Nox4 Mediates Hypoxia-Stimulated Myofibroblast ...

Original Paper Int Arch Allergy Immunol 2012;159:399–409 DOI: 10.1159/000337658

Received: August 29, 2011 Accepted after revision: February 22, 2012 Published online: July 26, 2012

Nox4 Mediates Hypoxia-Stimulated Myofibroblast Differentiation in Nasal Polyp-Derived Fibroblasts You-Mi Moon a Hee Joon Kang b Jung-Sun Cho a Il-Ho Park b, c Heung-Man Lee a–c a

Brain Korea 21 Project for Biomedical Science, b Department of Otorhinolaryngology-Head and Neck Surgery, College of Medicine, and c Medical Devices Clinical Trial Center, Guro Hospital, Korea University, Seoul, South Korea

Abstract Background: Chronic hypoxia is associated with remodeling in various organs. Reactive oxygen species (ROS) derived from NADPH oxidases (Nox), and transforming growth factor-␤1 (TGF-␤1) have been implicated in the pathogenesis of hypoxia-induced remodeling. The aims of this study were to determine in hypoxia-stimulated nasal polyp-derived fibroblasts (NPDF) the effect of hypoxia on the differentiation of myofibroblasts, the role of ROS, the major Nox homolog mediating myofibroblast differentiation, and the role of TGF-␤1. Methods: Eight primary cultures of NPDF were established from nasal polyps, which were incubated under hypoxic conditions. Reverse transcription polymerase chain reaction for ␣SMA, Nox1, Nox3, Nox4, Nox5, and fibronectin mRNA was performed. Western blotting for ␣-SMA and fibronectin was done. ROS production was detected using a fluorometer. NPDF were pretreated with ROS scavengers and transfected with siNox4. The TGF-␤1 protein level was measured by ELISA. The effect of treatment with TGF-␤1 type I tyrosine kinase inhibitor SB431542 on myofibroblast differentiation was ob-

© 2012 S. Karger AG, Basel 1018–2438/12/1594–0399$38.00/0 Fax +41 61 306 12 34 E-Mail [email protected] www.karger.com

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served. Results: Hypoxic stimulation of NPDF significantly increased ␣-SMA and fibronectin mRNA and protein expression. ROS production was increased by hypoxia, and ROS scavengers inhibited myofibroblast differentiation. Nox4 mRNA was the only Nox homolog increased by hypoxia. Transfection with siNox4 inhibited myofibroblast differentiation. TGF-␤1 was secreted endogenously by hypoxic NPDF. SB431542 significantly inhibited myofibroblast differentiation. Conclusions: Hypoxia induces myofibroblast differentiation of NPDF through a signaling pathway involving Nox4dependent ROS generation and TGF-␤1. Therapies targeting Nox4 may be effective against remodeling of nasal polyps. Copyright © 2012 S. Karger AG, Basel

Introduction

Nasal polyps are benign outgrowths that originate from the mucous membranes of the nasal cavity or paranasal sinuses. Although the pathophysiologic mechanisms leading to nasal polyp formation are still poorly understood, numerous studies suggest that differentia-

Y.-M. Moon and H.J. Kang contributed equally to this work.

Correspondence to: Dr. Heung-Man Lee Department of Otorhinolaryngology, Head and Neck Surgery Guro Hospital, Korea University College of Medicine 80 Guro-dong, Guro-gu, Seoul 152-703 (South Korea) Tel. +82 2 2626 3185, E-Mail lhman @ korea.ac.kr

Downloaded by: Univ. of Michigan, Taubman Med.Lib. 141.211.183.139 - 5/4/2016 3:58:49 PM

Key Words Fibroblast ⴢ Hypoxia ⴢ Nasal polyp ⴢ Nox4 ⴢ Remodeling ⴢ Transforming growth factor-␤1

