Protective Role of Mitochondrial Peroxiredoxin III against UVB-Induced Apoptosis of Epidermal Keratinocytes  Jin Young Baek, Sujin Park, Jiyoung Park, Ji Yong Jang, Su Bin Wang, Sin Ri Kim, Hyun Ae Woo, Kyung Min Lim, Tong-Shin Chang  Journal of Investigative Dermatology  Volume 137, Issue 6, Pages 1333-1342 (June 2017) DOI: 10.1016/j.jid.2017.01.027 Copyright © 2017 The Authors Terms and Conditions

Figure 1 UVB-induced increases in H2O2 in cytoplasm and mitochondria of HaCaT human keratinocytes are selectively detected with H2O2-specific indicators. (a, b) Irradiated with UVB (11 mJ/cm2), cells were incubated (a) for the indicated times or (b) for 15 minutes and stained with (a) CM-H2DCFDA or (b) PO-1, respectively. Fluorescent images were obtained and quantified at five regions randomly selected on each dish. Scale bar = 100 μm. Quantitative levels of (a) cytoplasmic ROS or (b) cytoplasmic H2O2 are shown as mean ± standard error of the mean (n = 5) of relative fluorescence intensity (RFI). ∗P < 0.05, ∗∗P < 0.01 compared with unexposed cells. (c) Cells expressing HyPer-Mito were irradiated as above. Representative pseudocolored fluorescent images obtained 15 minutes after UVB irradiation are shown. Scale bar = 10 μm. Real-time levels of mitochondrial H2O2 are shown as mean ± standard error of the mean (n = 5) of RFI ratio. CM-H2DCFDA, 5-(and 6-)chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate; min, minute; RFI, relative fluorescence intensity; ROS, reactive oxygen species. Journal of Investigative Dermatology 2017 137, 1333-1342DOI: (10.1016/j.jid.2017.01.027) Copyright © 2017 The Authors Terms and Conditions

Figure 2 PrxIII knockdown enhances accumulation of H2O2 in mitochondria of HaCaT keratinocytes after UVB exposure. (a) Western blotting of lysates from control (pSUPER) and PrxIII knockdown (pSUPER-siPrxIII) HaCaT cells. (b) Cells were transfected with pHyPer-Mito plasmid and irradiated with UVB (11 mJ/cm2). Representative pseudocolored fluorescent images obtained 60 minutes after UVB irradiation are shown. Scale bar = 10 μm. Real-time mitochondrial H2O2 levels are shown as mean ± standard error of the mean (n = 5) of the relative fluorescence intensity (RFI) ratio. (c, d) Irradiated as above, the cells were incubated for 8 hours and stained with (c) MitoPY1 or (d) PO-1. Fluorescent images were obtained and quantified at five regions randomly selected on each dish. Quantitative levels of (c) mitochondrial and (d) cytoplasmic H2O2 are shown as mean ± standard error of the mean (n = 5) of RFI. ∗P < 0.05; ∗∗P < 0.01. min, minute; MitoPY1, mitochondria peroxy yellow 1; PO-1, peroxy-orange-1; Prx, peroxiredoxin; RFI, relative fluorescence intensity. Journal of Investigative Dermatology 2017 137, 1333-1342DOI: (10.1016/j.jid.2017.01.027) Copyright © 2017 The Authors Terms and Conditions

Figure 3 PrxIII knockdown promotes UVB-induced apoptosis in HaCaT keratinocytes through a mitochondria-mediated pathway. Exposed to UVB (11 mJ/cm2), pSUPER and pSUPER-siPrxIII cells were incubated for (a, b) 8 hours or (c, d) indicated times. (a, b) Cells stained with (a) TMRE or (b) NAO. Quantification of fluorescent images shown as mean ± standard error of the mean (n = 5) of RFI with normalization to cell number at five regions randomly selected on each dish. Scale bar = 20 μm. ∗∗P < 0.01. (c) Cytochrome c was detected in cytosolic cell fractions (marked by PrxI). (d) Western blotting for caspase-9, caspase-3, PARP, or PrxIII. ∗Represents nonspecific bands. Immunoblots representative of three independent experiments. (e) Cell death measured as percentage of annexin-V–FITC or propidium iodide-positive cells (right panel) after analysis by FACS; mean ± standard error of the mean (n = 5). ∗∗P < 0.01. hr, hour; NAO, 10-N-nonyl-acridine orange; Prx, peroxiredoxin; RFI, relative fluorescence intensity; TMRE, tetramethylrhodamine ethyl ester. Journal of Investigative Dermatology 2017 137, 1333-1342DOI: (10.1016/j.jid.2017.01.027) Copyright © 2017 The Authors Terms and Conditions

Figure 4 PrxIII deficiency sensitizes primary epidermal keratinocytes to UVB-induced apoptosis through increased accumulation of mitochondrial H2O2 and mitochondria-dependent caspase activation. (a) PrxIII+/+, PrxIII+/–, and PrxIII–/– mouse genotyping. (b–f) Primary keratinocytes isolated from newborn PrxIII+/+ and PrxIII–/– mice. (b) Immunoblotting of cell lysates. UVB (20 mJ/cm2)-irradiated keratinocytes incubated for (c, d) 8 hours or (e, f) indicated times. (c, d) Cells stained with (c) both MitoPY1 and Mitotracker-Red or (d) PO-1. Fluorescent images quantified at five regions randomly selected on each dish. (c) Mitochondrial and (d) cellular H2O2 levels shown as mean ± standard error of the mean (n = 5) of RFI. ∗∗P < 0.01. Scale bar = (c) 10 μm or (d) 100 μm. (e) Representative (three independent experiments) immunoblotting for caspase-9, caspase-3, or PrxIII. (f) Cell death measured as percentage of annexin-V–FITC– or propidium iodide-positive cells; mean ± standard error of the mean (n = 5). ∗∗P < 0.01. bp, base pairs; hr, hour; MitoPY1, mitochondria peroxy yellow 1; PO-1, peroxy-orange-1; Prx, peroxiredoxin; RFI, relative fluorescence intensity. Journal of Investigative Dermatology 2017 137, 1333-1342DOI: (10.1016/j.jid.2017.01.027) Copyright © 2017 The Authors Terms and Conditions

Figure 5 Epidermis of PrxIII-deficient mice is highly sensitive to UVB-induced apoptosis. The dorsal skin of PrxIII+/+ and PrxIII–/– mice was irradiated with UVB (180 mJ/cm2). Skin was harvested for paraffin embedding and sectioning (a) 24 hours or (b–e) 12 hours after irradiation. (a) Hematoxylin and eosin-stained sections of epidermis. (b, c) Fluorescent detection of active caspase-3 (green, b), TUNEL staining (green, c), and DAPI nuclear staining (blue). Arrows show active caspase-3– and TUNEL-positive cells. Dotted lines demark the dermal-epidermal junction. Scale bars = 50 μm. (d, e) Fluorescence intensity of (d) active caspase-3–positive and (e) TUNEL-positive epidermal cells was quantified over a linear distance of 1.25 mm and then normalized to the number of DAPI-positive cells. Data are mean ± standard error of the mean of relative fluorescence intensity (RFI) of three mice from a representative experiment. ∗∗P < 0.01. H&E, hematoxylin and eosin; Prx, peroxiredoxin; RFI, relative fluorescence intensity. Journal of Investigative Dermatology 2017 137, 1333-1342DOI: (10.1016/j.jid.2017.01.027) Copyright © 2017 The Authors Terms and Conditions