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BH4 Axis Ameliorates Radiation-Induced Skin Injury by Modulating the ROS Cascade
Accepted Manuscript 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 PII:
S0022-202X(17)31604-4
DOI:
10.1016/j.jid.2017.05.019
Reference:
JID 898
To appear in:
The Journal of Investigative Dermatology
Received Date: 17 January 2017 Revised Date:
7 May 2017
Accepted Date: 16 May 2017
Please cite this article as: Xue J, Yu C, Sheng W, Zhu W, Luo J, Zhang Q, Yang H, Cao H, Wang W, Zhou J, Wu J, Cao P, Chen M, Ding W-Q, Cao J, Zhang S, The Nrf2/GCH1/BH4 axis ameliorates radiation-induced skin injury by modulating the ROS cascade, The Journal of Investigative Dermatology (2017), doi: 10.1016/j.jid.2017.05.019. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT TITLE PAGE The Nrf2/GCH1/BH4 axis ameliorates radiation-induced skin injury by modulating the ROS cascade
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Jiao Xue1,2,†, Chenxiao Yu1,2,†, Wenjiong Sheng1,2, Wei Zhu1,2, Judong Luo3, Qi Zhang1,2, Hongying Yang1,2, Han Cao1, Wenjie Wang1, Jundong Zhou4, Jinchang Wu4, Peng Cao5,6, Ming Chen7, Wei-Qun Ding8, Jianping Cao1,2,*, Shuyu
School of Radiation Medicine and Protection, Medical College of Soochow
University, Suzhou 215123, China 2
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1
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Zhang1,2,7,*
Collaborative Innovation Center of Radiation Medicine of Jiangsu Higher Education
Institutions and Jiangsu Provincial Key Laboratory of Radiation Medicine and
3
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Protection, Soochow University, Suzhou 215123, China
Department of Radiotherapy, Changzhou Tumor Hospital, Soochow University,
Changzhou, 213001, China
Suzhou Cancer Center Core Laboratory, Nanjing Medical University Affiliated
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4
5
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Suzhou Hospital, Suzhou 215001, China Affiliated Hospital of Integrated Traditional Chinese and Western Medicine, Nanjing
University of Chinese Medicine, Nanjing 210028, China 6
Laboratory of Cellular and Molecular Biology, Jiangsu Province Academy of
Traditional Chinese Medicine, Nanjing 210028, China, 7
Zhejiang Key Laboratory of Radiation Oncology, Zhejiang Cancer Hospital,
Hangzhou 310022, China 1
ACCEPTED MANUSCRIPT 8
Department of Pathology, University of Oklahoma Health Science Center, Oklahoma
City, OK 73104, USA These authors contributed equally to this work.
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†
Correspondence: School of Radiation Medicine and Protection, Medical College of Soochow University, No. 199 Ren’ai Rd, Suzhou 215123, China. (S. Zhang) Tel/Fax: E-mail:
[email protected];
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+86512-65880037, E-mail: [email protected].
(J.
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+86512-65883941,
Cao)
Tel/Fax:
Running title: Role of the Nrf2/GCH1/BH4 axis in radiation-induced skin injury
radiation,
skin
injury,
GTP
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Keywords:
cyclohydrolase
5,6,7,8-tetrahydrobiopterin (BH4), NF-E2-related factor 2 (Nrf2)
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ABBREVIATIONS
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GCH1: GTP cyclohydrolase I
BH4: 5,6,7,8-tetrahydrobiopterin Nrf2: NF-E2-related factor 2 ROS: reactive oxygen species RNS: reactive nitrogen species NOSs: nitric oxide synthases BH2: dihydrobiopterin 2
I
(GCH1),
ACCEPTED MANUSCRIPT ABSTRACT Radiation-induced skin injury is a common side effect of radiotherapy and can limit the duration and dose of radiotherapy. Most early work focused on elimination of
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reactive oxygen species (ROS) after radiation, however, less is known about the mechanisms underlying amplification of ROS and consequent skin injury by radiation. 5,6,7,8-tetrahydrobiopterin (BH4) is an essential co-factor for all nitric oxide
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synthases (NOSs). Inadequate availability of BH4 leads to uncoupling of NOSs and
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production of highly oxidative radicals. In this study, we demonstrated that radiation disrupted BH4, which resulted in NOS uncoupling and augmented radiation-induced ROS. Overexpression of GTP cyclohydrolase I (GCH1), the rate-limiting enzyme for BH4 synthesis, restored cellular BH4 levels and NO production and decreased
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radiation-induced ROS. GCH1 also protected skin cells and rat skins against radiation-induced damage. We found that GCH1 was regulated by NF-E2-related factor 2 (Nrf2), a key mediator of the cellular antioxidant response. Importantly, we
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identified GCH1 as a key effector for Nrf2-mediated protection against
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radiation-induced skin injury by inhibiting ROS production. Taken together, the findings of this study illustrate the key role of the Nrf2/GCH1/BH4 axis during radiation-induced skin damage.
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ACCEPTED MANUSCRIPT INTRODUCTION Radiotherapy is applied to over 50% of all cancer patients, either alone or in combination with other treatments (Hymes et al., 2006). Because the skin is the first
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tissue of entry for external radiation particles during radiation treatment, it is vulnerable to radiation-induced injury. In fact, radiation-induced skin syndrome remains a serious concern that may limit the duration and dose of radiotherapy (Di
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Franco et al., 2013, Hymes et al., 2006). In addition, the increasing use of radioactive
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materials in industry, science and military may also increase the likelihood of uncontrolled exposure to radiation (Hymes et al., 2006). Approximately 95 % of cancer patients receiving radiation therapy are estimated to have some forms of cutaneous reactions, including erythema, desquamation, dermatitis, ulcer and fibrosis
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(Ryan, 2012, Singh et al., 2016).
Ionizing radiation promotes reactive nitrogen and oxygen species (RNS/ROS) production due to radiolysis of water and direct ionization of target molecules (Leach
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et al., 2001, Spitz et al., 2004). These reactive molecules, such as hydroxyl radicals,
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superoxide anion, hydrogen peroxide and nitrogen dioxide, can result in oxidative damage to critical cellular biomolecules and cytotoxicity and consequently cause a series of pathophysiological changes that eventually lead to acute and/or chronic skin injuries. The involvement of an anti-oxidative response to radiation has been extensively reported, and the use of antioxidants could mitigate radiation-induced injury (Gu et al., 2013, Kodiyan and Amber, 2015, Yan et al., 2008, Zhang et al., 2014). For example, superoxide dismutases and their mimics reduced ROS levels and 4
ACCEPTED MANUSCRIPT ameliorated radiation-induced skin injury (Doctrow et al., 2013, Gu et al., 2013, Yan et al., 2008, Zhang et al., 2014). Most early studies focused on elimination of ROS after radiation (Doctrow et al., 2013, Gu et al., 2013, Kodiyan and Amber, 2015, Yan et al.,
the generation and amplification of ROS by radiation.
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2008, Zhang et al., 2014). However, less is known about the mechanisms underlying
Nitric oxide (NO) is a lipophilic messenger molecule that participates in various
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physiological and pathological activities, including neurotransmission, smooth muscle
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relaxation, cell migration, immune response and apoptosis (Forstermann and Sessa, 2012, Umar and van der Laarse, 2010). In skin, NO is important for the homeostasis of normal function and is emerging as a therapeutic target for specific cutaneous diseases (Cals-Grierson and Ormerod, 2004, Weller, 2003). In mammals, NO is
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generated by NO synthases (NOSs) from L-arginine, NADPH and oxygen. All three NOS isoforms are expressed in the skin, and 5,6,7,8-tetrahydrobiopterin (BH4) is an essential co-factor for all NOSs (Alderton et al., 2001). Limited availability of BH4
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leads to the uncoupling of NOSs and the production of highly oxidative radicals,
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including superoxide and peroxynitrite, at the cost of NO (Naylor et al., 2010). Stress-generated ROS production may decrease the availability of BH4 due to rapid oxidation of BH4 into dihydrobiopterin (BH2) (Heales et al., 1988), which may induce NOS uncoupling and increase the production of oxidative superoxide radicals (Berbee et al., 2010). In de novo synthesis of BH4, the major synthetic pathway, GTP cyclohydrolase I (GCH1) catalyzes the formation of dihydroneopterin triphosphate from GTP. Subsequently, dihydroneopterin triphosphate is converted to BH4 by 5
ACCEPTED MANUSCRIPT 6-pyruvoyl-tetrahydrobiopterin synthase and sepiapterin reductase (Thony et al., 2000). The rate of BH4 synthesis via a de novo pathway depends on the activity of its rate-limiting enzyme, GCH1 (Werner et al., 2011). In vivo inhibition of GCH1
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increased vascular oxidative stress and reduced white blood cell counts after irradiation (Pathak et al., 2014).
Although BH4 bioavailability is involved in NO/ROS transition, the role of BH4
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and its synthetic enzyme GCH1 in radiation-induced ROS and consequent skin injury
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is unclear. In this study, we elucidated the key role of BH4 and GCH1 in augmenting radiation-induced ROS. We further demonstrated that the Nrf2/GCH1/BH4 axis protects against radiation-induced skin damage, which provides strategies unreported
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before to our knowledge for ameliorating this injury.
