Chest
Volume 117, Issue 5, Supplement 1, May 2000, Pages 317S-320S
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Neutrophil Elastase Induces MUC5AC Messenger RNA Expression by an Oxidant-Dependent Mechanism

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Airway diseases such as cystic fibrosis, chronic bronchitis, and viral- or pollution-triggered asthma have two common pathologic features: mucus obstruction of the airways, and neutrophil-predominant airway inflammation. Neutrophils release high concentrations of elastase (neutrophil elastase [NE]), a serine protease, into the airways; exposure to elastase results in secretory metaplasia and increased production/secretion of mucin glycoproteins. We have previously shown that NE increases gene expression of a respiratory mucin, MUC5AC, in both A549, a lung adenocarcinoma cell line, and cultured normal human bronchial epithelial cells. In this study, we explored the intracellular signaling mechanisms required for NE-regulated MUC5AC gene expression. A549 cells were treated with dimethylthiourea (DMTU; 4 mM and 40 mM), a scavenger of hydroxyl radical, peroxynitrite, and other hydroxylated products, prior to and during NE stimulation. DMTU inhibited NE-induced MUC5AC expression. Furthermore, using dichlorodihydrofluorescein, an intracellular redox indicator, we showed that in both A549 cells and cultured normal human bronchial epithelial cells, NE treatment induced oxidative stress. These results support the role of reactive oxygen species mediating NE-induced MUC5AC gene expression.

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Cell Culture

Two respiratory epithelial model systems were utilized for these studies: A549 cells (American Type Culture Collection; Manassas, VA), a lung adenocarcinoma cell line that expresses both MUC5AC messenger RNA and glycoprotein; and normal human bronchial epithelial cells (NHBE; Clonetics; San Diego, CA) maintained in an air-liquid interface culture system. Both cell types were grown and maintained as previously described.3

Cell Stimulation

All studies were performed when A549 cells are 90 to 95% confluent. Cells

NE-Induced MUC5AC Messenger RNA Expression Is Inhibited by Oxidant Scavengers

NE increases MUC5AC messenger RNA expression by enhancing messenger RNA stability, but the signaling pathway of this mechanism is not known. We hypothesized that ROS mediate the NE-regulated increase in MUC5AC messenger RNA stability. To test this hypothesis, we treated cells with DMTU, a scavenger of hydroxyl radical, associated hydroxylated products, and peroxynitrite4 prior to and during NE stimulation. DMTU had no effect on baseline MUC5AC messenger RNA. Importantly, DMTU (40 mM) inhibited

Discussion

In this study, we demonstrated that NE enhancement of MUC5AC messenger RNA levels was dependent on the production of intracellular oxidants or an alteration in the redox state of the cell. DMTU inhibited NE-induced MUC5AC expression, suggesting a role for hydroxyl radical, hydroxylated products, or peroxynitrite in MUC5AC gene regulation. Recently, we have reported that NE increases MUC5AC messenger RNA levels by a posttranscriptional mechanism.3 Collectively, this suggests that ROS may play a

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Supported by the Cystic Fibrosis Foundation, The North Carolina Biotechnology Center, and Duke University Medical Center.

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