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Int Arch Allergy Immunol 2012;159:399–409

differentiation of cardiac fibroblasts to myofibroblasts [18]. However, to date, the role of Nox4 in hypoxia-associated nasal polyp remodeling has not been described. Transforming growth factor-␤ (TGF-␤) is involved in the pathogenesis of various airway diseases [21]. Among its many activities, TGF-␤ is able to induce fibroblast proliferation and differentiation into myofibroblasts. There are three isoforms of TGF-␤: TGF-␤1, TGF-␤2, and TGF␤3. TGF-␤1 has been shown to be the isoform most relevant to the pathogenesis of nasal polyps [22]. It is known that tissue hypoxia upregulates TGF-␤1, stimulating the development of tissue fibrosis [23]. In addition, hypoxiastimulated cells are known to secrete TGF-␤1, stimulating tissue fibrosis [24]. Thus, we investigated whether TGF-␤1 signaling participated in hypoxia-induced myofibroblast differentiation in NPDF. In this investigation, we sought to determine in hypoxia-stimulated NPDF the effect of hypoxia on the differentiation of myofibroblasts, the role of ROS, the major Nox homolog mediating myofibroblast differentiation, and the role of TGF-␤1.

Materials and Methods Reagents N-acetylcysteine (NAC), diphenyliodonium (DPI), and ebselen were purchased from Sigma (St. Louis, Mo., USA). Isolation and Induction of NPDF Fibroblasts were obtained from 8 patients (4 females and 4 males; 32.3 8 5.2 years of age) who underwent endoscopic sinus surgery for nasal polyps at the Department of Otorhinolaryngology, Korea University Medical Center. None of the patients were smokers, they had no history of nasal allergy, asthma, or aspirin hypersensitivity, and they had not been treated with oral or topical antiallergic agents during the previous 8 weeks. Written informed consent was obtained from all patients prior to surgery. This study was approved by the Ethics Committee of Korea University (KUGGR-2010-049). NPDF were isolated from surgical tissues by enzymatic digestion with collagenase (500 U/ml; Sigma), hyaluronidase (30 U/ml; Sigma), and DNAse (10 U/ml; Sigma). Briefly, following 2 h of incubation with under 5% CO2 at 37 ° C in culture plates, cells were collected by centrifugation, washed twice, and resuspended in Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, Calif., USA) containing 10% (v/v) heat-inactivated fetal bovine serum and antibiotics, 2-glutamate (Invitrogen), 100 ␮g/ml penicillin, and 100 ␮g/ml streptomycin. Cells were allowed to attach for 4 days. Nonadherent cells were removed by changing the medium. Fibroblasts were detached with trypsin-EDTA solution (Invitrogen). After the cells were washed, they were resuspended in medium and used for subsequent experiments. The purity of the fibroblast cultures was assessed using flow cytometry. Approximately 99% of cells in cultured NPDF were positive for the fibro

Moon /Kang /Cho /Park /Lee


tion of fibroblasts into myofibroblasts plays a key role [1, 2]. Besides expressing ␣-smooth muscle actin (␣-SMA), myofibroblasts produce large amounts of extracellular matrix (ECM) components [3]. In nasal polyps, the highest density of myofibroblasts is found in the pedicle area of the polyp. This is the area where the nasal polyp is thought to grow [4]. Differences in blood vessel density within the nasal cavity create an unequal distribution of blood flow [5]. When chronic inflammation of the tissue exists, such as that observed with chronic rhinosinusitis, these differences can lead to local tissue hypoxia. Nasal polyps usually originate in and around the middle meatus, which receives only a poor blood supply compared with other regions of the nasal cavity [6]. In addition, mucosal edema or nasal polyps can cause occlusion of paranasal sinus ostia and can also overwhelm neovascularization, further impacting local tissue hypoxia [5]. There is accumulating evidence supporting the idea that chronic hypoxia results in structural changes and accumulation of inflammatory and profibrotic factors surrounding the area of low oxygen [7–9]. Studies on hypoxic stimulation of nasal polyp-derived fibroblasts (NPDF) showed an increase in inflammatory mediators and fibrotic factors, promoting fibrosis of nasal polyps [6, 10]. Reactive oxygen species (ROS) initiate cellular signaling events that control a variety of cellular functions [11, 12]. It has been shown that ROS are released during the phenotypic transformation of fibroblasts to myofibroblasts and are important in the control of ECM protein deposition [13]. In animal models, ROS have been directly linked to the vascular remodeling associated with chronic hypoxia-induced pulmonary artery hypertension [14]. NADPH oxidases (Nox) are a family of multisubunit enzymes which catalyze the reduction of O2 to ROS [15]. The prototypical member of this family, Nox2 (gp91phox), is expressed in phagocytic cells [16]. There are multiple homologs in nonphagocytic cells with a wide range of tissue distributions: Nox1, Nox3, Nox4, Nox5, Duox1, and Duox2. In phagocytes ROS originating from Nox mediate microbicidal activities, whereas in nonphagocytic cells they are required for cellular responses to cytokines and growth factors [17]. Numerous studies have observed that Nox are involved in an increase in ROS generation under hypoxic conditions [18, 19]. Previous studies have shown the role of Nox4 in the pathogenesis of hypoxia-induced vascular remodeling [20]. Nox4 has also been implicated in the

