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Reactive oxygen species regulate Pseudomonas aeruginosa lipopolysaccharide-induced MUC5AC mucin expression via PKC-NADPH oxidase-ROS-TGF-α signaling pathways in human airway epithelial cells
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Biochemical and Biophysical Research Communications 366 (2008) 513–519 www.elsevier.com/locate/ybbrc
Reactive oxygen species regulate Pseudomonas aeruginosa lipopolysaccharide-induced MUC5AC mucin expression via PKC-NADPH oxidase-ROS-TGF-a signaling pathways in human airway epithelial cells Fugui Yan
a,1
, Wen Li
a,1
, Hirofumi Jono b, Qingmei Li a, Shuangmei Zhang a, Jian-Dong Li b, Huahao Shen a,*
a
b
Department of Respiratory Medicine, Second Affiliated Hospital, Zhejiang University School of Medicine, Zhejiang University Institute of Respiratory Diseases, Hangzhou 310009, China Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, NY 14642, USA Received 27 November 2007 Available online 20 December 2007
Abstract Mucin overproduction is a hallmark of chronic inflammatory airway diseases, such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis. Excessive production of mucin leads to airway mucus obstruction and contributes to morbidity and mortality in these diseases. The molecular mechanisms underlying mucin overproduction, however, still remain largely unknown. Here, we report that the bacterium P. aeruginosa, an important human respiratory pathogen causing cystic fibrosis, utilizes reactive oxygen species (ROS) to upregulate MUC5AC mucin expression. Pseudomonas aeruginosa lipopolysaccharide (PA-LPS) induces production of ROS through protein kinase C (PKC)-NADPH oxidase signaling pathway in human epithelial cells. Subsequently, ROS generation by PA-LPS releases transforming growth factor-a (TGF-a), which in turn, leads to up-regulate MUC5AC expression. These findings may bring new insights into the molecular pathogenesis of P. aeruginosa infections and lead to novel therapeutic intervention for inhibiting mucin overproduction in patients with P. aeruginosa infections. 2007 Elsevier Inc. All rights reserved. Keywords: MUC5AC mucin; Pseudomonas aeruginosa; Reactive oxygen species
Gram-negative bacterium Pseudomonas aeruginosa (P. aeruginosa) is an important and opportunistic human respiratory pathogen [1,2]. P. aeruginosa infections cause chronic inflammatory airway diseases, such as cystic fibrosis, asthma and chronic obstructive pulmonary disease [1–3]. P. aeruginosa is ineradicable by antibiotics and
Abbreviations: PKC, Protein kinase C; P. aeruginosa, Pseudomonas aeruginosa; PA-LPS, P. aeruginosa lipopolysaccharide; ROS, reactive oxygen species; TGF-a, transforming growth factor-a; TACE, TNF-aconverting enzyme. * Corresponding author. Fax: +86 571 87767122. E-mail address: [email protected] (H. Shen). 1 These authors contributed equally to this work. 0006-291X/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.11.172
responsible for airway mucus overproduction, a hallmark of chronic inflammatory airway diseases [4]. Mucus overproduction mainly results from up-regulation of mucin, a primary innate defensive response for mammalian airways [5,6]. Mucins, the major components of mucus secretions, are high-molecular weight and heavily glycosylated proteins, synthesized by the mucosal epithelial cells to protect the mucosal surface and trap the inhaled microbial particles, including bacteria and viruses for mucociliary clearance [7]. However, under diseased conditions, the mucociliary clearance mechanism becomes defective. In cystic fibrosis patients, the excessive production of mucin will cause airway mucus obstruction, which, in turn lead to death of patients [5–7]. To date, 20 mucin genes
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have been identified [6–10]. Among these, at least MUC2, MUC5AC, and MUC5B have been shown to play an important role in the pathogenesis of respiratory infectious diseases [6–12]. Recent studies have demonstrated that nontypeable Haemophilus influenzae, an important human respiratory pathogen, up-regulates MUC5AC expression via activation of p38 MAPK and MUC2 expression via activation of NF-jB signaling, respectively [13–15]. In addition, we recently also found that Streptococcus pneumoniae pneumolysin up-regulates MUC5AC via a novel IKK-ERK pathway [16]. Moreover, current studies have provided evidence that P. aeruginosa lipopolysaccharide (PA-LPS) up-regulates MUC2 mucin gene expression in epithelial cells via Src-dependent Ras-MEK1/2-ERK1/2pp90rsk-NF-jB pathway [17,18]. However, in contrast to the relatively well-known mechanism by which MUC2 mucin is up-regulated by PA-LPS, the signaling mechanism underlying PA-LPS-induced MUC5AC mucin expression is still largely unknown. Reactive oxygen species (ROS) are produced by epithelial cells lining the respiratory airways, and recognized as acute mediator for inflammation [19,20]. ROS have been implicated in the pathogenesis of a wide variety of respiratory diseases, such as asthma, emphysema and chronic obstructive pulmonary disease [21,22]. In addition to its known cytotoxic oxidative stress, growing evidence suggests that ROS play an important role in signal transduction [23]. Recent studies have provided evidence that ROS mediate mucin production induced by various stimuli, such as cigarette-smoked, Neutrophil elastase and phorbol 12-myristate 13-acetate [24–26]. Despite the critical role for ROS in the pathogenesis of respiratory disease, its role in regulating PA-LPS-induced MUC5AC mucin expression still remains elusive. Here, we show that ROS play crucial role in PA-LPSinduced MUC5AC expression. PA-LPS generates ROS through PKC-NADPH oxidase signaling pathway in human epithelial cells. ROS generation by PS-LPS releases TGF-a, which in turn, leads to up-regulate MUC5AC expression. These findings may provide new insights into the pathogenesis of P. aeruginosa infection and lead to novel therapeutic strategies for chronic inflammatory airway diseases.
