Introduction Cultures of diploid human fibroblasts can replicate only a finite number of times; rapid proliferation is followed by decline in replicative frequency and finally cells become senescent and are incapable of further proliferation. Senescencent cells display altered growth, morphology and deregulated metabolism; they express elevated levels of extracellular matrix (ECM) degrading enzymes, decreased levels of their inhibitors and decreased levels of ECM components such as elastin, laminin and collagen (1). The phenotype of senescent fibroblasts shifts from matix-synthesizing to matrix-degrading. In this study we use proteomic tools to characterise the secretome of young and senescent fibroblasts. Methods Three independent preparations of primary human foreskin fibroblasts were grown to senescence. Young, rapidly proliferating cells at passage 11 and cells from passage 28 displaying senescent morphology were plated in serum supplemented media at 2 million cells per 10 cm2 cell culture dish; after 24 hours of growth cells were washed with Hank's balanced salt solution (HBBS) and were incubated in serum free media for 72 hours. Proteins from cell culture media were precipitated and analysed by two complementary approaches: 1) Two-dimensional gel electro-phoresis (2DGE) was used to separate proteins and quantitate their expression; selected proteins were identified using mass spectrometry; 2) Proteins were digested into peptides and analysed using high performance liquid chromatography (HPLC) and mass spectrometry; peptide count was used to estimate protein abundance. Results 2DGE based analysis of secretion profiles of young and senescent cells derived from the same cell lineage revealed a number of protein spots differentially expressed. We have observed an increased secretion of matrix metalloproteinases (MMP1, 2 and 3), secreted protein acidic and rich in cysteine (SPARC) and two isoforms of metalloproteinase inhibitors (TIMP1 and 2). Major proteins decreased in senescent secretome were follistatin-like 1 and nucleobindin-1. Over 800 spots were separated by 2DGE of which many represent modified forms of the same protein. Using the gel free approach we were able to identify over 400 proteins and 29 of them showed significant differences in secretion between young and old fibroblasts. Among them MMP-1 was found up regulated and several ECM proteins were found down regulated: lamin A, collagen alpha-1(XII) chain and fibulin 3. Results obtained until now are in agreement with the suggested shift from matix - synthesizing to matrix - degrading phenotype in senescent fibroblasts except the increased secretion of metalloproteinase inhibitors. Work is going on to identify the remaining differentially regulated proteins and to strengthen the results by analysing two additional preparations of primary human fibroblasts. Innovative aspects Identification of proteins secreted by human primary fibroblasts Identification of difference in secretion patterns between young and senescent human primary fibroblasts References: (1) V.J. Cristofalo et al, Replicative senescence: a critical review. Mech Ageing Dev. 2004 Oct-Nov;125(10-11):827-48.
Secretome; Ageing; Fibroblasts
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Den Humane Proteom-Organisations 7. verdenskongres, 2008