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Aram Asatryan, Jorgelina M. Calandria, Nicolas G. Bazan; Neuroprotectin D1 Is A Transcriptional Modulator Of The Birc3 Gene That Encodes An Inhibitor Of Apoptosis Protein In Retinal Pigment Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2012;53(14):6424.
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Apoptosis in RPE cells and photoreceptors takes place in retinal degenerative diseases. Therefore, the understanding of endogenous apoptosis modulators is of high interest. The stereospecific docosahexaenoic acid (DHA)-derived messenger, neuroprotectin D1 (NPD1), prevents apoptosis in retinal pigment epithelial (RPE) cells and inhibits expression of pro-inflammatory genes, while enhancing the expression of anti-apoptotic proteins of the Bcl-2 family (PNAS, 101,8491-6,2004; J.Clin.Invest.,115,2774-83,2005). We hypothesized that NPD1 regulates a network of genes involved in apoptosis and that the integration of the evolving signaling is a cell fate decision maker.
ARPE-19 cells undergoing oxidative stress were treated with NPD1 (50nM). Affymetrix (Santa Clara, CA) U133 plus 2.0 data was used to determine whether NPD1 regulates gene expression triggered by oxidative stress. RT² Profiler™ PCR pathway focused array (SABiosciences, Frederick, MD) was used to determine the RNA expression of apoptosis-related genes regulated by NPD1. ARPE-19 cells were co-transfected with 5’ deletion mutants of BIRC3 promoter luciferase constructs.
Expression of BIRC3, a versatile modulator of inflammatory signaling and apoptosis, was increased more than four-fold in the presence of NPD1 in the cells undergoing oxidative stress, when compared with the controls (p<0.05). A deletion construct of the BIRC3 promoter containing 247 bases upstream of the transcription initiation site showed the highest levels of activation. Furthermore, promoter activity was twice as high when 200nM NPD1 was added after 4 and 6 hours of oxidative stress, whereas there was no noticeable change in the expression after 12 and 24 hours. In silica analysis of the promoter sequence, BIRC3 showed three binding sites for c-REL in the fragments that were responsive to NPD1.
These data demonstrate for the first time that NPD1 affects apoptosis by directly modulating gene expression of a pro-survival effector protein at a transcriptional level in RPE cells. These data will allow us to begin constructing a modulatory network of cell survival regulators targeted by NPD1.
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