Neutrophil elastase causes MUC5AC mucin synthesis via EGF receptor, ERK and NF-kB pathways in A549 cells

Abstract

Background: Neutrophil elastase (NE) was found to increase the respiratory mucin gene, MUC5AC, although the molecular mechanisms of this process remain unknown. We attempted to determine the signal transduction pathway through which NE induces MUC5AC gene expression in bronchial epithelial cells.

Methods: A fragment of 1.3 Kb MUC5AC promoter which had been cloned into the pGL3-Basic luciferase vector was transfected to the A549 cells. By measuring the luciferase activity, we were able to evaluate the MUC5AC promoter activity in A549 cells. The involvement of mitogen-activated protein kinases (MAPK) was confirmed by Western blotting. To confirm the involvement of nuclear factorkappaB (NF-kB), we used site-directed mutagenesis and electrophoretic mobility shift assay (EMSA) autoradiogram. The MUC5AC mRNA expression was confirmed by RT-PCR.

Results: NE increased the transcriptional activity of the MUC5AC promoter in A549 cells. The increased transcriptional activity of the MUC5AC promoter by NE was found to be associated with increased NF-kB activity. Site-directed mutagenesis showed that the transfection of the mutated NF-kB binding sites from the PGL3-MUC5AC-3752 promoter luciferase reporter plasmid decreased the luciferase activity after NE stimulation. Among the MAPKs, only extracellular signal-regulated kinases (ERK) were involved in this NE-induced MUC5AC mucin expression. RT-PCR also showed that NE increased MUC5AC mRNA. An EMSA autoradiogram revealed that NE induced NF-kB:DNA binding.

Conclusions: These results indicate that human NE induces MUC5AC mucin through the epidermal growth factor receptor (EGF-R), ERK, and NF-kB pathways in A549 cells.

Figure 1

Neutrophil elastase (NE) increases the transcriptional activity of MUC5AC promoter. MUC5AC reporter plasmid was transfected to the A549 cells, which then underwent treatment with vehicle or NE for 30 minutes at concentrations of 10, 25, 50, 100, and 200 nM concentrations. The cells were then harvested for measurement of luciferase activities. Significant difference in the transcriptional activity of MUC5AC promoter between control and 50 nM of NE stimulation to transfected A549 cells was detected are found. Values represent the average of six replicates normalized to co-transfected β-galactosidase luciferase activity, with standard deviation indicated with error bars (^*, p<0.05 compared with transfection with MUC5AC promoter without NE treatment).

Figure 2

Effects of inhibitors on the MUC5AC promoter activity in A549 cells. The nuclear factorkB (NF-kB) inhibitor (CAPE; 20 µg/mL), MAPK/ERK kinase (MEK) inhibitor (PD98059; 50 µM), and EGF-R inhibitor (AG1478; 50 µM), were added before treatments with neutrophil elastase (NE) (50 nM); all of these inhibitors markedly inhibited the promoter activity of MUC5AC in the A549 cells. Values represent the average of four assays, and standard deviation is provided by error bars (^*, p<0.01 compared with transfection with MUC5AC promoter without NE treatment).

Figure 3

Characterization of the nuclear factorkB (NF-kB) site and NF-kB in MUC5AC mucin induction by neutrophil elastase (NE). Two portions of the NF-kB binding sites of MUC5AC reporter plasmid were mutated (mutants 1, 2 and mutants 3, 4) and transfected into the A549 cells. Three base pairs of human MUC5AC regulatory regions (base pairs -971 to -939, mutant 1; -973 to -941, mutant 2; -235 to -203, mutant 3; -237 to -205; mutant 4) were mutated, as in table 1. Luciferase activity was measured from the control A549 cells and from each mutant-transfected A549 cell during the resting state and after stimulation with NE. Results shown are the average of four replicates, with standard deviation indicated by error bars (^*, p<0.05 with respect to controls).

Figure 4

RT-PCR analysis of MUC5AC expression in A549 cells. Neutrophil elastase (NE) (50 nM) increased the MUC5AC mRNA levels. Pretreatment with nuclear factor-kB (NF-kB) inhibitor (CAPE; 20 µg/mL), MAPK/ERK kinase (MEK) inhibitor (PD98059; 50 µM), or epidermal growth factor receptor (EGF-R) phosphorylation inhibitor (AG1478; 50 µM) suppressed the MUC5AC expression back to the levels of the control. MUC5AC mRNA levels were normalized to glyceraldehydes-3-phosphate dehydrogenase (GAPDH) levels by densitometry. Data is representative of four separate experiments (^*, p<0.05 with respect to controls).

Figure 5

Effects of neutrophil elastase (NE) and signal transduction inhibitors on the phosphorylation of extracellular signal-regulated kinases (ERKs), p38 MAPK, and JNKs were determined by Western blotting. NE (50 nM) phosphorylated the ERK1/2, whereas CAPE (20 µg/mL), PD98059 (50 µM), and AG1478 (50 µM) completely inhibited ERK1/2 phosphorylation. NE did not phosphorylate the p38 MAPK or the JNKs.

Figure 6

Representative electrophoretic mobility shift assay (EMSA) autoradiogram showing the neutrophil elastase (NE) (50 nM) induced nuclear factor-kB (NF-kB): DNA binding. Double-stranded oligonucleotide probes corresponding to the human MUC5AC NF-kB site were incubated with nuclear proteins (10 µg) from A549 cells pretreated with vehicle or NE for 30 minutes. Antibodies (Ab) used for supershift are indicated, as are the oligonucleotides used as cold competitors. NF-kB is involved in the induction of MUC5AC promoter activity by NE. Specificity of binding was ascertained by a supershift of the bands with antibodies to p50, p65, and c-Rel, as well as the reduced intensity of the signals with an excess amount of cold DNA probes (×100).