Nitric oxide induces MUC5AC mucin in respiratory epithelial cells through PKC and ERK dependent pathways.
BACKGROUND
Nitric oxide (NO) is generally increased during inflammatory airway diseases. This increased NO stimulates the secretion of mucin from the goblet cell and submucosal glands but the mechanism is still unknown precisely. In this study, we investigated potential signaling pathways involving protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) in the NO-induced MUC5AC mucin gene and protein expression in A549 cells.


METHODS
Nitric oxide was donated to the A549 cells by NOR-1. MUC5AC mucin levels were assayed by enzyme-linked immunosorbent assay (ELISA). MUC5AC promoter activity was determined by measuring luciferase activity after the lysing the transfected cells. Activation of PKC isoforms were measured by assessing the distribution of the enzyme between cytosolic and membrane fractions using immunoblotting. Immunoblotting experiments using a monoclonal antibody specific to PKC isoforms were performed in the cytosol and membrane fractions from A549 cells. Western blot analysis for pERK and p38 were performed using the corresponding antibodies from the cell lysates after donating NO to the A549 cells by NOR-1.


RESULTS
The transcriptional activity of MUC5AC promoter was maximal at the concentration of 0.1 mM NOR-1 for 1 hour incubation in transfected A549 cells. (+/-)-(E)-methyl-2-((E)-hydroxyimino)-5-nitro-6-methoxy-3-hexenamide (NOR-1) markedly displaced the protein kinase C (PKC)alpha and PKCdelta from the cytosol to the membrane. Furthermore, the PKC-alpha,betainhibitors, GO6976 (10 nM) and PKCdelta inhibitors, rottlerin (4 muM) inhibited the NOR-1 induced migration of PKCalpha and PKCdelta respectively. NOR-1 also markedly increased the MUC5AC promoter activity and mRNA expression, mucin synthesis and ERK1/2 phosphorylation. The PKC inhibitors also inhibited the NOR-1 induced MUC5AC mRNA and MUC5AC protein synthesis by inhibiting the activation of PKCalpha and PKCdelta with ERK1/2 pathways.


CONCLUSIONS
Exogenous NO induced the MUC5AC mucin gene and protein through the PKCalpha and PKCdelta-ERK pathways in A549 cells. Inhibition of PKC attenuated NO-mediated MUC5AC mucin synthesis. In view of this findings, PKC inhibitors might be useful in the treatment of bronchial asthma and chronic bronchitis patients where NO and mucus are increased in the bronchial airways.
Song
Jeong Sup
JS
Department of Internal Medicine, St Mary's hospital, Catholic University Medical College, Yeoi-Do Dong, Young Dung Po Gu, Seoul, Korea. jssong@catholic.ac.kr
Kang
Chun Mi
CM
Yoo
Moon Bin
MB
Kim
Seung Joon
SJ
Yoon
Hyung Kyu
HK
Kim
Young Kyoon
YK
Kim
Kwan Hyung
KH
Moon
Hwa Sik
HS
Park
Sung Hak
SH
D045744:Cell Line, Tumor; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D006898:Hydroxylamines / Q000494:pharmacology; D055271:Mucin 5AC; D009077:Mucins / Q000096:biosynthesis* / Q000235:genetics; D009569:Nitric Oxide / Q000494:pharmacology*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D011401:Promoter Regions, Genetic / Q000502:physiology; D011493:Protein Kinase C / Q000378:metabolism*; D051571:Protein Kinase C-alpha / Q000378:metabolism; D051745:Protein Kinase C-delta / Q000378:metabolism; D020545:Respiratory Mucosa / Q000187:drug effects* / Q000378:metabolism*; D014158:Transcription, Genetic
C477506:(+-)-E-4-methyl-2-((E)-hydroxylamino)-5-nitro-6-methoxy-3-hexanamide; D006898:Hydroxylamines; C524678:MUC5AC protein, human; D055271:Mucin 5AC; D009077:Mucins; D020030:Nitric Oxide Donors; D009569:Nitric Oxide; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D051745:Protein Kinase C-delta; D048049:Extracellular Signal-Regulated MAP Kinases