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Nrf2 Promotes Keratinocyte Proliferation in Psoriasis through Up-Regulation of Keratin 6, Keratin 16, and Keratin 17

  • Author Footnotes
    2 These authors contributed equally to this work.
    Luting Yang
    Footnotes
    2 These authors contributed equally to this work.
    Affiliations
    Department of Dermatology, Xijing Hospital, Fourth Military Medical University, Xi’an, China
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  • Author Footnotes
    2 These authors contributed equally to this work.
    Xueli Fan
    Footnotes
    2 These authors contributed equally to this work.
    Affiliations
    Department of Dermatology, Xijing Hospital, Fourth Military Medical University, Xi’an, China
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  • Tingting Cui
    Affiliations
    Department of Dermatology, Xijing Hospital, Fourth Military Medical University, Xi’an, China
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  • Erle Dang
    Affiliations
    Department of Dermatology, Xijing Hospital, Fourth Military Medical University, Xi’an, China
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  • Gang Wang
    Correspondence
    Correspondence: Gang Wang, Department of Dermatology, Xijing Hospital, Fourth Military Medical University, 127 West Changle Road, Xi’an.
    Affiliations
    Department of Dermatology, Xijing Hospital, Fourth Military Medical University, Xi’an, China
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  • Author Footnotes
    2 These authors contributed equally to this work.
Open ArchivePublished:May 30, 2017DOI:https://doi.org/10.1016/j.jid.2017.05.015
      Psoriasis is a chronic inflammatory skin disease characterized by keratinocyte hyperproliferation of epidermis. Although hyperproliferation-associated keratins K6, K16, and K17 are considered to be the hallmarks of psoriasis, the molecular basis underlying the overexpression of these keratins remains unclear. Nrf2 regulates cell proliferation. Therefore, we investigated whether Nrf2 regulates keratinocyte proliferation via promoting expression of K6, K16, and K17 in psoriasis. We initially found that psoriatic epidermis exhibited elevated expression of Nrf2. Furthermore, Nrf2 promoted expression of K6, K16, and K17 in both HaCaT cells and primary human keratinocytes by binding to the ARE domains located in the promoter of these genes. Additionally, upon stimulation with IL-17 or IL-22, Nrf2 translocated to the nucleus and initiated expression of targeted keratins. In mice of imiquimod-induced psoriasis-like dermatitis, topical application of Nrf2 small interfering RNA alleviated the epidermal hyperplasia with reduced expression of these keratins. More importantly, Nrf2 promoted the proliferation of human keratinocytes through up-regulation of K6, K16, or K17. These data suggested that inflammatory cytokines promoted Nrf2 nuclear translocation in psoriatic epidermis, which led to elevated expression of K6, K16, and K17, thus promoting keratinocyte proliferation and contributing to the pathogenesis of psoriasis.

      Abbreviations:

      ARE (antioxidant responsive element), ChIP (chromatin immunoprecipitation), EdU (5-ethynyl-2′-deoxyuridine), IMQ (imiquimod), K (keratin), pNrf2 (phosphorylated Nrf2), siRNA (small interfering RNA), TNF-α (tumor necrosis factor-α)

