Autophagy Declines with Premature Skin Aging resulting in Dynamic Alterations in Skin Pigmentation and Epidermal Differentiation

Abstract

Autophagy is a membrane traffic system that provides sustainable degradation of cellular components for homeostasis, and is thus considered to promote health and longevity, though its activity declines with aging. The present findings show deterioration of autophagy in association with premature skin aging. Autophagy flux was successfully determined in skin tissues, which demonstrated significantly decreased autophagy in hyperpigmented skin such as that seen in senile lentigo. Furthermore, an exacerbated decline in autophagy was confirmed in xerotic hyperpigmentation areas, accompanied by severe dehydration and a barrier defect, which showed correlations with skin physiological conditions. The enhancement of autophagy in skin ex vivo ameliorated skin integrity, including pigmentation and epidermal differentiation. The present results indicate that the restoration of autophagy can contribute to improving premature skin aging by various intrinsic and extrinsic factors via the normalization of protein homeostasis.

Keywords: aging; autophagy; hyperpigmentation; keratinocyte; melanosome; skin.

Figure 1

Enhancement of melanogenesis along with aberrant epidermal differentiation in senile lentigo regions. (a) Skin tissues were obtained from even-toned skin and senile lentigo regions of Japanese males (n = 8), and subjected to Fontana-Masson staining and immunofluorescent analysis. Merged images of PMEL17 (red), Ki67 (red), loricrin (green) and filaggrin (red), and magnified sets of filaggrin (FLG) (red) and loricrin (LOR) (green), transglutaminase 1 (TGM1) (green), and DAPI nuclear staining (blue) are shown. Bars = 100 μm or 20 μm (magnified for FLG/LOR). (b) Quantitative analysis of immunofluorescence of PMEL17-positve (left) and Ki67-positive (right) cells along the length of the basement membrane (BM). Values are shown as the mean ± SD (n = 8) *** p < 0.001 (paired t-test). Image analysis of immunofluorescence of loricrin (c), filaggrin (d) and transglutaminase 1 (TGM1) (e). Values are shown as the mean ± SD (n = 8) * p < 0.05 (paired t-test).

Figure 2

Changes in autophagy proteins in senile lentigo. (a) Skin tissues were obtained from even-toned skin and senile lentigo regions of Japanese males (n = 8), and subjected to immunofluorescent analysis. Merged image of LC3 (green), p62 (red), ATG9L1 (green), ATG5-ATG12 (green), ATG16L1 (red) and an overlaid set of ATG5-ATG12 (green), ATG16L1 (red), and DAPI nuclear staining (blue) are shown. Bars = 100 μm. Quantitative analysis of immunofluorescence of LC3 (b), p62 (c), ATG9L1 (d), ATG5-ATG12 (e) and ATG16L1 (f). Values are shown as the mean ± SD (n = 8) * p < 0.05 (paired t-test).

Figure 3

Decreased autophagy activity in senile lentigo. (a) Skin tissues obtained from even-toned and senile lentigo skin regions of Caucasian female subjects were cultured with or without 10 µM HCQ for 48 h, followed by western-blotting analysis using LC3- or p62-specific antibodies. β-actin = loading control. (b) After normalization with β-actin, the ratios of LC3-II (left panel) and p62 (right panel) under both conditions in the presence and absence of HCQ were compared. Values are shown as the mean ± SD from six independent subjects. ** p < 0.01; * p < 0.05 (paired t-test). (c) Correlations between ratios of LC3-II (left) and p62 (right), and chromameter-retrieved L* values. ** p < 0.01 (regression analysis).

Figure 4

Disruption of epidermal differentiation leads to extensive melanin accumulation and abnormal epidermal development in dark joint regions. Biopsied skin samples were obtained from upper inner arm, upper outer arm and elbow areas of AA and Caucasian subjects, then subjected to Fontana-Masson staining and immunofluorescence observations. Merged images of PMEL17 (red), Ki67 (red), a set of filaggrin (FLG) (red) and loricrin (LOR) (green) or transglutaminase 1 (TGM1) (green), and DAPI nuclear staining are shown. Lower insets show magnification of areas inside white rectangles. Bars = 100 μm or 50 μm (insets).

