Abstract
The extracellular matrix (ECM) provides an essential structural framework for cell attachment, proliferation, and differentiation, and undergoes progressive changes during senescence. To investigate changes in protein expression in the extracellular matrix between young and senescent fibroblasts, we compared proteomic data (LTQ-FT) with cDNA microarray results. The peptide counts from the proteomics analysis were used to evaluate the level of ECM protein expression by young cells and senescent cells, and ECM protein expression data were compared with the microarray data. After completing the comparative analysis, we grouped the genes into four categories. Class I included genes with increased expression levels in both analyses, while class IV contained genes with reduced expression in both analyses. Class II and Class III contained genes with an inconsistent expression pattern. Finally, we validated the comparative analysis results by examining the expression level of the specific gene from each category using Western blot analysis and semiquantitative RT-PCR. Our results demonstrate that comparative analysis can be used to identify differentially expressed genes.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Bartoloni, L., Blouin, J.L., Maiti, A.K., Sainsbury, A., Rossier, C., Gehrig, C., She, J.X., Marron, M.P., Lander, E.S., Meeks, M., et al. (2001). Axonemal beta heavy chain dynein DNAH9: cDNA sequence, genomic structure, and investigation of its role in primary ciliary dyskinesia. Genomics 72, 21–33.
Bernier, G., Pool, M., Kilcup, M., Alfoldi, J., De Repentigny, Y., and Kothary, R. (2000). Acf7 (MACF) is an actin and micro-tubule linker protein whose expression predominates in neural, muscle, and lung development. Dev. Dyn. 219, 216–225.
Bissell, M.J., Hall, H.G., and Parry, G. (1982). How does the extracellular matrix direct gene expression? J. Theor. Biol. 99, 31–68.
Boyce, S.T., and Ham, R.G. (1983). Calcium-regulated differentiation of normal human epidermal keratinocytes in chemically defined clonal culture and serum-free serial culture. J. Invest. Dermatol. 81, 33s–40s.
Campisi, J. (2005). Senescent cells, tumor suppression, and organismal aging: good citizens, bad neighbors. Cell 120, 513–522.
Capitanio, D., Vasso, M., Fania, C., Moriggi, M., Vigano, A., Procacci, P., Magnaghi, V., and Gelfi, C. (2009). Comparative proteomic profile of rat sciatic nerve and gastrocnemius muscle tissues in ageing by 2-D DIGE. Proteomics 9, 2004–2020.
Chen, H.L., Lu, C.Y., Hsu, Y.H., and Lin, J.J. (2004). Chromosome positional effects of gene expressions after cellular senescence. Biochem. Biophys. Res. Commun. 313, 576–586.
Chen, H., Suzuki, M., Nakamura, Y., Ohira, M., Ando, S., Iida, T., Nakajima, T., Nakagawara, A., and Kimura, H. (2005). Aberrant methylation of FBN2 in human non-small cell lung cancer. Lung Cancer 50, 43–49.
Cho, K.A., Ryu, S.J., Oh, Y.S., Park, J.H., Lee, J.W., Kim, H.P., Kim, K.T., Jang, I.S., and Park, S.C. (2004). Morphological adjustment of senescent cells by modulating caveolin-1 status. J. Biol. Chem. 279, 42270–42278.
Colige, A.C., Lambert, C.A., Nusgens, B.V., and Lapiere, C.M. (1992). Effect of cell-cell and cell-matrix interactions on the response of fibroblasts to epidermal growth factor in vitro. Expression of collagen type I, collagenase, stromelysin and tissue inhibitor of metalloproteinases. Biochem. J. 285, 215–221.
Coppe, J.P., Kauser, K., Campisi, J., and Beausejour, C.M. (2006). Secretion of vascular endothelial growth factor by primary human fibroblasts at senescence. J. Biol. Chem. 281, 29568–29574.
Demidenko, Z.N., and Blagosklonny, M.V. (2008). Growth stimulation leads to cellular senescence when the cell cycle is blocked. Cell Cycle 7, 3355–3361.
Dimri, G.P., Lee, X., Basile, G., Acosta, M., Scott, G., Roskelley, C., Medrano, E.E., Linskens, M., Rubelj, I., Pereira-Smith, O., et al. (1995). A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc. Natl. Acad. Sci. USA 92, 9363–9367.
Fidler, I.J. (2002). The organ microenvironment and cancer metastasis. Differentiation 70, 498–505.
Fodil-Bourahla, I., Drubaix, I., Robert, L., and Labat-Robert, J. (1999). Effect of in vitro aging on the modulation of protein and fibronectin biosynthesis by the elastin-laminin receptor in human skin fibroblasts. Gerontology 45, 23–30.
Fu, X., Gharib, S.A., Green, P.S., Aitken, M.L., Frazer, D.A., Park, D.R., Vaisar, T., and Heinecke. J.W. (2008). Spectral index for assessment of differential protein expression in shotgun proteomics. J. Proteome Res. 7, 845–854.
Harman, D. (1956). Aging: a theory based on free radical and radiation chemistry. J. Gerontol. 11, 298–300.
Hayflick, L., and Moorhead, P.S. (1961). The serial cultivation of human diploid cell strains. Exp. Cell Res. 25, 585–621.
Kang, J.Y., Kim, J.J., Jang, S.Y., and Bae, Y.S. (2009). The p53-p21Cip1/WAF1 pathway is necessary for cellular senescence induced by the inhibition of protein kinase CKII in human colon cancer cells. Mol. Cells 28, 489–494.
