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R. Antony, N. G. Bazan; Induction of Bcl-xL Expression by Neuroprotectin D1 in ARPE-19 Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):218.
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The survival of retinal pigment epithelial (RPE) cells is critical for photoreceptor integrity. Well orchestrated interactions between Bcl-2 family proteins (anti- and pro-apoptotic) are critical modulators of cell survival. Bcl-xL is one such molecule that protects cells against apoptosis. Neuroprotectin D1 (NPD1) inhibits oxidative stress-mediated pro-inflammatory gene induction and apoptosis, promotes RPE cell survival, and induces Bcl-xL. The precise molecular mechanism of NPD1 regulation is not understood. Here we have used constructs containing regions of the Bcl-xL promoter to address these issues.
ARPE-19 cells were maintained in DMEM/F12 with 10% fetal calf serum at 37°C and 5% CO2. Cells were transfected with mouse Bcl-xL promoter-driven luciferase (Luc) reporter plasmid pGL2-3.2, which contained a 3.2-kb fragment upstream of the Bcl-xL gene and a series of 5' deletion Luc reporter plasmids (-1160, -848, -822 ΔNF-ΚB, -631 ΔNF-ΚB ,Δsp-1 and -848 NF-ΚB(CC-GG mutation). pSV-β-galactosidase was used as an internal control. Cells were serum starved for 18 h to induce oxidative stress. Luciferase activities were normalized with β-galactosidase activities after 48 h of incubation in the presence and absence of 50 nM NPD1. Protein levels were analyzed using Western blot.
Transient transfection assays showed that Bcl-xLpromoter was transactivated in the presence of NPD1. Deletion or substitution of a putative NF-ΚB binding site identified in the Bcl-xL promoter increased NPD1 induced Bcl-xL expression. On the contrary, deletion of the transcription factor specificity protein 1 (Sp1) significantly decreased NPD-1 induced expression. Our findings suggest that Sp1 is the main regulator of NPD1-induced Bcl-xL gene in the retinal pigment epithelial cells.
NPD1-mediated modulation of Bcl-xLexpression may play an important role in RPE cell survival under normal conditions and when cells are exposed to oxidative stress. Therapeutic strategies targeting the NPD1 mediated Bcl-xL expression pathway may be useful to promote RPE cell survival and, in turn, photoreceptor integrity, in retinal degenerative diseases.
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