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Protein Kinase C-Dependent Upregulation of miR-203 Induces the Differentiation of Human Keratinocytes

  • Author Footnotes
    4 These authors contributed equally to this work
    Enikö Sonkoly
    Correspondence
    Molecular Dermatology Research Group, Unit of Dermatology and Venerology, Department of Medicine, Center for Molecular Medicine, L8:02, Karolinska Institutet, Stockholm SE-17176, Sweden.
    Footnotes
    4 These authors contributed equally to this work
    Affiliations
    Molecular Dermatology Research Group, Unit of Dermatology and Venerology, Department of Medicine, Center for Molecular Medicine, Karolinska Institutet, Stockholm, Sweden
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  • Author Footnotes
    4 These authors contributed equally to this work
    Tianling Wei
    Footnotes
    4 These authors contributed equally to this work
    Affiliations
    Molecular Dermatology Research Group, Unit of Dermatology and Venerology, Department of Medicine, Center for Molecular Medicine, Karolinska Institutet, Stockholm, Sweden
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  • Elizabeth Pavez Loriè
    Affiliations
    Department of Medical Sciences/Dermatology, Uppsala University, Uppsala, Sweden
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  • Hiroyuki Suzuki
    Affiliations
    Department of Experimental Pathology, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan
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  • Mitsuyasu Kato
    Affiliations
    Department of Experimental Pathology, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan
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  • Hans Törmä
    Affiliations
    Department of Medical Sciences/Dermatology, Uppsala University, Uppsala, Sweden
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  • Mona Ståhle
    Affiliations
    Molecular Dermatology Research Group, Unit of Dermatology and Venerology, Department of Medicine, Center for Molecular Medicine, Karolinska Institutet, Stockholm, Sweden
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  • Andor Pivarcsi
    Affiliations
    Molecular Dermatology Research Group, Unit of Dermatology and Venerology, Department of Medicine, Center for Molecular Medicine, Karolinska Institutet, Stockholm, Sweden
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  • Author Footnotes
    4 These authors contributed equally to this work
      Terminal differentiation of keratinocytes is a multistep process that requires a coordinated program of gene expression. We aimed to explore the possible involvement of a previously unreported class of non-coding RNA genes, microRNAs (miRNAs) in keratinocyte differentiation by using miRNA expression profiling. Out of 365 miRNAs tested, 7 showed significant change between keratinocytes cultured in low or high calcium concentration. The highest-ranked upregulated gene was miR-203, whose expression was significantly upregulated in response to calcium and other inducers of keratinocyte differentiation such as 12-O-tetradecanoylphorbol-13-acetate (TPA) and vitamin D3. Differentiation-induced upregulation of miR-203 expression was blocked by treatment with specific inhibitors of protein kinase C (PKC), GF109203X, and Ro31-8220. Moreover, our results showed that the activator protein-1 (AP-1) proteins c-Jun and JunB regulate miR-203 expression in keratinocytes. In contrast to inducers of keratinocyte differentiation, epidermal growth factor and keratinocyte growth factor suppressed miR-203 expression in keratinocytes below the basal level. Overexpression of miR-203 in keratinocytes resulted in enhanced differentiation, whereas inhibition of miR-203 suppressed calcium-induced terminal differentiation as judged by involucrin expression. These results suggest that upregulation of miR-203 in human keratinocytes is required for their differentiation and is dependent on the activation of the PKC/AP-1 pathway.

      Abbreviations

      AP-1
      activator protein-1
      EGF
      epidermal growth factor
      KGF
      keratinocyte growth factor
      miRNA
      microRNA
      PBS
      phosphate-buffered saline
      PKC
      protein kinase C
      RT
      reverse transcription
      SAM
      significance analysis of microarrays
      TLDA
      TaqMan Low Density Array
      TPA
      12-O-tetradecanoylphorbol-13-acetate

