| IGF-1-induced VEGF and IGFBP-3 secretion correlates with increased HIF-1 alpha expression and activity in retinal pigment epithelial cell line D407. | |
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MedLine Citation:
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PMID: 15277511 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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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. |
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Authors:
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Mark G Slomiany; Steven A Rosenzweig |
Publication Detail:
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Type: Journal Article; Research Support, U.S. Gov't, Non-P.H.S.; Research Support, U.S. Gov't, P.H.S. |
Journal Detail:
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Title: Investigative ophthalmology & visual science Volume: 45 ISSN: 0146-0404 ISO Abbreviation: Invest. Ophthalmol. Vis. Sci. Publication Date: 2004 Aug |
Date Detail:
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Created Date: 2004-07-27 Completed Date: 2004-08-31 Revised Date: 2007-11-15 |
Medline Journal Info:
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Nlm Unique ID: 7703701 Medline TA: Invest Ophthalmol Vis Sci Country: United States |
Other Details:
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Languages: eng Pagination: 2838-47 Citation Subset: IM |
Affiliation:
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Department of Cell and Molecular Pharmacology and Experimental Therapeutics and The Hollings Cancer Center, Medical University of South Carolina, Charleston, 29425, USA. |
Export Citation:
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| MeSH Terms | |
Descriptor/Qualifier:
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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*, metabolism Sodium-Potassium-Exchanging ATPase / metabolism Transcription Factors / metabolism* Up-Regulation Vascular Endothelial Growth Factor A / metabolism* |
| Grant Support | |
ID/Acronym/Agency:
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CA78887/CA/NCI NIH HHS |
| Chemical | |
Reg. No./Substance:
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0/HIF1A protein, human; 0/Hypoxia-Inducible Factor 1, alpha Subunit; 0/Insulin-Like Growth Factor Binding Protein 3; 0/Transcription Factors; 0/VEGFA protein, human; 0/Vascular Endothelial Growth Factor A; 67763-96-6/Insulin-Like Growth Factor I; EC 3.6.3.9/Sodium-Potassium-Exchanging ATPase |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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