Abstract
Purpose :
Glucocorticoids (GC) are required for photoreceptor survival, and are useful for retinal protection. However, little information is available about the role of GC in the retinal pigment epithelium (RPE). Therefore we tested ARPE-19 cells, a human RPE cell line, to find out whether survival and proliferation could be modified by dexamethasone (DEX), a specific agonist of GC receptor α (GRα), or mifepristone (MFP), a GC antagonist and a possible agonist of GC receptor β (GRβ).
Methods :
ARPE-19 cells were cultured for 48 h in DMEM/F12 with 10% fetal calf serum and antibiotics with 5% CO2 in air at 37°C. Cultures were then treated with DEX (0.08, 0.32, 1.28 mM) and/or MFP (1, 10 µM, both solubilized in ethanol). Controls were made in medium with or without ethanol. After 24 h, cultures were stained with acridine orange and ethidium bromide (AO/EB) for detection of apoptotic and necrotic cells. Other cultures were incubated in 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) during 4 h at 37°C to measure cell viability. The expression of GRβ was assayed in control cultures using Western blots or qPCR. Balb-c mice eye sections were immunostained with GRβ antibody.
Results :
The AO/EB procedure showed that 0.08 mM DEX cultures displayed 3% of necrotic/apoptotic cell nuclei, whereas 1.28 mM DEX exhibited 37% of cells with necrotic/apoptotic signs. MFP (1 and 10 µM) induced less than 5% of necrotic/apoptotic cell nuclei. Cell viability after 0.08 mM DEX was not different from controls, but it decreased after 0.32 or 1.28 mM DEX (p < 0.001). By contrast, MFP did not modify cell viability. Combination of 0.08 mM DEX with 1 or 10 µM MFP did not induce viability changes. Exposure to 1.28 mM DEX and MFP (1 or 10 µM) reduced viability in the same proportion as 1.28 mM DEX alone.
Both Western blots and qPCR demonstrated high expression of GRβ in ARPE-19 cells. Moreover, the mouse RPE showed a much higher GRβ immunoreactivity than the neural retina.
Conclusions :
Assays for cell death and cell viability demonstrated DEX cytotoxicity for ARPE-19 cells. These effects were not antagonized by MFP, suggesting that they would not be mediated by GRα receptors. Alternatively, the availability of MFP could be decreased by binding to GRβ. The selective localization of this receptor in RPE cells indicates that further studies should be carried out to understand its role in retinal physiology and protection.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.