The Role of FABP5 in Radiation-Induced Human Skin Fibrosis Author(s): Jianyuan Song,, Huojun Zhang, Zhenyu Wang, Wanglei Xu, Li Zhong, Jinming Cao, Jianfeng Yang, Ye Tian, Daojiang Yu, Jiang Ji, Jianping Cao and Shuyu Zhang Source: Radiation Research, 189(2):177-186. Published By: Radiation Research Society https://doi.org/10.1667/RR14901.1 URL: http://www.bioone.org/doi/full/10.1667/RR14901.1 BioOne (www.bioone.org) is a nonprofit, online aggregation of core research in the biological, ecological, and environmental sciences. BioOne provides a sustainable online platform for over 170 journals and books published by nonprofit societies, associations, museums, institutions, and presses. Your use of this PDF, the BioOne Web site, and all posted and associated content indicates your acceptance of BioOne’s Terms of Use, available at www.bioone.org/page/terms_of_use. Usage of BioOne content is strictly limited to personal, educational, and non-commercial use. 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DOI: 10.1667/RR14901.1 The Role of FABP5 in Radiation-Induced Human Skin Fibrosis Jianyuan Song,a,b,1 Huojun Zhang,c,1 Zhenyu Wang,b,1 Wanglei Xu,b Li Zhong,b Jinming Cao,b Jianfeng Yang,d Ye Tian,e Daojiang Yu,e Jiang Ji,e,2 Jianping Caob and Shuyu Zhangb,2 a Fujian Medical University Union Hospital, Fuzhou 350001, China; b School of Radiation Medicine and Protection and Collaborative Innovation Center of Radiation Medicine of Jiangsu Higher Education Institutions, Soochow University, Suzhou 215123, China; c Department of Radiation Oncology, Shanghai Changhai Hospital, the Second Military Medical University, Shanghai 200433, China; d Cyrus Tang Hematology Center, Soochow University, Suzhou 215123, China; and e The Second Affiliated Hospital of Soochow University, Suzhou, 215004, China human skin fibrosis and the critical role of FABP5 in Song, J., Zhang, H., Wang, Z., Xu, W., Zhong, L., Cao, J., activating the TGF-b signaling pathway. Ó 2018 by Radiation Yang, J., Tian, Y., Yu, D., Ji, J., Cao, J. and Zhang, S. The Role Research Society of FABP5 in Radiation-Induced Human Skin Fibrosis. Radiat. Res. 189, 177–186 (2018). Radiation-induced skin fibrosis is a detrimental and chronic disorder that occurs after radiation exposure. The molecular changes underlying the pathogenesis of radiation- INTRODUCTION induced fibrosis of human skin have not been extensively Radiation-induced skin fibrosis is a detrimental and reported. Technical advances in proteomics have enabled exploration of the biomarkers and molecular pathogenesis of chronic disorder that occurs weeks to years after radiation radiation-induced skin fibrosis, with the potential to broaden exposure (1–3). Despite the technological advances in our understanding of this disease. In this study, we compared radiotherapy, chronic radiation fibrosis is usually an protein expression in radiation-induced fibrotic human skin irreversible and progressive condition that occurs as an and adjacent normal tissues using iTRAQ-based proteomics unintentional effect of treatment, and may greatly impair the technology. We identified 186 preferentially expressed pro- conduct of radiotherapy and deteriorate the patient’s quality teins (53 upregulated and 133 downregulated) between of life. Exposure to ionizing radiation from potential radiogenic fibrotic and normal skin tissues. The differentially radiological accidents are a public health concern (4–6). expressed proteins included keratins (KRT5, KRT6A, KRT16 and KRT17), caspase-14, fatty acid-binding protein 5 Radiation-induced skin fibrosis is a complex response (FABP5), SLC2A14 and resistin. Through bioinformatic initiated to protect the skin from injury and involves a analysis of the proximal promoters, common motifs and multistage process. Skin fibrosis is characterized by the corresponding transcriptional factors were identified that increased production and deposition of extracellular matrix associate with the dysregulated proteins, including PAX5, (ECM) components and the accumulation of myofibro- TBX1, CLOCK and AP2D. In particular, FABP5 (2.15-fold blasts. Several mechanisms are established during the increase in fibrotic skin tissues), a transporter of hydrophobic progression of skin fibrosis, including fibroblast differenti- fatty acids, was investigated in greater detail. Immunohisto- ation (7), epithelial to mesenchymal transition (EMT) (8) chemistry confirmed that the protein level of FABP5 was increased in fibrotic human skin tissues, especially in the and leukocyte recruitment (neutrophils, lymphocytes, mac- epidermis. Overexpression of FABP5 resulted in nuclear rophages and fibrocytes) (9). translocation of SMAD2 and significant activation of the Numerous cytokines and chemokines have been report- profibrotic TGF-b signaling pathway in human fibroblast ed to be released in response to radiation injury in skin, WS1 cells. Moreover, exogenous FABP5 (FABP5-EGFP) most notably TGF-b, interleukin (IL)-1, IL-6, IL-18, could be incorporated by skin cells and intensify TGF-b CCL4 and CXCL10 (10–12). Resident skin keratinocytes, signaling, indicating a communication between the microen- fibroblasts and endothelial cells secrete cytokines and vironment and skin fibrosis. Taken together, our findings chemokines to initiate multiple events such as recruiting illustrate the molecular changes during radiation-induced circulating immune cells and fibroblast stimulation. For example, TGF-b signaling is transmitted by a pair of Editor’s note. The online version of this article (DOI: 10.1667/ transmembrane serine/threonine kinase receptors, known RR14901.1) contains supplementary information that is available to all authorized users. as the type I and type II TGF-b receptors (13). The 1 These authors contributed equally to this work. activated TGF-b receptor propagates the signal by 2 Address for correspondence: Department of Plastic Surgery, The phosphorylating intracellular Smad2/Smad3 proteins, Second Affiliated Hospital of Soochow University, Suzhou, 215004, China; emails: [email protected] and zhang.shuyu@hotmail. which shuttle into the nucleus and regulate profibrotic com. target genes. The TGF-b signaling pathway has been 177 178 SONG ET AL. emerging as a therapeutic target for radiation-induced skin Abcamt, Cambridge, MA) antibodies at 48C overnight followed by fibrosis (14, 15). incubation with anti-rabbit biotinylated secondary antibody (ZSGB- BIO Technology Co. Ltd., Beijing, China), diaminobenzidine The exact mechanism of fibrosis is not fully clear and substrate detection, washing, hematoxylin staining, dehydration and currently there is no effective treatment to prevent or mounting. mitigate radiation-induced skin fibrosis (6). Proteomics technology is an attractive discovery tool that can be applied Cell Culture and Irradiation to study large sets of biological molecules. Previously, we Human skin fibroblast WS1 cells were maintained in Dulbecco’s have reported proteomic profiling of radiation-induced skin modified Eagle’s medium (DMEM). Primary skin cells were fibrosis in a rat model (16). However, the proteomic maintained in DMEM. All culture media were supplemented with landscape of human skin in response to radiation has not 10% fetal bovine serum (FBS; Gibcot, Grand Island, NY). Cells were 8 been reported. In this study, we compared the protein grown at 37 Cin5%CO2 incubators. Cells were 0 or 5 Gy irradiated using an X-ray linear accelerator (Rad Source Technologies Inc., expression profiles of radiation-induced fibrotic human skin Suwanee, GA) at a fixed dose rate of 1.15 Gy/min. to normal skin tissues using iTRAQt-based protein quantitation, and observed that FABP5 was overexpressed Immunofluorescence Assay in fibrotic skin tissues, which promotes TGF-b signaling After treatment, WS1 cells were fixed with 4% paraformaldehyde, pathway. washed with PBS and permeabilized with 1% Tritone X-100 in PBS. Cells were blocked with blocking buffer [PBS, 1% Triton X-100 and 5% bovine serum albumin (BSA)] and incubated at 48C with anti- MATERIALS AND METHODS SMAD2 antibody (ImmunoWay, Staffordshire, UK) overnight. FITC- Human Skin Samples conjugated goat anti-mouse (1:100) was added for 30 min at room temperature. Nuclei were counterstained with DAPI. Human skin samples were obtained from a victim of an iridium 192 radiation accident. The patient picked up an iridium-192 ( Ir) metal FABP5 Overexpression Vector and Transfection chain (with an activity of 966.4 GBq or 26.1 Ci) with his right hand and placed it in the right pocket of his work coat, which resulted in The human FABP5 (accession no. ENST00000297258.10; Gen- radiation-induced skin injury. The estimated dose to the exposed right Bankt, NIH, Bethesda, MD) coding region was amplified by PCR limb was ;200 Gy at the center of the skin surface. The irradiated using a primer pair specific to FABP5. The amplified fragment was skin samples from the right limb were obtained 160 days inserted into the pcDNA3.1 vector. The plasmid was then sequenced postirradiation. The normal skin tissues were obtained when for