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

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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 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 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 (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 confirmation. For transfection, cells were transfected using performing skin grafting from dorsal myocutaneous flap. Informed FuGENEt HD transfection reagent (Promega Inc., Madison, WI). consent for sample collection was obtained from the patient. Western Blot Proteomic Analysis Cells were lysed in RIPA buffer. After centrifugation at 48C for 10 Radiation-induced fibrotic human skin and normal skin tissues were min (12,000g), supernatant was collected and subjected to Western collected (in duplicates). Both the epidermis and the dermis were used blotting. Protein (30 lm) was fractionated by 10% SDS-PAGE and for proteomic analysis. The detailed methods of proteomic analysis are electrophoretically transferred to polyvinylidene difluoride mem- described in the Supplementary Methods (http://dx.doi.org/10.1667/ branes (EMD Millipore, Bedford, MA). After blocking with 5% RR14901.1.S1). nonfat milk in PBS Tweene-20 for 1 h at room temperature, the membranes were blotted with FABP5 antibody (Abcam) at a 1:2,000 KEGG Annotation dilution. Alpha-tubulin was used as a loading control. After washing four times with TBST, the membranes were incubated with a We analyzed all of the dysregulated proteins with the Kyoto horseradish peroxidase-conjugated secondary antibody (Beyotime, Encyclopedia of Genes and Genomes (KEGG) pathway analysis using Nantong, China) for 2 h. The proteins were visualized using enhanced DAVID version 6.7 database (http://david.abcc.ncifcrf.gov/), as chemiluminescence (ECL; Beyotime). reported elsewhere (17). Assays Motif Search by MEME Software Program To measure the activity of TGF-b signaling pathway, luciferase One-kb upstream promoter sequences of the dysregulated proteins assay using TGF-b downstream effector SMAD2/3-responsive for locating known cis-regulatory elements were conducted using the luciferase reporter, which reflects TGF-b downstream transcriptional MEME sequence-analysis tool, described elsewhere (18), with a activity of Smad2/3, was performed, as reported elsewhere (20). The combined P value ,0.01 as threshold. The matrices generated by luciferase reporter with SMAD2/3-responsive elements was a kind gift MEME were then compared to the TRANSFACt database (GeneX- from Dr. Zengpeng Li (Third Institute of Oceanography, State Oceanic plain GmbH, Wolfenbu¨ttel, Germany) using the DNA binding motif Administration, Xiamen, China). Cells were transfected with the similarity tool STAMP (National Science Foundation, Alexandria, SMAD2/3-responsive reporter together with pRL-TK using FuGENE VA) (19). HD transfection reagent (Promega Inc.). Luciferase activity was measured with the Dual-Luciferaset Reporter Assay System (Promega Immunohistochemistry Inc.). Promoter activity was expressed as the ratio of firefly luciferase to Renilla luciferase activity. Skin tissues were fixed in 10% neutral-buffered formalin and embedded in paraffin. Paraffin sections, 3 lm, were deparaffinized Purification of FABP5-EGFP Fusion Protein and heat treated with citrate buffer (pH 6.0) for 7 min following an epitope retrieval protocol. Paraffin sections, 3 lm, were incubated The coding sequences of FABP5 and EGFP were subcloned into with the rabbit polyclone antibody against FABP5 (cat. no. ab37267; pET-28a vector (Novagent; EMD Biosciences, Madison, WI) to ROLE OF FABP5 IN RADIOGENIC SKIN FIBROSIS 179 generate pET-28a-FABP5-EGFP. Host E. coli BL21 (DE3) were Transcriptional Analysis of Dysregulated Proteins in transformed with the plasmid pET-28a-FABP5-EGFP and then the Fibrotic Skin transformants were selected on an LB plate containing kanamycin. BL21 (DE3) cells containing the expression plasmid were grown at Because the differentially expressed proteins are possi- 378C to an optical density OD600 of 0.8. Purification of FABP5-EGFP bly driven by transcriptional factors, which are mediated fusion protein was performed as described in the Supplementary Methods (http://dx.doi.org/10.1667/RR14901.1.S1). by the recruitment of transcription factors to cis-regulatory elements, we therefore analyzed the proximal promoter FABP5 Uptake by Skin Cells regions of the dysregulated proteins using MEME to identify motifs for transcriptional factors and co-activators. FABP5 fused with EGFP (FABP5-EGFP) recombinant protein was expressed and purified by Bioworld Biotech (Nanjing, China). The Evaluation of 2.0-kb upstream promoter regions (–1 to purity of FABP5-EGFP was over 85%. Skin cells were plated in six- –2,000 bp) of the dysregulated proteins with MEME well plates for 24 h, and then incubated with fresh media containing resulted in the identification of five common types of cis- FABP5-EGFP for 0–2 h at 378C in a 5% CO2 atmosphere. The cells regulatory elements. Multiple motifs with binding sites for were then washed with PBS three times. The cells were counterstained putative transcription factor were found for the upregulated using DAPI to visualize the cell nucleus and were observed with a confocal microscope (Olympus, Tokyo, Japan). or downregulated proteins (Fig. 2). For example, the motif 50-GGGAGGCA(C)GAGGCGGGNGGATCACNTGAG- 0 Statistical Analysis 3 was present in the promoters of upregulated proteins. This motif is likely to interact with transcriptional factors Data were expressed as the mean 6 SEM of at least three independent experiments. The results were evaluated via one-way EGR1, ZN219, TBX1, CLOCK and AP2D. There is one analysis of variance (ANOVA) to determine statistical significance. motif (no. 4 in the upregulated and downregulated The statistical analyses were performed using SPSSt software proteins), which is present in the promoters of both the (Chicago, IL). Differences were considered significant at P , 0.05. up- and downregulated proteins, indicating dual roles in radiation response depending on co-activators/co-suppres- RESULTS sors.