Reverse Transcription Polymerase Chain Reaction Reverse transcription was carried out with 2 ␮g of RNA for each sample. Total RNA was denatured at 65 ° C for 5 min. After cooling on ice, the following components were added to the samples: 10 ␮l of 5! reverse transcriptase buffer, 5 ␮l of 2.5 mM dNTP, 1 ␮l of RNase inhibitor, and 1 ␮l of Moloney murine leukemia virus reverse transcriptase. After 60 min at 37 ° C, reverse transcriptase was inactivated by heating the mixture at 95 ° C for 5 min. PCR was performed using primers for ␣-SMA, Nox1, Nox3, Nox4, Nox5, fibronectin, and GAPDH supplied by Bioneer (Daejeon, Korea).

Western Blot Analysis The cells were lysed in PRO-PREPTM protein extraction solution (iNtRON Biotechnology, Seongnam, Korea). Proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (PVDF; Millipore, Inc., Billerica, Mass., USA). The membranes were then incubated with antibodies for ␣-SMA (Millipore Molecular Biology, Billerica, Mass., USA), fibronectin (Santa Cruz Biotechnology, Inc., Santa Cruz, Calif., USA), and ␤actin (Santa Cruz Biotechnology). After incubation, the membrane was washed three times for 5 min and then treated with peroxidase-conjugated antimouse or anti-rabbit (Vector Laboratories, Burlingame, Calif., USA) for 1 h. After washing the membranes, substrate was added from an enhanced chemiluminescence reagent kit (Du Pont, Boston, Mass., USA) and exposed to x-ray film for 10 s. Measurement of ROS Generation Total ROS generation by cells was quantified using 2ⴕ,7ⴕdichlorfluorescein-diacetate (DCFH-DA) assay. Cells plated on 96-well cell culture plates were incubated under normoxic (21% O2, 5% CO2) or hypoxic (1% O2, 5% CO2) conditions for 60 min. Then the cells were washed and incubated with DCFH-DA (10 ␮ M) in serum-free culture medium under normoxic or hypoxic conditions for 30 additional minutes. The medium was removed and cells were washed three times with serum-free medium. The fluorescence measured at 535 nm with an excitation wavelength of 485 nm using a fluorometer (Beckman-Coulter, Fullerton, Calif., USA). Cells plated on 35-mm cell culture plates were cultured under normoxic or hypoxic conditions for 30 min. Then the cells were washed three times with serum-free medium. DCFH-DA was added in phosphate-buffered saline (PBS) and cells were incubated under normoxic or hypoxic conditions for 60 min. Cell culture plates were washed three times with PBS. Fluorescent signals were captured by fluorescent microscope (LSM700; Zeiss, Oberkochen, Germany).

