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.
Highlights
Fibrotic conditions are characterized by the excessive deposition and remodeling of extracellular matrix by fibroblasts residing within connective tissue
One of the key mediators of fibrosis is the profibrotic marker, CCN2 [4]. As this mRNA is potently induced by TGFβ [4, 14, 16], we first evaluated whether the ability of TGFβ1 to induce this transcript in HDFs and HGFs was sensitive to NAC
Consistent with the notion that NADPH oxidase (NOX) enzymes are required for the induction of a subset of fibrogenic responses in fibroblasts, we found that DPI impaired the ability of TGFβ1 to induce CCN2, CCN1 and αSMA mRNA expression in both HDF and HGF (Fig 2A and 2B)
Summary
Fibrotic conditions are characterized by the excessive deposition and remodeling of extracellular matrix by fibroblasts residing within connective tissue. Unlike in other adult tissues, fibrotic responses in the oral cavity do not elicit scar tissue deposition, but are instead characterized by gingival overgrowths, often in response to medications [2, 5]. The molecular mechanisms underlying the induction of the profibrotic mediator CCN2/CTGF appear to differ between dermal and gingival fibroblasts in terms of a requirement for c-Jun kinase [6,7]. Gingival fibroblasts appear to display a less potent response to both mechanical strain and TGFβ in terms of CCN2 induction [8]. As such, comparing the molecular mechanism underlying the signaling responses of dermal and gingival fibroblasts to fibrogenic stimuli, such as TGFβ, is of inherent value
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