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Regulation of pro-inflammatory cytokines TNFα and IL24 by microRNA-203 in primary keratinocytes
Cytokine 60 (2012) 741–748
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Regulation of pro-inflammatory cytokines TNFa and IL24 by microRNA-203 in primary keratinocytes Maria Nascimento Primo, Rasmus O. Bak, Beatrice Schibler, Jacob Giehm Mikkelsen ⇑ Department of Biomedicine, Aarhus University, DK-8000 Aarhus C, Denmark
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Article history: Received 9 March 2012 Received in revised form 20 June 2012 Accepted 26 July 2012 Available online 20 August 2012 Keywords: MicroRNA Cytokine Inflammation Skin
a b s t r a c t Cutaneous homeostasis and innate immunity is procured by a complex circuitry of intercellular cytokine signaling. MicroRNAs are important posttranscriptional regulators of keratinocyte gene expression and assist in modulating the fine balance between cell proliferation and differentiation in skin. A characteristic microRNA profile in inflammatory skin suggests putative functions of microRNAs in perturbed cytokine production and signaling during chronic inflammatory skin conditions such as psoriasis. It remains unclear, however, why certain microRNAs are aberrantly expressed during skin inflammation and if they serve pro- and/or anti-inflammatory functions. In this report, we focus on cytokine regulation by microRNA-203 (miR-203), which is highly abundant in keratinocytes and upregulated in psoriatic lesions. By screening a panel of cytokines that are upregulated in psoriatic skin for regulation by miR-203, we identify the genes encoding the pro-inflammatory cytokines TNFa and IL24 as direct targets of miR-203. Studies of miR-203 overexpression, inhibition, and mutagenesis validate posttranscriptional regulation of TNFa and IL24 by miR-203 in cell lines and primary keratinocytes. Our findings suggest that miR-203 serves to fine-tune cytokine signaling and may dampen skin immune responses by repressing key proinflammatory cytokines. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction The alertness of the immune system relies on sophisticated and highly controlled intercellular signaling among immune cells and between immune cells and differentiated somatic cells. Cytokines are key modulators of the innate immune response and play a prominent role during inflammation. Given the sensitivity and cellular impact of signal transmission, the production of cytokines needs to be highly flexible and is thus strongly regulated at the transcriptional level. Moreover, a tight regulation is conferred by regulatory mechanisms that operate during RNA processing and translation. Cytokine-encoding mRNAs are typically short-lived transcripts due to the presence of AU-rich elements located in the 30 untranslated regions (UTRs) of mRNA transcripts, which under normal cellular conditions promote rapid mRNA decay by attracting AU-rich elements-binding proteins and RNA degradation enzymes [1]. Moreover, components of AU-rich elements-mediated decay pathways may act together with the microRNA machinery to regulate the stability and translation of cytokine-encoding mRNAs [2]. ⇑ Corresponding author. Address: Department of Biomedicine, University of Aarhus, Wilh. Meyers Allé, Bldg. 1240, DK-8000 Aarhus C, Denmark. Tel.: +45 89421671. E-mail address: [email protected] (J.G. Mikkelsen). 1043-4666/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.cyto.2012.07.031
MicroRNAs (miRNAs) constitute a family of small (21nt), noncoding RNA molecules which regulate up to one-third of human genes by triggering either translational repression or mRNA degradation [3–5]. MicroRNA-directed gene suppression is accomplished in association with RNAi-induced silencing complex which recruits factors that facilitate mRNA destabilization or repression of the translation process [4,6–9]. Base-pairing at position 2–8 relative to the 50 end of the miRNA, termed the ‘seed region’, is of particular importance for gene suppression by miRNAs [10,11] and is in many cases sufficient to regulate gene expression. Chronic inflammatory diseases are characterized by aberrant cytokine signaling in the afflicted tissue. Such variations could potentially reflect abnormal expression of immune-regulatory miRNAs since a growing list of