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. 2016 Sep 9;11(9):e0162628.
doi: 10.1371/journal.pone.0162628. eCollection 2016.

Activation of Peroxisome Proliferator-Activated Receptor Alpha Improves Aged and UV-Irradiated Skin by Catalase Induction

Mi Hee Shin  1   2   3 , Se-Rah Lee  1   2   3 , Min-Kyoung Kim  1   2   3 , Chang-Yup Shin  1   2   3 , Dong Hun Lee  1   2   3 , Jin Ho Chung  1   2   3   4
Affiliations

Activation of Peroxisome Proliferator-Activated Receptor Alpha Improves Aged and UV-Irradiated Skin by Catalase Induction

Mi Hee Shin et al. PLoS One. .

Abstract

Peroxisome proliferator-activated receptor alpha (PPARα) is a nuclear hormone receptor involved in the transcriptional regulation of lipid metabolism, fatty acid oxidation, and glucose homeostasis. Its activation stimulates antioxidant enzymes such as catalase, whose expression is decreased in aged human skin. Here we investigated the expression of PPARα in aged and ultraviolet (UV)-irradiated skin, and whether PPARα activation can modulate expressions of matrix metalloproteinase (MMP)-1 and procollagen through catalase regulation. We found that PPARα mRNA level was significantly decreased in intrinsically aged and photoaged human skin as well as in UV-irradiated skin. A PPARα activator, Wy14643, inhibited UV-induced increase of MMP-1 and decrease of procollagen expression and caused marked increase in catalase expression. Furthermore, production of reactive oxygen species (ROS) was suppressed by Wy14643 in UV-irradiated and aged dermal fibroblasts, suggesting that the PPARα activation-induced upregulation of catalase leads to scavenging of ROS produced due to UV irradiation or aging. PPARα knockdown decreased catalase expression and abolished the beneficial effects of Wy14643. Topical application of Wy14643 on hairless mice restored catalase activity and prevented MMP-13 and inflammatory responses in skin. Our findings indicate that PPARα activation triggers catalase expression and ROS scavenging, thereby protecting skin from UV-induced damage and intrinsic aging.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. PPARα expression was decreased in aged and photoaged human skin and in UV-irradiated skin in vivo and in dermal fibroblasts.
(a) Levels of PPARα mRNA in inner arms and forearms of young and elderly subjects were determined by qPCR. Data are presented as means±SEM (n = 3), *p<0.05 vs. inner arms of young subjects; #p<0.05 vs. inner-arms of old subjects. (b) Young buttock skin was irradiated with UV light, and obtained after 24 h. PPARα mRNA expression following UV irradiation was compared with that of non-irradiated skin (control) by qPCR. Data are presented as means±SEM (n = 9), **p<0.01 vs. non-irradiated skin. (c) Cultured fibroblasts were starved with serum-free DMEM for 24 h, irradiated with UV (50 or 100 mJ/cm2), and harvested at the indicated time points. Levels of PPARα mRNA following UV irradiation were compared to those of non-irradiated controls. Data are presented as means±SEM (n = 3), *p<0.05 vs. time-matched control.
Fig 2
Fig 2. PPARα activator, Wy14643, could limit type I procollagen reduction and MMP-1 expression induced by UV in human dermal fibroblasts.
(a) Human dermal fibroblasts were treated with 1–100 μM Wy14643 for 72 h. Western blot analysis using the culture media was performed to determine the expression of procollagen and MMP-1 while cell lysates were used to determine β-actin protein level as a loading control. (b) Cultured fibroblasts were exposed to UV irradiation (100 mJ/cm2). Following irradiation, cells were treated with 1–100μM Wy14643 for 72 h. The western bands are representative of three separate experiments. Data are presented as means±SEM (n = 3), *p<0.05 vs. control; #p<0.05 vs. UV-irradiated cells.
Fig 3
Fig 3. PPARα agonist increased the expression of catalase and ameliorated UV-induced ROS generation in human dermal fibroblasts.
