The Molecular Mechanisms Underlying Pseudomonas Aeruginosa Lipopolysaccharide-induced MUC5AC Mucin Expression In Airway Epithelial Cells

IntroductionMucin overproduction is a hallmark of chronic inflammatory airway diseases, such as asthma,chronic obstructive pulmonary disease,and cystic fibrosis,while currently there is no effective treatment for mucin overproduction.Elucidating the mechanisms underlying the regulation of airway mucin gene expression could have important significance for finding potential therapy targets and developing new drugs against airway mucin overproduction.MUC5AC mucin is a major component of airway mucus,so it is very important to investigate the mechanisms underlying the regulation of MUC5AC.Many kinds of bacterias can induce upregulation of MUC5AC mucin,including Pseudomonas aeruginosa(PA)and its lipopolysaccharide(LPS)which is a main cause of respiratory infection.However,the molecular mechanisms underlying PA-LPS-induced MUC5AC overproduction still remain largely unknown.Previous studies have demonstrated that PA-LPS induced MUC5AC expression in human airway epithelial cells via tumor necrosis factorαconverting enzyme(TACE)→Transforming growth factor-α(TGF-α)→Epidermal growth factor receptor(EGFR) cascade,however,the molecular mechanisms by which PA-LPS leads to TACE activation still remain unclear.Recently a study showed that Reactive oxygen species (ROS)can induce TACE activation,and neutrophil elastase(NE)upregulates airway MUC5AC expression via signaling pathway ROS→TACE→TGF-α→EGF.Because previous studies have confirmed that LPS can induce ROS production of airway epithelial cells,we hypothesize that ROS may play an important role in PA-LPS-induced MUC5AC mucin expression in airway.NADPH oxidase(Nox)is one of main enzyme proteins that regulate cellular ROS production,and Nox family contain several homolog proteins,in which dual oxidase(duox,which includes two subtypes:duox1 and duox2)is expressed in airway and responsible for ROS production of airway epithelial cells.A newly study demonstrated that protein kinase C(PKC)can cause duox1 activation,and signaling pathway PKC→Duox1→ROS→TACE→TGF-α→EGFR was involved in the HNE-induced MUC5AC expression,therefore we investigate whether this pathway also invloves in PA-LPS-induced MUC5AC production in airway epithelial cells.Toll-like receptor 4(TLR4)mediates LPS-induced various pathophysiological reponses.Recently a series of studies found that there existed a cross-talk between TLR4 and Nox family,such as by using yeast two hybrid technology Park HS showed that the cross-talk between TLR4 and Nox 4 mediates LPS-induced ROS production of T lymphocytes,and Pacquelet S revealed the molecular mechanisms underlying this cross-talk:IRAK which is downstream of TLR4 can phosphorylate the cytosolic components p47phox of Nox.So we explore whether the cross-talk between TLR4 and Duox1 may mediate PA-LPS-induced airway MUC5AC expression.In conclusion,we investigated the role of TLR4-PKC-Duox1 signaling pathway in PA-LPS-induced MUC5AC mucin expression in human airway epithelial cells. Part 1 The role of ROS in PA-LPS-induced MUC5AC mucin expression in airway epithelial cellsObjectiveTo investigate the role of ROS in PA-LPS-induecd TGF-alpha production and MUC5AC mucin expression in human airway epithelial cells.MethodsNCI-H292 cells(a human pulmonary mucoepidermoid carcinoma cell line)were stimulated with PA-LPS for 2h to measure H₂O₂ productioon in the supematants of cells,for 4h to measure TGF-alpha production in the supematants of cells by ELISA, for 12h to measure MUC5AC mRNA expression of cells by RT-PCR,and for 24h to measure MUC5AC protein expression of cells by immunohistochemistry.NCI-H292 cells were pretreated with ROS scavengers DMTU for 30min and then stimulated together with PA-LPS to measure TGF-alpha and MUCJAC mRNA and protein expression.HM3(human colon epithelial)cells transfected with pMUC5AC 3.7kb-luc were stimulated with PA-LPS or different concentrations of H₂O₂ for 5h and then harvested for luciferase assay.For experiments with DMTU,cells were pretreated with DMTU for 30 min,then treated with PA-LPS or H₂O₂ for 5 h,and harvested for luciferase assays.ResultsAfter stimulated with PA-LPS,the H₂O₂ and TGF-apha production in the