      Introduction

      MicroRNAs (miRNAs) are short, typically ∼22 to 23nt long non-coding RNA molecules that regulate the expression of protein-coding genes at the post-transcriptional level by interfering with translation of mRNAs or by inducing their degradation (
      • Lee R.C.
      • Ambros V.
      An extensive class of small RNAs in Caenorhabditis elegans.
      ;
      • Bartel D.P.
      MicroRNAs: genomics, biogenesis, mechanism, and function.
      ;
      • He L.
      • Hannon G.J.
      MicroRNAs: small RNAs with a big role in gene regulation.
      ). An interesting aspect of miRNA function is that each miRNA potentially regulates hundreds of target genes simultaneously (
      • Baek D.
      • Villen J.
      • Shin C.
      • Camargo F.D.
      • Gygi S.P.
      • Bartel D.P.
      The impact of microRNAs on protein output.
      ). To date (March, 2009, miRBase 13.0) (
      • Griffiths-Jones S.
      • Grocock R.J.
      • van Dongen S.
      • Bateman A.
      • Enright A.J.
      miRBase: microRNA sequences, targets and gene nomenclature.
      ), 706 miRNA genes have been identified in the human genome and their predicted number is around 1,000. It is estimated that the majority of all protein-coding genes are subject to miRNA-mediated regulation. Therefore, miRNAs are the most abundant class of regulators of gene expression in the human genome. To date, little is known about the expression and regulation of miRNAs in human keratinocytes.
      Intensive research in recent years showed that miRNAs have pivotal functions in nearly all biological processes investigated. miRNAs have been shown to regulate embryonic development (
      • Ambros V.
      The functions of animal microRNAs.
      ), hematopoiesis (
      • Chen C.Z.
      • Li L.
      • Lodish H.F.
      • Bartel D.P.
      MicroRNAs modulate hematopoietic lineage differentiation.
      ), cell proliferation and apoptosis (
      • Croce C.M.
      • Calin G.A.
      miRNAs, cancer, and stem cell division.
      ), as well as several aspects of the immune response and inflammation (
      • Sonkoly E.
      • Stahle M.
      • Pivarcsi A.
      MicroRNAs: novel regulators in skin inflammation.
      ;
      • Sonkoly E.
      • Pivarcsi A.
      Advances in microRNAs: implications for immunity and inflammatory diseases.
      ). A function for miRNAs in cell differentiation is firmly established and has been shown in T cells (
      • Wu H.
      • Neilson J.R.
      • Kumar P.
      • Manocha M.
      • Shankar P.
      • Sharp P.A.
      • et al.
      miRNA profiling of naive, effector and memory CD8 T Cells.
      ), hematopoietic cells (
      • Shivdasani R.A.
      MicroRNAs: regulators of gene expression and cell differentiation.
      ), embryonic stem cells (
      • Tzur G.
      • Levy A.
      • Meiri E.
      • Barad O.
      • Spector Y.
      • Bentwich Z.
      • et al.
      MicroRNA expression patterns and function in endodermal differentiation of human embryonic stem cells.
      ), and osteoclasts (
      • Sugatani T.
      • Hruska K.A.
      Impaired micro-RNA pathways diminish osteoclast differentiation and function.
      ). miRNAs are also involved in skin development, as evidenced by morphological changes in the skin of mice with a conditional knockout of Dicer, an enzyme essential for miRNA biogenesis (
      • Yi R.
      • O’Carroll D.
      • Pasolli H.A.
      • Zhang Z.
      • Dietrich F.S.
      • Tarakhovsky A.
      • et al.
      Morphogenesis in skin is governed by discrete sets of differentially expressed microRNAs.
      ). In particular, one miRNA, miR-203, came into focus when our group identified it to be overexpressed in psoriasis and found that it has a unique expression profile by being expressed at the highest level in the skin for which it may be termed a “skin-specific miRNA” (
      • Sonkoly E.
      • Wei T.
      • Janson P.C.
      • Saaf A.
      • Lundeberg L.
      • Tengvall-Linder M.
      • et al.
      MicroRNAs: novel regulators involved in the pathogenesis of Psoriasis?.
      ). Within the major cellular constituents of healthy human skin, miR-203 was exclusively expressed by keratinocytes, suggesting a pivotal role for it in keratinocyte functions (
      • Sonkoly E.
      • Wei T.
      • Janson P.C.
      • Saaf A.
      • Lundeberg L.
      • Tengvall-Linder M.
      • et al.
      MicroRNAs: novel regulators involved in the pathogenesis of Psoriasis?.
      ). We found that miR-203 mediates the post-transcriptional suppression of Suppressor of Cytokine Signaling-3 (SOCS-3), a negative regulator of the JAK/signal transducer and activator of transcription (STAT) pathway (
      • Sonkoly E.
      • Wei T.
      • Janson P.C.
      • Saaf A.
      • Lundeberg L.
      • Tengvall-Linder M.
      • et al.
      MicroRNAs: novel regulators involved in the pathogenesis of Psoriasis?.
      ). Interestingly, miR-203 was not uniformly expressed in the healthy human epidermis, but formed a gradient with low expression in the basal cell layer and high expression in the more differentiated suprabasal layers (
      • Sonkoly E.
      • Wei T.
      • Janson P.C.
      • Saaf A.
      • Lundeberg L.
      • Tengvall-Linder M.
      • et al.
      MicroRNAs: novel regulators involved in the pathogenesis of Psoriasis?.
      ). This expression pattern suggested its involvement in keratinocyte differentiation. Compelling evidence for this came in 2008 when two independent groups showed that miR-203 induces cell-cycle exit and represses “stemness” in epidermal progenitors (
      • Lena A.M.
      • Shalom-Feuerstein R.
      • Rivetti di Val Cervo P.
      • Aberdam D.
      • Knight R.A.
      • Melino G.
      • et al.
      miR-203 represses ‘stemness’ by repressing DeltaNp63.
      ;
      • Yi R.
      • Poy M.N.
      • Stoffel M.
      • Fuchs E.
      A skin microRNA promotes differentiation by repressing ‘stemness’.
      ). In accordance with human data, miR-203 was also preferentially expressed in the suprabasal layers of healthy mouse epidermis (
      • Yi R.
      • Poy M.N.
      • Stoffel M.
      • Fuchs E.
      A skin microRNA promotes differentiation by repressing ‘stemness’.
      ). Moreover, transgenic overexpression of miR-203, driven by a keratin 14 promoter, resulted in the formation of a thinner epidermis and decreased proliferation, suggesting critical roles for miR-203 in epidermal morphogenesis by restricting the proliferative capacity of keratinocytes (
      • Yi R.
      • Poy M.N.
      • Stoffel M.
      • Fuchs E.
      A skin microRNA promotes differentiation by repressing ‘stemness’.
      ). Interestingly, miR-203 was found to be downregulated in esophageal cancer (
      • Feber A.
      • Xi L.
      • Luketich J.D.
      • Pennathur A.
      • Landreneau R.J.
      • Wu M.
      • et al.
      MicroRNA expression profiles of esophageal cancer.
      ).
      Keratinocyte differentiation is a highly coordinated multistep process regulated by growth factors, autocrine and paracrine intercellular signaling mechanisms and external stimuli. Several factors have been described as differentiating agents for keratinocytes in vitro and in vivo, such as calcium, vitamin D, and 12-O-tetradecanoylphorbol-13-acetate (TPA) (
      • Hawley-Nelson P.
      • Stanley J.R.
      • Schmidt J.
      • Gullino M.
      • Yuspa S.H.
      The tumor promoter, 12-O-tetradecanoylphorbol-13-acetate accelerates keratinocyte differentiation and stimulates growth of an unidentified cell type in cultured human epidermis.
      ;
      • Pillai S.
      • Bikle D.D.
      • Mancianti M.L.
      • Cline P.
      • Hincenbergs M.
      Calcium regulation of growth and differentiation of normal human keratinocytes: modulation of differentiation competence by stages of growth and extracellular calcium.
      ;
      • Gibbs S.
      • Backendorf C.
      • Ponec M.
      Regulation of keratinocyte proliferation and differentiation by all-trans-retinoic acid, 9-cis-retinoic acid and 1,25-dihydroxy vitamin D3.
      ). Calcium is the best characterized differentiation agent. In vitro, increasing the extracellular calcium concentration above 0.1mM (calcium switch) leads to the expression of differentiation-related genes and morphological changes (
      • Yuspa S.H.
      • Kilkenny A.E.
      • Steinert P.M.
      • Roop D.R.
      Expression of murine epidermal differentiation markers is tightly regulated by restricted extracellular calcium concentrations in vitro.
      ). The expression and function of most miRNAs in human keratinocytes and during their differentiation has not been explored.
      We investigated the expression of miRNAs in a keratinocyte model of differentiation and identified miR-203 as the most upregulated miRNA during keratinocyte differentiation. Furthermore, we characterized the signal pathways involved in the regulation of miR-203 and the functional consequence of miR-203 dysregulation during keratinocyte differentiation.

