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Mark G. Slomiany, Steven A. Rosenzweig; IGF-1–Induced VEGF and IGFBP-3 Secretion Correlates with Increased HIF-1α Expression and Activity in Retinal Pigment Epithelial Cell Line D407. Invest. Ophthalmol. Vis. Sci. 2004;45(8):2838-2847. doi: 10.1167/iovs.03-0565.
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purpose. To examine insulin-like growth factor (IGF)-1 stimulation of expression of hypoxia inducible factor (HIF)-1α 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-1α 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-1α, VEGF, and IGFBP-3, and visualized by epifluorescence microscopy. Cells grown on Transwell inserts were probed with antibodies to the Na+/K+-ATPase α-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-1α protein. Epifluorescence microscopy demonstrated a positive correlation between HIF-1α 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 α-1 subunit, characteristic of polarized RPE, with IGF-1 receptor α and β subunits exhibiting a nonpolarized distribution.
conclusions. IGF-1 stimulates increased HIF-1α 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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