Characterization of TNF-α– and IL-17A–Mediated Synergistic Induction of DEFB4 Gene Expression in Human Keratinocytes through IκBζ  Claus Johansen, Trine Bertelsen, Christine Ljungberg, Maike Mose, Lars Iversen  Journal of Investigative Dermatology  Volume 136, Issue 8, Pages 1608-1616 (August 2016) DOI: 10.1016/j.jid.2016.04.012 Copyright © 2016 The Authors Terms and Conditions

Figure 1 TNF-α and IL-17A–induced synergistic induction of hBD2 in keratinocytes. Cultured human keratinocytes were stimulated with TNF-α (10 ng/ml), IL-17A (100 ng/ml), or a combination of the two cytokines for the indicated time points. (a) DEFB4 mRNA expression was analyzed by quantitative real-time reverse transcriptase PCR (n = 5). RPLP0 was used as a reference gene for normalization. (b) Protein level of hBD2 was measured by ELISA (n = 4). (c) Cultured human keratinocytes were transfected with a luciferase plasmid containing 2662 base pairs of the DEFB4 promoter together with an internal control (renilla). After transfection, cells were stimulated as indicated. The activity was determined as a ratio between firefly and renilla luciferase activity (n = 4). Results are expressed as mean ± standard deviation. ∗P < 0.05 compared with vehicle-treated cells. ∗∗P < 0.05 compared with the additive values of TNF-α and IL-17A separately. h, hours; hBD2, human β-defensin 2; mRNA, messenger RNA; TNF-α, tumor necrosis factor-α; Veh, vehicle. Journal of Investigative Dermatology 2016 136, 1608-1616DOI: (10.1016/j.jid.2016.04.012) Copyright © 2016 The Authors Terms and Conditions

Figure 2 Characterization of TNF-α/IL-17A–induced hBD2 expression. (a, b) Human keratinocytes were preincubated with the indicated inhibitors for 45 minutes before being stimulated with TNF-α and/or IL-17A for 24 hours. (a) DEFB4 mRNA expression was measured by quantitative real-time reverse transcriptase PCR (n = 5). RPLP0 expression was used for normalization. (b) The hBD2 protein level was measured by ELISA (n = 4). ∗P < 0.05 compared with IL-17A-treated cells. ∗∗P < 0.05 compared with TNFα+IL-17A-treated cells. (c, d) Keratinocytes were transfected with siRNA directed against p38α and p38β (sip38α+β), p65 (sip65), JNK1 and JNK2 (siJNK1/2), control siRNA (siCon) or transfection reagent alone (Mock) before stimulation for 24 hours. (c) Quantitative real-time reverse transcriptase PCR was used to analyze DEFB4 mRNA expression (n = 5). (d) hBD2 protein level was measured by ELISA (n = 4). Results are expressed as mean ± standard deviation. ∗P < 0.05. (e) Keratinocytes were stimulated with TNF-α, IL-17A, or a combination for the indicated time points. The indicated proteins were analyzed by Western blotting. β-Actin was used as a loading control (n = 3). ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; hBD2, human β-defensin 2; MAPK, mitogen-activated protein kinase; min., minutes; mRNA, messenger RNA; siRNA, small interfering RNA; TNF-α, tumor necrosis factor-α; Veh, vehicle. Journal of Investigative Dermatology 2016 136, 1608-1616DOI: (10.1016/j.jid.2016.04.012) Copyright © 2016 The Authors Terms and Conditions

Figure 3 Mutations in specific transcription factor binding sites abolished DEFB4 promoter activity. (a) Cells were transfected with different DEFB4-promoter plasmids as indicated together with an internal control (renilla). After transfection, cells were stimulated with a combination of TNF-α and IL-17A for 24 hours. Results are expressed as mean ± standard deviation (n = 3). (b) The DEFB4_DL4 promoter constructs are shown. One C/EBP site, one NF-κB site, two OCT1 sites, and one AP-1 site in the DEFB4_DL4 promoter were mutated in different combinations as indicated. (c) Keratinocytes were transfected with DEFB4_DL4 or mutated forms of DEFB4_DL4-promoter plasmids together with an internal control (renilla) before being stimulated with TNF-α and/or IL-17A for 24 hours (n = 4). Bars represent mean ± standard deviation. ∗P < 0.05 compared with DEFB4_DL4-transfected cell. AP-1, activator protein 1; C/EBP, CCAAT-enhancer-binding protein-β; LUC, luciferase; Mut, mutation; OCT1, organic cation transporter 1; TNF-α, tumor necrosis factor-α; Veh, vehicle. Journal of Investigative Dermatology 2016 136, 1608-1616DOI: (10.1016/j.jid.2016.04.012) Copyright © 2016 The Authors Terms and Conditions

