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Yafeng Li, Ying Song, Delu Song, Liangliang Zhao, Milan Theurl, Joshua L Dunaief; Signaling Mechanism of Iron-induced Complement Component 3 (C3) Expression and Activation in the Retinal Pigment Epithelium (RPE). Invest. Ophthalmol. Vis. Sci. 2014;55(13):76.
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Retinal iron accumulation and complement dysregulation are implicated in the pathogenesis of age-related macular degeneration (AMD). To assess the impact of iron overload on retinal pathophysiology, we performed microarray analysis of gene expression changes following addition of iron to cultured RPE cells. Interestingly, one of the top “hits” was C3. This work aims to better understand the molecular link between iron accumulation and C3 dysregulation in the context of AMD.
In a model of iron overload in the retina, ARPE-19 cells were treated with ferric salt for time points up to 48h. RNA was extracted, reverse transcribed, and followed by microarray and qPCR studies. Protein was also extracted, followed by a protein antibody array and Western analyses. The C3 promoter cloned into the pGL3 vector was used to determine iron-induced transcriptional activity in a luciferase assay. Intravitreal injections of low dose ferric salt in murine eyes and an equimolar control were performed. C3 mRNA levels were assessed in the RPE of ceruloplasmin/hephaestin double knockout mice.
In cultured RPE cells exposed to iron, activation of some members of the TGF-β superfamily of ligands and downstream effectors, including BMP4, occurred in conjunction with a strong increase in C3 expression. The signaling pathway involved cross-talk with MAPK pathways, as determined by inhibitor and knockdown studies. Western analysis detected activated C3 cleavage products, even when cells were grown in serum-free medium. The iron-responsive portion of the C3 promoter was defined using a luciferase assay. Murine intravitreal injections of low doses of iron resulted in localized abnormalities on retinal imaging after 2 or 4 days and featured RPE and photoreceptor degeneration. Isolated RPE cells from these animals demonstrated an increase in C3 mRNA levels, as did RPE cells from ceruloplasmin/hephaestin double knockout mice, which have increased RPE iron.
Iron appears to up-regulate expression of C3 within the RPE cell, as well as proteases that cleave C3. This iron-induced local production of complement cascade components within the RPE may contribute to pathologic complement activation and deposition in the retinas of AMD patients. Analysis of the detailed molecular pathway may identify therapeutic targets by which RPE pathophysiology in AMD could be regulated.
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