help button home button
AJRCMB
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Published ahead of print on June 6, 2008, doi:10.1165/rcmb.2007-0262OC
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
2007-0262OCv1
39/5/598    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kim, H. J.
Right arrow Articles by Yoon, J.-H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kim, H. J.
Right arrow Articles by Yoon, J.-H.
American Journal of Respiratory Cell and Molecular Biology. Vol. 39, pp. 598-609, 2008
© 2008 American Thoracic Society
DOI: 10.1165/rcmb.2007-0262OC

The Role of Nox4 in Oxidative Stress–Induced MUC5AC Overexpression in Human Airway Epithelial Cells

Hyun Jik Kim1,6, Young-Duck Park4, Uk Yeol Moon4, Jun-Hee Kim1, Ju Hyun Jeon2, Jeung-Gweon Lee2, Yun Soo Bae7 and Joo-Heon Yoon2,3,4,5

1 Department of Otolaryngology—Head and Neck Surgery, Chung-Ang University College of Medicine, Seoul, Korea; 2 Department of Otorhinolaryngology, 3 The Airway Mucus Institute, 4 BK 21 Project for Medical Science, 5 Research Center for Natural Human Defense System, 6 Department of Medicine the Graduate School, Yonsei University College of Medicine, Seoul, Korea; 7 The Center for Cell Signaling Research, Division of Molecular Life Sciences, and Department of Biological Science, Ewha Womans University, Seoul, Korea

Correspondence and requests for reprints should be addressed to Joo-Heon Yoon, M.D., Ph.D., Department of Otorhinolaryngology, Yonsei University College of Medicine, 134 Shinchon-dong, Seodaemun-gu, Seoul, Korea 120-752. E-mail: jhyoon{at}yuhs.ac


   Abstract
Top
 Abstract
CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Mucus hypersecretion is a prominent manifestation in patients with chronic inflammatory airway diseases, and MUC5AC is a major airway mucin. It is well known that reactive oxygen species (ROS) may be involved in the pathogenesis of various inflammatory airway diseases. The purpose of this study was to identify which secreted mucin genes are induced by exogenous hydrogen peroxide and the mechanism by which these genes are up-regulated in normal human nasal epithelial (NHNE) cells. Exogenous H2O2 induced the ligand-independent activation of epidermal growth factor receptors (EGFR) and the subsequent activation of ERK1 mitogen-activated protein kinase, resulting in the induction of intracellular ROS generation. Through this signal pathway, exogenous H2O2 markedly induced overexpression of the MUC5AC gene alone. In addition, Nox4, a subtype of nonphagocytic NADPH oxidase, was found to play a key role in intracellular ROS generation and exogenous H2O2–induced MUC5AC gene expression in NHNE cells.

Key Words: hydrogen peroxide • MUC5AC • epidermal growth factor receptor • Nox4



   CLINICAL RELEVANCE
Top
 Abstract
 CLINICAL RELEVANCE
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Exogenous H2O2 induces intracellular reactive oxygen species (ROS) generation via a signal pathway involving EGFR-ERK1 mitogen-activated protein kinase and Nox4, resulting in MUC5AC gene expression in normal human nasal epithelial (NHNE) cells, and Nox4 plays a key role in intracellular ROS generation in NHNE cells.

 
Mucin hypersecretion is commonly observed in patients with respiratory diseases such as rhinitis, sinusitis, otitis media, nasal allergy, chronic bronchitis, and cystic fibrosis (1–3). To date, 20 different mucin genes have been identified and subdivided into two groups: the membrane-bound and secreted mucins. MUC5AC, MUC5B, MUC6, MUC7, and MUC19 are the secreted mucins (4–8). MUC5AC and 5B, the major secreted mucins, are highly expressed in the goblet cells of human airway epithelium and the submucosal glands (9–11).

Oxidative injury triggered by either inhaled or locally generated reactive oxygen species (ROS) elicits an inflammatory response that can profoundly impair the structural integrity and biological properties of bronchial epithelium (3, 9–12). There are several potential sources of ROS in most cells, including nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, xanthine oxidase, uncoupled nitric oxide (NO) synthase, and the mitochondrial respiratory chain reaction. ROS can also be produced in response to a variety of extracellular stimuli such as air pollutants or cigarette smoking. A large number of studies have demonstrated that ROS such as hydrogen peroxide (H2O2), superoxide anion (O2–), and hydroxyl radical, play a role in the progression of many airway diseases and mucin gene expression in human airway epithelial cells (13, 14). The mechanism of ROS generation has been studied extensively in phagocytic cells, in which O2– is produced via the one-electron reduction of O2 by the multicomponent NADPH oxidase (Nox) system (15). In contrast, the mechanism behind ROS generation in nonphagocytic cells remains unclear. Evidence suggests that the system in nonphagocytic cells is functionally similar to, and yet structurally and genetically distinct from, the Nox system of phagocytes (16, 17). To date, seven homologs of gp91phox (Nox2), the core component of Nox, have been identified in various non-phagocytic cells, including Nox1, Nox3, Nox4, Nox5, Duox1, and Duox2. Recently, Duox1 was identified in normal human bronchial epithelial cells and shown to generate ROS (18, 19). Nox4 is highly expressed in endothelial cells (20), but there have been few reports about its expression in airway epithelium.

The regulation of gene expression by oxidative stress involves numerous signaling pathways, including mitogen-activated protein (MAP) kinase, triggered by receptor tyrosine kinases such as epidermal growth factor receptor (EGFR) (14, 21, 22). Aside from ligand-dependent activation, EGFR activation may also be caused by oxidative stress induced by activated neutrophils or exogenous H2O2, and this activation of EGFR may result in the stimulation of mucin synthesis in NCI-H292 cells (3, 14).

In the present study, we first examined which secreted mucin genes, including MUC5AC, MUC5B, MUC6, MUC7, and MUC19, were induced by exogenous H2O2 in NHNE cells. Second, we examined the signal pathway responsible for up-regulating these genes and confirmed that EGFR and ERK1/2 MAP kinase were associated with H2O2 stimulation in NHNE cells. Finally, we investigated and measured intracellular ROS generation after stimulating exogenous oxidative stress and examined which Nox subtype was involved in the endogenous generation of ROS in NHNE cells.

We found that exogenous H2O2 specifically induced the gene expression of MUC5AC alone in a dose- and time-dependent manner through the ligand-independent activation of EGFR and phosphorylation of ERK1 MAP kinase. Exogenous H2O2 did not induce MUC5B, MUC6, MUC7, or MUC19 expression. In addition, exogenous H2O2 induced intracellular ROS generation through Nox4, one homolog of gp91phox, and Nox4 protein was expressed predominantly in the cell membrane and cytoplasm of goblet cells. The inhibition of Nox4-based intracellular ROS generation suppressed MUC5AC gene overexpression. Our findings provide new evidence that exogenous H2O2 generates intracellular ROS and that Nox4 may play critical roles downstream of EGFR and ERK1 MAP kinase in ROS-induced MUC5AC gene overexpression in chronic airway diseases.


