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M. McGahan, J. Ferrell, L. N. Fleisher, S. Nagar, M. M. Lall, J. Harned; Proteins Integral to Iron Metabolism are Regulated by Hypoxia Inducible Factor-1 in Retinal Pigmented Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3970. doi: https://doi.org/.
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Hypoxia-inducible factor 1 (HIF-1) is a transcription factor regulating dozens of genes, including vascular endothelial growth factor (VEGF) and many genes involved in iron metabolism. In turn, HIF-1 activity is regulated by iron availability, with a decrease in iron availability causing an increase in HIF-1 activity. HIF-1 plays important roles in retinal ischemia and other retinal pathologies by increasing the production of VEGF and other proteins. Significantly, iron metabolism is dysregulated in retinal degenerative diseases. Therefore, the purpose of this study was to determine how a decrease in HIF-1 activity, using siRNA knockdown, affects the levels of heme oxygenase-1 (HO-1),the iron transport protein, transferrin and the assembly of the iron storage protein, ferritin. In addition, the effects of iron chelation on HIF-1 activity were measured.
Primary cultures of RPE cells were prepared from canine eyes obtained from dogs euthanized at the Johnston County, NC pound. siRNA for the essential HIF-1 subunit HIF-1a was obtained from Dharmacon, Lafayette, CO. Measurements were made 48h after transfection. HIF-1 activity was determined using a luciferase reporter gene assay (Panomics). VEGF and transferrin were measured in the cell-conditioned medium by ELISA. HO-1 levels were measured in cell lysates by ELISA. Synthesis of ferritin subunits was determined by metabolic labeling followed by gel electrophoresis of proteins from the RPE cytosol.
Transfection of the RPE cells with siRNA for HIF-1a resulted in a significant decrease in HIF-1 activity as determined indirectly by a 35% decrease in VEGF secretion into the medium. In addition, treatment of RPE cells with the iron chelator dipyridyl almost doubled (191% of control) HIF-1 activity as measured in the luciferase assay. siRNA knockdown of HIF-1 activity significantly altered transferrin secretion (18% decrease) into the media, HO-1 levels in RPE (28% decrease) and ferritin subunit assembly. All of these changes likely have significant downstream effects on iron metabolism, uptake and storage in RPE cells.
Clearly there is a significant interaction between regulation of HIF-1 activity by iron and the reciprocal regulation of iron metabolism by HIF-1 in RPE cells. A fuller understanding of this complex relationship is necessary in order to determine the pathophysiological connection between iron dysregulation and HIF-1 activity in retinal disease.
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