Figure 4 Knockdown of OCT1 expression decreases the expression of hBD2. Cultured human keratinocytes were transfected with OCT1 siRNA (siOCT1), control siRNA (siCon) or transfection reagent alone (Mock) before being stimulated with a combination of TNF-α and IL-17A for 24 hours. (a) RNA and protein were isolated from the cells, and the expression of POU2F1 mRNA and OCT1 protein was measured by quantitative real-time reverse transcriptase PCR and Western blotting, respectively (n = 3). (b) Cells were treated as described, and the mRNA expression of DEFB4 was analyzed by quantitative real-time reverse transcriptase PCR (n = 3). RPLP0 was used as a reference gene for normalization. (c) The hBD2 protein level was measured in the cell culture medium using ELISA (n = 3). Results are expressed as mean ± standard deviation. ∗P < 0.05. hBD2, human β-defensin 2; mRNA, messenger RNA; OCT1, organic cation transporter 1; TNF-α, tumor necrosis factor-α; Veh, vehicle. Journal of Investigative Dermatology 2016 136, 1608-1616DOI: (10.1016/j.jid.2016.04.012) Copyright © 2016 The Authors Terms and Conditions

Figure 5 Binding of NF-κB, OCT1, and AP-1 to their specific binding sites in the human DEFB4 promoter. Cultured human keratinocytes were stimulated with TNF-α, IL-17A, or a combination of TNF-α and IL-17A for 1 hour before the DNA binding activity of (a) NF-κB, (b) OCT1, and (c) AP-1 to the DEFB4 promoter was analyzed by electrophoretic mobility shift assay. Antibodies against p50, p65, OCT1, c-Fos, and c-Jun were tested for their ability to cause a supershift of the nuclear complex associated with the probe. Representative results of three separate experiments are shown. AP-1, activator protein 1; NS, nonspecific; OCT1, organic cation transporter 1; oligo, oligonucleotides; TNF-α, tumor necrosis factor-α. Journal of Investigative Dermatology 2016 136, 1608-1616DOI: (10.1016/j.jid.2016.04.012) Copyright © 2016 The Authors Terms and Conditions

Figure 6 IκBζ expression is regulated by IL-17A through a p38 MAPK-dependent mechanism. (a, b) Human keratinocytes were pretreated with SB202190 for 45 minutes before being stimulated with TNF-α and/or IL-17A for 1.5 hours. (a) NFKIBZ mRNA expression was analyzed by quantitative real-time reverse transcriptase PCR (n = 3). RPLP0 expression was used for normalization. Results are expressed as mean ± standard deviation. ∗P < 0.05 compared with vehicle-treated cells. ∗∗P < 0.05 compared with IL-17A–treated cells. ∗∗∗P < 0.05 compared with TNF-α/IL-17A–treated cells. (b) IκBζ protein levels were analyzed by Western blotting (n = 3). (c) Keratinocytes were transfected with IκBζ siRNA (siIκBζ), control siRNA (siCon) or transfection reagent alone (Mock) before stimulation with TNF-α and IL-17A for 24 hours. hBD2 protein levels were analyzed by ELISA (n = 3). (d) Working model of the signaling mechanism through which TNF-α and IL-17A mediate their synergistic induction of hBD2 in human keratinocytes. AP-1, activator protein 1; hBD2, human β-defensin 2; JNK, c-Jun N-terminal kinase; mRNA, messenger RNA; OCT1, organic cation transporter 1; TNF-α, tumor necrosis factor-α; Veh, vehicle. Journal of Investigative Dermatology 2016 136, 1608-1616DOI: (10.1016/j.jid.2016.04.012) Copyright © 2016 The Authors Terms and Conditions