Iron-induced Local Complement Component 3 (C3) Up-regulation via Non-canonical Transforming Growth Factor (TGF)-β Signaling in the Retinal Pigment Epithelium

J Biol Chem. 2015 May 8;290(19):11918-34. doi: 10.1074/jbc.M115.645903. Epub 2015 Mar 23.


Dysregulation of iron homeostasis may be a pathogenic factor in age-related macular degeneration (AMD). Meanwhile, the formation of complement-containing deposits under the retinal pigment epithelial (RPE) cell layer is a pathognomonic feature of AMD. In this study, we investigated the molecular mechanisms by which complement component 3 (C3), a central protein in the complement cascade, is up-regulated by iron in RPE cells. Modulation of TGF-β signaling, involving ERK1/2, SMAD3, and CCAAT/enhancer-binding protein-δ, is responsible for iron-induced C3 expression. The differential effects of spatially distinct SMAD3 phosphorylation sites at the linker region and at the C terminus determined the up-regulation of C3. Pharmacologic inhibition of either ERK1/2 or SMAD3 phosphorylation decreased iron-induced C3 expression levels. Knockdown of SMAD3 blocked the iron-induced up-regulation and nuclear accumulation of CCAAT/enhancer-binding protein-δ, a transcription factor that has been shown previously to bind the basic leucine zipper 1 domain in the C3 promoter. We show herein that mutation of this domain reduced iron-induced C3 promoter activity. In vivo studies support our in vitro finding of iron-induced C3 up-regulation. Mice with a mosaic pattern of RPE-specific iron overload demonstrated co-localization of iron-induced ferritin and C3d deposits. Humans with aceruloplasminemia causing RPE iron overload had increased RPE C3d deposition. The molecular events in the iron-C3 pathway represent therapeutic targets for AMD or other diseases exacerbated by iron-induced local complement dysregulation.

Keywords: AMD; C3; CCAAT/Enhancer-binding Protein (C/EBP); Extracellular Signal-regulated Kinase (ERK); Iron; RPE; SMAD3; Signaling; Transforming Growth Factor β (TGF-β).

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • CCAAT-Enhancer-Binding Protein-delta / metabolism
  • Cell Line
  • Complement C3 / metabolism*
  • Gene Expression Regulation
  • Gene Knockdown Techniques
  • Humans
  • Iron / chemistry
  • Iron Overload
  • Ligands
  • Macular Degeneration / metabolism*
  • Male
  • Mice
  • Mice, Inbred C57BL
  • Mice, Transgenic
  • Middle Aged
  • Retinal Pigment Epithelium / metabolism*
  • Signal Transduction
  • Smad3 Protein / metabolism
  • Transforming Growth Factor beta1 / metabolism*
  • Up-Regulation


  • Complement C3
  • Ligands
  • SMAD3 protein, human
  • Smad3 Protein
  • Smad3 protein, mouse
  • TGFB1 protein, human
  • Tgfb1 protein, mouse
  • Transforming Growth Factor beta1
  • CCAAT-Enhancer-Binding Protein-delta
  • Iron