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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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The TGZ inhibition effect is independent to PPARγ activation. A: ARPE-19 cells were pretreated with different doses of GW9662 (5–20 μM, 2 h) before TGZ combing with TGFβ2 treatment for additional 48 h. Immunoblot results are from a representative experiment with β-actin as loading control. B: ARPE-19 cell monolayers were either treated with TGFβ2 or pretreated with 10 μM GW9662 before TGZ combing with TGFβ2 treatment and wounded with a P-200 pipette tip immediately. Wells were photographed at 48 h adjacent to a reference line scraped on the bottom of the plate.
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f7: The TGZ inhibition effect is independent to PPARγ activation. A: ARPE-19 cells were pretreated with different doses of GW9662 (5–20 μM, 2 h) before TGZ combing with TGFβ2 treatment for additional 48 h. Immunoblot results are from a representative experiment with β-actin as loading control. B: ARPE-19 cell monolayers were either treated with TGFβ2 or pretreated with 10 μM GW9662 before TGZ combing with TGFβ2 treatment and wounded with a P-200 pipette tip immediately. Wells were photographed at 48 h adjacent to a reference line scraped on the bottom of the plate.

Mentions: To investigate whether TGZ acts through the activation of its receptor PPARγ to suppress TGFβ2-mediated reactions, ARPE-19 cells were pretreatment for 2 h with 5–20 μM GW9662, a specific PPARγ antagonist, and then assayed for PPARγ levels, fibronectin levels and cell migration in the presence of 10 μM TGZ combining with TGFβ2. As showed in Figure 7A, western blots showed that there was no change in the PPARγ protein levels with exposure to TGZ in the presence of TGFβ2. In addition, GW9662 did not reverse the inhibition of TGFβ2-induced fibronectin production (Figure 7A). in vitro wound healing assay also revealed that TGZ suppressed TGFβ2-promoted cell migration can not be reversed by GW9662 (Figure 7B, compared TGFβ2+TGZ with GW9662+TGFβ2+TGZ-treated cells at 48 h post-wounding). Therefore, the results led us to suggest that TGZ-mediated inhibitions are through a PPARγ-independent mechanism.


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)

The TGZ inhibition effect is independent to PPARγ activation. A: ARPE-19 cells were pretreated with different doses of GW9662 (5–20 μM, 2 h) before TGZ combing with TGFβ2 treatment for additional 48 h. Immunoblot results are from a representative experiment with β-actin as loading control. B: ARPE-19 cell monolayers were either treated with TGFβ2 or pretreated with 10 μM GW9662 before TGZ combing with TGFβ2 treatment and wounded with a P-200 pipette tip immediately. Wells were photographed at 48 h adjacent to a reference line scraped on the bottom of the plate.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f7: The TGZ inhibition effect is independent to PPARγ activation. A: ARPE-19 cells were pretreated with different doses of GW9662 (5–20 μM, 2 h) before TGZ combing with TGFβ2 treatment for additional 48 h. Immunoblot results are from a representative experiment with β-actin as loading control. B: ARPE-19 cell monolayers were either treated with TGFβ2 or pretreated with 10 μM GW9662 before TGZ combing with TGFβ2 treatment and wounded with a P-200 pipette tip immediately. Wells were photographed at 48 h adjacent to a reference line scraped on the bottom of the plate.
Mentions: To investigate whether TGZ acts through the activation of its receptor PPARγ to suppress TGFβ2-mediated reactions, ARPE-19 cells were pretreatment for 2 h with 5–20 μM GW9662, a specific PPARγ antagonist, and then assayed for PPARγ levels, fibronectin levels and cell migration in the presence of 10 μM TGZ combining with TGFβ2. As showed in Figure 7A, western blots showed that there was no change in the PPARγ protein levels with exposure to TGZ in the presence of TGFβ2. In addition, GW9662 did not reverse the inhibition of TGFβ2-induced fibronectin production (Figure 7A). in vitro wound healing assay also revealed that TGZ suppressed TGFβ2-promoted cell migration can not be reversed by GW9662 (Figure 7B, compared TGFβ2+TGZ with GW9662+TGFβ2+TGZ-treated cells at 48 h post-wounding). Therefore, the results led us to suggest that TGZ-mediated inhibitions are through a PPARγ-independent mechanism.

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