miRNAs has been associated with the pathogenesis of immune diseases. In psoriatic skin lesions, keratinocytes contribute to a chronic inflammatory state by responding to and producing cytokines that affect cell proliferation and differentiation, promote angiogenesis, and attract skin-homing immune cells [12]. MicroRNA-203 is highly expressed in keratinocytes and plays a role in skin morphogenesis, as supported by its suppression of the transcription factor p63 [13,14]. This miRNA is upregulated in psoriatic lesions and is thought to play a potential part in disease progression through its repression of suppressor of cytokine signaling 3 (SOCS3) [15,16], which is a negative regulator of STAT3 [17,18]. In addition, miR-203 was recently found to
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target SOCS6 [19]. Together, these findings indicate that upregulation of miR-203 facilitates repression of immunosuppressive genes and plays a potential part in disease progression. However, there is no formal proof of this assumption, and miR-203 could have additional targets in a complex network of immune-modulatory interactions in which the miRNA under certain conditions acts as an activator and under other circumstances as a suppressor of immune signaling. In this report, we address the hypothesis that miR-203 is a modulator of cytokine signaling with the ability to both accelerate and suppress an innate immune response. To do so, we challenged the notion that cytokine-encoding mRNAs are rare primary miRNA targets [20] and screened a panel of 30 UTRs from mRNAs, all encoding cytokines upregulated in human psoriatic skin, for sensitivity to miR-203-directed regulation. We demonstrate that the pro-inflammatory cytokines tumor necrosis factor a (TNFa) and interleukin24 (IL24) are direct targets of miR-203 in a keratinocyte cell line and primary keratinocytes. Our findings lend support to the notion that miR-203 serves to fine-tune, or balance, cytokine signaling by down-regulating members of the SOCS gene family as well as proinflammatory cytokines, possibly indicating that enhanced production of miR-203 in psoriatic skin could be part of an anti-inflammatory response. 2. Materials and methods
protocol. Primary keratinocytes were seeded in 6-well plates (6 104 cells/well) 1 day before transduction performed with an MOI of 10. Seventy-two hours post-transduction, primary keratinocyte culture supernatants were harvested for quantification of secreted protein, and total RNA was extracted using the SV Total RNA Isolation System (Promega, Madison, WI, USA) according to manufacturer’s protocol. 2.3. Bioinformatics miR-203 target sites in SOCS6, TNFa and IL24 mRNAs were predicted by PicTar 5, RNAhybrid and TargetScan 5.2 softwares available at http://pictar.mdc-berlin.de/, http://bibiserv.techfak. uni-bielefeld.de/rnahybrid/ and http://www.targetscan.org/, respectively. 2.4. Statistical analyses All experiments were performed in triplicates and data are depicted as mean + SEM. All p-values were calculated by a two-tailed Student’s T-test to test the null hypothesis of no difference between two compared groups. The assumption of equal variances was tested by the F-test. In all statistical analyses, p-values <0.05 were considered significant. Further details related to materials and methods are provided in the Supplementary Materials and Methods.
2.1. Cell lines and primary keratinocytes HaCaT, HeLa, HEK293 and 293T cells were cultured at 37 °C in 5% (v/v) CO2 and maintained in Dulbeccoo’s modified Eagle’s medium (Cambrex, Verviers, Belgium) supplemented with 10% fetal calf serum, penicillin (100 U/mL), streptomycin (0.1 mg/mL), and L-glutamine (265 mg/L). Primary keratinocytes were cultured at 37 °C in 5% (v/v) CO2 and maintained in Serum-free Keratinocyte medium (Gibco BRL-Life Technologies, Eggenstein, Germany) supplemented with bovine pituitary extract (25 lg/mL) and recombinant epidermal growth factor (0.1–0.2 ng/mL). 