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W. Samuel, R. Kutty, T. Duncan, B. Wiggert; Regulation of Gadd153 Gene Expression by Retinoic Acid Receptors in Human Retinal Pigment Epithelial (RPE) Cells During N–(4–Hydroxyphenyl)Retinamide Induced Apoptosis . Invest. Ophthalmol. Vis. Sci. 2006;47(13):4856.
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The growth arrest and DNA damage–inducible transcription factor 153 (Gadd153), a member of CCAAT/enhancer binding protein (C/EBP) family of transcription factors, is transcriptionally activated and is often increased when cells are subjected to a stressful environment by a variety of growth arrest and/or DNA–damaging factors, endoplasmic stress and reactive oxygen species (ROS) generation. Gadd153 serves as a key control point and regulator of apoptosis mediated by ROS. Earlier we have shown that N–(4–hydroxyphenyl)retinamide (4HPR) induced apoptosis in human retinal pigment epithelial cells (ARPE–19) is mediated through ROS generation via the retinoic acid receptor (RAR) pathway. In the present study we investigated the expression of Gadd153 and its regulation by RARs in 4HPR–induced apoptosis in ARPE–19 cells.
Human RPE cells (ARPE–19) in culture were treated with 4HPR (1–20 µM) in the presence or absence of retinoid receptor antagonists for various time intervals. Progression of apoptosis was measured in cell lysates by ELISA using anti–histone antibodies directed against mono–and oligonuclesomes. RNA extracted from ARPE–19 cells was used for measuring Gadd153 transcript by real–time RT–PCR. Dual Luciferase Reporter Assay system was used for measuring Gadd153 promoter activity. The intracellular generation of ROS was measured using oxidation–sensitive fluorescent dye DCF–DA.
4HPR induced apoptosis and ROS generation was time–dependent in ARPE–19 cells. The expression of Gadd153 mRNA was increased 10–fold after 4HPR treatment. 4HPR increased the Gadd153 promoter activity (6–fold) indicating that 4HPR regulates Gadd153 expression at the transcriptional level. The increase in both Gadd153 expression and its promoter activity were inhibited by RAR pan–antagonists (AGN194310 and AGN193109) and by an RARα–specific antagonist (AGN194301). Cells pretreated with pyrrolidinedithiocarbamate, a free–radical scavenger, not only inhibited ROS generation and apoptosis but also inhibited the 4HPR–induced expression of Gadd153 gene and its promoter activity.
The inhibition of Gadd153 expression and its promoter activity by RAR antagonists indicates that Gadd153 mediates apoptosis induced by 4HPR in RPE cells through an RAR–dependent signaling pathway.
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