Regulated secretion of complement factor H by RPE and its role in RPE migration
Regulated secretion of complement factor H by RPE and its role in RPE migration
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DOI：
10.1007/s00417-009-1049-y
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发表时间：
2009-05-01
影响因子：
2.7
通讯作者：
Hinton, David R.
中科院分区：
文献类型：
--
作者：
Kim, Yeong Hoon;He, Shikun;Hinton, David R.
补体因子 H (CFH) 基因的变异被认为是导致年龄相关性黄斑变性 (AMD) 的主要危险因素。然而，关于视网膜色素上皮细胞 (RPE) 调节 CFH 局部表达和分泌的因素知之甚少。培养的人早期传代 RPE 细胞、高度分化、极化的人 RPE 培养物和牛 RPE 外植体在存在或不存在重组人或牛干扰素-γ (IFN-γ；25 ng/ml) 的情况下孵育。通过蛋白质印迹检查细胞裂解物中的 CFH 表达以及分泌到培养物上清液中。通过共聚焦显微镜分析 CHF 表达和定位。在改良的 Boyden 室中，用重组 CFH 蛋白 (1-100 ng/ml) 刺激后的早期传代人 RPE 细胞进行迁移测定。CFH 在 RPE 细胞的细胞裂解物中表达，并且该表达被 IFN-γ 显着上调。在牛外植体的 RPE 细胞和高度分化的人 RPE 单层细胞中检测到 CFH 的免疫反应性，并且在 IFN-γ 刺激后免疫反应性水平增加。共聚焦显微镜显示 CFH 主要位于极化的人 RPE 的顶端细胞质中。 Western blot 证实 IFN-γ 增加了 CFH 分泌到 RPE 上清液中。 CFH诱导RPE细胞呈剂量依赖性趋化性迁移。IFN-γ促进RPE细胞顶端区室中CFH的表达，并增加CFH向RPE培养上清液中的分泌。此外，CFH 促进 RPE 的趋化迁移。这项研究表明 CFH 和 IFN-γ 之间的相互作用有可能在 AMD 的发病机制中发挥作用。
Variants in the gene for complement factor H (CFH) have been implicated as a major risk factor for the development of age-related macular degeneration (AMD). Little is known, however, about the factors regulating local expression and secretion of CFH by retinal pigment epithelial cells (RPE).Cultured human early passage RPE cells, highly differentiated, polarized human RPE cultures, and bovine RPE explants were incubated in the presence or absence of recombinant human or bovine interferon-gamma (IFN-gamma; 25 ng/ml). CFH expression in cell lysates, and secretion into culture supernatants were examined by Western blot. CHF expression and localization was analyzed by confocal microscopy. Migration assay was performed in a modified Boyden chamber with early passage human RPE cells after stimulation with recombinant CFH protein (1-100 ng/ml).CFH was expressed in the cell lysates of RPE cells, and this expression was significantly upregulated by IFN-gamma. Immunoreactivity for CFH was detected in RPE cells of bovine explants and highly differentiated human RPE monolayers, and the level of immunoreactivity increased after IFN-gamma stimulation. Confocal microscopy revealed that CFH was predominantly localized in the apical cytoplasm of polarized human RPE. Western blot confirmed that IFN-gamma increased CFH secretion into RPE supernatants. Dose-dependent RPE cell chemotactic migration was induced by CFH.IFN-gamma promotes CFH expression in the apical compartment of RPE cells and increases secretion of CFH into RPE culture supernatants. Furthermore, CFH promotes chemotactic migration of RPE. This study suggests that interactions between CFH and IFN-gamma have the potential to play a role in the pathogenesis of AMD.