   MATERIALS AND METHODS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Materials
Hydrogen peroxide (H2O2) and aprotinin were purchased from Sigma Aldrich (St. Louis, MO). Anti–phospho-EGFR (Tyr1068), anti-EGFR, anti–phospho-ERK1/2 MAP kinase (Thr202/Tyr204), anti-total ERK MAP kinase, anti–phospho-p38 MAP kinase (Thr180/Tyr182), and anti–phospho-SAPK/JNK MAP kinase (Thr183/Tyr185) antibodies were purchased from Cell Signaling (Beverly, MA). Anti-Nox4 and anti-MUC5AC antibodies, and ERK1 MAP kinase and ERK2 MAP kinase siRNA, were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti–{alpha}-tubulin antibody and GM6001 were purchased from Calbiochem (San Diego, CA). Control siRNA (scramble RNA) for Nox4 was purchased from Dharmacon (Cat# D-001210–01–20; Dharmacon, Lafayette, CO) and siRNA for Nox4 was provided by Y.S.B. (Ewha Womans University, Seoul, Korea). A 21-nucleotide sequence (GTCAACATCCAGCTGTACCdTdT) specific to human Nox4 cDNA (nucleotide residues, 1474–1492) was selected for siRNA synthesis. The depletion of endogenous Nox4 by siRNA was confirmed by reverse transcriptase PCR (RT-PCR).

Cell Culture
After approval of the study protocol by the Institutional Review Board of the Yonsei University College of Medicine, human nasal middle turbinate specimens were obtained from two healthy volunteers. Normal human nasal epithelial (NHNE) cells were cultured as described previously (23, 24). In brief, passage-2 NHNE cells (1 x 105 cells/culture) were seeded in 0.5 ml of culture medium onto a 24.5-mm, 0.45-µm-pore Transwell-clear (Costar Co, Cambridge, MA, USA) culture insert. Cells were cultured in a 1:1 mixture of basal epithelial growth medium and Dulbecco's Modified Eagle's Medium (DMEM) containing all the supplements described previously (24). Cultures were grown while submerged for the first 9 days, during which time the culture medium was changed on Day 1 and every other day thereafter. The air–liquid interface (ALI) was created on Day 9 by removing the apical medium and feeding the cultures from only the basal compartment. The culture medium was changed daily after the ALI was initiated, and all experiments used NHNE cells on Day 14 after the creation of the ALI.

The human lung mucoepidermoid carcinoma cell line NCI-H292 was purchased from the American Type Culture Collection (CRL-1848; Manassas, VA). The cells were cultured in RPMI 1640 (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin at 39°C in a humidified chamber with 5% CO2. Cells were grown to confluence in 6-well plates (Falcon, Flanklin Lakes, NJ). For 24-hour deprivation, confluent cells were washed twice with phosphate-buffered saline (PBS) and recultured in RPMI 1640 with 0.2% FBS to deprive them of serum (9).

Experimental Conditions
H2O2 was diluted in PBS to stock concentrations of 100, 250, 500, and 1,000 mM. The H2O2 stocks were further diluted in DMEM and bronchial epithelial cell basal medium (BEBM) to experimental concentrations of 100, 250, 500, and 1,000 µM. NHNE cells were treated with H2O2 (100, 250, 500, and 1,000 µM) for 2, 4, 8, 12, and 24 hours for dose- and time-dependent studies, and the expression profiles of MUC5AC, MUC5B, MUC6, MUC7, and MUC19 were evaluated using RT-PCR. For Western blot or intracellular ROS assays, cells were treated with 250 µM H2O2 for 10, 30, 60, 120, and 180 minutes.

RT-PCR
Total RNA was isolated from NHNE cells treated with H2O2 (100, 250, 500, and 1,000 µM) using TRIzol (Invitrogen). cDNA was synthesized with random hexamer primers (PerkinElmer Life Sciences, Waltham, MA, and Roche Applied Science, Indianapolis, IN) using Molony murine leukemia virus-reverse transcriptase (PerkinElmer Life Sciences). Oligonucleotide PCR primers were designed based on the MUC5AC, MUC5B, MUC6, MUC7, and MUC19 Genbank sequences and Nox subunit sequences (Tables 1 and 2). We used comparative kinetic analysis to compare the mRNA levels of each gene for each set of culture conditions as described previously (24). PCR products were resolved on 2% agarose gels (FMC, Rockland, ME) and visualized with ethidium bromide under a transilluminator. When reverse transcriptase was omitted, no PCR products were observed, confirming that the amplified products were from mRNA, not genomic DNA contamination. The specific amplification of target genes was confirmed by sequencing the PCR products (dsDNA Cycle Sequencing System; GibcoBRL, Rockville, MD).


View this table:
[in this window]
[in a new window]

  		TABLE 1. POLYMERASE CHAIN REACTION EXPERIMENTAL CONDITIONS AND OLIGONUCLEOTIDE SEQUENCES

 

View this table:
[in this window]
[in a new window]

  		TABLE 2. POLYMERASE CHAIN REACTION EXPERIMENTAL CONDITIONS AND OLIGONUCLEOTIDE SEQUENCES

 
Real-Time PCR
Primers and probes were designed with PerkinElmer Life Sciences Prime Express software and purchased from PE Biosystems (Foster City, CA). Commercial reagents (Taqman PCR Universal PCR Master Mix; PerkinElmer Life Sciences) were used according to the manufacturer's protocol. One microgram of cDNA (reverse transcription mixture), oligonucleotide primers at a final concentration of 800 nM, and 200 nM TaqMan hybridization probes were incubated in a final volume of 25 µl. The real-time PCR probe was labeled with carboxyfluoroscein (FAM) at the 5' end and the quencher carboxytetramethylrhodamine (TAMRA) at the 3' end. The MUC5AC and β2-microglobulin primers and TaqMan probe used are described in Table 3. Real-time PCR was performed using a PerkinElmer Life Sciences ABI PRISM 7700 Sequence Detection System. The reaction parameters were 50°C for 2 minutes, 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. The relative MUC5AC mRNA quantity was obtained using a comparative cycle threshold method and was normalized to β2-microglobulin as an endogenous control.


View this table:
[in this window]
[in a new window]

  		TABLE 3. EXPERIMENTAL PRIMER AND TAQMAN OLIGONUCLEOTIDE SEQUENCES USED IN REAL-TIME PCR

 
Western Blot Analysis
NHNE cells were grown to confluence in 6-well plates. After treatment with 250 µM H2O2 for 10, 30, 60, 120, or 180 minutes, the cells were lysed with 2x lysis buffer (250 mM Tris-Cl [pH 6.5], 2% SDS, 4% β-mercaptoethanol, 0.02% bromphenol blue, and 10% glycerol). Equal amounts of whole cell lysate were resolved using 10% SDS-PAGE and transferred to a PVDF membrane in Tris-buffered saline (TBS; 50 mM Tris-Cl [pH 7.5], 150 mM NaCl) for 90 or 180 minutes at room temperature. The blot was incubated overnight with primary antibody in TTBS (0.5% Tween 20 in TBS). After washing with TTBS, the blot was incubated for 1 hour at room temperature with anti-rabbit or anti-mouse antibody (Cell Signaling) in TTBS and visualized using the ECL system (Amersham, Little Chalfont, UK). Optical densities of each band were measured for total and phosphorylated-forms of EGFR, ERK1/2 MAP kinase, tubulin, and Nox using a densitometer (LAS 4000; Fuji, Tokyo, Japan). The fold increase of phosphorylated form was calculated by dividing phosphorylated into total form, and densitometric values were reported as fold relative to the control.

