PEDF Downregulates Genes Involved in Neuronal Cell Death

Abstract

Abstract: : Purpose: PEDF protects retinal cells from stressful stimuli including chronic light exposure, retinal detachment, and oxidative stress, though the mechanism by which it acts has not yet been determined. In this study we analysed the effects of PEDF on two retinal cell lines and retinal explants by transcriptional profiling to identify the intracellular pathways by which PEDF might be neuroprotective. Methods: Human ARPE19 and Y79 retinoblastoma cells were adapted into serum-free medium for 24 hrs. Mouse retinas were explanted directly into serum-free medium. Cultures were treated with 100 ng/ml PEDF for 48 hrs and RNA isolated for cDNA synthesis. cDNA was radioactively labeled and used to hybridize 8K human gene arrays (Clontech), or fluorescently labeled for hybridization to 9K mouse retinal arrays. Expression signals and ratio differences between PEDF treated and controls were plotted after global normalization and background subtraction using appropriate software packages. Based on their expression profiles, a number of genes were selected for quantitative RT-PCR analysis. Results: By microarray analysis PEDF down-regulated a cluster of genes, including caspase 2, calpain 1, and MAP kinase kinase 1, involved in different apoptotic pathways. Quantitative RT-PCR showed that after 48 hrs, PEDF was effective in down regulating the transcription of these genes by about 3 fold in both the RPE and Y79 cells. By analyzing the spectrum of changes induced by PEDF in different cells, we have begun to determine how the physiological composition of the target can influence the responses to PEDF application. Conclusions: Calpain 1 and Caspase 2 are intracellular cysteine proteases regulated by calcium influx, oxidative stress, and ischemic injury. Their overexpression is linked to various necrotic and apoptotic conditions associated with acute neurodegenerative processes. MAP kinases become activated by MKKs following a variety of injury stimuli including, focal cerebral ischemia. These signaling molecules play a key role in convergent signaling pathways that effect growth or death of neurons and their regulation may be one mechanism by which PEDF is neuroprotective in the retina.

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