E-Cadherin–Mediated Cell Contact Controls the Epidermal Damage Response in Radiation Dermatitis  Guojiang Xie, Xiulan Ao, Tianmiao Lin, Guixuan Zhou, Ming Wang, Hanwei Wang, Yuangui Chen, Xiaobo Li, Benhua Xu, Wangzhong He, Hao Han, Yuval Ramot, Ralf Paus, Zhicao Yue  Journal of Investigative Dermatology  Volume 137, Issue 8, Pages 1731-1739 (August 2017) DOI: 10.1016/j.jid.2017.03.036 Copyright © 2017 The Authors Terms and Conditions

Figure 1 IR disrupts cell-cell contact in the mouse footpad model of RD. (a) Loss of cell contact and disruption of adherens junctions in the epidermis as shown by TEM analysis, and F-actin/β-catenin/E-cadherin staining. Dotted lines are quantified in c–e. (b) γ-H2AX positive cell counts showing the DNA damage in the epidermis. n ≥ 5 for each condition. (c–e) Quantification of the staining intensity of F-actin, β-catenin, and E-cadherin. ∗∗∗P < 0.001. Scale bar = 10 μm. IR, ionizing radiation; RD, radiation dermatitis; T, day; TEM, transmission electron microscopy. Journal of Investigative Dermatology 2017 137, 1731-1739DOI: (10.1016/j.jid.2017.03.036) Copyright © 2017 The Authors Terms and Conditions

Figure 2 TEM analysis of ultrastructural changes in the mouse footpad model of RD. (a) Basal keratinocytes in normal skin. (b) T3. Notice the increased intercellular spaces, extensive tonofibrils (arrow head), and nuclear invagination (arrow). (c) T3. An apoptotic cell (asterisk) surrounded by normal viable cells. (d) T6. Notice the fragmented nucleus (arrow), detached basement membrane (asterisk), and increased intercellular spaces. The cytoplasm appeared homogeneous with no obvious vacuoles. (e) A suprabasal keratinocyte at T6. Other than increased intercellular spaces and extensive tonofibrils, this cell appeared normal and viable. (f) T9. This basal keratinocyte lost contact with neighboring cells and partially detached from the basement membrane (asterisk). Arrowhead, perinuclear and cell junctional tonofibrils. Scale bar = 2 μm. RD, radiation dermatitis; T, day; TEM, transmission electron microscopy. Journal of Investigative Dermatology 2017 137, 1731-1739DOI: (10.1016/j.jid.2017.03.036) Copyright © 2017 The Authors Terms and Conditions

Figure 3 Activation of Hippo and Wnt signaling in the mouse footpad model of RD. (a) Additional marker analysis of the mouse footpad skin after 40 Gy irradiation. Notice the expanded expression of K14 and K17 at T9. Disruption of cell-cell contact at T9 was obvious by K17 staining. Nuclear Yap1 staining indicated activation of Hippo signaling, which (c) is quantified. (b) Activation of Wnt signaling as shown by LacZ staining in whole-mount and sectioned skin samples from irradiated TOPGAL mice. (d) Changes of the epidermal cell number during the damage and repair process. ∗P < 0.1, ∗∗P < 0.01, ∗∗∗P < 0.001. Scale bar = 20 μm in a, 50 μm in b. K, keratin; RD, radiation dermatitis; T, day. Journal of Investigative Dermatology 2017 137, 1731-1739DOI: (10.1016/j.jid.2017.03.036) Copyright © 2017 The Authors Terms and Conditions

Figure 4 ROS-mediated degradation of β-catenin/E-cadherin and its contribution in RD. (a) IR, H2O2 treatment of mouse footpad skin, and H2O2 treatment of MCF7 cells all down-regulated E-cadherin and β-catenin expression. Each experiment was repeated at least three times, and representative results are shown. ∗P < 0.1, ∗∗P < 0.01, ∗∗∗P < 0.001. (b) H2O2 treatment disrupted cytoskeleton and AJs in MCF7 cell culture. Scale bar = 100 μm. (c) β-catenin binding is required for E-cadherin degradation. An eGFP expression plasmid was used as a control for electroporation in MCF7 cells. (d) A blocking peptide (peptide A, 10 μmol/L; n = 8) or a neutralizing antibody (DECMA-1, 2 mg/kg; n = 10) works synergistically with IR (30 Gy) to induce desquamation. The control group received 30 Gy irradiation only (n = 10). AJ, adherens junction; Cyto, cytoplasmic domain; EC, extracellular domain; eGFP, enhanced green fluorescent protein; h, hour; IR, ionizing radiation; M, mol/L; RD, radiation dermatitis; ROS, reactive oxygen species; T, day; TM, transmembrane domain. Journal of Investigative Dermatology 2017 137, 1731-1739DOI: (10.1016/j.jid.2017.03.036) Copyright © 2017 The Authors Terms and Conditions

Figure 5 Tyrosine kinase inhibitors rescue ROS-induced disassembly of AJs in vitro and RD in vivo. (a) Increased overall and β-catenin–specific tyrosine phosphorylation after H2O2 treatment in MCF7 cells. Activation of Src/Abl kinases was also shown (p-Src). (b) RNAi knockdown of SRC or ABL1 impeded H2O2-induced degradation of β-catenin and its tyrosine phosphorylation. The RNAi efficiencies were determined by Western blot analysis. (c–d) Genistein (Ge) and dasatinib (Da) rescued H2O2-induced disruption of AJs and increased the levels of β-catenin and E-cadherin. Scale bar = 100 μm. ∗P < 0.1, ∗∗P < 0.01. (e) Ge and Da rescue RD in vivo. Shown are representative images and statistics of the different damage levels (n = 10 for the control and Ge groups, n = 12 for the Da group). AJ, adherens junction; h, hour; IR, ionizing radiation; M, mol/L; RD, radiation dermatitis; RNAi, RNA interference; ROS, reactive oxygen species. Journal of Investigative Dermatology 2017 137, 1731-1739DOI: (10.1016/j.jid.2017.03.036) Copyright © 2017 The Authors Terms and Conditions

Figure 6 Clinical specimen analysis and a model for the acute epidermal damage response in RD. (a–c) γ-H2AX and Yap1 nuclear localization were increased after 23 fractioned (Fx) IR exposures, 2.0 Gy for each Fx. Disruption of AJs was indicated by the diffusive β-catenin and E-cadherin distribution. Scale bar = 10 μm. (b, c) Quantification of (b) γ -H2AX and (c) Yap1 nuclear localization. ∗∗∗P < 0.001. (d, e) IR disrupts AJs in skin epidermis and results in loss of cell contact, which is likely mediated by ROS-induced activation of Src/Abl kinases and degradation of β-catenin/E-cadherin. This then leads to the activation of Wnt and Hippo signaling to guide the subsequent repair and regeneration process. AJ, adherens junction; IR, ionizing radiation; ROS, reactive oxygen species. Journal of Investigative Dermatology 2017 137, 1731-1739DOI: (10.1016/j.jid.2017.03.036) Copyright © 2017 The Authors Terms and Conditions