Role of reactive oxygen species in transforming growth factor beta1-induced alpha smooth-muscle actin and collagen production in nasal polyp-derived fibroblasts.
BACKGROUND
Myofibroblasts are detected in nasal polyps and are involved in nasal polyp formation by inducing extracellular matrix accumulation. Reactive oxygen species (ROS) are released during the differentiation of fibroblasts to myofibroblasts. The purpose of this study was to investigate ROS production and nicotinamide adenine dinucleotide phosphate oxidase (NOX) expression in nasal polyp-derived fibroblasts (NPDFs) and to evaluate whether ROS from NOX mediates transforming growth factor (TGF)-β1-induced production of alpha smooth-muscle actin (α-SMA) and collagen production.


METHODS
NPDFs were incubated and treated with TGF-β1. The mRNA expression of NOXs, α-SMA, and collagen type I and IV was determined by reverse transcription-polymerase chain reaction, and the expression of α-SMA protein was determined by immunofluorescence microscopy. The amount of total soluble collagen production was analyzed by the SirCol assay. The ROS generation of cells was investigated using the 2',7'-dichlorfluorescein-diacetate. The fluorescence was captured by fluorescent microscope and measured using a fluorometer.


RESULTS
Stimulation with TGF-β1 increased ROS production by NPDFs compared with NPDFs not treated with TGF-β1. Stimulation with TGF-β1 increased the expression of NOX4 mRNA most potently among various Nox enzymes. siNOX4 was able to decrease the level of ROS production. Myofibroblast differentiation and the production of collagen in NPDFs were prevented by inhibition of ROS generation with diphenyliodonium, N-acetylcysteine, ebselen, and siNox4.


CONCLUSIONS
This study showed that NOX4 and ROS have a role in myofibroblast differentiation and collagen production of TGF-β1-induced NPDFs and that these processes are inhibited by the elimination of ROS.
Department of Otorhinolaryngology-Head and Neck Surgery, Guro Hospital, Korea University College of Medicine, Seoul, South Korea.
Il-Ho
IH
Park
Se-Jin
SJ
Park
Jung-Sun
JS
Cho
You-Mi
YM
Moon
Tae Hoon
TH
Kim
Sang Hag
SH
Lee
Heung-Man
HM
Lee
D000111:Acetylcysteine / Q000494:pharmacology; D000199:Actins / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D000328:Adult; D001393:Azoles / Q000494:pharmacology; D001713:Biphenyl Compounds / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D024042:Collagen Type I / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D024141:Collagen Type IV / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D005260:Female; D005347:Fibroblasts / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D005452:Fluoresceins; D005456:Fluorescent Dyes; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D054833:Isoindoles; D008297:Male; D008856:Microscopy, Fluorescence; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors* / Q000235:genetics; D009298:Nasal Polyps / Q000378:metabolism* / Q000473:pathology; D009861:Onium Compounds / Q000494:pharmacology; D016566:Organoselenium Compounds / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000235:genetics; D017382:Reactive Oxygen Species / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology*
D000199:Actins; D001393:Azoles; D001713:Biphenyl Compounds; D024042:Collagen Type I; D024141:Collagen Type IV; D005452:Fluoresceins; D005456:Fluorescent Dyes; D054833:Isoindoles; D009861:Onium Compounds; D016566:Organoselenium Compounds; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D053773:Transforming Growth Factor beta1; C031291:diphenyliodonium; C029569:diacetyldichlorofluorescein; C042986:ebselen; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C498910:NOX4 protein, human; D000111:Acetylcysteine