of MUC5AC expression. HM3 (human colon epithelial) cells stably transfected with pMUC5AC 3.7kb-luc were maintained as described previously [14]. PA-LPS treatment. After 24 h of serum starvation, cells were stimulated with PA-LPS (10 lg/mL). For inhibitor studies, serum starved cells were pretreated with inhibitors for 30 min before exposure to stimuli, then the cells were cultured for various times with PA-LPS and inhibitors. RT-PCR. Total RNA was isolated from NCI-H292 cells using TRIzol Reagent (Invitrogen) according to the manufacturer’s instruction. For RT-PCR, cDNA was generated by reverse transcription using 2 lg total RNA. A 529-bp fragment of human MUC5AC was amplified using primers (forward, 5 0 -ACC TGC CCA GCC GAC AAG-3 0 ; reverse, 5 0 GGT ACA GGG TCC CGT TGA TG-3 0 ). As internal controls, a 527-bp fragment of human b-actin was amplified using primers (forward, 5 0 -CTA CAA TGA GCT GCG TGT GG-3 0 ; reverse, 5 0 -AAG GAA GGC TGG AAG AGT GC-3 0 ). The PCR mixture was denatured at 94 C for 5 min, followed by 30 cycles at 94 C for 30 s, 55 C for 45 s, and 72 C for 45 s. The PCR products were resolved by gel electrophoresis on 1.5% agarose gels containing ethidium bromide. Lack of DNA contamination was verified by RT-PCR with presence or absence of RT. Luciferase assay. HM3 cells stably transfected with MUC5AC-luciferase plasmid were treated with PA-LPS or H2O2 for 5 h and then harvested for luciferase assay. For experiments with inhibitors, cells were pretreated with inhibitors for 30 min, then treated with PA-LPS or H2O2 for 5 h, and harvested for luciferase assays. Luciferase assays were performed on a Monolight 3010 luminometer [27,28]. Immunohistochemistry. Cells were fixed in 4% paraformaldehyde solution for 15 min, permeabilized with 0.1% Tween 20 in PBS for 10 min, peroxidase-blocked in 0.3% H2O2 for 10 min, blocked by normal goat serum for 10 min subsequently incubated with mouse monoclonal antibody to MUC5AC (clone 45M1, 1:100) for 1 h followed by 10 min in biotinylated rabbit anti-mouse antibody (1:300) and another 10 min in streptavidin–biotin horseradish peroxidase (1:50). Cells were developed for 2 min with diaminobenzidine as chromogen substrate (DAKO Ltd.), counterstained with hematoxylin, and mounted in a xylene-based mountant (BDH-Merck, UK). H2O2 measurements. For inhibitory studies, cells were pretreated with inhibitors for 30 min before exposure to stimuli. After Cells were treated with PA-LPS (10 lg/mL) for 2 h, H2O2 production in the cell supernatants was measured by using the Amplex Red Hydrogen Peroxide/Peroxidase Assay kit (Invitrogen) according to the manufacturer’s instructions. ELISA. After reaching confluence and being serum starved for 24 h, cells were stimulated with PA-LPS (10 lg/mL) for 4 h. For inhibitor studies, cells were pretreated with the inhibitors for 30 min before PA-LPS was added. Cell supernatants were collected and concentrated 10-fold using an Ultracel YM-3 Centrifugal Filter Device (Millipore), and the levels of TGF-a in concentrated cell supernatants were quantified using ELISA kits (R&D Systems Inc., Minneapolis, MN).