centration of 200 nM with OligofectamineTM (Invitrogen). After an incubation period of 24 h under normoxic conditions, the cells were exposed to normoxic or hypoxic conditions for an additional 24 h. Immunofluoescent Staining of ␣-SMA Protein Cells were fixed in PBS containing 4% paraformaldehyde for 30 min, blocked with 3% bovine serum albumin, incubated with an anti-␣-SMA (1:200) for 2 h, and washed 3 times with PBS for 5 min. Cells were then incubated in goat anti-mouse IgG Alexa Fluor 488 (Invitrogen) at 1: 200 for 1 h and mounted on Vectashield (Vector Laboratories) with 4ⴕ,6-diamidino-2-phenylindole. TGF-␤1 Measurements TGF-␤1 was measured using an ELISA kit (R&D Systems, Minneapolis, Minn., USA) according to the manufacturer’s protocol. In brief, fibroblasts were incubated with 2.5% fetal bovine serum, and the culture media were collected at the end of the incubation. For active TGF-␤1, a 96-well microplate was coated with capture antibody overnight at room temperature. After three washes with wash buffer (0.05% Tween 20 in PBS), the plates were blocked with the blocking buffer for a minimum of 1 h. Aliquots of samples or standards were added to each well, covered with an adhesive strip, and incubated for 2 h at room temperature. The detection antibody was added and incubated for 2 h. A working dilution of streptavidin was added to each well and incubated for 20 min at room temperature in the dark. The substrate solution was added and incubated for 20 min. After the addition of stop solution, the optical density of each well was determined immediately with the use of a microplate reader set to 450 nm with wavelength correction set to 540 nm. The total amount of TGF-␤1 protein was measured after the culture media were preactivated by addition of acid. Measurement of Cellular Proliferation In order to determine the proliferation rate of NPDF, the colorimetric MTT-[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoliumbromide (Sigma)] based test was performed. NPDF of 4 ! 105 cells/ml were seeded into 96-well tissue culture plates. After seeding, the medium was replaced with the various doses of histamine. The MTT test was performed after 36 h. The optical density of each well was measured using an automatic plate reader (F2000; Hitachi Ltd., Tokyo, Japan) with a 590-nm wavelength. Statistical Analysis The statistical significance of the difference between the control and experimental data was analyzed using Student’s t test. p ! 0.05 was considered statistically significant.

Results

Inhibition of Nox4 by Small Interference RNA NPDF were plated on 100-mm cell culture plates. NPDF were transfected with small interference RNA (siRNA) specific for human Nox4, 5ⴕ-ACUGAGGUACAGCUGGAUGUU-3ⴕ, and its complementary sequence. As a control, scrambled siRNA (siScr) was used. Transfection with siRNA was carried out at a final con-

Hypoxia Increases ␣-SMA and Fibronectin Expression To investigate the effect of hypoxia on ␣-SMA mRNA and protein levels, NPDF were incubated under hypoxia. Reverse transcription polymerase chain reaction (RTPCR) and Western blotting showed that ␣-SMA mRNA and protein expression were significantly increased in

Hypoxia in Nasal Polyps


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blast markers vimentin and Thy-1 but negative for the epithelial marker E-cadherin. Experimental cells were obtained from the fourth cell passage.

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Fig. 1. Hypoxia increases ␣-SMA mRNA and protein expression in NPDF. a RTPCR of ␣-SMA mRNA. b Western blot of ␣-SMA protein. c Immunofluorescent

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NPDF incubated under hypoxic conditions compared with NPDF incubated under normoxic conditions (fig. 1a, b). The increased myofibroblast differentiation (␣-SMA) was confirmed by immunofluorescent staining (fig. 1c). These results indicate that hypoxia induces myofibroblast differentiation in NPDF. RT-PCR and Western blotting showed significantly increased expression of fibronectin mRNA and protein in hypoxic NPDF (fig. 2a, b). This shows that hypoxia induces NPDF to differentiate into myofibroblasts, which secrete ECM protein. ROS Mediates Hypoxia-Induced ␣-SMA mRNA Expression To investigate the role of ROS in ␣-SMA expression, NPDF were pretreated with ROS scavengers NAC (a glutathione precursor and scavenger of H2O2), DPI (an inhibitor of flavin-containing oxidoreductases such as NADPH oxidases), and ebselen (a nonspecific H2O2 scav402


