title: NADPH oxidase-2 is a key regulator of human dermal fibroblasts: a potential therapeutic strategy for the treatment of skin fibrosis.
abstract: The proliferation of human skin dermal fibroblasts (HDFs) is a critical step in skin fibrosis, and transforming growth factor-beta1 (TGF-β1) exerts pro-oxidant and fibrogenic effects on HDFs. In addition, the oxidative stress system has been implicated in the pathogenesis of skin disease. However, the role of NADPH oxidase as a mediator of TGF-β1-induced effects in HDFs remains unknown. Thus, our aim was to investigate the role of NADPH in human skin dermal fibroblasts. Primary fibroblasts were cultured and pretreated with various stimulants. Real-time Q-PCR and Western blotting analyses were used for mRNA and protein detection. In addition, siRNA technology was applied for gene knock-down analysis. Hydrogen peroxide production and 2',7'-dichlorofluorescein diacetate (DCFDA) measurement assay were performed. Here, our findings demonstrated that HDFs express key components of non-phagocytic NADPH oxidase mRNA. TGF-β1 induced NOX2 and reactive oxygen species formation via NADPH oxidase activity. In contrast, NOX3 was barely detectable, and other NOXs did not display significant changes. In addition, TGF-β1 phosphorylated MAPKs and increased activator protein-1 (AP-1) in a redox-sensitive manner, and NOX2 suppression inhibited baseline and TGF-β1-mediated stimulation of Smad2 phosphorylation. Moreover, TGF-β1 stimulated cell proliferation, migration, collagen I and fibronectin expression, and bFGF and PAI-1 secretion: these effects were attenuated by diphenylene iodonium (DPI), an NADPH oxidase inhibitor, and NOX2 siRNA. Importantly, NOX2 siRNA suppresses collagen production in primary keloid dermal fibroblasts. These findings provide the proof of concept for NADPH oxidase as a potential target for the treatment of skin fibrosis.
authors: Department of Hand and Plastic Surgery, The Second Affiliated Hospital of Wenzhou Medical University, Wenzhou, China; Department of Plastic and Reconstructive Surgery, Shanghai Ninth People's Hospital of Shanghai Jiaotong University School of Medicine, Shanghai, China.
authors: Guo-You
authors: GY
authors: Zhang
authors: Liang-Cai
authors: LC
authors: Wu
authors: Tao
authors: T
authors: Dai
authors: Shi-Yi
authors: SY
authors: Chen
authors: An-Yuan
authors: AY
authors: Wang
authors: Kang
authors: K
authors: Lin
authors: Da-Mu
authors: DM
authors: Lin
authors: Jing-Quan
authors: JQ
authors: Yang
authors: Biao
authors: B
authors: Cheng
authors: Li
authors: L
authors: Zhang
authors: Wei-Yang
authors: WY
authors: Gao
authors: Zhi-Jie
authors: ZJ
authors: Li
mesh_terms: D002478:Cells, Cultured; D003094:Collagen / Q000096:biosynthesis / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005347:Fibroblasts / Q000201:enzymology*; D005355:Fibrosis; D055785:Gene Knockdown Techniques; D006801:Humans; D007627:Keloid / Q000201:enzymology / Q000235:genetics / Q000628:therapy; D020935:MAP Kinase Signaling System; D008562:Membrane Glycoproteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D009861:Onium Compounds / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D017382:Reactive Oxygen Species / Q000378:metabolism; D012867:Skin / Q000201:enzymology*; D051785:Smad Proteins / Q000378:metabolism; D018808:Transcription Factor AP-1 / Q000378:metabolism; D053773:Transforming Growth Factor beta1 / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; D008562:Membrane Glycoproteins; D009861:Onium Compounds; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D051785:Smad Proteins; D018808:Transcription Factor AP-1; D053773:Transforming Growth Factor beta1; C007517:diphenyleneiodonium; D003094:Collagen; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases
keywords: NADPH; TGF-β1; fibroblasts; fibrosis; wound healing