Cell Transfection with ERK1 MAP Kinase siRNA, ERK2 MAP Kinase siRNA, or Nox4 siRNA
Specific siRNA (Santa Cruz) against ERK1 MAP kinase and ERK2 MAP kinase was used to suppress their respective expressions. The transfection rates of ERK1 MAP kinase siRNA or ERK2 MAP kinase siRNA were verified to be over 90% in NCI-H292 cells. One microgram of each siRNA and 1{lambda} Lipofectamine were mixed with RPMI without serum and antibiotics, respectively, and then transfection was performed onto 6-well NCI-H292 cell plates. This procedure did not affect cell viability (data not shown), and after 48 hours of transfection, cellular deprivation was performed according to standard methods (9). The same procedure was performed with control siRNA (sc-37007; Santa Cruz).

NCI-292 cells were transiently transfected with Nox4 siRNA and control siRNA (200 nmol/L) using oligofectamineTM reagent following the manufacturer's instructions (Invitrogen). After 48 hours of transfection with siRNA, NCI-H292 cells were used for the described experiments (25).

Intracellular ROS Assay
Intracellular ROS production was assessed as reported by Ohba and coworkers (26). Plastic dishes (100 mm diameter) of confluent cells were stimulated with H2O2 (250 µM) for 30, 60, 120, and 180 minutes, washed with Modified Eagle's Medium (HBSS) without phenol red, and incubated for 10 minutes in the dark in Krebs-Ringer solution containing 5 µM 2',7'-dichlorofluorescin diacetate (DCF-DA). DCF-DA is a nonpolar compound that readily diffuses into cells, where it is hydrolyzed to the nonfluorescent polar derivative DCF and trapped within the cells (27). We washed the cells with 1 ml of HBSS at least five times to get rid of extracellular H2O2. In the presence of a proper oxidant, this compound is converted into 2'7'-dichlorofluorescin (DCF) by intracellular esterases and then oxidized to the highly fluorescent 2'7'-DCF by ROS or reactive nitrogen species (RNS). Culture dishes were viewed on a Zeiss Axiovert 135 inverted confocal microscope equipped with a x20 Neofluor objective and Zeiss LSM 410 confocal attachment (Carl Zeiss, Minneapolis, MN). DCF fluorescence was measured at an excitation wavelength of 488 nm and emission at 515 to 540 nm. Seven fields of each dish were randomly selected and the fluorescence intensity was measured with the Karl Zeiss vision system (KS400, version 3.0). The seven values were averaged to obtain the mean relative fluorescence intensity, and the mean relative fluorescence intensities were used for comparisons. All experiments were repeated at least three times.

Immunocytofluorescence Study
Human nasal polyps were obtained from patients with chronic sinusitis, and cytospin slides were prepared for immunostaining. Double immunostaining was performed using anti-Nox4 and anti-MUC5AC antibodies. The samples were fixed and incubated with rabbit anti-Nox4 antibody (diluted 1:100 in PBS) and mouse anti-MUC5AC antibody (diluted 1:1,000 in PBS) overnight at 4°C. The samples were then washed repeatedly with PBS and incubated with a secondary FITC-conjugated affinity-purified goat anti-mouse IgG (1:250, for MUC5AC) or rhodamine-conjugated affinity-purified goat anti-rabbit IgG (1:250, for Nox4) for 1 hour at room temperature. After extensive washing, glass coverslips were mounted, and the slides were examined with a confocal microscope. The same procedures were performed using nonimmunized mouse IgG (purified IgG; Sigma) instead of primary antibody as a negative control.

Statistical Analysis
At least three separate experiments were performed for each measurement and the data are expressed as mean ± SD of triplicate cultures. Differences between treatment groups were assessed by ANOVA with a post hoc test. Differences were considered statistically significant at P < 0.05.


   RESULTS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
H2O2 Induces MUC5AC Gene Expression in a Dose- and Time-Dependent Manner, but Not MUC5B, MUC6, MUC7, or MUC19 Expression
To examine which secreted mucin genes could be induced by exogenous H2O2, RT-PCR was performed after treating cells (1 x 106/ml) with H2O2 (100, 250, 500, and 1,000 µM) for 24 hours. MUC5AC mRNA levels increased after H2O2 treatmentin a dose-dependent manner, but MUC5B, MUC6, MUC7, and MUC19 mRNA levels did not (Figure 1A). This finding demonstrates that, among secreted mucin genes, exogenous H2O2 specifically induces MUC5AC gene expression. Real-time PCR revealed that MUC5AC gene expression was significantly higher after treatment with 250 µM (4.25- ± 0.59-fold over control; P < 0.05), 500 µM (4.17- ± 0.04-fold over control; P < 0.05), and 1 mM H2O2 (5.08- ± 0.49-fold over control; P < 0.05) (Figure 1B). After H2O2 treatment for 2, 4, 8, 12, and 24 hours, we performed real-time PCR. MUC5AC gene expression was higher starting from 8 hours after treatment (8 h: 4.47- ± 0.10-, 12 h: 4.94- ± 0.15-, 24 h: 4.86- ± 0.02-fold over control; P < 0.05; Figure 1C). No corresponding change was found in β2-microglobulin expression (the internal control). We used 250 µM H2O2 for all subsequent experiments.


Figure 1
View larger version (16K):
[in this window]
[in a new window]

  		Figure 1. H2O2-induced mucin gene expression. (A) Confluent normal human nasal epithelial (NHNE) cells were treated with H2O2 (100 µM, 250 µM, 500 µM, or 1 mM) for 24 hours. Cont, control. β2-microglobulin (β2 M) was used as an internal control. (B) Real-time PCR demonstrating the dose-dependent effect of H2O2 on MUC5AC gene expression after 24 hours. (C) Real-time PCR demonstrating the time-dependent effect of H2O2 (250 µM) on MUC5AC gene expression. The results are presented as mean ± SD of triplicate cultures (#P < 0.05 when compared with control).

 
Exogenous H2O2 Induces MUC5AC Gene Expression through the Ligand-Independent Activation of EGFR
EGFR can be activated by oxidative stress and influence MUC5AC overexpression in NCI-H292 cells (14). We examined whether EGFR was activated after stimulation by exogenous H2O2 in NHNE cells. Maximum activation of EGFR occurred 10 minutes (4.91- ± 0.07-fold over control) after stimulation with exogenous H2O2 (250 µM) and gradually decreased thereafter (Figure 2A). We next evaluated the mechanism behind EGFR activation. NHNE cells were treated with galardin (GM6001, 10 µM), a broad-spectrum matrix metalloproteinase inhibitor, and aprotinin (1 µM), a serine protease inhibitor, for 1 hour and then stimulated with exogenous H2O2. Pretreatment of NHNE cells with galardin or aprotinin did not inhibit increased phosphorylation of EGFR after stimulation with exogenous H2O2 (4.72- ± 0.21- versus 4.98- ± 0.61- or 4.72- ± 0.21- versus 4.63- ± 0.82-fold over control, respectively; Figure 2B), indicating that exogenous H2O2 increased the phosphorylation of EGFR through a ligand-independent mechanism.