      Introduction

      Nrf2 is a transcription factor known for its role in regulating cellular defenses against a variety of oxidative stresses and environmental insults (
      • Sykiotis G.P.
      • Bohmann D.
      Stress-activated cap'n'collar transcription factors in aging and human disease.
      ). Normally, Nrf2 is sequestered in cytoplasm by binding to the Kelch domain of Keap1, a substrate adaptor protein for ubiquitin ligase complex (
      • Knatko E.V.
      • Higgins M.
      • Fahey J.W.
      • Dinkova-Kostova A.T.
      Loss of Nrf2 abrogates the protective effect of Keap1 downregulation in a preclinical model of cutaneous squamous cell carcinoma.
      ,
      • Zhang Y.
      • Hou Y.
      • Liu C.
      • Li Y.
      • Guo W.
      • Wu J.L.
      • et al.
      Identification of an adaptor protein that facilitates Nrf2-Keap1 complex formation and modulates antioxidant response.
      ). However, when cells encounter oxidative stresses and other stimuli, Nrf2 is dissociated from Keap1, allowing its translocation to the nucleus, and thereafter activates its downstream target genes. Several antioxidant genes, as well as proteostasis genes and metabolic genes are regulated by Nrf2 through the antioxidant responsive element (ARE) located in their promoter regions (
      • Hayes J.D.
      • Dinkova-Kostova A.T.
      The Nrf2 regulatory network provides an interface between redox and intermediary metabolism.
      ,
      • Lacher S.E.
      • Lee J.S.
      • Wang X.
      • Campbell M.R.
      • Bell D.A.
      • Slattery M.
      Beyond antioxidant genes in the ancient Nrf2 regulatory network.
      ,
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      • Taguchi K.
      • Kawatani Y.
      • Shibata T.
      • Nukiwa T.
      • Aburatani H.
      • et al.
      Nrf2 redirects glucose and glutamine into anabolic pathways in metabolic reprogramming.
      ).
      Very recently, studies from several groups showed that the extension of Nrf2 target genes is actually much wider than previously thought and encompasses some additional genes that regulate both cell proliferation and differentiation (
      • Al-Sawaf O.
      • Fragoulis A.
      • Rosen C.
      • Keimes N.
      • Liehn E.A.
      • Holzle F.
      • et al.
      Nrf2 augments skeletal muscle regeneration after ischaemia-reperfusion injury.
      ,
      • Murakami S.
      • Motohashi H.
      Roles of Nrf2 in cell proliferation and differentiation.
      ). For instance, Nrf2 has been proven to enhance cancer cell proliferation through up-regulation of metabolic genes (
      • Singh A.
      • Happel C.
      • Manna S.K.
      • Acquaah-Mensah G.
      • Carrerero J.
      • Kumar S.
      • et al.
      Transcription factor NRF2 regulates miR-1 and miR-206 to drive tumorigenesis.
      ). Evidences from keratinocyte cell line HaCaT cells also showed that in allergic contact dermatitis, Nrf2 translocated to the nucleus and induced the release of inflammatory mediators (e.g., IL-6, IL-8, and IL-1β), but the underlying mechanism has not been clarified yet (
      • Martin S.F.
      • Esser P.R.
      • Weber F.C.
      • Jakob T.
      • Freudenberg M.A.
      • Schmidt M.
      • et al.
      Mechanisms of chemical-induced innate immunity in allergic contact dermatitis.
      ). What is more important, the chemopreventive agent sulforaphane, which activates Nrf2, up-regulated epidermal K16 and K17. More directly, Nrf2 itself also activates K16 in human keratinocytes (
      • Endo H.
      • Sugioka Y.
      • Nakagi Y.
      • Saijo Y.
      • Yoshida T.
      A novel role of the NRF2 transcription factor in the regulation of arsenite-mediated keratin 16 gene expression in human keratinocytes.
      ,
      • Kerns M.
      • DePianto D.
      • Yamamoto M.
      • Coulombe P.A.
      Differential modulation of keratin expression by sulforaphane occurs via Nrf2-dependent and -independent pathways in skin epithelia.
      ). However, the expression profile and putative roles of Nrf2 in epidermal proliferation remain unknown.
