title: Increased expression of NAPDH oxidase 4 in systemic sclerosis dermal fibroblasts: regulation by transforming growth factor β.
abstract: OBJECTIVE
  Systemic sclerosis (SSc) is characterized by severe and often progressive fibrosis of the skin and multiple internal organs. The mechanisms responsible for these alterations remain obscure, although excessive reactive oxygen species (ROS)-mediated oxidative stress has been implicated. NOX-4 is 1 of 7 isoforms of NADPH oxidase responsible for the generation of ROS. The purpose of this study was to examine NOX-4 expression in skin and cultured dermal fibroblasts from SSc patients and to examine its regulation by transforming growth factor β1 (TGFβ1).
  
  
  METHODS
  NOX-4 was assessed in normal and SSc skin by immunohistologic analysis and in normal and SSc cultured dermal fibroblasts by quantitative polymerase chain reaction analysis, fluorescence microscopy, and Western blotting. ROS levels were assessed by fluorescence measurement of H2 O2 production. Specific kinase inhibitors were used to study the TGFβ1 signaling involved in NOX-4 stimulation. NOX-4 inhibition/down-regulation was induced with a selective NOX-4 small-molecule inhibitor and NOX-4 small interfering RNA (siRNA).
  
  
  RESULTS
  In contrast with normal skin fibroblasts, those from SSc skin showed intense NOX-4 staining. Cultured SSc fibroblasts displayed increased NOX-4 expression. TGFβ1 caused potent NOX-4 protein and messenger RNA stimulation in normal and SSc fibroblasts, which was mediated by the protein kinase Cδ (PKCδ) and Smad2/3 pathways. NOX-4 knockdown in SSc fibroblasts reduced the production of ROS and lowered the expression of type I collagen.
  
  
  CONCLUSIONS
  NOX-4 expression and production were found to be constitutively elevated in SSc skin and cultured SSc dermal fibroblasts. TGFβ1 stimulated NOX-4 expression in normal and SSc fibroblasts through PKCδ and Smad2/3 signaling pathways. A small-molecule NOX-4 inhibitor decreased collagen and fibronectin production by normal and SSc fibroblasts, and NOX-4 siRNA knockdown reduced ROS and collagen production by SSc fibroblasts. These results demonstrate the involvement of NOX-4 in SSc-associated fibrosis and indicate NOX-4 inhibitors as novel therapeutic approaches for SSc.
authors.affiliation: Thomas Jefferson University, Philadelphia, Pennsylvania.
authors.forename: Sonsoles
authors.initials: S
authors.lastname: Piera-Velazquez
authors.affiliation: Thomas Jefferson University, Philadelphia, Pennsylvania.
authors.forename: Alma
authors.initials: A
authors.lastname: Makul
authors.affiliation: Thomas Jefferson University, Philadelphia, Pennsylvania.
authors.forename: Sergio A
authors.initials: SA
authors.lastname: Jiménez
mesh_terms: D016022:Case-Control Studies; D002478:Cells, Cultured; D003094:Collagen / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D005353:Fibronectins / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000235:genetics* / Q000378:metabolism*; D018384:Oxidative Stress / Q000187:drug effects; D051745:Protein Kinase C-delta / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D012867:Skin / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D016212:Transforming Growth Factor beta / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D005353:Fibronectins; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D016212:Transforming Growth Factor beta; D003094:Collagen; D006861:Hydrogen Peroxide; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C498910:NOX4 protein, human; D051745:Protein Kinase C-delta