The Nrf2/GCH1/BH4 Axis Ameliorates Radiation-Induced Skin Injury by Modulating the ROS Cascade  Jiao Xue, Chenxiao Yu, Wenjiong Sheng, Wei Zhu, Judong Luo, Qi Zhang, Hongying Yang, Han Cao, Wenjie Wang, Jundong Zhou, Jinchang Wu, Peng Cao, Ming Chen, Wei-Qun Ding, Jianping Cao, Shuyu Zhang  Journal of Investigative Dermatology  Volume 137, Issue 10, Pages 2059-2068 (October 2017) DOI: 10.1016/j.jid.2017.05.019 Copyright © 2017 The Authors Terms and Conditions

Figure 1 Radiation disrupted BH4 and decreased NO production. (a) UV-Vis spectra of BH4 and its irradiated derivatives before and after 20 Gy irradiation. Two groups of BH4 aqueous solution were exposed to 0 or 20 Gy of X-ray irradiation and optical properties were examined immediately or 24 hours after irradiation. (b) HPLC analysis was employed to separate and detect BH4 and its derivatives (e.g., BH2) in the raw BH4 and the 20 Gy-irradiated liquids. (c) Intracellular BH4/BH2 ratio 24 hours after treatment with 0 or 20 Gy of X-ray irradiation in HaCaT and WS1 cells. (d) Western blot analysis of GCH1 expression in HaCaT and WS1 cells 24 hours after the indicated doses of radiation. (e) The GCH1 level of irradiated human and rat skin tissues was detected using immunohistochemistry. Scale bar = 100 μm. Radiation decreased cellular NO levels in HaCaT and WS1 cells. The NO concentration was measured using an NO-sensitive probe (DAF-FM DA) in (f) HaCaT and (h) WS1 cells. The fluorescence intensity of DAF-FM was measured by flow cytometry. The NO concentration at different time points after irradiation in (g) HaCaT and (i) WS1 cells was measured using the Griess assay as described in the Supplementary Materials and Methods online. (j) HaCaT and (k) WS1 cells were preinfected with control adenovirus or GCH1 adenovirus followed by 0 or 20 Gy irradiation. The NO concentration was measured using the Griess assay. The ROS levels in (l) HaCaT and (m) WS1 cells were determined using flow cytometry. * P < 0.05 and ** P < 0.01, compared with the control group. BH2, dihydrobiopterin; BH4, 5,6,7,8-tetrahydrobiopterin; DAF-FM DA, 3-amino,4-aminomethyl-2′,7′-difluorescein, diacetate; GCH1, GTP cyclohydrolase I; NO, nitric oxide; ROS, reactive oxygen species. Journal of Investigative Dermatology 2017 137, 2059-2068DOI: (10.1016/j.jid.2017.05.019) Copyright © 2017 The Authors Terms and Conditions

Figure 2 The effect of GCH1 on radiation-induced damage in skin cells. Cells were preinfected with control adenovirus or GCH1 adenovirus followed by irradiation. (a) The dynamic repair process of DNA DSBs was measured by detecting nuclear γH2AX foci at several time points after 5 Gy of X-ray irradiation. * P < 0.05 and ** P < 0.01, compared with the control group. (b) The mitochondrial membrane potential in HaCaT cells was evaluated using a JC-1 staining assay. Scale bar = 50 μm. (c) The ER structure was visualized using ER-Tracker Red. (d) The proliferation of HaCaT and WS1 cells was measured in an EdU incorporation assay at 48 hours after radiation. Scale bar = 20 μm (e) The apoptosis rate of HaCaT and WS1 was detected using Annexin-V/7-AAD staining. The cells were collected 24 or 48 hours after irradiation. The data are shown as means ± SEM for three independent experiments. * P < 0.05 compared with the control cells. 7-AAD, 7-amino-actinomycin D; DSB, double-strand break; ER, endoplasmic reticulum; GCH1, GTP cyclohydrolase I; SEM, standard error of the mean. Journal of Investigative Dermatology 2017 137, 2059-2068DOI: (10.1016/j.jid.2017.05.019) Copyright © 2017 The Authors Terms and Conditions

