA and protein for MUC5AC in each group were detected by RT-PCR and ELISA respectively; also the content of reactive oxygen species(ROS) were measured using a special kit. (2) A549 airway epithelial cells were either incubated with 20% CSE alone or with the addition of TNF-αprotease inhibitor(TAPI)-1,also some were preincubated with ROS scavenger DMTU before exposure to CSE.The MUC5AC mRNA levels were analyzed by RT-PCR,MUC5AC mucin protein and soluble transforming growth factor(TGF)-αwere detected by enzyme linked immunosorbent assay( ELISA) respectively,and phosphorylated epidermal growth factor receptor(p-EGFR),extracelluar signal-regulated kinase(ERK) protein production was assayed by western blot.(3) Also the A549 airway epithelial cells line were pretreated with SP600125,which was a potent and selective c-Jun N-terminal kinase(JNK) inhibitor or with Src kinase selective inhibitor PP2,then cultured in the presence of CSE,The levels of MUC5AC protein in culture supernatant,activated EGFR,c-Src and MUC5AC mRNA in culture cells were measured by ELISA,Western Blot and RT-PCR,respectively.(4)A549 cells divided into 4 groups::TNF-αgroup;10%CSE group;10%CSE,TNF-αcombination group and serum free medium as negative control,after 24 hours incubation,MUC5AC protein and mRNA levels were measured by ELISA and RT-PCR, p-EGFR,p-c-Src protein were analyzed by western blot,the expression of MUC5AC and nuclear factor(NF)-κBp65 protein were assayed with immunocytochemistry(ICC ).Results (1)At low concentrations, CSE increased proliferation of A549 cells, whereas high concentrations were inhibitory as a result of cytotoxicity.CSE significantly increased MUC5AC gene expression and protein synthesis dose dependently.the highest effect observed in the 30%CSE-treated group,with a high concentration of ROS.(2)the expression of soluble TGF-α,p-EGFR and p-ERK protein were also upregulated by CSE obviously.Pretreatment of the cells with TACE inhibitor TAPI-1 decreased CSE-induced MUC5AC gene expression,MUC5AC mucin production,soluble TGF-α,p-EGFR,p-ERK and production in a dose dependent manner,but have no significant effect on JNK phosphorylation.DMTU could also inhibited CSE-induced mucous hypersecretion and EGFR,ERK and JNK activation(all p<0.05) . Pretreated with DMTU decreased the level of MUC5AC mRNA,MUC5AC protein,and soluble TGF-α(all p<0.05,compared with CSE-stimulated alone),however,it has little effect on the high expression of MUC5AC mRNA and MUC5AC protein in exogenous TGF-αtreated group(p>0.05).(3)SP600125 significantly reduced CSE-induced mucin production at both secretional and transcriptional level,but no significant changes in EGFR phosphorylation were observed.Src kinase selective inhibitor PP2 had the similar effect of SP600125,primarily by decreasing the phosphorylation of JNK.Also DMTU reduced c-Src kinase activation obviously.(4)MUC5AC protein,MUC5AC mRNA and p-EGFR increased in each group exposed to CSE,TNF-αalone,and the response was enhanced when CSE was used in combination with TNF-α.Immunocytochemistriy demonstrated high expression of NF-κB p65 in resting A549 cells,with the protein homogeneously distributed throughout the cytoplasm and absent from the nucleus. However, upon TNF-αtreatment, this pattern shifted, and NF-κBp65 was found primarily around or within the nucleus.Nuclear translocation was clear after 3 h,By 24 h,NF-κBp65 was again localized primarily in the cytoplasm and not in the nucleus.All the cells were undergone serum-free starvation for 24 h before any intervention in order to retain its basal expression.Conclusions (1)CSE can induce MUC5AC expression in the airway epithelial cells A549,with a high concentration of Reactive Oxygen Species.(2)CSE can induce MUC5AC expression through a signaling pathway involving ROS/TACE/EGFR.(3)Apart from the EGFR tyrosinase autophosphorylation dependent way,ROS/Src/JNK signal pathway also play a part in the CSE-induced MUC5AC expression and mucous hypersecretion,in a EGFR independent manner.(4)TNF-αsynergistically increased gene expression and protein production of MUC5AC mucin induced by CSE in human airway epithelial A549 cells,with the nuclear translocation and activation of nuclear factor-κB.