2.2. Lentiviral vector production and transduction For production of lentiviral vectors, 293T cells were seeded in 10-cm dishes 1 day before transfection (4 106 cells/dish). Cells were transfected by the calcium phosphate method with 3.75 lg pMD.2G, 3 lg pRSV-Rev, 13 lg pMDLg/pRRE, and 13 lg lentiviral transfer vector. Forty-eight hours after transfection the viral supernatants were harvested and passed through 0.45 lm filters (Sarstedt, Nümbrecht, Germany). Titer assays based on colony formation were performed in HaCaT cells seeded in 6-well plates (5 104 cells/well) 1 day before transduction. Lentiviral supernatants for transduction of naïve HaCaT and HaCaT-203 cells were serially diluted and supplemented with polybrene (8 lg/mL; Sigma–Aldrich, Milwaukee, WI, USA). Transduced HaCaT cells were grown in 1 lg/mL puromycin-containing medium (Sigma–Aldrich, Milwaukee, WI, USA) for 10 days after which the number of colonies was counted and the titer was calculated. HaCaT-203 and naïve HaCaT cells were seeded in 6-well plates (6 104 cells/well) 1 day before transduction. In high-dose experiments, transductions were performed at an MOI of 50, and total RNA was extracted 72 h post-transduction using the SV Total RNA Isolation System (Promega, Madison, WI, USA) according to manufacturer’s protocol. For stable transduction experiments, transductions were performed at an MOI of 5, followed by puromycin selection (final concentration of 1 lg/mL; Sigma–Aldrich, Milwaukee, WI, USA) for 14 days. Total RNA was extracted using the SV Total RNA Isolation System (Promega, Madison, WI, USA) according to manufacturer’s
3. Results 3.1. Identification of cytokine-encoding genes as potential targets of miR-203 Skin inflammation is characterized by deregulated expression of a wealth of cytokines and several miRNAs. The abundance of miR203 in lesional psoriatic skin could suggest that this particular miRNA contributes to the progression of the disease and/or is aberrantly expressed as part of a protective response. To study the regulatory properties of miR-203 in cytokine production, we set out to screen a panel of genes associated with the cytokine network in psoriasis as possible targets of miR-203 using a dual luciferase reporter assay. Candidate target sequences were fused with the Renilla luciferase (Rluc) reporter gene in context of the psiCHECK-2 reporter plasmid, allowing the use of Rluc expression as a measure for potential miRNA targeting of the fusion mRNA. Expression of firefly luciferase from a separate expression cassette in the same plasmid allowed for normalization in case of transfection differences. Different reporter constructs were generated containing 30 UTRs from six genes encoding cytokines that are upregulated in psoriatic skin (TNFa, IL12B, IL15, IL17, IL20 and IL24) and from the gene encoding SOCS6 (which was identified as a miR-203 target gene during the course of these studies; [19]). In addition, we included three positive controls, a single perfect target site for miR-203 (psiCHECK.miR203target) and the 30 UTRs derived from the genes encoding transcription factor p63 and SOCS3 which are both validated targets of miR-203 [14,21]. MicroRNA-203 was expressed from a 335-bp human genomic DNA segment encoding the miR-203 precursor transcribed by a CMV promoter contained within a Sleeping Beauty (SB) DNA transposon plasmid (pT2/CMV-pri-miR-203.SV40-neo; Fig. 1a), allowing studies of both transiently and stably expressed miR-203. The reporter plasmids were co-transfected into HEK293 cells that do not have endogenous expression of miR-203, along with either pT2/CMV-pri-miR-203.SV40-neo or a negative control plasmid, pUC19. As shown in Fig. 1b, the functionality of plasmid-encoded miR-203 was verified as it was found to potently
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Fig. 1. Functional screening of potential miR-203 target genes. (a) Schematic representation of an SB DNA transposon-based vector expressing the pri-miR-203 sequence. The processing of pri-miR-203 to pre-miR-203, and the RNA-induced silencing complex (RISC) loaded with mature miR-203 is schematically shown below the vector. (b) Identification of SOCS6, TNFa, and IL24 as potential target genes of miR-203. HEK293 cells were co-transfected with either pUC19 (negative control) or the pri-miR-203encoding vector and the psiCHECK reporter plasmid containing a luciferase gene fused to a fragment of the indicated gene. ‘Control’ indicates a psiCHECK plasmid without a target sequence, whereas a psiCHECK plasmid with a perfect target sequence for miR-203 was included in transfections labeled ‘miR-203 target’. p < 0.01; p < 0.001. Experiments were performed in triplicates, and the data are depicted as mean + SEM.
