Zusammenfassung
PURPOSE. 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 mu M hydrogen peroxide (H2O2) for 1 and 2 hours or treated with 1.0 ng/ mL TGF-beta 1 or -beta 2 for 12, 24, and 48 hours. ...
Zusammenfassung
PURPOSE. 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 mu M hydrogen peroxide (H2O2) for 1 and 2 hours or treated with 1.0 ng/ mL TGF-beta 1 or -beta 2 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-beta 1, -beta 2, and -beta 3 isoforms and the TGF-beta II receptor. RESULTS. H2O2 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-beta 1 and -beta 2 showed similar changes. H2O2 and TGF-beta 1 and -beta 2 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-beta 1, -beta 2, and -beta 3 isoforms and the TGF-beta II receptor prevented the oxidative stress-mediated elevation of senescence-associated biomarkers. CONCLUSIONS. Oxidative stress, TGF-beta 1, and TGF-beta 2 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. (Invest Ophthalmol Vis Sci. 2009; 50: 926-935) DOI: 10.1167/iovs.07-1003
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