      Psoriasis is a common, chronic inflammatory skin disease characterized by hyperproliferation and aberrant differentiation of keratinocytes. Studies on changes in the epidermis of psoriasis have shown evidence for the presence of three hyperproliferation-associated keratins: keratin (K) 6, K16, and K17. K16 serves as the type I keratin partner of K6 in the formation of intermediated filament heterodimers (
      • Moll R.
      • Divo M.
      • Langbein L.
      The human keratins: biology and pathology.
      ). This K6/K16 keratin pair is induced in hyperproliferative cells, such as psoriatic epidermis, but not found in normal epidermis (
      • Korver J.E.
      • van Duijnhoven M.W.
      • Pasch M.C.
      • van Erp P.E.
      • van de Kerkhof P.C.
      Assessment of epidermal subpopulations and proliferation in healthy skin, symptomless and lesional skin of spreading psoriasis.
      ,
      • Thewes M.
      • Stadler R.
      • Korge B.
      • Mischke D.
      Normal psoriatic epidermis expression of hyperproliferation-associated keratins.
      ). Recently, we identified the role of another keratin, K17, in the pathogenesis of psoriasis (
      • Chang T.
      • Sun L.
      • Wang Y.
      • Wang D.
      • Li W.
      • Li C.
      • et al.
      Inhibition of keratin 17 expression with antisense and RNAi strategies: exploring novel therapy for psoriasis.
      ,
      • Fu M.
      • Wang G.
      Keratin 17 as a therapeutic target for the treatment of psoriasis.
      ,
      • Jin L.
      • Wang G.
      Keratin 17: a critical player in the pathogenesis of psoriasis.
      ). K17, which is a cytoskeletal protein, displayed the same expression pattern as K6 and K16: overexpression in psoriatic lesional epidermis but not in healthy epidermis. Moreover, we confirmed a positive feedback regulatory loop, K17-T cell-inflammatory cytokine (IFN-γ, IL-17, and IL-22), in psoriasis in that K17 expression could be induced by IFN-γ, IL-17, and IL-22 in epidermis. K17 then in turn promotes the proliferation of psoriatic T cells and production of these cytokines (
      • Shen Z.
      • Chen L.
      • Liu Y.F.
      • Gao T.W.
      • Wang G.
      • Fan X.L.
      • et al.
      Altered keratin 17 peptide ligands inhibit in vitro proliferation of keratinocytes and T cells isolated from patients with psoriasis.
      ,
      • Shi X.
      • Jin L.
      • Dang E.
      • Chang T.
      • Feng Z.
      • Liu Y.
      • et al.
      IL-17A upregulates keratin 17 expression in keratinocytes through STAT1- and STAT3-dependent mechanisms.
      ,
      • Zhang W.
      • Dang E.
      • Shi X.
      • Jin L.
      • Feng Z.
      • Hu L.
      • et al.
      The pro-inflammatory cytokine IL-22 up-regulates keratin 17 expression in keratinocytes via STAT3 and ERK1/2.
      ). Hence, K6, K16, and K17 are considered to be the hallmarks of psoriasis.
      To our knowledge, few studies have focused on the role of Nrf2 in promoting keratinocyte proliferation or have investigated its target genes in psoriasis. Given that AREs matching the consensus for Nrf2 binding could be identified in the region of the human genome that houses K6, K16, and K17 genes, we speculated that Nrf2 regulated hyperproliferation-related keratins and that the change of these keratins might serve as an important mechanism that affects keratinocyte proliferation in psoriasis. To verify our hypothesis, we examined the expression of Nrf2 in psoriatic lesions. Through knockdown and overexpression of Nrf2, we analyzed the effects of Nrf2 on expression of K6, K16, and K17 and proliferation of keratinocytes. Furthermore, we used chromatin immunoprecipitation (ChIP) assay to determine whether K6, K16, and K17 are associated target genes of Nrf2. In addition, we evaluated the impacts of Nrf2 blockade on an imiquimod (IMQ)-induced psoriasis-like dermatitis mouse model. We showed that upon the stimulation with proinflammatory cytokines such as IL-17 or IL-22, Nrf2 translocated to the nucleus and up-regulated the expression of K6, K16, and K17 genes via ARE-binding region in psoriasis. Furthermore, Nrf2 promoted proliferation of keratinocytes by up-regulating the expression of K6, K16, and K17. Our study provides important insights into the role of Nrf2 in the pathogenesis of psoriasis.

