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J. Gnana Prakasam, M. Thangaraju, P. M. Martin, V. Ganapathy; Absence of Iron-regulatory Protein Hfe Results in Hyperproliferation of Retinal Pigment Epithelium Possibly Through Induction of Cystine/glutamate Transporter. Invest. Ophthalmol. Vis. Sci. 2009;50(13):2914.
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Hemochromatosis (HH) is an autosomal disorder of iron overload. Functional loss of HFE, (Histocompatability leukocyte antigen class I-like protein involved in iron (FE) homeostasis), due to a mutation in HFE is responsible for ~ 90% of cases with HH. We recently demonstrated the expression of HFE in the basal membrane of RPE. To analyze further the role of HFE in retina, we isolated RPE cells from HFE knockout mice and checked for biochemical changes and differential gene expression.
The rate of cell proliferation was analyzed using Giemsa staining, thymidine incorporation, and MTT assay. Microarray analysis was done to examine the gene expression pattern. RT-PCR and functional assays were done to monitor the expression of cystine/glutamate exchanger (a heterodimeric amino acid transporter consisting of two subunits, xCT and 4F2hc). Intracellular glutathione levels were measured using a glutathione assay kit.
Giemsa staining indicated that HFE-null primary RPE cells proliferated faster than wild type RPE cells. Thymidine incorporation and MTT assay also confirmed this finding. Microarray analysis showed that many cell cycle-related genes were differentially regulated in HFE-null primary RPE cells. One of the genes upregulated in HFE-null RPE cells was cystine/glutamate exchanger, which is responsible for maintenance of cellular levels of glutathione. We confirmed the microarrray data by monitoring xCT and 4F2hc mRNA levels and also by measuring transport function. We also found increased levels of glutathione in HFE-null RPE cells.
HFE expression in the basal membrane of RPE, which is in contact with blood, indicates its essential role in retinal iron homeostasis. HFE mutations have already been attributed to many forms of cancer. The present study indicating hyperproliferation of primary RPE from HFE-null mouse retina confirms the vital role played by HFE in maintaining retinal health. Increased glutathione levels due to overexpression of cystine/glutamate exchanger may facilitate cell cycle progression in HFE-null RPE cells.
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