      Results

      Calcium-induced differentiation alters miRNA expression in keratinocytes

      To investigate whether calcium-induced differentiation of keratinocytes affects miRNA expression, we carried out a comprehensive analysis of miRNA expression in primary human keratinocytes cultured under low-calcium (0.06mM) or high-calcium (1.5mM) conditions. Using the TaqMan Low Density Array (TLDA) platform, we analyzed the expression of the mature, biologically active form of 365 human miRNAs 48hours after calcium treatment. The 25 most abundant miRNAs in proliferating and in differentiated keratinocytes could be classified into several gene families: the miR-17 family (miR-20a, miR-19a and miR-19b, miR-92, and miR-93), miR-221 family (miR-221 and miR-222), miR-26 family (miR-26a and miR-26b), miR-99 family (miR-99a and miR-100), the miR-125 family (miR-125a and miR-125b), and the miR-15 family (miR-15b and miR-16). In addition to these miRNA families, miR-205, miR-200c, miR-31, miR-24, and miR-21 were identified as particularly abundant miRNAs in both undifferentiated and differentiated human keratinocytes (Figure 1a and b). Moreover, miR-203 was among the abundant miRNAs in differentiated but not in undifferentiated keratinocytes (Figure 1b).
      Figure thumbnail gr1
      Figure 1MicroRNA expression profiling in proliferating and differentiated keratinocytes. Primary human keratinocytes were cultured in the presence of 1.5mM CaCl2 (n=3, high calcium) or low-calcium medium (n=3, low calcium) for 48hours, and miRNA expression was analyzed using TaqMan Low Density Array. (a) Relative abundance of miRNAs in low-calcium-grown keratinocytes. The 25 most abundant miRNAs and U48 (RNU48) are shown. Data are expressed in relative units (RU) compared with U48 RNA. (b) Relative abundance of miRNAs in high-calcium-grown keratinocytes. The 25 most abundant miRNAs and the housekeeping small nucleolar RNA U48 (RNU48) are shown. (c) Differentially expressed miRNAs in high-calcium-grown keratinocytes versus low-calcium-grown keratinocytes according to the SAM algorithm (fold change >1.5, q<20%). (d) Relative abundance of miRNAs in keratinocytes treated with high calcium and fold change of miRNAs in high-calcium-grown keratinocytes versus low-calcium-grown keratinocytes. (e) The expressions of involucrin and miR-203 were analyzed using quantitative real-time PCR in low-calcium-grown and high-calcium-grown keratinocytes. Data are expressed in RU compared with 18S RNA and U48 RNA, respectively.
      Comparing the miRNA expression profile between low-calcium-grown and high-calcium-grown keratinocytes using the significance analysis of microarrays (SAM), we identified five miRNAs (miR-203, miR-429, miR-98, miR-200a, and miR-365) to be upregulated and two miRNAs (miR-200c and miR-210) to be downregulated (Figure 1c). Interestingly, the miRNA showing the highest upregulation was miR-203, a miRNA that we previously identified to be preferentially expressed in the skin out of 21 human organs analyzed and shown to be expressed specifically by keratinocytes among the cellular constituents of the skin (
      • Sonkoly E.
      • Wei T.
      • Janson P.C.
      • Saaf A.
      • Lundeberg L.
      • Tengvall-Linder M.
      • et al.
      MicroRNAs: novel regulators involved in the pathogenesis of Psoriasis?.
      ). miR-203 was upregulated 8.3-fold in high-calcium-grown keratinocytes compared with low-calcium-grown controls (Figure 1c–e). Involucrin, an early marker of keratinocyte terminal differentiation known to be induced by calcium treatment (
      • Watt F.M.
      Involucrin and other markers of keratinocyte terminal differentiation.
      ), was upregulated 6.9-fold in high-calcium-grown compared with low-calcium-grown keratinocytes (Figure 1f), indicating that differentiation was indeed induced in high-calcium-treated samples.

      miR-203 is upregulated during calcium- and TPA-induced keratinocyte differentiation

      As miR-203 showed the highest upregulation in high-calcium-treated keratinocytes, we next focused on characterizing the regulation of miR-203 by agents that are known to affect keratinocyte functions. First, we aimed to analyze the kinetics of miR-203 upregulation after treatment with calcium and another known inducer of keratinocyte differentiation, the phorbol ester TPA (
      • Hawley-Nelson P.
      • Stanley J.R.
      • Schmidt J.
      • Gullino M.
      • Yuspa S.H.
      The tumor promoter, 12-O-tetradecanoylphorbol-13-acetate accelerates keratinocyte differentiation and stimulates growth of an unidentified cell type in cultured human epidermis.
      ) in time-course experiments. To this end, we treated keratinocytes with calcium or TPA for 1, 4, 24, and 48hours, monitored morphological changes in the cell cultures, and measured miR-203 expression by miRNA-specific quantitative real-time PCR (Figure 2a and b). After 24–48hours of treatment with TPA or calcium, keratinocytes showed morphological signs of terminal differentiation (data not shown). miR-203 expression was significantly (P<0.05) induced 24hours after exposure of keratinocytes to high calcium (Figure 2a). We observed further upregulation of miR-203 48hours after calcium treatment (Figure 2a). Stimulation with TPA resulted in a detectable and significant (P<0.01) upregulation of miR-203 expression already after 4hours, and this effect was even stronger 24 and 48hours post treatment (Figure 2b). These results showed that the upregulation of miR-203 expression in differentiating keratinocytes is not specific for calcium-induced differentiation but can also be achieved by other inducers of keratinocyte differentiation.
      Figure thumbnail gr2
      Figure 2Inducers of keratinocyte differentiation upregulate, whereas growth factors suppress miR-203 expression. (a) The expression of miR-203 was analyzed using quantitative real-time PCR in keratinocytes treated with CaCl2 or medium. (b) The expression of miR-203 was analyzed using quantitative real-time PCR in keratinocytes treated with TPA or DMSO. (c and d) Primary human keratinocytes were cultured in the presence or absence of vitamin D3 for 3, 6, 12, 24 or 48hours. The expressions of hCAP18 (c) and the functionally active, mature form of miR-203 (d) were analyzed using quantitative real-time PCR. (e and f) Primary human keratinocytes were analyzed when 25% confluent, 50% confluent (pre-confluent), 100% confluent, and 4 days after reaching confluence (post-confluent, 100%+4 days). The expressions of involucrin (e) and the functionally active, mature form of miR-203 (f) were analyzed using quantitative real-time PCR. Data are expressed as relative units (RU) compared with the 25% confluence culture. (g) Primary human keratinocytes were cultured in the presence or absence of epidermal growth factor (EGF; 10ngml−1) or keratinocyte growth factor (KGF; 20ngml−1) for 24 and 48hours. The expression of the functionally active, mature form of miR-203 was analyzed using quantitative real-time PCR. The expression of miR-203 was normalized to U48 RNA, and the expression of hCAP18 and involucrin was normalized to 18S RNA. Data are expressed as RU. Means±SD of three independent experiments are shown. *P<0.05, **P<0.01, and ***P<0.001.

      miR-203 is upregulated by vitamin D in human keratinocytes

      Next, we aimed to investigate whether vitamin D, which induces keratinocyte differentiation through a different pathway, regulates miR-203 expression. To determine possible early and late effects, we treated keratinocytes with vitamin D3 (1,25(OH)2D3) for 3, 6, 12, 24, and 48hours and analyzed the expression of miR-203 and a positive control gene known to be directly regulated by vitamin D3, hCAP18 (
      • Weber G.
      • Heilborn J.D.
      • Chamorro Jimenez C.I.
      • Hammarsjo A.
      • Torma H.
      • Stahle M.
      Vitamin D induces the antimicrobial protein hCAP18 in human skin.
      ). In accordance with literature data, vitamin D3 induced hCAP18 expression in a time-dependent manner. hCAP18 mRNA levels were significantly (P<0.01) increased as early as 3hours after treatment, and this induction was even stronger at 6, 12, 24, and 48hours post treatment (Figure 2c). In comparison, quantitative real-time PCR results showed significantly increased miR-203 expression only after 24 (P<0.001) and 48hours after vitamin D3 treatment (P<0.001) (Figure 2d).

