Altered Expression of the Iron Transporter, Slc11a1, in the Retinal Pigment Epithelium (RPE) of Microphthalmia-Associated Transcription Factor (Mitf) Mouse Mutants

Abstract

Purpose:: Microphthalmia-associated Transcription Factor (Mitf) is expressed in the RPE during ocular development. Mitf^mi mouse mutants lack pigmentation, and are microphthalmic, while Mitf^vit mouse mutants display abnormal RPE pigmentation, and progressive retinal degeneration. Microarray analysis was used to identify novel downstream gene targets/pathways in the RPE that are altered by mutations in the transcription factor Mitf.

Methods:: Using the Affymetrix platform, gene expression profiles were generated using the eyes of E13.5 mouse fetuses that were wildtype, heterozygous, or homozygous for the Mitf^mi mutation. In a separate experiment, eyes from E13.5 mouse fetuses homozygous for the Mitf^vit mutation were compared to eyes from the C57BL/6 control background strain. Statistical analyses were performed using Robust Multiarray Average, mixed-effects ANOVA and random-variance t-tests, and GENECLUSTER was used to cluster coordinately regulated genes.

Results:: Altered expression of genes involved in melanosome biogenesis/transport, pigment formation, and iron homeostasis were observed. 12 genes were commonly mis-regulated in the eyes of both Mitf mutants: 10 of these genes were downregulated in both mutants, while 2 of the genes (Ephx1 and Slc11a1) were downregulated in Mitf^mi mutants, and conversely, upregulated in Mitf^vit mutants. Quantitative RT-PCR and immunohistochemistry were used to confirm altered gene/protein expression in the RPE.

Conclusions:: Expression of Slc11a1 in the RPE has not previously been reported. Slc11a1 (Nramp1) transports ferrous iron (Fe⁺²), a co-factor utilized in melanin formation. Accumulation of Fe⁺² in the RPE has been associated with oxidative stress and age-related macular degeneration. Increased activity of Slc11a1 leading to iron overload in the RPE may result in oxidative stress and impaired melanogenesis, contributing to the pathology and progressive retinal degeneration observed in Mitf^vit mutants.

Keywords: gene microarray • retinal pigment epithelium • transcription factors