Abstract
Purpose:
Complement dysregulation is strongly implicated in the pathogenesis of AMD. We sought to determine whether C3 might be produced and activated locally in the RPE. Our lab has previously shown that mice carrying mutations causing retinal iron overload have increased RPE C3 mRNA levels and activated C3 deposition in Bruch’s membrane. This work aims to probe the molecular mechanism regulating RPE C3 production in the context of iron accumulation and oxidative stress.
Methods:
Immunolabeling for C3 was performed in retinas from mice with RPE-specific conditional knockout of the iron transporter hephaestin. ARPE-19 cells were differentiated for one month and then treated with iron for up to 48h. RNA was extracted and studied by microarray and qPCR. Protein was extracted and assessed by protein antibody array and Western blotting. Inhibitor and knockdown studies were performed to study the role of ERK1/2 and SMAD3 in the iron signaling pathway that promotes C3 expression. The C3 promoter was cloned into the luciferase vectors to define regions necessary for iron-induced transcriptional up-regulation. Cell culture supernatants were used in ELISAs to detect activated C3 and activated Factor B in the alternative pathway.
Results:
Immunolabeling in mice with a mosaic pattern of RPE iron overload showed that iron accumulation corresponded to C3 deposition. For cultured RPE cells exposed to iron, DAVID analysis of the microarray data showed that the TGF-β pathway was among the most strongly affected biological processes occurring concurrently with increased C3 expression. Pharmacologic inhibitor and knockdown experiments implicated a non-canonical TGF-β signaling pathway, specifically cross-talk between ERK1/2 and SMAD3, triggered by iron. Luciferase assays showed transcriptional up-regulation by iron within a 500bp region of the proximal C3 promoter. In parallel, ELISAs revealed an iron dose-dependent increase in C3 protein expression, C3a production, and Factor Ba formation, a marker of alternative pathway activation.
Conclusions:
Our data show that iron can up-regulate RPE C3 expression through a non-canonical TGF-β signaling pathway involving ERK1/2 and SMAD3. The molecular events in this pathway may represent therapeutic targets for AMD or other retinal diseases exacerbated by ocular complement expression.