Materials and methods
ROS are positively involved in PA-LPS-induced MUC5AC expression
Materials. PA-LPS from serotype 10 was purchased from Sigma. Bisindolylmaleimide I, 1,3-dimethyl-2-thiourea (DMTU) and Apocynin were purchased from Calbiochem. Monoclonal MUC5AC (45M1) antibody was obtained from Lab Vision. Biotinylated rabbit anti-mouse antibody and streptavidin-biotin horseradish peroxidase were purchased from DAKO, UK Ltd. Cell culture. NCI-H292 cells (a human pulmonary mucoepidermoid carcinoma cell line) were purchased from the American Type Culture Collection (ATCC, Manassas, VA) and cultured in RPMI-1640 medium supplemented with 10% (vol/vol) fetal bovine serum, 50 U/ml penicillin, and 50 lg/ml streptomycin (Gibco-BRL, Grand Island, NY) in a humidified atmosphere of 5% CO2 at 37 C. Before experiments, confluent NCI-H292 cells were serum-starved for 24 h to maintain low basal levels
Results and discussion
To determine whether ROS are involved in PA-LPSinduced MUC5AC expression, we assessed the effect of perturbing ROS generation on PA-LPS-induced MUC5AC expression in human epithelial cells. As shown in Fig. 1A, DMTU, an ROS scavenger, attenuated PA-LPSinduced MUC5AC expression at the endogenous mRNA level by performing RT-PCR analysis. To further confirm the involvement of ROS production in PA-LPS-induced MUC5AC expression, we then assessed the effect of DMTU on the MUC5AC expression at the transcriptional
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Fig. 1. ROS are positively involved in PA-LPS-induced MUC5AC expression. (A) NCI-H292 cells were pretreated with DMTU (20 mM) for 30 min and then stimulated with PA-LPS for 12 h. RT-PCR was performed to measure the changes in steady-state mRNA levels. b-actin served as a control for the amount of RNA used in each reaction. (B) HM3 cells stably transfected with pMUC5AC 3.7kb-luc were pretreated with DMTU (20 mM) for 30 min and then stimulated with PA-LPS. Luciferase assays were carried out in triplicate. (C) H2O2 up-regulates MUC5AC transcription in HM3 cells stably transfected with pMUC5AC 3.7kb-luc in dose-dependent manner. (D) DMTU blocks H2O2-induced MUC5AC transcription in dose-dependent manner. (E) PA-LPS-induced MUC5AC protein expression was inhibited by DMTU. After pretreatment with DMTU for 30 min, NCI-H292 cells were stimulated with PA-LPS for 24 h to measure MUC5AC protein expression by Immunohistochemistry (200·). Values are means ± SD (n = 3).
level by performing Luciferase assay. As shown in Fig. 1B, PA-LPS-induced MUC5AC transcriptional activity was blocked by DMTU in HM3 cells stably transfected with pMUC5AC 3.7kb-luc. Consistent with these results, hydrogen peroxide (H2O2), which is known as ROS, up-regulates MUC5AC transcriptional activity in dose-dependent manner (Fig. 1C). H2O2-induced MUC5AC transcription was also blocked by DMTU in dose-dependent manner (Fig. 1D). Furthermore, DMTU abrogated PA-LPSinduced MUC5AC protein expression by performing Immunohistochemistry in human epithelial cells (Fig. 1E). Taken together, these data indicate that ROS are positively involved in PA-LPS-induced MUC5AC expression.
PA-LPS up-regulates MUC5AC expression through NADPH Oxidase Because NADPH oxidase, known as the respiratory burst oxidase, generates ROS in response to bacteria infection [29], we next investigated whether NADPH oxidase is required for PA-LPS-induced MUC5AC expression. Interestingly, apocynin, which is known to block NADPH oxidase assembly, inhibited PA-LPS-induced MUC5AC expression at both mRNA and transcriptional level in human epithelial cells (Fig. 2A and B). Moreover, as shown in Fig. 2C, apocynin attenuated PA-LPS-induced MUC5AC protein expression by performing Immunohistochemistry in human epithelial cells. Concomitantly, PA-LPS
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Fig. 2. PA-LPS up-regulates MUC5AC expression through NADPH oxidase. (A) Apocynin, an NADPH oxidase (Nox) inhibitor attenuates PA-LPSinduced MUC5AC expression. NCI-H292 cells were pretreated with apocynin (1 mM) for 30 min, then treated with PA-LPS for 12 h to analyze MUC5AC expression by RT-PCR. (B) Apocynin blocks PA-LPS-induced transcription in HM3 cells stably transfected with pMUC5AC 3.7kb-luc. (C) PA-LPSinduced MUC5AC protein expression was abolished by apocynin. After pretreatment with apocynin (1 mM) for 30 min, NCI-H292 cells were stimulated with PA-LPS for 24 h to measure MUC5AC protein expression by Immunohistochemistry (200·). (D) NCI-H292 cells were pretreated with apocynin (1 mM) for 30 min. Then, the cells were stimulated with PA-LPS (10 lg/mL) for 2 h to measure H2O2 production in the supernatants of cells. Data are expressed as means ± SD (n = 3). *p < 0.01, compared with PA-LPS alone.
induced ROS generation was indeed blocked by apocynin (Fig. 2D). Thus, these results suggest that NADPH oxidase generates ROS in response to PA-LPS stimulation, which in turn, leads to up-regulate MUC5AC expression. PKC mediates PA-LPS-induced MUC5AC expression via ROS generation Having identified that ROS generation by NADPH oxidase is involved in PA-LPS-induced MUC5AC expression, still unknown is which upstream signaling molecules regulate PA-LPS-induced MUC5AC expression through ROS.