Figure 2
Figure 2
Figure 2
View larger version (103K):
[in this window]
[in a new window]

  		Figure 2. Effect of exogenous H2O2 on the activation of epidermal growth factor receptor (EGFR) and ERK1 mitogen-activated protein (MAP) kinase. (A) NHNE cells were treated with H2O2 (250 µM) for 5, 10, 30, or 60 minutes. Control cells were maintained in basal growth medium with PBS. Western blot analysis demonstrates the effect of H2O2 on EGFR. (B) NHNE cells were treated with galardin (GM6001, 1 µM) and aprotinin (1 µM) for 1 hour before treatment with H2O2 (250 µM). Western blot analysis shows that pretreatment with galardin and aprotinin did not inhibit the increased phosphorylation of EGFR. (C) NHNE cells were treated with H2O2 (250 µM) for 5, 10, 30, or 60 minutes. Western blot analysis shows that exogenous H2O2 increased the phosphorylation of ERK1/2 MAP kinase. (D) NHNE cells were treated with AG1478 (10 µM) for 30 minutes, then stimulated with H2O2 (250 µM) for 10 minutes. Western blot analysis and real-time PCR illustrate the effect of AG1478. (E) Western blot and real-time PCR show the expression of ERK1/2 MAP kinase and MUC5AC gene expression after transfection with ERK1 MAP kinase siRNA and ERK2 MAP kinase siRNA, respectively. The results of Western blot analyses are representative of three separate experiments, and the results of densitometry and real-time PCR are presented as mean ± SD of triplicate cultures (#P < 0.05 when compared with control, * P < 0.05 when compared with H2O2 treatment group).

 
Phosphorylation of EGFR and ERK1 MAP Kinase Is Involved in Exogenous H2O2-Induced MUC5AC Gene Expression
As a next step, we examined the involvement of the MAP kinase signal pathway as a downstream signal of EGFR. ERK1/2 MAP kinase was maximally activated after 10 minutes of stimulation with exogenous H2O2 (4.71- ± 0.71-fold over control; P < 0.05) and gradually decreased thereafter in NHNE cells (Figure 2C). No significant change was detected in p38 and JNK activation (data not shown). To investigate the involvement of EGFR and ERK1/2 MAP kinase in H2O2-induced MUC5AC gene expression, we first pretreated NHNE cells with AG1478 (10 uM), a tyrosine kinase inhibitor, for 1 h before treatment with H2O2. Western blot analysis and real time-PCR clearly showed that AG1478 pretreatment inhibited the phosphorylation of ERK1/2 MAP kinase and H2O2-induced MUC5AC gene expression (4.80- ± 0.58- versus 1.61- ± 0.16-fold over control; P < 0.05; Figure 2D).

To study the specificity of ERK1 and ERK2 MAP kinase, cells were transfected transiently with ERK1 or ERK2 MAP kinase siRNA, respectively. Interestingly, transfection with ERK1 MAP kinase siRNA specifically reduced exogenous H2O2-induced MUC5AC gene expression compared with control siRNA transfection (sc-37007) (4.67- ± 0.70- versus 1.82- ± 0.10-fold over control; P < 0.05), while transfection with ERK2 MAP kinase siRNA did not (4.67- ± 0.70- versus 4.11- ± 0.18-fold over control, Figure 2E). These results indicate that exogenous H2O2-induced MUC5AC gene expression requires the activation of EGFR and subsequent phosphorylation of ERK1 MAP kinase in NHNE cells.

Exogenous H2O2 Induces Intracellular ROS Generation through NADPH Oxidase
NHNE cells were stimulated with media containing 250 µM H2O2 for 30, 60, 120, and 180 minutes. The production of ROS was measured using a fluorescence-based assay with 2',7'-DCF-DA and laser-scanning confocal microscopy. The stimulation of NHNE cells with exogenous H2O2 resulted in a time-dependent increase in the intensity of DCF fluorescence, with maximal increase (4.6-fold) apparent 60 minutes after stimulation. Fluorescence had diminished by 180 minutes (Figure 3A).


Figure 3
Figure 3
Figure 3
View larger version (98K):
[in this window]
[in a new window]

  		Figure 3. Production of intracellular reactive oxygen species (ROS) through Nox. (A) NHNE cells were stimulated with media containing 250 µM H2O2 for 30, 60, 120, or 180 minutes, and the production of intracellular ROS was measured using a fluorescence-based assay with 2',7'–DCF-DA and laser-scanning confocal microscopy. The figures of fluorescent intensity are representative of three separate experiments, and the graph of intensity depicts mean ± SD of triplicate cultures (#P < 0.05). (B) After pretreatment with NMEA (10 µM), allopurinol (100 µM), dicumarol (30 µM), and DPI (30 µM), we measured the change of intracellular ROS (#P < 0.05 when compared with control, *P < 0.05 when compared with H2O2 treatment group). (C) Real-time PCR shows the effect of each inhibitor on exogenous H2O2 (250 µM)-induced MUC5AC gene expression. The results are presented as mean ± SD of triplicate cultures (#P < 0.05 when compared with control, *P < 0.05 when compared with H2O2 treatment group).

 
We next examined whether Nox is involved in exogenous H2O2-induced intracellular ROS generation. To determine the involvement of other enzymes, we investigated the effect of the inhibitors of each enzymes, including Nox (diphenyleneiodium chloride, DPI; Sigma), NO synthase (NG-Monoethyl-L-arginine, NMEA; Calbiochem), xanthine oxidase (allopurinol; Sigma), and NADPH:quinine oxidoreductase (dicumarol; Sigma). After pretreating NHNE cells with DPI 30 µM, NMEA 10 µM, allopurinol 100 µM, or dicumarol 30 µM, we measured the change of intracellular ROS and performed real-time PCR to examine MUC5AC gene expression. NMEA, allopurinol, and dicumarol did not have a significant inhibitory effect on exogenous H2O2-induced intracellular ROS or MUC5AC gene overexpression (Figures 3B and 3C). In contrast, pretreatment with DPI suppressed exogenous H2O2-induced intracellular ROS (4.66- ± 0.22- versus 1.62- ± 0.38-fold over control, P < 0.05) and MUC5AC gene expression (4.72- ± 0.89- versus 1.52- ± 0.23-fold over control, P < 0.05; Figures 3B and 3C). These results suggest that exogenous H2O2 can produce intracellular ROS through Nox and that Nox may affect MUC5AC gene overexpression through the generation of intracellular ROS.

Ligand-Independent Activation of EGFR and Phosphorylation of ERK1 MAP Kinase Mediates Intracellular ROS Generation after Stimulation with Exogenous H2O2
Next, we investigated whether EGFR and ERK1 MAP kinase are involved in exogenous H2O2-induced intracellular ROS generation. NHNE cells were pretreated with AG1478 (10 µM) or an MEK1 inhibitor (PD98059, 30 µM) for 30 minutes before stimulation with exogenous H2O2 for 60 minutes, and intracellular ROS production was measured using confocal fluorescence microscopy. The generation of intracellular ROS was suppressed after pretreatment with AG1478 (4.85- ± 1.16- versus 1.31- ± 0.28-fold over control, P < 0.05) and PD98059 (4.85- ± 1.16- versus 1.32- ± 0.20-fold over control, P < 0.05; Figure 4), demonstrating that exogenous H2O2-induced intracellular ROS generation was promoted by the activation of EGFR and ERK1 MAP kinase, which may be involved in Nox-related generation of intracellular ROS.


Figure 4
View larger version (32K):
[in this window]
[in a new window]

  		Figure 4. The role of EGFR and ERK1 MAP kinase in exogenous H2O2-induced intracellular ROS generation. NHNE cells were pretreated with AG1478 (10 µM) or PD98059 (30 µM) for 30 minutes, then stimulated with H2O2 (250 µM) for 60 minutes. Intracellular ROS production was measured using a fluorescence-based assay with 2',7'–DCF-DA and laser-scanning confocal microscopy. Individual data points are quantified as fluorescent intensity units and are presented as a percentage of control. The figures of fluorescent intensity are representative of three separate experiments, and the graph of intensity depicts mean ± SD of triplicate cultures (#P < 0.05 when compared with control, *P < 0.05 when compared with H2O2 treatment group).