Proteomic Analysis of Radiation-Induced Fibrosis of Radiation Induces the Expression of FABP5 Human Skin Tissue Among the dysregulated proteins, we focus on FBAP5, an We obtained a specimen from a human patient who had important fatty acid chaperone in skin homeostasis (21, 22), been accidentally exposed to a source of radioactive because the increased expression of FABP5 is consistent iridium. Representative fibrotic skin tissue and normal skin with our previously reported work using radiogenic skin tissue are shown in Fig. 1A. Dermal hyperplasia and skin fibrosis from a rat model (16). Using immunohistochemis- fibrosis were evident. To elucidate the molecular changes, try, we sought to confirm the expression of FABP5 in we investigated the proteomic profiles of the radiogenic human skin tissues. The results revealed that in the fibrotic fibrotic and normal skin tissues (in duplicates) using TMT skin tissues, the expression of FABP5 was increased (Fig. labeling and HPLC fractionation followed by high-resolu- 3A), which is consistent with the result from proteomic tion LC-MS/MS analysis. In total, 3,365 proteins were analysis by iTRAQ technology. Higher expression of quantified from the two groups of skin tissues (Fig. 1B and FABP5 was pronounced in the epidermis. C and Supplementary Table S1; http://dx.doi.org/10.1667/ We next explored the expression of FABP5 in response to RR14901.1.S1). When the test value was set at ,0.05, t P ionizing radiation. Human skin fibroblast WS1 cells were and the quantification ratio was set at .1.5 for the exposed to different doses of radiation and then assayed for upregulated threshold and ,0.83 for the downregulated FABP5 protein expression by Western blotting. The results threshold, 53 upregulated and 133 downregulated proteins revealed that radiation induces the expression of FABP5 were obtained between radiogenic fibrotic and normal skin (Fig. 3B), indicative of its involvement in cellular response tissues (Fig. 1B and Table 1). The identified differentially to the radiation. expressed proteins included keratins (KRT5, KRT6A, KRT16 and KRT17), caspase-14, fatty acid-binding protein FABP5 Regulates TGF-b Signaling Pathway in Skin Cells 5 (FABP5), SLC2A14 and resistin. As a reference knowledge base, KEGG pathway analyses is widely used Because the TGF-b signaling pathway plays a central in molecular interaction networks and it can provide higher- role in tissue fibrosis, we next investigated whether order functional meanings and genome information on cells FABP5 affected the activity of the TGF-b pathway. We and organisms (17). The significantly enriched pathways constructed an FABP5 overexpression vector and trans- include transcriptional dysregulation, systemic lupus eryth- fected it into WS1 cells. The results showed that the ematosus, complement and coagulation cascades, leishman- FABP5 protein level was markedly increased by Western iasis, leukocyte transendothelial migration, staphylococcus blotting (Fig. 4A). To explore the role of FABP5 in skin aureus infection, hematopoietic cell lineage and hagosome fibrosis, we focused on TGF-b signaling. Immunofluores- (Fig. 1D). cence assays were used to determine cellular distribution 180 SONG ET AL.