supernatants of NCI-H292 cells as well as the MUC5AC mRNA and protein expression in cells were all significantly upregulated(p＜0.01),and which were obviously blocked by ROS scavengers DMTU(p＜0.01).Both PA-LPS and different concentrations of H₂O₂ can significantly upregulate transcriptional activity of MUC5AC in HM3 cells stably transfected with pMUC5AC 3.7kb-luc,and which was blocked by DMTU.ConclusionROS mediate PA-LPS-induced MUC5AC mucin expression via regulation of TGF-alpha production in human airway epithelial cells.This study suggests that oxidative stress play an important role in PA-LPS-induced airway mucin overproduction.Part 2 The role of TLR4-PKC-Duox1 signaling pathway in PA-LPS-induced MUC5AC mucin expression in airway epithelial cellsObjectiveTo investigate the role of TLR4-PKC-Duox1 signaling pathway in PA-LPS-induecd ROS and TGF-alpha production as well as MUC5AC mucin expression in human airway epithelial cells.MethodsNCI-H292 cells were pretreated with Nox inhibitors Apocynin and PKC inhibitors Bisindolylmaleimide I for 30min respectively and then stimulated together with PA-LPS for 2h to measure H₂O₂ productioon in the supematants of cells,for 4h to measure TGF-alpha production in the supematants of cells by ELISA,for 12h to measure MUC5AC mRNA expression of cells by RT-PCR,and for 24h to measure MUC5AC protein expression of cells by immunohistochemistry.HM3 cells transfected with pMUC5AC 3.7kb-luc were pretreated with Apocynin and Bisindolylmaleimideâ… for 30min respectively,then treated with PA-LPS for 5 h,and harvested for luciferase assays.A549 cells were stimulated with PA-LPS for 6h,12h, 24h respectively and then the Duox1 mRNA expression was examined by RT-PCR. A549 cells were transfected with Duox1 siRNA or TLR4 siRNA or negative siRNA for 48h and then harvested for Duox1 and TLR4 mRNA assay to estimate the knockdown effects.At 48h after transfected with selected Duox1 and TLR4 siRNA respectively,the A549 cells were stimulated with PA-LPS for various timeto measure H₂O₂ and TGF-alpha production as well as MUC5AC mucin expression.ResultsThe H₂O₂ and TGF-apha production in the supematants of NCI-H292 cells as well as the MUC5AC mRNA and protein expression in NCI-H292 cells in response to PA-LPS were all significantly blocked by Nox inhibitors Apocynin and PKC inhibitors Bisindolylmaleimide I respectively(p＜0.01).PA-LPS-induced MUC5AC transcriptional activity was also blocked by Apocynin and Bisindolylmaleimide I in HM3 cells stably transfected with pMUC5AC 3.7kb-luc.After A549 cells were stimulated with PA-LPS for 6h,12h,and 24h respectively,no significant changes were detected in the Duox1 mRNA expression.After transfection with Duox1 and TLR4 siRNA respectively,the Duox1 and TLR4 mRNA expression in A549 cells were down-regulated about 80%,and the H₂O₂ and TGF-apha production in the supernatants of A549 cells as well as the MUC5AC mRNA and protein expression in A549 cells in response to PA-LPS were all significantly blocked.ConclusionTLR4,PKC,and Duox1 can all mediate PA-LPS-induced MUC5AC mucin expression via regulation of ROS and TGF-alpha production in human airway epithelial cells;Both PKC and Duox1 can regulate PA-LPS-induced MUC5AC transcriptional activity;This study suggests that TLR4-PKC-Duox1 signaling pathway is involved in the regulation of ROS production,and then ROS mediate MUC5AC expression via TACE—TGF-alpha—EGFR cascade in human airway epithelial cells. We have investigated the molecular mechanisms underlying Pseudomonas aeruginosa lipopolysaccharide-induced MUC5AC mucin expression in human airway epithelial cells,and suggest that:1)ROS mediate PA-LPS-induced MUC5AC mucin expression via regulation of TGF-alpha production in human airway epithelial cells,and oxidative stress play an important role in PA-LPS-induced airway mucin overproduction.2)TLR4,PKC,and Duox1 can all mediate PA-LPS-induced MUC5AC mucin expression via regulation of ROS and TGF-alpha production in human airway epithelial cells;Both PKC and Duox1 can regulate PA-LPS-induced MUC5AC transcriptional activity.3)This study suggests that TLR4—PKC—Duox1 signaling pathway is involved in the regulation of ROS production,and then ROS mediate MUC5AC expression via TACE—TGF-alpha—EGFR cascade in human airway epithelial cells.