      Cell density regulates miR-203 expression in human keratinocytes

      To further investigate the regulation of miR-203 expression in keratinocytes, we set out to measure miR-203 levels in monolayer keratinocyte cultures at different cell densities. As cultures grew to confluence, cells became tightly packed, maintained epithelial morphology, and close association to one another, eventually establishing close contacts as a large continuous monolayer sheet of cells. After confluence, cells became stratified and few mitoses were observed. We chose to determine the expression of involucrin as a marker for the differentiation stage of keratinocytes in this model system. Involucrin expression began to increase when cells reached confluence and showed a more than six-fold increase in cells that are kept confluent for 4 days (Figure 2e). In accordance with our previous results, miR-203 was expressed already at low cell densities at a relatively high level in keratinocytes. We observed more than 10-fold increase in miR-203 expression in cells that are kept in confluence for 4 days, compared with subconfluent keratinocytes (Figure 2f). These results further confirmed the notion that miR-203 expression is tightly linked to the differentiation program in keratinocytes.

      EGF and KGF suppress miR-203 expression in keratinocytes

      Next, we aimed to study whether growth factors that maintain keratinocytes in an undifferentiated, proliferative state can regulate miR-203 expression. To this end, primary human keratinocytes were treated with recombinant human epidermal growth factor (EGF) or keratinocyte growth factor (KGF) or medium for 24 and 48hours and miR-203 expression was analyzed (Figure 2g). Quantitative real-time PCR results showed a significant (P<0.001), 2.4-fold decrease in miR-203 expression 24hours after treatment with KGF (Figure 2g). Similar to KGF, we observed a significant, 1.8-fold (P<0.05) decrease in miR-203 expression in keratinocytes treated with EGF for 24hours (Figure 2g). The suppressive effect of EGF on miR-203 expression in keratinocytes was more pronounced 48hours after treatment (P<0.05; 7.1-fold decrease) (Figure 2g). Moreover, KGF treatment significantly (P<0.05; 8.1-fold) suppressed miR-203 48hours after treatment (Figure 2g). These results show that KGF and EGF suppress miR-203 expression in keratinocytes.
      In addition to differentiation- and proliferation-inducing agents, we also analyzed the regulation of miR-203 expression by cytokines and microbial compounds known to modulate keratinocyte functions. MiR-203 expression was not regulated by any of the cytokines or microbial compounds tested (Supplementary Figure S1a and b).

      Activation of protein kinase C is required for differentiation-induced upregulation of miR-203 expression

      Next, we sought to explore the signal transduction pathways involved in the differentiation-induced regulation of miR-203 in keratinocytes. TPA-induced terminal differentiation of keratinocytes is mediated by the activation of protein kinase C (PKC) (
      • Yuspa S.H.
      • Ben T.
      • Hennings H.
      • Lichti U.
      Divergent responses in epidermal basal cells exposed to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate.
      ). In vitro, PKC inhibitors effectively block the terminal differentiation program induced by calcium or TPA in keratinocytes (
      • Dlugosz A.A.
      • Yuspa S.H.
      Coordinate changes in gene expression which mark the spinous to granular cell transition in epidermis are regulated by protein kinase C.
      ). Therefore, we set out to investigate whether the induction of miR-203 during keratinocyte differentiation is dependent on PKC signaling by measuring miR-203 expression in primary human keratinocytes treated with specific PKC inhibitors, GF109203X and Ro31-8220, together with TPA or DMSO alone (Figure 3a).
      Figure thumbnail gr3
      Figure 3miR-203 expression during keratinocyte differentiation is dependent on protein kinase C (PKC) activity and is regulated by the AP-1 transcription factor complex. (a) The expression of involucrin (left panel) and miR-203 (right panel) in human keratinocytes cultured in the presence of DMSO (control), phorbol ester TPA, TPA+GF109203X, or TPA+Ro31-8220. The expression of involucrin was analyzed using quantitative real-time PCR and expression data were normalized to 18S RNA. The expression of the functionally active, mature form of miR-203 was analyzed using quantitative real-time PCR. Expression data were normalized to U48 RNA. Mean±SD of three independent experiments is shown. (b and c) Nuclear extracts were prepared from keratinocytes treated with DMSO, TPA, TPA+GF109203X, or TPA+Ro31-8220, and (b) JunB- or (c) c-Jun-dependent AP-1 DNA-binding activity was measured using transcription factor assays. Mean±SD of triplicates is shown. (d) Stable HaCaT keratinocyte cell lines overexpressing the dominant-negative mutant form of JunB (JunBΔN6 and JunBΔN8) were established and the expression of miR-203 was measured by quantitative real-time PCR. (e) Stable HaCaT keratinocyte cell lines overexpressing c-Jun (c-Jun2 and c-Jun12) were established and the expression of miR-203 was measured by quantitative real-time PCR. *P<0.05, **P<0.01, ***P<0.001.
      At 24hours after administration of TPA, keratinocytes formed dendritic structures, became elongated with spindle-like phenotype or prominent cell rounding, all of which were reversed by treatment with the specific PKC inhibitors (data not shown). Quantitative real-time PCR results showed a significant upregulation of involucrin expression, which was equally reversed by both PKC inhibitors (Figure 3a). Moreover, TPA treatment induced a fivefold, significant (P<0.001) increase in the expression of miR-203 in comparison with DMSO-treated cells 24hours after treatment (Figure 3a). Treatment of keratinocytes with the specific PKC inhibitor, GF109203X, not only significantly (P<0.001) blocked TPA-induced miR-203 expression but also suppressed it to below the basal level (Figure 3a). Similar to GF109203X, another specific inhibitor of PKC, Ro31-8220, significantly (P<0.001) inhibited TPA-induced miR-203 expression (Figure 3a).
      A downstream target for PKC action in keratinocytes is activator protein-1 (AP-1), which is a transcription factor consisting of homodimers or heterodimers of the Jun and Fos families of nuclear proteins (
      • Rutberg S.E.
      • Saez E.
      • Glick A.
      • Dlugosz A.A.
      • Spiegelman B.M.
      • Yuspa S.H.
      Differentiation of mouse keratinocytes is accompanied by PKC-dependent changes in AP-1 proteins.
      ;
      • Shaulian E.
      • Karin M.
      AP-1 in cell proliferation and survival.
      ). AP-1 proteins have essential roles in the regulation of keratinocyte growth and differentiation, although different members of the AP-1 family can have antagonistic effects. While c-Jun is primarily a positive regulator of proliferation, JunB suppresses proliferation and promotes differentiation (
      • Shaulian E.
      • Karin M.
      AP-1 in cell proliferation and survival.
      ;
      • Zenz R.
      • Wagner E.F.
      Jun signalling in the epidermis: from developmental defects to psoriasis and skin tumors.
      ;
      • Ikebe D.
      • Wang B.
      • Suzuki H.
      • Kato M.
      Suppression of keratinocyte stratification by a dominant negative JunB mutant without blocking cell proliferation.
      ). To explore the regulation of Jun family members by TPA in primary keratinocytes, we measured JunB and c-Jun activity in TPA-treated keratinocytes using transcription factor assays. TPA treatment led to significantly increased JunB activation (P<0.05, Figure 3b), which was prevented by the PKC inhibitors GF109203X and Ro31-8220 (P<0.001 and P<0.01, respectively, Figure 3b). In contrast to JunB, the activation of c-Jun was decreased in TPA-treated keratinocytes (P<0.01, Figure 3c), and this decrease was prevented by GF109203X (P<0.05, Figure 3c) but not by Ro31-8220 (Figure 3c).

