ABSTRACT
Neutrophil-predominant airway inflammation and mucus obstruction of airways are major pathological features of cystic fibrosis, chronic bronchitis, and acute exacerbations of asthma. The severity of airflow obstruction and respiratory morbidity in patients with chronic inflammatory airway diseases have been directly related to the intensity of neutrophilic inflammation (1). Neutrophils release elastase (NE), a serine protease, that impairs mucociliary clearance by several mechanisms. NE activates mucin secretion (2), triggers goblet cell metaplasia (3), and increases mucin granule production (4). Thus, NE may be a key molecule linking neutrophilic inflammation and overproduction of mucus in chronic inflammatory airway diseases.The molecular mechanisms by which NE increases mucus production are beginning to be elucidated. The major macromolecular constituents of mucus are the mucin glycoproteins. Of the mucin genes expressed in the respiratory tract, MUCA and MUC5AC are two of the predominant mucin genes expressed in superficial tracheobronchial epithelium. MUCA is expressed in both superficial se211
cretory and ciliated tracheobronchial cells (5). The MUC4 cDNA encodes for both a transmembrane domain and a proteolytic cleavage site, suggesting that it may be present as a cell surface mucin and/or a component of airway secretions (6). MUC5AC is primarily expressed in goblet cells but also can be detected in submucosal gland cells (5). MUC5AC glycoprotein is present in airway secretions from normal subjects, patients with bronchial asthma (7) and with cystic fibrosis (8). Therefore, we have investigated the effect of NE on the regulation of MUC5AC and MUC4. METHODS AND RESULTS
To study the effect of NE on airway epithelial cell mucin gene expression, we have used two cell culture models: A549 cells and normal human bronchial epithelial cells (NHBE). A549 (ATCC) is a lung carcinoma cell line that expresses both MUC5AC mRNA and glycoprotein (9). NHBE (Clonetics), grown in submerged conditions on plastic tissue culture dishes (undifferentiated NHBE), express MUC4 mRNA. When NHBE are cultured in an air/liquid interface culture system (10), the cells develop a mucociliary phenotype (differentiated NHBE) and express both MUC4 and MUC5AC.Regulation of MUC5AC and MUC4 gene expression by NE was assessed by northern analysis. In A549 cells, NE increased MUC5AC mRNA expression in a concentration-and time-dependent manner. NE also increased MUC5AC expression in a time-dependent manner in differentiated NHBE (11). In undifferentiated NHBE, NE increased MUC4 mRNA levels. A comparison of NE-regulated mucin expression revealed that both MUC4 and MUC5AC mRNA levels increased in response to the same NE treatment conditions (50 nM, 24 hours).To determine the mechanism of NE-regulated mucin expression, we tested whether NE increased MUC5AC gene expression by transcriptional or posttran-scriptional regulation. NE did not increase transcription of MUC5AC as determined by nuclear run-on assay (11). In contrast, NE did regulate both MUC5AC and MUC4 by a posttranscriptional mechanism. A549 and undifferentiated NHBE cells were stimulated with neutrophil elastase or control vehicle and then treated with an RNA transcription inhibitor, actinomycin D. The decay rate of MUC5AC and MUC4 mRNA was followed over time. Treatment with NE tripled the half-lives of both MUC5AC and MUC4 mRNA. Thus, these studies suggest that NE increases expression of both MUC5AC and MUC4 by a novel regulatory mechanism for mucin genes: stabilization of mRNA transcripts.The stability of mRNA transcripts is controlled by the interaction of RNA-binding proteins and mRNA cis stability sequences. This interaction affects the activity of RNases. Several intracellular signaling molecules, such as protein kinase C, calcium, iron, and reactive oxygen species, alter mammalian gene mRNA stability (12). We hypothesized that NE may regulate the stability of both
MUC5AC and MUC4 mRNA by activating similar intracellular signals, specifically reactive oxygen species. To test this hypothesis, A549 and undifferentiated NHBE were treated with antioxidants prior to and during NE stimulation. The effect of antioxidants on NE-induced mucin expression was evaluated by Northern analyses for MUC5AC and MUC4. Dimethylthiourea, a broad-spectrum antioxidant that scavenges hydroxyl radical, hydroxylated products, and peroxyni-trite, attenuated the NE-induced increase in both MUC5AC and MUC4 mRNA. Furthermore, in A549, polyethylene glycol-conjugated catalase, a hydrogen peroxide scavenger, inhibited the NE-induced increase in MUC5AC mRNA levels. Together, these experiments suggest that reactive oxygen species are critical intracellular signals that mediate NE-induced mucin gene expression.To confirm the presence of reactive oxygen species in respiratory epithelial cells following NE treatment, A549 and both differentiated and undifferentiated NHBE were evaluated by fluorescent microscopy for changes in fluorescence of an oxidation marker, dichlorodihydrofluorescein (DCF). Cells were loaded with DCF, a molecule that is retained intracellularly and fluoresces when exposed to hydrogen peroxide or hydroxylated products (13). Following DCF loading, cells were stimulated with NE or control vehicle and then examined by fluorescent microscopy. Cells fluoresced only after NE treatment and not after control vehicle treatment. In the absence of DCF, there was no increase in autofluorescence following NE or control vehicle treatment. Thus, NE treatment increases oxidant stress in epithelial cells. DISCUSSION AND CONCLUSIONS
NE regulates MUC5AC and MUC4 by a novel mechanism, stabilization of mRNA transcripts. There is a growing body of evidence that mucin genes are regulated by posttranscriptional as well as transcription mechanisms. Rat Muc4 is regulated at the posttranscriptional level by transforming growth factor (3 in mammary epithelial cells (14). MUC2 is regulated posttranscriptionally by phor-bol ester in a human colon carcinoma cell line (15). Tumor necrosis factor a increases the mRNA stability of MUC5AC in lung carcinoma cells (16). Our data support the concept that posttranscriptional regulation is an important mechanism to amplify mucin gene expression. Furthermore, this is the first report to demonstrate that two different mucin genes, localized to different chromosomes, are regulated by the same mediator with similar kinetics and mechanisms.Although NE has been reported to regulate expression of several mammalian genes (reviewed in Ref. 11), little is known about the molecular mechanisms of NE action. We demonstrate that NE triggers oxidant stress in respiratory epithelial cells, and this oxidant stress is an important signal in mucin gene regulation. There are several potential sources of oxidant stress that are triggered by NE activity. NE has been reported to catalyze the conversion of xanthine dehy
drogenase to xanthine oxidase, a superoxide generator (17). Alternatively, NE may promote oxidative stress in respiratory epithelial cells by proteolytically degrading antioxidant defenses such as extracellular superoxide dismutase (18), thus diminishing the antioxidant capacity of the cell.In conclusion, by defining the molecular pathways utilized by NE to regulate respiratory mucin genes, we will identify specific targets for more effective therapies to treat mucus obstruction in chronic airway inflammatory diseases. REFERENCES
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