Abstract
Purpose::
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.
Methods::
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.
Results::
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.
Conclusions::
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.
Keywords: age-related macular degeneration • growth factors/growth factor receptors • retinal pigment epithelium