Hypoxia-Inducible Factor Expression in Human RPE Cells
- Farzin Forooghian (farzin.forooghian{at}utoronto.ca),
- Rozita Razavi,
- Lee Timms
- Hospital for Sick Children, Canada
- Hospital for Sick Children, Canada
- Hospital for Sick Children, Canada
- Published Online First 13 June 2007
Abstract
Background: Hypoxia-inducible factor (HIF) is a common transcription factor for many angiogenic proteins. Retinal pigment epithelial (RPE) cells are an important source of angiogenic factors in the retina. The expression of HIF, its' regulation by proline hydroxylase (PHD) enzymes, and its' downstream regulation of angiogenic factors like VEGF and erythropoietin (EPO) was studied in RPE cells in order to delineate some of the molecular mechanisms underlying ischemic retinal disease.
Methods: ARPE-19 cells were cultured for various times under hypoxic conditions. Cellular HIF and PHD isoforms were analyzed and quantified using Western blot and densitometry. VEGF and EPO secreted into the media was assayed using enzyme-linked immunosorbent assay (ELISA). Messenger RNA (mRNA) was quantified using real-time quantitative reverse transcriptase polymerase chain reaction (qPCR). RNA interference was achieved using siRNA techniques.
Results: HIF-1á was readily produced by ARPE-19 cells under hypoxia, but HIF-2α and HIF-3α could not be detected even after HIF-1á silencing. HIF-1α protein levels showed an increasing trend for the first 24 hours while HIF-1α mRNA levels fluctuated during this time. After 36 hours HIF-1α protein levels declined to baseline levels, a change that was coincident with a rise in both PHD2 and PHD3. Silencing of HIF-1α significantly decreased VEGF secretion. Significant production of EPO could not be detected at the protein or mRNA level.
Conclusions: HIF-1α appears to be the main isoform of HIF functioning in ARPE-19 cells. Under hypoxia, HIF-1α levels are likely self-regulated by a feedback loop that involves both transcriptional and post-translational mechanisms. VEGF production by human RPE cells is regulated by HIF-1α. EPO was not produced in significant amounts by RPE cells under hypoxic conditions, suggesting that other cells and/or transcription factors in the retina are responsible for its production.