      The AP-1 transcription factor complex is involved in the regulation of miR-203

      To investigate whether TPA-induced changes in the activity of AP-1 proteins can have an effect on miR-203 expression, we analyzed miR-203 expression in HaCaT keratinocytes stably overexpressing c-Jun, or a dominant-negative mutant of JunB, JunBΔN (
      • Ikebe D.
      • Wang B.
      • Suzuki H.
      • Kato M.
      Suppression of keratinocyte stratification by a dominant negative JunB mutant without blocking cell proliferation.
      ). In HaCaT-JunBΔN cell lines (JunBΔN6 and JunBΔN8), miR-203 expression was suppressed compared with mock-transfected HaCaT cells (Figure 3d), suggesting that JunB is a positive regulator of miR-203 expression. Moreover, in c-Jun-overexpressing HaCaT cell lines, miR-203 was suppressed compared with mock transfectants (Figure 3e), suggesting that c-Jun is a negative regulator of miR-203 expression. Hence, reduced JunB or increased c-Jun activity led to decreased miR-203 expression in HaCaT keratinocytes, indicating the involvement of AP-1 in the regulation of miR-203.

      Overexpression of miR-203 induces keratinocyte differentiation

      To explore whether miR-203 has a functional role in keratinocyte differentiation, we transiently overexpressed miR-203 in primary keratinocytes by transfection of the precursor molecule of miR-203 (pre-miR-203), and analyzed the expression of involucrin in the transfected cells. Immunofluorescence (Figure 4a) as well as western blot (Figure 4b) analyses showed the upregulation of involucrin in miR-203-overexpressing keratinocytes compared with those transfected with a scrambled control, indicating that overexpression of miR-203 induces differentiation of keratinocytes also in the absence of calcium or TPA.
      Figure thumbnail gr4
      Figure 4Overexpression of miR-203 induces keratinocyte differentiation, whereas inhibition of miR-203 suppresses calcium-induced keratinocyte differentiation. (a and b) Normal human keratinocytes cultured in low-calcium medium were transfected with a synthetic precursor molecule for miR-203 (pre-miR-203) or scrambled oligos as negative control (pre-miR-CON). (a) The expression of involucrin was visualized by immunofluorescent staining (red color) in the keratinocytes 96hours after transfection. Cell nuclei were visualized by DAPI (blue color). Bar=100μm. (b) Western blotting was used to analyze the expression of involucrin in the keratinocytes 96hours after transfection with pre-miR-203 or pre-miR-CON. (c and d) Primary human keratinocytes were transiently transfected with a specific miR-203 inhibitor (anti-miR-203) or a scrambled inhibitor (anti-miR-CON) and treated with 1.5mM calcium for 72hours. (c) The expression of the keratinocyte-specific differentiation marker involucrin was visualized by immunofluorescent staining (red color) 72hours after calcium treatment. Cell nuclei were visualized by DAPI (blue color). Bar=100μm. (d) The expression of involucrin was measured by western blotting.

      Inhibition of miR-203 suppresses calcium-induced keratinocyte differentiation

      Next, we investigated the effect of miR-203 inhibition on keratinocyte differentiation. To this end, we transfected keratinocytes with locked nucleic acid (LNA) antagomirs specific for miR-203 (anti-miR-203) or nonspecific scrambled control LNA antagomir (anti-miR CON) and subsequently treated the cells with calcium. Knockdown of miR-203 by antagomir resulted in decreased involucrin expression after calcium treatment, in comparison with controls, as shown by immunofluorescence (Figure 4c) and western blotting (Figure 4d). These results suggest that miR-203 expression is required for keratinocyte differentiation.

