IGFBP5增龄性表达促进胸腺上皮细胞线粒体自噬和凋亡

Age-related increase of IGFBP5 promotes mitophagy and apoptosis in thymic epithelial cells

目的 探讨在胸腺上皮细胞中随年龄增长而上调表达的胰岛素样生长因子结合蛋白5(Insulin Like Growth Factor Binding Protein 5,IGFBP5)在胸腺衰退中的作用和机制。方法 使用基因差异表达分析方法从已发表人胸腺基质细胞单细胞数据集(GSE147520)中筛选出随年龄增长表达上调程度最高的基因IGFBP5;Mfuzz趋势分析筛选出与IGFBP5具有相同表达趋势的基因;富集分析注释与IGFBP5共趋势基因集;使用蛋白序列比对工具研究IGFBP5蛋白氨基酸序列的人鼠同源性;使用NIH基因数据库研究IGFBP5在人鼠器官组织中表达模式;免疫组化研究IGFBP5在不同年龄C57/BL6小鼠胸腺中的表达模式;构建IGFBP5过表达胸腺上皮细胞系;透射电镜观察IGFBP5过表达胸腺上皮细胞系中线粒体的形态变化;免疫蛋白印记(Western Blot, WB)对线粒体及全细胞组分线粒体自噬标志物微管相关蛋白1B(Microtubule Associated Protein 1 Light Chain 3 Beta, LC3B)、PTEN诱导激酶1(Pten Induced Kinase 1, PINK1)、Parkin泛素蛋白连接酶(Parkin Rbr E3 Ubiquitin Protein Ligase, Parkin)、BCL2互作蛋白3(BCL2 Interacting Protein 3, BNIP3),细胞凋亡标记物活化型胱天蛋白酶3 (Cleaved-Caspase3)表达水平进行检测;细胞双重免疫荧光对线粒体标志物与线粒体探针(MitoTracker)胞内定位进行检测;使用流式细胞术(Flow Cytometry, FCM)对细胞系凋亡水平进行检测。结果 IGFBP5是人胸腺上皮细胞中随年龄增长表达上调程度最大的基因,与其具有共同表达模式的基因集显著富集在线粒体相关通路上。人与小鼠IGFBP5蛋白序列高度保守,其在人、小鼠多器官组织中表达模式相近,且IGFBP5在人、小鼠胸腺中均呈现中等表达水平;此外,IGFBP5在人和小鼠胸腺上皮中随年龄增长表达升高;在IGFBP5过表达小鼠胸腺上皮细胞系中,透射电镜结果表明线粒体自噬体增多,WB结果表明线粒体组分和细胞组分LC3B、PINK1、Parkin蛋白表达水平上调,BNIP3无明显差异;细胞双重免疫荧光结果表明LC3B、PINK1、Parkin蛋白胞质表达增多且与线粒体共定位;WB表明IGFBP5过表达鼠胸腺上皮细胞系中细胞凋亡标志物Cleaved-Caspase3水平升高;FCM表明IGFBP5过表达细胞系凋亡细胞比例增加。结论 人、小鼠胸腺上皮细胞中随年龄增长而表达升高的IGFBP5促进线粒体自噬和细胞凋亡,从而导致胸腺上皮细胞减少和胸腺功能衰退,因此,IGFBP5可成为干预胸腺衰老、重构人体免疫力的潜在靶标。

Objective To investigate the role and underlying mechanism of insulin like growth factor binding protein 5(IGFBP5),a gene up-regulated with age in thymic epithelial cells,in the pathogenesis of thymic involution.Methods Different gene expression analysis was applied to published human thymic stromal cells single-cell RNA-seq data set(GSE147520)to identify genes up-regulated with aging,and IGFBP5 was screened out as the most significant gene.Mfuzz trends analysis was employed to fine the genes with similar expression pattern as IGFBP5.Gene ontology(GO)enrichment analysis was conducted to annotate the function of the common trend gene set associated with IGFBP5.Protein sequence alignment tool was utilized to analyze the human-mouse homology of IGFBP5 protein sequence.Then NIH gene database was consulted to investigate the expression pattern of IGFBP5 in various organs and tissues of both humans and mice.Immunohistochemical assay was performed on mouse thymus samples to examine the expression pattern of IGFBP5.After a mouse thymic epithelial cell line with IGFBP5 overexpression was constructed,transmission electron microscopy was used to observe the morphological changes in the mitochondria,Western blotting was employed to detect altered protein components related to mitochondrial autophagy,including microtubule associated protein 1 light chain 3 beta(LC3B),PTEN induced kinase 1(PINK1),Parkin RBR E3 ubiquitin protein ligase(Parkin),and BCL2 interacting protein 3(BNIP3),in both whole cell extracts and isolated mitochondria,and cleaved-Capase3 was used to estimate cell apoptosis.Double immunofluorescence staining was performed to observe the intracellular localization of the mitophagy markers as described above with mitochondria probe(MitoTracker).Flow cytometry was used to detect the apoptotic rates of the cell lines.Results IGFBP5 was identified with the highest increase in gene expression in human thymic epithelial cells with ageing,and the genes with similar expression pattern as IGFBP5 were related to mitochondrial metabolism pathways.Protein sequence alignment demonstrated a high conservation between human and mouse IGFBP5 protein sequences.IGFBP5 was moderately expressed in both human and mouse thymus tissues.Mouse thymus showed an age-dependent increase in IGFBP5 expression in thymic epithelial cells like human.In the mouse thymic epithelial cell line with IGFBP5 overexpression,transmission electron microscopy indicated an increased mitophagy bodies,Western blotting revealed up-regulated protein levels of LC3B,PINK1,and Parkin in the mitochondrial and cellular components,while no significant difference was observed for BNIP3.Double immunofluorescence staining demonstrated that increased cytoplasmic expression of LC3B,PINK1,and Parkin were co-localized with mitochondria.Western blotting confirmed that Cleaved-Caspase3 level was elevated,as a marker of cell apoptosis in IGFBP5 overexpression mouse thymic epithelial cell line.Flow cytometry indicated that IGFBP5 overexpression cell line had an increased proportion of apoptotic cells.Conclusion Increased expression of IGFBP5 with aging promotes mitochondrial autophagy and apoptosis in thymic epithelial cells,and then leads to thymic involution.IGFBP5 might be a potential target in the intervention of thymic aging and reconstruction of human immune system.