Characterization of Skin Aging–Associated Secreted
Proteins (SAASP) Produced by Dermal Fibroblasts
Isolated from Intrinsically Aged Human Skin
Daniel M. Waldera Lupa1,2, Faiza Kalfalah3, Kai Safferling4, Petra Boukamp5, Gereon Poschmann1,2,
Elena Volpi6, Christine Götz-Rösch6, Francoise Bernerd7, Laura Haag3, Ulrike Huebenthal6, Ellen Fritsche6,
Fritz Boege3, Niels Grabe4,9, Julia Tigges6,9, Kai Stühler1,2,9 and Jean Krutmann6,8,9
Most molecular hallmarks of cellular senescence have been identified in studies of cells aged in vitro by driving them into replicative or stress-induced senescence. Comparatively, less is known about the characteristic features of cells that have aged in vivo. Here we provide a systematic molecular analysis of normal human dermal fibroblasts (NHDFs) that were isolated from intrinsically aged human skin of young versus middle aged versus old donors. Intrinsically aged NHDFs in culture exhibited more frequently nuclear foci positive for p53 binding protein 1 and promyelocytic leukemia protein reminiscent of ‘DNA segments with chromatin alterations reinforcing senescence (DNA-SCARS)’. Formation of such foci was neither accompanied by increased DNA double strand breaks, nor decreased cell viability, nor telomere shortening. However, it was associated with the development of a secretory phenotype, indicating incipient cell senescence. By quantitative analysis of the entire secretome present in conditioned cell culture supernatant, combined with a multiplex cytokine determination, we identified 998 proteins secreted by intrinsically aged NHDFs in culture. Seventy of these proteins exhibited an age-dependent secretion pattern and were accordingly denoted ‘skin aging–associated secreted proteins (SAASP)’. Systematic comparison of SAASP with the classical senescence-associated secretory phenotype (SASP) revealed that matrix degradation as well as proinflammatory processes are common aspects of both conditions.
However, secretion of 27 proteins involved in the biological processes of ‘metabolism’ and ‘adherens junction interactions’ was unique for NHDFs isolated from intrinsically aged skin. In conclusion, fibroblasts isolated from intrinsically aged skin exhibit some, but not all, molecular hallmarks of cellular senescence. Most importantly, they secrete a unique pattern of proteins that is distinct from the canonical SASP and might reflect specific processes of skin aging.
Journal of Investigative Dermatology advance online publication, 14 May 2015; doi:10.1038/jid.2015.120
Skin aging is characterized by structural and functional alterations of the dermis (Gilchrest and Krutmann, 2006).
The dermal stroma consists mainly of fibroblasts and their surrounding matrix (Boukamp, 2005; Parrinello et al., 2005;
Tigges et al., 2014) and is a mostly post-mitotic tissue, which allows for dysfunctional cells to accumulate over time.
Therefore, the homeostasis of the dermis depends strongly
ORIGINAL ARTICLE 1Molecular Proteomics Laboratory, Heinrich-Heine-University, Düsseldorf, Germany; 2Biomedical Research Center (BMFZ), Heinrich-Heine-University, Düsseldorf,
Germany; 3Institute of Clinical Chemistry and Laboratory Diagnostics, Heinrich-Heine-University, Med. Faculty, Düsseldorf, Germany; 4Hamamatsu Tissue Imaging and Analysis Center, BIOQUANT, Ruprecht-Karls-University, Heidelberg, Germany; 5German Cancer Research Centre, Heidelberg, Germany; 6IUF - Leibniz
Research Institute for Environmental Medicine at the Heinrich-Heine-University Düsseldorf gGmbH, Düsseldorf, Germany; 7L’Oréal Recherche, Clichy, Paris, France and 8University of Düsseldorf, Medical Faculty, Deanery of Medicine, Düsseldorf, Germany
Correspondence: Jean Krutmann, IUF - Leibniz Research Institute for Environmental Medicine at the Heinrich-Heine-University Düsseldorf gGmbH, Auf'm
Hennekamp 50, 40225 Düsseldorf, Germany. E-mail: firstname.lastname@example.org 9These authors contributed equally to this work.
Received 18 June 2014; revised 12 March 2015; accepted 18 March 2015; accepted article preview online 27 March 2015
Abbreviations: BP, binding protein; CDH, cadherin; CDNB, 1-chloro-2,4-dinitrobenzene; CXCL, C-X-C motif chemokine; DNA-SCARS, DNA segments with chromatin alterations reinforcing senescence; ECM, extracellular matrix; ENO2, enolase 2; ERBB2, human epidermal growth factor receptor 2 (HER2); GGCT, γ-glutamylcyclo-transferase; GST, glutathione s-transferase; H2AX, Histon 2AX; IGF, insulin-like growth factor; IGFBP, insulin-like growth factor binding protein;
IL, interleukin; kbp, kilo base pair; LC, liquid chromatography; LGALS1, Galectin; LMAN2, Lectin, mannose-binding protein 2; MMP, matrix metalloproteinase;
MS, mass spectrometry; MUC, Mucin; NEU1, Sialidase-1; NHDF, normal human dermal fibroblast; PI, propidium iodide; PML, promyelocytic leukemia protein;
PN, passage number; PRELP, prolargin; PSAP, prosaposin; qRT–PCR, quantitativereverse-transcription PCR; RT, room temperature; SAASP, skin aging–associated secreted protein; SASP, senescence associated secretory phenotype; SERPINB2, plasminogen activator inhibitor 2 (PAI-2); SUMF1, sulfatase-modifying factor 1;
TFPI, tissue factor pathway inhibitor; TRFL, telomere restriction fragment length © 2015 The Society for Investigative Dermatology www.jidonline.org 1 on cellular adaptation and damage clearance. A welldescribed adaptive/maladaptive response of fibroblasts subjected to aging is a genetic program termed cellular senescence that limits cell proliferation (d'Adda di Fagagna et al., 2003;
Campisi and d'Adda di, 2007). In vitro in cultured skin fibroblasts, cellular senescence has been found to be induced by replicative telomere shortening (= replicative senescence; d'Adda di Fagagna et al., 2003) or stress (= stress induced premature senescence; Toussaint et al., 2000; Dierick et al., 2002; Campisi and d'Adda di, 2007). The resulting senescent phenotypes encompass irreversible cell cycle arrest (Hayflick and Moorhead, 1961; d'Adda di Fagagna et al., 2003; Herbig et al., 2004; ), flat and enlarged cell morphology (Elzi et al., 2012), enhanced heterochromatinization (Narita et al., 2003;