Extracellular Vesicles Derived from Senescent Fibroblasts Attenuate the Dermal Effect on Keratinocyte Differentiation

The skin is a multilayered and primary defensive organ. Intimate intercellular communication in the skin is necessary to ensure effective surveillance. Extracellular vesicles (EVs) are being explored for their involvement in intercellular skin communication. The aim of this study was to evaluate how human dermal fibroblasts (HDFs) accelerate EV production during senescence and the effects of senescence-associated EVs on epidermal homeostasis. Replicative senescent HDFs were assessed with senescence-associated β-galactosidase staining and the expression of senescence-related markers. Isolated EVs were characterized by dynamic light scattering and EV marker expression. EVs secreted from untreated young or senescent HDFs, or from those treated with a nSMase inhibitor, antioxidant, and lysosomal activity regulators, were determined by sandwich ELISA for CD81. Human epidermal keratinocytes were treated with young- and senescent HDF-derived EVs. Compared to young HDFs, senescent HDFs produced relatively high levels of EVs due to the increased nSMase activity, oxidative stress, and altered lysosomal activity. The nSMase inhibitor, antioxidant, and agents that recovered lysosomal activity reduced EV secretion in senescent HDFs. Relative to young HDF-derived EVs, senescent HDF-derived EVs were less supportive in keratinocyte differentiation and barrier function but increased proinflammatory cytokine IL-6 levels. Our study suggests that dermis-derived EVs may regulate epidermal homeostasis by reflecting cellular status, which provides insight as to how the dermis communicates with the epidermis and influences skin senescence.


Western blotting
Human dermal fibroblasts (HDFs) or human epidermal keratinocytes (HEKs) were lysed in RIPA cell lysis buffer (EMD Millipore, Billerica, MA, USA) containing a protease-and phosphatase inhibitor cocktail (Sigma-Aldrich Corp., St. Louis, MO, USA). The cell lysate was collected by centrifugation at 13,000 rpm for 10 min and the protein concentration was measured by a BCA assay (Thermo Fisher Scientific, Loughborough, UK). Proteins were separated by SDS-PAGE, transferred to PVDF membranes (EMD Millipore, Billerica, MA, USA), and detected by the following antibodies: anti-p21 (Cell Signaling Technology

Sandwich ELISA for EV detection
Mouse monoclonal anti-CD81 antibody (Abcam; clone No. B1.3.3.22) was immobilized on 96well microtiter plates (Greiner Bio-one, Frickenhausen, Germany) overnight and blocked with 1% BSA in PBS for 1.5 h. HDFs were treated with vehicle or reagents for 24 h (GW4869, NAC, and bafilomycin A) or 25 d (KU60019). The culture supernatants were pre-cleared by sequential centrifugation at 500 × g for 10 min and 3,000 × g for 20 min, added to pre-coated 96-well microtiter plates containing anti-CD81 antibody, and incubated for 2 h. They were incubated with biotinylated mouse monoclonal anti-CD81 antibody (BD Biosciences; clone No. JS-81) for another 2 h and streptavidin (R&D Systems, Minneapolis, MN, USA) was added to each well. After 30 min of incubation, luminescence was measured on a Wallace Victor 1420 multilabel plate reader (PerkinElmer, Norwalk, CT, USA). All steps were performed at room temperature.

Measurement of lysosomal pH
Lysosomal pH was measured using LysoSensor Yellow/Blue DND-160 (Molecular Probes, Eugene, OR, USA) according to the manufacturer's protocol 1 Supplementary Fig. S1: Expression of senescence-associated secretory phenotypes in human dermal fibroblasts. Young HDFs (PDL < 10) and senescent HDFs (PDL > 50) were analyzed for IL-8 and MMP1 mRNA expression (a) and phosphorylated (phosphor-p65) and total NF-κB (p65) protein levels (b) by qRT-PCR and western blot, respectively. The mRNA levels were normalized to that of RPL13A. GAPDH was the loading control for western blot. MMP1, matrix metalloproteinase 1.

Supplementary Fig. S2: EV secretion was increased by UV irradiation and in aged fibroblasts. a
HDFs (PDL < 10) were irradiated with UVB (20 mJ cm -2 ). The numbers of EVs derived from equal numbers (1 × 10 6 cells) of UVB-treated or untreated HDFs were determined from a BCA protein assay. b Culture media for equal numbers (1 × 10 6 cells) of neonatal and adult (33-y) HDFs at the same passage (p16) were subjected to EV purification. Purified EV levels were determined by BCA protein assay. Representative data shown are fold differences between pairs of groups. Supplementary Fig. S3: The effect of KU60019 in senescent HDFs. a Senescent HDFs (PDL > 50) were treated with various concentrations of KU60019 for 25 d and SA-β-gal staining in young (PDL < 10) or senescent HDFs was performed. Scale bar = 500 μm. b, c Senescent HDFs were treated with KU60019 (0.5 µM) for 25 d. b Western blot analyses for LAMP1, p16INK, and p21CIP were performed in young or senescent HDFs treated with or without KU60019. GAPDH, loading control. c nSMase activity was determined by the nSMase activity assay kit for equal amounts of protein or equal numbers of senescent HDFs. Data are means ± SD of three independent experiments using a senescent cell line (***p < 0.001).
Supplementary Fig. S4: The effect of bafilomycin A on EV secretion. Young HDFs were treated with vehicle (-) or bafilomycin A (100 nM) for 24 h. The secreted EV levels from young HDFs with or without bafilomycin A or senescent HDFs were determined using a sandwich ELISA for CD81 form each conditioned medium. The data are expressed as the mean ± SD of three independent experiments (***p < 0.001).

Supplementary Fig. S5: Expression of epidermal differentiation markers in human keratinocytes.
HEKs were cultured for 1 d or 4 d. a, b Cells were analyzed daily by qRT-PCR for mRNA expression of keratinocyte differentiation-related (KRT1, LOR, BLMH) or cell cycle inhibition-related (p16, p21) markers. The mRNA levels were normalized to that of RPL13A. c Conditioned media were harvested at 1 d or 4 d. The IL-6 and IL-8 levels were measured with specific ELISA kits. Data are means ± SD of three independent experiments (***p < 0.001; ns, not significant). Supplementary Fig. S7: Effect of p53 on the increase in EV secretion in senescent HDFs. a Young (PD < 10) or senescent (PD > 50) HDFs were subjected to western blot using anti-p53 or anti-p21 antibodies. GAPDH, loading control. b Senescent HDFs were treated for 24 h with various concentrations of the p53 inhibitor pifithrin-α. EV levels were determined by sandwich ELISA for CD81. Data are means ± SD of three independent experiments using a senescent cell line (***p < 0.001).