Antioxidants and NOX1/NOX4 inhibition blocks TGFβ1-induced CCN2 and α-SMA expression in dermal and gingival fibroblasts

• Published in: PloS one 2017-10, Vol.12 (10), p.e0186740-e0186740
• Main Authors: Murphy-Marshman, Hannah, Quensel, Katherine, Shi-Wen, Xu, Barnfield, Rebecca, Kelly, Jacalyn, Peidl, Alex, Stratton, Richard J, Leask, Andrew
• Format: Article
• Language: English
• Subjects: Acetylcysteine; Actin; Actins - metabolism; Antioxidants; Antioxidants - pharmacology; Arthritis; Biology and Life Sciences; Blocking; Cell adhesion & migration; Cell culture; Cells, Cultured; Collagen; Collagen (type I); Connective tissue growth factor; Connective Tissue Growth Factor - metabolism; Contraction; Dentistry; Endothelin; Endothelin 1; Endothelins; Extracellular matrix; Fibroblasts; Fibroblasts - metabolism; Fibrosis; Gene expression; Gingiva; Gingiva - cytology; Gingiva - metabolism; Growth factors; Humans; Inhibition; Inhibitors; Kinases; Medicine; Medicine and Health Sciences; NAD(P)H oxidase; NADPH Oxidase 1 - antagonists & inhibitors; NADPH Oxidase 4 - antagonists & inhibitors; NADPH-diaphorase; Nicotinamide; Nicotinamide adenine dinucleotide; NOX4 protein; Oxidase; Oxygen; Pharmacology; Phosphates; Physiology; Polymerase chain reaction; Pulmonary fibrosis; Reactive oxygen species; Research and Analysis Methods; Rheumatology; Scleroderma; Skin; Skin - cytology; Skin - metabolism; Smooth muscle; Transforming Growth Factor beta1 - antagonists & inhibitors; Transforming Growth Factor beta1 - physiology; Transforming growth factor-b1
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0186740

TGFbeta induces fibrogenic responses in fibroblasts. Reactive oxygen species (ROS)/nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) may contribute to fibrogenic responses. Here, we examine if the antioxidant N-acetylcysteine (NAC), the NOX inhibitor diphenyleneiodonium (DPI) and the selective NOX1/NOX4 inhibitor GKT-137831 impairs the ability of TGFbeta to induce profibrotic gene expression in human gingival (HGF) and dermal (HDF) fibroblasts. We also assess if GKT-137831 can block the persistent fibrotic phenotype of lesional scleroderma (SSc) fibroblasts. We use real-time polymerase chain reaction and Western blot analysis to evaluate whether NAC and DPI impair the ability of TGFbeta1 to induce expression of fibrogenic genes in fibroblasts. The effects of GKT-137831 on TGFbeta-induced protein expression and the persistent fibrotic phenotype of lesional scleroderma (SSc) fibroblasts were tested using Western blot and collagen gel contraction analyses. In HDF and HGF, TGFbeta1 induces CCN2, CCN1, endothelin-1 and alpha-smooth muscle actin (SMA) in a fashion sensitive to NAC. Induction of COL1A1 mRNA was unaffected. Similar results were seen with DPI. NAC and DPI impaired the ability of TGFbeta1 to induce protein expression of CCN2 and alpha-SMA in HDF and HGF. GKT-137831 impaired TGFbeta-induced CCN2 and alpha-SMA protein expression in HGF and HDF. In lesional SSc dermal fibroblasts, GKT-137831 reduced alpha-SMA and CCN2 protein overexpression and collagen gel contraction. These results are consistent with the hypothesis that antioxidants or NOX1/4 inhibition may be useful in blocking profibrotic effects of TGFbeta on dermal and gingival fibroblasts and warrant consideration for further development as potential antifibrotic agents.