down-regulate the Rluc fusion transcript carrying a perfect miR203 target site (about 90% knockdown). Also, the two previously reported targets of miR-203, p63 and SOCS3, were targeted by transiently expressed miR-203, resulting in both cases in approximately 50% knockdown. Among the remaining target sequences, transient expression of miR-203 resulted in targeting of Rluc transcripts fused to sequences derived from SOCS6, TNFa and IL24, resulting in 25, 45, and 20% knockdown, respectively, compared to the negative control (p < 0.01). These findings suggest that mRNAs encoding SOCS6, TNFa and IL24 are targeted by miR-203. 3.2. SOCS6, TNFa, and IL24 mRNA transcripts are direct targets of miR203 Human miRNAs mainly target the 30 UTRs of mRNAs, predominantly by complete sequence complementarity within the ‘seed region’. Thus, to identify potential miR-203 target sites within mRNAs encoding SOCS6, TNFa, and IL24, we performed a bioinformatic search for miR-203 seed match sequences using PicTar 5, RNAhybrid and TargetScan 5.2 softwares. As a result, we identified four potential miR-203 target sites within each of the 30 UTRs of SOCS6 and IL24 mRNA transcripts and two within the 30 UTR of TNFa mRNA (Fig. 2a, upper panel). To decipher the importance of these potential miR-203 target sites, we generated mutated 30 UTR sequences for each of the three genes in which all the predicted miR-203 seed binding sites were abolished by introducing a 4-bp mutation (Fig. 2a, bottom panel). The seed match mutation sequences were designed strictly on the basis of computational prediction of sequences that would have limited complementarity to the miR-203 sequence and at the same time would not match the seed region
of any other human miRNA. The mutated 30 UTR sequences were fused to the Rluc gene in the psiCHECK reporter plasmid, and the susceptibility of the fusion mRNAs to miR-203 regulation was compared with the corresponding mRNAs containing the native SOCS6, TNFa, and IL24 30 UTRs. As shown in Fig. 2b–d, the mutated seed matches completely abolished miR-203-mediated repression of expression from mRNA transcripts containing the SOCS6, TNFa, and IL24 sequences, suggesting that miR-203 interacted directly with one or more of the computationally predicted target sites within the 30 UTRs of SOCS6-, TNFa-, and IL24-encoding mRNAs. To further confirm that the regulation of the Rluc fusion transcripts by miR-203 was mediated by the predicted target sites in the mutated 3’ UTRs, we generated a mutant miR-203 (miR-203mut) with ‘seed region’-complementarity to the mutated 30 UTR target sites carrying the 4-bp mutations. Due to the presence of computationally predicted target sites for miR-203-mut within the Rluc gene, we replaced the Rluc reporter gene within the psiCHECK.IL24-mut plasmid with the eGFP gene. HEK293 cells were co-transfected with the modified reporter vector (psiCHECK.eGFP-IL24-mut) and with plasmids expressing either primiR-203 or pri-miR-203-mut. As shown in Fig. 2e, over-expression of pri-miR-203-mut led to repression of eGFP expression from an mRNA fusion transcript containing the mutant IL24 sequence, resulting in approximately 20% knockdown compared to co-transfections with plasmids encoding either the unmodified pri-miR203 or pri-miR-125b (the latter included as an additional negative control). Taken together, these findings validated the importance of mRNA-miRNA complementarity, demonstrating that the generegulatory effects of miR-203 were achieved by direct interaction with target sequences within the 30 UTRs of the affected genes.
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Fig. 2. Site-directed mutagenesis validates SOCS6, TNFa and IL24 mRNAs as direct targets of miR-203. (a, top panel) Schematic representation of the 30 UTRs of SOCS6, TNFa and IL24 mRNA transcripts (grey bars) with indication of the predicted target sites for miR-203 (black boxes). Numbers underlying the black boxes represent the first and the last basepair position within the NCBI reference. (a, bottom panel) Schematic representation of the miR-203 seed match mutations and miR-203 mutations (bold nucleotides are written in bold). (b–e) Disruption of miR203-directed gene regulation by introduced mutations. In panels (b–d) HEK293 cells were co-transfected with the indicated psiCHECK vectors and either pUC19 (negative control) or the pri-miR-203-encoding vector; in panel (e), HEK293 cells were transfected with psiCHECK.eGFP-IL24-mut and the vectors indicated. p < 0.001; p < 0.0001. Experiments were performed in triplicates, and the data are depicted as mean + SEM.
