Senescent fibroblasts drive ageing pigmentation: ​A potential therapeutic target for senile lentigo

Abstract

Cutaneous ageing is an important extrinsic process that modifies the pigmentary system. Because cellular senescence is a fundamental ageing mechanism, we examined the role of senescent cells in ageing pigmentation. Methods: Biopsies obtained from senile lentigo and perilesional normal skin were assayed for a marker of cellular senescence, p16^INK4A. To determine the secretory phenotypes of senescent fibroblasts, we performed microarray, RNA sequencing and methylation array analyses in senile lentigo and senescent fibroblasts. To further investigate the impact of senescent cells on ageing-related pigmentation, an intervention that targeted senescent cells using radiofrequency was performed. Results:In vivo, senescent fibroblasts accumulated at the sites of age-related pigmentation. Phenotype switching of the cells resulted in the repression of stromal-derived factor 1 (SDF1) by promoter methylation. SDF1 induced melanocyte differentiation via stromal-epithelial interactions, ultimately driving skin pigmentation. Furthermore, the elimination of senescent fibroblasts from pigmented skin using radiofrequency was accompanied by skin lightening, rendering it a potential target for treatment. Conclusion: Aged pigmented skin contains an increasing proportion of senescent fibroblasts. Cells with phenotype switching exhibited a loss of SDF1, which stimulates the melanogenic process and thereby contributes to aging pigmentation. These data may promote the development of new therapeutic paradigms, such as a stroma-targeting therapy for pigmentary disorders.

Keywords: SDF1; Skin pigmentation; senescent fibroblasts; senile lentigo.

Figure 1

Cellular senescence in ageing pigmented skin. (A) Biopsies obtained from the facial senile lentigo (SL) and perilesional normal skin of 17 volunteers were immunostained for p16^INK4A. The proportion of p16^INK4A-positive cells is shown according to the depth of the skin. The bar graph indicates the percentage of p16^INK4A-positive cells in the dermis. The superficial dermis is defined as cells within 500 μm of the epidermal-dermal junction. (B) The number of p16^INK4A-positive cells is presented in a dotted graph. Lines in the graph indicate the mean values. (p value, Wilcoxon test). (C) Frozen tissues were stained with SA-β-Gal/nuclear fast red (NFR) or for p16^INK4A (n=4). (D) Ki67 and p16^INK4A co-immunostaining. Arrowheads and arrows indicate Ki67⁺/p16^INK4A- keratinocytes and Ki67^-/p16^INK4A+ fibroblasts, respectively. (E) Immunostaining for vimentin and p16^INK4A (n=6) (scale bars, 50 μm).

Figure 2

Senescent fibroblasts promote skin pigmentation. (A) Replicative senescent (RS) and UVA-induced senescent (UVAiS) fibroblasts. Primary human fibroblasts were sub-cultured for 6 months or irradiated with 5 J/cm² UVA or sham for 7 days. Two-hundred cells were counted in each experiment, and the number of SA-β-Gal positive fibroblasts (arrow) is presented as a percentage (left, n=5). Real-time PCR analysis of p16^INK4A mRNA levels (right). (B) Melanocytes were co-cultured with senescent fibroblasts (RS or UVAiS) or control cells (young or sham). Melanin content and tyrosinase (TYR) activity were measured. (C) mRNA and protein expression levels of MITF and tyrosinase. (D) Pigmentation of ex vivo human skin visualized with Fontana-Masson staining (Left). The pigmented area/epidermal area (PA/EA) ratio was measured by image analysis (Right). Data are presented as the mean ± SD (p value, t-test) (scale bars, 50 μm).

Figure 3

Senescent fibroblasts exhibit SDF1 deficiency caused by altered DNA methylation. (A) Real-time PCR and (B) ELISA analyses of SDF1. N.D.: not detected. (C) SDF1 immunostaining of SL (scale bars: upper, 100 μm; lower, 50 μm). The expression levels were graded as none, weak, moderate, or strong (p value, χ² test). (D) SDF1 promoter methylation analysis in senescent fibroblasts (RS and UVAiS). The CpG islands (-741 to -477) in the human SDF1 promoter were analysed in ten clones from each cell. Blue circles indicate an unmethylated status, and red circles denote methylation. The methylation % of each CpG island is presented, with red numbers indicating a more than a 1.5-fold increase in CpG island methylation. (E) Real-time PCR analysis of SDF1 mRNA levels in the presence of 5-aza-deoxycytidine (5-Aza). (F) DNA methylation status of the SDF1 promoter in SL patients (n=5). Pt # indicates the patient number.

Figure 4

Effects of SDF1 on skin pigmentation. (A) SDF1 and CXCR4 expression in cultured melanocytes (MC), keratinocytes (KC) and fibroblasts (FB). Real-time PCR (left upper), ELISA (left lower), and immunocytochemical (right, scale bar, 10 μm) analyses. N.D., not detected. (B) Immunohistochemical staining performed in normal human skin. SDF1 (red)/vimentin (green) double-immunostained fibroblasts and CXCR4 (green)-/MITF (red)-stained melanocytes were visualized (scale bars, 50 μm). (C) Pigmentation in melanocytes co-cultured with fibroblasts infected with shSDF1 lentivirus or controls. (D) Pigmentation in melanocytes co-cultured with SDF1-overexpressing fibroblasts. (E) Effect of treatment with recombinant human SDF1α (rhSDF1, 100 ng/mL) in melanocytes. (F) Real-time PCR analysis of MITF and tyrosinase mRNA expression in cells incubated in the presence of a CXCR4 inhibitor (AMD3100). Melanocytes were cultured with conditioned media (CM) obtained from young or RS fibroblasts incubated with/without 1 μM AMD3100 for 2 days. The CM-treated cells were then analysed for MITF and tyrosinase mRNA expression. (G) Pigmentation of ex vivo human skin treated with rhSDF1 (scale bar, 100 μm). (H) SDF1 overexpression in senescent fibroblasts reversed the stimulatory effects of the cells on melanogenesis. (I) Expression of phospho-CREB (upper) and cAMP formation (lower) in melanocytes treated with an rhSDF1- or SDF1-expressing lentivirus. Data are shown as the mean ± SD (p value, t-test). N.S. indicates no significance.

Figure 5

The elimination of senescent fibroblasts induces skin lightening. (A) Scheme of the intervention performed to target senescent fibroblasts. Ten volunteers with senile lentigo (SL) underwent radiofrequency (RF) treatment for 6 weeks. Matched biopsies obtained from 4 individual subjects in whom a pair (SL and perilesional normal) of samples were obtained from untreated facial tissues and a second pair of samples were obtained from facial tissue treated with RF were assayed to determine the expression profiles of p16^INK4A (B) and cleaved caspase 3 (C) by immunohistochemistry. (D) Skin lightness was assessed using L* (lightness) values measured by a chromameter and using histological evaluation. Circles and quadrangles indicate SL lesions (left panel). The pigmented area/epidermal area (PA/EA) ratio was measured by image analysis (right lower). (E) SL and perilesional normal tissue obtained from facial tissues that were untreated and treated with RF were assayed to determine the expression levels of SDF1 and procollagen type 1 by immunohistochemistry (scale bars: 50 μm).