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enger) and then incubated under hypoxia for 72 h. Then total ROS production was determined using a fluorometer, and ␣-SMA and fibronectin mRNA expressions were determined by RT-PCR. Exposure to hypoxia significantly increased the total ROS production, which was potently inhibited by pretreatment with ROS scavengers (fig. 3a). RT-PCR showed a significant increase in ␣-SMA and fibronectin mRNA expressions, which were significantly inhibited by pretreatment with ROS scavengers (fig. 3b). Western blotting for ␣-SMA and fibronectin showed similar results (fig. 3c). These results indicate that ROS mediates hypoxia-induced NPDF differentiation Hypoxia Increases Expression of Nox4 mRNA but Not Nox1, Nox3, or Nox5 mRNA NADPH oxidases are important sources of ROS in human fibroblasts. To identify which Nox is upregulated, RT-PCR was performed for Nox1, Nox3, Nox4, and Nox5. Moon /Kang /Cho /Park /Lee


staining of myofibroblasts (scale bar = 100 ␮m). The results were obtained from at least three independent experiments. * p ! 0.05.

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Endogenously-Produced Factors Mediate HypoxiaInduced Myofibroblast Differentiation in NPDF To determine whether hypoxia directly increases Nox4 expression in NPDF or acts through a mediator, culture media were removed from NPDF that were exposed to hypoxia for 24 h, and then added to normoxic NPDF. The ␣-SMA protein expression level was assessed 72 h later. Western blotting showed that normoxic NPDF grown in hypoxia-conditioned culture media showed a

significant increase in ␣-SMA protein and fibronectin protein expression compared with NPDF grown in normoxic culture media (fig. 5a). This indicates that hypoxic NPDF produce soluble mediators that cause myofibroblast differentiation. It has been shown previously that hypoxic cells secrete TGF-␤1 [9]. To explore whether NPDF exposed to hypoxia also secrete TGF-␤1, the TGF-␤1 levels in hypoxic NPDF were assessed by ELISA. Exposure of NPDF to hypoxia for 48 h resulted in a significant increase in TGF-␤1 production compared with media from NPDF cultured under normoxic conditions (fig. 5b). This shows that TGF-␤1 is endogenously secreted by hypoxic NPDF. The above findings suggest that hypoxia may increase Nox4-mediated myofibroblast differentiation through endogenous production of TGF-␤1. To investigate the functional relevance of TGF-␤1 produced by hypoxic NPDF, NPDF were incubated with and without the TGF␤1 type I tyrosine kinase inhibitor SB431542. RT-PCR showed that treatment with SB431542 significantly inhibited Nox4 mRNA expression (fig. 5c). To investigate the effect of SB431542 on ROS production, NPDF were pretreated with SB431542, and then incubated under normoxia and hypoxia. Then the total ROS production was determined using a fluorometer. The total ROS production by hypoxic NPDF was significantly inhibited by pretreatment with SB431542 (fig. 5d). Western blotting for ␣-SMA and fibronectin showed that SB431542 treatment significantly inhibited ␣-SMA and fibronectin protein expression (fig. 5e). This suggests that TGF-␤1 regulates Nox4 expression and ROS production, and subsequent myofibroblast differentiation.



No significant differences in the expression of Nox1, Nox3, and Nox5 mRNA were observed between normoxic and hypoxic NPDF, but a significant increase in Nox4 mRNA was seen in hypoxic NPDF (fig. 4a). This indicates that Nox4 is the major NADPH homolog in hypoxia-induced myofibroblast differentiation. Nox4 Mediates Hypoxia-Induced Myofibroblast Differentiation To further determine the role of Nox4, we transfected NPDF with small interference oligonucleotide RNA directed against Nox4 (siNox4) prior to hypoxic exposure. RT-PCR for Nox4 mRNA, a fluorometer for total ROS production, and Western blot for ␣-SMA protein were used. Transfection with siNox4 significantly inhibited the expression of Nox4 mRNA (fig. 4b), significantly reduced the total ROS production (fig. 4c), and potently inhibited ␣-SMA and fibronectin protein expression (fig. 4d) in hypoxic NPDF compared with NPDF transfected with siScr. These results indicate that Nox4 is the source of ROS mediating hypoxia-induced myofibroblast differentiation.