Because PKC has recently been shown to activate NADPH oxidase to generate ROS [25], we next explored the possibility that PKC is also involved in PA-LPS-induced MUC5AC expression. Bisindolylmaleimide I, a PKC inhibitor, blocked PA-LPS-induced MUC5AC expression at both mRNA and transcriptional level in human epithelial cells (Fig. 3A and B). Consistent with these data, PALPS-induced MUC5AC protein expression was abolished by Bisindolylmaleimide I (Fig. 3C). Furthermore, Bisindolylmaleimide I also inhibited PA-LPS induced ROS generation (Fig. 3D). Collectively, these data indicates that PKC mediates PA-LPS-induced MUC5AC expression via ROS generation.
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Fig. 3. PKC mediates PA-LPS-induced MUC5AC expression via ROS generation. (A) NCI-H292 cells were pretreated with Bisindolylmaleimide I (5 lM), a PKC inhibitor, for 30 min, then treated with PA-LPS for 12 h to measure MUC5AC gene expression by RT-PCR. (B) Bisindolylmaleimide I blocks PALPS-induced MUC5AC transcription in HM3 cells stably transfected with pMUC5AC 3.7kb-luc. (C) PA-LPS-induced MUC5AC protein expression was attenuated by Bisindolylmaleimide I. After pretreatment with bisindolylmaleimide I (5 lM) for 30 min, NCI-H292 cells were stimulated with PA-LPS for 24 h to measure MUC5AC protein expression by immunohistochemistry (200·). (D) NCI-H292 cells were pretreated with PKC inhibitor bisindolylmaleimide I (5 lM) for 30 min. Then, the cells were stimulated with PA-LPS (10 lg/mL) for 2 h to measure H2O2 production in the supernatants of cells. Data are expressed as means ± SD (n = 3). *p < 0.01, compared with PA-LPS alone.
PA-LPS up-regulates MUC5AC expression via TGF-a released by ROS Although we have demonstrated that ROS generated by PA-LPS play an important role in MUC5AC expression, one key issue that has yet to be addressed is how ROS transmit signal to up-regulate MUC5AC expression. Based on recent studies showing that TGF-a released by TNF-aconverting enzyme (TACE) activation regulates mucin expression through EGF receptor transactivation and that ROS mediate the activation of TACE-EGF receptor pathway [24–26], we sought to determine whether ROS generated by PA-LPS trigger TGF-a release to mediate MUC5AC expression. As shown in Fig. 4A, apocynin sig-
nificantly blocked PA-LPS-induced TGF-a release, suggesting the involvement of TGF-a in ROS mediated MUC5AC induction by PA-LPS. In conclusion, our studies demonstrated that ROS play a crucial role in PA-LPS-induced MUC5AC expression through PKC-NADPH oxidase-ROS-TGF-a signaling pathway (Fig. 4B). PA-LPS generates ROS through PKC-NADPH oxidase signaling pathway in human epithelial cells. Subsequently, ROS generation by PA-LPS releases TGF-a, which in turn, leads to up-regulate MUC5AC expression. A major finding in the present study is the direct evidence that bacterial PA-LPS utilizes ROS for transmitting signals to provoke the host defense response and
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Fig. 4. PA-LPS up-regulates MUC5AC expression via TGF-a released by ROS. (A) NCI-H292 cells were pretreated with Apocynin (1 mM) for 30 min. Then, the cells were stimulated with PA-LPS for 4 h to measure soluble TGF-a in the supernatants. Data are expressed as means ± SD (n = 3). *p < 0.01, compared with PA-LPS alone. (B) Schematic representation depicting how PA-LPS induces MUC5AC expression in human epithelial cells.