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ACCEPTED MANUSCRIPT RESULTS Radiation reduces GCH1 expression and BH4 availability, which impairs NO homeostasis in skin tissues and cells
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NO synthesis is important for the homeostasis of skin function (Cals-Grierson and Ormerod, 2004, Weller, 2003). BH4, a co-factor of NOSs, is shown to be converted to BH2 by oxidative stress (Naylor et al., 2010). To determine whether the structure of
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BH4 was disrupted following ionizing radiation, we used UV-Vis spectra and
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high-performance liquid chromatography (HPLC) analysis. The results showed that the optical properties of BH4 aqueous solution were not changed immediately after 20 Gy of X-ray irradiation (Figure 1a). Twenty-four hours after irradiation, the absorptions of BH4 (at 217 to 230 nm and 315 to 330 nm) gradually decreased over
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time, while the absorptions of BH4 (at 275 nm) increased. Moreover, absorption of BH4 24 h after irradiation was blue-shifted slightly compared to the bulk value (at 375 nm; Figure 1a), indicating that radiation alters the structure of BH4. Next, HPLC
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analysis was used to separate and detect BH4 and its derivatives (e.g. BH2) in the raw
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BH4 and the 20 Gy-irradiated liquids. As shown in Figure 1b, weak BH2 peaks were observed in the fresh new BH4 liquid. In contrast, the content of BH2 and other derivatives was substantially increased after 20 Gy irradiation. The results demonstrated that the unstable BH4 can be easily oxidized into multiple derivatives in addition to BH2. In cell extracts of human keratinocyte HaCaT and skin fibroblast WS1 cells, the BH4/BH2 ratio was significantly reduced after radiation (Figure 1c), confirming that radiation could decrease the bioavailability of BH4. 7
ACCEPTED MANUSCRIPT Then, the expression of the BH4 rate-limiting enzyme GCH1 was measured by Western blotting. Results from Western blot analysis showed that radiation decreased GCH1 expression in a dose-dependent manner in HaCaT and WS1 cells (Figure 1d),
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indicative of reduced BH4 synthesis after radiation. Using immunohistochemistry, we found that GCH1 expression in irradiated human skin tissue was lower than that in the normal counterparts (Figure 1e). We further measured cellular NO levels by flow
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cytometric-based analysis of a NO-sensitive probe (DAF-FM DA) and Griess assay of
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NaNO2 levels. The results consistently showed that NO levels in 20 Gy-irradiated skin cells were significantly reduced in both HaCaT and WS1 cells compared with those of nonirradiated counterparts (Figure 1f-1i). The above results suggested that radiation interferes with de novo synthesis of BH4 and consequent NO homeostasis.
restores NO level
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Overexpression of GCH1 decreases NOS uncoupling-associated ROS and
Since lack of BH4 has been reported to induce NOS uncoupling, which consequently
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generates RNS and ROS (Naylor et al., 2010), we investigated whether RNS
and
ROS
can
be
modulated
by
GCH1.
A
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radiation-induced
GCH1-overexpression adenovirus (Ad-GCH1) was constructed and introduced into cultured cells, leading to a marked increase in the protein levels of GCH1 in HaCaT (Figure 1j) and WS1 cells (Figure 1k). Ad-GCH1 infection induced a significant ~ 1.5-fold increase of cellular NO levels in HaCaT and WS1 cells without radiation (Figure 1j and 1k). Compared with the Ad-NC groups, NO levels in Ad-GCH1-infected HaCaT and WS1 cells after 20 Gy irradiation were increased by 8
ACCEPTED MANUSCRIPT 2.2- and 3.5-fold, respectively (Figure 1j and 1k). These results suggested that GCH1 overexpression restores NO level in skin cells. We further explored the effect of GCH1 silencing on baseline and radiation-induced ROS in skin cells. We found that
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silencing of GCH1 did not affect baseline ROS levels without radiation but aggravated radiation-induced ROS in both HaCaT and WS1 cell lines (Figure S1), indicating that GCH1 is involved in the amplification of radiation-induced ROS.
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Conversely, forced expression of GCH1 significantly reduced radiation-induced ROS
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in HaCaT and WS1 cells (Figure 1l and 1m). We also measured the formation of 3-nitrotyrosine (3-NT), a biomarker of peroxynitrite (ONOO-). We found that GCH1 overexpression also significantly reduced intracellular 3-NT levels, indicative of decreased oxidative damage mediated by peroxynitrite (Figure S2). These results
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suggested that radiation-induced RNS and ROS could be attenuated by inhibiting NOS uncoupling through GCH1.
GCH1 protects skin cells from radiation-induced damage
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Since GCH1 overexpression could inhibit radiation-induced NOS uncoulpling and
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consequently reduce ROS levels, we next investigated whether increasing BH4 synthesis via GCH1 overexpression could alleviate radiation-induced damage in skin cells. DNA repair of DSBs was measured by detecting nuclear γH2AX foci at several time points after irradiation of HaCaT cells at 5 Gy. The number of foci formed in GCH1 adenovirus-infected cells was significantly reduced to an average of 54.44% (49 foci per cell) of the control adenovirus-treated cells at 1 h after irradiation. At 2 and 4 h after irradiation, the number of γH2AX foci in GCH1 adenovirus-treated 9
ACCEPTED MANUSCRIPT groups decreased to 35.12% (P < 0.01) and 35.07% (P < 0.01) of the control adenovirus-treated cells, respectively (Figure 2a and Figure S3). These results demonstrated that overexpression of GCH1 could attenuate the radiation-induced
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DNA damage of human skin cells. Mitochondrial functional failure, including the loss of mitochondrial integrity and change of mitochondrial membrane potential, is considered to be one of the most
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important factors causing cell death (Green and Kroemer, 2004). To explore the
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protective role of GCH1 on mitochondrial function, mitochondrial potential was examined by JC-1 staining. Non-irradiated HaCaT cells predominantly showed red fluorescence, whereas a substantial proportion of cells shifted to green fluorescence after 20 Gy of irradiation, indicating that the mitochondrial membrane potential was
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reduced (Figure 2b). However, cells with forced expression of GCH1 exhibited a decreased shift from red to green fluorescence, indicating that the mitochondrial membrane potential was maintained after exposure to ionizing radiation (Figure 2b),
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indicative of the protection of the mitochondria against ionizing radiation. Next, we
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explored the protective role of GCH1 on the endoplasmic reticulum (ER) structure using ER-Tracker Red. As shown in Figure 2c, non-irradiated HaCaT cells presented robust ER-Tracker Red staining that was attenuated, and a clear ER structure that was morphologically
altered,
after
irradiation
at
20
Gy.
However,
GCH1
adenovirus-infected cells showed stronger ER-Tracker Red staining after radiation and were morphologically similar to the non-irradiated cells, indicating that the integrity of the ER was maintained after ionizing radiation exposure. 10
ACCEPTED MANUSCRIPT GCH1 modulates the radiosensitivity of human skin cells Next, we sought to investigate whether increasing BH4 synthesis via GCH1 overexpression could modulate the radiosensitivity of skin cells. Skin cells were
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pre-infected with control adenovirus or GCH1 adenovirus followed by 20 Gy of irradiation. The results from the EdU assay revealed that HaCaT and WS1 cells that were infected with GCH1 adenovirus exhibit a significantly higher percentage of
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EdU-positive cells than those of untreated or control adenovirus-infected cells after
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radiation (P < 0.01 for HaCaT and P < 0.01 for WS1; Figure 2d).
We next investigated whether GCH1 overexpression was associated with decreased apoptosis. Annexin-V/7-AAD staining-based flow cytometric analysis of apoptosis was performed at 24 and 48 h after radiation. Overexpression of GCH1 significantly
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reduced the apoptosis of HaCaT and WS1 cells after 20 Gy of irradiation compared with that of cells infected with the control adenovirus (Figure 2e). These results demonstrated that GCH1 reduces radiation-induced apoptotic cell death of skin cells.
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skin injury
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GCH1 overexpression or BH4 supplementation ameliorates radiation-induced
To determine whether GCH1 overexpression could attenuate radiation-induced skin injury, rat models were used as described previously (Zhang et al., 2014). After 45 Gy of electron beam exposure, rats received subcutaneous injection of GCH1 adenovirus or BH4 (1 mg/kg). The radiation-induced skin injury recovery process in the Ad-GCH1-infected group, the BH4-supplemented group and the control group was evaluated for 75 d. At 24 d after irradiation, radiation-induced skin injury was 11
ACCEPTED MANUSCRIPT significantly alleviated in the GCH1 adenovirus-infected and BH4-supplemented groups compared with that of the control group (Figure 3a and 3b). Subcutaneous supplementation of BH4 also mitigated the radiation-induced skin injury (Figure 3a
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and 3b), which was similar to the effect of GCH1. The clinically used chitosan showed no therapeutic effect on rat skin irradiated by the 45 Gy electron beam (Figure S4). Although infection with GCH1 adenovirus and BH4 supplementation groups
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showed similar skin wounds as those of PBS-treated or control adenovirus-infected
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groups at 75 d post-irradiation, GCH1 overexpression or BH4 supplementation attenuated the epidermal hyperplasia (white arrow) and maintained skin appendages (blue arrow; Figure 3c-3e), which were often destroyed by irradiation. Taken together, these
results
demonstrated
that
increasing
BH4
level
could
ameliorate
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radiation-induced skin injury in vivo.