      Discussion

      In this study, we identified the typical miRNA signature of proliferating, undifferentiated (cultured in low calcium), and differentiated (cultured in high calcium) human keratinocytes and also identified miR-203 as a regulator of human keratinocyte differentiation.
      Interestingly, the most abundant miRNAs in human keratinocytes could be classified into several gene families sharing 5′ seed sequences (the highly conserved 7- or 8-mer sequence within a miRNA that establishes target specificity). Expressing multiple miRNAs with shared seed sequences may enable the suppression of miRNA targets more effectively and rapidly. Moreover, it may ensure that mutation of one miRNA gene will not have detrimental effect on cells as other family members may compensate for the loss of function. In addition to the coordinated expression of these gene families, miR-205, miR-200c, miR-31, miR-24, and miR-21 were identified as particularly abundant miRNAs in human keratinocytes. To date, virtually nothing is known about the roles of most of these miRNAs in keratinocytes. Thus, investigation of the function of these highly expressed miRNAs will be very relevant to keratinocyte biology.
      Out of 365 miRNAs analyzed, miR-203 was the most upregulated by keratinocyte differentiation. In addition to calcium, miR-203 expression was upregulated by other inducers of keratinocyte differentiation such as TPA, high cell density, and vitamin D3 (
      • Hawley-Nelson P.
      • Stanley J.R.
      • Schmidt J.
      • Gullino M.
      • Yuspa S.H.
      The tumor promoter, 12-O-tetradecanoylphorbol-13-acetate accelerates keratinocyte differentiation and stimulates growth of an unidentified cell type in cultured human epidermis.
      ). The induction of miR-203 during keratinocyte differentiation is in line with the expression pattern previously reported by us, where miR-203 was mainly expressed by keratinocytes in the suprabasal layers in the epidermis of healthy human skin (
      • Sonkoly E.
      • Wei T.
      • Janson P.C.
      • Saaf A.
      • Lundeberg L.
      • Tengvall-Linder M.
      • et al.
      MicroRNAs: novel regulators involved in the pathogenesis of Psoriasis?.
      ).
      Using TPA-induced keratinocyte differentiation as a model, we showed that activation of PKC is required for differentiation-induced upregulation of miR-203 in keratinocytes. Two specific PKC inhibitors (
      • Toullec D.
      • Pianetti P.
      • Coste H.
      • Bellevergue P.
      • Grand-Perret T.
      • Ajakane M.
      • et al.
      The bisindolylmaleimide GF 109203X is a potent and selective inhibitor of protein kinase C.
      ;
      • Cuenda A.
      • Alessi D.R.
      Use of kinase inhibitors to dissect signaling pathways.
      ) suppressed miR-203 expression below the basal level, suggesting that endogenous PKC activation is required for homeostatic miR-203 expression. A downstream target for PKC action in keratinocytes is the AP-1 transcription factor (
      • Rutberg S.E.
      • Saez E.
      • Glick A.
      • Dlugosz A.A.
      • Spiegelman B.M.
      • Yuspa S.H.
      Differentiation of mouse keratinocytes is accompanied by PKC-dependent changes in AP-1 proteins.
      ), which is composed of the Jun and Fos family of nuclear proteins (
      • Zenz R.
      • Wagner E.F.
      Jun signalling in the epidermis: from developmental defects to psoriasis and skin tumors.
      ). AP-1 activity has been shown to have essential roles in the regulation of keratinocyte growth and differentiation (
      • Zenz R.
      • Eferl R.
      • Kenner L.
      • Florin L.
      • Hummerich L.
      • Mehic D.
      • et al.
      Psoriasis-like skin disease and arthritis caused by inducible epidermal deletion of Jun proteins.
      ;
      • Zenz R.
      • Wagner E.F.
      Jun signalling in the epidermis: from developmental defects to psoriasis and skin tumors.
      ). We here show that TPA treatment of primary keratinocytes increases the activity of JunB, whereas it suppresses c-Jun activation, in accordance with previous data showing differential effects of different AP-1 components: JunB has been shown to suppress proliferation and promotes differentiation of keratinocytes, whereas c-Jun is a positive regulator of proliferation (
      • Welter J.F.
      • Eckert R.L.
      Differential expression of the fos and jun family members c-fos, fosB, Fra-1, Fra-2, c-jun, junB and junD during human epidermal keratinocyte differentiation.
      ;
      • Zenz R.
      • Wagner E.F.
      Jun signalling in the epidermis: from developmental defects to psoriasis and skin tumors.
      ;
      • Ikebe D.
      • Wang B.
      • Suzuki H.
      • Kato M.
      Suppression of keratinocyte stratification by a dominant negative JunB mutant without blocking cell proliferation.
      ). Analysis of the putative promoter region of the miR-203 gene showed the presence of an AP-1 transcription factor-binding site (unpublished observation), suggesting that AP-1 is directly involved in the transcriptional regulation of miR-203. Our results showed that in stably transfected HaCaT keratinocytes, both suppression of JunB and the overexpression of c-Jun decreased miR-203 expression, suggesting that JunB is a positive whereas c-Jun is a negative regulator of miR-203 expression, which is in line with their antagonistic roles in keratinocyte functions. In conclusion, our results suggest that differentiation-induced miR-203 upregulation is dependent on the activation of PKC and an altered balance between AP-1 family members (Figure 5).
      Figure thumbnail gr5
      Figure 5A proposed model for the regulation and role of miR-203 in keratinocyte differentiation. Differentiation-inducing stimuli (TPA and calcium) activate PKC and lead to an altered balance between AP-1 proteins (JunB and c-Jun) and increased miR-203 expression in keratinocytes. miR-203 induces keratinocyte differentiation through post-transcriptional suppression of its target mRNAs (p63, SOCS-3, and yet-unidentified targets).
      Previously, we identified miR-203 to be significantly overexpressed in psoriasis in comparison with healthy skin and atopic eczema (
      • Sonkoly E.
      • Wei T.
      • Janson P.C.
      • Saaf A.
      • Lundeberg L.
      • Tengvall-Linder M.
      • et al.
      MicroRNAs: novel regulators involved in the pathogenesis of Psoriasis?.
      ). As psoriasis is characterized by hyperproliferation and disturbed differentiation, one may expect decreased levels of miR-203 during differentiation; which is clearly not the case. However, psoriasis keratinocytes do not show an undifferentiated (basal) phenotype but undergo an alternative differentiation program and early terminal differentiation markers such as involucrin are indeed overexpressed (
      • Bernard B.A.
      • Robinson S.M.
      • Vandaele S.
      • Mansbridge J.N.
      • Darmon M.
      Abnormal maturation pathway of keratinocytes in psoriatic skin.
      ).
      In contrast to differentiation-inducing agents, the growth factors EGF and KGF suppressed the expression of miR-203. Both EGF and KGF have profound effects on keratinocytes. Interestingly, growth factors also modulate the PKC/AP-1 pathway (
      • Sharma G.D.
      • Kakazu A.
      • Bazan H.E.
      Protein kinase C alpha and epsilon differentially modulate hepatocyte growth factor-induced epithelial proliferation and migration.
      ), suggesting that this pathway might not only be involved in the induction of miR-203 by differentiation but also in its suppression by growth factors. Our finding showing the suppression of miR-203 by EGF and KGF identifies a previously unknown pathway by which growth factors affect keratinocyte functions. Investigations of miRNA-mediated effects of growth factors may represent a previously unreported line of research in keratinocyte biology.
      Previously, we observed that miR-203 has a unique expression pattern among miRNAs and can be termed a “skin-specific” miRNA (
      • Sonkoly E.
      • Wei T.
      • Janson P.C.
      • Saaf A.
      • Lundeberg L.
      • Tengvall-Linder M.
      • et al.
      MicroRNAs: novel regulators involved in the pathogenesis of Psoriasis?.
      ). Within the major cellular constituents of healthy human skin, miR-203 was expressed only by keratinocytes in relevant quantities (
      • Sonkoly E.
      • Wei T.
      • Janson P.C.
      • Saaf A.
      • Lundeberg L.
      • Tengvall-Linder M.
      • et al.
      MicroRNAs: novel regulators involved in the pathogenesis of Psoriasis?.
      ). This suggested that miR-203 has a skin- and/or keratinocyte-specific function. We here show that overexpression of miR-203 in human keratinocytes promotes their differentiation even in the absence of other differentiating agents, as evidenced by increased involucrin expression. Furthermore, our data show that inhibition of endogenous miR-203 using antagomirs can suppress calcium-induced keratinocyte differentiation, suggesting that miR-203 upregulation is required for complete keratinocyte differentiation. Although overexpression of miR-203 led to increased involucrin expression, this effect is likely to be indirect, as miRNAs have negative effects on protein output and because there are no predicted binding sites for miR-203 in the 3′-UTR of involucrin mRNA (unpublished observation). We hypothesize that miR-203-induced involucrin expression is mediated by miR-203 targets that have negative effects on differentiation (Figure 5). Overexpression of miR-203 would eventually lead to the post-transcriptional suppression of these genes, which in turn would allow keratinocyte differentiation (Figure 5). To date, SOCS-3 and p63 have been identified as direct targets of miR-203 (
      • Sonkoly E.
      • Wei T.
      • Janson P.C.
      • Saaf A.
      • Lundeberg L.
      • Tengvall-Linder M.
      • et al.
      MicroRNAs: novel regulators involved in the pathogenesis of Psoriasis?.
      ;
      • Yi R.
      • Poy M.N.
      • Stoffel M.
      • Fuchs E.
      A skin microRNA promotes differentiation by repressing ‘stemness’.
      ). However, additional target genes that may serve as a link between miR-203 and differentiation still await discovery.
      Recently,
      • Yi R.
      • Poy M.N.
      • Stoffel M.
      • Fuchs E.
      A skin microRNA promotes differentiation by repressing ‘stemness’.
      found that transgenic mice overexpressing miR-203 driven by keratin 14 promoter had a thinner epidermis with lower p63 expression in comparison with wild-type mice. Moreover, transgenic mouse keratinocytes overexpressing miR-203 had a reduced proliferation rate and reduced colony-forming capacity in comparison with wild-type keratinocytes (
      • Yi R.
      • Poy M.N.
      • Stoffel M.
      • Fuchs E.
      A skin microRNA promotes differentiation by repressing ‘stemness’.
      ). However, epidermal differentiation markers were not induced by overexpression of miR-203 as judged by the expression of K10, Filaggrin, and Loricrin mRNAs (
      • Yi R.
      • Poy M.N.
      • Stoffel M.
      • Fuchs E.
      A skin microRNA promotes differentiation by repressing ‘stemness’.
      ). In contrast, we found that ectopic overexpression of miR-203 led to increased keratinocyte differentiation judged by involucrin protein level. Moreover, inhibition of endogenous miR-203 impaired calcium-induced increase of involucrin protein. Further investigation and probably the identification of additional miR-203 targets will be necessary to determine whether differences between these observations are due to different methods and markers used (the measurement of gene expression at the protein or mRNA level, the use of different differentiation markers, the use of synthetic miRNA precursors instead of viral vectors, the use of antagomir controls) or perhaps can be attributed to the expression of a different set of targets in human and mouse. Although miR-203 is highly conserved between mice and humans, this does not necessarily imply that all of their targets are conserved as it is indicated by the obvious differences between the human and the mouse epidermis.
      Taken together, our results identify miR-203 as the most upregulated miRNA during keratinocyte differentiation. Moreover, we show that the differentiation-induced upregulation of miR-203 is dependent on the activation of the PKC/AP-1 pathway. In contrast, growth factors suppressing the keratinocyte differentiation program suppress miR-203 expression below the basal level, and one can hypothesize that this suppression may be required for their function. Furthermore, our results show that upregulation of miR-203 in human keratinocytes is required for their differentiation.