      Results

      Nrf2 is up-regulated and activated in the epidermis of psoriatic lesions

      To determine the role of Nrf2 in psoriasis, we first assessed the subcellular localization and phosphorylation status of Nrf2 by immunofluorescence and immunohistochemical staining. Significant increases in nuclear-localized Nrf2 and phosphorylated Nrf2 (pNrf2) were detected in psoriatic lesional epidermis compared with normal skin (Figure 1a, 1b). In addition, Nrf2 mRNA levels were elevated by 4.3-fold in the epidermis of psoriatic lesions, compared with the normal control samples (Figure 1c). Consistently, protein levels of total Nrf2 and pNrf2 were markedly increased in psoriatic lesional epidermis but with a minimal detectable expression in normal skin (Figure 1d). Taken together, these results indicated an elevated level and activation of Nrf2 in psoriatic epidermis, suggesting its potential role in the pathogenesis of psoriasis.
      Figure thumbnail gr1
      Figure 1Nrf2 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.

      Nrf2 induces psoriasis-related K6, K16, and K17 expressions in vitro

      To determine whether Nrf2 might affect the expression of K6, K16, and K17 in psoriasis, we treated HaCaT cells with small interfering RNA (siRNA) or pCMV6-XL5-Nrf2 to knock down or overexpress Nrf2. The efficiency of all three Nrf2 siRNAs and pCMV6-XL5-Nrf2 was verified by real-time PCR and Western blot (see Supplementary Figure S1 online), and we randomly used Nrf2 siRNA1 in all the subsequent experiments. We found that mRNA and protein levels of all these three keratins were decreased after Nrf2 siRNA knockdown but increased in pCMV6-XL5-Nrf2 transfected cells (Figure 2a, 2b). Furthermore, knockdown of Nrf2 significantly reduced the fluorescence intensities of all these keratins, whereas overexpression of Nrf2 increased K6, K16, and K17 fluorescence signals (Figure 2c, Supplementary Figure S2 online). To further confirm this, we performed these experiments in primary keratinocytes isolated from normal human foreskin and found that Nrf2 also induced expression of K6, K16, and K17 (see Supplementary Figure S3a, S3b online). These results indicated that Nrf2 could up-regulate the expressions of K6, K16, and K17 in both primary and immortalized keratinocytes in vitro.
      Figure thumbnail gr2
      Figure 2Nrf2 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.

      Nrf2 associates with the promoters of K6, K16, and K17 genes

      Next, we conducted ChIP assays to investigate whether Nrf2 could be recruited to the promoter regions of K6, K16, and K17 genes. ChIP assays showed that Nrf2 bound to the –284 to +12 fragment of the K6 promoter (Figure 2d, left, Supplementary Figure S4a online). Additionally, Nrf2 was also found to be recruited to the –475 to –184 base pair of the K17 promoter (Figure 2d, right, Supplementary Figure S4b). Similar to the previous data from electrophoretic mobility shift assays (
      • Endo H.
      • Sugioka Y.
      • Nakagi Y.
      • Saijo Y.
      • Yoshida T.
      A novel role of the NRF2 transcription factor in the regulation of arsenite-mediated keratin 16 gene expression in human keratinocytes.
      ), we also confirmed that Nrf2 could be recruited to the ARE site from –312 to –28 region of K16 promoter (Figure 2e). Collectively, our data showed that Nrf2 could interact with the promoter regions of all K6, K16, and K17 genes, and this provides a possible explanation for the activation of these hyperproliferation-associated keratins.

      IL-17 and IL-22 enhance the expression of hyperproliferation-related keratins mediated by Nrf2

      IL-17, IFN-γ, and IL-22 have been reported to induce K17 up-regulation in psoriasis (
      • Shen Z.
      • Chen L.
      • Liu Y.F.
      • Gao T.W.
      • Wang G.
      • Fan X.L.
      • et al.
      Altered keratin 17 peptide ligands inhibit in vitro proliferation of keratinocytes and T cells isolated from patients with psoriasis.
      ,
      • Shi X.
      • Jin L.
      • Dang E.
      • Chang T.
      • Feng Z.
      • Liu Y.
      • et al.
      IL-17A upregulates keratin 17 expression in keratinocytes through STAT1- and STAT3-dependent mechanisms.
      ,
      • Zhang W.
      • Dang E.
      • Shi X.
      • Jin L.
      • Feng Z.
      • Hu L.
      • et al.
      The pro-inflammatory cytokine IL-22 up-regulates keratin 17 expression in keratinocytes via STAT3 and ERK1/2.
      ). As such, we tested whether these factors could signal through the Nrf2 pathway to induce K6, K16, and K17 up-regulation in psoriasis. IL-17 and IL-22 showed potent effects on the expression of total Nrf2 and pNrf2, which correlated well with elevated levels of K6, K16, and K17 in keratinocytes, with the maximal effect observed at 50 ng/ml. However, only slightly increased levels of Nrf2 and pNrf2 were observed after IFN-γ treatment (Figure 3a, Supplementary Figure S5a online). Likewise, these effects of IL-17 and IL-22 on induction of Nrf2 were also confirmed at the mRNA level and by immunofluorescence analysis (Figure 3b, Supplementary Figure S5b).
      Figure thumbnail gr3
      Figure 3IL-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.
      We next examined Nrf2 localization by cellular fractionation after treatment with IL-17, IL-22, and IFN-γ. We found that the Nrf2 translocation into the nucleus was markedly increased after treatment with either 50 ng/ml of IL-17 or of IL-22, but not IFN-γ (Figure 3c). To test whether Nrf2 regulated the expressions of K6, K16, and K17 in response to IL-17 and IL-22, we measured K6, K16, and K17 expressions in HaCaT transfected with Nrf2 siRNA after treatment with IL-17 or IL-22. Indeed, Nrf2 knockdown inhibited the increase of K6, K16, and K17 induced by IL-17 and IL-22 (Figure 3d, Supplementary Figure S5c). Furthermore, Nrf2 blockade markedly reduced the IL-22–induced proliferation of keratinocyte, although IL-22 only slightly induced proliferation of keratinocytes (see Supplementary Figure S6 online). Collectively, the cooperation of Nrf2 with IL-17 and IL-22 signaling to induce K6, K16, and K17 expressions suggest that IL-17 and IL-22 are positive regulators that target the intermediate Nrf2, thus regulating hyperproliferation-related keratins.

