Goblet-cell hyperplasia is a critical pathological feature in hypersecretory diseases of airways. However, the underlying mechanisms are unknown, and no effective therapy exists. Here we show that stimulation of epidermal growth factor receptors (EGF-R) by its ligands, EGF and transforming growth factor ␣ (TGF␣), causes MUC5AC expression in airway epithelial cells both in in vitro and in vivo. We found that a MUC5AC-inducing epithelial cell line, NCI-H292, expresses EGF-R constitutively; EGF-R gene expression was stimulated further by tumor necrosis factor ␣ (TNF␣). EGF-R ligands increased the expression of MUC5AC at both gene and protein levels, and this effect was potentiated by TNF␣. Selective EGF-R tyrosine kinase inhibitors blocked MUC5AC expression induced by EGF-R ligands. Pathogenfree rats expressed little EGF-R protein in airway epithelial cells; intratracheal instillation of TNF␣ induced EGF-R in airway epithelial cells, and subsequent instillation of EGF-R ligands increased the number of goblet cells, Alcian blueperiodic acid-Schiff staining (ref lecting mucous glycoconjugates), and MUC5AC gene expression, whereas TNF␣, EGF, or TGF␣ alone was without effect. In sensitized rats, three intratracheal instillations of ovalbumin resulted in EGF-R expression and goblet-cell production in airway epithelium. Pretreatment with EGF-R tyrosine kinase inhibitor, BIBX1522, prevented goblet-cell production both in rats stimulated by TNF␣-EGF-R ligands and in an asthma model. These findings suggest potential roles for inhibitors of the EGF-R cascade in hypersecretory diseases of airways.

Oxidative stress has been implicated in the pathogenesis of inflammatory diseases of airways. Here we show that oxidative stress causes ligand-independent activation of epidermal growth factor receptors (EGFR) and subsequent activation of mitogen-activated protein kinase kinase (MEK)-p44/42 mitogen-activated protein kinase (p44/42mapk), resulting in mucin synthesis in NCI-H292 cells. Exogenous hydrogen peroxide and neutrophils activated by IL-8, FMLP, or TNF-α increased EGFR tyrosine phosphorylation and subsequent activation of p44/42mapk and up-regulated the expression of MUC5AC at both mRNA and protein levels in NCI-H292 cells. These effects were blocked by selective EGFR tyrosine kinase inhibitors (AG1478, BIBX1522) and by a selective MEK inhibitor (PD98059), whereas a selective platelet-derived growth factor receptor tyrosine kinase inhibitor (AG1295), a selective p38 MAPK inhibitor (SB203580), and a negative compound of tyrosine kinase inhibitors (A1) were without effect. Neutrophil supernatant-induced EGFR tyrosine phosphorylation, activation of p44/42mapk, and MUC5AC synthesis were inhibited by antioxidants (N-acetyl-l-cysteine, DMSO, dimethyl thiourea, or superoxide dismutase); neutralizing Abs to EGFR ligands (EGF and TGF-α) were without effect, and no TGF-α protein was found in the neutrophil supernatant. In contrast, the EGFR ligand, TGF-α, increased EGFR tyrosine phosphorylation, activation of p44/42mapk, and subsequent MUC5AC synthesis, but these effects were not inhibited by antioxidants. These results implicate oxidative stress in stimulating mucin synthesis in airways and provide new therapeutic approaches in airway hypersecretory diseases.

Mucus hypersecretion is a prominent manifestation in patients with chronic inflammatory airway diseases. MUC5AC mucin is a major component of airway mucus, and its expression is modulated by a TNF-␣-converting enzyme (TACE)-EGF receptor pathway that can be activated by reactive oxygen species (ROS). Dual oxidase 1 (Duox1), a homologue of glycoprotein p91 phox , is expressed in airway epithelium and generates ROS. We hypothesize that Duox1 activates TACE, cleaving pro-TGF-␣ into soluble TGF-␣, resulting in mucin expression. To examine this hypothesis, we stimulated both normal human bronchial epithelial cells and NCI-H292 airway epithelial cells with phorbol 12-myristate 13-acetate and with human neutrophil elastase. These stimuli induced TACE activation, TGF-␣ release, and mucin expression, effects that were inhibited by ROS scavengers, implicating ROS in TACE activation. Inhibition of epithelial NADPH oxidase or knockdown of Duox1 expression with small interfering RNA prevented ROS generation, TGF-␣ release, and mucin expression by these stimuli, implicating Duox1 in TACE activation and mucin expression. Furthermore, the PKC␦͞PKC inhibitor rottlerin prevented the effects induced by phorbol 12-myristate 13-acetate and human neutrophil elastase, suggesting that PKC␦ and PKC are involved in Duox1 activation. From these results, we conclude that Duox1 plays a critical role in mucin expression by airway epithelial cells through PKC␦͞PKC-Duox1-ROS-TACE-pro-ligand-EGF receptor cascade.EGF receptor ͉ mucus hypersecretion ͉ reactive oxygen species ͉ TNF-␣-converting enzyme ͉ type ␣ TGF

An unresolved question in cystic fibrosis (CF) research is how mutations of the CF transmembrane conductance regulator, a Cl ion channel, cause airway mucus obstruction leading to fatal lung disease. Recent evidence has linked the CF transmembrane conductance regulator mutation to the onset and persistence of Pseudomonas aeruginosa infection in the airways, and here we provide evidence directly linking P. aeruginosa infection to mucus overproduction. We show that P. aeruginosa lipopolysaccharide profoundly upregulates transcription of the mucin gene MUC 2 in epithelial cells via inducible enhancer elements and that this effect is blocked by the tyrosine kinase inhibitors genistein and tyrphostin AG 126. These findings improve our understanding of CF pathogenesis and suggest that the attenuation of mucin production by lipopolysaccharide antagonists and tyrosine kinase inhibitors could reduce morbidity and mortality in this disease.