3.3. Potent knockdown of endogenously expressed SOCS6, TNFa, and IL24 mRNAs in HaCaT cells with engineered over-expression of miR203 To investigate miR-203 suppression of endogenously expressed full-length mRNA transcripts encoding SOCS6, TNFa, and IL24, we utilized the bipartite SB DNA transposon system to establish HaCaT keratinocyte cell lines with engineered over-expression of miR203. HaCaT cells were co-transfected with pT2/CMV-pri-miR203.SV40-neo (Fig. 1a) and either a plasmid encoding an inactive mutated SB transposase or the hyperactive SB100X transposase [22]. The number of neomycin-resistant colonies was significantly increased in the presence of SB100X relative to the mutated transposase (Supplementary Fig. S1a), indicating that genomic integration of the bicistronic vector was efficiently accomplished by a transposase-directed mechanism. We next isolated and expanded three individual clones containing the transposon (HaCaT-203 clones A, B, and C) and detected increased expression of miR-203 in all three HaCaT clones, when compared to the naïve HaCaT cell line, ranging from 8.7 to 12.4-fold increase (Supplementary Fig. S1b). MicroRNA-203-directed regulation of endogenously expressed SOCS6, TNFa, and IL24 were evaluated by qRT-PCR on naïve HaCaT cells and on the HaCaT-203 clone with highest miR-203 expression (clone C, from now on designated HaCaT-203). For all three target genes, over-expression of miR-203 repressed the mRNA levels, resulting in levels that were reduced to 40%, 5%, and 10% of the levels of mRNA transcripts present in naïve HaCaT cells for SOCS6, TNFa, and IL24 mRNA, respectively (Fig. 3a–c, grey bars). To validate that post-transcriptional suppression by miR-203 resulted in reduced levels of protein in keratinocytes, we investigated TNFa
and IL24 protein levels in both naïve HaCaT and HaCaT-203 cells. Indeed, over-expression of miR-203 led to a marked reduction of TNFa, resulting in 80% knockdown of both TNFa and IL24 protein levels (Fig. 3b and c, black bars). These findings confirmed the potential of miR-203 to alter expression levels of cytokines and possibly interact directly with distinct components of the inflammatory response in keratinocytes. To investigate whether expression of the potential miR-203 target genes varied with the miR-203 expression level, we analyzed the IL24 mRNA levels in the other two HaCaT-203 clones (clones A and B). As illustrated in Fig. 3d, IL24 mRNA levels inversely correlated with miR-203 levels in the four HaCaT keratinocyte cell lines, indicating that suppression of IL24 mRNA transcripts was directly dependent on miR-203 levels. 3.4. Establishment of miR-203 inhibition by lentiviral vector-encoded ‘antagomiRs’ We have previously provided evidence of potent and sustained in vivo knockdown of cytokine-encoding genes by the use of lentiviral vectors encoding small interfering RNAs [23,24]. Hence, we decided to explore the same lentiviral platform for construction of viral vectors expressing short, non-coding RNAs that were complementary to miRNAs as a method for persistently antagonizing miRNA function in vitro. A cassette expressing such an RNA ‘antagomiR’ directed against miR-203 (antagomiR-203) from an H1 promoter was cloned into a lentiviral vector plasmid (previously described in [24]) and analyzed for its ability to target miR-203. HaCaT-203, HaCaT, and HeLa cells were co-transfected with the Rluc reporter plasmid encoding the Rluc gene fused with the perfect miR-203 target site (psiCHECK.miR203target) and with either
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Fig. 3. Stable over-expression of miR-203 leads to marked reduction of endogenous SOCS6, TNFa, and IL24 in HaCaT cells. (a–c) Reduced levels of SOCS6, TNFa, and IL24 in HaCaT-203 cells. mRNA levels were evaluated by qRT-PCR in naïve HaCaT cells and a HaCaT-203 clone stably over-expressing miR-203. TNFa and IL24 protein levels (panels (b) and (c), respectively) were measured by ELISA from naïve HaCaT and HaCaT-203 cell supernatants. (d) IL24 mRNA levels (black line) and miR-203 levels (light-grey columns) were evaluated by qRT-PCR in all HaCaT cell lines. For all experiments naïve HaCaT cells and HaCaT-203 cells were incubated with anisomycin (500 ng/mL) for 18 h before both RNA and protein collection. p < 0.001; p < 0.0001. Experiments were performed in triplicates, and the data depicted as mean + SEM.
