Regulation of retinal pigment epithelial cell phenotype by Annexin A8.
Regulation of retinal pigment epithelial cell phenotype by Annexin A8.
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DOI：
10.1038/s41598-017-03493-3
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发表时间：
2017-07-05
影响因子：
4.6
通讯作者：
Moss SE
中科院分区：
文献类型：
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作者：
Lueck K;Carr AF;Stampoulis D;Gerke V;Rescher U;Greenwood J;Moss SE
维甲酸衍生物芬维A胺（FR）能够将培养的视网膜色素上皮（RPE）细胞转分化为神经元样表型，但其潜在机制尚不清楚。为了鉴定参与这一过程的基因，我们对FR处理前后的RPE细胞进行了微阵列分析，并观察到转分化细胞中膜联蛋白A8（AnxA 8）的显著下调。为了确定AnxA 8是否在维持RPE细胞表型中起作用，我们使用siRNA介导的基因抑制和AnxA 8-GFP的过表达结合暴露于FR直接操纵培养的和原代RPE细胞中的AnxA 8表达。用AnxA 8 siRNA处理RPE细胞重现了暴露于FR，伴随细胞周期停滞，神经元转分化，以及伴随的神经元标记物钙视网膜蛋白和钙结合蛋白的上调，如通过实时PCR和免疫荧光所评估的。相反，ARPE-19细胞中AnxA 8的瞬时过表达阻止了FR诱导的分化。AnxA 8缺失细胞中AnxA 8的异位表达导致神经元标记物染色减少，并且通过磷酸组蛋白H3染色、细胞计数和切割的半胱天冬酶-3水平判断细胞生长正常。这些数据表明，下调AnxA 8对于RPE细胞的神经元转分化是必要的和充分的，并揭示了AnxA 8作为RPE表型的关键调节因子的重要作用。
The retinoic acid derivative fenretinide (FR) is capable of transdifferentiating cultured retinal pigment epithelial (RPE) cells towards a neuronal-like phenotype, but the underlying mechanisms are not understood. To identify genes involved in this process we performed a microarray analysis of RPE cells pre- and post-FR treatment, and observed a marked down-regulation of AnnexinA8 (AnxA8) in transdifferentiated cells. To determine whether AnxA8 plays a role in maintaining RPE cell phenotype we directly manipulated AnxA8 expression in cultured and primary RPE cells using siRNA-mediated gene suppression, and over-expression of AnxA8-GFP in conjunction with exposure to FR. Treatment of RPE cells with AnxA8 siRNA recapitulated exposure to FR, with cell cycle arrest, neuronal transdifferentiation, and concomitant up-regulation of the neuronal markers calretinin and calbindin, as assessed by real-time PCR and immunofluorescence. In contrast, AnxA8 transient over-expression in ARPE-19 cells prevented FR-induced differentiation. Ectopic expression of AnxA8 in AnxA8-depleted cells led to decreased neuronal marker staining, and normal cell growth as judged by phosphohistone H3 staining, cell counting and cleaved caspase-3 levels. These data show that down-regulation of AnxA8 is both necessary and sufficient for neuronal transdifferentiation of RPE cells and reveal an essential role for AnxA8 as a key regulator of RPE phenotype.