up-regulate MUC5AC mucin expression. Since ROS have been implicated in the pathogenesis of a wide variety of respiratory diseases, such as asthma, emphysema and chronic obstructive pulmonary disease [21,22], this finding should enhance our understanding of bacteria-induced signaling mechanism under these diseased conditions. Future studies will focus on identifying which cell surface receptor is responsible for ROS-mediated MUC5AC expression induced by PA-LPS. Another important finding in the present study is elucidation of the signaling mechanism involved in PA-LPSinduced MUC5AC expression, because the molecular mechanisms by which P. aeruginosa cause mucin overproduction have remained largely unknown. In contrast to the relatively well-known mechanism by which MUC2 mucin is up-regulated by PA-LPS, the signaling mechanism underlying PA-LPS-induced MUC5AC mucin expression is still elusive. In this study, we provided direct evidence that PA-LPS up-regulates MUC5AC expression via PKCNADPH oxidase-ROS-TGF-a signaling pathway. These results imply that the molecular mechanism underlying up-regulation of different mucin gene may be quite different in P. aeruginosa infection and may help us to develop more feasible therapeutic strategy for inhibiting mucin overproduction. Take together, our finding may provide the novel insight into modulating mucin overproduction in P. aeruginosa infection and may lead to novel intervention for chronic inflammatory airway diseases. Acknowledgments This work was supported in part by grants from National Natural Science Foundation of China (No. 30570807 and 30770948) and High-level Innovation Personnel Project of Department of Health of Zhejiang
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Biochemical and Biophysical Research Communications 366 (2008) 513–519 www.elsevier.com/locate/ybbrc
Reactive oxygen species regulate Pseudomonas aeruginosa lipopolysaccharide-induced MUC5AC mucin expression via PKC-NADPH oxidase-ROS-TGF-a signaling pathways in human airway epithelial cells Fugui Yan
a,1
, Wen Li
a,1
, Hirofumi Jono b, Qingmei Li a, Shuangmei Zhang a, Jian-Dong Li b, Huahao Shen a,*
a
b
Department of Respiratory Medicine, Second Affiliated Hospital, Zhejiang University School of Medicine, Zhejiang University Institute of Respiratory Diseases, Hangzhou 310009, China Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, NY 14642, USA Received 27 November 2007 Available online 20 December 2007
Abstract Mucin overproduction is a hallmark of chronic inflammatory airway diseases, such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis. Excessive production of mucin leads to airway mucus obstruction and contributes to morbidity and mortality in these diseases. The molecular mechanisms underlying mucin overproduction, however, still remain largely unknown. Here, we report that the bacterium P. aeruginosa, an important human respiratory pathogen causing cystic fibrosis, utilizes reactive oxygen species (ROS) to upregulate MUC5AC mucin expression. Pseudomonas aeruginosa lipopolysaccharide (PA-LPS) induces production of ROS through protein kinase C (PKC)-NADPH oxidase signaling pathway in human epithelial cells. Subsequently, ROS generation by PA-LPS releases transforming growth factor-a (TGF-a), which in turn, leads to up-regulate MUC5AC expression. These findings may bring new insights into the molecular pathogenesis of P. aeruginosa infections and lead to novel therapeutic intervention for inhibiting mucin overproduction in patients with P. aeruginosa infections. 2007 Elsevier Inc. All rights reserved. Keywords: MUC5AC mucin; Pseudomonas aeruginosa; Reactive oxygen species
Gram-negative bacterium Pseudomonas aeruginosa (P. aeruginosa) is an important and opportunistic human respiratory pathogen [1,2]. P. aeruginosa infections cause chronic inflammatory airway diseases, such as cystic fibrosis, asthma and chronic obstructive pulmonary disease [1–3]. P. aeruginosa is ineradicable by antibiotics and
Abbreviations: PKC, Protein kinase C; P. aeruginosa, Pseudomonas aeruginosa; PA-LPS, P. aeruginosa lipopolysaccharide; ROS, reactive oxygen species; TGF-a, transforming growth factor-a; TACE, TNF-aconverting enzyme. * Corresponding author. Fax: +86 571 87767122. E-mail address: [email protected] (H. Shen). 1 These authors contributed equally to this work. 0006-291X/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.11.172
responsible for airway mucus overproduction, a hallmark of chronic inflammatory airway diseases [4]. Mucus overproduction mainly results from up-regulation of mucin, a primary innate defensive response for mammalian airways [5,6]. Mucins, the major components of mucus secretions, are high-molecular weight and heavily glycosylated proteins, synthesized by the mucosal epithelial cells to protect the mucosal surface and trap the inhaled microbial particles, including bacteria and viruses for mucociliary clearance [7]. However, under diseased conditions, the mucociliary clearance mechanism becomes defective. In cystic fibrosis patients, the excessive production of mucin will cause airway mucus obstruction, which, in turn lead to death of patients [5–7]. To date, 20 mucin genes
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have been identified [6–10]. Among these, at least MUC2, MUC5AC, and MUC5B have been shown to play an important role in the pathogenesis of respiratory infectious diseases [6–12]. Recent studies have demonstrated that nontypeable Haemophilus influenzae, an important human respiratory pathogen, up-regulates MUC5AC expression via activation of p38 MAPK and MUC2 expression via activation of NF-jB signaling, respectively [13–15]. In addition, we recently also found that Streptococcus pneumoniae pneumolysin up-regulates MUC5AC via a novel IKK-ERK pathway [16]. Moreover, current studies have provided evidence that P. aeruginosa lipopolysaccharide (PA-LPS) up-regulates MUC2 mucin gene expression in epithelial cells via Src-dependent Ras-MEK1/2-ERK1/2pp90rsk-NF-jB pathway [17,18]. However, in contrast to the relatively well-known mechanism by which MUC2 mucin is up-regulated by PA-LPS, the signaling mechanism underlying PA-LPS-induced MUC5AC mucin expression is still largely unknown. Reactive oxygen species (ROS) are produced by epithelial cells lining the respiratory airways, and recognized as acute mediator for inflammation [19,20]. ROS have been implicated in the pathogenesis of a wide variety of respiratory diseases, such as asthma, emphysema and chronic obstructive pulmonary disease [21,22]. In addition to its known cytotoxic oxidative stress, growing evidence suggests that ROS play an important role in signal transduction [23]. Recent studies have provided evidence that ROS mediate mucin production induced by various stimuli, such as cigarette-smoked, Neutrophil elastase and phorbol 12-myristate 13-acetate [24–26]. Despite the critical role for ROS in the pathogenesis of respiratory disease, its role in regulating PA-LPS-induced MUC5AC mucin expression still remains elusive. Here, we show that ROS play crucial role in PA-LPSinduced MUC5AC expression. PA-LPS generates ROS through PKC-NADPH oxidase signaling pathway in human epithelial cells. ROS generation by PS-LPS releases TGF-a, which in turn, leads to up-regulate MUC5AC expression. These findings may provide new insights into the pathogenesis of P. aeruginosa infection and lead to novel therapeutic strategies for chronic inflammatory airway diseases.
