Upregulation of Alpha-2-Macroglobulin in Replicative Senescence

  • Li Wei Ma
  • Guo Dong Li
  • Tan Jun Tong
Part of the Tumor Dormancy and Cellular Quiescence and Senescence book series (DOQU, volume 2)


Replicative senescence is a well-established model system for studying the molecular basis of aging. Using high-density complementary deoxyribonucleic acid (cDNA) arrays, we identified 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 fibroblasts. The levels of α2M increased in senescence cells, but not in quiescent state of cultured fibroblasts, and remained stable in immortal HeLa cells. Moreover, the mRNA level of α2M in leucocytes showed significant 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 identified 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 fibroblasts. 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 core sequence 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.


Alpha-2-macroglobulin (α2Μ) Electrophoretic mobility shift assays (EMSA) Functional promoter region determination α2M transcription enhancement element (ATEE) Positive element identification Replicative senescence Reporter gene assay Trans-acting factors 



This work was supported by grants 2013CB530801 from National Basic Research of China and grants 31000609,81170319 from National Natural Science Foundation of China.


  1. Alder MJ, Coronel C, Shelton E, Seegmiller JE, Dewji NN (1991) Increased gene expression of Alzheimer disease beta-amyloid precursor protein in senescent cultured fibroblasts. Proc Natl Acad Sci U S A 88:16–20CrossRefGoogle Scholar
  2. Allsopp RC, Vaziri H, Patterson C, Goldstein S, Younglai EV, Futcher AB, Greider CW, Harley CB (1992) Telomere length predicts replicative capacity of human fibroblasts. Proc Natl Acad Sci U S A 89:10114–10118PubMedCrossRefGoogle Scholar
  3. Caldini R, Chevanne M, Mocali A, Tombaccini D, Paoletti F (1998) Premature induction of aging in sublethally H2O2-treated young MRC5 fibroblasts correlates with increased glutathione peroxidase levels and resistance to DNA breakage. Mech Ageing Dev 105:137–150PubMedCrossRefGoogle Scholar
  4. Chen QM, Prowse KR, Tu VC, Purdom S, Linskens MH (2001) Uncoupling the senescent phenotype from telomere shortening in hydrogen peroxide-treated fibroblasts. Exp Cell Res 265:294–303PubMedCrossRefGoogle Scholar
  5. Choi AMK, Pignolo RJ, Rhys CMJ, Cristifalo VJ, Holbrook NJ (1995) Alterations in the molecular response to DNA damage during cellular aging of cultured fibroblasts: reduced Ap-1activation and collagenase gene expression. J Cell Physiol 164:65–74PubMedCrossRefGoogle Scholar
  6. Crookston KP, Webb DJ, Wolf BB, Gonias SLJ (1994) Classification of alpha 2-macroglobulin-cytokine interactions based on affinity of noncovalent association in solution under apparent equilibrium conditions. J Biol Chem 269:1533–1540PubMedGoogle Scholar
  7. Dimri GP, Lee X, Basile G, Acosta M, Scott G, Roskelley C, Medrano EE, Linskens M, Rubelj I, Pereira-Smith O (1995) A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc Natl Acad Sci U S A 92:9363–9367PubMedCrossRefGoogle Scholar
  8. DiPaolo BR, Pignolo RJ, Cristifalo VJ (1992) Overexpression of two-chain form of cathepsin B in senescent WI-38 cells. Exp Cell Res 201:500–505PubMedCrossRefGoogle Scholar
  9. Duan JM, Zhang ZY, Tong TJ (2001) Senescence delay of human diploid fibroblast induced by anti-sense p16INK4a expression. J Biol Chem 276:48325–48331PubMedCrossRefGoogle Scholar
  10. Frippiat C, Chen QM, Zdanov S, Magalhaes JP, Remacle J, Toussaint O (2001) Subcytotoxic H2O2 stress triggers a release of transforming growth factor-beta 1, which induces biomarkers of cellular senescence of human diploid fibroblasts. J Biol Chem 276:2531–2537PubMedCrossRefGoogle Scholar
  11. Hocke GM, Barry D, Fey GH (1992) Synergistic action of interleukin-6 and glucocorticoids is mediated by the interleukin-6 response element of the rat alpha 2 macroglobulin gene. Mol Cell Biol 12:2282–2294PubMedGoogle Scholar
  12. Kondo T, Sakaguchi M, Namba M (2001) Two-dimensional gel electrophoretic studies on the cellular aging: accumulation of alpha-2-macroglobulin in human fibroblasts with aging. Exp Gerontol 36:487–495PubMedCrossRefGoogle Scholar