GCH1 transcription and NO level are restored by Nrf2 NF-E2-related factor 2 (Nrf2), a member of the NF-E2 family of basic leucine zipper
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transcription factors, is considered a key mediator of the antioxidant response (Itoh et
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al., 2004, Itoh et al., 1999, Motohashi and Yamamoto, 2004). The relationship of GCH1/BH4 with Nrf2 antioxidant pathways remains unknown. Bioinformatics analysis predicted a putative Nrf2 binding site in the proximal promoter of the GCH1 gene (Figure 4a). Luciferase constructs containing wild-type (pGL3-GCH1-Pro WT) or ARE-deleted GCH1 promoter (pGL3-GCH1-Pro Del) were each transfected into skin cells. Overexpression of Nrf2 by adenovirus increased the GCH1 promoter activity, which was repressed by Nrf2 knockdown (Figure 4b and 4c). However, 12
ACCEPTED MANUSCRIPT Nrf2-mediated activation was not observed when either HaCaT or WS1 cells were transfected with the GCH1 promoter without ARE (pGL3-GCH1-Pro Del; Figure 4b and 4c). Moreover, in primary Nrf2-/- skin cells, GCH1 promoter activity was
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significantly lower than that of the Nrf2 wild-type cells and was recovered by infection of Ad-Nrf2 (Figure 4d). To further confirm that Nrf2 directly interacts with the GCH1 promoter, chromatin immunoprecipitation (ChIP) was performed to
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examine the occupancy of Nrf2 at the GCH1 promoter region in vivo. In HaCaT cells,
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the promoter region of GCH1 was specifically precipitated with Nrf2 antibody but not IgG, similar to its binding to the well-established HO-1 promoter, indicating that Nrf2 could directly interact with the GCH1 promoter (Figure 4e). Western blot analysis further confirmed that GCH1 expression is positively associated with the Nrf2
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expression level (Figure 4f and 4g) in these cells. Immunohistochemistry showed that subcutaneous infection of Ad-Nrf2 increased GCH1 expression in the epidermis and dermis of rat skin tissues (Figure 4h). In addition, GCH1 expression in skin tissues of
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Nrf2-/- mice was substantially lower than that in WT mice (Figure 4i). Since GCH1
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catalyzes the generation of BH4, which is required for NO production, we evaluated whether Nrf2 could elevate NO level. As shown in Figure 4j and 4k, overexpression of Nrf2 significantly increased the level of NO with or without radiation. Taken together, these results clearly indicated that Nrf2 is a direct positive regulator of GCH1 expression and NO production. GCH1 mediates the protective role of Nrf2 against radiation-induced damage in skin cells 13
ACCEPTED MANUSCRIPT The above results showed that GCH1 overexpression exhibited antioxidant and radio-protective roles in skin cells that mimicked those of Nrf2. We therefore explored whether GCH1 mediates the radio-protective role of Nrf2. Skin cells were transfected
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with siRNA control (siNC) or siRNA targeting GCH1 (siGCH1), together with adenovirus infection. After irradiation, the elimination of radiation-induced ROS by Nrf2 overexpression was abrogated after the transfection of siRNA targeting GCH1 in
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both HaCaT and WS1 cells (Figure 5a). The combined treatment of Nrf2 adenovirus
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and GCH1 knockdown failed to inhibit the radiation-generated 3-NT (Figure S5). These results indicated that the RNS and ROS scavenging ability of Nrf2 depends on GCH1.
Furthermore, silencing of GCH1 in Nrf2 adenovirus-infected cells failed to
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maintain the mitochondrial membrane potential from shifting, indicating that GCH1 may play a critical role in the mitochondrial protection of Nrf2 (Figure 5b). Additionally, the combined treatment of Nrf2 adenovirus and GCH1 knockdown
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failed to protect cells from radiation-induced cell death and apoptosis (Figure 5c, 5d).
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Taken together, these results demonstrated that the protective effect of Nrf2 against radiation-induced damage is likely mediated through GCH1.
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ACCEPTED MANUSCRIPT DISCUSSION Radiation-induced skin reaction remains a serious concern for radiotherapy such as in the management of skin and breast cancers (Richardson et al., 2005, Ryan, 2012).
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Thus, exploring the mechanisms involved in the pathophysiology of radiation-induced skin injury is needed to develop new strategies against this disease. BH4 is an essential co-factor for all NOSs and BH4 bioavailability is a critical factor in the
balance
between
NO
and
superoxide
production
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regulating
(NOS
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coupling/uncoupling). As a vulnerable co-factor, BH4 was shown to be converted to BH2 in previous reports (Crabtree et al., 2011, Pathak et al., 2014). We found that radiation converts BH4 to many different compounds besides BH2 as determined by HPLC (Figure 1b), indicating the difference between radiation and other oxidative
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stresses. Mice treated with an oral dose of BH4 show reduced vascular ROS production (Pathak et al., 2014). BH4 has been emerging a potential strategy for the treatment of cardiac remodeling, fibrosis and diastolic dysfunction, which are all
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associated with ROS (Cai et al., 2002, Cai et al., 2005). Conversely, inhibition of BH4
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by GCH1 feedback regulatory protein (GFRP) promoted radiation-induced oxidative stress (Pathak et al., 2014). Diminished BH4 availability likely induces NOS uncoupling and increases O2− production, thereby promoting oxidative stress and aggravating radiation-induced
damage to
cells
(Schmidt
and
Alp,
2007,
Sethumadhavan et al., 2016). Our results showed that GCH1 expression and BH4 concentration in irradiated human skin tissue are lower than that of the non-irradiated counterparts, and cellular NO level was decreased after radiation. To ascertain 15
ACCEPTED MANUSCRIPT whether restoring NOS uncoupling could decrease ROS generation, we overexpressed GCH1 in human skin cells and found that radiation-generated ROS were substantially reduced. Our results may offer a general model that NOS uncoupling due to BH4
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disruption by radiation is an important source of radiation-induced secondary ROS. As a natural extension, we further demonstrated that overexpression of GCH1 confers radio-protection in vitro and in vivo. Thus, increasing BH4 level through
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overexpression of GCH1 constitutes a strategy to ameliorate radiation-induced skin
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injury, which is not reported before to our knowledge.
Ionizing radiation induces several types of free radicals (Leach et al., 2001, Spitz et al., 2004), which may be rapidly converted to each other (Mikkelsen and Wardman, 2003). Nrf2 is considered one of the most important general switches of cellular
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antioxidant power (Itoh et al., 2004, Itoh et al., 1999, Kaspar et al., 2009, Motohashi and Yamamoto, 2004) and plays a crucial role in the defense against exogenous oxidative stress. Nrf2 is conventionally considered to exert an antioxidant role
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through activating ROS-eliminating enzymes, including SOD2, NQO1, GST and
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HO-1 (Itoh et al., 1999, Jaiswal, 2004). The most interesting finding from this study is that we identified the GCH1/BH4 pathway as a direct target of Nrf2 in skin cells, which mediates Nrf2’s protective action against radiation-induced injury, and provides a molecular mechanism, not reported before to our knowledge, by which Nrf2 protects skin cells. Because ROS generation occurs in advance of ROS elimination, it is plausible that GCH1 mediates the antioxidant and radio-protective role of Nrf2. In contrast to ROS-eliminating enzymes, GCH1 activated by Nrf2 increases BH4 and 16
ACCEPTED MANUSCRIPT inhibits NOS uncoupling, which reduces the generation of ROS, rather than eliminating ROS. In conclusion, we demonstrated that radiation disrupts BH4, which augments the
attenuated
radiation-induced
radio-protective
capacity,
ROS
primarily
generation. through
Nrf2
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ROS cascade. GCH1 restored radiation-induced disruption of cellular BH4 level and overexpression
transcriptionally
exerted
activating
the
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GCH1/BH4 pathway (Figure 5e). Taken together, the current study demonstrates the
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key role of the Nrf2/GCH1/BH4 axis during radiation-induced skin injury and reveals molecular mechanisms by which strategies can be developed to ameliorate skin
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damage, which is not reported before to our knowledge.
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ACCEPTED MANUSCRIPT MATERIALS AND METHODS Animal studies Protocols for experiments involving animals were approved by the Animal
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Experimentation Ethics Committee at Soochow University (Suzhou, China). Male Sprague Dawley (SD) rats (4 weeks of age) were purchased from the Shanghai SLAC Laboratory Animal Co., Ltd.. These animals were housed in a pathogen-free
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environment at the facilities of Medical School of Soochow University. Irradiation
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was administered to the treatment area at a dose rate of 750 cGy/min using a 6-MeV electron beam accelerator (Clinac 2100EX, Varian Medical Systems, Palo Alto, CA) as reported previously (Zhang et al., 2014).
The C57BL/6 Nrf2 knockout (Nrf2-/-) mice were a kind gift from Dr. Peng Cao
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(Jiangsu Research Institute of Traditional Chinese Medicine, Nanjing, China). The mice were originally obtained from The Jackson Laboratory (stock number: 017009). Nrf2-/- mice were genotyped by PCR.
