Proteomic analysis reveals novel common genes modulated in both replicative and stress-induced senescence

Abstract

Cellular senescence causes profound changes in gene expression profile. In this study, we used a combined 2D-DIGE and nanoLC-ESI-LIT-MS/MS approach to evaluate the proteomic changes occurring both in replicative and stress-induced senescence of human IMR90 cells. Twenty protein spots were identified as shifting their quantitative representation in the same direction (over- or down-represented) in both conditions of senescence, which were associated with 25 sequence entries. Dedicated experiments demonstrated that the decreased representation of a set of these proteins is associated with the down-regulation of the corresponding mRNAs, indicating that the regulation of these genes during the senescence process occurs at a transcriptional level. We also performed functional studies by silencing nine of these genes in young cells, which demonstrated that RNA interference-mediated knockdown of LEPRE1, LIMA1/EPLIN, MAGOHA and MAGOHB induces a premature senescent phenotype in IMR90 cells. Chromatin immunoprecipitation experiments indicated that the reduced expression of these four genes is associated with changes in the histone methylation pattern of their promoters, as proved by the occurrence of increased repressive H3K27me3 along with decreased active H3K4me3 marks, respectively.

Biological significance: Cellular senescence, a stable form of cell cycle arrest, is recognized as a phenomenon related to aging and age-related pathologies as well as interfering with tumor progression. Gene expression changes are closely associated with the onset of senescence but the molecular pathways regulating this process are still poorly understood. By using proteomics coupled to functional studies, we here show that both replicative and stress-induced senescence share quantitative modification of four novel proteins, in addition to others already reported in the literature. When ectopically down-regulated, corresponding four genes induce a premature senescence in young cells. The observed parallelism concerning the down-regulation of these genes both in vitro and in vivo senescent cells may foresee a possible biomarker role of the corresponding proteins in monitoring the progression of both aging and age-related diseases. In conclusion, these results for the first time highlight a possible role of LEPRE1, LIMA1/EPLIN, MAGOHA and MAGOHB in the biology of cellular senescence/aging, thus contributing to gain a deeper knowledge of the molecular mechanisms involved in the senescence program.

Keywords: 2D-DIGE; Cellular senescence; Chromatin immunoprecipitation; Gene regulation; Human primary fibroblasts; Oxidative stress.