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(Chest. 2000;117:317S-320S.)
© 2000 American College of Chest Physicians

Neutrophil Elastase Induces MUC5AC Messenger RNA Expression by an Oxidant-Dependent Mechanism*

Bernard Fischer, DVM, PhD and Judith Voynow, MD

* From the Division of Pediatric Pulmonary Diseases, Duke University Medical Center, Durham, NC.

Correspondence to: Judith A. Voynow, MD, Division of Pediatric Pulmonary Diseases, Duke University Medical Center, Box 2994, Durham, NC 27710; e-mail: voyno001{at}mc.duke.edu


   Abstract
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 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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.

Key Words: airway epithelium • elastase • MUC5AC • oxidative stress


   Introduction
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 Abstract
 Introduction
Materials and Methods
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Abbreviations: DCHF = dichlorodihydrofluorescein diacetate; DMTU = dimethylthiourea; NE = neutrophil elastase; NHBE = normal human bronchial epithelial cells; ROS = reactive oxygen species

Patients with chronic airway diseases such as cystic fibrosis, chronic bronchitis, and asthma are plagued by recurrent airway inflammation and obstruction leading to respiratory impairment/failure. In these airway diseases, the predominance of a neutrophilic inflammation suggests an important role for neutrophils and their associated products in the pathogenesis of the airway injury. Neutrophils release a variety of inflammatory mediators, including reactive oxygen species (ROS) and neutrophil elastase (NE). NE is a serine protease that has been shown to impair ciliary function, stimulate mucus production and hypersecretion, and induce mucus cell hypertrophy and hyperplasia.1 However, the mechanism(s) of NE-induced mucus production are not known. We suggest that NE increases mucus production by inducing the expression of mucin genes.

Mucin glycoproteins are the major macromolecular components of mucus. Mucins are large, heavily glycosylated molecules; to date, at least nine different mucin genes have been identified. Of the mucin genes expressed in the airway tissues, MUC5AC appears to be one of the major respiratory mucins.2 We have recently reported that NE increases respiratory epithelial expression of MUC5AC messenger RNA by increasing MUC5AC messenger RNA stability.3 In this study, we explored the intracellular signaling mechanisms required for regulation of MUC5AC expression by NE. We report that NE induces the production of ROS that mediate the regulation of MUC5AC gene expression.


   Materials and Methods
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 Abstract
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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 were changed to serum-free medium. A549 cells were pre- and co-incubated with dimethylthiourea (DMTU; Sigma; St. Louis, MO), 4 mM and 40 mM, and then treated with 100 nm (2.6 U) NE or vehicle control (Elastin Products; Owensville, MO) for 6 h. Total cellular RNA was isolated, and MUC5AC messenger RNA expression was evaluated by Northern analysis.

RNA Isolation and Northern Analysis
Total cellular RNA was isolated from cell cultures as previously described by the guanidinium thiocyanate-cesium chloride method.3 Total RNA was separated by agarose-formaldehyde gel electrophoresis, transferred to nylon membrane (Nytran plus; Schleicher & Schuell; Keene, NH), cross-linked, and probed with 32P-labeled complementary DNA probe for MUC5AC as previously described.3

Assessment of Cellular Oxidative Stress Utilizing Dichlorodihydrofluorescein
For the evaluation of NE-induced cellular oxidative stress, cells were loaded with dichlorodihydrofluorescein diacetate (DCHF; A549, 2.5 µM; NHBE, 10 µM; Molecular Probes; Eugene, OR). Cells were exposed to NE or vehicle control. (A549, 100 nm, 30 min.; NHBE, 500 nm, 60 min.) and then evaluated for evidence of oxidative stress by fluorescent photomicroscopy. Hydrogen peroxide (1 mM) was used as a positive control stimulus for oxidative stress.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
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 NE-induced MUC5AC expression (Fig 1 ).



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  		Figure 1. NE-induced MUC5AC expression: inhibition by ROS scavenger DMTU. A549 cells were treated with DMTU (0, 4, or 40 mM) 30 min prior to and during NE treatment (0 nm or 100 nm, 6 h). MUC5AC messenger RNA expression was evaluated by Northern analysis (top, A). Ethidium stain of agarose gel (bottom, B).

 
NE-Induced Oxidant Stress in Respiratory Epithelial Cells
The effect of DMTU on NE-induced MUC5AC expression suggests that NE may function by inducing cellular oxidative stress as part of the regulatory mechanisms important for enhancing MUC5AC messenger RNA levels. Therefore, to evaluate if NE triggers ROS production and cellular oxidative stress in A549 cells and NHBE, we used a fluorescent intracellular redox indicator, DCHF. Cells were loaded with DCHF. In the presence of ROS such as hydrogen peroxide, DCHF is oxidized to the fluorescent product dichlorofluorescein.5 Fluorescent microscopy revealed that NE and exogenous hydrogen peroxide increased cellular fluorescence. There was no increase in fluorescence in cells treated with control vehicle, and there was no autofluorescence in cells not loaded with DCHF. These studies together implicate the role of oxidative stress in the regulation of NE-induced MUC5AC expression.