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Cell culture and irradiation
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Human keratinocyte HaCaT and human skin fibroblast WS1 cells were kind gifts from Prof Hongying Yang (Soochow University). The cell lines were confirmed by STR. The cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Thermo, Shanghai, China). Primary skin cells of mice were isolated from skin of adult mice (5-6 weeks of age) using the procedures as reported. Primary skin cells were maintained in DMEM. All culture media were supplemented with 10% FBS (Gibco, Grand Island, NY). Cells were exposed to different dosages (5 or 20 Gy) of ionizing 18
ACCEPTED MANUSCRIPT radiation using X-ray linear accelerator (RadSource, Suwanee, GA) at a fixed dose rate of 1.15 Gy/min. Reagents and adenoviruses
(BSA)
and
4,
6-diamidino-2-phenylindole
(DAPI)
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BH4 was purchased from the Cayman Chemical Company. Bovine serum albumin were
purchased
from
Sigma-Aldrich (St. Louis, MO). The Nrf2 overexpression adenovirus (Ad-Nrf2) was
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designed and constructed by GeneChem. The control adenovirus and GCH1
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overexpression adenovirus (Ad-GCH1) were obtained from Vigene Biosciences (Jinan, China). shRNA targeting Nrf2 was designed and constructed by Sangon Biotech (Shanghai, China). siRNA targeting GCH1 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
ROS
levels
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ROS generation assay were
determined
7-dichlorofluoresceindiacetate
using
(DCF-DA)
the
(Nanjing
ROS-sensitive Jiancheng
dye
2,
Bioengineering
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Institute, Nanjing, China). The level of DCF fluorescence, reflecting the concentration
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of ROS, was measured by a fluorescence microscope. The cellular level of DCF fluorescence was measured using flow cytometry (BD Biosciences, Franklin Lakes, NJ).
Intracellular NO determination Cells were collected by trypsinization, and intracellular NO content was detected by the probe 3-amino, 4-aminomethyl-2’, 7’-difluorescein, diacetate (DAF-FM DA; Beyotime, Shanghai, China) according to the manufacturer’s protocol. In brief, the 19
ACCEPTED MANUSCRIPT collected cells were incubated with 5 mM DAF-FM DA for 30 min at 37°C in the dark, and the fluorescence intensity of DAF-FM was measured by flow cytometry (BD Biosciences, Franklin Lakes, NJ) with excitation and emission wavelengths of
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488 and 530 nm, respectively. Total NO concentration in cultured cells was detected by measuring the concentration of nitrate and nitrite by a modified Griess reaction method. After lysis
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of cells, a total Nitric Oxide Assay Kit (Beyotime, Shanghai, China) was used to
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detect NO. Cell apoptosis assay
Cells were pre-infected with the adenovirus 24 h before receiving irradiation. Apoptosis was measured using the 7-AAD/Annexin-V double staining apoptosis kit
Lakes, NJ). Statistical analysis
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(BD Biosciences, Franklin Lakes, NJ) by flow cytometry (BD Biosciences, Franklin
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The data were evaluated using either unpaired two-sided Student’s t-tests or one-way
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ANOVA to determine statistical significance after confirming that the data met appropriate assumptions (normality, homogenous variance, and independent sampling). For all in vitro experiments, three biological replicates were analyzed. For all in vivo experiments, five biological replicates were analyzed for each condition. Statistical analysis was performed using Prism 6 software (GraphPad Software, Inc. La Jolla, CA). Data are expressed as means ± SEM and considered significant if P < 0.05 (*) and P < 0.01 (**). For the animal study, skin injury was scored in a 'blinded' 20
ACCEPTED MANUSCRIPT manner. Other methods are detailed in the Supplementary Material.
The authors declare no conflict of interest. ACKNOWLEDGMENTS
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CONFLICT OF INTEREST
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This work is supported by the National Natural Science Foundation of China
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(81522039, 81274150, 81573080 and 31400720), the Key Scientific Development Program of China (2016YFC0904702), the Suzhou Administration of Science & Technology (SYS201416), the Suzhou Key Medical Center (SZZX201506) and the Priority Academic Program Development of Jiangsu Higher Education Institutions
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(PAPD).
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Cals-Grierson MM, Ormerod AD. Nitric oxide function in the skin. Nitric Oxide
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Di Franco R, Sammarco E, Calvanese MG, De Natale F, Falivene S, Di Lecce A, et al. Preventing the acute skin side effects in patients treated with radiotherapy for breast cancer: the use of corneometry in order to evaluate the protective effect of moisturizing creams. Radiat Oncol 2013;8:57. 22
ACCEPTED MANUSCRIPT Doctrow SR, Lopez A, Schock AM, Duncan NE, Jourdan MM, Olasz EB, et al. A synthetic superoxide dismutase/catalase mimetic EUK-207 mitigates radiation dermatitis and promotes wound healing in irradiated rat skin. J Invest
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ACCEPTED MANUSCRIPT to the amino-terminal Neh2 domain. Genes Dev 1999;13:76-86. Jaiswal AK. Nrf2 signaling in coordinated activation of antioxidant gene expression. Free Radic Biol Med 2004;36:1199-207.
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Naylor AM, Pojasek KR, Hopkins AL, Blagg J. The tetrahydrobiopterin pathway and pain. Curr Opin Investig Drugs 2010;11:19-30.
Pathak R, Pawar SA, Fu Q, Gupta PK, Berbee M, Garg S, et al. Characterization of transgenic Gfrp knock-in mice: implications for tetrahydrobiopterin in modulation of normal tissue radiation responses. Antioxid Redox Signal 2014;20:1436-46. Richardson J, Smith JE, McIntyre M, Thomas R, Pilkington K. Aloe vera for 24
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2012;132:985-93. Schmidt TS, Alp NJ. Mechanisms for the role of tetrahydrobiopterin in endothelial function and vascular disease. Clin Sci (Lond) 2007;113:47-63.
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Sethumadhavan S, Whitsett J, Bennett B, Ionova IA, Pieper GM, Vasquez-Vivar J.
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Increasing tetrahydrobiopterin in cardiomyocytes adversely affects cardiac redox state and mitochondrial function independently of changes in NO production. Free Radic Biol Med 2016;93:1-11.
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Spitz DR, Azzam EI, Li JJ, Gius D. Metabolic oxidation/reduction reactions and cellular responses to ionizing radiation: a unifying concept in stress response
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Thony B, Auerbach G, Blau N. Tetrahydrobiopterin biosynthesis, regeneration and functions. Biochem J 2000;347 Pt 1:1-16.
Umar S, van der Laarse A. Nitric oxide and nitric oxide synthase isoforms in the normal, hypertrophic, and failing heart. Mol Cell Biochem 2010;333:191-201.
Weller R. Nitric oxide: a key mediator in cutaneous physiology. Clin Exp Dermatol 2003;28:511-4. Werner
ER,
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biochemistry
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ACCEPTED MANUSCRIPT pathophysiology. Biochem J 2011;438:397-414. Yan S, Brown SL, Kolozsvary A, Freytag SO, Lu M, Kim JH. Mitigation of radiation-induced skin injury by AAV2-mediated MnSOD gene therapy. J
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Gene Med 2008;10:1012-8. Zhang S, Wang W, Gu Q, Xue J, Cao H, Tang Y, et al. Protein and miRNA profiling of radiation-induced skin injury in rats: the protective role of peroxiredoxin-6
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against ionizing radiation. Free Radic Biol Med 2014;69:96-107.
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ACCEPTED MANUSCRIPT FIGURE LEGENDS Figure 1. Radiation disrupted BH4 and decreased NO production. (a) UV-Vis spectra of BH4 and its irradiated derivatives before and after 20 Gy
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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 h after irradiation. (b) HPLC analysis was employed to separate and detect BH4 and its derivatives (e.g.
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BH2) in the raw BH4 and the 20 Gy-irradiated liquids. (c) Intracellular BH4/ BH2
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ratio 24 h post post-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 h after the indicated doses of radiation. (e) The GCH1 level of irradiated human and rat skin tissues was detected using immunohistochemistry. Scale bar = 100µm. Radiation
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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
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NO concentration at different time points after irradiation in (g) HaCaT and (i) WS1
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cells was measured using the Griess assay as described in the Materials and Methods. (j) HaCaT and (k) WS1 cells were pre-infected 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.
27
ACCEPTED MANUSCRIPT Figure 2. The effect of GCH1 on radiation-induced damage in skin cells. Cells were pre-infected with control adenovirus or GCH1 adenovirus followed by irradiation. (a) The dynamic repair process of DNA DSBs was measured by detecting
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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 JC-1 staining assay. Scale bar = 50µm.
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(c) The ER structure was visualized using ER-Tracker Red. (d) The proliferation of
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HaCaT and WS1 cells was measured in an EdU incorporation assay at 48 h after radiation. Scale bar = 20µm (e) The apoptosis rate of HaCaT and WS1 was detected using Annexin-V/7-AAD staining. The cells were collected 24 or 48 h post irradiation. The data are shown as means ± SEM for three independent experiments. * P < 0.05
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compared with the control cells.
Figure 3. GCH1 and BH4 ameliorated radiation-induced skin injury.
