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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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TGZ decreases TGFβ2-induced expression of fibronectin and collagen type I mRNA in ARPE-19 cells.ARPE-19 cells were either left untreated or were treated with 4 ng/ml TGFβ2 for 24 h, or were pretreated with 10 μM TGZ, DMSO (0.05%), 10 ng/ml Actinomycin D, or 10 μM SB203580 for 1 h and then incubated with TGFβ2 for additional 24 h. Total RNA was extracted, and RT–PCR analysis for fibronectin and collagen type I (COLI) was performed. Glyceraldehyde-3-phosphate dehydrogenase (G3PDH) expression was examined for normalization purposes. Experiments were repeated twice and the results were reproducible.
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f3: TGZ decreases TGFβ2-induced expression of fibronectin and collagen type I mRNA in ARPE-19 cells.ARPE-19 cells were either left untreated or were treated with 4 ng/ml TGFβ2 for 24 h, or were pretreated with 10 μM TGZ, DMSO (0.05%), 10 ng/ml Actinomycin D, or 10 μM SB203580 for 1 h and then incubated with TGFβ2 for additional 24 h. Total RNA was extracted, and RT–PCR analysis for fibronectin and collagen type I (COLI) was performed. Glyceraldehyde-3-phosphate dehydrogenase (G3PDH) expression was examined for normalization purposes. Experiments were repeated twice and the results were reproducible.

Mentions: Next, we investigated whether TGZ was capable of inhibition of TGFβ2-induced collagen type I and fibronectin mRNA expression. As shown in Figure 3, RT–PCR analysis demonstrated that ARPE-19 cells cultured in serum-free medium expressed a basal level of collagen type I and fibronectin genes; treatment with exogenous TGFβ2 for 24 h markedly induced expression of both mRNAs. Cells treated with TGFβ2 for 8 h also displayed an increased expression of both mRNAs but at levels less than following stimulation for 24 h (approximately threefold). The TGFβ2-mediated induction effect was completely blocked by actinomycin D pretreatment, suggesting that the increased mRNA expression was transcription dependent. Cells pretreated for 1 h with 10 μM TGZ before TGFβ2 treatment suppressed TGFβ2-induced collagen type I and fibronectin mRNA to basal levels that were comparable to untreated cells. Pretreatment with DMSO had no such effect. Similarly, 10 μM SB203580 pretreatment partially prevented TGFβ2-induced collagen type I mRNA expression (compare Figure 2 with Figure 3).


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)

TGZ decreases TGFβ2-induced expression of fibronectin and collagen type I mRNA in ARPE-19 cells.ARPE-19 cells were either left untreated or were treated with 4 ng/ml TGFβ2 for 24 h, or were pretreated with 10 μM TGZ, DMSO (0.05%), 10 ng/ml Actinomycin D, or 10 μM SB203580 for 1 h and then incubated with TGFβ2 for additional 24 h. Total RNA was extracted, and RT–PCR analysis for fibronectin and collagen type I (COLI) was performed. Glyceraldehyde-3-phosphate dehydrogenase (G3PDH) expression was examined for normalization purposes. Experiments were repeated twice and the results were reproducible.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: TGZ decreases TGFβ2-induced expression of fibronectin and collagen type I mRNA in ARPE-19 cells.ARPE-19 cells were either left untreated or were treated with 4 ng/ml TGFβ2 for 24 h, or were pretreated with 10 μM TGZ, DMSO (0.05%), 10 ng/ml Actinomycin D, or 10 μM SB203580 for 1 h and then incubated with TGFβ2 for additional 24 h. Total RNA was extracted, and RT–PCR analysis for fibronectin and collagen type I (COLI) was performed. Glyceraldehyde-3-phosphate dehydrogenase (G3PDH) expression was examined for normalization purposes. Experiments were repeated twice and the results were reproducible.
Mentions: Next, we investigated whether TGZ was capable of inhibition of TGFβ2-induced collagen type I and fibronectin mRNA expression. As shown in Figure 3, RT–PCR analysis demonstrated that ARPE-19 cells cultured in serum-free medium expressed a basal level of collagen type I and fibronectin genes; treatment with exogenous TGFβ2 for 24 h markedly induced expression of both mRNAs. Cells treated with TGFβ2 for 8 h also displayed an increased expression of both mRNAs but at levels less than following stimulation for 24 h (approximately threefold). The TGFβ2-mediated induction effect was completely blocked by actinomycin D pretreatment, suggesting that the increased mRNA expression was transcription dependent. Cells pretreated for 1 h with 10 μM TGZ before TGFβ2 treatment suppressed TGFβ2-induced collagen type I and fibronectin mRNA to basal levels that were comparable to untreated cells. Pretreatment with DMSO had no such effect. Similarly, 10 μM SB203580 pretreatment partially prevented TGFβ2-induced collagen type I mRNA expression (compare Figure 2 with Figure 3).

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