Increased Expression of NAPDH Oxidase 4 in Systemic Sclerosis Dermal Fibroblasts Regulation by Transforming Growth Factor β

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 beta 1 (TGF beta 1). 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 H2O2 production. Specific kinase inhibitors were used to study the TGF beta 1 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. TGF beta 1 caused potent NOX-4 protein and messenger RNA stimulation in normal and SSc fibroblasts, which was mediated by the protein kinase C delta (PKC delta) and Smad2/3 pathways. NOX-4 knockdown in SSc fibroblasts reduced the production of ROS and lowered the expression of type I collagen. Conclusion. NOX-4 expression and production were found to be constitutively elevated in SSc skin and cultured SSc dermal fibroblasts. TGF beta 1 stimulated NOX-4 expression in normal and SSc fibroblasts through PKC delta 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 knock-down 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.