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Rat gluteal skin was irradiated with an electron beam followed by subcutaneous
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injection of control adenovirus, GCH1 adenovirus, PBS and BH4 (six animals per group). (a) Skin injury in these groups was measured using a semi-quantitative 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 d after irradiation. (c) Representative hematoxylin and eosin (H&E) staining of rat skins at 75 d after irradiation. Scale bar = 200µm. (d) Calculated number of skin appendages in each group. (e) Calculated epidermal thickness in each group. ** P < 0.01, compared with 28
ACCEPTED MANUSCRIPT the control group.
Figure 4. Nrf2 overexpression activated GCH1 expression and recovered NO
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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
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of a luciferase reporter gene. (b) HaCaT, (c) WS1 and (d) primary mouse skin cells
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were co-transfected 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 h after transfection. The firefly luciferase activity of each sample was normalized to Renilla
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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
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infection with Nrf2 adenovirus or transfection with siRNA-targeting GCH1 in (f)
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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 = 200µm. (i) The GCH1 expression in the skin of WT and Nrf2 knock-out (Nrf2-/-) mice. Scale bar = 200µm. (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. NS, non-significant. 29
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Figure 5. The radio-protective role of Nrf2 was mediated by GCH1. Skin cells were pre-infected with control adenovirus or Nrf2 adenovirus together with
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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
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microscope. Scale bar = 50µm. (b) The mitochondrial membrane potential in cells
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was measured using JC-1 staining. Representative images of fluorescent signals based on fluorescence microscopy under equivalent conditions. Scale bar = 50µm. (c) Cell proliferation was detected using an EdU incorporation assay at 24 h after irradiation as described in Materials and Methods. (d) After 20 Gy of irradiation, cell apoptosis
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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
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uncoupling and augmented radiation-induced secondary ROS. Overexpression of
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Nrf2 transcriptionally activates GCH1, which restores BH4, relieves NOS uncoupling and reduces ROS. GCH1 confers radio-protection for skin cells.
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S0022-202X(17)31604-4
DOI:
10.1016/j.jid.2017.05.019
Reference:
JID 898
To appear in:
The Journal of Investigative Dermatology
Received Date: 17 January 2017 Revised Date:
7 May 2017
Accepted Date: 16 May 2017
Please cite this article as: Xue J, Yu C, Sheng W, Zhu W, Luo J, Zhang Q, Yang H, Cao H, Wang W, Zhou J, Wu J, Cao P, Chen M, Ding W-Q, Cao J, Zhang S, The Nrf2/GCH1/BH4 axis ameliorates radiation-induced skin injury by modulating the ROS cascade, The Journal of Investigative Dermatology (2017), doi: 10.1016/j.jid.2017.05.019. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT TITLE PAGE The Nrf2/GCH1/BH4 axis ameliorates radiation-induced skin injury by modulating the ROS cascade
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Jiao Xue1,2,†, Chenxiao Yu1,2,†, Wenjiong Sheng1,2, Wei Zhu1,2, Judong Luo3, Qi Zhang1,2, Hongying Yang1,2, Han Cao1, Wenjie Wang1, Jundong Zhou4, Jinchang Wu4, Peng Cao5,6, Ming Chen7, Wei-Qun Ding8, Jianping Cao1,2,*, Shuyu
School of Radiation Medicine and Protection, Medical College of Soochow
University, Suzhou 215123, China 2
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1
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Zhang1,2,7,*
Collaborative Innovation Center of Radiation Medicine of Jiangsu Higher Education
Institutions and Jiangsu Provincial Key Laboratory of Radiation Medicine and
3
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Protection, Soochow University, Suzhou 215123, China
Department of Radiotherapy, Changzhou Tumor Hospital, Soochow University,
Changzhou, 213001, China
Suzhou Cancer Center Core Laboratory, Nanjing Medical University Affiliated
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4
5
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Suzhou Hospital, Suzhou 215001, China Affiliated Hospital of Integrated Traditional Chinese and Western Medicine, Nanjing
University of Chinese Medicine, Nanjing 210028, China 6
Laboratory of Cellular and Molecular Biology, Jiangsu Province Academy of
Traditional Chinese Medicine, Nanjing 210028, China, 7
Zhejiang Key Laboratory of Radiation Oncology, Zhejiang Cancer Hospital,
Hangzhou 310022, China 1
ACCEPTED MANUSCRIPT 8
Department of Pathology, University of Oklahoma Health Science Center, Oklahoma
City, OK 73104, USA These authors contributed equally to this work.
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†
Correspondence: School of Radiation Medicine and Protection, Medical College of Soochow University, No. 199 Ren’ai Rd, Suzhou 215123, China. (S. Zhang) Tel/Fax: E-mail:
[email protected];
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+86512-65880037, E-mail: [email protected].
(J.
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+86512-65883941,
Cao)
Tel/Fax:
Running title: Role of the Nrf2/GCH1/BH4 axis in radiation-induced skin injury
radiation,
skin
injury,
GTP
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Keywords:
cyclohydrolase
5,6,7,8-tetrahydrobiopterin (BH4), NF-E2-related factor 2 (Nrf2)
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ABBREVIATIONS
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GCH1: GTP cyclohydrolase I
BH4: 5,6,7,8-tetrahydrobiopterin Nrf2: NF-E2-related factor 2 ROS: reactive oxygen species RNS: reactive nitrogen species NOSs: nitric oxide synthases BH2: dihydrobiopterin 2
I
(GCH1),
ACCEPTED MANUSCRIPT ABSTRACT Radiation-induced skin injury is a common side effect of radiotherapy and can limit the duration and dose of radiotherapy. Most early work focused on elimination of
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reactive oxygen species (ROS) after radiation, however, less is known about the mechanisms underlying amplification of ROS and consequent skin injury by radiation. 5,6,7,8-tetrahydrobiopterin (BH4) is an essential co-factor for all nitric oxide
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synthases (NOSs). Inadequate availability of BH4 leads to uncoupling of NOSs and
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production of highly oxidative radicals. In this study, we demonstrated that radiation disrupted BH4, which resulted in NOS uncoupling and augmented radiation-induced ROS. Overexpression of GTP cyclohydrolase I (GCH1), the rate-limiting enzyme for BH4 synthesis, restored cellular BH4 levels and NO production and decreased
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radiation-induced ROS. GCH1 also protected skin cells and rat skins against radiation-induced damage. We found that GCH1 was regulated by NF-E2-related factor 2 (Nrf2), a key mediator of the cellular antioxidant response. Importantly, we
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identified GCH1 as a key effector for Nrf2-mediated protection against
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radiation-induced skin injury by inhibiting ROS production. Taken together, the findings of this study illustrate the key role of the Nrf2/GCH1/BH4 axis during radiation-induced skin damage.
3
ACCEPTED MANUSCRIPT INTRODUCTION Radiotherapy is applied to over 50% of all cancer patients, either alone or in combination with other treatments (Hymes et al., 2006). Because the skin is the first
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tissue of entry for external radiation particles during radiation treatment, it is vulnerable to radiation-induced injury. In fact, radiation-induced skin syndrome remains a serious concern that may limit the duration and dose of radiotherapy (Di
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Franco et al., 2013, Hymes et al., 2006). In addition, the increasing use of radioactive
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materials in industry, science and military may also increase the likelihood of uncontrolled exposure to radiation (Hymes et al., 2006). Approximately 95 % of cancer patients receiving radiation therapy are estimated to have some forms of cutaneous reactions, including erythema, desquamation, dermatitis, ulcer and fibrosis
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(Ryan, 2012, Singh et al., 2016).
Ionizing radiation promotes reactive nitrogen and oxygen species (RNS/ROS) production due to radiolysis of water and direct ionization of target molecules (Leach
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et al., 2001, Spitz et al., 2004). These reactive molecules, such as hydroxyl radicals,
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superoxide anion, hydrogen peroxide and nitrogen dioxide, can result in oxidative damage to critical cellular biomolecules and cytotoxicity and consequently cause a series of pathophysiological changes that eventually lead to acute and/or chronic skin injuries. The involvement of an anti-oxidative response to radiation has been extensively reported, and the use of antioxidants could mitigate radiation-induced injury (Gu et al., 2013, Kodiyan and Amber, 2015, Yan et al., 2008, Zhang et al., 2014). For example, superoxide dismutases and their mimics reduced ROS levels and 4
ACCEPTED MANUSCRIPT ameliorated radiation-induced skin injury (Doctrow et al., 2013, Gu et al., 2013, Yan et al., 2008, Zhang et al., 2014). Most early studies focused on elimination of ROS after radiation (Doctrow et al., 2013, Gu et al., 2013, Kodiyan and Amber, 2015, Yan et al.,
the generation and amplification of ROS by radiation.