the lentiviral vector plasmid encoding antagomiR-203 (pLV/antagomiR-203) or pUC19 plasmid (negative control). The Rluc fusion transcript was thus targeted by endogenously expressed miR203, and transient expression of antagomiR-203 was suspected to relieve this suppression. As shown in Fig. 4a, the presence of antagomiR-203 led to increased luciferase activities in all three cell lines. We detected a 4.6-fold increase in HaCaT-203 cells, a 3.3-fold increase in HaCaT cells and a 2.1-fold increase in HeLa cells. We also evaluated miR-203 levels by qRT-PCR analysis (Fig. 4a, insert) and found that the miR-203 expression pattern in the three cell lines correlated directly with the degree of miR-203 inhibition. Hence, the impact of antagomiR-203 was most pronounced in the cells with highest levels of miR-203. We also evaluated the dose-dependency of antagomiR-203 by co-transfecting the three cell lines with psiCHECK.miR203target and increasing amounts of the antagomiR-203-encoding lentiviral plasmid. A dose–response relationship was obtained in the three cell lines, as increased amounts of antagomiR-203-encoding plasmid resulted in increased Rluc expression (Fig. 4b). Taken together,
our results showed robust miR-203 inhibition by introduction of antagomiR-203 and demonstrated that the level of inhibition was dependent on both the endogenous miR-203 expression level and the antagomiR dose. To evaluate the potency of lentiviral vector-delivered antagomiRs, HaCaT cells were transduced with lentiviral vectors encoding antagomiR-203 (LV/antagomiR-203). As negative controls, we included a vector which did not carry an antagomiR (LV/vehicle) and a vector encoding an antagomiR predicted not to target any known miRNA (LV/antagomiR-neg). For all three vectors, cells were transduced at a multiplicity of infection (MOI) of approximately 120. On the following day, cells were transfected either with psiCHECK.miR203target or pUC19 (negative control), and luciferase activities were measured 48 h post-transfection. As shown in Fig. 4c, expression of antagomiR-203 from lentiviral vectors resulted in almost complete relief of miR-203-mediated suppression of the reporter (p < 0.01). This demonstrated that lentiviral delivery of antagomiR-203 efficiently inhibited the functional properties of miR-203.
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Fig. 4. Inhibition of miR-203 function by plasmid or lentiviral delivery of DNA-encoded antagomiR-203. (a) Impact of antagomiR-203 in cells with variable levels of miR-203. Cell lines were co-transfected with the psiCHECK.miR203target vector and either pUC19 (negative control) or the lentiviral plasmid expressing the antagomiR-203 (pLV/ antagomiR-203). miR-203 Ct values from qRT-PCR are shown in the small insert (all cell lines showed similar Ct values of the reference small RNA, RU48). (b) Suppression of miR-203 activity is increased with enhanced dosages of antagomiR-203. Cell lines were co-transfected with the psiCHECK.miR203target vector and with increasing amounts of pLV/antagomiR-203. (c) Repression of miR-203 activity by lentivirally delivered antagomiR-203. HaCaT cells were transduced at an MOI of 120 with LV/antagomiR-203, LV/ antagomiR-neg, or LV/vehicle and transfected with either pUC19 (negative control) or psiCHECK.miR203target vector (miR-203 target). p < 0.01; p < 0.001; p < 0.0001. Experiments were performed at least in triplicates, and the data are depicted as mean + SEM.
3.5. miR-203 regulates TNFa and IL24 protein levels in primary keratinocytes During the course of our studies, SOCS6 was reported to be a target for miR-203 [19]. Here, we chose to focus our analyses on the pro-inflammatory cytokines IL24 and TNFa as targets of miR203 in a potential cytokine-regulating response. We analyzed the levels of IL24 mRNA in naïve HaCaT and HaCaT-203 cells after exposure of the cells to LV/antagomir-203 and measured a significant increase in the levels of IL24 mRNA in both cell lines (Supplementary Fig. S2). Notably, the effect of antagomiR-203 was persistent, allowing long-term repression of miR-203 function by antagomiR-203 expressed from genomically integrated lentiviral vectors (Supplementary Fig. S2). We also observed that the levels of detectable miR-203 in HaCaT-203 cells were unaffected by the treatment with antagomiR-203, lending support to the notion that the miRNA was antagonized, and not degraded, by the antagomiR (Supplementary Fig. S2).
