Chiang Cheng-Kang, Tworak Aleksander, Kevany Brian M, Xu Bo, Mayne Janice, Ning Zhibin, Figeys Daniel, Palczewski Krzysztof
From the Ottawa Institute of Systems Biology and Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, Ontario K1H 8M5, Canada.
the Department of Chemistry, National Dong Hwa University, No. 1 Sec. 2 Da Hsueh Road, Shoufeng, Hualien 97401, Taiwan.
J Biol Chem. 2017 Dec 1;292(48):19826-19839. doi: 10.1074/jbc.M117.812677. Epub 2017 Oct 4.
One of the major biological functions of the retinal pigmented epithelium (RPE) is the clearance of shed photoreceptor outer segments (POS) through a multistep process resembling phagocytosis. RPE phagocytosis helps maintain the viability of photoreceptors that otherwise could succumb to the high metabolic flux and photo-oxidative stress associated with visual processing. The regulatory mechanisms underlying phagocytosis in the RPE are not fully understood, although dysfunction of this process contributes to the pathogenesis of multiple human retinal degenerative disorders, including age-related macular degeneration. Here, we present an integrated transcriptomic, proteomic, and phosphoproteomic analysis of phagocytosing RPE cells, utilizing three different experimental models: the human-derived RPE-like cell line ARPE-19, cultured murine primary RPE cells, and RPE samples from live mice. Our combined results indicated that early stages of phagocytosis in the RPE are mainly characterized by pronounced changes in the protein phosphorylation level. Global phosphoprotein enrichment analysis revealed involvement of PI3K/Akt, mechanistic target of rapamycin (mTOR), and MEK/ERK pathways in the regulation of RPE phagocytosis, confirmed by immunoblot analyses and phagocytosis assays. Most strikingly, phagocytosis of POS by cultured RPE cells was almost completely blocked by pharmacological inhibition of phosphorylation of Akt. Our findings, along with those of previous studies, indicate that these phosphorylation events allow the RPE to integrate multiple signals instigated by shed POS at different stages of the phagocytic process.
视网膜色素上皮（RPE）的主要生物学功能之一是通过类似于吞噬作用的多步骤过程清除脱落的光感受器外段（POS）。RPE吞噬作用有助于维持光感受器的活力，否则光感受器可能会因与视觉处理相关的高代谢通量和光氧化应激而受损。尽管这一过程的功能障碍会导致包括年龄相关性黄斑变性在内的多种人类视网膜退行性疾病的发病机制，但RPE吞噬作用的调控机制尚未完全了解。在这里，我们利用三种不同的实验模型：人源RPE样细胞系ARPE-19、培养的小鼠原代RPE细胞和活体小鼠的RPE样本，对吞噬RPE细胞进行了转录组学、蛋白质组学和磷酸蛋白质组学的综合分析。我们的综合结果表明，RPE吞噬作用的早期阶段主要表现为蛋白质磷酸化水平的显著变化。全局磷酸化蛋白质富集分析显示，PI3K/Akt、雷帕霉素机制靶点（mTOR）和MEK/ERK信号通路参与了RPE吞噬作用的调控，免疫印迹分析和吞噬作用试验证实了这一点。最引人注目的是，培养的RPE细胞对POS的吞噬作用几乎完全被Akt磷酸化的药理学抑制所阻断。我们的研究结果与之前的研究结果表明，这些磷酸化事件使RPE能够整合吞噬过程不同阶段脱落的POS引发的多种信号。