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2008, Zhang et al., 2014). However, less is known about the mechanisms underlying
Nitric oxide (NO) is a lipophilic messenger molecule that participates in various
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physiological and pathological activities, including neurotransmission, smooth muscle
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relaxation, cell migration, immune response and apoptosis (Forstermann and Sessa, 2012, Umar and van der Laarse, 2010). In skin, NO is important for the homeostasis of normal function and is emerging as a therapeutic target for specific cutaneous diseases (Cals-Grierson and Ormerod, 2004, Weller, 2003). In mammals, NO is
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generated by NO synthases (NOSs) from L-arginine, NADPH and oxygen. All three NOS isoforms are expressed in the skin, and 5,6,7,8-tetrahydrobiopterin (BH4) is an essential co-factor for all NOSs (Alderton et al., 2001). Limited availability of BH4
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leads to the uncoupling of NOSs and the production of highly oxidative radicals,
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including superoxide and peroxynitrite, at the cost of NO (Naylor et al., 2010). Stress-generated ROS production may decrease the availability of BH4 due to rapid oxidation of BH4 into dihydrobiopterin (BH2) (Heales et al., 1988), which may induce NOS uncoupling and increase the production of oxidative superoxide radicals (Berbee et al., 2010). In de novo synthesis of BH4, the major synthetic pathway, GTP cyclohydrolase I (GCH1) catalyzes the formation of dihydroneopterin triphosphate from GTP. Subsequently, dihydroneopterin triphosphate is converted to BH4 by 5
ACCEPTED MANUSCRIPT 6-pyruvoyl-tetrahydrobiopterin synthase and sepiapterin reductase (Thony et al., 2000). The rate of BH4 synthesis via a de novo pathway depends on the activity of its rate-limiting enzyme, GCH1 (Werner et al., 2011). In vivo inhibition of GCH1
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increased vascular oxidative stress and reduced white blood cell counts after irradiation (Pathak et al., 2014).
Although BH4 bioavailability is involved in NO/ROS transition, the role of BH4
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and its synthetic enzyme GCH1 in radiation-induced ROS and consequent skin injury
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is unclear. In this study, we elucidated the key role of BH4 and GCH1 in augmenting radiation-induced ROS. We further demonstrated that the Nrf2/GCH1/BH4 axis protects against radiation-induced skin damage, which provides strategies unreported
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before to our knowledge for ameliorating this injury.
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ACCEPTED MANUSCRIPT RESULTS Radiation reduces GCH1 expression and BH4 availability, which impairs NO homeostasis in skin tissues and cells
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NO synthesis is important for the homeostasis of skin function (Cals-Grierson and Ormerod, 2004, Weller, 2003). BH4, a co-factor of NOSs, is shown to be converted to BH2 by oxidative stress (Naylor et al., 2010). To determine whether the structure of
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BH4 was disrupted following ionizing radiation, we used UV-Vis spectra and
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high-performance liquid chromatography (HPLC) analysis. The results showed that the optical properties of BH4 aqueous solution were not changed immediately after 20 Gy of X-ray irradiation (Figure 1a). Twenty-four hours after irradiation, the absorptions of BH4 (at 217 to 230 nm and 315 to 330 nm) gradually decreased over
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time, while the absorptions of BH4 (at 275 nm) increased. Moreover, absorption of BH4 24 h after irradiation was blue-shifted slightly compared to the bulk value (at 375 nm; Figure 1a), indicating that radiation alters the structure of BH4. Next, HPLC
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analysis was used to separate and detect BH4 and its derivatives (e.g. BH2) in the raw
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BH4 and the 20 Gy-irradiated liquids. As shown in Figure 1b, weak BH2 peaks were observed in the fresh new BH4 liquid. In contrast, the content of BH2 and other derivatives was substantially increased after 20 Gy irradiation. The results demonstrated that the unstable BH4 can be easily oxidized into multiple derivatives in addition to BH2. In cell extracts of human keratinocyte HaCaT and skin fibroblast WS1 cells, the BH4/BH2 ratio was significantly reduced after radiation (Figure 1c), confirming that radiation could decrease the bioavailability of BH4. 7
ACCEPTED MANUSCRIPT Then, the expression of the BH4 rate-limiting enzyme GCH1 was measured by Western blotting. Results from Western blot analysis showed that radiation decreased GCH1 expression in a dose-dependent manner in HaCaT and WS1 cells (Figure 1d),
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indicative of reduced BH4 synthesis after radiation. Using immunohistochemistry, we found that GCH1 expression in irradiated human skin tissue was lower than that in the normal counterparts (Figure 1e). We further measured cellular NO levels by flow
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cytometric-based analysis of a NO-sensitive probe (DAF-FM DA) and Griess assay of
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NaNO2 levels. The results consistently showed that NO levels in 20 Gy-irradiated skin cells were significantly reduced in both HaCaT and WS1 cells compared with those of nonirradiated counterparts (Figure 1f-1i). The above results suggested that radiation interferes with de novo synthesis of BH4 and consequent NO homeostasis.
restores NO level
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Overexpression of GCH1 decreases NOS uncoupling-associated ROS and
Since lack of BH4 has been reported to induce NOS uncoupling, which consequently
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generates RNS and ROS (Naylor et al., 2010), we investigated whether RNS
and
ROS
can
be
modulated
by
GCH1.
A
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radiation-induced
GCH1-overexpression adenovirus (Ad-GCH1) was constructed and introduced into cultured cells, leading to a marked increase in the protein levels of GCH1 in HaCaT (Figure 1j) and WS1 cells (Figure 1k). Ad-GCH1 infection induced a significant ~ 1.5-fold increase of cellular NO levels in HaCaT and WS1 cells without radiation (Figure 1j and 1k). Compared with the Ad-NC groups, NO levels in Ad-GCH1-infected HaCaT and WS1 cells after 20 Gy irradiation were increased by 8
ACCEPTED MANUSCRIPT 2.2- and 3.5-fold, respectively (Figure 1j and 1k). These results suggested that GCH1 overexpression restores NO level in skin cells. We further explored the effect of GCH1 silencing on baseline and radiation-induced ROS in skin cells. We found that
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silencing of GCH1 did not affect baseline ROS levels without radiation but aggravated radiation-induced ROS in both HaCaT and WS1 cell lines (Figure S1), indicating that GCH1 is involved in the amplification of radiation-induced ROS.
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Conversely, forced expression of GCH1 significantly reduced radiation-induced ROS
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in HaCaT and WS1 cells (Figure 1l and 1m). We also measured the formation of 3-nitrotyrosine (3-NT), a biomarker of peroxynitrite (ONOO-). We found that GCH1 overexpression also significantly reduced intracellular 3-NT levels, indicative of decreased oxidative damage mediated by peroxynitrite (Figure S2). These results
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suggested that radiation-induced RNS and ROS could be attenuated by inhibiting NOS uncoupling through GCH1.
GCH1 protects skin cells from radiation-induced damage
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Since GCH1 overexpression could inhibit radiation-induced NOS uncoulpling and
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consequently reduce ROS levels, we next investigated whether increasing BH4 synthesis via GCH1 overexpression could alleviate radiation-induced damage in skin cells. DNA repair of DSBs was measured by detecting nuclear γH2AX foci at several time points after irradiation of HaCaT cells at 5 Gy. The number of foci formed in GCH1 adenovirus-infected cells was significantly reduced to an average of 54.44% (49 foci per cell) of the control adenovirus-treated cells at 1 h after irradiation. At 2 and 4 h after irradiation, the number of γH2AX foci in GCH1 adenovirus-treated 9
ACCEPTED MANUSCRIPT groups decreased to 35.12% (P < 0.01) and 35.07% (P < 0.01) of the control adenovirus-treated cells, respectively (Figure 2a and Figure S3). These results demonstrated that overexpression of GCH1 could attenuate the radiation-induced
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DNA damage of human skin cells. Mitochondrial functional failure, including the loss of mitochondrial integrity and change of mitochondrial membrane potential, is considered to be one of the most
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important factors causing cell death (Green and Kroemer, 2004). To explore the
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protective role of GCH1 on mitochondrial function, mitochondrial potential was examined by JC-1 staining. Non-irradiated HaCaT cells predominantly showed red fluorescence, whereas a substantial proportion of cells shifted to green fluorescence after 20 Gy of irradiation, indicating that the mitochondrial membrane potential was
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reduced (Figure 2b). However, cells with forced expression of GCH1 exhibited a decreased shift from red to green fluorescence, indicating that the mitochondrial membrane potential was maintained after exposure to ionizing radiation (Figure 2b),
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indicative of the protection of the mitochondria against ionizing radiation. Next, we
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explored the protective role of GCH1 on the endoplasmic reticulum (ER) structure using ER-Tracker Red. As shown in Figure 2c, non-irradiated HaCaT cells presented robust ER-Tracker Red staining that was attenuated, and a clear ER structure that was morphologically
altered,
after
irradiation
at
20
Gy.
However,
GCH1
adenovirus-infected cells showed stronger ER-Tracker Red staining after radiation and were morphologically similar to the non-irradiated cells, indicating that the integrity of the ER was maintained after ionizing radiation exposure. 10
ACCEPTED MANUSCRIPT GCH1 modulates the radiosensitivity of human skin cells Next, we sought to investigate whether increasing BH4 synthesis via GCH1 overexpression could modulate the radiosensitivity of skin cells. Skin cells were
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pre-infected with control adenovirus or GCH1 adenovirus followed by 20 Gy of irradiation. The results from the EdU assay revealed that HaCaT and WS1 cells that were infected with GCH1 adenovirus exhibit a significantly higher percentage of
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EdU-positive cells than those of untreated or control adenovirus-infected cells after
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radiation (P < 0.01 for HaCaT and P < 0.01 for WS1; Figure 2d).