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tein. The results were obtained from at least three independent experiments. * p ! 0.05.

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Fig. 2. Hypoxia increases fibronectin expression in NPDF. a RT-PCR of fibronectin mRNA. b Western blot of fibronectin pro-

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(1 mM), diphenyleneiodonium chloride (DPI, 2 ␮ M), and ebselen (10 ␮ M) prior to incubation under normoxic and hypoxic conditions. a Representative cytofluorescent images of intracellular ROS production detected by DCFH-DA (scale bar = 100 ␮m). b RTPCR of ␣-SMA mRNA and fibronectin mRNA. c Western blot of ␣-SMA and fibronectin protein. The results were obtained from at least three independent experiments. * p ! 0.05 versus normoxia; † p ! 0.05 versus hypoxia alone.

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Hypoxia Stimulates NPDF Proliferation through the Nox4 Pathway To investigate whether Nox4-derived ROS are relevant for cellular proliferation, NPDF were transfected with siNox4 and then incubated under hypoxia. MTT assay was then performed. A statistically significant decrease in proliferation was seen in siNox4-transfected NPDF compared with NPDF transfected with siScr (fig. 6). This suggests the pro-proliferative role of Nox4-derived ROS in hypoxia-stimulated NPDF.

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In the present study, we have demonstrated in NPDF that hypoxia-induced myofibroblast differentiation and ECM production are regulated by Nox4-dependent generation of ROS. Also, hypoxia induced endogenous secretion of TGF-␤1, which mediated an increase in Nox4 gene expression, with subsequent myofibroblast differentiation. The events leading to nasal polyp formation are not fully known, but differentiation of fibroblasts into myo-

fibroblasts with a resultant accumulation of ECM proteins and fibrosis are considered to be key processes [1]. Most myofibroblasts are found in the peduncle area, where fibrosis is most evident. The nasal polyps are thought to grow from this area [4]. Nasal polyps most commonly originate in and around the middle meatus, where the tissue receives only a scant bloody supply compared with other parts of the nose, resulting in local tissue hypoxia [6]. Mucosal edema due to chronic rhinosinusitis or a nasal polyp can result in occlusion of the sinus ostium, aggravating the hypoxia [25]. Because myofibroblasts are mainly found in the peduncle of the nasal polyp, they can be stimulated by local hypoxia in the middle meatus or the sinus ostia. There is accumulating evidence that chronic hypoxia is a clinically important stimulus for structural changes such as pulmonary vascular remodeling, pulmonary fibrosis, liver fibrosis, and kidney fibrosis [8–10, 14, 26]. In the present study, NPDF cultured under hypoxic conditions expressed significantly higher levels of ␣-SMA mRNA and protein compared with NPDF cultured under normoxic conditions (fig. 1a, b). Hypoxic incubation also significantly increased fibronectin mRNA and pro-



Discussion

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200

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myofibroblast differentiation and fibronectin production. a RT-PCR for Nox1, Nox3, Nox4, and Nox5 mRNA in NPDF incubated under normoxic and hypoxic conditions. b RT-PCR for Nox4 mRNA following transfection with siNox4 and siScr. c Fluorometer for ROS production following transfection with siNox4 and siScr. d Western blot for fibronectin protein following transfection with siNox4 and siScr. The results were obtained from at least three independent experiments. * p ! 0.05 versus normoxia; † p ! 0.05 versus siScr.