 
Nox4 Is the Primary Nox Homolog Involved in Exogenous H2O2-Induced Intracellular ROS Generation and MUC5AC Gene Overexpression in NHNE Cells
To determine whether H2O2 causes a significant increase in the expression of Nox subtypes and to identify which Nox subtypes may be involved in intracellular ROS generation within NHNE cells, cells were treated with exogenous H2O2 in a time-dependent manner and RT-PCR for Nox subtypes was performed. Interestingly, only Nox4 gene expression increased significantly 30 minutes after stimulation with exogenous H2O2 (4.39- ± 0.39-fold over control, P < 0.05; Figure 5A). Expression of Nox1, Nox2, Duox1, and Duox2 genes did not increase, and Nox3 and Nox5 were not expressed in NHNE cells after stimulation with exogenous H2O2 (Figure 5A). We then performed Western blot analysis to examine whether Nox4 protein is activated after stimulation by exogenous H2O2 in NHNE cells. The expression of Nox4 protein increased after stimulation with exogenous H2O2 (250 µM) and peaked 60 and 120 minutes (3.79- ± 0.51- and 3.80- ± 0.28-fold over control, respectively, P < 0.05; Figure 5B) after stimulation.


Figure 5
Figure 5
View larger version (67K):
[in this window]
[in a new window]

  		Figure 5. Nox4 gene and protein expression after stimulation with exogenous H2O2. NHNE cells were stimulated with exogenous H2O2 (250 µM) for 10, 30, 60, 120, or 180 minutes. (A) RT-PCR shows that only Nox4 gene expression increased significantly 30 minutes after stimulation with exogenous H2O2. (B) The maximum increase of Nox4 protein was observed 60 min and 120 minutes after stimulation with exogenous H2O2. The results of Western blot analyses and RT-PCR are representative of three separate experiments, and the results of densitometry are presented as mean ± SD of triplicate cultures (#P < 0.05 when compared with control).

 
We obtained the same results when the experiments (Figures 3A, 3B, and 4) were performed using NCI-H292 cells (data not shown). Accordingly, we subjected NCI-H292 cells to transient transfection with Nox4 siRNA to verify the critical function of Nox4 in exogenous H2O2-induced intracellular ROS generation. Transfection with Nox4 siRNA specifically reduced exogenous H2O2-increased Nox4 gene expression compared with transfection using control siRNA (Figure 6A). Importantly, our data showed that cells transfected with Nox4 siRNA did not undergo exogenous H2O2-induced intracellular ROS generation and inhibited MUC5AC gene overexpression, whereas cells transfected with control siRNA exhibited the expected increase in ROS and MUC5AC gene expression in response to exogenous H2O2 (Figures 6B and 6C). These results suggest that Nox4 is a critical Nox homolog in NHNE cells in response to exogenous H2O2 and is essential for exogenous H2O2-induced intracellular ROS generation and MUC5AC gene expression.


Figure 6
Figure 6
View larger version (61K):
[in this window]
[in a new window]

  		Figure 6. The role of Nox4 in exogenous H2O2-induced intracellular ROS generation and MUC5AC gene overexpression. (A) The increased Nox4 gene expression was suppressed after transfection with Nox4 siRNA. Similar results were obtained in three separate experiments. (B) Real-time PCR shows that MUC5AC gene expression was suppressed after transfection with Nox4 siRNA. The results are presented as mean ± SD of triplicate cultures (#P < 0.05 when compared with control, *P < 0.05 when compared with H2O2 treatment group). (C) The increased fluorescence intensity after stimulation with exogenous H2O2 was reduced after transfection with Nox4 siRNA. The figures of fluorescent intensity are representative of three separate experiments, and the graph of intensity depicts mean ± SD of triplicate cultures (#P < 0.05 when compared with control, *P < 0.05 when compared with H2O2 treatment group).

 
Nox4 Is Involved in Exogenous H2O2-Induced Intracellular ROS Generation Downstream of EGFR/ERK1 MAP Kinase
Having established the role of Nox4 in exogenous H2O2-intracellular ROS generation and MUC5AC gene overexpression, we examined the signal transduction sequence among EGFR, ERK1 MAP kinase, and Nox4. NHNE cells were pretreated with DPI (30 µM) and stimulated with exogenous H2O2 (250 µM), followed by Western blot analysis. No significant change in the phosphorylation of ERK1/2 MAP kinase was found after DPI pretreatment compared with exogenous H2O2 stimulation alone (Figure 7A). Next, cells were transfected transiently with either ERK1 or ERK2 MAP kinase siRNA. Transfection with ERK1 MAP kinase siRNA specifically suppressed the exogenous H2O2-dependent induction of Nox4 mRNA expression compared with control siRNA transfection (4.66- ± 0.54- versus 1.33- ± 0.14-fold over control, P < 0.05), and no change in Nox4 gene expression was observed following transfection with ERK2 MAP kinase siRNA (4.66- ± 0.54- versus 4.61- ± 0.49-fold over control; Figure 7B). We obtained similar results with Western blot analysis. These results show that exogenous H2O2-induced MUC5AC gene expression requires the activation of EGFR, the phosphorylation of ERK1 MAP kinase, and subsequent Nox4 overexpression in NHNE cells.


Figure 7
View larger version (33K):
[in this window]
[in a new window]

  		Figure 7. Nox4 produced intracellular ROS through the activation of EGFR/ERK1 MAP kinase. (A) After pretreatment with DPI (30 µM), NHNE cells were stimulated with exogenous H2O2 (250 µM). Western blot analysis demonstrates no significant change in the phosphorylation of ERK1/2 MAP kinase. (B) NCI-H292 cells were transfected with ERK1 MAP kinase siRNA and ERK2 MAP kinase siRNA. The results of Western blot analyses and RT-PCR are representative of three separate experiments, and the results are presented as mean ± SD of triplicate cultures (#P < 0.05 when compared with control).

 
Nox4 Is Localized Predominantly in the Cell Membrane and Cytoplasm of Goblet Cells in Human Nasal Epithelial Cells
Nox4 protein is reportedly localized in both paranuclear and nuclear lesions of human aortic smooth muscle cells or human airway smooth muscle cells (28, 29). We performed double immunocytofluorescence staining using anti-Nox4 and MUC5AC antibodies to examine the localization of Nox4 protein in human nasal epithelial cells. MUC5AC was used as a marker for goblet cells. Nox4 was expressed in the cell membrane and cytoplasm of all kinds of cells (Figure 8, red color, right panels) in the cytospin slide, though its degree of expression varied. We also observed some MUC5AC-positive goblet cells (Figure 8, green color, right panels). Interestingly, Nox4 was strongly expressed in goblet cells containing an eccentric nucleus, which stained yellow from the colocalization of Nox4 (red) with MUC5AC (green) in a merged image (Figure 8, arrow). No staining was detected when the primary anti-Nox4 and MUC5AC antibodies were omitted and replaced with purified IgG (Figure 8, left panels). This finding suggests that Nox4 protein is expressed predominantly in the cell membrane and cytoplasm of goblet cells in human nasal epithelium.


Figure 8
View larger version (45K):
[in this window]
[in a new window]

  		Figure 8. The localization of Nox4 in NHNE cells. The cellular localization of Nox4 proteins in human nasal epithelial cells was analyzed by double immunocytofluorescence staining with anti-Nox4 polyclonal and anti-MUC5AC monoclonal antibody. MUC5AC-positive staining (green) was noted in goblet cells and Nox4-positive staining (red) was noted predominantly in the cell membrane and cytoplasm of goblet cells (yellow [arrow] in merged image) in human nasal epithelial cells (right panels). No staining was detected when the primary Nox4 and MUC5AC antibodies were omitted and replaced with purified IgG (left panels).

