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Troglitazone suppresses transforming growth factor beta-mediated fibrogenesis in retinal pigment epithelial cells.

Cheng HC, Ho TC, Chen SL, Lai HY, Hong KF, Tsao YP - Mol. Vis. (2008)

Bottom Line: The inhibitory effects of TGZ on collagen type I (COLI) and fibronectin (FN) expression induced by TGFbeta2 was evaluated by reverse transcriptase-polymerase chain reaction.TGZ pretreatment can significantly prevent TGFbeta2-induced epithelial- mesenchymal transition of RPE cells, and retards cell migration.On the other hand, TGZ does not alter the levels of TGFbeta2-induced p38 MAPK phosphorylation, the effect of TGZ is unlikely to be mediated by p38 MAPK signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Mackay Memorial Hospital, Taipei, Taiwan.

ABSTRACT

Purpose: Transforming growth factor (TGF)-beta2 induction of epithelial-mesenchymal transition of retinal pigment epithelium (RPE) cells has been implicated to be an important event during the development of proliferative vitreoretinopathy. The present study was conducted to examine whether troglitazone (TGZ) can inhibit TGFbeta2-mediated fibrosis of RPE cells. The mechanism of the TGZ effect was also investigated by studying major TGFbeta2-induced signaling including activation of Smad and p38 mitogen activated protein kinase (MAPK).

Methods: Human RPE cells (ARPE-19) were exposed to various concentrations of TGZ in the presence of TGFbeta2. The inhibitory effects of TGZ on collagen type I (COLI) and fibronectin (FN) expression induced by TGFbeta2 was evaluated by reverse transcriptase-polymerase chain reaction. COLI synthesis was evaluated by the concentration of the C-terminal propeptide of COLI in the medium. The protein levels of FN and the phosphorylation of p38 MAPK and Smad2 and Smad3 were assessed by immunoblotting. TGZ inhibition of TGFbeta2-promoted ARPE-19 cell migration was evaluated by an in vitro wound-healing assay. The influence of TGZ on cell viability was evaluated by the colorimetric conversion of 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide.

Results: TGZ dose-dependently inhibited TGFbeta2-induced COLI and FN overexpression at the levels of mRNA and protein manufacture. A dose-dependent TGZ inhibition was also apparent in TGFbeta2-induced cell migration; cell viability was unaffected. TGFbeta2 induced sequential phosphorylation of Smad2 and Smad3 and p38 MAPK. TGZ inhibited TGFbeta2-induced early Smad2 and Smad3 and late Smad3 phosphorylation but had no influence on TGFbeta2-induced p38 MAPK activation.

Conclusions: TGZ pretreatment can significantly prevent TGFbeta2-induced epithelial- mesenchymal transition of RPE cells, and retards cell migration. This may be achieved through the prevention of TGFbeta2-induced Smad2 and Smad3 phosphorylation and subsequent nuclear accumulation. On the other hand, TGZ does not alter the levels of TGFbeta2-induced p38 MAPK phosphorylation, the effect of TGZ is unlikely to be mediated by p38 MAPK signaling.

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Related in: MedlinePlus

TGFβ2-induced translocation of Smads2/3 from the cytosolic to nucleus fractions is inhibited by TGZ.ARPE-19 cells were treated with 4 ng/ml TGFβ2 alone or pretreated with 10 μM TGZ, 10 μM SB203580 or DMSO for 1 h and then stimulated with TGFβ2 for 2 h. Cells were harvested and aliquots containing equal amounts of protein from cytosolic (C) and nucleus (N) fractions were subjected to SDS–PAGE and western blot analysis. The result of one representative assay from two similar independent experiments is shown.
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f10: TGFβ2-induced translocation of Smads2/3 from the cytosolic to nucleus fractions is inhibited by TGZ.ARPE-19 cells were treated with 4 ng/ml TGFβ2 alone or pretreated with 10 μM TGZ, 10 μM SB203580 or DMSO for 1 h and then stimulated with TGFβ2 for 2 h. Cells were harvested and aliquots containing equal amounts of protein from cytosolic (C) and nucleus (N) fractions were subjected to SDS–PAGE and western blot analysis. The result of one representative assay from two similar independent experiments is shown.

Mentions: TGFβ binding to its receptor causes phosphorylation of Smad2 and Smad3 to form a hetero-oligomeric complex with Smad4, which then translocates into the nucleus to regulate transcription of target genes [25]. We thus examined if TGZ prevented Smad2 and 3 phosphorylation that could also block cytoplasmic Smad translocation to nucleus. Since TGZ can suppress TGFβ2-induced early (2 h) Smad 2 and 3 phosphorylation, the treated cells were subjected to subcellular fractionation and the TGZ effect was examined by western blotting. As shown in Figure 10, as predicted, TGZ prevented TGFβ2-induced Smad2 and Smad3 nuclear translocation (compare the nuclear fractions of TGFβ2 and TGFβ2+TGZ). DMSO or SB203580 pretreatment had no effect.


