Impairment of Ubiquitin–Proteasome Pathway in Retinal Pigment Epithelial Cells Enhances Expression and Secretion of Vascular Endothelial Growth Factor

Abstract

Abstract: : Purpose: Choroidal neovascularization (CNV) is a hallmark of age–related macular degeneration (AMD). Angiogenic factors produced by retinal pigment epithelial cells (RPE) are major contributors to CNV development. The ubiquitin–proteasome pathway (UPP) plays critical roles in many cellular processes, such as protein quality control, signal transduction and transcription. Hypoxia–induced factor (HIFα) is the transcription factor that controls the expression of vascular endothelial growth factor (VEGF) and it is known that levels of HIFα are controlled by the UPP. The objective of this study is to determine roles of the UPP in controlling the levels of HIFα in RPE as well as in expression and secretion of VEGF. Methods: Cultural RPE (ARPE–19) were treated with or without 10mM MG132, a proteasome inhibitor, for 8–16 hours. The media were collected to determine the levels of VEGF–A165 by ELISA assay. Levels of HIFα were determined by Western Blotting. Total RNA was extracted from the cells and levels of VEGF mRNA were assessed by real–time RT–PCR. Results: As in many other types of cells, HIFα was not detectable in ARPE–19 cells that were cultured under normal conditions. However, when proteasome was inhibited, HIFα accumulated in RPE in a time–dependent manner. Consistent with the HIFα accumulation in the cells, levels of VEGF mRNA in RPE cells treated with proteasome inhibitor were up to 6.8–fold higher than those determined in control RPE cells. In contrast to unconditioned–media where VEGF–A165 was negligible, RPE–conditioned media contain as high as 2.5ng/ml VEGF–A165. Inhibition of proteasome activity increased the secretion of VEGF–A165 by 3.5–fold. Conclusions: These data demonstrate that RPE have an active UPP and that HIFα is one of the substrates for this proteolytic pathway. Consequences of impairment of the UPP include accumulation of HIFα and enhanced expression and secretion of VEGF, one of the major factors that contribute to CNV. Since the UPP is vulnerable to oxidative stress, the data indicate that impairment of the UPP by oxidative stress may underlie the molecular mechanism of CNV and AMD.

Keywords: neovascularization • gene/expression • proteolysis