Neutrophil elastase causes MUC5AC mucin synthesis via EGF receptor, ERK and NF-kB pathways in A549 cells.
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.
Song
Jeong-Sup
JS
Department of Internal Medicine, St Mary's Hospital, The Catholic University of Korea College of Medicine, Seoul, Korea. jssong@catholic.ac.kr
Cho
Kyung-Sook
KS
Yoon
Hyung-Kyu
HK
Moon
Hwa-Sik
HS
Park
Sung-Hak
SH
D001980:Bronchi / Q000166:cytology; D045744:Cell Line, Tumor; D004847:Epithelial Cells; D066246:ErbB Receptors / Q000378:metabolism*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D005786:Gene Expression Regulation; D006801:Humans; D019272:Leukocyte Elastase / Q000378:metabolism*; D055271:Mucin 5AC; D009077:Mucins / Q000096:biosynthesis* / Q000235:genetics; D016328:NF-kappa B / Q000378:metabolism*; D015398:Signal Transduction; D014158:Transcription, Genetic
C524678:MUC5AC protein, human; D055271:Mucin 5AC; D009077:Mucins; D016328:NF-kappa B; D066246:ErbB Receptors; D048049:Extracellular Signal-Regulated MAP Kinases; D019272:Leukocyte Elastase