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D. Zhu, S. J. Ryan, D. R. Hinton; BMP-4: A Mediator of RPE Cell Senescence. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2205.
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Bone morphogenetic protein-4 (BMP-4) is involved in a variety of important biologic processes, including programmed cell death and cell senescence. We have shown previously that BMP-4 is highly expressed in the retinal pigment epithelium (RPE) of patients with dry age-related macular degeneration (AMD). We sought to determine the effects of BMP4 on the promotion of programmed death and/or senescence of RPE cells.
1). ARPE-19 and human fetal RPE (fRPE) cells were treated with tert--butylhydroperoxide (tBH, 30 uM) and hydrogen peroxide (H2O2, 150 uM) in culture medium containing 10% fetal bovine serum for 2 hours for each stress. The cells were serially stressed five times with tBH treatment and three times with H2O2 treatment. 2). BMP4 overexpressed ARPE-19 stable cell lines (ARPE-BMP4) were established by transducing BMP4-lentiviral constructs into ARPE-19 cells. BMP4-lentiviral vector was constructed by inserting BMP4 cDNA into the pLenti6/V5-TOPO vector (Invitrogen). 3). Western blot and real time RT-PCR were used to semiquantitate or quantitate the expression levels of target genes. 4). SA-ß-Gal staining assay was used to detect the senescent cells.
Western blot showed significantly increased expression and secretion of recombinant BMP4 protein in ARPE-BMP4 cells. ß-galactosidase activity was dramatically increased in ARPE-BMP4 cells when the cells were cultured in serum free ARPE medium for one week. BMP4 expression in ARPE-19 and fRPE cells increased about eight-fold after tBH or H2O2 treatment compared to that of the non-stressed cells. tBH and H2O2 induced irreversible cell growth arrest and premature senescence of ARPE-19 and fRPE cells.
Both BMP4 and oxidative stress induce RPE cell senescence. Oxidative stress also increases the BMP4 expression in human RPE cells. The results indicate that the expression of RPE senescence-related genes or biomarkers under oxidative stress may be mediated by the BMP4 pathway.
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