      Nrf2 promotes the proliferation of human keratinocytes through up-regulation of K6, K16, and K17

      Because Nrf2 has been shown to promote the proliferation of a variety of cell lines, including hepatocytes and cancer cells (
      • Beyer T.A.
      • Xu W.
      • Teupser D.
      • auf dem Keller U.
      • Bugnon P.
      • Hildt E.
      • et al.
      Impaired liver regeneration in Nrf2 knockout mice: role of ROS-mediated insulin/IGF-1 resistance.
      ,
      • Singh A.
      • Happel C.
      • Manna S.K.
      • Acquaah-Mensah G.
      • Carrerero J.
      • Kumar S.
      • et al.
      Transcription factor NRF2 regulates miR-1 and miR-206 to drive tumorigenesis.
      ), our next question was to ask whether Nrf2 promotes the proliferation of keratinocytes. Accordingly, we performed Cell Counting Kit-8 and 5-ethynyl-2′-deoxyuridine (EdU) labeling to elucidate the role of Nrf2 in human keratinocyte proliferation. Overexpression of Nrf2 led to an increased proliferation ratio of EdU-stained HaCaT cells, whereas knockdown of Nrf2 significantly reduced EdU-positive cells (Figure 4a, 4b). Consistently, similar results were observed from Cell Counting Kit-8 analysis, with the maximal effect observed at 72 hours after transfection (Figure 4c). Moreover, we have verified that in primary keratinocytes, Nrf2 also promoted cell proliferation (see Supplementary Figure S3c). Collectively, these data showed the role of Nrf2 in promoting the proliferation of both primary and human keratinocytes in vitro.
      Figure thumbnail gr4
      Figure 4Nrf2 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.
      Next, we aimed to determine whether Nrf2 promoted the proliferation of human keratinocytes through up-regulation of K6, K16, or K17. After co-transfection of siRNAs for K6, K16, or K17 with pCMV6-XL5-Nrf2, we tested the ability of Nrf2 to promote the proliferation of HaCaT cells after knockdown of these keratins (see Supplementary Figure S7a, S7b online). As shown by both EdU assay and Cell Counting Kit-8 analysis, knockdown of either K6, K16, or K17 decreased the proliferation of HaCaT cells (Figure 4d–f). These data suggested that Nrf2 promoted the proliferation of human keratinocyte through up-regulation of K6, K16, or K17.
      Along with the change in keratin expression, Nrf2 also affected the production of proinflammatory cytokines and chemokines. mRNA levels of IL-17A, tumor necrosis factor (TNF)-α, CXCL1, and IL-1β were decreased after Nrf2 knockdown, whereas IL-17A, TNF-α, and CXCL1, but not IL-1β, were increased after Nrf2 overexpression (see Supplementary Figure S8a, S8b online). To investigate whether Nrf2 induced up-regulation of these cytokines and chemokines also through K6, K16, and K17, siRNAs for these keratins were co-transfected with pCMV6-XL5-Nrf2. None of K6, K16, or K17 knockdowns induced a significant decrease in IL-17A, TNF-α, and CXCL1 mRNA levels (see Supplementary Figure S8c), which indicated the involvement of mechanisms other than K6, K16, and K17 in Nrf2-induced up-regulation of IL-17A, TNF-α, and CXCL1 in psoriasis.

