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

Abstract

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.

A Potential Link Between Oxidative Stress and Endothelial-to-Mesenchymal Transition in Systemic Sclerosis

“…In addition, ligand-mediated receptor activation by cytokines and growth factors can also increase ROS generation ( 2 , 35 , 37 ). For example, TGF-β is a profibrotic cytokine that plays a key role in the ligand-mediated receptor process that triggers the onset and progression of SSc ( 2 , 37 – 41 ). Other putative factors involved in the pathogenesis of SSc include the platelet-derived growth factors (PDGF), vascular endothelial growth factor (VEGF), connective tissue growth factor (CTGF), angiotensin II, interleukin 3, interleukin 6, tumor necrosis factor-alpha (TNF-α), nerve growth factor, and fibroblast growth factor (FGF).…”

Role of NADPH oxidase and its therapeutic intervention in TGF-β-mediated EMT progression: an in vitro analysis on HeLa cervical cancer cells

“…4b) of the HeLa cells was significantly diminished. The difference in effect imposed by the above-mentioned therapeutic agents might be attributed to the disparity in their mode of action in inhibiting NOX isoforms and other endogenous sources of ROS as follows: While NAC being radical scavenger through aiding in the synthesis of glutathione (GSH), a powerful antioxidant [1], GKT acts as specific NOX1/4 inhibitor [11]. Apocynin and DPI is said to non-specifically inhibit ROS production from diverge sources (such as mitochondrial ROS production, cytochrome P450 mono-oxygenase, Nitric oxide synthase, Xanthine oxidase, NOX enzymes and other flavoproteins).…”

CCN proteins as potential actionable targets in scleroderma

“…The role of CCN1 has not been extensively studied in fibrosis, although its role in promoting tissue repair in wound healing is becoming clearer . One study has found that CCN1 is induced in dermal fibroblasts by TGF‐β along with CCN2 and α‐SMA …”

“…[13] One study has found that CCN1 is induced in dermal fibroblasts by TGFβ along with CCN2 and α-SMA. [43] CCN5 is the most abundant CCN in the dermis in terms of mRNA expression. [25] Contrary to CCN2, CCN5 has been found to be downregulated after TGFβ exposure in human skin fibroblasts.…”