Regulation of retinal endothelial cell apoptosis through activation of the IGFBP-3 receptor.
Regulation of retinal endothelial cell apoptosis through activation of the IGFBP-3 receptor.
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DOI：
10.1007/s10495-012-0793-3
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发表时间：
2013-03
期刊：
影响因子：
7.2
通讯作者：
Steinle, Jena J.
中科院分区：
文献类型：
--
作者：
Zhang, Qiuhua;Soderland, Carl;Steinle, Jena J.
本研究的目的是探讨胰岛素样生长因子结合蛋白-3受体（IGFBP-3 receptor）是否是IGFBP-3抑制视网膜内皮细胞（REC）凋亡所必需的。REC在正常葡萄糖（5 mM）或高葡萄糖培养基（25 mM）中生长3天。一旦细胞达到汇合，将它们用1 μg/ml的内皮特异性IGFBP-3质粒DNA（非IGF结合; IGFBP-3 NB）转染24小时。提取细胞蛋白并通过Western印迹分析IGFBP-3受体表达，或用于免疫共沉淀或共定位实验以检测IGFBP-3和IGFBP-3受体结合。在转染IGFBP-3 NB质粒DNA之前，用或不用IGFBP-3受体siRNA转染REC。将细胞裂解物处理用于细胞死亡ELISA、切割的半胱天冬酶3ELISA和蛋白质印迹以测量关键的促凋亡和抗凋亡标志物：Bcl-xL、Bax、细胞色素C和Akt。REC中存在IGFBP-3受体，REC中IGFBP-3的过表达显著增加IGFBP-3受体的蛋白水平（p < 0.05）。与未处理的样品相比，在用IGFBP-3受体siRNA转染的细胞中发现细胞死亡的显著增加（p < 0.05）。数据表明，IGFBP-3通过在高血糖环境中激活IGFBP-3受体来抑制视网膜内皮细胞死亡。这是IGFBP-3受体参与抑制REC细胞死亡的首次证明。未来的研究将探讨IGFBP-3受体抑制视网膜内皮细胞死亡的机制。
The goal of this study was to investigate whether insulin-like growth factor binding protein-3 receptor (IGFBP-3 receptor) is required for IGFBP-3 to inhibit retinal endothelial cell (REC) apoptosis. REC were grown in normal glucose (5 mM) or high glucose medium (25 mM) for 3 days. Once cells reached confluence, they were transfected with an endothelial- specific IGFBP-3 plasmid DNA (non-IGF binding; IGFBP-3 NB) at 1 μg/ml for 24 h. Cell proteins were extracted and analyzed for IGFBP-3 receptor expression by Western blotting or use in coimmunoprecipitation or co-localization experiments for detection of IGFBP-3 and IGFBP-3 receptor binding. REC were also transfected with or without IGFBP-3 receptor siRNA before IGFBP-3NB plasmid DNA transfection. Cell lysates were processed for a cell death ELISA, a cleaved caspase 3 ELISA, and Western blotting to measure key pro- and anti-apoptotic markers: Bcl-xL, Bax, Cytochrome C and Akt. The IGFBP-3 receptor is present on REC. Overexpression of IGFBP-3 in REC significantly increased protein levels of IGFBP-3 receptor (p < 0.05). Significant increases in cell death were found in cells transfected with IGFBP-3 receptor siRNA versus not treated samples (p < 0.05). Data suggest that IGFBP-3 inhibits retinal endothelial cell death through activation of an IGFBP-3 receptor in a hyperglycemic environment. This is the first demonstration of the involvement of IGFBP-3 receptor in inhibition of REC cell death. Future studies will investigate the mechanism by which IGFBP-3 receptor may inhibit retinal endothelial cell death.
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