 

   DISCUSSION
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
In the present study, we showed that exogenous H2O2 stimulated intracellular ROS generation in NHNE cells, and that MUC5AC gene expression was induced by intracellular ROS. Oxidative stress has detrimental effects on the host, since the molecules involved are potentially toxic to host cells, and the effect on host tissue may manifest as inflammatory, allergic, or autoimmune diseases (30). It has been generally understood that endogenous ROS may activate some signal pathways (31–33). At the same time, airway epithelium is continuously exposed to exogenous oxidants. Therefore, exogenous ROS may induce cellular damage or molecular changes in the airway epithelium and contribute to pathogenesis in chronic inflammatory airway disorders (22, 34, 35). We found that exogenous H2O2 specifically increased only MUC5AC mRNA expression in a dose- and time-dependent manner in NHNE cells. These results suggest that H2O2 stimulation induces the expression of MUC5AC mucoprotein in goblet cells, while MUC5B, MUC6, MUC7, and MUC19 mucoproteins may not be affected.

ROS-induced signaling has been established in airway epithelial cells (14, 34–39). The intracellular signal pathways responsible for the response to exogenous H2O2 have been evaluated in relation to EGFR and ERK1/2 MAP kinase in NCI-H292 cells (14, 34). In particular, in normal human bronchial epithelial cells, it has been suggested that the activation of EGFR is necessary for exogenous H2O2 signal transduction (34, 36).

Including direct activation by its ligands, various other mechanisms may activate EGFR, such as ligand-dependent transactivation and ligand-independent activation. Ligand-dependent transactivation of EGFR is related to metalloproteinase or serine protease–induced cleavage of membrane-anchored EGFR ligands, and inhibitors of metalloproteinase or serine protease can suppress the cleavage of transmembrane ligands and the transactivation of EGFR. Ligand-independent activation of EGFR can be induced directly by exposure to ROS, smoke, bacterial toxins, and ultraviolet radiation (40, 41). In our NHNE cell experiments, exogenous H2O2 increased the phosphorylation of EGFR. These results parallel those of studies using NCI-H292 cells and normal human bronchial epithelial cells. We also found that galardin (GM6001), a broad-spectrum matrix metalloproteinase inhibitor, and aprotinin, a serine protease inhibitor, did not inhibit the exogenous H2O2-induced phosphorylation of EGFR in NHNE cells. These findings indicate that exogenous H2O2 participates in the ligand-independent activation of EGFR and implicate H2O2 as a biological stimulator in EGFR activation and regulation of the downstream signaling cascades, especially ERK1 MAP kinase and MUC5AC gene overexpression in NHNE cells.

The signal pathways behind the response to exogenous or environmental H2O2 and the generation of intracellular ROS have not been fully evaluated in airway epithelial cells. We observed that stimulation with exogenous H2O2 for 60 minutes promoted the generation of intracellular ROS through the activation of Nox, and EGFR/ERK1 MAP kinase signal transduction mediated the Nox-induced intracellular ROS generation in NHNE cells. In other words, exogenous H2O2-induced MUC5AC gene expression may be associated with intracellular ROS generation through the EGFR/ERK1 MAP kinase signal pathway in NHNE cells.

The activity of Nox is significantly increased by various specific stimuli (16, 17), and Nox homolog expression has diverse cell-specific associations (42). It has been suggested that Duox is an important source of regulated ROS production in the respiratory tract (19, 34, 43–46). Duox1 can be activated by PMA or neutrophil elastase, producing ROS and resulting in MUC5AC mucin overproduction in human bronchial epithelial cells (30). In addition, differential cytokine regulation of Duox1 and Duox2 leads to distinct functions in airway epithelial host defense through the production of ROS in human primary tracheobronchial epithelial cells (46).

Intracellular ROS generation of Nox4 has been characterized in vascular endothelial cells and smooth muscle cells of the heart (25, 47). Nox4-derived ROS induce inflammatory signaling in response to LPS in human aortic endothelial cells (25), and Nox4 activates arachidonic acid through ROS generation in cardiac fibroblasts (48).

Interestingly, we found that, along with the increase of intracellular ROS generation, gene expression of Nox4 alone was higher after treatment with exogenous H2O2 in NHNE cells. Expressions of Nox1, Nox2, Duox1, and Duox2 were unchanged. Moreover, treatment with exogenous H2O2 increased the expression of Nox4 protein. Furthermore, the specific inhibition of Nox4 resulted in a significant reduction in intracellular ROS generation and MUC5AC gene overexpression in NHNE cells. These results indicate a key role for Nox4 in the generation of intracellular ROS and, consequently, MUC5AC gene expression after stimulation with exogenous H2O2 in NHNE cells. In contrast with other reports (19, 43–46) describing the importance of Duox1 or Duox2 in respiratory epithelial cells, we found that Nox4 was essential for generating intracellular ROS in NHNE cells. This discrepancy may be due to differences in stimulants among reports or the use of upper versus lower airway epithelial cells.

Until now, all Nox isoforms were predicted to have transmembrane domains and have been identified in the cell membrane of various cells (41). The intracellular localization of Nox4 protein, however, has been reported to be different. Sturrock and colleagues reported that Nox4 protein expression is higher in the ER and perinuclear regions of human airway smooth muscle cells after stimulation with TGF-β1 (29). Pedruzzi and coworkers reported that treatment with 7-ketocholesterol induces Nox4 protein expression in both paranuclear and nuclear regions of aortic smooth muscle cells (28). In our study, we found that Nox4 was localized predominantly in the cell membrane and cytoplasm of goblet cells in human nasal epithelial cells. The location of Nox4 expression may vary in cell- and tissue-specific ways. We speculate, however, that Nox4 is located constitutively in the cell membrane, and exposure to various stimuli may increase the expression of Nox4 protein in the ER or ribosomes in the cytoplasm. Nox4 protein may be detected in the cell membranes of unstimulated cells and in the ER, perinuclear region, or cytoplasm of cells after exposure to various stimuli.

In summary, exogenous H2O2 induces intracellular ROS generation via a signal pathway involving EGFR-ERK1 MAP kinase and Nox4, resulting in MUC5AC gene expression in NHNE cells. Nox4, one subunit of the nonphagocytic Nox system, is located predominantly in the cell membrane and cytoplasm of goblet cells and plays a key role in intracellular ROS generation in NHNE cells.


   Footnotes
 
This work was supported by a grant (R01-2006-000-10100-0) from the Basic Research Program of the Korea Science and Engineering Foundation, and by a KOSEF SRC grant funded by the Korean government (MOST) (R11-2007-040-02001-0).