      Local depletion of Nrf2 repressed K6, K16, and K17 expressions and alleviated IMQ-induced psoriasiform lesion

      Next, we applied an IMQ-induced psoriasis-like mouse model to uncover whether Nrf2 had similar effects in vivo. We found that topical application of Nrf2 siRNA to the ear skin of IMQ-treated mice efficiently ameliorated psoriasis-like lesions, showing a reduced ear thickness and alleviated erythema (Figure 5a, 5b). Similarly, hematoxylin and eosin staining of tissue sections from mouse ears after topical treatment with Nrf2 siRNA showed evident epidermal changes with reduced thickness and decreased hyperplasia (Figure 5c).
      Figure thumbnail gr5
      Figure 5Blocking 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.
      We were then interested in whether knockdown of Nrf2 could affect the expression levels of K6, K16, and K17 in vivo and the proliferation status of mice epidermis. Immunofluorescence analysis showed an almost undetectable level of K6, K16, K17, and Nrf2 in normal mice, whereas IMQ strongly induced the expressions of these keratins as well as Nrf2 in epidermis of mice in a temporal manner, which also confirmed the success of IMQ-induced psoriasis-like dermatitis (see Supplementary Figure S9 online). In the IMQ-induced mouse model, Nrf2 knockdown resulted in rapid attenuation of K6, K16, and K17 expressions at the mRNA and protein levels and immunofluorescence intensities (Figure 5d–f, Supplementary Figure S10b online). In addition, the percentage of proliferating cell nuclear antigen-positive cells was also reduced in the mice with Nrf2 blockade, indicating that Nrf2 might affect the proliferation status of mice epidermis (see Supplementary Figure S10a). Meanwhile, a concurrent reduction in mRNA levels of psoriasis-related IL-17A, TNF-α, IL-1β, and CXCL1 was noted (see Supplementary Figure S11 online). These results suggested the pivotal role of Nrf2 in promoting hyperproliferation-related K6, K16, and K17 expressions in vivo.

