Angori Silvia, Capanni Cristina, Faulkner Georgine, Bean Camilla, Boriani Giuseppe, Lattanzi Giovanna, Cenni Vittoria
Institute of Molecular Genetics (IGM)-CNR, Unit of Bologna, Bologna, Italy.
Laboratory of Musculoskeletal Cell Biology, Rizzoli Orthopaedic Institute, Bologna, Italy.
Cell Physiol Biochem. 2017;42(1):169-184. doi: 10.1159/000477309. Epub 2017 May 25.
Ankrd2 is a stress responsive protein mainly expressed in muscle cells. Upon the application of oxidative stress, Ankrd2 translocates into the nucleus where it regulates the activity of genes involved in cellular response to stress. Emery-Dreifuss Muscular Dystrophy 2 (EDMD2) is a muscular disorder caused by mutations of the gene encoding lamin A, LMNA. As well as many phenotypic abnormalities, EDMD2 muscle cells also feature a permanent basal stress state, the underlying molecular mechanisms of which are currently unclear.
Experiments were performed in EDMD2-lamin A overexpressing cell lines and EDMD2-affected human myotubes. Oxidative stress was produced by H2O2 treatment. Co-immunoprecipitation, cellular subfractionation and immunofluorescence analysis were used to validate the relation between Ankrd2 and forms of lamin A; cellular sensibility to stress was monitored by the analysis of Reactive Oxygen Species (ROS) release and cell viability.
Our data demonstrate that oxidative stress induces the formation of a complex between Ankrd2 and lamin A. However, EDMD2-lamin A mutants were able to bind and mislocalize Ankrd2 in the nucleus even under basal conditions. Nonetheless, cells co-expressing Ankrd2 and EDMD2-lamin A mutants were more sensitive to oxidative stress than the Ankrd2-wild type lamin A counterpart.
For the first time, we present evidence that in muscle fibers from patients affected by EDMD2, Ankrd2 has an unusual nuclear localization. By introducing a plausible mechanism ruling this accumulation, our data hint at a novel function of Ankrd2 in the pathogenesis of EDMD2-affected cells.
Ankrd2是一种应激反应蛋白，主要在肌肉细胞中表达。在施加氧化应激时，Ankrd2会转移到细胞核中，在那里它调节参与细胞应激反应的基因的活性。埃默里-德赖富斯肌营养不良症2型（EDMD2）是一种由编码核纤层蛋白A（LMNA）的基因突变引起的肌肉疾病。除了许多表型异常外，EDMD2肌肉细胞还具有永久性的基础应激状态，其潜在的分子机制目前尚不清楚。
在过表达EDMD2-核纤层蛋白A的细胞系和受EDMD2影响的人肌管中进行实验。通过H2O2处理产生氧化应激。采用免疫共沉淀、细胞亚组分分离和免疫荧光分析来验证Ankrd2与核纤层蛋白A各形式之间的关系；通过分析活性氧（ROS）释放和细胞活力来监测细胞对应激的敏感性。
我们的数据表明，氧化应激诱导Ankrd2与核纤层蛋白A形成复合物。然而，即使在基础条件下，EDMD2-核纤层蛋白A突变体也能够在细胞核中结合并使Ankrd2定位错误。尽管如此，共表达Ankrd2和EDMD2-核纤层蛋白A突变体的细胞比Ankrd2-野生型核纤层蛋白A对应的细胞对氧化应激更敏感。
我们首次提供证据表明，在受EDMD2影响的患者的肌肉纤维中，Ankrd2具有异常的核定位。通过引入一种解释这种积累的合理机制，我们的数据暗示了Ankrd2在EDMD2影响细胞的发病机制中的新功能。
