Antioxidants and NOX1/NOX4 inhibition blocks TGFβ1-induced CCN2 and α-SMA expression in dermal and gingival fibroblasts
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Published on: October 27, 2020
Hannah Murphy-Marshman1, Katherine Quensel1, Xu Shi-Wen2
1Department of Dentistry, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada.
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.
Insights
Antioxidants and NOX1/4 inhibitors, like N-acetylcysteine (NAC) and GKT-137831, can block transforming growth factor-beta (TGFbeta)-induced fibrotic gene expression in fibroblasts. These agents may offer potential antifibrotic therapies.
Area of Science:
- Fibrosis research
- Cellular signaling pathways
- Pharmacological interventions
Background:
- Transforming growth factor-beta (TGFbeta) is a key mediator of fibrotic responses in fibroblasts.
- Reactive oxygen species (ROS), particularly those generated by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) enzymes, are implicated in TGFbeta-driven fibrogenesis.
- Understanding the role of ROS/NOX in fibroblast activation is crucial for developing antifibrotic strategies.
Purpose of the Study:
- To investigate the efficacy of the antioxidant N-acetylcysteine (NAC) and NOX inhibitors (diphenyleneiodonium [DPI] and GKT-137831) in mitigating TGFbeta-induced profibrotic gene expression in human gingival fibroblasts (HGF) and dermal fibroblasts (HDF).
- To determine if GKT-137831 can reverse the persistent fibrotic phenotype in scleroderma (SSc) fibroblasts.
- To explore the potential of targeting ROS/NOX pathways as an antifibrotic therapeutic approach.
Main Methods:
- Real-time polymerase chain reaction (PCR) and Western blot analysis were used to assess gene and protein expression.
- Fibroblast cultures (HGF, HDF, and lesional SSc fibroblasts) were treated with TGFbeta1 in the presence or absence of NAC, DPI, or GKT-137831.
- Collagen gel contraction assays were employed to evaluate the functional fibrotic capacity of SSc fibroblasts.
Main Results:
- TGFbeta1-induced expression of CCN2, CCN1, endothelin-1, and alpha-smooth muscle actin (SMA) in HGF and HDF was sensitive to NAC and DPI.
- NAC and DPI inhibited TGFbeta1-induced protein levels of CCN2 and alpha-SMA.
- GKT-137831 effectively reduced TGFbeta-induced CCN2 and alpha-SMA protein expression in HGF and HDF.
- In lesional SSc fibroblasts, GKT-137831 decreased alpha-SMA and CCN2 overexpression and attenuated collagen gel contraction.
Conclusions:
- Antioxidants and NOX1/4 inhibition demonstrate potential in blocking TGFbeta-mediated profibrotic gene expression and activity in dermal and gingival fibroblasts.
- GKT-137831 shows promise in reversing the fibrotic phenotype of scleroderma fibroblasts.
- Targeting the ROS/NOX pathway warrants further investigation as a therapeutic strategy for fibrotic diseases.
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