FIG. 1. Overview of proteomic analysis. Panel A: Representative H&E staining of normal and irradiated skin tissue. Panel B: Venn diagram showing significant differentially expressed proteins in the two groups. Panel C: Heatmap of dysregulated proteins between the two groups. Expression levels are indicated by color change. Red indicates higher expression, whereas green indicates lower expression. Irr.: irradiated skin tissue; N: normal skin tissue. Panel D: KEGG analysis of dysregulated proteins between normal and fibrotic skin tissues. ROLE OF FABP5 IN RADIOGENIC SKIN FIBROSIS 181

TABLE 1 Preferentially Expressed Proteins (Top 20 Fibrotic Skin Tissues/Normal Skin Tissues) No. Gene name Fold change P value Description Upregulated 1 KRT6A 6.62 9.73E-06 Keratin, type II cytoskeletal 6A 2 KRT17 4.10 1.35E-07 Keratin, type I cytoskeletal 17 3 TRIM29 3.99 3.64E-03 Tripartite motif-containing protein 29 4 KRT5 3.20 6.52E-10 Keratin, type II cytoskeletal 5 5 KRT16 3.09 9.84E-09 Keratin, type I cytoskeletal 16 6 DSP 3.05 4.52E-15 Desmoplakin 7 KRT14 2.69 2.33E-13 Keratin, type I cytoskeletal 14 8 IVL 2.57 8.98E-03 Involucrin OS 9 CASP14 2.43 2.84E-03 Caspase-14 10 PKP1 2.41 1.17E-03 Plakophilin-1 11 JUP 2.27 5.84E-05 Junction plakoglobin 12 MT2A 2.17 5.11E-04 Metallothionein-2 13 SFN 2.15 1.38E-04 14-3-3 protein sigma 14 FABP5 2.15 3.57E-06 Fatty acid-binding protein, epidermal 15 FOLR2 2.05 3.37E-03 Folate receptor beta 16 CPA3 1.94 6.08E-04 Mast cell carboxypeptidase A 17 MX1 1.91 4.01E-03 Interferon-induced GTP-binding protein Mx1 18 DSG1 1.89 3.32E-03 Desmoglein-1 19 APCS 1.84 6.55E-08 Serum amyloid P-component 20 NPL 1.79 8.53E-03 N-acetylneuraminate lyase Downregulated 1 CXCL5 0.16 3.05E-04 C-X-C motif chemokine 5 2 TNNC2 0.19 1.38E-05 Troponin C, skeletal muscle 3 SLC2A14 0.19 9.82E-05 Solute carrier family 2, facilitated glucose transporter member 14 4 LCN2 0.21 1.21E-07 Neutrophil gelatinase-associated lipocalin 5 CAMP 0.24 1.60E-05 Cathelicidin antimicrobial peptide 6 S100A12 0.24 1.24E-06 Protein S100-A12 7 CLC 0.25 4.01E-03 Galectin-10 8 KLHL41 0.26 4.74E-03 Kelch-like protein 41 9 ANXA3 0.27 3.50E-21 Annexin A3 10 MYLPF 0.27 1.07E-04 Myosin regulatory light chain 2, skeletal muscle isoform 11 MYL3 0.28 6.24E-04 Myosin light chain 3 12 LTF 0.28 8.19E-76 Lactotransferrin 13 BPI 0.29 3.15E-03 Bactericidal permeability-increasing protein 14 CSRP3 0.29 1.16E-04 Cysteine and glycine-rich protein 3 15 PADI4 0.31 5.20E-04 Protein-arginine deiminase type-4 16 FGR 0.32 9.40E-04 Tyrosine-protein kinase Fgr 17 NCF1B 0.34 8.04E-10 Putative neutrophil cytosol factor 1B 18 S100A1 0.34 9.67E-03 Protein S100-A1 19 MMP9 0.34 1.87E-11 Matrix metalloproteinase-9 20 CKM 0.34 6.96E-10 Creatine kinase M-type

of SMAD2, a TGF-b signaling effector (14, 15). We found Exogenous FABP5 from the Microenvironment can be that forced expression of FABP5 promoted nuclear Incorporated into Skin Cells and Activates the TGF-b translocation of SMAD2. Moreover, overexpression of Downstream Pathway this gene also enhanced SMAD2 nuclear distribution after Since several FABP family member proteins possess the 5 Gy irradiation (Fig. 4B). To confirm the functional ability to cross the cellular membrane and shuttle between significance of SMAD2 nuclear translocation, SMAD2/3- cells (23), we investigated whether FABP5 from the responsive luciferase reporter was used. The results microenvironment could be incorporated by skin fibro- showed that transfection of FABP5 significantly increased blasts. Recombinant FABP5 fused with EGFP (FABP5- the luciferase activity of SMAD2/3-responsive reporter by EGFP) was purified from E. coli (.85% purity; Fig. 5A). ;4-fold without irradiation (Fig. 4C) and by ;3.2-fold WS1 cells were incubated with fresh media containing PBS, with 5 Gy irradiation (Fig. 4D). Taken together, these EGFP or EGFP-tagged FABP5 protein (FABP5-EGFP). It results indicated that FABP5 enhanced the TGF-b was observed that FABP5-EGFP could be incorporated into signaling in skin fibroblasts. skin cells in time- and dose-dependent manners (Fig. 5C–F). 182 SONG ET AL.

