Nrf2 Promotes Keratinocyte Proliferation in Psoriasis through Up-Regulation of Keratin 6, Keratin 16, and Keratin 17  Luting Yang, Xueli Fan, Tingting Cui, Erle Dang, Gang Wang  Journal of Investigative Dermatology  Volume 137, Issue 10, Pages 2168-2176 (October 2017) DOI: 10.1016/j.jid.2017.05.015 Copyright © 2017 The Authors Terms and Conditions

Figure 1 Nrf2 expression is elevated in psoriatic epidermis. (a) Immunohistochemical staining of Nrf2 and pNrf2 in three normal and psoriatic lesional skin samples. Scale bar = 50 μm. (b) Immunofluorescence staining of Nrf2 and pNrf2 in three normal and psoriatic lesional skin samples. Scale bar = 50 μm. (c) mRNA levels of Nrf2 and (d) protein levels of Nrf2 and pNrf2 were measured in epidermis of skin biopsy samples from healthy control subjects and psoriatic patients. White arrows represent cytoplasmic Nrf2, and green arrows represent nuclear Nrf2. Results are represented as mean ± standard error of the mean. N, normal skin; p, phosphorylated; Pso, Psoriasis lesion. **P < 0.01. Journal of Investigative Dermatology 2017 137, 2168-2176DOI: (10.1016/j.jid.2017.05.015) Copyright © 2017 The Authors Terms and Conditions

Figure 2 Nrf2 up-regulates K6, K16, and K17 expressions in HaCaT cell lines. (a) mRNA and (b) protein levels of K6, K16, and K17 were measured after transfection with Nrf2 siRNA or pCMV6-XL5-Nrf2 in HaCaT cells. Data are expressed as mean ± standard error of the mean from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. (c) Immunofluorescence analysis of Nrf2 (red) and K6, K16, and K17 (green) expressions. Scale bar = 50 μm. (d) ChIP assays to show that Nrf2 binds to K6 (left) and K17 (right) promoters. M, DNA marker; lanes 1–12, PCR product derived from direct input DNA template without immunoprecipitation (upper), normal IgG (middle), and anti-Nrf2 antibody (lower). (e) Chromatin immunoprecipitation assays to show that Nrf2 binds to K16 promoter. K, keratin; p, phosphorylated; siRNA, small interfering RNA. Journal of Investigative Dermatology 2017 137, 2168-2176DOI: (10.1016/j.jid.2017.05.015) Copyright © 2017 The Authors Terms and Conditions

Figure 3 IL-17 and IL-22 promote nuclear translocation of Nrf2 and initiate downstream K6, K16, and K17 gene expressions. (a, b) HaCaT cells were pretreated with IL-17, IL-22, or IFN-γ in different concentrations as indicated for 48 hours. (a) Nrf2, K6, K16, and K17 proteins were analyzed by Western blot. (b) Immunofluorescence analysis of Nrf2 and K17 expressions. Scale bar = 10 μm. (c) Nuclear fraction was prepared, and Nrf2 protein was analyzed and quantitated by Western blot. Lamin B and tubulin-β were used as loading controls for nuclear and cytosol fractions, respectively. *P < 0.05. (d) HaCaT cells were stimulated with 50 ng/ml IL-17 or IL-22 at 24 hours after transfection with Nrf2 siRNA. Western blot analysis of Nrf2, K6, K16, and K17 protein levels. K, keratin; p, phosphorylated; siRNA, small interfering RNA. Journal of Investigative Dermatology 2017 137, 2168-2176DOI: (10.1016/j.jid.2017.05.015) Copyright © 2017 The Authors Terms and Conditions

Figure 4 Nrf2 promotes proliferation of human keratinocyte through up-regulation of K6, K16, or K17. (a–c) HaCaT cells were transfected with Nrf2 siRNA or pCMV6-XL5-Nrf2. Proliferation was analyzed by (a, b) EdU assay 48 hours after transfection and by (c) Cell Counting Kit-8 at 24 hours, 48 hours, and 72 hours after transfection. The cells with red fluorescence are in the S phase of mitosis, and the cells with blue fluorescence represent all the cells. (d–f) siRNAs for K6, K16, or K17 and control siRNA were cotransfected with pCMV6-XL5-Nrf2. Proliferation was analyzed (d, e) by EdU assay and (f) by Cell Counting Kit-8. Data are represented as mean ± standard error of the mean. *P < 0.05; **P < 0.01; ***P < 0.001. EdU, 5-ethynyl-2′-deoxyuridine; K, keratin; OD, optical density; p, phosphorylated; siRNA, small interfering RNA. Journal of Investigative Dermatology 2017 137, 2168-2176DOI: (10.1016/j.jid.2017.05.015) Copyright © 2017 The Authors Terms and Conditions

Figure 5 Blocking Nrf2 expression ameliorates epidermal hyperplasia and reduced expression of K6, K16, and K17 in IMQ-treated mice. Nrf2 siRNA or scrambled siRNA was applied topically every 48 hours to the ears of mice treated with IMQ every 24 hours for 7 days. (a) Phenotypical presentation of mouse ears skin. (b) Ear thickness was measured on day 8. (c) Hematoxylin and eosin staining of ear tissues from mice (left). Upper, original magnification ×100; lower, original magnification ×400. Epidermal thickness was measured (right). (d) Immunofluorescence staining of Nrf2, pNrf2, K6, K16, and K17. Scale bar = 50 μm. (e) Protein and (f) mRNA levels of Nrf2, K6, K16, and K17 were measured in each group of mice. **P < 0.01; ***P < 0.001. IMQ, imiquimod; K, keratin; p, phosphorylated; siRNA, small interfering RNA. Journal of Investigative Dermatology 2017 137, 2168-2176DOI: (10.1016/j.jid.2017.05.015) Copyright © 2017 The Authors Terms and Conditions

Figure 6 Schematic proposal of the Nrf2-ARE/K6, K16, and K17 pathways in psoriasis keratinocytes under stimuli of inflammatory cytokines IL-17 and IL-22. Upon stimulation of inflammatory cytokines in psoriatic keratinocytes, Nrf2 dissociates from Keap1, and phosphorylated Nrf2 translocates to the nucleus, where it binds to the AREs of K6, K16, and K17 genes and up-regulates their expressions. Nrf2 activation promotes the proliferation of human keratinocyte through up-regulation of K6, K16, or K17. Concurrently, Nrf2 could promote the release of inflammatory cytokines and chemokines IL-17, TNF-α, and CXCL1, leading to Th1- and Th17-dominant inflammatory responses in psoriatic lesions. The breakdown of homeostasis by Nrf2 contributes to epidermal hyperplasia in psoriasis. Solid line indicates the results from this study, and dotted line indicates our speculations. ARE, antioxidant responsive element; K, keratin; P, phosphorylation; Th, T helper type; TNF, tumor necrosis factor. Journal of Investigative Dermatology 2017 137, 2168-2176DOI: (10.1016/j.jid.2017.05.015) Copyright © 2017 The Authors Terms and Conditions