To verify this type of regulation in primary cells, we transferred antagomiR-203-encoding lentiviral vectors to primary keratinocytes at an MOI of 10 and subsequently, 3 days after transduction, measured levels of mRNA and protein by qRT-PCR and ELISA, respectively. As depicted in Fig. 5a, we did not detect a statistically significant decrease in the levels of miR-203 in LV/antagomirR203-treated primary keratinocytes, when compared to cells transduced with LV/antagomiR-neg, in accordance with our observations in HaCaT and HaCaT-203 cells (Supplementary Fig. S2). Nevertheless, we measured a 1.4-fold increase of both TNFa and IL24 mRNA levels in the presence of vector-encoded antagomiR203 in primary keratinocytes (p < 0.0001) (grey bars, Fig. 5b and c). In agreement with the detection of enhanced mRNA levels for both cytokines, we measured increased levels of both proteins in the presence of miR-203 (1.8 and 1.5-fold for TNFa and IL24, respectively) (black bars, Fig. 5b and c). These data demonstrated that the antagomiRs did not facilitate miRNA degradation, but that suppression of miR-203 activity correlated with increased levels of
Fig. 5. miR-203 is a regulator of TNFa and IL24 in primary keratinocytes. Primary keratinocytes were transduced at an MOI of 10 with LV/vehicle, LV/antagomiR-neg or LV/ antagomiR-203 and RNA and protein samples were collected 3 days after transduction. (a) Levels of miR-203 in lentiviral vector-treated keratinocytes. miR-203 levels were evaluated by qRT-PCR. (b) Increased expression of TNFa in primary keratinocytes treated with antagomirR-203. TNFa mRNA and protein levels were evaluated by qRT-PCR and ELISA, respectively. (c) Enhanced expression of IL24 in primary keratinocytes treated with antagomirR-203. IL24 mRNA and protein levels were evaluated by qRT-PCR and ELISA, respectively. For all experiments primary keratinocytes were incubated with anisomycin (500 ng/mL) for 18 h before RNA and protein collection. p < 0.05; p < 0.001; p < 0.0001. All experiments were performed in triplicates and data depicted as mean + SEM.
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both TNFa and IL24 mRNA transcripts and protein. In summary, our data demonstrate that expression of TNFa and IL24 is directly regulated by miR-203 suggesting that miR-203 may act as a suppressor as well as an activator of immune responses in skin through regulation of SOCS3 and SOCS6 as well as the pro-inflammatory cytokines TNFa and IL24.
4. Discussion Damaged epidermal tissue is continuously repaired by mechanisms that govern homeostasis of the skin. Self-renewing epidermal stem cells serve as a source of keratinocytes replacing injured keratinocytes as well as keratinocytes that are lost during epidermal differentiation and the constant turnover of skin tissue. Skin homeostasis is maintained through complex interactive processes that involve intercellular communication and intracellular signaling facilitated by a complex circuitry of activities by chemokines and cytokines as well as growth factors and activated transcription factors. Together, these factors control the balance between proliferation, differentiation, and apoptosis of keratinocytes. Due to the potential of pro-inflammatory cytokines to induce continuous tissue inflammation and damage, it is vital that the production of cytokines by keratinocytes and skin-homing lymphocytes is tightly regulated during skin homeostasis as well as during inflammatory responses toward infections and injury. MicroRNAs are considered to have a significant role during epidermal remodeling and skin development, as specific gene ablation of the Dicer enzyme, which is essential for processing of miRNAs, leads to loss of miRNA expression, abnormal hair follicle development, and impaired epidermal differentiation [25,26]. Several studies have identified miR-203 as a skin-specific miRNA expressed in differentiated murine and human epidermal keratinocytes [13,27], and global miRNA analyses have shown that miR-203 is upregulated in lesional psoriatic skin [15,28]. In this report, we investigated putative anti-inflammatory functions of miR-203 mediated through direct regulation of the expression of pro-inflammatory cytokines in keratinocytes. Among a panel of cytokines that are central factors in chronic skin inflammation leading to psoriasis, we identified TNFa- and IL24-encoding mRNAs as specific targets of miR-203. By managing the levels of miR-203 in keratinocytes, either by over-expressing miR-203 or by inhibiting miR-203 function, we found that miR-203 plays an active role in modulating inflammatory cytokines in skin cells. Hence, together with the findings of other