Tumor Necrosis Factor-α Triggers Mucus Production in Airway Epithelium through an IκB Kinase β-dependent Mechanism

José M Lora,Dong Mei Zhang,Sha Mei Liao,Timothy Burwell,Anne Marie King,Philip A Barker,Latika Singh,Marie Keaveney,Jay Morgenstern,José Carlos Gutiérrez-Ramos,Anthony J Coyle,Christopher C Fraser

doi:10.1074/jbc.m507977200

Abstract

Excessive mucus production by airway epithelium is a major characteristic of a number of respiratory diseases, including asthma, chronic bronchitis, and cystic fibrosis. However, the signal transduction pathways leading to mucus production are poorly understood. Here we examined the potential role of IκB kinase β (IKKβ) in mucus synthesis in vitro and in vivo. Tumor necrosis factor-α (TNF-α) or transforming growth factor-α stimulation of human epithelial cells resulted in mucus secretion as measured by MUC5AC mRNA and protein. TNF-α stimulation induced IKKβ-dependent p65 nuclear translocation, mucus synthesis, and production of cytokines from epithelial cells. TNF-α, but not transforming growth factor-α, induced mucus production dependent on IKKβ-mediated NF-κB activation. In vivo, TNF-α induced NF-κB as determined by whole mouse body bioluminescence. This activation was localized to the epithelium as revealed by LacZ staining in NF-κB-LacZ transgenic mice. TNF-α-induced mucus production in vivo could also be inhibited by administration into the epithelium of an IKKβ dominant negative adenovirus. Taken together, our results demonstrated the important role of IKKβ in TNF-α-mediated mucus production in airway epithelium in vitro and in vivo.

Highlights

Despite the clinical importance of excessive mucus production, the signal transduction pathways leading to its synthesis and release from the epithelium are poorly understood
Following a combination of cell culture and in vivo experiments, we show the following: first, TNF-␣ induces mucin and cytokine production and activates NF-␬B in a human lung epithelial cell line; second, IKK␤ is required for TNF-␣-induced mucin and cytokine production but not for TGF-␣-dependent mucus production by this human lung epithelial cell line; and third, TNF-␣ activates NF-␬B in mouse airway epithelium in vivo and leads to goblet cell metaplasia and mucus production in an IKK␤-dependent manner
TNF-␣ Induces Mucin and Cytokine Production by NCI-H292 Human Lung Epithelial Cells—In order to investigate if mucus and/or cytokine production can be modulated by TNF-␣ or TGF-␣ in human lung epithelial cells, NCI-H292 cells were cultured in the presence of TNF-␣ (50 ng/ml) or TGF-␣ (50 ng/ml) alone or in combination for 12

Summary

Introduction

Despite the clinical importance of excessive mucus production, the signal transduction pathways leading to its synthesis and release from the epithelium are poorly understood. Proximal regulatory regions from MUC2 and MUC5AC genes have been cloned and characterized in detail [11, 12] Sequence analyses of these regions have indicated the presence of NF-␬B binding sequences, and electromobility shift assays and supershift experiments with specific antibodies have demonstrated binding of p50/p65 complexes to these sequences upon stimulation of cell cultures with Pseudomonas aeruginosa-derived LPS. P38 MAPK has been shown to play a role in MUC5AC up-regulation upon stimulation of normal human nasal epithelial cells and human epithelial cell lines with IL-1, TNF-␣, and cytoplasmic proteins of nontypeable Haemophilus influenzae (16 –18). TGF-␣-mediated EGFR tyrosine phosphorylation has been found to play an important role in mucin elaboration in human airway epithelial cells (19 –21). In the present work we have investigated the role of IKK␤ in transducing the pro-inflammatory signal delivered by TNF-␣ and TGF-␣ to trigger mucus production by airway epithelial cells. Following a combination of cell culture and in vivo experiments, we show the following: first, TNF-␣ induces mucin and cytokine production and activates NF-␬B in a human lung epithelial cell line; second, IKK␤ is required for TNF-␣-induced mucin and cytokine production but not for TGF-␣-dependent mucus production by this human lung epithelial cell line; and third, TNF-␣ activates NF-␬B in mouse airway epithelium in vivo and leads to goblet cell metaplasia and mucus production in an IKK␤-dependent manner

Methods

Results

Conclusion