Originally Published in Press as DOI: 10.1165/rcmb.2007-0262OC on June 6, 2008

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form July 11, 2007

Accepted in final form April 12, 2008


   References
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

  1. Yuta A, Ali M, Sabol M, Gaumond E, Baraniuk JN. Mucoglycoprotein hypersecretion in allergic rhinitis and cystic fibrosis. Am J Physiol 1997;273:1203–1207.
  2. Kim SS, Kim KS, Lee JG, Park IY, Koo JS, Yoon JH. Levels of intracellular protein and messenger RNA of mucin and lysozyme in normal human nasal and polyp epithelium. Laryngoscope 2000;112:276–280.
  3. Nadel JA. Role of epidermal growth factor receptor activation in regulating mucin synthesis. Respir Res 2001;2:85–89.[CrossRef][Medline]
  4. Van de Bovenkamp JH, Hau CM, Strous GJ, Buller HA, Dekker J, Einerhand AW. Molecular cloning of human gastric mucin MUC5AC reveals conserved cysteine-rich D- domain and a putative leucin zipper motif. Biochem Biophys Res Commun 1998;245:853–859.[CrossRef][Medline]
  5. Keates AC, Nunes DP, Afdhal NH, Troxler RF, Offner GD. Molecular cloning of major human gall bladder mucin: complete C-terminal sequence and genomic organization of MUC5B. Biochem J 1997;324:295–303.[Medline]
  6. Ho SB, Roberton AM, Shekels LL, Lyftogt CT, Niehans GA, Toribara NW. Expression cloning of gastric mucin complementary DNA and localization of mucin gene expression. Gastroenterology 1995;109:735–747.[CrossRef][Medline]
  7. Bobek LA, Tsai H, Biesbrock AR, Levine MJ. Molecular cloning, sequence, and specificity of expression of the gene encoding the low molecular weight human salivary mucin (MUC7). J Biol Chem 1993;268:20563–20569.[Abstract/Free Full Text]
  8. Chen Y, Zhao YH, Kalaslavadi TB, Hamati E, Nehrke K, Le AD, Ahn DK, Wu R. Genome-wide search and identification of a novel-gel forming mucin MUC19/muc19 in glandular tissues. Am J Respir Cell Mol Biol 2004;30:155–165.[Abstract/Free Full Text]
  9. Song KS, Lee WJ, Chung KC, Koo JS, Yang EJ, Choi JY, Yoon JH. Interleukin-1β and tumor necrosis factor-{alpha} induce MUC5AC overexpression through a mechanism involving ERK/p38 mitogen-activated protein kinases-MSK1-CREB activation in human airway epithelial cells. J Biol Chem 2003;276:23243–23250.
  10. Hewson CA, Edbrooke MR, Johnston SL. PMA induces the MUC5AC respiratory mucin in human bronchial epithelial cells, via PKC, EGF/TGF-{alpha}, Ras/Raf, MEK, ERK and Sp1-dependent mechanisms. J Mol Biol 2004;344:683–695.[CrossRef][Medline]
  11. Hovenberg HW, Davies JR, Carlstedt I. Different mucins are produced by the surface epithelium and the submucosa in human trachea: identification of MUC5AC as a major mucin from the goblet cells. Biochem J 1996;318:319–324.[Medline]
  12. Wang B, Lim DJ, Han J, Kim YS, Basbaum CB, Li JD. Novel cytoplasmic proteins of nontypeable Haemophilus influenzae up-regulate human MUC5AC mucin transcription via a positive p38 mitogen-activated protein kinase pathway and a negative phosphoinositide 3-kinase-Akt pathway. J Biol Chem 2002;277:949–957.[Abstract/Free Full Text]
  13. Barnes PJ. Reactive oxygen species and airway inflammation. Free Radic Biol Med 1990;9:235–245.[CrossRef][Medline]
  14. Takeyama K, Dabbagh K, Shim JJ, Pick TD, Ueki IF, Nadel JA. Oxidative stress causes mucin synthesis via transactivation of epidermal growth factor receptor: role of neutrophils. J Immunol 2000;164:1546–1552.[Abstract/Free Full Text]
  15. Thelen M, Dewald B, Baggiolini M. Neutrophil signal transduction and activation of the respiratory burst. Physiol Rev 1998;73:797–821.
  16. Pagano PJ, Clark JK, Cifuentes-Pagano ME, Clark SM, Callis GM, Quinn MT. Localization of a constituitively active, phagocyte-like NADPH oxidase in rabbit aortic adventitia: enhancement by angiotensin II. Proc Natl Acad Sci USA 1997;94:14483–14488.[Abstract/Free Full Text]
  17. Emmendorffer A, Roesler J, Elsner J, Raeder E, Lohmann-Matthes ML, Meier B. Production of oxygen radicals by fibroblasts and neutrophils from a patient with x–linked chronic granulomatous disease. Eur J Haematol 1993;51:223–227.[Medline]
  18. De Deken X, Wang D, Many MC, Costagliola S, Libert F, Vassart G, Dumont JE, Miot F. Cloning of two human thyroid cDNAs encoding new members of the NADPH oxidase family. J Biol Chem 2000;275:23227–23233.[Abstract/Free Full Text]
  19. Geiszt M, Witta J, Baffi J, Lekstrom K, Leto TL. Dual oxidases represent novel hydrogen peroxide sources supporting mucosal surface host defense. FASEB J 2003;17:1502–1504.[Abstract/Free Full Text]
  20. Ago T, Kitazono T, Ooboshi H, Iyama T, Han YH, Takada J, Wakisaka M, Ibayashi S, Utsumi H, Iida M. Nox4 as the major catalytic component of an endothelial NAD(P)H oxidase. Circulation 2004;109:227–233.[Abstract/Free Full Text]
  21. Garrington TP, Johnson GL. Organization and regulation of mitogen-activated protein kinase signaling pathways. Curr Opin Cell Biol 1999;11:211–218.[CrossRef][Medline]
  22. Pelaia G, Cuda G, Vatrella A, Gallelli L, Fratto D, Gioffre V, D'Agostino B, Caputi M, Maselli R, Rossi F, et al. Effects of hydrogen peroxide on MAPK activation, IL-8 production and cell viability in primary cultures of human bronchial epithelial cells. J Cell Biochem 2004;93:142–152.[CrossRef][Medline]
  23. Yoon JH, Gray T, Guzman K, Koo JS, Nettesheim P. Regulation of the secretory phenotype of human airway epithelium by retinoic acid, triiodothyronine, and extracellular matrix. Am J Respir Cell Mol Biol 1997;l6:724–731.
  24. Yoon JH, Kim KS, Kim SS, Lee JG, Park IY. Secretory differentiation of serially-passed normal human nasal epithelial cells by retinoic acid: expression of mucin and lysozyme. Ann Otol Rhinol Laryngol 2000;109:594–601.[Medline]
  25. Park HS, Chun JN, Jung HY, Choi C, Bae YS. Role of NADPH oxidase 4 in lipopolysaccharide-induced proinflammatory response by human aortic endothelial cells. Cardiovasc Res 2006;72:447–455.[Abstract/Free Full Text]
  26. Ohba M, Shibanuma M, Kuroki K, Nose K. Production of hydrogen peroxide by transforming growth factor-beta 1 and its involvement in induction of egr-1 in mouse osteoblastic cells. J Cell Biol 1994;126:1079–1088.[Abstract/Free Full Text]
  27. Bae YS, Kang SW, Seo MS, Baines IC, Tekle E, Chock PB, Rhee SG. Epidermal growth factor (EGF)-induced generation of hydrogen peroxide. J Biol Chem 1997;272:217–221.[Abstract/Free Full Text]
  28. Pedruzzi E, Ghichrd C, Ollivier V, Driss F, Fay M, Prunet C, Maire J-C, Pouzet C, Samadi M, Elbim C, et al. NAD(P)H oxidase Nox-4 mediates 7-Ketocholesterol-induced endoplasmic reticulum stress and apoptosis in human aortic smooth muscle cells. Mol Cell Biol 2004;24:10713–10717.
  29. Sturrock A, Huecksteadt TP, Norman K, Sanders K, Murphy TM, Chitano P, Wilson K, Hoidal JR, Kennedy TP. Nox4 mediates TGF-β1-induced retinoblastoma protein phosphorylation, proliferation, and hypertrophy in human airway smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 2007;292:1543–1555.[CrossRef]
  30. Folkerts G, Kloek J, Muijsers RBR, Nijkamp FP. Reactive nitrogen and oxygen species in airway inflammation. Eur J Pharmacol 2001;429:251–262.[CrossRef][Medline]
  31. Arnold RS, Shi J, Murad E, Whalen AM, Sun CQ, Polavarapu R, Parthasarathy S, Petros JA, Lambeth JD. Hydrogen peroxide mediates the cell growth and transformation caused by the mitogenic oxidase Nox1. Proc Natl Acad Sci USA 2001;98:5550–5555.[Abstract/Free Full Text]
  32. Kuroki M, Voest EE, Amano S, Beerepoot LV, Takashima S, Tolentino M, Kim RY, Rohan RM, Colby KA, Yeo KT, et al. Reactive oxygen intermediates increase vascular endothelial growth factor expression in vitro and in vivo. J Clin Invest 1996;98:1667–1675.[Medline]
  33. Esposito F, Cuccovillo F, Vanoni F, Cimino CW, Anderson EA, Russo T. Redox-mediated regulation of p21waf1/cip1 expression involves a post-transcriptional mechanism and activation of the mitogen-activated protein kinase pathway. Eur J Biochem 1997;245:730–737.[Medline]
  34. Shao MXG, Nadel JA. Dual oxidase 1-dependent MUC5AC mucin expression in cultured human airway epithelial cells. Proc Natl Acad Sci USA 2005;102:767–772.[Abstract/Free Full Text]
  35. Pelaia G, Cuda G, Vatrella A, Grembiale RD, Fratto D, Tagliaferri P, Marselli R, Costanzo FS, Marsoco SA. Effects of transforming growth factor-β and budesonide of mitogen-activated protein kinase activation and apoptosis in airway epithelial cells. Am J Respir Cell Mol Biol 2003;29:12–18.[Abstract/Free Full Text]
  36. Nabeyrat E, Jones GE, Fenwick PS, Barnes PJ, Donnelly LE. Mitogen-activated protein kinases mediate peroxynitrite-induced cell death in human bronchial epithelial cells. Am J Physiol Lung Cell Mol Physiol 2003;284:1112–1120.
  37. Guyton KZ, Liu Y, Gorospe M, Xu Q, Holbrook NJ. Activation of mitogen-activated protein kinase by H2O2: role in cell survival following oxidant injury. J Biol Chem 1996;271:4138–4142.[Abstract/Free Full Text]
  38. Stevenson MA, Pollock SS, Coleman CN, Calderwood SK. X-irradiation, phorbol esters, and H2O2 stimulate mitogen-activated protein kinase activity in NIH-3T3 cells through the formation of reactive oxygen intermediates. Cancer Res 1994;54:12–15.[Abstract/Free Full Text]
  39. Abe MK, Kartha S, Karpova AY, Liu JLPT, Kuo WL, Hershenson MB. Hydrogen peroxide activates extracellular signal-regulated kinase via protein kinase C, Raf-1, and MEK1. Am J Respir Cell Mol Biol 1998;18:562–569.[Abstract/Free Full Text]
  40. Lemjabbar H, Li D, Gallup M, Sidhu S, Drori E, Basbaum C. Tobacco smoke-induced lung cell proliferation mediated by tumor necrosis factor {alpha}-converting enzyme and amphiregulin. J Biol Chem 2003;278:26202–26207.[Abstract/Free Full Text]
  41. Peus D, Vasa RA, Meves A, Pott M, Beyerle A, Squillace K, Pittelkow MR. H2O2 is an important mediator of UVB-induced EGF-receptor phosphorylation in cultured keratinocytes. J Invest Dermatol 1998;110:966–971.[CrossRef][Medline]
  42. Dworakowski R, Anilkumar N, Zhang M, Shah AM. Redox signaling involving NADPH oxidase-derived reactive oxygen species. Biochem Soc Trans 2006;34:960–964.[CrossRef][Medline]
  43. Forteza R, Salathe M, Miot F, Conner GE. Regulated hydrogen peroxide production by Duox in human airway epithelial cells. Am J Respir Cell Mol Biol 2005;32:462–469.[Abstract/Free Full Text]
  44. Geiszt M, Leto TL. The Nox family of NAD(P)H oxidases: host defence and beyond. J Biol Chem 2004;279:51715–51718.[Free Full Text]
  45. Caillou B, Dupuy C, Lacroix L, Nocera M, Talbot M, Ohayon R, Deme D, Bidart JM, Schlumberger M, Virion A. Expression of reduced nicotinamide adenine dincleotide phosphate oxidase (ThoX, LNOX, Duox) genes and proteins in human thyroid tissue. J Clin Endocrinol Metab 2001;86:3351–3358.[Abstract/Free Full Text]
  46. Harper RW, Xu C, Eiserich JP, Chen Y, Kao C-Y, Thai P, Setiadi H, Wu R. Differential regulation of dual NADPH oxidases/Peroxidases, Duox1 and Duox2, by Th1 and Th2 cytokines in respiratory tract epithelium. FEBS Lett 2005;579:4911–4917.[CrossRef][Medline]
  47. Sturrock A, Cahill B, Norman K, Huecksteadt TP, Hill K, Sanders K, Karwande SV, Stringham JC, Bull DA, Gleich M, et al. Transforming growth factor-β1 induces Nox4 NADPH oxidase and reactive oxygen species-dependent proliferation in human pulmonary artery smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 2006;290:661–673.[CrossRef]
  48. Colston JT, Rossa SD, Strader JR, Anderson MA, Freeman GL. H2O2 activates Nox4 through PLA2-dependent arachidonic acid production in adult cardiac fibroblasts. FEBS Lett 2005;579:2533–2540.[CrossRef][Medline]