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
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 role in MUC5AC messenger RNA stability. Posttranscriptional regulation of gene expression by ROS is not unique to MUC5AC. ROS have been reported to mediate increased messenger RNA stability of vascular endothelial growth factor,6 catalase,7 and the transferrin receptor.8 It is possible that NE treatment of respiratory epithelial cells affects similar redox or ROS-sensitive protein interactions with MUC5AC messenger RNA stability sequences.

Using a fluoroscopic assay to detect intracellular oxidant stress, we demonstrate that in both A549 cells and NHBE, NE treatment induced oxidant stress. The sources of NE-triggered ROS production and oxidant stress are not known in epithelial cells, but there are several potential mechanisms including: generation by oxidases, lipoxygenases, P450 activity or "leakage" of mitochondrial oxidants.9 NE has also been reported to stimulate intracellular oxidant stress in endothelial cells by catalyzing the conversion of xanthine dehydrogenase to xanthine oxidase, a superoxide generator.10 There may be a similar mechanism activated by NE in epithelial cells. Our report suggests that NE should be included in a growing list of inflammatory mediators—including platelet-activating factor,11 tumor necrosis factor-{alpha}, and lipopolysaccharide12 —that stimulate increased intracellular oxidant stress.

In summary, NE treatment resulted in increased MUC5AC messenger RNA expression mediated by ROS. In addition, NE induced cellular oxidative stress. These results suggest that ROS may play an important role in NE regulation of MUC5AC messenger RNA stability.


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


   References
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Stockley, RA (1994) The role of proteinases in the pathogenesis of chronic bronchitis. Am J Respir Crit Care Med 150,S109-S113
  2. Rose, MC, Gendler, SJ (1997) Mucin genes and gene products. Rogers, D Lethem, M eds. Airway mucus: basic mechanisms and clinical perspectives ,41-66 Birkhauser Verlag Basel, Switzerland.
  3. Voynow, JA, Young, LR, Wang, Y, et al (1999) Neutrophil elastase increases MUC5AC mRNA and protein expression in respiratory epithelial cells. Am J Physiol 276,L835-L843[Abstract/Free Full Text]
  4. Whiteman, M, Halliwell, B (1997) Thiourea and dimethylthiourea inhibit peroxynitrite-dependent damage: nonspecificity as hydroxyl radical scavengers. Free Radic Biol Med 22,1309-1312[CrossRef][ISI][Medline]
  5. Scott, JA, Homcy, CJ, Khaw, B-A, et al (1988) Quantitation of intracellular oxidation in a renal epithelial cell line. Free Radic Biol Med 4,79-83[CrossRef][ISI][Medline]
  6. Kuroki, M, Voest, EE, Amano, S, et al (1996) Reactive oxygen intermediates increase vascular endothelial growth factor expression in vitro and in vivo. J Clin Invest 98,1667-1675[ISI][Medline]
  7. Clerch, LB, Massaro, D (1992) Oxidation-reduction-sensitive binding of lung protein to rat catalase mRNA. J Biol Chem 267,2853-2855[Abstract/Free Full Text]
  8. Hentze, MW, Rouault, RA, Harford, JB, et al (1989) Oxidation-reduction and the molecular mechanism of a regulatory RNA-protein interaction. Science 244,357-9[Abstract/Free Full Text]
  9. Schraufstatter, IU, Cochrane, CG (1997) Oxidants: types, sources and mechanisms of injury. Crystal, RG West, JB eds. The lung ,2251-2258 Lippincott-Raven Publishers Philadelphia, PA.
  10. Phan, SH, Gannon, DE, Ward, PA, et al (1992) Mechanism of neutrophil-induced xanthine dehydrogenase to xanthine oxidase conversion in endothelial cells: evidence of a role for elastase. Am J Respir Cell Mol Biol 6,270-278
  11. Kinnula, VL, Adler, KB, Ackley, NJ, et al (1992) Release of reactive oxygen species by guinea pig tracheal epithelial cells in vitro. Am J Physiol 262,L708-L712[Abstract/Free Full Text]
  12. Rochelle, LG, Fischer, BM, Adler, KB (1998) Concurrent production of reactive oxygen and nitrogen species by airway epithelial cells in vitro. Free Radic Biol Med 24,863-868[CrossRef][ISI][Medline]



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