IGF-1-induced VEGF and IGFBP-3 secretion correlates with increased HIF-1 alpha expression and activity in retinal pigment epithelial cell line D407

Invest Ophthalmol Vis Sci. 2004 Aug;45(8):2838-47. doi: 10.1167/iovs.03-0565.


Purpose: To examine insulin-like growth factor (IGF)-1 stimulation of expression of hypoxia inducible factor (HIF)-1 alpha and secretion of vascular endothelial growth factor (VEGF) and IGF binding protein (IGFBP)-3 in human retinal pigment epithelial (RPE) cell line D407.

Methods: D407 cells cultured in dishes or Transwell inserts were treated with cobalt chloride or varying doses of IGF-1. Whole cell lysates were assayed by immunoblot for HIF-1 alpha expression, whereas conditioned medium was TCA precipitated and assayed by immunoblot for VEGF and ligand blot for IGFBP-3. Cells grown on coverslips were similarly treated and probed with antibodies to HIF-1 alpha, VEGF, and IGFBP-3, and visualized by epifluorescence microscopy. Cells grown on Transwell inserts were probed with antibodies to the Na(+)/K(+)-ATPase alpha-1 subunit and either the alpha or beta subunits of the IGF-1 receptor and visualized in Z-section using confocal microscopy.

Results: Immunoblot analysis of whole cell lysates from IGF-1-treated D407 cells revealed the upregulation of HIF-1 alpha protein. Epifluorescence microscopy demonstrated a positive correlation between HIF-1 alpha expression and nuclear localization, VEGF and IGFBP-3 synthesis and export, and IGF-1 action. Western and ligand blot analyses of RPE cell-conditioned medium indicated that IGF-1 induced increases in VEGF and IGFBP-3 secretion. Cells grown on Transwell inserts exhibited constitutive apical secretion of VEGF and IGFBP-3, which increased on apical or basolateral treatment with IGF-1. Confocal analysis of Transwell-cultured D407 cells confirmed the apical localization of the Na(+)/K(+)-ATPase alpha-1 subunit, characteristic of polarized RPE, with IGF-1 receptor alpha and beta subunits exhibiting a nonpolarized distribution.

Conclusions: IGF-1 stimulates increased HIF-1 alpha expression as well as VEGF and IGFBP-3 secretion in D407 cells. Similar to their in vivo counterparts, D407 cells maintain reversed epithelial polarity. Apical secretion of VEGF and IGFBP-3 increases in response to either apical or basolateral IGF-1 stimulation consistent with the nonpolarized distribution of IGF-1 receptors.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Cell Line
  • Cell Polarity
  • Humans
  • Hypoxia-Inducible Factor 1, alpha Subunit
  • Immunoblotting
  • Insulin-Like Growth Factor Binding Protein 3 / metabolism*
  • Insulin-Like Growth Factor I / pharmacology*
  • Microscopy, Fluorescence
  • Pigment Epithelium of Eye / drug effects*
  • Pigment Epithelium of Eye / metabolism
  • Sodium-Potassium-Exchanging ATPase / metabolism
  • Transcription Factors / metabolism*
  • Up-Regulation
  • Vascular Endothelial Growth Factor A / metabolism*


  • HIF1A protein, human
  • Hypoxia-Inducible Factor 1, alpha Subunit
  • Insulin-Like Growth Factor Binding Protein 3
  • Transcription Factors
  • VEGFA protein, human
  • Vascular Endothelial Growth Factor A
  • Insulin-Like Growth Factor I
  • Sodium-Potassium-Exchanging ATPase