We next investigated whether GCH1 overexpression was associated with decreased apoptosis. Annexin-V/7-AAD staining-based flow cytometric analysis of apoptosis was performed at 24 and 48 h after radiation. Overexpression of GCH1 significantly
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reduced the apoptosis of HaCaT and WS1 cells after 20 Gy of irradiation compared with that of cells infected with the control adenovirus (Figure 2e). These results demonstrated that GCH1 reduces radiation-induced apoptotic cell death of skin cells.
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skin injury
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GCH1 overexpression or BH4 supplementation ameliorates radiation-induced
To determine whether GCH1 overexpression could attenuate radiation-induced skin injury, rat models were used as described previously (Zhang et al., 2014). After 45 Gy of electron beam exposure, rats received subcutaneous injection of GCH1 adenovirus or BH4 (1 mg/kg). The radiation-induced skin injury recovery process in the Ad-GCH1-infected group, the BH4-supplemented group and the control group was evaluated for 75 d. At 24 d after irradiation, radiation-induced skin injury was 11
ACCEPTED MANUSCRIPT significantly alleviated in the GCH1 adenovirus-infected and BH4-supplemented groups compared with that of the control group (Figure 3a and 3b). Subcutaneous supplementation of BH4 also mitigated the radiation-induced skin injury (Figure 3a
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and 3b), which was similar to the effect of GCH1. The clinically used chitosan showed no therapeutic effect on rat skin irradiated by the 45 Gy electron beam (Figure S4). Although infection with GCH1 adenovirus and BH4 supplementation groups
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showed similar skin wounds as those of PBS-treated or control adenovirus-infected
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groups at 75 d post-irradiation, GCH1 overexpression or BH4 supplementation attenuated the epidermal hyperplasia (white arrow) and maintained skin appendages (blue arrow; Figure 3c-3e), which were often destroyed by irradiation. Taken together, these
results
demonstrated
that
increasing
BH4
level
could
ameliorate
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radiation-induced skin injury in vivo.
GCH1 transcription and NO level are restored by Nrf2 NF-E2-related factor 2 (Nrf2), a member of the NF-E2 family of basic leucine zipper
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transcription factors, is considered a key mediator of the antioxidant response (Itoh et
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al., 2004, Itoh et al., 1999, Motohashi and Yamamoto, 2004). The relationship of GCH1/BH4 with Nrf2 antioxidant pathways remains unknown. Bioinformatics analysis predicted a putative Nrf2 binding site in the proximal promoter of the GCH1 gene (Figure 4a). Luciferase constructs containing wild-type (pGL3-GCH1-Pro WT) or ARE-deleted GCH1 promoter (pGL3-GCH1-Pro Del) were each transfected into skin cells. Overexpression of Nrf2 by adenovirus increased the GCH1 promoter activity, which was repressed by Nrf2 knockdown (Figure 4b and 4c). However, 12
ACCEPTED MANUSCRIPT Nrf2-mediated activation was not observed when either HaCaT or WS1 cells were transfected with the GCH1 promoter without ARE (pGL3-GCH1-Pro Del; Figure 4b and 4c). Moreover, in primary Nrf2-/- skin cells, GCH1 promoter activity was
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significantly lower than that of the Nrf2 wild-type cells and was recovered by infection of Ad-Nrf2 (Figure 4d). To further confirm that Nrf2 directly interacts with the GCH1 promoter, chromatin immunoprecipitation (ChIP) was performed to
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examine the occupancy of Nrf2 at the GCH1 promoter region in vivo. In HaCaT cells,
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the promoter region of GCH1 was specifically precipitated with Nrf2 antibody but not IgG, similar to its binding to the well-established HO-1 promoter, indicating that Nrf2 could directly interact with the GCH1 promoter (Figure 4e). Western blot analysis further confirmed that GCH1 expression is positively associated with the Nrf2
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expression level (Figure 4f and 4g) in these cells. Immunohistochemistry showed that subcutaneous infection of Ad-Nrf2 increased GCH1 expression in the epidermis and dermis of rat skin tissues (Figure 4h). In addition, GCH1 expression in skin tissues of
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Nrf2-/- mice was substantially lower than that in WT mice (Figure 4i). Since GCH1
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catalyzes the generation of BH4, which is required for NO production, we evaluated whether Nrf2 could elevate NO level. As shown in Figure 4j and 4k, overexpression of Nrf2 significantly increased the level of NO with or without radiation. Taken together, these results clearly indicated that Nrf2 is a direct positive regulator of GCH1 expression and NO production. GCH1 mediates the protective role of Nrf2 against radiation-induced damage in skin cells 13
ACCEPTED MANUSCRIPT The above results showed that GCH1 overexpression exhibited antioxidant and radio-protective roles in skin cells that mimicked those of Nrf2. We therefore explored whether GCH1 mediates the radio-protective role of Nrf2. Skin cells were transfected
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with siRNA control (siNC) or siRNA targeting GCH1 (siGCH1), together with adenovirus infection. After irradiation, the elimination of radiation-induced ROS by Nrf2 overexpression was abrogated after the transfection of siRNA targeting GCH1 in
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both HaCaT and WS1 cells (Figure 5a). The combined treatment of Nrf2 adenovirus
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and GCH1 knockdown failed to inhibit the radiation-generated 3-NT (Figure S5). These results indicated that the RNS and ROS scavenging ability of Nrf2 depends on GCH1.
Furthermore, silencing of GCH1 in Nrf2 adenovirus-infected cells failed to
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maintain the mitochondrial membrane potential from shifting, indicating that GCH1 may play a critical role in the mitochondrial protection of Nrf2 (Figure 5b). Additionally, the combined treatment of Nrf2 adenovirus and GCH1 knockdown
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failed to protect cells from radiation-induced cell death and apoptosis (Figure 5c, 5d).
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Taken together, these results demonstrated that the protective effect of Nrf2 against radiation-induced damage is likely mediated through GCH1.
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ACCEPTED MANUSCRIPT DISCUSSION Radiation-induced skin reaction remains a serious concern for radiotherapy such as in the management of skin and breast cancers (Richardson et al., 2005, Ryan, 2012).
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Thus, exploring the mechanisms involved in the pathophysiology of radiation-induced skin injury is needed to develop new strategies against this disease. BH4 is an essential co-factor for all NOSs and BH4 bioavailability is a critical factor in the
balance
between
NO
and
superoxide
production
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regulating
(NOS
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coupling/uncoupling). As a vulnerable co-factor, BH4 was shown to be converted to BH2 in previous reports (Crabtree et al., 2011, Pathak et al., 2014). We found that radiation converts BH4 to many different compounds besides BH2 as determined by HPLC (Figure 1b), indicating the difference between radiation and other oxidative
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stresses. Mice treated with an oral dose of BH4 show reduced vascular ROS production (Pathak et al., 2014). BH4 has been emerging a potential strategy for the treatment of cardiac remodeling, fibrosis and diastolic dysfunction, which are all
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associated with ROS (Cai et al., 2002, Cai et al., 2005). Conversely, inhibition of BH4
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by GCH1 feedback regulatory protein (GFRP) promoted radiation-induced oxidative stress (Pathak et al., 2014). Diminished BH4 availability likely induces NOS uncoupling and increases O2− production, thereby promoting oxidative stress and aggravating radiation-induced
damage to
cells
(Schmidt
and
Alp,
2007,
Sethumadhavan et al., 2016). Our results showed that GCH1 expression and BH4 concentration in irradiated human skin tissue are lower than that of the non-irradiated counterparts, and cellular NO level was decreased after radiation. To ascertain 15
ACCEPTED MANUSCRIPT whether restoring NOS uncoupling could decrease ROS generation, we overexpressed GCH1 in human skin cells and found that radiation-generated ROS were substantially reduced. Our results may offer a general model that NOS uncoupling due to BH4
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disruption by radiation is an important source of radiation-induced secondary ROS. As a natural extension, we further demonstrated that overexpression of GCH1 confers radio-protection in vitro and in vivo. Thus, increasing BH4 level through
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overexpression of GCH1 constitutes a strategy to ameliorate radiation-induced skin
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injury, which is not reported before to our knowledge.
Ionizing radiation induces several types of free radicals (Leach et al., 2001, Spitz et al., 2004), which may be rapidly converted to each other (Mikkelsen and Wardman, 2003). Nrf2 is considered one of the most important general switches of cellular
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antioxidant power (Itoh et al., 2004, Itoh et al., 1999, Kaspar et al., 2009, Motohashi and Yamamoto, 2004) and plays a crucial role in the defense against exogenous oxidative stress. Nrf2 is conventionally considered to exert an antioxidant role
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through activating ROS-eliminating enzymes, including SOD2, NQO1, GST and
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HO-1 (Itoh et al., 1999, Jaiswal, 2004). The most interesting finding from this study is that we identified the GCH1/BH4 pathway as a direct target of Nrf2 in skin cells, which mediates Nrf2’s protective action against radiation-induced injury, and provides a molecular mechanism, not reported before to our knowledge, by which Nrf2 protects skin cells. Because ROS generation occurs in advance of ROS elimination, it is plausible that GCH1 mediates the antioxidant and radio-protective role of Nrf2. In contrast to ROS-eliminating enzymes, GCH1 activated by Nrf2 increases BH4 and 16
ACCEPTED MANUSCRIPT inhibits NOS uncoupling, which reduces the generation of ROS, rather than eliminating ROS. In conclusion, we demonstrated that radiation disrupts BH4, which augments the
attenuated
radiation-induced
radio-protective
capacity,
ROS
primarily
generation. through
Nrf2
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ROS cascade. GCH1 restored radiation-induced disruption of cellular BH4 level and overexpression
transcriptionally
exerted
activating
the
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GCH1/BH4 pathway (Figure 5e). Taken together, the current study demonstrates the
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key role of the Nrf2/GCH1/BH4 axis during radiation-induced skin injury and reveals molecular mechanisms by which strategies can be developed to ameliorate skin
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damage, which is not reported before to our knowledge.
