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D. Zhu, C. Spee, S. J. Ryan, D. R. Hinton; Smad and P38 Pathways Are Involved in BMP4-Mediated RPE Cell Senescence. Invest. Ophthalmol. Vis. Sci. 2008;49(13):225.
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The senescence of RPE cells may be a critical step in the pathogenesis of age-related macular degeneration (AMD). Previously we have shown that Bone Morphogenetic Protein-4 (BMP4) mediates oxidative stress-induced RPE cell senescence. The current study aims to determine more detailed mechanisms involved in BMP4-mediated RPE cell senescence under oxidative stress.
1). Oxidative stressor and BMP4 treatment: ARPE-19 cells were treated with tert--butylhydroperoxide (tBH, 10-50 uM), or hydrogen peroxide (H2O2, 50-250 uM) in culture medium supplemented with 10% FBS,and with 0, 10, 25 and 50 ng/ml of recombinant BMP4 protein (R&D System) for 1 hours or 24 hours in medium containing 1% FBS. 2).Inhibitor treatment: ARPE-19 cells were treated with 0.1ug/ml of recombinant Chordin-like protein (R&D system) or with 10 uM SB203580 (EMD bioscience) during and after stressor treatments in ARPE medium with 10% FBS. 3). Western blot and real time RT-PCR were used to quantitate the expression levels of target genes. 4). Senescence associated beta-galactosidase (SA-β-Gal) staining assay was used to detect senescent cells.
Western blot analyses showed that P21Cip/WAF1 and P53 proteins were increased, and phosphorylated Rb protein was decreased in RPE cells treated with BMP4 or oxidative stressors (H2O2 and tBH), and in lentivirally transduced RPE cells that over-express BMP4 (ARPE-BMP4), when compared to untreated ARPE-19 cells; phospho-Smad1/5/8 and phospho-P38 were also increased in those RPE cells treated with BMP4 or oxidative stressors. After blocking BMP4 with Chordin-like recombinant protein or inhibiting phospho-P38 with SB203580, phosphorylated Rb was increased, while P21 and P53 proteins, the senescence marker gene, apoJ, and the number of SA-β-Gal positive cells were all significantly decreased.
Under oxidative stress, RPE cells increase the expression of BMP4, which further activates its down stream signaling pathway to induce P53 and P21Cip1/WAF1, but suppress phosphorylated Rb via Smad and P38 MAPK pathways. The increased P53 and decreased phospho-Rb triggers RPE cell senescence. This BMP4-mediated RPE cell senescence can be partly blocked by BMP4 antagonists or P38 inhibitors.
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