Figure 5

Impaired autophagy proteins in hyperpigmented skin. (a) Skin tissues from upper inner arm, upper outer arm and elbow regions of AA and Caucasian subjects were subjected to immunofluorescent analysis. Merged images of LC3 (green), p62 (red), ATG9L1 (green), ATG5-ATG12 (green) or ATG16L1 (red), and DAPI nuclear staining are presented. Lower insets show magnification of areas inside white rectangles. Bars = 100 μm or 50 μm (insets).

Figure 6

Depression of autophagic flux through stimulation of mTORC1 activity has significant impact on skin color and skin hydration. (a) Skin tissues obtained from upper outer arm (arm) and elbow regions of AA subjects were cultured with or without 10 µM HCQ for 48 h, followed by western-blotting analysis using an LC3-specific antibody. β-actin = loading control. (b) Following normalization with β-actin, the amount of LC3-II was compared between the presence and absence of HCQ. Values are shown as the mean ± SD from five independent AA subjects. ** p < 0.01 (paired t-test). (c) Correlations shown between LC3-II flux and C* (left), skin capacitance (center) and TEWL (right). Samples were obtained from the upper outer arm and elbow skin regions of five AA subjects. (d) Proteins were harvested from punch-biopsy-obtained skin tissues from the upper outer arm (A) and elbow (E) regions, and subjected to western blotting for phosphorylated-p70S6 kinase (p-p70S6K) or total p70S6K. β-actin = loading control. (e) Graphs show relative intensity of p-p70S6K (left), p70S6K (center) and p-p70S6K/p70S6K (right) after normalization. Values are shown as the mean ± SD (n = 5) and expressed as a ratio as compared with the upper outer arm region. * p < 0.05 (paired t-test).

Figure 7

Inhibition of mTORC1 improved hyperpigmentation through restoration of autophagy. (a) Tissues obtained from AA elbow samples were treated with or without 1 μM Torin 1 for five days. Skin tissues cultured ex vivo were then subjected to hematoxylin and eosin (HE), and Fontana-Masson melanin staining for immunofluorescent analyses of LC3 (green), p62 (red), ATG5-ATG12 (green), ATG16L1 (red), FLG (red) and LOR (green) together, TGM1 (green), and Ki67 (red). Merged images with DAPI nuclear staining are shown. Bars = 100 μm. (b) Stained areas of aggregated p62 protein lumps were measured using the Image J software package. Values for the total areas of the p62 lumps are expressed as the mean ± SD (n = 4). * p < 0.05 (paired t-test). (c) Tissues with Fontana-Masson staining were subjected to Image J analysis to determine the thickness of the whole epidermis. Values are shown as the mean ± SD (n = 4). * p < 0.05 (paired t-test). (d) Relative areas positive for melanin per the length of stratum corneum were analyzed. Values show means ± SD (n = 4). * p < 0.05 (paired t-test). (e) Immunofluorescent analysis (green, top panels) and phosphorylation (green, bottom panels) of mTOR (p-mTOR) were performed after subjecting to nuclear staining with DAPI. Bars = 100 μm. (f) Fluorescent intensity was quantified and normalized based on the length of the basement membrane (BM). The ratio of p-mTOR and mTOR was compared between Torin 1-treated and -untreated tissues. Values are shown as the mean ± SD from 4 samples. * p < 0.05 (paired t-test). (g) Human normal epidermal keratinocytes (NHEKs) were treated with Torin 1 at the indicated doses for 24 h. mRNA transcript levels of TGM1 (left), IVL (center) and SKP2 (right) were determined using TaqMan real-time PCR, and their gene expression levels were normalized with those of RPLP0 (ribosomal protein large P0). Values are shown as the mean ± SD from 3 samples. ** p < 0.01; * p < 0.05 (ANOVA, Dunnett’s test).