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Expression and Activity in Retinal Pigment Epithelial Cell Line D407
From the Department of Cell and Molecular Pharmacology and Experimental Therapeutics and The Hollings Cancer Center, Medical University of South Carolina, Charleston, South Carolina.
PURPOSE. To examine insulin-like growth factor (IGF)-1 stimulation of expression of hypoxia inducible factor (HIF)-1
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 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, VEGF, and IGFBP-3, and visualized by epifluorescence microscopy. Cells grown on Transwell inserts were probed with antibodies to the Na+/K+-ATPase -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 protein. Epifluorescence microscopy demonstrated a positive correlation between HIF-1 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 -1 subunit, characteristic of polarized RPE, with IGF-1 receptor and ß subunits exhibiting a nonpolarized distribution.
CONCLUSIONS. IGF-1 stimulates increased HIF-1 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.
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