TSLP Down-Regulates S100A7 and ß-Defensin 2 Via the JAK2/STAT3-Dependent Mechanism  Hana Lee, Woo-In Ryu, Hee Joo Kim, Hyun Cheol Bae, Hwa Jung Ryu, Jung Jin Shin, Kwon-Ho Song, Tae Woo Kim, Sang Wook Son  Journal of Investigative Dermatology  Volume 136, Issue 12, Pages 2427-2435 (December 2016) DOI: 10.1016/j.jid.2016.07.027 Copyright © 2016 The Authors Terms and Conditions

Figure 1 Down-regulation of S100A7 and hBD2 expression by TSLP. NHEKs were stimulated with different TSLP doses (0, 1, 10, and 100 ng/ml) and incubated for 24 hours. (a) S100A7 and hBD2 mRNA levels were down-regulated by TSLP. (b) Protein expression and (c) promoter activity were decreased under the same conditions. (d) S100A7 and hBD2 expression level was down-regulated by TSLP in the HSEMs. The HSEMs were stimulated with TSLP (500 ng/ml) and incubated for 24 hours, and an immunohistochemical analysis was performed to detect S100A7 and hBD2. Data are mean ± standard deviation. ∗P < 0.05. Scale bar = 50 μm. hBD2, human β-defensin 2; HSEM, human skin equivalent model; NHEK, normal human epidermal keratinocyte; S100A7, S100 calcium-binding protein A7; SB, stratum basale; SC, stratum corneum; SG, stratum granulosum; SP, stratum spinosum; TSLP, thymic stromal lymphopoietin. Journal of Investigative Dermatology 2016 136, 2427-2435DOI: (10.1016/j.jid.2016.07.027) Copyright © 2016 The Authors Terms and Conditions

Figure 2 JAK-2/STAT3 regulates the TSLP-induced reduction in S100A7 and hBD2 expression. (a) Keratinocytes were treated with TSLP (100 ng/ml) for 24 hours. JAK-2 and STAT3 protein expressions were examined by Western blot analysis. (b) Blocking JAK activation with a specific JAK inhibitor (pyridone 6) substantially restored S100A7 and hBD2 expression. (c) NHEKs were transfected with pcDNA3.1/STAT3. A Western blot analysis was performed to characterize S100A7 and hBD2 expression. (d) STAT3 overexpression down-regulated S100A7 and hBD2 promoter activity. ∗P < 0.05. (e) Western blot analysis of S100A7 and hBD2 expression levels after TSLP treatment with or without a STAT3 small interfering RNA. (f) Western blot analysis of S100A7 and hBD2 expression levels after TSLP treatment with or without STA-21, a STAT3 inhibitor. hBD2, human β-defensin 2; NHEK, normal human epidermal keratinocyte; pSTAT-3, phospho-STAT3; S100A7, S100 calcium-binding protein A7; scram, scramble; siRNA, small interfering RNA; STAT3, signal transducer and activator of transcription 3; TSLP, thymic stromal lymphopoietin. Journal of Investigative Dermatology 2016 136, 2427-2435DOI: (10.1016/j.jid.2016.07.027) Copyright © 2016 The Authors Terms and Conditions

Figure 3 STAT3 is highly expressed in the skin of patients with AD. (a) (upper) H&E staining and (lower) immunohistochemical staining with anti-pSTAT3 (as indicated by red arrow) of human skin specimens. Left, healthy subject; right, lesional skin of AD patients. Scale bars = 200 μm. (b) The intensities of the staining for pSTAT3 were graded visually on a scale from 0 (no staining) to 3 (the most intense staining). AD, atopic dermatitis; H&E, hematoxylin and eosin; pSTAT3, phospho-STAT3. Journal of Investigative Dermatology 2016 136, 2427-2435DOI: (10.1016/j.jid.2016.07.027) Copyright © 2016 The Authors Terms and Conditions

Figure 4 STAT3 directly regulates the TSLP-induced decrease in S100A7 and hBD2 expression. (a) Expression of pGL3/S100A7 or pGL3/S100A7 mutant 1– 3 or pGL3/hBD2 or pGL3/hBD2 mutant 1– 3 after treatment with TSLP (n = 3). ∗P < 0.05. (b) EMSA and supershift assays. Oligonucleotide probe from the S100A7 and hBD2 promoter, which contained consensus STAT3 binding elements (1–3). Nuclear extracts prepared from the nontreated keratinocytes (lane 1) or TSLP-treated keratinocytes (lanes 2–4). The supershift assay (lane 3) was performed with an anti-pSTAT3 antibody. Competition assays were performed with unlabeled oligonucleotides (lane 4). (c) The S100A7 and hBD2 expression after TSLP treatment with or without a Sin3a siRNA were detected. (d) Chromatin was incubated with anti-Sin3a and pSTAT3 antibody. The 250 bp fragment contained STAT3 elements 1–3 in the S100A7 and hBD2 promoters. ∗P < 0.05. bp, base pairs; EMSA, electrophoretic mobility shift assays; hBD2, human β-defensin 2; S100A7, S100 calcium-binding protein A7; si, small interfering; STAT3, signal transducer and activator of transcription 3; TSLP, thymic stromal lymphopoietin. Journal of Investigative Dermatology 2016 136, 2427-2435DOI: (10.1016/j.jid.2016.07.027) Copyright © 2016 The Authors Terms and Conditions