PEDF Expression and Promoter Activity is Regulated by Retinoic Acid

Abstract

Abstract: : Purpose: Retinoic acid (RA) can control differentiation and apoptosis in the developing nervous system. RA effects are mediated through a group of cytoplasmic receptors (RAR) that regulate transcription of genes containing RAR binding motifs. An RAR motif is present in the PEDF promoter. The purpose of this study was to determine the effects of all trans RA (ATRA) on PEDF promoter activity and also to test the effects of PEDF on the expression of RARs in PEDF-producing and PEDF-target cell populations. Methods: Two human cell lines expressing PEDF, ARPE19 and Y79, and primary mouse Müller glial cells, were treated for 5 days with 1-10 nM of ATRA in serum-free medium and PEDF expression analyzed. Cells were also treated with PEDF for 48 hrs, harvested, and RNA extracted for transcriptional profiling using 8K human gene and 9K mouse retinal arrays. To test the specific effect of RA on the PEDF promoter, the 5’ flanking region of the PEDF gene, -869/+59, was cloned into the pGL3 basic vector upstream of the firefly luciferase gene. D407 human RPE cells were transfected with this plasmid and luciferase activity assayed after treatment of the transfected cells with varying concentrations of ATRA. Results: PEDF levels in conditioned-medium of treated cells were elevated by approximately 3X as compared to controls. PEDF mRNA levels in treated cells showed a similar increase. Increased luciferase activity was observed in cell lines that were treated with ATRA following transfection with the PEDF promoter pGL3 plasmid. Array data indicated that PEDF induces expression of the RAR gamma and RAR orphan C by 5 fold and 2 fold respectively, but downregulates the RAR alpha by 2 fold. Conclusions: All trans retinoic acid and PEDF show an interesting relationship. Both agents are known to regulate cell differentiation and cell death. By regulating the activity of each other, they may have complementary effects in these processes.

Keywords: retinal degenerations: cell biology • neuroprotection • gene microarray