Identification of Biomarkers of Human Skin Ageing in Both Genders. Wnt Signalling - A Label of Skin Ageing?

<div><p>The goal of our work has been to investigate the mechanisms of gender-independent human skin ageing and examine the hypothesis of skin being an adequate model of global ageing. For this purpose, whole genome gene profiling was employed in sun-protected skin obtained from European Caucasian young and elderly females (mean age 26.7±4 years [n1 = 7] and 70.75±3.3 years [n2 = 4], respectively) and males (mean age 25.8±5.2 years [n3 = 6] and 76±3.8 years [n4 = 7], respectively) using the Illumina array platform. Confirmation of gene regulation was performed by real-time RT-PCR and immunohistochemistry. 523 genes were significantly regulated in female skin and 401 genes in male skin for the chosen criteria. Of these, 183 genes exhibited increased and 340 decreased expression in females whereas 210 genes showed increased and 191 decreased expression in males with age. In total, 39 genes were common in the target lists of significant regulated genes in males and females. 35 of these genes showed increased (16) or decreased (19) expression independent of gender. Only 4 overlapping genes (<em>OR52N2</em>, <em>F6FR1OP2</em>, <em>TUBAL3</em> and <em>STK40</em>) showed differential regulation with age. Interestingly, Wnt signalling pathway showed to be significantly downregulated in aged skin with decreased gene and protein expression for males and females, accordingly. In addition, several genes involved in central nervous system (CNS) ageing (f.i. <em>APP</em>, <em>TAU</em>) showed to be expressed in human skin and were significanlty regulated with age. In conclusion, our study provides biomarkers of endogenous human skin ageing in both genders and highlight the role of Wnt signalling in this process. Furthermore, our data give evidence that skin could be used as a good alternative to understand ageing of different tissues such as CNS.</p> </div

Hierarchical clustering of genes that overlap in aged samples, irrespective of sex (A); Age dependent changes in WNT signalling of males and females (B); Hierachical clustering showing a similar expression profile in young subjects for genes regulated by age in WNT-signalling (C).

<p>(A) All relevant genes are listed according to their average expression values. Each column corresponds to one gene, each row to 4 replicates for each group sample. Green indicates a low expression value and red a high expression value. Black corresponds to a median expression value in relation to all shown values. Euclidean Distance was used to calculate average linkage clustering of the sample and gene tree. (B) The KEGG-pathway shows genes which are negative regulated with age in green colour. (C) All relevant genes are listed according to their expression values shown as log2. Each row corresponds to one gene, each column to 4 different microarray experiments. Green indicates a low expression value and red a high expression values. Black corresponds to a median expression value in relation to all shown values.</p

Protein expression of target genes via immunohistochemistry.

<p>A comparison between young and aged sun-protected skin provided by female and male healthy donors (n = 7, accordingly). Localization of WIF1 (A,B) and FZD7 (C,D) in aged and young skin, respectively [DAB staining, Dako]. The expression of both proteins was negative in skin biopsies obtained from elderly subjects. On the other hand, the young group showed a significant higher expression of both proteins (p = 0.0019 and p = 0.013, respectively) only in the basal cell layer of the epidermis. No significant gender differences were observed. E, F: Localization of PPAR-δ in aged and young skin, respectively [LSAB, REAL Detection System, Dako]. Strong immune reaction of the sebaceous glands in the skin of both groups and significantly stronger reaction of the sebaceous duct in the skin of the aged group (p<0.019). All experiments have been performed in triplicate.</p

Confirmation of microarray data via real time RT-PCR.

<p>Examination of expression levels of candidate genes: <i>TGFβ</i>, <i>AXIN2</i>, <i>WIF1</i>, <i>SIRT6</i>, <i>MIB1</i>, <i>APP</i>, <i>TAU</i>, <i>PSEN1</i>, <i>PARK2</i>, <i>NLGN2</i>, <i>B3GALT3</i> and <i>ATXN1</i> in female and male young and elderly donors. The figure shows the logarithmic ratios aged vs. young with base two (log2) of the selected genes whose expression was deduced by microarray and real-time RT–PCR. A ratio of 1 represents a twofold change with age. Values greater than zero mean higher expression in aged and values less than zero, higher expression in young donors. All experiments have been performed in triplicate. (p<0.05: *, p<0.01:**, p<0.001: ***).</p

Results of the analysis of statistically significant pathways at <i>P</i>-value<0.1 in skin biopsies of males and females with age.

<p>Pathways were taken from the KEGG database (human pathways). Genes that were judged as non-detectable by the BG value criterion were excluded from analysis (up: number of genes that were taken into account for computing the statistical test showing upregulated expression with age; down: number of genes that were taken into account for computing the statistical test showing downregulated expression with age in females and males, respectively). The full lists of pathways of all regulated genes (up-and downregulated) in both genders are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050393#pone.0050393.s006" target="_blank">Table S5</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050393#pone.0050393.s007" target="_blank">S6</a>, accordingly.</p