Cultured fibroblasts were serum-starved for 24 h, and treated with 1–100 μM Wy14643 for 72 h. (a) Total soluble proteins were isolated from the cells, and levels of catalase protein were measured by western blot analysis and normalized to β-actin. (b) Catalase mRNA expression was determined by qPCR. Data are presented as means±SEM (n = 3), *p<0.05 vs. control. (c) Catalase activity was measured by a spectrophotometric method. Catalase enzyme unit is equivalent to 1 μmol of substrate disappearance or product formation per min. Data are presented as mean±SEM (n = 3). (d) Human dermal fibroblasts were treated with Wy14643 for 72 h, and then irradiated with UV. Subsequently, cells were treated with 2,7-dichlororofluorescein diacetate (DCFDA) and assayed using a fluorescence reader. Data are presented as means±SEM (n = 6), *p<0.05, ***p<0.001 vs. control, #p<0.05 vs. UV-treated cells.
Fig 4
Fig 4. PPARα siRNA transfection decreased catalase expression and abolished beneficial effects of Wy14643 in human dermal fibroblasts.
(a) Human fibroblasts were transfected with scrambled siRNA as a negative control (NC) or siRNAs selectively targeting PPARα (100 nM) for 48 h. Cells were subjected to serum-starvation for 24 h. Levels of PPARα and catalase mRNAs were measured by qPCR. Data are presented as mean±SEM (n = 3), *p<0.05, ***p<0.001 vs. cells transfected with scrambled siRNA (b) Cultured fibroblasts were transfected with scrambled siRNA or PPARα siRNA for 48 h, and serum-starved for 24 h. Then, they were exposed to UV irradiation (100 mJ/cm2), followed by 1–100 μM Wy14643 treatment for 72 h. Levels of procollagen and MMP-1 proteins released into culture media were measured by western blotting. The bands are representative of results from three independent experiments. (c) Procollagen and MMP-1 mRNA expression was measured by qPCR. Data are presented as mean±SEM (n = 3), *p<0.05, **p<0.01 vs. UV-treated cells; #p<0.05, ##p<0.01 vs. NC siRNA-transfected UV-treated cells.
Fig 5
Fig 5. PPARα activator increased the expression of procollagen and catalase protein while decreased the expression of MMP-1 and ROS in aged human dermal fibroblasts.
(a) Wy14643 (1–100 μM) was treated for 72 h to human dermal fibroblasts obtained from forearm skin of elderly subjects (mean age 78.6 year; n = 3). Procollagen and MMP-1 levels were measured using the culture media, and catalase and β-actin expression was using cell lysates by western blotting. The western bands are representative of three separate experiments. (b) Aged fibroblasts were treated with Wy14643 for 72 h, and cells were treated with 2,7-dichlororofluorescein diacetate (DCFDA) for 30 min, and assayed using a fluorescence reader. Data are presented as means±SEM (n = 6), *p<0.05 vs. control. (c) Aged fibroblasts were transfected with scrambled siRNA as a negative control (NC), or PPARα siRNA for 48 h. The transfected cells were serum starved for 24 h, and then, treated with 1–100 μM Wy14643 for 72 h. The western bands are representative of three separate experiments. (d) Procollagen and MMP-1 mRNA expression was measured by qPCR. Data are presented as mean±SEM (n = 3), *p<0.05, vs. cells transfected with NC siRNA.
Fig 6
Fig 6. Topical application of Wy14643 prevented the induction of MMP-13, COX-2, IL-1β, and IL-6 expressions and the reduction of catalase activity by UV in hairless mouse skin in vivo.
Dorsal skin of HR-1 hairless mice was topically treated with vehicle (ethanol:polyethylene glycol = 30:70) only or Wy14643 (2 mM or 10 mM), at 24 h prior to, immediately, and 24 h following UV irradiation. Skin biopsy was carried out at 48 h after UV irradiation. (a) MMP-13, (b) COX-2, (c) IL-1β and (d) IL-6 mRNA expression was quantified by qPCR. (e) Catalase activity was measured using total soluble protein by a spectrophotometric method. Catalase enzyme unit is equivalent to 1 μmol of substrate disappearance or product formation per min. Data are presented as mean±SEM (n = 8 for each group). *p<0.05, **p<0.01 vs. vehicle only; #p<0.05 vs. vehicle-treated UV-irradiated skin.
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Grants and funding

This research was supported by a grant of Korea Research Foundation and Basic Science Research program through the National Research Foundation (NRF) of Korea (2009-2-E00032) and by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number: HI14C0089).
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