Hypoxia-inducible factor expression in human RPE cells.
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 vascular endothelial growth factor (VEGF) and erythropoietin (EPO) was studied in RPE cells in order to determine some of the molecular mechanisms underlying ischaemic retinal disease.


METHODS
ARPE-19 cells were cultured for various times under hypoxic conditions. Cellular HIF and PHD isoforms were analysed and quantified using western blot and densitometry. VEGF and EPO secreted into the media were 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-1 alpha was readily produced by ARPE-19 cells under hypoxia, but HIF-2 alpha and HIF-3 alpha could not be detected even after HIF-1 alpha silencing. HIF-1 alpha protein levels showed an increasing trend for the first 24 h while HIF-1 alpha mRNA levels fluctuated during this time. After 36 h HIF-1 alpha protein levels declined to baseline levels, a change that was coincident with a rise in both PHD2 and PHD3. Silencing HIF-1 alpha significantly decreased VEGF secretion. Significant production of EPO could not be detected at the protein or mRNA level.


CONCLUSIONS
HIF-1 alpha appears to be the main isoform of HIF functioning in ARPE-19 cells. Under hypoxia, HIF-1 alpha 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-1 alpha. 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.
Forooghian
Farzin
F
Department of Ophthalmology and Vision Sciences and Institute of Medical Sciences, University of Toronto, Toronto, ON, Canada. farzin.forooghian@utoronto.ca
Razavi
Rozita
R
Timms
Lee
L
D002478:Cells, Cultured; D004921:Erythropoietin / Q000032:analysis; D005786:Gene Expression Regulation / Q000235:genetics; D006801:Humans; D000860:Hypoxia / Q000235:genetics / Q000378:metabolism; D051793:Hypoxia-Inducible Factor 1 / Q000032:analysis* / Q000235:genetics; D007536:Isomerism; D010857:Pigment Epithelium of Eye / Q000737:chemistry*; D011393:Procollagen-Proline Dioxygenase / Q000032:analysis; D012333:RNA, Messenger / Q000032:analysis; D042461:Vascular Endothelial Growth Factor A / Q000032:analysis
D051793:Hypoxia-Inducible Factor 1; D012333:RNA, Messenger; D042461:Vascular Endothelial Growth Factor A; D004921:Erythropoietin; D011393:Procollagen-Proline Dioxygenase