Effects Of Neutrophil Elastase On The MUC5AC Respiratory Mucin Expression And Regulation In Pulmonary A549 Cell Line

Objective:Mucus hypersecretion and neutrophil-predominant inflammation are the major pathological features of many inflammatory airway diseases. The excessive mucus in the airways overwhelms the normal mucociliary clearance mechanisms, leading to obstruction and impaired pulmonary function. In addition, mucus obstruction of airways is associated with recurrent airway inflammation and infection. The neutrophils release several mediators during inflammation, that causes increased mucin production and secretion by several mechanisms. Of these mediators,neutrophil elastase (NE) , a major component of primary or azurophilic granules, is the most widely studied with regard to enhanced mucus secretion. MUC5AC glycoprotein was recently shown to be a major component of respiratory secretions in subjects with lung disorders. MUC5AC has been shown to be stimulated by a wide variety of stimuli, including pro-inflammatory cytokines and air-pollutants. The molecular mechanism of MUC5AC production is due to increased MUC5AC mRNA expression. However, the effect of NE on MUC5AC gene regulation has not been investigated fully. Such knowledge is required to guide the development of new therapies for mucus hypersecretion in chronic inflammatory airway diseases.Methods:Expression of MUC5AC in A549 cells: The levels of MUC5AC protein secreted into the supernatants were determined by ELISA. The expression of MUC5AC in A549 cells was detected with RT-PCR on transcript levels.Design and construction of luciferase reporter gene plasmids containing different length of human MUC5AC gene promoter: Using the restriction enzymes,PCR and gene recombination techniques,four luciferase reporter gene plasmids containing different length of human MUC5AC gene promoter were constructed.The plasmids and intracontrol plasmid pRL-TK were co-transfected into the human pulmonary A549 cells with eukaryotic gene transfection techniques,and the relative luciferase activities were detected in the transfected A549 cells.Construction of luciferase reporter gene plasmids containing site-directed mutants of NF-κB and Sp-1 in MUC5AC promotor: With sequence analysis,site-directed mutagenesis technique was used to establish mutants of Sp-l and NF-кB site in MUC5AC gene promoter. The mutated bases was introduced into the cis-elements by PCR primer.Above plasmids containing mutants and intracontrol plasmid pRL-TK were co-transfected into human pulmonary A549 cells with eukaryotic gene transfection techniques,and the relative luciferase activities were detected in the transfected A549 cells.Results:Expression of MUC5AC in A549 cells :The expression of MUC5AC protein was elevated significantly in NE stimulating groups compared with the group without NE stimulation. The expressions of MUC5AC protein in groups treated with NE in diferent dose were higher than that of the control group(t=3.406-20.909, P<0.01).NE increased MUC5AC levels in a dose- and time-dependent manner. Specific band could be amplified from total RNA of A549 cells by RT-PCR.NE (100 nM, 30min ) increased MUC5AC mRNA levels in A549 cells were higher than that of the control group (t=10.299,P<0.01).Construction of luciferase reporter gene plasmids containing different length of human MUC5AC gene promoter:Restriction enzyme digestion and DNA sequencing were performed to confirm the successful construction of four luciferase reporter gene containing different sequences of human MUC5AC promotor.NE could increase the expression of luciferase reporter gene plasmid containing -1300bp,-689bp and-324bp version of MUC5AC promoter in the transfected A549 cells (P<0.05),but could not increase the expression of luciferase reporter gene plasmid containing -64bp version of MUC5AC promoter.Construction of luciferase reporter gene plasmids containing site-directed mutants of NF-κB and Sp-in MUC5AC promotor: Restriction enzyme digestion and DNA sequencing was performed to confirm the successful construction of site-directed mutants of NF-кB and Sp-l in MUC5AC pmmotor.NE could increase the expression of luciferase reporter gene plasmid containing -324bp and mutated NF-кB version (P<0.05 vs contro1)of MUC5AC promoter in the transfected A549 cells,The induction by NE decreased markedly when the Sp-l elements in MUC5AC promoter have been mutated.Conclusions:1.NE could increase the level of MUC5AC protein and mRNA respectively in a concentration and a time dependent manner by ELISA and RT-PCR.2.In order to define the regulatory effect of promoter sequences on the transcription of MUC5AC gene in A549 cells treated with NE , we constructed a series of luciferase reporter gene containing different length sequences of human MUC5AC promotor and site-directed mutants of NF-κB and Sp-1 in promotor by gene recombinant technique and PCR. The constructed luciferasereporter gene plasmids include pGL3E-MUC5AC-1300/+48,pGL3E-MUC5AC-689/+48,pGL3E-MUC5AC-324/+48, pGL3E-MUC5AC-64/+48, pGL3E-MUC5AC-NF-κB-MU and pGL3E-MUC5AC-Sp-1-MU.3.After above luciferase reporter plasmid and intracontrol plasmid pRL-TK were co-transfected into A549 cells, it is observed that NE could increase the expression of luciferase reporter gene plasmid containing 1300bp(PGL3E-1300/+48);-689bp(PGL3E-689/+48),-324bp(PGL3E-324/+48) of MUC5AC promotor in the transfected A549 cells, whereas deletion derivative(PGL3E-64/+48) of MUC5AC promotor prevented NE-induced increase in the expression of luciferase reporter gene plasmid. These results suggest that there are NE-responsive region between -324bp and-64bp of promoter.The region may be involved in NE mediated induction of MUC5AC.4.NE could increase the expression of luciferase reporter gene plasmid containing -324bp and mutated NF-кB version (P<0.05 vs contro1)of MUC5AC promoter in the transfected A549 cells,The induction by NE decreased markedly when the Sp-l elements in MUC5AC promoter have been mutated. Sp-l site of promotor mediated NE-induced MUC5AC expression in human A549 cells.