Kim, J.Y., Lee, J.H., Park, G.W., Cho, K., Kwon, K.H., Park, Y.M., Cho, S.Y., Paik, Y.K., and Yoo. J.S. (2005). Utility of electrophoretically derived protein mass estimates as additional constraints in proteome analysis of human serum based on MS/MS analysis. Proteomics 5, 3376–3385.
Kim, S.Y., Ryu, S.J., Ahn, H.J., Choi, H.R., Kang, H.T., and Park, S.C. (2010) Senescence-related functional nuclear barrier by down-regulation of nucleo-cytoplasmic trafficking gene expression. Biochem. Biophys. Res. Commun. 391, 28–32.
Labat-Robert, J. (2004). Cell-matrix interactions in aging: role of receptors and matricryptins. Ageing Res. Rev. 3, 233–247.
Labat-Robert, J., and Robert, L. (2007). The effect of cell-matrix interactions and aging on the malignant process. Adv. Cancer Res. 98, 221–259.
Lin, J.J., Hegmann, T.E., and Lin, J.L. (1988). Differential localization of tropomyosin isoforms in cultured nonmuscle cells. J. Cell Biol. 107, 563–572.
Lin, J.J., Eppinga, R.D., Warren, K.S., and McCrae, K.R. (2008). Human tropomyosin isoforms in the regulation of cytoskeleton functions. Adv. Exp. Med. Biol. 644, 201–222.
Nishio, K., Inoue, A., Qiao, S., Kondo, H., and Mimura, A. (2001). Senescence and cytoskeleton: overproduction of vimentin induces senescent-like morphology in human fibroblasts. Histochem. Cell Biol. 116, 321–327.
Pagani, F., Zagato, L., Vergani, C., Casari, G., Sidoli, A., and Baralle, F.E. (1991). Tissue-specific splicing pattern of fibronectin messenger RNA precursor during development and aging in rat. J. Cell Biol. 113, 1223–1229.
Petersen, O.W., Ronnov-Jessen, L., Howlett, A.R., and Bissell, M.J. (1992). Interaction with basement membrane serves to rapidly distinguish growth and differentiation pattern of normal and malignant human breast epithelial cells. Proc. Natl. Acad. Sci. USA 89, 9064–9068.
Pflieger, D., Chabane, S., Gaillard, O., Bernard, B.A., Ducoroy, P., Rossier, J., and Vinh, J. (2006). Comparative proteomic analysis of extracellular matrix proteins secreted by two types of skin fibroblasts. Proteomics 6, 5868–5879.
Pieples, K., and Wieczorek, D.F. (2000). Tropomyosin 3 increases striated muscle isoform diversity. Biochemistry 39, 8291–8297.
Ren, J.L., Pan, J.S., Lu, Y.P., Sun, P., and Han, J. (2009). Inflammatory signaling and cellular senescence. Cell Signal. 21, 378–383.
Rhim, J.H., Jang, I.S., Choi, J.S., Kwon, H.J., Yeo, E.J., and Park, S.C. (2009). Time-dependent differential gene expression in lysophosphatidic acid-treated young and senescent human diploid fibroblasts. Mech. Ageing Dev. 130, 648–651.
Rodier, F., Coppe, J.P., Patil, C.K., Hoeijmakers, W.A., Munoz, D.P., Raza, S.R., Freund, A., Campeau, E., Davalos, A.R., and Campisi, J. (2009). Persistent DNA damage signalling triggers senescence-associated inflammatory cytokine secretion. Nat. Cell Biol. 11, 973–979.
Ryu, S.J., An, H.J., Oh, Y.S., Choi, H.R., Ha, M.K., and Park, S.C. (2008). On the role of major vault protein in the resistance of senescent human diploid fibroblasts to apoptosis. Cell Death Differ. 15, 1673–1680.
Sell, D.R., and Monnier, V.M. (1989). Structure elucidation of a senescence cross-link from human extracellular matrix. Implication of pentoses in the aging process. J. Biol. Chem. 264, 21597–21602.
Verzar, F. (1964). Ageing of connective tissue. G. Gerontol. 12, 915–921.
Wienkoop, S., Larrainzar, E., Niemann, M., Gonzalez, E.M., Lehmann, U., and Weckwerth, W. (2006). Stable isotope-free quantitative shotgun proteomics combined with sample pattern recognition for rapid diagnostics. J. Sep. Sci. 29, 2793–2801.
Yeo, E.J., Hwang, Y.C., Kang, C.M., Choy, H.E., and Park, S.C. (2000). Reduction of UV-induced cell death in the human senescent fibroblasts. Mol. Cells 10, 415–422.
Yoshisue, H., and Hasegawa, K. (2004). Effect of MMP/ADAM inhibitors on goblet cell hyperplasia in cultured human bronchial epithelial cells. Biosci. Biotechnol. Biochem. 68, 2024–2031.
Zhao, C.Q., Wang, L.M., Jiang, L.S., and Dai, L.Y. (2007) The cell biology of intervertebral disc aging and degeneration. Ageing Res. Rev. 6, 247–261.
Author information
Authors and Affiliations
Corresponding authors
Additional information
These authors contributed equally to this work.
About this article
Cite this article
Yang, K.E., Kwon, J., Rhim, JH. et al. Differential expression of extracellular matrix proteins in senescent and young human fibroblasts: A comparative proteomics and microarray study. Mol Cells 32, 99–106 (2011). https://doi.org/10.1007/s10059-011-0064-0
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10059-011-0064-0