of MUC5AC expression. HM3 (human colon epithelial) cells stably transfected with pMUC5AC 3.7kb-luc were maintained as described previously [14]. PA-LPS treatment. After 24 h of serum starvation, cells were stimulated with PA-LPS (10 lg/mL). For inhibitor studies, serum starved cells were pretreated with inhibitors for 30 min before exposure to stimuli, then the cells were cultured for various times with PA-LPS and inhibitors. RT-PCR. Total RNA was isolated from NCI-H292 cells using TRIzol Reagent (Invitrogen) according to the manufacturer’s instruction. For RT-PCR, cDNA was generated by reverse transcription using 2 lg total RNA. A 529-bp fragment of human MUC5AC was amplified using primers (forward, 5 0 -ACC TGC CCA GCC GAC AAG-3 0 ; reverse, 5 0 GGT ACA GGG TCC CGT TGA TG-3 0 ). As internal controls, a 527-bp fragment of human b-actin was amplified using primers (forward, 5 0 -CTA CAA TGA GCT GCG TGT GG-3 0 ; reverse, 5 0 -AAG GAA GGC TGG AAG AGT GC-3 0 ). The PCR mixture was denatured at 94 C for 5 min, followed by 30 cycles at 94 C for 30 s, 55 C for 45 s, and 72 C for 45 s. The PCR products were resolved by gel electrophoresis on 1.5% agarose gels containing ethidium bromide. Lack of DNA contamination was verified by RT-PCR with presence or absence of RT. Luciferase assay. HM3 cells stably transfected with MUC5AC-luciferase plasmid were treated with PA-LPS or H2O2 for 5 h and then harvested for luciferase assay. For experiments with inhibitors, cells were pretreated with inhibitors for 30 min, then treated with PA-LPS or H2O2 for 5 h, and harvested for luciferase assays. Luciferase assays were performed on a Monolight 3010 luminometer [27,28]. Immunohistochemistry. Cells were fixed in 4% paraformaldehyde solution for 15 min, permeabilized with 0.1% Tween 20 in PBS for 10 min, peroxidase-blocked in 0.3% H2O2 for 10 min, blocked by normal goat serum for 10 min subsequently incubated with mouse monoclonal antibody to MUC5AC (clone 45M1, 1:100) for 1 h followed by 10 min in biotinylated rabbit anti-mouse antibody (1:300) and another 10 min in streptavidin–biotin horseradish peroxidase (1:50). Cells were developed for 2 min with diaminobenzidine as chromogen substrate (DAKO Ltd.), counterstained with hematoxylin, and mounted in a xylene-based mountant (BDH-Merck, UK). H2O2 measurements. For inhibitory studies, cells were pretreated with inhibitors for 30 min before exposure to stimuli. After Cells were treated with PA-LPS (10 lg/mL) for 2 h, H2O2 production in the cell supernatants was measured by using the Amplex Red Hydrogen Peroxide/Peroxidase Assay kit (Invitrogen) according to the manufacturer’s instructions. ELISA. After reaching confluence and being serum starved for 24 h, cells were stimulated with PA-LPS (10 lg/mL) for 4 h. For inhibitor studies, cells were pretreated with the inhibitors for 30 min before PA-LPS was added. Cell supernatants were collected and concentrated 10-fold using an Ultracel YM-3 Centrifugal Filter Device (Millipore), and the levels of TGF-a in concentrated cell supernatants were quantified using ELISA kits (R&D Systems Inc., Minneapolis, MN).