Troglitazone suppresses transforming growth factor beta-mediated fibrogenesis in retinal pigment epithelial cells.

Cheng HC, Ho TC, Chen SL, Lai HY, Hong KF, Tsao YP - Mol. Vis. (2008)

TGFβ2-induced translocation of Smads2/3 from the cytosolic to nucleus fractions is inhibited by TGZ.ARPE-19 cells were treated with 4 ng/ml TGFβ2 alone or pretreated with 10 μM TGZ, 10 μM SB203580 or DMSO for 1 h and then stimulated with TGFβ2 for 2 h. Cells were harvested and aliquots containing equal amounts of protein from cytosolic (C) and nucleus (N) fractions were subjected to SDS–PAGE and western blot analysis. The result of one representative assay from two similar independent experiments is shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2254970&req=5

f10: TGFβ2-induced translocation of Smads2/3 from the cytosolic to nucleus fractions is inhibited by TGZ.ARPE-19 cells were treated with 4 ng/ml TGFβ2 alone or pretreated with 10 μM TGZ, 10 μM SB203580 or DMSO for 1 h and then stimulated with TGFβ2 for 2 h. Cells were harvested and aliquots containing equal amounts of protein from cytosolic (C) and nucleus (N) fractions were subjected to SDS–PAGE and western blot analysis. The result of one representative assay from two similar independent experiments is shown.
Mentions: TGFβ binding to its receptor causes phosphorylation of Smad2 and Smad3 to form a hetero-oligomeric complex with Smad4, which then translocates into the nucleus to regulate transcription of target genes [25]. We thus examined if TGZ prevented Smad2 and 3 phosphorylation that could also block cytoplasmic Smad translocation to nucleus. Since TGZ can suppress TGFβ2-induced early (2 h) Smad 2 and 3 phosphorylation, the treated cells were subjected to subcellular fractionation and the TGZ effect was examined by western blotting. As shown in Figure 10, as predicted, TGZ prevented TGFβ2-induced Smad2 and Smad3 nuclear translocation (compare the nuclear fractions of TGFβ2 and TGFβ2+TGZ). DMSO or SB203580 pretreatment had no effect.

Bottom Line: The inhibitory effects of TGZ on collagen type I (COLI) and fibronectin (FN) expression induced by TGFbeta2 was evaluated by reverse transcriptase-polymerase chain reaction.TGZ pretreatment can significantly prevent TGFbeta2-induced epithelial- mesenchymal transition of RPE cells, and retards cell migration.On the other hand, TGZ does not alter the levels of TGFbeta2-induced p38 MAPK phosphorylation, the effect of TGZ is unlikely to be mediated by p38 MAPK signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Mackay Memorial Hospital, Taipei, Taiwan.

ABSTRACT

Purpose: Transforming growth factor (TGF)-beta2 induction of epithelial-mesenchymal transition of retinal pigment epithelium (RPE) cells has been implicated to be an important event during the development of proliferative vitreoretinopathy. The present study was conducted to examine whether troglitazone (TGZ) can inhibit TGFbeta2-mediated fibrosis of RPE cells. The mechanism of the TGZ effect was also investigated by studying major TGFbeta2-induced signaling including activation of Smad and p38 mitogen activated protein kinase (MAPK).

Methods: Human RPE cells (ARPE-19) were exposed to various concentrations of TGZ in the presence of TGFbeta2. The inhibitory effects of TGZ on collagen type I (COLI) and fibronectin (FN) expression induced by TGFbeta2 was evaluated by reverse transcriptase-polymerase chain reaction. COLI synthesis was evaluated by the concentration of the C-terminal propeptide of COLI in the medium. The protein levels of FN and the phosphorylation of p38 MAPK and Smad2 and Smad3 were assessed by immunoblotting. TGZ inhibition of TGFbeta2-promoted ARPE-19 cell migration was evaluated by an in vitro wound-healing assay. The influence of TGZ on cell viability was evaluated by the colorimetric conversion of 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide.

Results: TGZ dose-dependently inhibited TGFbeta2-induced COLI and FN overexpression at the levels of mRNA and protein manufacture. A dose-dependent TGZ inhibition was also apparent in TGFbeta2-induced cell migration; cell viability was unaffected. TGFbeta2 induced sequential phosphorylation of Smad2 and Smad3 and p38 MAPK. TGZ inhibited TGFbeta2-induced early Smad2 and Smad3 and late Smad3 phosphorylation but had no influence on TGFbeta2-induced p38 MAPK activation.

Conclusions: TGZ pretreatment can significantly prevent TGFbeta2-induced epithelial- mesenchymal transition of RPE cells, and retards cell migration. This may be achieved through the prevention of TGFbeta2-induced Smad2 and Smad3 phosphorylation and subsequent nuclear accumulation. On the other hand, TGZ does not alter the levels of TGFbeta2-induced p38 MAPK phosphorylation, the effect of TGZ is unlikely to be mediated by p38 MAPK signaling.

Show MeSH
Related in: MedlinePlus