      Discussion

      In this study, we showed that Nrf2 was overexpressed and activated in the epidermis of psoriatic lesions. Concurrently, an elevated level of pNrf2 was also noted in psoriatic dermis, which might be due to increased levels of inflammatory cytokines. Under stimuli of several psoriasis-related proinflammatory cytokines, such as IL-17 or IL-22, Nrf2 translocated to the nucleus and activated its target genes K6, K16, and K17 by interaction with ARE domains located in the promoter regions of these genes. In this study, the role of Nrf2 in promoting keratinocyte proliferation through K6, K16, and K17 has been characterized, which might be an important mechanism underlying the pathogenesis of psoriasis. Along with this, Nrf2 induced production of IL-17A, TNF-α, and CXCL1 in psoriatic keratinocyte. We speculated that Nrf2 might also induce inflammation in psoriasis, but the precise mechanism requires further investigation (Figure 6).
      Figure thumbnail gr6
      Figure 6Schematic 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.
      Nrf2 belongs to the CNC family that contains leucine zipper. Recent studies proved that AREs could be specifically bound by several basic leucine zipper transcription factors, including Nrf2 (
      • Zhang G.
      • Li S.
      • Kang Y.
      • Che J.
      • Cui R.
      • Song S.
      • et al.
      Enhancement of dopaminergic activity and region-specific activation of Nrf2-ARE pathway by intranasal supplements of testosterone propionate in aged male rats.
      ). As accumulating evidences have clarified the functional roles of Nrf2 beyond the cytoprotection against a set of detoxifying and antioxidant genes, increasing attempts have been made to dig deep into the effect of Nrf2 on physiological and pathological process. Because AREs matching the consensus for Nrf2 binding can be identified in the promoter regions of K6, K16, and K17, the putative role of Nrf2 was to regulate their expression via ARE-binding domain. In vitro studies showed that Nrf2 promoted expressions of K6, K16, and K17 in both primary human keratinocyte and immortalized keratinocyte cell line HaCaT cells. Moreover, ChIP analysis showed that Nrf2 in the nucleus was bound with the ARE sequence located in the promoter regions of K6, K16, and K17. These keratins such as K16 act not only as Nrf2 target genes, but also power a positive feedback loop toward Nrf2 activation in skin keratinocyte (
      • Kerns M.L.
      • Hakim J.M.
      • Lu R.G.
      • Guo Y.
      • Berroth A.
      • Kaspar R.L.
      • et al.
      Oxidative stress and dysfunctional NRF2 underlie pachyonychia congenita phenotypes.
      ,
      • Lessard J.C.
      • Coulombe P.A.
      Keratin 16-null mice develop palmoplantar keratoderma, a hallmark feature of pachyonychiacongenita and related disorders.
      ). Our study gains a deeper insight into the diversity of Nrf2 functions, and this Nrf2-keratin underlying pathway may serve as a model for keratinocyte activation in psoriasis. Additional studies are needed to further probe this intriguing phenomenon.
      The discovery of K6, K16, and K17 as markers of keratinocyte hyperproliferation in psoriasis drew widespread attention to the importance of these keratins in the pathogenesis of psoriasis (
      • Korge B.
      • Stadler R.
      • Mischke D.
      Effect of retinoids on hyperproliferation-associated keratins K6 and K16 in cultured human keratinocytes: a quantitative analysis.
      ,
      • Leigh I.M.
      • Navsaria H.
      • Purkis P.E.
      • McKay I.A.
      • Bowden P.E.
      • Riddle P.N.
      Keratins (K16 and K17) as markers of keratinocyte hyperproliferation in psoriasis in vivo and in vitro.
      ,
      • Paramio J.M.
      • Casanova M.L.
      • Segrelles C.
      • Mittnacht S.
      • Lane E.B.
      • Jorcano J.L.
      Modulation of cell proliferation by cytokeratins K10 and K16.
      ). Since then, increasing studies have been performed to clarify the mechanism underlying the regulation of these keratins. The K17/T cell/cytokine positive feedback regulatory loop in the psoriatic epidermis described by our group implies a possible mechanism for regulation of K17 expression in psoriasis, in which IFN-γ, IL-22, and IL-17 have all been identified as regulators of K17 expression (
      • Shen Z.
      • Chen L.
      • Liu Y.F.
      • Gao T.W.
      • Wang G.
      • Fan X.L.
      • et al.
      Altered keratin 17 peptide ligands inhibit in vitro proliferation of keratinocytes and T cells isolated from patients with psoriasis.
      ,
      • Shi X.
      • Jin L.
      • Dang E.
      • Chang T.
      • Feng Z.
      • Liu Y.
      • et al.
      IL-17A upregulates keratin 17 expression in keratinocytes through STAT1- and STAT3-dependent mechanisms.
      ,
      • Zhang W.
      • Dang E.
      • Shi X.
      • Jin L.
      • Feng Z.
      • Hu L.
      • et al.
      The pro-inflammatory cytokine IL-22 up-regulates keratin 17 expression in keratinocytes via STAT3 and ERK1/2.
      ). Our previous in vitro experiments also confirmed the participation of signal transducer and activator of transcription-1 and -3 pathways in this loop. However, neither of these pathways can fully account for the mechanism by which K17 is regulated. In this study, we added on a regulator Nrf2 in the positive loop K17/T cell/cytokine in psoriasis. Furthermore, we investigated the mechanism accounting for Nrf2 activation in psoriasis. We found out that apart from oxidative stress, psoriasis-related inflammatory cytokines IL-17 and IL-22 were also inducers of Nrf2. We substantiated that Nrf2 can be induced by inflammatory cytokines IL-17 and IL-22, further shedding light on the importance of Nrf2 in inflammatory skin diseases. A role for Nrf2 as a regulator of K6, K16, and K17 expressions, which advance our knowledge of pathogenesis of psoriasis, makes Nrf2 an attractive target for therapeutic intervention of psoriasis in the future.

      Materials and Methods

      Cell culture and reagents

      This study was approved by the Ethics Committee of Xijing Hospital. Written informed consent was obtained from all donors. Human keratinocyte cell line HaCaT cells were cultured in RPMI 1640 media supplemented with 10% fetal bovine serum at 37 °C with 5% CO2. Cells at 40–60% confluence were stimulated with IL-17, IL-22, and IFN-γ (10, 25, 50 ng/ml; Peprotech, Rocky Hill, NJ). To inhibit Nrf2, Nrf2 siRNA was transfected into cells 24 hours before stimulation with each cytokine.