This article has been cited by other articles:


Home page
 Am. J. Respir. Crit. Care Med.Home page
H. S. Deshmukh, A. McLachlan, J. J. Atkinson, W. D. Hardie, T. R. Korfhagen, M. Dietsch, Y. Liu, P. Y. P. Di, S. C. Wesselkamper, M. T. Borchers, et al.
Matrix Metalloproteinase-14 Mediates a Phenotypic Shift in the Airways to Increase Mucin Production
Am. J. Respir. Crit. Care Med., November 1, 2009; 180(9): 834 - 845.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
A. W. Boots, M. Hristova, D. I. Kasahara, G. R. M. M. Haenen, A. Bast, and A. van der Vliet
ATP-mediated Activation of the NADPH Oxidase DUOX1 Mediates Airway Epithelial Responses to Bacterial Stimuli
J. Biol. Chem., June 26, 2009; 284(26): 17858 - 17867.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
K. S. Song, Y. H. Choi, J.-M. Kim, H. Lee, T.-J. Lee, and J.-H. Yoon
Suppression of prostaglandin E2-induced MUC5AC overproduction by RGS4 in the airway
Am J Physiol Lung Cell Mol Physiol, April 1, 2009; 296(4): L684 - L692.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
2007-0262OCv1
39/5/598    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kim, H. J.
Right arrow Articles by Yoon, J.-H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kim, H. J.
Right arrow Articles by Yoon, J.-H.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Proc. Am. Thorac. Soc. Am. J. Respir. Crit. Care Med.
Copyright © 2008 American Thoracic Society.