Materials and methods
ROS are positively involved in PA-LPS-induced MUC5AC expression
Materials. PA-LPS from serotype 10 was purchased from Sigma. Bisindolylmaleimide I, 1,3-dimethyl-2-thiourea (DMTU) and Apocynin were purchased from Calbiochem. Monoclonal MUC5AC (45M1) antibody was obtained from Lab Vision. Biotinylated rabbit anti-mouse antibody and streptavidin-biotin horseradish peroxidase were purchased from DAKO, UK Ltd. Cell culture. NCI-H292 cells (a human pulmonary mucoepidermoid carcinoma cell line) were purchased from the American Type Culture Collection (ATCC, Manassas, VA) and cultured in RPMI-1640 medium supplemented with 10% (vol/vol) fetal bovine serum, 50 U/ml penicillin, and 50 lg/ml streptomycin (Gibco-BRL, Grand Island, NY) in a humidified atmosphere of 5% CO2 at 37 C. Before experiments, confluent NCI-H292 cells were serum-starved for 24 h to maintain low basal levels
Results and discussion
To determine whether ROS are involved in PA-LPSinduced MUC5AC expression, we assessed the effect of perturbing ROS generation on PA-LPS-induced MUC5AC expression in human epithelial cells. As shown in Fig. 1A, DMTU, an ROS scavenger, attenuated PA-LPSinduced MUC5AC expression at the endogenous mRNA level by performing RT-PCR analysis. To further confirm the involvement of ROS production in PA-LPS-induced MUC5AC expression, we then assessed the effect of DMTU on the MUC5AC expression at the transcriptional
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Fig. 1. ROS are positively involved in PA-LPS-induced MUC5AC expression. (A) NCI-H292 cells were pretreated with DMTU (20 mM) for 30 min and then stimulated with PA-LPS for 12 h. RT-PCR was performed to measure the changes in steady-state mRNA levels. b-actin served as a control for the amount of RNA used in each reaction. (B) HM3 cells stably transfected with pMUC5AC 3.7kb-luc were pretreated with DMTU (20 mM) for 30 min and then stimulated with PA-LPS. Luciferase assays were carried out in triplicate. (C) H2O2 up-regulates MUC5AC transcription in HM3 cells stably transfected with pMUC5AC 3.7kb-luc in dose-dependent manner. (D) DMTU blocks H2O2-induced MUC5AC transcription in dose-dependent manner. (E) PA-LPS-induced MUC5AC protein expression was inhibited by DMTU. After pretreatment with DMTU for 30 min, NCI-H292 cells were stimulated with PA-LPS for 24 h to measure MUC5AC protein expression by Immunohistochemistry (200·). Values are means ± SD (n = 3).
level by performing Luciferase assay. As shown in Fig. 1B, PA-LPS-induced MUC5AC transcriptional activity was blocked by DMTU in HM3 cells stably transfected with pMUC5AC 3.7kb-luc. Consistent with these results, hydrogen peroxide (H2O2), which is known as ROS, up-regulates MUC5AC transcriptional activity in dose-dependent manner (Fig. 1C). H2O2-induced MUC5AC transcription was also blocked by DMTU in dose-dependent manner (Fig. 1D). Furthermore, DMTU abrogated PA-LPSinduced MUC5AC protein expression by performing Immunohistochemistry in human epithelial cells (Fig. 1E). Taken together, these data indicate that ROS are positively involved in PA-LPS-induced MUC5AC expression.
PA-LPS up-regulates MUC5AC expression through NADPH Oxidase Because NADPH oxidase, known as the respiratory burst oxidase, generates ROS in response to bacteria infection [29], we next investigated whether NADPH oxidase is required for PA-LPS-induced MUC5AC expression. Interestingly, apocynin, which is known to block NADPH oxidase assembly, inhibited PA-LPS-induced MUC5AC expression at both mRNA and transcriptional level in human epithelial cells (Fig. 2A and B). Moreover, as shown in Fig. 2C, apocynin attenuated PA-LPS-induced MUC5AC protein expression by performing Immunohistochemistry in human epithelial cells. Concomitantly, PA-LPS
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Fig. 2. PA-LPS up-regulates MUC5AC expression through NADPH oxidase. (A) Apocynin, an NADPH oxidase (Nox) inhibitor attenuates PA-LPSinduced MUC5AC expression. NCI-H292 cells were pretreated with apocynin (1 mM) for 30 min, then treated with PA-LPS for 12 h to analyze MUC5AC expression by RT-PCR. (B) Apocynin blocks PA-LPS-induced transcription in HM3 cells stably transfected with pMUC5AC 3.7kb-luc. (C) PA-LPSinduced MUC5AC protein expression was abolished by apocynin. After pretreatment with apocynin (1 mM) for 30 min, NCI-H292 cells were stimulated with PA-LPS for 24 h to measure MUC5AC protein expression by Immunohistochemistry (200·). (D) NCI-H292 cells were pretreated with apocynin (1 mM) for 30 min. Then, the cells were stimulated with PA-LPS (10 lg/mL) for 2 h to measure H2O2 production in the supernatants of cells. Data are expressed as means ± SD (n = 3). *p < 0.01, compared with PA-LPS alone.
induced ROS generation was indeed blocked by apocynin (Fig. 2D). Thus, these results suggest that NADPH oxidase generates ROS in response to PA-LPS stimulation, which in turn, leads to up-regulate MUC5AC expression. PKC mediates PA-LPS-induced MUC5AC expression via ROS generation Having identified that ROS generation by NADPH oxidase is involved in PA-LPS-induced MUC5AC expression, still unknown is which upstream signaling molecules regulate PA-LPS-induced MUC5AC expression through ROS.
