Troglitazone suppresses transforming growth factor beta-mediated fibrogenesis in retinal pigment epithelial cells.
OBJECTIVE
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.
Cheng
Huey-Chuan
HC
Department of Ophthalmology, Mackay Memorial Hospital, Taipei, Taiwan.
Ho
Tsung-Chuan
TC
Chen
Show-Li
SL
Lai
Huei-Yi
HY
Hong
Kuo-Fu
KF
Tsao
Yeou-Ping
YP
D002460:Cell Line; D002465:Cell Movement / Q000187:drug effects; D002839:Chromans / Q000494:pharmacology*; D024042:Collagen Type I / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics; D004789:Enzyme Activation; D005353:Fibronectins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics; D005355:Fibrosis; D006801:Humans; D047495:PPAR gamma / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D010857:Pigment Epithelium of Eye / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology*; D012333:RNA, Messenger / Q000037:antagonists & inhibitors / Q000378:metabolism; D015398:Signal Transduction; D051899:Smad2 Protein / Q000378:metabolism; D051900:Smad3 Protein / Q000378:metabolism; D045162:Thiazolidinediones / Q000494:pharmacology*; D016212:Transforming Growth Factor beta / Q000378:metabolism*; D000077288:Troglitazone; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D002839:Chromans; D024042:Collagen Type I; D005353:Fibronectins; D047495:PPAR gamma; D012333:RNA, Messenger; C497782:SMAD2 protein, human; C497790:SMAD3 protein, human; D051899:Smad2 Protein; D051900:Smad3 Protein; D045162:Thiazolidinediones; D016212:Transforming Growth Factor beta; D048051:p38 Mitogen-Activated Protein Kinases; D000077288:Troglitazone