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Fig. 5. Endogenously produced factors mediate hypoxia-induced myofibroblast differentiation. a Western blot for ␣-SMA protein and fibronectin protein in hypoxia-conditioned media and in normoxic media. b ELISA for TGF-␤1 in NPDF incubated under hypoxic conditions and normoxic conditions. c RT-PCR for Nox4 mRNA with and without SB431542. d Fluorometer for ROS production following treatment with SB431542. e Western

tein (fig. 2a, b). These results indicate that hypoxia induces myofibroblast differentiation with ECM protein secretion in NPDF. ROS initiate cellular signaling events that control a variety of cellular functions, including regulation of ECM [12]. Activation of NADPH oxidases and the subsequent production of ROS have been reported during the phenotypic transformation of fibroblasts into myofibroblasts in pulmonary artery smooth muscle cells, cardiac fibroblasts, and alveolar epithelial cells, and are important in the control of ECM protein deposition [10, 18, 19]. Nonphagocytic NADPH oxidases have recently been suggested to play a major role in the regulation of physi406


ological and pathophysiological processes, in particular hypertrophy, remodeling, and angiogenesis in the systemic circulation [20]. Chronic hypoxia induces activation of the NADPH oxidases, and the subsequent production of ROS has been reported in heart failure, pulmonary vascular remodeling, and liver fibrosis [8, 13, 14, 20]. In our study, incubation of NPDF under hypoxic conditions induced an increase in ROS production as well as ␣-SMA mRNA and protein expression. Preincubation of NPDF with ROS scavengers potently inhibited hypoxiainduced ROS production (fig. 3a), ␣-SMA and fibronectin mRNAs (fig. 3b), and ␣-SMA and fibronectin protein expression (fig. 3c). This indicates that hypoxia induces Moon /Kang /Cho /Park /Lee


blot for ␣-SMA and fibronectin protein following treatment with SB431542. The results were obtained from at least three independent experiments. * p ! 0.05 versus normoxia conditioned media; † p ! 0.5 versus hypoxia alone.

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Fig. 6. Hypoxia stimulates NPDF proliferation through the Nox4

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pathway. MTT assays for cellular proliferation following transfection with siNox4. * p ! 0.05 versus normoxia; † p ! 0.05 versus hypoxia alone.

Fig. 7. Proposed schematic signaling pathway of hypoxia-stimulated Nox4-dependent NPDF differentiation. Hypoxia stimulates NPDF to endogenously secrete TGF-␤1, which induces the Nox4 gene. ROS produced from Nox4 mediates differentiation of NPDF to myofibroblasts, resulting in ECM protein secretion.

NPDF differentiation and ECM protein production through a signaling pathway involving ROS. NADPH oxidases are well-characterized sources of ROS in human fibroblasts [14]. The inhibition of hypoxiainduced ROS by ROS scavengers suggested a role for an NADPH oxidase. It was recently reported that Nox4 may contribute to the pathogenesis of hypoxia-induced vascular remodeling in human pulmonary artery smooth muscle cells [19, 20]. In the present investigation, we found that Nox4 is the only Nox subunit significantly upregulated in hypoxic NPDF (fig. 4a), which indicates that Nox4 is the major Nox homolog in hypoxia-induced NPDF differentiation. Furthermore, transfection of NPDF with siNox4 significantly inhibited Nox4 mRNA expression, as well as ROS production, cellular proliferation, and ␣SMA and fibronectin protein levels (fig. 4b–d, 6). This gives stronger support to the role of Nox4 as a mediator. Therefore, we have shown that Nox4 is the major Nox homolog mediating fibrosis in hypoxic NPDF. Taken together, the inhibitory effects of ROS scavengers and siNox4 indicate that hypoxia produces NPDF differentiation through an ROS-dependent signaling pathway mediated by an increased expression of Nox4. TGF-␤ belong to a family of dimeric polypeptide growth factors which regulate cell activation and differentiation and are involved in the pathogenesis of various airway diseases [21]. Among its many activities, TGF-␤ is able to induce fibroblast proliferation and differentiation into myofibroblasts [3]. Recently, TGF-␤ has also been