FIG. 2. Transcriptional analysis of proteomics change of radiogenic skin fibrosis. The differentially expressed proteins were analyzed by MEME for the –1 bp to –2.0 kb (relative to the transcription start site) sequences of the promoters. Shown were the top five common motifs and the putative transcriptional factors for the upregulated or the downregulated proteins.

FIG. 3. Radiation induces the expression of FABP5. Panel A: The expression of FABP5 in normal and irradiated human skin tissues was measured by immunohistochemistry. Scale bar ¼ 100 lm. Panel B: At 24 h after WS1 cells were 0, 5 and 10 Gy X-ray irradiated, the expression of FABP5 in WS1 was determined by Western blot. Relative FABP5 expression was calculated by SMARTView image analysis system from the Western blot results.

! FIG. 4. FABP5 regulates TGF-b signaling in skin cells. Panel A: WS1 cells were transfected with pcDNA3.1 or FABP5 overexpression vector (pcDNA3.1-FABP5). At 24 h after transfection, Western blot analysis was used to measure FABP5 expression in WS1 cells. Panel B: WS1 cells were pre-transfected with pcDNA3.1 or pcDNA3.1-FABP5 followed by sham or 5 Gy irradiation. The cellular distribution of SMAD2 was measured by immunofluorescence assay. Panel C: Luciferase assay of SMAD2/3-responsive luciferase reporter activity after transfection with pcDNA3.1 or pcDNA3.1-FABP5. Panel D: Luciferase assay of SMAD2/3-responsive luciferase reporter activity after 5 Gy irradiation. Data are shown as the mean 6 SEM for three independent experiments. **P , 0.01. ROLE OF FABP5 IN RADIOGENIC SKIN FIBROSIS 183 184 SONG ET AL.

FIG. 5. Exogenous FABP5 can be incorporated into skin cells and intensifies the TGF-b signaling pathway. Panel A: Expression and purification of FABP5-EGFP recombinant protein. Expressed and purified fusion proteins were analyzed by 12% SDS-PAGE followed by Coomassie Blue staining. Lanes are as follows: lane 1, non-induced cell lysis (transformed with the plasmid pET-28a-FABP5-EGFP); lane 2, induced cell lysis (transformed with pET-28a-FABP5-EGFP); lane 3, supernatant after sonication; lane 4, eluent buffer with 5 mM imidazole; lane 5, eluent buffer with 10 mM imidazole; lane 6, eluent buffer with 20 mM imidazole. Panel B: FABP5-EGFP recombinant protein regulated the TGF-b signaling pathway. Luciferase assay was performed after transfection of SMAD2/3-responsive luciferase reporter and the addition of 10 lM FABP5-EGFP or EGFP. **P , 0.01. Panel C: Dose-dependent uptake of FABP5-EGFP recombinant protein into WS1 cells. WS1 cells were treated with FABP5-EGFP at different concentrations for 2 h. After the addition of FABP5-EGFP, cells were washed three times with PBS to remove free protein. Fluorescent signals were obtained under the same conditions. Panel D: Percentage of FABP5-EGFP-transduced cells. Data are shown as mean 6 SEM for three independent experiments. Panel E: Time-dependent uptake of FABP5-EGFP recombinant protein into WS1 cells. WS1 cells were treated with 10 lM FABP5-EGFP for 0 to 2 h. Panel F: Percentage of FABP5-EGFP- transduced cells. Data are shown as mean 6 SEM for three independent experiments.