Figure 3 GCH1 and BH4 ameliorated radiation-induced skin injury. Rat gluteal skin was irradiated with an electron beam followed by subcutaneous injection of control adenovirus, GCH1 adenovirus, PBS, and BH4 (six animals per group). (a) Skin injury in these groups was measured using a semiquantitative score of 1 (no damage) to 5 (severe damage). *P < 0.05, compared with the control group. (b) Representative skin images of the indicated groups at 15 and 30 days after irradiation. (c) Representative hematoxylin and eosin (H&E) staining of rat skins at 75 days after irradiation. Scale bar = 200 μm. (d) Calculated number of skin appendages in each group. (e) Calculated epidermal thickness in each group. **P < 0.01, compared with the control group. BH4, 5,6,7,8-tetrahydrobiopterin; GCH1, GTP cyclohydrolase I; PBS, phosphate buffered saline. Journal of Investigative Dermatology 2017 137, 2059-2068DOI: (10.1016/j.jid.2017.05.019) Copyright © 2017 The Authors Terms and Conditions

Figure 4 Nrf2 overexpression activated GCH1 expression and recovered NO level. (a) Bioinformatics analysis predicted a putative Nrf2 binding site in the proximal promoter of GCH1. The promoter region of the GCH1 gene was cloned downstream of a luciferase reporter gene. (b) HaCaT, (c) WS1, and (d) primary mouse skin cells were cotransfected with the firefly luciferase reporter of WT (pGL3-GCH1-WT) or ARE-mutated GCH1 promoter (pGL3-GCH1-Mut) together with either Nrf2 adenovirus or its shRNA (shNrf2). Luciferase activity was assayed 24 hours after transfection. The firefly luciferase activity of each sample was normalized to Renilla luciferase activity. The normalized luciferase activity in the control group was set as 100%. (e) Determination of the direct interaction between Nrf2 and the GCH1 promoter region using a ChIP assay. Western blot analysis of GCH1 expression after infection with Nrf2 adenovirus or transfection with siRNA-targeting GCH1 in (f) HaCaT and (g) WS1 cells. (h) Immunohistochemistry analysis of GCH1 expression in the skin of control adenovirus or Nrf2 overexpression adenovirus-injected rat skins. Scale bar = 200 μm. (i) The GCH1 expression in the skin of WT and Nrf2 knockout (Nrf2−/−) mice. Scale bar = 200 μm. (j) HaCaT and (k) WS1 cells were infected with Nrf2 (Ad-Nrf2) or control adenovirus (Ad-NC). The NO concentration was measured using NO-sensitive probe (DAF-FM DA) by flow cytometry. *P < 0.05; **P < 0.01, compared with the control group. Ad-Nrf2, Nrf2-overexpression adenovirus; ChIP, chromatin immunoprecipitation; DAF-FM DA, 3-amino,4-aminomethyl-2′,7′-difluorescein, diacetate; GCH1, GTP cyclohydrolase I; NO, nitric oxide; Nrf2, NF-E2-related factor 2; NS, nonsignificant; WT, wild-type. Journal of Investigative Dermatology 2017 137, 2059-2068DOI: (10.1016/j.jid.2017.05.019) Copyright © 2017 The Authors Terms and Conditions

Figure 5 The radioprotective role of Nrf2 was mediated by GCH1. Skin cells were preinfected with control adenovirus or Nrf2 adenovirus together with control siRNA (siNC) or GCH1-silencing siRNA (siGCH1). (a) The cellular ROS levels of each group of cells were determined using a DCF-DA probe. Fluorescent signals reflecting the concentration of ROS were measured using a fluorescence microscope. Scale bar = 50 μm. (b) The mitochondrial membrane potential in cells was measured using JC-1 staining. Representative images of fluorescent signals based on fluorescence microscopy under equivalent conditions. Scale bar = 50 μm. (c) Cell proliferation was detected using an EdU incorporation assay at 24 hours after irradiation as described in Supplementary Materials and Methods online. (d) After 20 Gy of irradiation, cell apoptosis was measured using flow cytometry. The data are shown as means ± SEM for three independent experiments. (e) Schematic representation of the Nrf2/GCH1/BH4 axis in the radiation response of skin cells. Radiation disrupts BH4, which results in NOS uncoupling and augmented radiation-induced secondary ROS. Overexpression of Nrf2 transcriptionally activates GCH1, which restores BH4, relieves NOS uncoupling and reduces ROS. GCH1 confers radioprotection for skin cells. BH4, 5,6,7,8-tetrahydrobiopterin; DCF-DA, 2,7-dichlorofluorescein diacetate; GCH1, GTP cyclohydrolase I; NOS, nitric oxide synthase; Nrf2, NF-E2-related factor 2; ROS, reactive oxygen species; SEM, standard error of the mean. Journal of Investigative Dermatology 2017 137, 2059-2068DOI: (10.1016/j.jid.2017.05.019) Copyright © 2017 The Authors Terms and Conditions