Activation of TGF-β1 by AQP3-Mediated H2O2 Transport into Fibroblasts of a Bleomycin-Induced Mouse Model of Scleroderma  Jingying Luo, Xin Liu, Jie Liu, Miao Jiang, Miao Luo, Jingjun Zhao  Journal of Investigative Dermatology  Volume 136, Issue 12, Pages 2372-2379 (December 2016) DOI: 10.1016/j.jid.2016.07.014 Copyright © 2016 The Authors Terms and Conditions

Figure 1 AQP3 levels in the dermis from skin samples and in dermal fibroblasts in BLM and PBS group mice (n = 10). (a) Evaluation of hydroxyproline content. (b) H2O2 levels in skin samples. (c) AQP3 mRNA and (d, e) protein expression in the dermis from skin samples obtained from mice after 7, 14, and 28 days of injection with PBS or BLM. (f) Immunohistochemical staining of skin samples collected 28 days after injection of PBS or BLM. The arrows indicate fibroblasts (scale bar = 20 μm). (g) AQP3 mRNA and (h) protein levels in PMF and BMF. (i) AQP3 localization in PMF and BMF determined by confocal microscopy (scale bar = 50 μm). All data are presented as means ± SD. #P < 0.05; *P < 0.005. AQP3, aquaporin 3; BLM, bleomycin; BMF, BLM mice fibroblasts; H2O2, hydrogen peroxide; PBS, phosphate-buffered saline; PMF, PBS mice fibroblasts; SD, standard deviation. Journal of Investigative Dermatology 2016 136, 2372-2379DOI: (10.1016/j.jid.2016.07.014) Copyright © 2016 The Authors Terms and Conditions

Figure 2 BLM-mediated increased inflow of H2O2 in BMF is dependent on AQP3 (n = 4). (a) Western blot evaluation of the efficiency of AQP3 silencing. (b) H2O2 inflow into BMF relies on AQP3. At 48 hours after transfection with shNC or AQP3 shRNA, BMF were incubated with H2O2 (0–300 μM) for 30 minutes, and intracellular H2O2 levels were detected by DCFH-DA fluorescence. (c, d) Increased H2O2 inflow into BLM-treated BMF is dependent on AQP3. BMF at 48 hours after transfection with shNC or AQP3 shRNA or at 30 minutes after catalase or DPI treatment were incubated with BLM (1 μg/ml) for 24 hours, and DCFH-DA fluorescence was used to determine intracellular H2O2 levels (scale bar = 50 μm). All experiments were repeated three times. All data are presented as means ± SD. #P < 0.05; *P < 0.005. AQP3, aquaporin 3; BLM, bleomycin; BMF, BLM mice fibroblasts; COL1, collagen type I; COL3, collagen type III; DCFH-DA, 2′,7′-dichlorofluorescein diacetate; H2O2, hydrogen peroxide; SD, standard deviation; shNC, negative control shRNA; shRNA, short hairpin RNA. Journal of Investigative Dermatology 2016 136, 2372-2379DOI: (10.1016/j.jid.2016.07.014) Copyright © 2016 The Authors Terms and Conditions

Figure 3 Silencing AQP3 can reduce BLM-induced TGF-β1 expression by decreasing intracellular H2O2. (a) At 48 hours after transfection with shNC or AQP3 shRNA, BMF were incubated with 0–1,000 μM H2O2 for 24 hours, and TGF-β1, COL1, and COL3 protein levels were analyzed by western blot (n = 3). (b, c) At 48 hours after transfection with shNC or AQP3 shRNA or at 30 minutes after catalase or DPI treatment, BMF were incubated with BLM (1 μg/ml) for 24 hours. Expression of TGF-β1 was examined by real-time PCR (b) and western blot (c) (n = 4). All experiments were repeated three times. All data are presented as means ± SD. *P < 0.005. AQP3, aquaporin 3; BLM, bleomycin; BMF, BLM mice fibroblasts; COL1, collagen type I; COL3, collagen type III; DPI, diphenyleneiodonium chloride; H2O2, hydrogen peroxide; SD, standard deviation; shNC, negative control shRNA; shRNA, short hairpin RNA; TGF-β1, transforming growth factor-β1. Journal of Investigative Dermatology 2016 136, 2372-2379DOI: (10.1016/j.jid.2016.07.014) Copyright © 2016 The Authors Terms and Conditions

Figure 4 Silencing AQP3 can reduce BLM-induced COL1 and COL3 expression by decreasing intracellular H2O2. (a, b) At 48 hours after transfection with shNC or AQP3 shRNA or at 30 minutes after catalase or DPI treatment, BMF were incubated with BLM (1 μg/ml) for 24 hours. Expression of COL1 and COL3 was examined by real-time PCR (a) and western blot (b) (n = 4). All experiments were repeated three times. All data are presented as means ± SD. *P < 0.005. AQP3, aquaporin 3; BLM, bleomycin; BMF, BLM mice fibroblasts; COL1, collagen type I; COL3, collagen type III; DPI, diphenyleneiodonium chloride; H2O2, hydrogen peroxide; SD, standard deviation; shNC, negative control shRNA; shRNA, short hairpin RNA. Journal of Investigative Dermatology 2016 136, 2372-2379DOI: (10.1016/j.jid.2016.07.014) Copyright © 2016 The Authors Terms and Conditions

Figure 5 Silencing AQP3 inhibited fibrosis in the scleroderma mouse model (n = 10). (a) hematoxylin and eosin (H&E) and Masson’s trichrome staining for skin tissue samples. The red arrow indicates dermal thickness (scale bar = 50 μm). (b) Dermal thickness of skin tissue samples. (c) H2O2 levels in skin samples. (d) TGF-β1, COL1, and COL3 mRNA levels were determined by real-time PCR analysis. (e) Western blot analysis was used to determine TGF-β1, COL1, and COL3 protein levels. (f) Model of H2O2/AQP3 pathway-mediated activation of TGF-β1 and synthesis of COL1 and COL3 in the scleroderma mouse model. All data are presented as means ± SD. #P < 0.05, *P ≤ 0.005. AQP3, aquaporin 3; COL1, collagen type I; COL3, collagen type III; H2O2, hydrogen peroxide; SD, standard deviation; TGF-β1, transforming growth factor-β1. Journal of Investigative Dermatology 2016 136, 2372-2379DOI: (10.1016/j.jid.2016.07.014) Copyright © 2016 The Authors Terms and Conditions