reported to be involved in the pathogenesis of chronic upper airway disorders, such as nasal polyposis and chronic rhinosinusitis [1, 27]. Increased production of TGF-␤ leads to stromal fibrosis, with fibroblast activation and differentiation [1]. Among the three isoforms of TGF-␤, TGF-␤1 is considered to be the isoform most relevant to the pathogenesis of nasal polyps [22]. TGF-␤1 is known to induce Nox4 expression and ROS production in human pulmonary artery smooth muscle cells, and Nox4 was found to be causally related to the differentiation of cardiac fibroblasts into myofibroblasts in response to TGF-␤1 [18, 19]. These observations strongly suggest its importance in pulmonary artery remodeling in pulmonary artery hypertension or cardiac fibrosis in heart failure. We thus investigated whether TGF-␤1 also participated in hypoxia-induced NPDF differentiation. Significantly increased expression of ␣-SMA protein was observed in hypoxia-conditioned culture media (fig. 5a), indicating that hypoxic NPDF produce soluble mediators that cause myofibroblast differentiation. In addition, we found elevated levels of TGF-␤1 in hypoxiaconditioned culture media (fig. 5b) which suggests that TGF-␤1 is endogenously secreted by hypoxic NPDF. These observations suggest that hypoxia may drive Nox4mediated myofibroblast differentiation by stimulating endogenous production of TGF-␤1 by NPDF. Furthermore, preincubation with SB431542 significantly inhibited Nox4 mRNA expression (fig. 5c), total ROS production (fig. 5d), and ␣-SMA and fibronectin protein ex-



407


Normoxia

pression (fig. 5e). Therefore, this suggests that TGF-␤1 regulates Nox4 expression and subsequent myofibroblast differentiation and ECM protein secretion. TGF-␤ is known to suppress the immune system and induce ECM components. Overproduction of TGF-␤ can result in excessive deposition of scar tissue and fibrosis [3]. Little et al. [28] investigated the roles of TGF-␤1 in NPDF and observed an increase in profibrotic factors and downregulation of several proinflammatory cytokines by TGF-␤1. They suggested that the combination of profibrotic and anti-inflammatory effects of TGF-␤1 on NPDF may represent a signaling pathway shift from nasal inflammation to fibrosis and polyp formation. This may explain our findings that increased levels of TGF-␤1 in hypoxic NPDF results in increased ECM protein secretion. Our investigation showed that hypoxic incubation of NPDF resulted in an increase in fibronectin production, but not collagen. Fibronectin, which is constitutively expressed in polyp-derived fibroblast cultures, is an important component of ECM in nasal polyps [29]. By providing binding sites for other ECM components, fibronectin helps create a cross-linked network within the ECM, and it is an important mediator of wound healing and fibrosis in a variety of tissues [28]. Studies on hypoxic stimulation of NPDF by other researchers [10, 28] have also found increased expression of fibronectin, but not collagen. Further studies are necessary to determine the stimuli and pathways regulating collagen production by NPDF.

Based on the findings of our investigation, we propose a schematic signaling pathway for hypoxia-stimulated Nox4-dependent differentiation of NPDF in figure 7. Hypoxia stimulates NPDF to endogenously secrete TGF-␤1, which induces the Nox4 gene. ROS derived from Nox4 mediates the differentiation of NPDF to myofibroblasts, resulting in ECM protein secretion. In summary, we have demonstrated the functional significance of Nox4 as a mediator of NPDF differentiation in response to hypoxia. Hypoxia induces myofibroblast differentiation of NPDF through a signaling pathway involving TGF-␤1 and Nox4-dependent ROS generation. This is the first study to implicate Nox4 in the fibrogenesis of nasal polyps, which will contribute to a greater understanding of the mechanisms involved in nasal polyp growth. Genetic or pharmacologic targeting Nox4 may prove to be an effective therapeutic strategy for protecting against fibrosis and growth of nasal polyps. Additional studies are needed to determine the precise mechanism by which Nox4 mediates fibrosis in nasal polyps.

Acknowledgements This study was supported by a grant from the Korea Healthcare Technology R&D Project, Ministry for Health, Welfare and Family Affairs, Republic of Korea (A090084), and by the Health Fellowship Foundation.

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