Slomiany Mark G, Rosenzweig Steven A
Department of Cell and Molecular Pharmacology and Experimental Therapeutics and The Hollings Cancer Center, Medical University of South Carolina, Charleston, 29425, USA.
Invest Ophthalmol Vis Sci. 2004 Aug;45(8):2838-47. doi: 10.1167/iovs.03-0565.
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.
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.
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.
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.
研究胰岛素样生长因子（IGF）-1对人视网膜色素上皮（RPE）细胞系D407中缺氧诱导因子（HIF）-1α表达、血管内皮生长因子（VEGF）分泌及IGF结合蛋白（IGFBP）-3分泌的刺激作用。
将培养在培养皿或Transwell小室中的D407细胞用氯化钴或不同剂量的IGF-1处理。全细胞裂解物通过免疫印迹法检测HIF-1α表达，而条件培养基经三氯乙酸沉淀后通过免疫印迹法检测VEGF，通过配体印迹法检测IGFBP-3。在盖玻片上生长的细胞进行类似处理，并用抗HIF-1α、VEGF和IGFBP-3抗体进行探测，通过落射荧光显微镜观察。在Transwell小室中生长的细胞用抗Na(+)/K(+)-ATP酶α-1亚基以及IGF-1受体的α或β亚基的抗体进行探测，并使用共聚焦显微镜在Z轴切片中观察。
对经IGF-1处理的D407细胞的全细胞裂解物进行免疫印迹分析显示HIF-1α蛋白上调。落射荧光显微镜显示HIF-1α表达与核定位、VEGF和IGFBP-3合成及分泌以及IGF-1作用之间呈正相关。对RPE细胞条件培养基的蛋白质免疫印迹和配体印迹分析表明，IGF-1诱导VEGF和IGFBP-3分泌增加。在Transwell小室中生长的细胞表现出VEGF和IGFBP-3的组成性顶端分泌，在用IGF-1进行顶端或基底外侧处理后增加。对Transwell培养的D407细胞的共聚焦分析证实了Na(+)/K(+)-ATP酶α-1亚基的顶端定位，这是极化RPE的特征，而IGF-1受体的α和β亚基表现出非极化分布。
IGF-1刺激D407细胞中HIF-像其体内对应细胞一样，D407细胞保持反转的上皮极性。VEGF和IGFBP-3的顶端分泌在顶端或基底外侧IGF-1刺激下增加，这与IGF-1受体的非极化分布一致。 1α表达以及VEGF和IGFBP-3分泌增加。
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