Upregulation of Alpha-2- macroglobulin in replicative senescence

Abstract

Replicative senescence is a well- established model system for studying the molecular basis of aging. Using high-density complementary deoxyribonucleic acid (cDNA) arrays, we identifi ed alpha-2- macroglobulin (α2M) as a differentially expressed gene in replicative senescence. Our study demonstrated a positive linear-correlation of the mRNA level of α2M with cumulative population doublings (PDL) of human fi broblasts. The levels of α2M increased in senescence cells, but not in quiescent state of cultured fi broblasts, and remained stable in immortal HeLa cells. Moreover, the mRNA level of α2M in leucocytes showed signifi cant difference between newborn and old human body. These results indicate that the up-regulated expression of α2M could be a universal age- related phenotype and the mRNA level of α2M may be used as a biomarker of aging in vitro and in vivo. To further explore the mechanism responsible for the up-regulation of α2M in senescent, We identifi ed a novel transcriptional regulatory element, the α2M transcription enhancement element (ATEE), within the α2M promoter. This element differentially activates α2M expression in senescent versus young fi broblasts. Electrophoretic mobility shift assays revealed abundant complexes in senescent cell nuclear extracts compared with young cell nuclear extracts. The DNase I footprint revealed the protein-binding coresequence through which the protein binds the ATEE. Mutation within ATEE selectively abolished α2M promoter activity in senescent (but not young) cells. These results indicated the ATEE, as a positive transcription regulatory element, contributes to the up-regulation of α2M during replicative senescence.