      Materials and Methods

      miRNA expression profiling

      miRNAs were reverse transcribed and amplified (PCR) using the multiplex reverse transcription (RT) TaqMan MicroRNA Low Density Array (TLDA) (Applied Biosystems, Foster City, CA). An amount of 640ng of starting total RNA (80ng for each of the eight RT-PCR) was used for each sample. The global miRNA profiling for 365 human miRNAs was carried out using the TaqMan LDA Human microRNA Panel v1.0 (MicroFluidic card, Applied Biosystems). TLDAs were run on the ABI7900 HT analyzer with TLDA upgrade and analysed with RQ Manager software provided by Applied Biosystems. All the quality control tests were validated: blanks and reproducibility (standard deviation of cycle threshold (CT) <1) of the two small nucleolar housekeeping RNAs RNU48 (SNORD48) and RNU44 (SNORD44). The amount of RNA from each sample was calibrated to the more stable (between the different arrays) small nucleolar housekeeping RNA, RNU48. To find consistently differentially expressed genes, the data were subjected to SAM analysis as described (
      • Sonkoly E.
      • Wei T.
      • Janson P.C.
      • Saaf A.
      • Lundeberg L.
      • Tengvall-Linder M.
      • et al.
      MicroRNAs: novel regulators involved in the pathogenesis of Psoriasis?.
      ). Genes showing at least 1.5-fold regulation and a q-value <20% were considered to be differentially expressed.

      Cells and transfections

      Human adult skin epidermal keratinocytes (obtained from Cascade Biologics, Portland, OR) were cultured in EpiLife serum-free keratinocyte growth medium including Human Keratinocyte Growth Supplement at a final Ca2+ concentration of 0.06mM (Cascade Biologics). Third passage keratinocytes were used at 50–70% confluence for all experiments, except when assessing cell-density-dependant miR203 expression.
      To investigate the effects of miR-203 on keratinocyte differentiation, human keratinocytes at 70% confluence were transfected with 10nM Pre-miR 203 miRNA Precursor (Ambion, Foster City, CA), 10nM Pre-miR miRNA Precursor Negative Control #1 (Ambion), 5nM LNA-based miR-203 inhibitor (Santaris Pharma, Hoersholm, Denmark), or 5nM Universal LNA-based negative control (Santaris Pharma) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA), following the manufacturer's instruction. The expression of the mature, biologically active form of miR-203 in transfected keratinocytes was confirmed by real-time PCR. In the inhibitor experiments, the cells were exposed to 1.5mM CaCl2 (Cascade Biologics) after 16hours. Transfection with anti-miR-203 prevented calcium-induced upregulation of endogenous miR-203 as evidenced by the results of real-time PCR analysis. At 72–96hours post-transfection, the cells were assayed for involucrin protein expression by western blotting and immunofluorescence. HaCaT keratinocytes were cultured and transfected with JunBΔN and c-Jun construct as previously described (
      • Ikebe D.
      • Wang B.
      • Suzuki H.
      • Kato M.
      Suppression of keratinocyte stratification by a dominant negative JunB mutant without blocking cell proliferation.
      ).

      Keratinocyte differentiation models

      To investigate the effects of keratinocyte differentiation inducers in regulating miRNAs expression, keratinocytes were subcultured into six-well plates at a density of 24,000 cells per well and exposed to 1.5mM CaCl2 (Cascade Biologics), 50ngml−1 TPA, and 100nM vitamin D3 (1,25(OH)2D3) (both from Sigma, St Louis, MO). Medium was used as a control for CaCl2, and DMSO was used as a control for TPA and for vitamin D3. Plates to be used for RNA preparation, real-time PCR, and LDA profiling (Applied Biosystems) were washed twice with phosphate-buffered saline (PBS) at 1, 4, 24, and 48hours after calcium and TPA treatment, and at 3, 6, 12, 24, and 48hours after vitamin D3 treatment. To avoid effects of changes in keratinocyte physiology during culture, each time point includes treated and corresponding untreated, control samples. To examine whether confluent status of keratinocytes influences miR-203 expression, RNA were collected from the cells at 25, 50, and 100% confluence and at 4 days after confluence.