After 10 lM FABP5-EGFP fusion protein treatment for 2 h, microenvironment or from distal tissues contributes to nearly 100% of WS1 cells were incorporated with this radiation-induced skin fibrosis. exogenous protein (Fig. 5C–F). Exogenous FABP5-EGFP was mainly located in the cytosol (Supplementary Fig. S1; DISCUSSION http://dx.doi.org/10.1667/RR14901.1.S1). Moreover, com- pared to EGFP-treated cells, the addition of FABP5-EGFP Radiation-induced skin fibrosis remains a serious concern significantly increased the activity of TGF-b downstream for radiotherapy, radiation accidents and occupational effector SMAD2/3-responsive reporter by 3.1-fold (Fig. exposure (4–6). To detect novel biomarkers and molecular 5B), suggesting that the exogenous FABP5 in the targets in radiation-induced skin fibrosis, we identified 186 ROLE OF FABP5 IN RADIOGENIC SKIN FIBROSIS 185 preferentially expressed proteins (53 upregulated and 133 This phenomenon has also been reported in other skin downregulated) between radiogenic fibrotic and normal skin disorders, such as arsenic-induced skin keratosis (35)and tissues by iTRAQ-based proteomic approach. This study wound healing (36, 37), with unknown mechanisms. FABP5 provides an overview of the altered protein expression in has also been shown to enhance the phosphorylation of radiation-induced skin fibrosis. The altered protein abun- EGFR in cells (38). The involvement of EGFR in dance might result in physiological adaptations that contrib- tissue fibrosis has been well-documented. ROS can mediate ute to radiogenic fibrosis. Several keratin family proteins the activation of EGFR. Inhibition of EGFR tyrosine kinase (KRT5, KRT6A, KRT16 and KRT17) were significantly activity by antibodies or compounds prevents renal (39, 40), upregulated, which may affect the structure and repair of peritoneal (41) and liver fibrosis (42). Therefore, EGFR may damaged skin cells. It has been reported that injury to the mediate the activation of TGF-b pathway by FABP5. skin results in an induction of keratin-6, -16 and -17, which Moreover, we found that FABP5 could be incorporated into are required to activate the reepithelialization of keratinocytes receptor cells, which enhanced the TGF-b pathway. These (24). Some of the proteins were consistent with our findings indicate that FABP5, enriched in epidermis, may previously reported work using radiation-induced skin also contribute to radiation-induced fibrosis in the dermis fibrosis in a rat model (16). For example, FABP5 and (16). Thus, exogenous FABP5 in the microenvironment or epididymal secretory protein E1 (NPC2) were both overex- from distal tissues contributes to the expansion of radiation- pressed in fibrotic skin. In contrast, fatty acid binding protein induced skin fibrosis. 4 (FABP4) was decreased in fibrotic skin tissues. Our study In conclusion, this study is the first to report proteomic also identified novel protein regulation in fibrotic human change during radiogenic human skin fibrosis, which tissues induced by radiation. C-X-C motif chemokine 5 provides potential biomarkers and targets for this disease. (CXCL5) was decreased in fibrotic skin, which may affect Overexpression of intracellular or exogenous FABP5 results the recruitment of neutrophils (25). Caspase-14 was in the activation the profibrotic TGF-b downstream upregulated, and it is involved in terminal differentiation of signaling in human skin cells. Our findings illustrate the keratinocytes and in barrier function (26, 27). This difference molecular changes during radiation-induced skin fibrosis between the two studies may be partially due to different and the critical role of FABP5 in activating the TGF-b stages after radiation exposure and partially due to difference signaling pathway. between species. Further studies are required to understand the biological role of these proteins in the pathogenesis of SUPPLEMENTARY INFORMATION radiation-induced skin fibrosis. Transcriptional regulation of gene expression has Supplementary Methods. emerged as an important mechanism for cellular response Table S1. Identified proteins in the normal and the to ionizing radiation (28). We therefore analyzed the fibrotic skin tissues. proximal promoter regions of the dysregulated proteins. Fig. S1. Cellular distribution of exogenous FABP5. WS1 Ten motifs with corresponding transcriptional factors were cells were treated with 10 lM FABP5-EGFP for 2 h. The predicted using a bioinformatic approach. In particular, cells were counter stained using DAPI to visualize the cell EGR1 has been shown to mediate cell apoptosis in response nucleus and were observed by a confocal microscope. to radiation. EGR1 activates apoptosis-related genes including TNF-a, p53 and Bax, all of which mediate cell ACKNOWLEDGMENTS apoptosis (29). In another published study, it was found that silencing EGR1 attenuates radiation-induced apoptosis in This work is supported by the National Natural Science Foundation of normal cells, but reduces the growth of cancer cells (30). China (81522039, 31770911 and 81673096). The Development Plan of Science & Technology (SZS201509) and Key Scientific Development EGR1 has also been reported to facilitate the transcription of Program of China (2016YFC0904700 and 2016YFC0904702) and China diacylglycerol kinase alpha (DGKA) and promote radiation- Health Promotion Foundation (THC2015001). induced skin fibrosis (31). 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