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ACCEPTED MANUSCRIPT MATERIALS AND METHODS Animal studies Protocols for experiments involving animals were approved by the Animal
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Experimentation Ethics Committee at Soochow University (Suzhou, China). Male Sprague Dawley (SD) rats (4 weeks of age) were purchased from the Shanghai SLAC Laboratory Animal Co., Ltd.. These animals were housed in a pathogen-free
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environment at the facilities of Medical School of Soochow University. Irradiation
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was administered to the treatment area at a dose rate of 750 cGy/min using a 6-MeV electron beam accelerator (Clinac 2100EX, Varian Medical Systems, Palo Alto, CA) as reported previously (Zhang et al., 2014).
The C57BL/6 Nrf2 knockout (Nrf2-/-) mice were a kind gift from Dr. Peng Cao
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(Jiangsu Research Institute of Traditional Chinese Medicine, Nanjing, China). The mice were originally obtained from The Jackson Laboratory (stock number: 017009). Nrf2-/- mice were genotyped by PCR.
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Cell culture and irradiation
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Human keratinocyte HaCaT and human skin fibroblast WS1 cells were kind gifts from Prof Hongying Yang (Soochow University). The cell lines were confirmed by STR. The cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Thermo, Shanghai, China). Primary skin cells of mice were isolated from skin of adult mice (5-6 weeks of age) using the procedures as reported. Primary skin cells were maintained in DMEM. All culture media were supplemented with 10% FBS (Gibco, Grand Island, NY). Cells were exposed to different dosages (5 or 20 Gy) of ionizing 18
ACCEPTED MANUSCRIPT radiation using X-ray linear accelerator (RadSource, Suwanee, GA) at a fixed dose rate of 1.15 Gy/min. Reagents and adenoviruses
(BSA)
and
4,
6-diamidino-2-phenylindole
(DAPI)
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BH4 was purchased from the Cayman Chemical Company. Bovine serum albumin were
purchased
from
Sigma-Aldrich (St. Louis, MO). The Nrf2 overexpression adenovirus (Ad-Nrf2) was
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designed and constructed by GeneChem. The control adenovirus and GCH1
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overexpression adenovirus (Ad-GCH1) were obtained from Vigene Biosciences (Jinan, China). shRNA targeting Nrf2 was designed and constructed by Sangon Biotech (Shanghai, China). siRNA targeting GCH1 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
ROS
levels
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ROS generation assay were
determined
7-dichlorofluoresceindiacetate
using
(DCF-DA)
the
(Nanjing
ROS-sensitive Jiancheng
dye
2,
Bioengineering
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Institute, Nanjing, China). The level of DCF fluorescence, reflecting the concentration
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of ROS, was measured by a fluorescence microscope. The cellular level of DCF fluorescence was measured using flow cytometry (BD Biosciences, Franklin Lakes, NJ).
Intracellular NO determination Cells were collected by trypsinization, and intracellular NO content was detected by the probe 3-amino, 4-aminomethyl-2’, 7’-difluorescein, diacetate (DAF-FM DA; Beyotime, Shanghai, China) according to the manufacturer’s protocol. In brief, the 19
ACCEPTED MANUSCRIPT collected cells were incubated with 5 mM DAF-FM DA for 30 min at 37°C in the dark, and the fluorescence intensity of DAF-FM was measured by flow cytometry (BD Biosciences, Franklin Lakes, NJ) with excitation and emission wavelengths of
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488 and 530 nm, respectively. Total NO concentration in cultured cells was detected by measuring the concentration of nitrate and nitrite by a modified Griess reaction method. After lysis
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of cells, a total Nitric Oxide Assay Kit (Beyotime, Shanghai, China) was used to
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detect NO. Cell apoptosis assay
Cells were pre-infected with the adenovirus 24 h before receiving irradiation. Apoptosis was measured using the 7-AAD/Annexin-V double staining apoptosis kit
Lakes, NJ). Statistical analysis
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(BD Biosciences, Franklin Lakes, NJ) by flow cytometry (BD Biosciences, Franklin
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The data were evaluated using either unpaired two-sided Student’s t-tests or one-way
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ANOVA to determine statistical significance after confirming that the data met appropriate assumptions (normality, homogenous variance, and independent sampling). For all in vitro experiments, three biological replicates were analyzed. For all in vivo experiments, five biological replicates were analyzed for each condition. Statistical analysis was performed using Prism 6 software (GraphPad Software, Inc. La Jolla, CA). Data are expressed as means ± SEM and considered significant if P < 0.05 (*) and P < 0.01 (**). For the animal study, skin injury was scored in a 'blinded' 20
ACCEPTED MANUSCRIPT manner. Other methods are detailed in the Supplementary Material.
The authors declare no conflict of interest. ACKNOWLEDGMENTS
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CONFLICT OF INTEREST
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This work is supported by the National Natural Science Foundation of China
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(81522039, 81274150, 81573080 and 31400720), the Key Scientific Development Program of China (2016YFC0904702), the Suzhou Administration of Science & Technology (SYS201416), the Suzhou Key Medical Center (SZZX201506) and the Priority Academic Program Development of Jiangsu Higher Education Institutions
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(PAPD).
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ACCEPTED MANUSCRIPT FIGURE LEGENDS Figure 1. Radiation disrupted BH4 and decreased NO production. (a) UV-Vis spectra of BH4 and its irradiated derivatives before and after 20 Gy
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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 h after irradiation. (b) HPLC analysis was employed to separate and detect BH4 and its derivatives (e.g.
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BH2) in the raw BH4 and the 20 Gy-irradiated liquids. (c) Intracellular BH4/ BH2
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ratio 24 h post post-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 h after the indicated doses of radiation. (e) The GCH1 level of irradiated human and rat skin tissues was detected using immunohistochemistry. Scale bar = 100µm. Radiation
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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
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NO concentration at different time points after irradiation in (g) HaCaT and (i) WS1
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cells was measured using the Griess assay as described in the Materials and Methods. (j) HaCaT and (k) WS1 cells were pre-infected 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.
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ACCEPTED MANUSCRIPT Figure 2. The effect of GCH1 on radiation-induced damage in skin cells. Cells were pre-infected with control adenovirus or GCH1 adenovirus followed by irradiation. (a) The dynamic repair process of DNA DSBs was measured by detecting
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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 JC-1 staining assay. Scale bar = 50µm.
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(c) The ER structure was visualized using ER-Tracker Red. (d) The proliferation of
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HaCaT and WS1 cells was measured in an EdU incorporation assay at 48 h after radiation. Scale bar = 20µm (e) The apoptosis rate of HaCaT and WS1 was detected using Annexin-V/7-AAD staining. The cells were collected 24 or 48 h post irradiation. The data are shown as means ± SEM for three independent experiments. * P < 0.05
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Figure 3. GCH1 and BH4 ameliorated radiation-induced skin injury.
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Rat gluteal skin was irradiated with an electron beam followed by subcutaneous
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injection of control adenovirus, GCH1 adenovirus, PBS and BH4 (six animals per group). (a) Skin injury in these groups was measured using a semi-quantitative 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 d after irradiation. (c) Representative hematoxylin and eosin (H&E) staining of rat skins at 75 d after irradiation. Scale bar = 200µm. (d) Calculated number of skin appendages in each group. (e) Calculated epidermal thickness in each group. ** P < 0.01, compared with 28
ACCEPTED MANUSCRIPT the control group.
Figure 4. Nrf2 overexpression activated GCH1 expression and recovered NO
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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
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of a luciferase reporter gene. (b) HaCaT, (c) WS1 and (d) primary mouse skin cells
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were co-transfected 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 h after transfection. The firefly luciferase activity of each sample was normalized to Renilla
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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
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infection with Nrf2 adenovirus or transfection with siRNA-targeting GCH1 in (f)
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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 = 200µm. (i) The GCH1 expression in the skin of WT and Nrf2 knock-out (Nrf2-/-) mice. Scale bar = 200µm. (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. NS, non-significant. 29
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Figure 5. The radio-protective role of Nrf2 was mediated by GCH1. Skin cells were pre-infected with control adenovirus or Nrf2 adenovirus together with
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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
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microscope. Scale bar = 50µm. (b) The mitochondrial membrane potential in cells
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was measured using JC-1 staining. Representative images of fluorescent signals based on fluorescence microscopy under equivalent conditions. Scale bar = 50µm. (c) Cell proliferation was detected using an EdU incorporation assay at 24 h after irradiation as described in Materials and Methods. (d) After 20 Gy of irradiation, cell apoptosis
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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
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uncoupling and augmented radiation-induced secondary ROS. Overexpression of
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Nrf2 transcriptionally activates GCH1, which restores BH4, relieves NOS uncoupling and reduces ROS. GCH1 confers radio-protection for skin cells.
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