      PKC inhibitors, growth factors, and cytokines

      Two PKC inhibitors, 100nM Ro31-8220 (Calbiochem, Darmstadt, Germany) and 10μM GF109203X (Calbiochem) were applied to keratinocytes, followed by 50ngml−1 TPA stimulation. The cells were washed with PBS twice after 24hours and approached to RNA extraction. To explore regulation of growth factors and cytokines in miR-203 expression, cells were treated with 10ngml−1 EGF (Sigma) and 20ngml−1 KGF (Sigma), and harvested 24 or 48hours after treatment. Furthermore, keratinocytes were treated with IL-4 (50ngml−1), IFN-γ (50ngml−1), GM-CSF (50ngml−1), IL-6 (100ngml−1), lipopolysaccharide (1μgml−1; Sigma), or zymosan (80μgml−1; Sigma) for 4 or 24hours, or left untreated. Moreover, keratinocytes were treated with 50ngml−1 tumor necrosis factor-α for 1, 4, 24, or 48hours, or left untreated. All cytokines were purchased from R&D Systems (Minneapolis, MN, USA).

      RNA extraction and RT

      Total RNA was prepared using Trizol (Invitrogen). The RNA concentration was determined by spectrophotometry. Total RNA (1.0μg) was reversed transcribed into cDNA in a 20-μl reaction by RevertAid First Strand cDNA Synthesis Kit (Fermentas, Burlington, CA). The resulting cDNA was diluted with 80μl dH2O to obtain a concentration of 10ngμl−1 cDNA.

      Quantitative real-time PCR

      Quantification of miRNAs by TaqMan Real-Time PCR was carried out as described by the manufacturer (Applied Biosystems). Briefly, 80ng of template RNA was reverse transcribed using the TaqMan MicroRNA Reverse Transcription Kit and the multiplex RT primer pools containing miRNA-specific stem-loop primers (Applied Biosystems). Diluted RT product (1.5μl) was introduced into the 20-μl PCR reactions, which were incubated in 384-well plates on the ABI 7900HT thermocycler (Applied Biosystems) at 95°C for 10minutes, followed by 40 cycles of 95°C for 15seconds and 60°C for 1minute. Target gene expression was normalized between different samples based on the values of U48 small nucleolar RNA expression.
      For the quantification of involucrin, 20ng of cDNA was amplified per reaction in the presence of SYBR green master mix, (Applied Biosystems) and the following specific primers: involucrin—F: 5′-ACCCATCAGGAGCAAATGAAA-3′ and R: 5′-GCTCGACAGGCACCTTCTGGCA-3′ (Stage 1: 95°C for 5minutes, stage 2: 95°C for 15seconds, 60°C for 1minute, repeated 40 times, stage 3: 95°C for 1minute, stage 4: 55°C for 1minute; stage 5: 55°C for 10seconds, increasing the set point temperature after cycle 2 by 0.5°C, repeated 80 times). For the quantification of hCAP18, 20ng of cDNA was amplified per reaction in the presence of TaqMan universal master mix (Applied Biosystems) and the following specific primers and probe: 5′-GTCACCAGAGGATTGTGACTTCAA-3′ and 5′-TTGAGGGTCACTGTCCCCATA-3′ for the primers, and 6-FAM–5′-CCGCTTCACCAGCCCGTCCTT-3′–BHQ1 for the fluorogenic probe. Gene-specific PCR products were measured by means of an ABI PRISM 7000 Sequence Detection Systems (Applied Biosystems) (stage 1: 50°C for 2minutes; stage 2: 95°C for 10minutes; and stage 3: 95°C for 15seconds, 60°C for 1minute, repeated 40 times). Target gene expression was normalized based on the values of the expression of 18S RNA (18S-F: 5′-CGGCTACCACATCCAAGGAA-3′ and 18S-R: 5′-GCTGGAATTACCGCGGCT-3′; 18S TaqMan probe: 5′-FAM/TGCTGGCACCAGACTTGCCCTC-3′).

      Western blotting

      Keratinocyte lysates were analyzed for protein expression by western blotting with anti-human involucrin antibody at a concentration of 1:1,000 (Sigma Aldrich, St Louis, MO). The protein levels were visualized by ECL (GE Healthcare, Niskayuna, NY) using horseradish peroxidase-conjugated Protein A/G (Pierce Chemical and Thermo Fisher Scientific, Waltham, MA).

      Immunofluorescence

      Upon harvest, the cells were washed in PBS, followed by 10minutes fixation in 4% formaldehyde and 5minutes blocking in serum-free protein block (Dako, Denmark) at room temperature. The primary antibody (anti-human involucrin antibody, Sigma Aldrich) diluted at 1:200 in 1%BSA/PBS was added and incubated overnight. The secondary antibody, 1μgml−1 Alexa Fluor 594 donkey anti-mouse IgG (Invitrogen), was diluted in 1%BSA/PBS and incubated with the cells for 17minutes at room temperature without exposing to direct light. Sections were mounted using 4′-6-diamidino-2-phenylindole (DAPI)-containing mounting medium (Vector Laboratories, Burlington, CA).

      AP-1 transcription factor assay

      To examine the involvement of the AP1 transcription factor complex in the regulation of miR-203, keratinocytes were cultured in 60mm Petri dishes at a density of 50,000 cells per well and cultured in EpiLife serum-free keratinocyte growth medium excluding Human Keratinocyte Growth Supplement when reaching to 70% confluence. One day after starvation, 100nM Ro31-8220 (Calbiochem) or 10μM GF109203X (Calbiochem) were added to keratinocytes, followed by 50ngml−1 TPA stimulation. The nuclear fraction was extracted by nuclear extract kit (Active Motif, Carlsbad, CA), and activation of JunB and c-Jun were measured using the TransAM AP-1 Family Transcription Factor Assay Kit (Active Motif) according to the manufacturer's instruction.

      Statistical analysis

      In the analysis of the TLDA data, differentially expressed genes were identified by were subjecting data to SAM analysis as described (
      • Sonkoly E.
      • Wei T.
      • Janson P.C.
      • Saaf A.
      • Lundeberg L.
      • Tengvall-Linder M.
      • et al.
      MicroRNAs: novel regulators involved in the pathogenesis of Psoriasis?.
      ). Genes showing at least 1.5-fold regulation and a q-value <20% were considered to be differentially expressed. Using Student's t-test, statistical differences between means of quantitative real-time PCR and transcription factor assay data was determined. A P-value <0.05 was considered statistically significant.

      Conflict of Interest

      The authors state no conflict of interest.

      ACKNOWLEDGMENTS

      This work was supported by the National Psoriasis Foundation, the Swedish Research Council, Medical Research Council (#K2008-74x-07133-21A), Karolinska Institutet, the Swedish Psoriasis Association (Psoriasisförbundet), the Welander Finsens Foundations, Tore Nilsons Foundation, Konsul ThC Bergh Foundations, and the Stockholm County Council. Andor Pivarcsi has been supported by the Marie Curie Intra-European Fellowship.

      SUPPLEMENTARY MATERIAL

      Supplementary material is linked to the online version of the paper at http://www.nature.com/jid

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