Angiogenin interacts with ribonuclease inhibitor regulating PI3K/AKT/mTOR signaling pathway in bladder cancer cells
Angiogenin interacts with ribonuclease inhibitor regulating PI3K/AKT/mTOR signaling pathway in bladder cancer cells
复制标题
血管生成素与核糖核酸酶抑制剂相互作用调节膀胱癌细胞中的 PI3K/AKT/mTOR 信号通路
DOI：
10.1016/j.cellsig.2014.08.021
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发表时间：
2014-12-01
影响因子：
4.8
通讯作者：
Chen, Junxia
中科院分区：
文献类型：
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作者：
Peng, Yuan;Li, Lin;Chen, Junxia
血管生成素（Angiogenin，ANG）是RNase A超家族成员，是唯一具有核糖核酸裂解活性的血管生成因子。近年来的研究表明，ANG在多种肿瘤中的表达均升高。越来越多的证据表明，ANG通过刺激癌细胞增殖和肿瘤血管生成在癌症进展中起重要作用。人核糖核酸酶抑制因子（RI）是一种细胞质蛋白，几乎完全由富含亮氨酸的重复序列（LRR）构成，其存在于一个大家族的蛋白质中，其特征在于其显示巨大的表面积以促进蛋白质-蛋白质相互作用。RI除抑制RNase A活性外，还可能参与其他未知的生物学效应。实验证明，RI在体外也能与血管生成素（ANG）紧密结合，从而抑制ANG的活性。PI 3 K/AKT/mTOR信号通路在细胞生长、存活、增殖、凋亡和血管生成中发挥关键作用。我们最近报道上调RI可通过抑制血管生成素和PI 3 K/AKT信号通路抑制小鼠黑色素瘤细胞的生长并诱导其凋亡。然而，迄今为止，ANG受体尚未被鉴定，其相关的信号转导途径尚不完全清楚，RI和ANG之间的相互作用机制在很大程度上仍然未知。因此，我们推测RI可能通过与细胞内ANG联合收割机，阻断其核转位，调节PI 3 K/AKT/mTOR信号通路，从而抑制ANG的生物学功能。本文首次报道了利用免疫共沉淀技术（Co-IP）和GST pull-down技术，ANG可以与RI发生内源性和外源性相互作用。在激光共聚焦显微镜下，用免疫荧光染色法观察血管生成素和RI在细胞内的共定位。此外，通过荧光共振能量转移（FRET）分析，我们进一步证实了这两种蛋白在活细胞中具有物理相互作用。随后，我们证明了上调ANG包括ANG His 37 Ala突变体明显降低RI表达并激活PI 3 K/AKT/mTOR信号通路关键下游靶分子的磷酸化。最后，上调ANG导致促进体内肿瘤血管生成、肿瘤发生和转移。这些结果为ANG通过RI调控PI 3 K/AKT/mTOR信号通路提供了新的机制，为肿瘤的治疗提供了新的靶点。(C)2014 Elsevier Inc. All rights reserved.
Angiogenin (ANG), a member of RNase A superfamily, is the only angiogenic factor that possesses ribonucleolytic activity. Recent studies showed that the expression of ANG was elevated in various types of cancers. Accumulating evidence indicates that ANG plays an essential role in cancer progression by stimulating both cancer cell proliferation and tumor angiogenesis. Human ribonuclease inhibitor (RI), a cytoplasmic protein, is constructed almost entirely of leucine rich repeats (LRRs), which are present in a large family of proteins that are distinguished by their display of vast surface areas to foster protein-protein interactions. RI might be involved in unknown biological effects except inhibiting RNase A activity. The experiment demonstrated that RI also could suppress activity of angiogenin (ANG) through closely combining with it in vitro. PI3K/AKT/mTOR signaling pathway exerts a key role in cell growth, survival, proliferation, apoptosis and angiogenesis. We recently reported that up-regulating RI inhibited the growth and induced apoptosis of murine melanoma cells through repression of angiogenin and PI3K/AKT signaling pathway. However, ANG receptors have not yet been identified to date, its related signal transduction pathways are not fully clear and underlying interacting mechanisms between RI and ANG remain largely unknown. Therefore, we hypothesize that RI might combine with intracellular ANG to block its nuclear translocation and regulate PI3K/AKT/mTOR signaling pathway to inhibit biological functions of ANG. Here, we reported for the first time that ANG could interact with RI endogenously and exogenously by using co-immunoprecipitation (Co-IP) and GST pull-down. Furthermore, we observed the colocalization of ANG and RI in cells with immunofluorescence staining under laser confocal microscope. Moreover, through fluorescence resonance energy transfer (FRET) assay, we further confirmed that these two proteins have a physical interaction in living cells. Subsequently, we demonstrated that up-regulating ANG including ANG His37Ala mutant obviously decreased RI expression and activated phosphorylation of key downstream target molecules of PI3K/AKT/mTOR signaling pathway. Finally, up-regulating ANG led to the promotion of tumor angiogenesis, tumorigenesis and metastasis in vivo. Taken together, our data provided a novel mechanism of ANG in regulating PI3K/AKT/mTOR signaling pathway via RI, which suggested a new therapeutic target for cancer therapy. (C) 2014 Elsevier Inc. All rights reserved.