  13. Lerner L, Henriksen MA, Zhang X, Darnell JJ (2003) STAT3-dependent enhanceosome assembly and disassembly: synergy with GR for full transcriptional increase of the alpha 2-macroglobulin gene. Genes Dev 17:2564–2577PubMedCrossRefGoogle Scholar
  14. Li RZ, Ma LW, Huang Y, Zhang ZY, Tong TJ (2011) Characterization of a novel positive transcription regulatory element that differentially regulates the alpha-2-macroglobulin gene in replicative senescence. Biogerontology 12:517–525PubMedCrossRefGoogle Scholar
  15. Lin VK, Wang SY, Boetticher NC, Vazquez DV, Saboorian H, McConnell JD, Roehrborn CG (2005) Alpha(2) macroglobulin, a PSA binding protein, is expressed in human prostate stroma. Prostate 63:299–308PubMedCrossRefGoogle Scholar
  16. Liu Q, Ling TY, Shieh HS, Johnson FE, Huang SS (2001) Identification of the high affinity binding site in transforming growth factor-beta involved in complex formation with alpha 2-macroglobulin. Implications regarding the molecular mechanisms of complex formation between alpha 2-macroglobulin and growth factors, cytokines, and hormones. J Biol Chem 276:46212–46218PubMedCrossRefGoogle Scholar
  17. Ma H, Li RZ, Zhang ZY, Tong TJ (2004) mRNA level of alpha-2-macroglobulin as an aging biomarker of human fibroblasts in culture. Exp Gerontol 39:415–421PubMedCrossRefGoogle Scholar
  18. Misra UK, Pizzo SV (2002) Regulation of cytosolic phospholipase A2 activity in macrophages stimulated with receptor-recognized forms of alpha 2-macroglobulin: role in mitogenesis and cell proliferation. J Biol Chem 277:4069–4078PubMedCrossRefGoogle Scholar
  19. Qiu Z, Strickland DK, Hyman BT, Rebeck GW (1999) Alpha 2-macroglobulin enhances the clearance of endogenous soluble beta-amyloid peptide via low-density lipoprotein receptor-related protein in cortical neurons. J Neurochem 73:1393–1398PubMedCrossRefGoogle Scholar
  20. Scottrup-Jensen L, Sand O, Kristensen L, Fey GH (1989) The alpha-macroglobulin bait region. Sequence diversity and localization of cleavage sites for proteinases in five mammalian alpha-macroglobulins. J Biol Chem 264:15781–15789Google Scholar
  21. Seshadri T, Campisi J (1990) Repression of c-fos transcription and an altered genetic program in senescent human fibroblasts. Science 247:205–209PubMedCrossRefGoogle Scholar
  22. Severino J, Allen RG, Balin S, Balin A, Cristifalo VJ (2000) Is â-galactosidase staining a maker of senescence in vitro and in vivo. Exp Cell Res 257:162–171PubMedCrossRefGoogle Scholar
  23. Sottile J, Mann DM, Diemer V, Millis AJ (1989) Regulation of collagenase and collagenase mRNA production in early- and late-passage human diploid fibroblasts. J Cell Physiol 138:281–290PubMedCrossRefGoogle Scholar
  24. Sun Y, Li RZ, Zhang ZY, Tong TJ (2005) Functions of á-2-macroglobulin in human diploid fibroblasts during aging. Chin J Geriatr 24:858–861Google Scholar
  25. Toussaint O, Remacle J, Dierick JF, Pascal T, Frippiat C, Zdanov S, Magalhaes JP, Royer V, Chainiaux F (2002) From the Hayflick mosaic to the mosaics of ageing. Role of stress-induced premature senescence in human ageing. Int J Biochem Cell Biol 34:1415–1429PubMedCrossRefGoogle Scholar
  26. Tresini M, Pignolo RJ, Allen RG, Cristifalo VJ (1999) Effects of donor age on the expression of a marker of replicative senescence (EPC-1) in human dermal fibroblasts. J Cell Physiol 179:11–17PubMedCrossRefGoogle Scholar
  27. Wager V, Wagnerova M, Zvara K (1982) Age correlation of alpha-2-macroglobulin levels in healthy subjects. Physiol Bohemoslov 31:359–364Google Scholar
  28. Yoo JY, Wang W, Desiderio S, Nathans D (2001) Synergistic activity of STAT3 and c-Jun at a specific array of DNA elements in the alpha 2-macroglobulin promoter. J Biol Chem 276:26421–26429PubMedCrossRefGoogle Scholar

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© Springer Science+Business Media Dordrecht 2014

Authors and Affiliations

  1. 1.Research Center on Aging, Department of Biochemistry and Molecular BiologyPeking University Health Science CenterBeijingPeople’s Republic of China

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