title: Targeting of NADPH oxidase in vitro and in vivo suppresses fibroblast activation and experimental skin fibrosis.
abstract: Although there is increasing evidence that oxidative stress is involved in collagen synthesis and myofibroblast activation, the NADPH oxidase (Nox) system is incompletely investigated in the context of human dermal fibroblasts (HDFs) and skin fibrosis. Using the pan-Nox inhibitor diphenyleneiodonium (DPI) as an initial tool, we show that gene expression of collagen type I, α-smooth muscle actin (α-SMA) and fibronectin 1 is suppressed in HDFs. Detailed expression analysis of all Nox isoforms and adaptors revealed expression of RNA and protein expression of Nox4, p22phoxand Poldip2 but neither Nox1 nor Nox2. Nox4 could be immunolocalized to the endoplasmic reticulum. Importantly, TGF-β1had a dose- and time-dependent upregulating effect on NADH activity and Nox4 gene expression in HDFs. Genetic silencing of Nox4 as demonstrated by siRNA in HDFs as well as in murine fibroblasts established from Nox4 knockout mice confirmed that TGF-β1-mediated collagen type I gene, α-SMA and fibronectin 1 gene expressions were Nox4-dependent. This TGF-β1effect was mediated by Smad3 as shown by in silico promoter analysis, pharmacological inhibition and gene silencing of Smad3. The relevance of these findings is highlighted in the bleomycin-induced scleroderma mouse model. DPI treatment attenuated skin fibrosis and myofibroblast activation. Moreover, Nox4 knockdown by siRNA reduced skin collagen synthesis, α-SMA and fibronectin 1 expression in vivo. Finally, analyses of HDFs from patients with systemic sclerosis confirmed the expression of Nox4 and its adaptors, whereas Nox1 and Nox2 were not detectable. Our findings indicate that Nox4 targeting is a promising future treatment for fibrotic skin diseases.
authors: Department of Dermatology, University of Münster, Münster, Germany.|Department of Botany and Microbiology, Alexandria University, Alexandria, Egypt.
authors: Heba
authors: H
authors: Dosoki
authors: Department of Dermatology, University of Münster, Münster, Germany.
authors: Agatha
authors: A
authors: Stegemann
authors: Institute of Anatomy & Vascular Biology, University of Münster, Münster, Germany.
authors: Muna
authors: M
authors: Taha
authors: Institute of Anatomy & Vascular Biology, University of Münster, Münster, Germany.
authors: Hans
authors: H
authors: Schnittler
authors: Department of Dermatology, University of Münster, Münster, Germany.
authors: Thomas A
authors: TA
authors: Luger
authors: Institute of Cardiovascular Physiology, Goethe-University, Frankfurt, Germany.
authors: Katrin
authors: K
authors: Schröder
authors: Department of Internal Medicine 3 and Institute for Clinical Immunology, University of Erlangen-Nuremberg, Erlangen, Germany.
authors: Jörg H W
authors: JH
authors: Distler
authors: Department of Biomedical Sciences, School of Human Sciences, University of Osnabrück, Osnabrück, Germany.
authors: Claus
authors: C
authors: Kerkhoff
authors: Department of Dermatology, University of Münster, Münster, Germany.
authors: Markus
authors: M
authors: Böhm
mesh_terms: D000199:Actins / Q000235:genetics; D000328:Adult; D000818:Animals; D002478:Cells, Cultured; D024042:Collagen Type I / Q000235:genetics / Q000378:metabolism; D000090602:Collagen Type I, alpha 1 Chain; D003198:Computer Simulation; D016207:Cytokines / Q000235:genetics; D004195:Disease Models, Animal; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D005347:Fibroblasts / Q000201:enzymology*; D005353:Fibronectins; D005355:Fibrosis; D015870:Gene Expression / Q000187:drug effects; D020869:Gene Expression Profiling; D020868:Gene Silencing; D006801:Humans; D007231:Infant, Newborn; D007527:Isoenzymes / Q000235:genetics; D008297:Male; D051379:Mice; D008875:Middle Aged; D009097:Multienzyme Complexes / Q000378:metabolism; D058628:Myofibroblasts; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism; D000074624:NADPH Oxidase 1 / Q000235:genetics; D000074662:NADPH Oxidase 2 / Q000235:genetics; D000074663:NADPH Oxidase 4 / Q000235:genetics* / Q000378:metabolism; D009861:Onium Compounds / Q000494:pharmacology; D061251:Primary Cell Culture; D012333:RNA, Messenger / Q000378:metabolism; D012595:Scleroderma, Systemic / Q000139:chemically induced / Q000201:enzymology* / Q000473:pathology; D012867:Skin / Q000201:enzymology* / Q000473:pathology*; D016212:Transforming Growth Factor beta / Q000494:pharmacology; D055815:Young Adult
chemical_list: C541116:ACTA2 protein, human; D000199:Actins; C000619309:Col1a2 protein, mouse; D024042:Collagen Type I; D000090602:Collagen Type I, alpha 1 Chain; D016207:Cytokines; D004791:Enzyme Inhibitors; C067625:FN1 protein, human; D005353:Fibronectins; D007527:Isoenzymes; D009097:Multienzyme Complexes; D009861:Onium Compounds; D012333:RNA, Messenger; D016212:Transforming Growth Factor beta; C544013:alpha-smooth muscle actin, mouse; C007517:diphenyleneiodonium; C023107:NADH oxidase; D009247:NADH, NADPH Oxidoreductases; C056558:CYBB protein, human; D000074624:NADPH Oxidase 1; D000074662:NADPH Oxidase 2; D000074663:NADPH Oxidase 4; C402799:NOX1 protein, human; C498910:NOX4 protein, human; C422943:Nox4 protein, mouse
keywords: 
  NADPH
  ; Myofibroblast; collagen; dermal fibroblasts; fibrosis