title: Differential effects of TGFbeta and vitreous on the transformation of retinal pigment epithelial cells.
abstract: OBJECTIVE
  In proliferative vitreoretinopathy retinal pigment epithelial (RPE) cells undergo epithelial-mesenchymal transformation (EMT). Vitreous and transforming growth factor-beta (TGFbeta) have been implicated in this EMT. The role of TGFbeta in the vitreous-mediated transformation of low-passage human RPE cells was investigated.
  
  
  METHODS
  Cells were treated with vitreous or TGFbeta2. SB431542 was used to inhibit TGFbeta signaling. Morphology was investigated using phase-contrast or confocal microscopy. Motility was measured using a monolayer-wounding assay. Invasion was determined using basement membrane matrix-based assays. Gene expression was measured by quantitative PCR, immunohistochemistry, or immunoblotting.
  
  
  RESULTS
  Changes in phosphorylation or cellular localization of Smad -2, -3, or -4 indicated a TGFbeta-like activity in vitreous. Cortical actin filaments in untreated cells were replaced by stress fibers after TGFbeta treatment, but peripheral actin aggregates were seen in vitreous-treated cells. SB431542 did not block the morphologic change induced by vitreous. Vitreous-treated cells exhibited increased motility and invasion, whereas TGFbeta-treated cells did not. However, SB431542 decreased vitreous-meditated changes in motility and invasion. The levels of mRNA for genes indicative of myofibroblast differentiation (alpha-SMA and CTGF) were increased by treatment with TGFbeta but suppressed by vitreous. TGFbeta or vitreous caused increased expression of Snail1.
  
  
  CONCLUSIONS
  Vitreous or TGFbeta caused a fibroblast-like morphology and induced Snail1, a marker of EMT. TGFbeta activity in vitreous was necessary but not sufficient for the vitreous-induced motile, invasive phenotype. However, differences in the cytoskeletal organization and in the expression of CTGF and alpha-SMA suggested that TGFbeta-treatment caused differentiation along a myofibroblast pathway, whereas vitreous treatment suppressed myofibroblast formation.
authors: Department of Pathology, Microbiology, and Immunology, University of South Carolina School of Medicine, Columbia, South Carolina;
authors: Sunil K
authors: SK
authors: Parapuram
authors: Binyue
authors: B
authors: Chang
authors: Lei
authors: L
authors: Li
authors: Ren A
authors: RA
authors: Hartung
authors: Kakarla V
authors: KV
authors: Chalam
authors: Joyce U
authors: JU
authors: Nair-Menon
authors: D Margaret
authors: DM
authors: Hunt
authors: Richard C
authors: RC
authors: Hunt
mesh_terms: D000199:Actins / Q000235:genetics; D001549:Benzamides / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects; D002461:Cell Line, Transformed; D002465:Cell Movement / Q000187:drug effects; D002478:Cells, Cultured; D055513:Connective Tissue Growth Factor / Q000235:genetics; D004149:Dioxoles / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D018613:Microscopy, Confocal; D008858:Microscopy, Phase-Contrast; D010766:Phosphorylation; D012333:RNA, Messenger / Q000378:metabolism; D018125:Receptors, Transforming Growth Factor beta / Q000037:antagonists & inhibitors; D055213:Retinal Pigment Epithelium / Q000166:cytology* / Q000502:physiology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000071250:Snail Family Transcription Factors; D014157:Transcription Factors / Q000235:genetics; D053781:Transforming Growth Factor beta2 / Q000494:pharmacology*; D014822:Vitreous Body / Q000502:physiology*
chemical_list: C459179:4-(5-benzo(1,3)dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)benzamide; C541116:ACTA2 protein, human; D000199:Actins; D001549:Benzamides; C526317:CCN2 protein, human; D004149:Dioxoles; D012333:RNA, Messenger; D018125:Receptors, Transforming Growth Factor beta; C000604407:SNAI1 protein, human; D000071250:Snail Family Transcription Factors; D014157:Transcription Factors; D053781:Transforming Growth Factor beta2; D055513:Connective Tissue Growth Factor