Abstract
Purpose: :
Pigment epithelium-derived factor (PEDF), is a member of the serine protease inhibitor (SERPIN) gene family. It is neurotrophic and protective for neurons cultured from the cerebellum, hippocampus, spinal cord (motor neurons), and retina. PEDF is an agonist of neuroprotectin D1 (NPD1) synthesis that in turn induces homeostatic survival regulation (PNAS, 104, 13152, 2007). Antioxidant responsive elements (ARE), cis-acting transcriptional enhancers, mediate the transcriptional induction of a battery of genes that comprise the chemoprotective response system and mediate antioxidant defense. Previous studies have identified that oxidative stress enhances expression of ARE in ARPE-19 cells, and NPD1 further enhances ARE expression by 6-10 fold. Here we report a study on the effects of PEDF and DHA on ARE expression in ARPE-19 cells.
Methods: :
ARPE-19 cells, grown overnight, were transfected with an ARE construct (linked to Luciferase) together with PEDF expression vector using Fugene-6 transfection method for 24h. A β-galactosidase plasmid was co-transfected as a transfection control. Transfected cells were then serum-starved overnight. The serum-starved cells were induce oxidative stress and challenged with NPD1 (100nM) for 6h. In another set of experiment, ARPE-19 cells were transfected with ARE construct, serum starved, and then treated with either PEDF (10 ng/ml) alone or PEDF and DHA (100nM) for 6h. Cell extracts were made, and luciferase enzyme assays were then performed to measure ARE expression using luciferin as a substrate.
Results: :
Our results indicate that oxidative stress promotes upregulation of ARE expression. PEDF alone resulted in upgregulation of ARE expression in PEDF transfected ARPE-19 cells. DHA together with PEDF potentiate upregulation (5-6 fold) in ARE transfected cells. In contrast, oxidative stress had no additional effect on upregulation of ARE in PEDF-transfected cells other than the PEDF effect. However, NPD1 potentiated further upregulation (at least 5-fold) of ARE in PEDF transfected cells under oxidative stress. Moreover, NPD1-induced upregulation of ARE in PEDF transfected cells are additive. Experiments are underway using two PEDF peptides, one anti-angiogenic and the other anti-apoptotic, under similar conditions to validate our results.
Conclusions: :
The results demonstrate that PEDF alone activates ARE expression in RPE cells, and PEDF/DHA potentiate this ARE upregulation. Furthermore, our findings demonstrate that PEDF/DHA mediated upregulation of ARE may involved NPD1 synthesis in ARPE-19 cells and contribute to cell survival through gene antioxidant defense signaling.
Keywords: antioxidants • stress response • retinal pigment epithelium