TIEG1 Represses Smad7-Mediated Activation of TGF-β1/Smad Signaling in Keloid Pathogenesis  Zhi-Cheng Hu, Fen Shi, Peng Liu, Jian Zhang, Dong Guo, Xiao-Ling Cao, Chu-Fen Chen, Shan-Qiang Qu, Jia-Yuan Zhu, Bing Tang  Journal of Investigative Dermatology  Volume 137, Issue 5, Pages 1051-1059 (May 2017) DOI: 10.1016/j.jid.2016.12.019 Copyright © 2017 The Authors Terms and Conditions

Figure 1 Increased expression of TIEG1 and decreased expression of Smad7 in keloids. (a, b) Immunohistochemistry staining of TIEG1 and Smad7 in normal skin and keloids. Scale bar = 100 μm (upper), 50 μm (lower). The right panel presents the immunoreactive score (IRS) (Mann-Whitney test). (c) Immunofluorescence staining for TIEG1 and Smad7 in normal fibroblasts (NFs) (n = 4) and keloid fibroblasts (KFs) (n = 5). Scale bar = 50 μm. (d–g) The mRNA and protein levels of TIEG1, Smad7, and collagen I were detected by real-time PCR and Western blotting in normal skin (n = 3), keloids (n = 5), NFs (n = 3), and KFs (n = 5). Data are mean ± standard error of the mean. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, unpaired t test. IRS, immunoreactive score; KF, keloid fibroblast; NF, normal human fibroblast. Journal of Investigative Dermatology 2017 137, 1051-1059DOI: (10.1016/j.jid.2016.12.019) Copyright © 2017 The Authors Terms and Conditions

Figure 2 TIEG1 is important for KF proliferation, viability, migration, invasion, and collagen synthesis. KFs were transfected with negative control RNA (NC) or TIEG1-targeted siRNAs (siTIEG1) for 24 hours. (a–e) (b) The mRNA levels of collagen I and III were detected by real-time PCR (n = 4); the protein levels of (a) TIEG1, (c) collagen I and III, (d) proliferating cell nuclear antigen (PCNA) and cyclin D1, and (e) fibronectin, matrix metalloproteinase (MMP)-2, and MMP-9 were tested by Western blotting (n = 3). (f, g) The viability and proliferation were detected by (f) MTS assay and (g) BrdU incorporation assay (n = 4). (h, i) The (h) migration and (i) invasion are shown at various time points up to 24 hours (n = 4). Data are mean ± standard error of the mean. ∗P < 0.05, ∗∗P < 0.01; unpaired t test. h, hours; KF, keloid fibroblast; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; siRNA, small interfering RNA. Journal of Investigative Dermatology 2017 137, 1051-1059DOI: (10.1016/j.jid.2016.12.019) Copyright © 2017 The Authors Terms and Conditions

Figure 3 TIEG1 promotes phosphorylation of Smad2 by down-regulating Smad7 in KFs. (a, b) KFs transfected with negative control RNA (NC) or TIEG1-targeted siRNAs (siTIEG1) were treated with TGF-β1 (5 ng ml−1) for 1 hour or 24 hours. (a) The levels of TIEG1, phosphorylated Smad2 (P-Smad2), and Smad2 were detected by Western blot (n = 3); (b) cell lysates were immunoprecipitated (IP) with anti-TGF-β type I receptor (TβRI) antibody, and IP and lysate were analyzed by immunoblotting (n = 3). (c, d) KFs were transfected with (c) siTIEG1 and NC or (d) TIEG1-expressing plasmid (TIEG1) and empty vector (Vector); the levels of TIEG1, Smad7, and Smad6 were detected by Western blotting (n = 3), and Smad7 mRNA was detected by real-time PCR (n = 4). P-Smad2, phosphorylated Serine (P-Ser), and Smad7 were quantified. Data are mean ± standard error of the mean. ∗P < 0.05, ∗∗P < 0.01; unpaired t test. KF, keloid fibroblast; h, hours; siRNA, small interfering RNA. Journal of Investigative Dermatology 2017 137, 1051-1059DOI: (10.1016/j.jid.2016.12.019) Copyright © 2017 The Authors Terms and Conditions

Figure 4 TIEG1 inhibits Smad7 promoter activity in KFs. (a) Human normal fibroblasts (NFs) and KFs (n = 4), (b) KFs transfected with negative control RNA (NC) or TIEG1-targeted siRNA (siTIEG1) (n = 3), and (c) KFs transfected with empty vector (Vector) or TIEG1-expressing plasmid (TIEG1) (n = 3) were each transfected with 1 μg of Smad7-Luc. After 12 hours, dual-luciferase reporter assays were performed. The luciferase activity was normalized to Renilla luciferase activity, and luciferase activities of NFs, NC, or Vector control were set to 1. Data are mean ± standard error of the mean. ∗P < 0.05, ∗∗P < 0.01; unpaired t test. KF, keloid fibroblast; siRNA, small interfering RNA. Journal of Investigative Dermatology 2017 137, 1051-1059DOI: (10.1016/j.jid.2016.12.019) Copyright © 2017 The Authors Terms and Conditions

Figure 5 Luciferase activity was detected when an intact GC-box/Sp1 site at –1392 to –1382 base pairs in the Smad7 promoter was maintained. (a) Smad7 promoter 5′ sequential deletion constructs. Different lengths of the Smad7 promoter with the same 3′ end were cloned into pGL3-Basic. Human normal fibroblasts (NFs) and keloid fibroblasts (KFs) were transfected with 1 μg of each of the Smad7 promoter-based reporters for 12 hours (n = 3). (b) The wild-type GC-box/Sp1 site at –1392 / –1382 base pairs, GGGGGCGGGGG (WT), was mutated to GTTGGCGGGGT (MUT). NFs and KFs were transfected with 1 μg of Smad7-Luc containing the WT or MUT sites (n = 3). Dual-luciferase reporter assays were performed. Data are mean ± standard error of the mean. ∗P < 0.05, ∗∗P < 0.01; unpaired t test. C, cytosine; G, guanine; MUT, mutated; WT, wild type. Journal of Investigative Dermatology 2017 137, 1051-1059DOI: (10.1016/j.jid.2016.12.019) Copyright © 2017 The Authors Terms and Conditions

Figure 6 TIEG1 directly binds to GC-box/Sp1 sites in the Smad7 promoter. Cell lysates from normal fibroblasts (NFs) and keloid fibroblasts (KFs) were subjected to chromatin immunoprecipitation analysis using the anti-TIEG1 antibody. (a) Chromatins were sonicated into 500-bp fragments. (b) Western blot analysis with anti-TIEG1 antibody was able to precipitate TIEG1 from the cross-linked and fragmented protein-DNA complexes, showing the immunoprecipitation (IP) specificity and efficiency. (c) DNA isolated and purified from immunoprecipitated material was detected by PCR, the amplified PCR fragments were analyzed on a 2% agarose gel. Equal amounts of total genomic DNA (Input) were used for immunoprecipitation in each condition. Data shown are representative of three independent and reproducible experiments. bp, base pairs. Journal of Investigative Dermatology 2017 137, 1051-1059DOI: (10.1016/j.jid.2016.12.019) Copyright © 2017 The Authors Terms and Conditions