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Kai Stühler: The secretory phenotype of aged fibroblasts

Wann 14.10.2013
von 13:15 bis 14:00
Wo FRIAS Lecture Hall, Albertstr. 19, 79104 Freiburg
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Kai Stühler, Heinrich-Heine-Universität Düsseldorf, Germany: 

"The secretory phenotype of aged fibroblasts"

Aging is a multi-factorial process, where endogenous changes and exogenous factors are discussed to play relevant role. It has been observed that exposing aged stem cells to a young environment leads to rejuvenation of these cells. This suggests that the aging process of an organ is influenced by its stroma, with fibroblasts as the dominate cell type. This indicates that the driving force for skin ageing is not the constantly self-renewing epidermis, but the much more static dermis with its interplay of fibroblasts and their matrix. Proteins are the key players in cell biology and involved in relevant aging processes like e.g. proteostasis, detoxification or protein degradation. Furthermore, secreted proteins have a particular impact on the surrounding cells in the tissue. Because of these facts, an exhaustive differential proteomics study of both proteome and secretome of aged fibroblasts is the appropriate opportunity to find the key proteins in stromal aging.

For the identification of key proteins of stromal aging dermal fibroblasts of 15 female donors of three different age groups (20-26, 40-49, 60-67) were cultivated under standardized conditions till passage three for proteome and passage six for secretome analysis. To describe the stromal proteome as well as the secretome and to identify novel proteins potentially involved in stromal aging, we have performed a label-free proteomics approach using high resolution MS in combination with HPLC separation. Briefly, for LC-MS/MS analysis the cells were lysed and proteins extracted and the supernatants were concentrated and desalted. Subsequently, the proteins were digested with trypsin using FASP as well as in-gel digestion. For differential analysis two approaches were used: ANOVA analysis with age groups (p<0.05; ratio>1.5) and Pearson correlation without grouping (p<0.05; ratio>1.5).

For the fibroblast’s proteome 2351 proteins were identified resulting in 1564 quantifiable proteins. Differential analysis with ANOVA revealed 16 differentially regulated proteins and Pearson correlation revealed 51 differentially regulated proteins. The regulated proteins were involved in metabolism, intracellular transport, cell communication, cell cycle, immune system process and homeostasis. About 970 of the quantifiable proteins showed stable abundances (CV<40%) within the different samples. The enrichment analysis of these proteins resulted in protein biosynthesis, RNA-binding, actin-cytoskeleton and chaperones. For the fibroblast’s secretome 1331 proteins were identified resulting in 1036 quantifiable proteins. Differential analysis with ANOVA revealed 37 differentially regulated proteins and Pearson correlation revealed 44 differentially regulated proteins. The proteins were classified as hydrolases, extracellular matrix proteins, receptors, signaling and cell adhesion molecules and proteases.

Taken together, the label-free proteomics approach allowed the detection of novel senescence associated and aging-related changes in protein expression in human fibroblasts involved in process of stromal aging.