      RNA interference

      Three siRNAs targeting Nrf2 (Nrf2 siRNAs 1, 2, and 3) and control siRNA (scrambled siRNA) were used as previously described (
      • Jian Z.
      • Li K.
      • Liu L.
      • Zhang Y.
      • Zhou Z.
      • Li C.
      • et al.
      Heme oxygenase-1 protects human melanocytes from H2O2-induced oxidative stress via the Nrf2-ARE pathway.
      ). siRNAs for K6, K16, and K17 were designed and synthesized by Ribobio Biotechnologies (Guangzhou, China).

      RNA extraction and real-time PCR

      Total RNA from HaCaT cells or epidermal tissues were extracted using TRIzol reagent (Invitrogen, Carlsbad, CA). Real-time PCR was performed as described (
      • Jian Z.
      • Li K.
      • Liu L.
      • Zhang Y.
      • Zhou Z.
      • Li C.
      • et al.
      Heme oxygenase-1 protects human melanocytes from H2O2-induced oxidative stress via the Nrf2-ARE pathway.
      ) on a Chromo 4 continuous fluorescence detector with a PTC-200 DNA Engine Cycler (Bio-Rad, Hercules, CA). Primers are listed in Supplementary Table S1.

      Western blot analysis

      Western blot was performed as previously described (
      • Jian Z.
      • Li K.
      • Liu L.
      • Zhang Y.
      • Zhou Z.
      • Li C.
      • et al.
      Heme oxygenase-1 protects human melanocytes from H2O2-induced oxidative stress via the Nrf2-ARE pathway.
      ) using antibodies as follows: K6, K16, K17, Nrf2, pNrf2 (Abcam, Cambridge, UK), β-tubulin (Proteintech, Rosemont, IL) and lamin-B1 (Santa Cruz Biotechnology, Dallas, TX). Intensities of the bands were quantified by Image Lab, version 5.2.1 (Bio-Rad). β-Actin was used as a loading control. Relative intensities of the bands are normalized to loading control.

      EdU proliferation assay

      HaCaT cells were seeded in quadruplicate at 4 × 103 cells per well in 96-well plates. The cells were incubated for 24 hours and transfected with siRNAs or overexpression plasmid. Forty-eight hours after transfection, cell proliferation was detected by EdU assay with the EdU Cell Proliferation Assay Kit (Ribobio, Guangzhou, China). Briefly, the cells were exposed to 50 μM EdU for 4 hours before fixation and permeabilization. The cell nuclei were stained with 100 μl of Hoechst 33342 (5 μg/ml) for 30 minutes. The proportion of the cells that incorporated EdU was determined with fluorescence microscopy (Olympus, Tokyo, Japan). For each group, EdU-positive cells were counted by taking five random fields of vision.

      ChIP analysis

      Analysis was performed using the Pierce Agarose Assay Kit (26156; Thermo Fisher, Waltham, MA). Details are in the Supplementary Materials online. Primers are listed in Supplementary Table S2.

      Mouse studies

      Seven-week-old female BALB/c mice were purchased from the Department of Laboratory Animal Medicine of Fourth Military Medical University (Xi’an, China). All experimental procedures involving mice were in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Review Committee for the Use of Animals of the Fourth Military Medical University. Mice were topically treated with IMQ cream (iNova, Chatswood, Australia) on both ears once a day for 7 days to induce a psoriasis-like dermatitis. To evaluate the effect of Nrf2 inhibition on the development of psoriasis, 2.5 nmol siRNA targeting Nrf2 (Ribobio, Guangzhou, China) was mixed with 5 mg emulsion matrix and applied topically to the left ear of mice every other day for 7 days. The right ear of mice was treated with negative control siRNA. Mice were killed on day 8, and ear skin was harvested for section analysis.

      Statistical analysis

      All the data in our study were obtained from at least three independent experiments and presented as mean ± standard error of the mean. Data between the two groups were analyzed with unpaired two-tailed Student t test, and comparisons between groups were performed using one-way analysis of variance followed by the Newman-Keuls multiple comparison test. P-values less than 0.05 were considered statistically significant.

      Conflict of Interest

      The authors state no conflict of interest.

      Acknowledgments

      We thank all patients who assisted with this study. The study was supported by the grants from National Natural Science Foundation of China (81430073, 81602749, 81502716, and 81402620).

      Supplementary Material

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