Subtoxic oxidative stress induces senescence in retinal pigment epithelial cells via TGF-β release

Abstract

PURPOSE. The goal of the present study was to determine whether oxidative stress and transforming growth factor (TGF)-β induce cellular senescence in human retinal pigment epithelial (RPE) cells. METHODS. Cultured human RPE cells were exposed to 50 to 150 μM hydrogen peroxide (H2O2) for 1 and 2 hours or treated with 1.0 ng/mL TGF-β1 or -β2 for 12, 24, and 48 hours. Senescence-associated β-galactosidase (SA-β-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-β blocking on the oxidative stress-induced expression of senescence-associated biomarkers were investigated by simultaneous incubation with neutralizing antibodies against the TGF-β1, -β2, and -β3 isoforms and the TGF-βII receptor. RESULTS. H2O 2 markedly increased the number of SA-β-Galpositive cells to up to 89% and the expression of Apo J, CTGF, fibronectin, and SM22 by approximately three to fourfold. Treatment with TGF-β1 and -β2 showed similar changes. H2O2 and TGF-β1 and -β2 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-β1, -β2, and -β3 isoforms and the TGF-βII receptor prevented the oxidative stress-mediated elevation of senescence-associated biomarkers. CONCLUSIONS. Oxidative stress, TGF-β1, and TGF-β2 are capable of inducing cellular senescence in cultured human RPE cells. Therefore, reduction of oxidative stress and minimizing TGF-β may help to prevent senescence-associated changes in the RPE as seen in early age-related macular degeneration. Copyright © Association for Research in Vision and Ophthalmology.