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Antioxidants and NOX1/NOX4 inhibition blocks TGF?1-induced CCN2 and ?-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.

SUBMITTER: Murphy-Marshman H

PROVIDER: S-EPMC5648211 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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

Murphy-Marshman Hannah H Quensel Katherine K Shi-Wen Xu X Barnfield Rebecca R Kelly Jacalyn J Peidl Alex A Stratton Richard J RJ Leask Andrew A

PloS one 20171019 10

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 bl ...[more]

PMID: 29049376

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Project description:Fibroblasts are widely used cells for regenerative medicine in clinics, such as gingival or facial skin treatment. In fact, fibroblasts are considered as a mixture of various types of cells with "spindle shape" and there is no available clear marker. Gingival and dermal fibroblasts are similar in their morphology and function; however it is considered that the cultured cells retain their original characteristics depending on the origin, which may contribute to the differential therapeutic effects. For example, gingival wounds are known to heal relatively quickly with less scar formation compared with skin, which may imply their higher capability for regeneration as a therapeutic effect. The reason for this phenomenon may be partly due to characteristic differences between gingival and dermal fibroblasts including the expression of migration stimulating factor and matrix formation but these differences remain largely unknown. Recently, the characteristics of dermal fibroblasts have been reported to be different depending on body sites, such as face, trunk and plamoplantar skin. Although the expression of fibronectin and their splicing variants were known to be different between trunk and oral mucosal fibroblasts, there is still no detailed report on the functional differences between gingival and dermal fibroblasts. In this study, we investigated differential gene expression in normal gingival and dermal fibroblasts using DNA microarray to investigate the difference between the vague fibroblast-type cells from different tissue origin to achieve higher therapeutic effect in cell therapy. Gingival and dermal tissues were collected from healthy patients. After successive stages primary culture and RNA extraction and hybridization on Affymetrix microarrays (Genome Focus Array). Dermal and gingival tissues were obtained from healthy volunteers (8 cases for dermal tissue; 6 females, 2 males , average age 48 and 8 cases for buccal gingival tissue; 6 females, 2 males, average age 43) whose informed consent was obtained according to a protocol approved by the ethics committee of Nagoya University Hospital. After enzymatic digestion, tissues were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum at 37Â°C in the presence of 5% CO2 for about 4 weeks as reported previously. Total mRNAs were extracted from cells between passages 4-5 by Trizol reagent (Invitrogen, Carlsbad, CA, USA) and were applied to Human Focus Arrays (Affymetrix, Santa Clara, CA, USA) for microarray analysis according to the manufacturer's protocol (http://www.affymetrix.com/support/technical/manuals.affx). The gene expression data were analyzed by Arrayassist (Stratagene, La Jolla, CA, USA). Briefly, 8,500 probes on the array, normalization and scaling (MAS5), flag-positive gene selection, unpaired t-test, and CV selection (<20) resulted in 5,284 genes to analyze. GO (Gene ontology) analysis was performed using the software default settings to find the gene group related to the same category of biological function by searching common key terms that were reported for each gene.

Project description:ObjectivesThe inhibitory action of the superficial gingival connective tissues may limit the regenerative potential of alveolar bone in periodontal therapy or dental implant applications. The aims of this study were to investigate the hypothesis that gingival fibroblasts (GF) can inhibit bone morphogenetic protein (BMP)-induced osteoblastic differentiation, to determine their expression of BMP inhibitors, and finally to determine whether reduction of these inhibitors can relieve suppression of osteoblastic differentiation.MethodsGingival fibroblasts were co-cultured either directly or indirectly with calvarial osteoblasts to assess alkaline phosphatase inhibitory activity, a marker of osteoblastic differentiation. To test total BMP-inhibitory activity of rat GF, conditioned media (GFCM) were collected from cultures. ROS 17/2.8 osteoblastic cells were stimulated with BMP2, together with GFCM. Inhibitor expression was tested using RT-qPCR, Western blotting and in situ hybridization. Removal of inhibitors was carried out using immunoprecipitation beads.ResultsCo-culture experiments showed GF-secreted factors that inhibit BMP-stimulated ALP activity. 10 ng/ml BMP2 increased alkaline phosphatase expression in ROS cells by 41%. GFCM blocked BMP activity which was equivalent to the activity of 100 ng/ml Noggin, a well-described BMP inhibitor. Cultured gingival fibroblasts constitutively expressed BMP antagonist genes from the same subfamily, Grem1, Grem2 and Nbl1 and the Wnt inhibitor Sfrp1. Gremlin1 (6.7 × reference gene expression) had highest levels of basal expression. ISH analysis showed Gremlin1 expression was restricted to the inner half of the gingival lamina propria and the PDL. Removal of Gremlin1 protein from GFCM eliminated the inhibitory effect of GFCM on ALP activity in ROS cells. Subsequent addition of recombinant Gremlin1 restored the inhibitory activity.ConclusionsFactors secreted by gingival fibroblasts inhibit BMP-induced bone formation and a range of BMP inhibitors are constitutively expressed in gingival connective tissues. These inhibitors, particularly Gremlin1, may limit coronal alveolar bone regenerative potential during oral and periodontal surgery.

Dataset Information

Antioxidants and NOX1/NOX4 inhibition blocks TGF?1-induced CCN2 and ?-SMA expression in dermal and gingival fibroblasts.

Publications

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

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