Age-related increase of <italic style="font-style: italic">IGFBP</italic>5 promotes mitophagy and apoptosis in thymic epithelial cells

Xichan CHEN; Jitao ZENG; Siming QU; Weiwu GAO; Yuzhang WU

doi:10.16016/j.2097-0927.202312068

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

Basic Medicine | 更新时间：2026-01-09

- Age-related increase of IGFBP5 promotes mitophagy and apoptosis in thymic epithelial cells
- Journal of Army Medical University Vol. 46, Issue 6, Pages: 567-577(2024)
- 作者机构：
  
  1.400038 重庆，陆军军医大学(第三军医大学)：基础医学院免疫学教研室
  2.400038 重庆，陆军军医大学(第三军医大学)：第一附属医院生殖科
  3.650032 昆明，昆明医科大学第一附属医院器官移植中心
- 作者简介：
- 基金信息：
  
  the National Natural Science Foundation for Young Scholars of China(82200679)
- DOI：10.16016/j.2097-0927.202312068
  CLC： R322.53;R329.25;R394.2
- Received：13 December 2023，
  
  Revised：2024-01-07，
  
  Published：30 March 2024
- 稿件说明：
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Xichan CHEN, Jitao ZENG, Siming QU, et al. Age-related increase of IGFBP5 promotes mitophagy and apoptosis in thymic epithelial cells[J]. Journal of Army Medical University, 2024, 46(6): 567-577.
DOI：

Xichan CHEN, Jitao ZENG, Siming QU, et al. Age-related increase of IGFBP5 promotes mitophagy and apoptosis in thymic epithelial cells[J]. Journal of Army Medical University, 2024, 46(6): 567-577. DOI： 10.16016/j.2097-0927.202312068.

摘要

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

IGFBP

5)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

IGFBP

5；Mfuzz趋势分析筛选出与

IGFBP

5具有相同表达趋势的基因；富集分析注释与

IGFBP

5共趋势基因集；使用蛋白序列比对工具研究

IGFBP

5蛋白氨基酸序列的人鼠同源性；使用NIH基因数据库研究

IGFBP

5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

IGFBP

5过表达胸腺上皮细胞系；透射电镜观察

IGFBP

5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

IGFBP

5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

IGFBP

5在人、小鼠胸腺中均呈现中等表达水平；此外，

IGFBP

5在人和小鼠胸腺上皮中随年龄增长表达升高；在

IGFBP

5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

IGFBP

5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

IGFBP

5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

IGFBP

5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，

IGFBP

5可成为干预胸腺衰老、重构人体免疫力的潜在靶标。

Abstract

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

IGFBP

5 was scree

ned 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

IGFBP

5. 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

IGFBP

5 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

IGFBP

5 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

IGFBP

5 was identified with the highest increase in gene expression in human thymic epithelial cells with ageing

and the genes with similar expression pattern as

IGFBP

5 were related to mitochondrial metabolism pathways. Protein sequence alignment demonstrated a high conservation between human and mouse IGFBP5 protein sequences.

IGFBP

5 was moderately expressed in both human and mouse thymus tissues. Mouse thymus showed an age-dependent increas

e in IGFBP5 expression in thymic epithelial cells like human. In the mouse thymic epithelial cell line with

IGFBP

5 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

IGFBP

5 overexpression mouse thymic epithelial cell line. Flow cytometry indicated that

IGFBP

5 overexpression cell line had an increased proportion of apoptotic cells.

Conclusion

Increased expression of

IGFBP

5 with aging promotes mitochondrial autophagy and apoptosis in thymic epithelial cells

and then leads to thymic involution.

IGFBP

5 might be a potential target in the intervention of thymic aging and reconstruction of human immune system.

关键词

Keywords

references

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⁰

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

DOI：10.16016/j.2097-0927.202312068

摘要

Abstract

关键词

Keywords

references