Because PKC has recently been shown to activate NADPH oxidase to generate ROS [25], we next explored the possibility that PKC is also involved in PA-LPS-induced MUC5AC expression. Bisindolylmaleimide I, a PKC inhibitor, blocked PA-LPS-induced MUC5AC expression at both mRNA and transcriptional level in human epithelial cells (Fig. 3A and B). Consistent with these data, PALPS-induced MUC5AC protein expression was abolished by Bisindolylmaleimide I (Fig. 3C). Furthermore, Bisindolylmaleimide I also inhibited PA-LPS induced ROS generation (Fig. 3D). Collectively, these data indicates that PKC mediates PA-LPS-induced MUC5AC expression via ROS generation.
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Fig. 3. PKC mediates PA-LPS-induced MUC5AC expression via ROS generation. (A) NCI-H292 cells were pretreated with Bisindolylmaleimide I (5 lM), a PKC inhibitor, for 30 min, then treated with PA-LPS for 12 h to measure MUC5AC gene expression by RT-PCR. (B) Bisindolylmaleimide I blocks PALPS-induced MUC5AC transcription in HM3 cells stably transfected with pMUC5AC 3.7kb-luc. (C) PA-LPS-induced MUC5AC protein expression was attenuated by Bisindolylmaleimide I. After pretreatment with bisindolylmaleimide I (5 lM) for 30 min, NCI-H292 cells were stimulated with PA-LPS for 24 h to measure MUC5AC protein expression by immunohistochemistry (200·). (D) NCI-H292 cells were pretreated with PKC inhibitor bisindolylmaleimide I (5 lM) for 30 min. Then, the cells were stimulated with PA-LPS (10 lg/mL) for 2 h to measure H2O2 production in the supernatants of cells. Data are expressed as means ± SD (n = 3). *p < 0.01, compared with PA-LPS alone.
PA-LPS up-regulates MUC5AC expression via TGF-a released by ROS Although we have demonstrated that ROS generated by PA-LPS play an important role in MUC5AC expression, one key issue that has yet to be addressed is how ROS transmit signal to up-regulate MUC5AC expression. Based on recent studies showing that TGF-a released by TNF-aconverting enzyme (TACE) activation regulates mucin expression through EGF receptor transactivation and that ROS mediate the activation of TACE-EGF receptor pathway [24–26], we sought to determine whether ROS generated by PA-LPS trigger TGF-a release to mediate MUC5AC expression. As shown in Fig. 4A, apocynin sig-
nificantly blocked PA-LPS-induced TGF-a release, suggesting the involvement of TGF-a in ROS mediated MUC5AC induction by PA-LPS. In conclusion, our studies demonstrated that ROS play a crucial role in PA-LPS-induced MUC5AC expression through PKC-NADPH oxidase-ROS-TGF-a signaling pathway (Fig. 4B). PA-LPS generates ROS through PKC-NADPH oxidase signaling pathway in human epithelial cells. Subsequently, ROS generation by PA-LPS releases TGF-a, which in turn, leads to up-regulate MUC5AC expression. A major finding in the present study is the direct evidence that bacterial PA-LPS utilizes ROS for transmitting signals to provoke the host defense response and
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Fig. 4. PA-LPS up-regulates MUC5AC expression via TGF-a released by ROS. (A) NCI-H292 cells were pretreated with Apocynin (1 mM) for 30 min. Then, the cells were stimulated with PA-LPS for 4 h to measure soluble TGF-a in the supernatants. Data are expressed as means ± SD (n = 3). *p < 0.01, compared with PA-LPS alone. (B) Schematic representation depicting how PA-LPS induces MUC5AC expression in human epithelial cells.
up-regulate MUC5AC mucin expression. Since ROS have been implicated in the pathogenesis of a wide variety of respiratory diseases, such as asthma, emphysema and chronic obstructive pulmonary disease [21,22], this finding should enhance our understanding of bacteria-induced signaling mechanism under these diseased conditions. Future studies will focus on identifying which cell surface receptor is responsible for ROS-mediated MUC5AC expression induced by PA-LPS. Another important finding in the present study is elucidation of the signaling mechanism involved in PA-LPSinduced MUC5AC expression, because the molecular mechanisms by which P. aeruginosa cause mucin overproduction have remained largely unknown. In contrast to the relatively well-known mechanism by which MUC2 mucin is up-regulated by PA-LPS, the signaling mechanism underlying PA-LPS-induced MUC5AC mucin expression is still elusive. In this study, we provided direct evidence that PA-LPS up-regulates MUC5AC expression via PKCNADPH oxidase-ROS-TGF-a signaling pathway. These results imply that the molecular mechanism underlying up-regulation of different mucin gene may be quite different in P. aeruginosa infection and may help us to develop more feasible therapeutic strategy for inhibiting mucin overproduction. Take together, our finding may provide the novel insight into modulating mucin overproduction in P. aeruginosa infection and may lead to novel intervention for chronic inflammatory airway diseases. Acknowledgments This work was supported in part by grants from National Natural Science Foundation of China (No. 30570807 and 30770948) and High-level Innovation Personnel Project of Department of Health of Zhejiang
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