Absence of iron-regulatory protein Hfe results in hyperproliferation of retinal pigment epithelium: role of cystine/glutamate exchanger.

Haemochromatosis is an iron-overload disorder with age-dependent oxidative stress and dysfunction in a variety of tissues. Mutations in HFE (histocompatability leucocyte antigen class I-like protein involved in iron homoeostasis) are responsible for most cases of haemochromatosis. We demonstrated recently that HFE is expressed exclusively in the basal membrane of RPE (retinal pigment epithelium). In the present study, we used Hfe-/- mice to examine ferritin levels (an indirect readout for iron levels) and morphological changes in retina. We found increased ferritin accumulation in retina in 18-month-old, but not in 2-month-old, mice with considerable morphological damage compared with age-matched controls. The retinal phenotype included hypertrophy and hyperplasia of RPE. RPE cells isolated from Hfe-/- mice exhibited a hyperproliferative phenotype. We also compared the gene expression profile between wild-type and Hfe-/- RPE cells by microarray analysis. These studies showed that many cell cycle-related genes were differentially regulated in Hfe-/- RPE cells. One of the genes up-regulated in Hfe-/- RPE cells was Slc7a11 (where Slc is solute carrier) which codes for the 'transporter proper' xCT in the heterodimeric cystine/glutamate exchanger (xCT/4F2hc). This transporter plays a critical role in cellular glutathione status and cell-cycle progression. We confirmed the microarrray data by monitoring xCT mRNA levels by RT (reverse transcription)-PCR and also by measuring transport function. We also found increased levels of glutathione and the transcription factor/cell-cycle promoter AP1 (activator protein 1) in Hfe-/- RPE cells. Wild-type mouse RPE cells and human RPE cell lines, when loaded with iron by exposure to ferric ammonium citrate, showed increased expression and activity of xCT, reproducing the biochemical phenotype observed with Hfe-/- RPE cells.