Iejima Daisuke, Itabashi Takeshi, Kawamura Yuich, Noda Toru, Yuasa Shinsuke, Fukuda Keiichi, Oka Chio, Iwata Takeshi
From the Division of Molecular and Cellular Biology, National Institute of Sensory Organs, and.
the Division of Ophthalmology, National Hospital Organization Tokyo Medical Center, Tokyo 152-8902, Japan.
J Biol Chem. 2015 Jan 30;290(5):2784-97. doi: 10.1074/jbc.M114.593384. Epub 2014 Dec 17.
Dry age-related macular degeneration (AMD) accounts for over 85% of AMD cases in the United States, whereas Japanese AMD patients predominantly progress to wet AMD or polypoidal choroidal vasculopathy. Recent genome-wide association studies have revealed a strong association between AMD and an insertion/deletion sequence between the ARMS2 (age-related maculopathy susceptibility 2) and HTRA1 (high temperature requirement A serine peptidase 1) genes. Transcription regulator activity was localized in mouse retinas using heterozygous HtrA1 knock-out mice in which HtrA1 exon 1 was replaced with β-galactosidase cDNA, thereby resulting in dominant expression of the photoreceptors. The insertion/deletion sequence significantly induced HTRA1 transcription regulator activity in photoreceptor cell lines but not in retinal pigmented epithelium or other cell types. A deletion construct of the HTRA1 regulatory region indicated that potential transcriptional suppressors and activators surround the insertion/deletion sequence. Ten double-stranded DNA probes for this region were designed, three of which interacted with nuclear extracts from 661W cells in EMSA. Liquid chromatography-mass spectrometry (LC-MS/MS) of these EMSA bands subsequently identified a protein that bound the insertion/deletion sequence, LYRIC (lysine-rich CEACAM1 co-isolated) protein. In addition, induced pluripotent stem cells from wet AMD patients carrying the insertion/deletion sequence showed significant up-regulation of the HTRA1 transcript compared with controls. These data suggest that the insertion/deletion sequence alters the suppressor and activator cis-elements of HTRA1 and triggers sustained up-regulation of HTRA1. These results are consistent with a transgenic mouse model that ubiquitously overexpresses HtrA1 and exhibits characteristics similar to those of wet AMD patients.
在美国，干性年龄相关性黄斑变性（AMD）占AMD病例的85%以上，而日本AMD患者主要进展为湿性AMD或息肉状脉络膜血管病变。最近的全基因组关联研究表明，AMD与ARMS2（年龄相关性黄斑病变易感性2）和HTRA1（高温需求A丝氨酸蛋白酶1）基因之间的插入/缺失序列存在强关联。利用杂合型HtrA1基因敲除小鼠（其中HtrA1外显子1被β-半乳糖苷酶cDNA取代，从而导致光感受器的显性表达），将转录调节活性定位在小鼠视网膜中。插入/缺失序列在光感受器细胞系中显著诱导HTRA1转录调节活性，但在视网膜色素上皮或其他细胞类型中则不然。HTRA1调节区域的缺失构建体表明，潜在的转录抑制因子和激活因子围绕着插入/缺失序列。针对该区域设计了10个双链DNA探针，其中3个在电泳迁移率变动分析（EMSA）中与661W细胞的核提取物相互作用。随后，对这些EMSA条带进行液相色谱-质谱联用（LC-MS/MS）分析，鉴定出一种与插入/缺失序列结合的蛋白质，即富含赖氨酸的癌胚抗原相关细胞黏附分子1共分离蛋白（LYRIC）。此外，与对照组相比，携带插入/缺失序列的湿性AMD患者的诱导多能干细胞显示HTRA1转录本显著上调。这些数据表明，插入/缺失序列改变了HTRA1的抑制因子和激活因子顺式元件，并触发了HTRA1的持续上调。这些结果与一个普遍过表达HtrA1并表现出与湿性AMD患者相似特征的转基因小鼠模型一致。
