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

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 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.

Keywords

• thymic epithelial cells
• insulin like growth factor binding protein 5
• mitophagy
• apoptosis
• thymic involution