Abstract
P 077
Regulation of pigment epithelial-derived factor in retinal glial (Müller) cells
XiuMei Yang1, Wolfram Eichler1, Andreas Reichenbach2, Peter Wiedemann1
1Klinik und Poliklinik für Augenheilkunde, Universitätsklinikum Leipzig, Leipzig; 2Paul-Flechsig-Institut für Hirnforschung, Universität Leipzig A.ö.R., Leipzig
Objective
Pigment epithelium-derived factor (PEDF), a glycoprotein with pleiotropic functions, is naturally occurring in the eye and considered as crucial to prevent pathological angiogenesis. Since retinal glial (Müller) cells produce PEDF, we investigated the regulation of PEDF production by these cells, especially under ischemic/ hypoxic conditions.
Methods
PEDF was determined in primary cultured guinea pig as well as rat Müller cells exposed to the protein synthesis inhibitor, cycloheximide (CHX), the transcription inhibitor actinomycin D (Act D), the selective proteasome inhibitor MG132, and to a hypoxia-inducible factor (HIF)-1 inhibitor. Suppression of HIF-1 alpha expression using RNA interference (RNAi) was additionally used to determine the impact of HIF-1 alpha on PEDF expression. PEDF production and secretion were studied by semi-quantitative Realtime-PCR, ELISA, and Western blotting.
Results
CHX, Act D and MG132 treatment of Müller cells resulted in an increase of PEDF mRNA and protein levels under both normoxia and hypoxia. Both HIF-1 inhibitor and HIF-1 alpha siRNA were able to up-regulate PEDF expression at the mRNA level with a decrease of PEDF release. In parallel, vascular endothelial growth factor (VEGF), up-regulated under hypoxia, could be down-regulated by CHX, Act D and HIF-1 inhibitor whereas it was up-regulated by MG 132.
Conclusions
These results suggest that PEDF in Müller cells is largely regulated post-transcriptionally. HIF-1 alpha, which is a master regulator of VEGF expression, appears to keep Müller cell-associated PEDF levels in check.
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