title: Subtoxic oxidative stress induces senescence in retinal pigment epithelial cells via TGF-beta release.
abstract: OBJECTIVE
  The goal of the present study was to determine whether oxidative stress and transforming growth factor (TGF)-beta induce cellular senescence in human retinal pigment epithelial (RPE) cells.
  
  
  METHODS
  Cultured human RPE cells were exposed to 50 to 150 microM hydrogen peroxide (H(2)O(2)) for 1 and 2 hours or treated with 1.0 ng/mL TGF-beta1 or -beta2 for 12, 24, and 48 hours. Senescence-associated beta-galactosidase (SA-beta-Gal) activity was detected by histochemical staining. Expression of senescence-associated genes (apolipoprotein J [Apo J], connective tissue growth factor [CTGF], fibronectin, and SM22) was examined by real-time PCR and induction of signal transduction proteins (p21, p16, and pRb) by Western blot analysis. The effects of TGF-beta blocking on the oxidative stress-induced expression of senescence-associated biomarkers were investigated by simultaneous incubation with neutralizing antibodies against the TGF-beta1, -beta2, and -beta3 isoforms and the TGF-betaII receptor.
  
  
  RESULTS
  H(2)O(2) markedly increased the number of SA-beta-Gal-positive cells to up to 89% and the expression of Apo J, CTGF, fibronectin, and SM22 by approximately three to fourfold. Treatment with TGF-beta1 and -beta2 showed similar changes. H(2)O(2)and TGF-beta1 and -beta2 markedly enhanced the expression of p21 but downregulated pRb. In contrast, they had no effect on p16 expression. Simultaneous treatment with neutralizing antibodies against the TGF-beta1, -beta2, and -beta3 isoforms and the TGF-betaII receptor prevented the oxidative stress-mediated elevation of senescence-associated biomarkers.
  
  
  CONCLUSIONS
  Oxidative stress, TGF-beta1, and TGF-beta2 are capable of inducing cellular senescence in cultured human RPE cells. Therefore, reduction of oxidative stress and minimizing TGF-beta may help to prevent senescence-associated changes in the RPE as seen in early age-related macular degeneration.
authors.affiliation: Department of Ophthalmology, Ludwig-Maximilians-University, Munich, Germany.
authors.forename: Alice L
authors.initials: AL
authors.lastname: Yu
authors.forename: Rudolf
authors.initials: R
authors.lastname: Fuchshofer
authors.forename: Daniel
authors.initials: D
authors.lastname: Kook
authors.forename: Anselm
authors.initials: A
authors.lastname: Kampik
authors.forename: Hans
authors.initials: H
authors.lastname: Bloemendal
authors.forename: Ulrich
authors.initials: U
authors.lastname: Welge-Lüssen
mesh_terms: D000328:Adult; D015153:Blotting, Western; D002478:Cells, Cultured; D016922:Cellular Senescence / Q000187:drug effects*; D051152:Clusterin / Q000235:genetics; D055513:Connective Tissue Growth Factor / Q000235:genetics; D005353:Fibronectins / Q000235:genetics; D006801:Humans; D006861:Hydrogen Peroxide / Q000633:toxicity*; D015227:Lipid Peroxidation; D008840:Microfilament Proteins / Q000235:genetics; D009124:Muscle Proteins / Q000235:genetics; D018384:Oxidative Stress; D012333:RNA, Messenger / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D055213:Retinal Pigment Epithelium / Q000166:cytology* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D053773:Transforming Growth Factor beta1 / Q000378:metabolism* / Q000494:pharmacology; D053781:Transforming Growth Factor beta2 / Q000378:metabolism* / Q000494:pharmacology; D001616:beta-Galactosidase / Q000096:biosynthesis
chemical_list: D051152:Clusterin; D005353:Fibronectins; D008840:Microfilament Proteins; D009124:Muscle Proteins; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D053773:Transforming Growth Factor beta1; D053781:Transforming Growth Factor beta2; C051615:transgelin; D055513:Connective Tissue Growth Factor; D006861:Hydrogen Peroxide; D001616:beta-Galactosidase