Abstract
Purpose: :
We have shown earlier that the synthetic retinoid N–(4–hydoxyphenyl)–retinamide (4HPR) induces apoptosis in human retinal pigment epithelial cells in culture. Reactive oxygen species (ROS) appears to be a key mediator of this process. Heme oxygenase (HO–1), the rate limiting enzyme in the heme degradative pathway, is known to be regulated by ROS. Therefore, our present study is aimed at understanding the role of HO–1 in 4HPR–induced ROS generation and apoptosis in RPE cells.
Methods: :
Human RPE cells (ARPE–19) in culture were treated with 5 µM 4HPR in the presence or absence of retinoid receptor antagonists for various time intervals. Progression of apoptosis was estimated by a sandwich–enzyme immunoassay using anti–histone antibody directed against mono– and oligo–nucleosomes. The expression of HO–1 mRNA and protein were measured by real–time RT–PCR and by ELISA, respectively. The intracellular generation of ROS was measured using oxidation–sensitive fluorescent dye DCF–DA.
Results: :
HO–1 mRNA expression was increased more than a 100–fold in ARPE–19 cells undergoing apoptosis following 4HPR treatment (5µM, 24 h). This was associated with a 4–fold increase in HO–1 protein. In addition, 4HPR–induced HO–1 expression in treated cells was preceded by the generation of ROS. Both HO–1 mRNA and protein expression induced by 4HPR were inhibited by the RAR pan–antagonists (AGN194310 and AGN193109) and by an RARα–specific antagonist (AGN194301).
Conclusions: :
These results demonstrate that HO–1 expression is induced in RPE cells undergoing apoptosis following 4HPR treatment, probably in response to ROS generation. The regulation of HO–1 expression by ROS may play an important role in RPE pathophysiology.
Keywords: retinal pigment epithelium • apoptosis/cell death • antioxidants