laboratories, our data suggest that miR-203 possesses properties of both anti- and pro-inflammatory character and, thus, may serve a buffering function in skin homeostasis. TNFa is a central cytokine in innate and adaptive immunity, and its vital role during chronic inflammation has been supported by the fact that systemically administered TNFa inhibitors are currently successfully employed for treatment of rheumatoid arthritis and severe psoriasis [29,30]. In accordance, we recently demonstrated amelioration of psoriatic lesions in a skin xenotransplantation model by local, lentiviral administration of small hairpin RNAs targeting TNFa-encoding mRNAs through the RNA interference pathway [23]. Given the regulatory actions of miR-203 on TNFa mRNA in primary keratinocytes, it may seem contradictory that the levels of both TNFa protein and miR-203 are increased in lesional psoriatic skin. However, our findings lend support to the hypothesis that the upregulation of miR-203 in psoriasis is a probable consequence of the disease development rather than a causative factor in the development or progression of the disease. Notably, increased levels of TNFa protein in lesional psoriatic skin are not the result of increased mRNA levels but rather of modulated posttranscriptional regulation of expression [31]. Whether such balanced levels of TNFa mRNA in psoriatic skin involve the
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potential degradation of TNFa-encoding mRNA by increased levels of miR-203 is not clear at the present stage. However, together with well-defined signaling cascades, such as the p38 MAPK and NF-rB pathways, miR-203 may serve to modulate the expression of TNFa. With our current knowledge it is not clear whether the increase of miR-203 observed in psoriatic skin is sufficient to influence or control the production of TNFa and IL24. However, by antagonizing miR-203 function in primary keratinocytes by lentivirus-encoded antagomiRs, we observed a significant influence on the expression of both TNFa and IL24, indicating a direct correlation with miR-203 activity. Future studies based on managing of miR-203 in the xenotransplantation skin model will reveal whether pro-inflammatory cytokines in a similar fashion can be regulated through lentiviral delivery of expression cassettes expressing miR-203 or inhibitors of miR-203 function. Such experiments will address also to which extent small changes in the cellular content of active miR-203 have an impact on the expression on TNFa and IL24. IL24 is one of several cytokines which is known to directly mediate the cross-talk between skin-infiltrating T cells and resident cells in skin [32]. This cytokine is constitutively expressed by proliferating keratinocytes in vitro, although it is preferentially expressed in inflamed skin by Th2 cells and activated monocytes, resulting in persistent STAT3 activation in keratinocytes, which leads to expression of inflammatory mediators in skin [32]. Moreover, the gene expression profile in IL24-induced keratinocytes is consistent with the profile that is evident during the re-epithelialization phase in wound healing, establishing IL24 as an important modulator in epidermal remodeling [33]. In accordance, it has been demonstrated recently that IL24-transgenic mice die neonatally, with increased epidermal thickness and significant macrophage infiltration in the dermis [34]. Interestingly, transgenic mice over-expressing miR-203 also frequently die shortly after birth, but are shown by histological evaluation to present with a thinner epidermis relative to wild-type mice [13]. These findings suggest that miR-203 and IL24 have antagonistic roles in skin homeostasis, lending support to our finding of IL24-encoding mRNA as a direct target of miR-203 in primary keratinocytes. Taken together, our results demonstrate that the inflammatory cytokines TNFa and IL24 are direct targets of miR-203 in keratinocytes, suggesting a new level of complexity in epidermal remodeling and skin homeostasis. Even though the physiological function of miR-203 and the reason for its enhanced expression during inflammation in skin is still debatable, our findings propose that miR-203 plays a vital role in epidermal remodeling and cytokine signaling. MicroRNA-203 is known to promote epidermal differentiation by repressing expression of p63 [14]. Combined with evidence of miR-203-directed regulation of members of the SOCS gene family [16,19], our findings of pro-inflammatory cytokines as additional targets define both pro- and anti-inflammatory functions of miR-203, suggesting that miR-203 is a potential key player in fine-tuning the innate immune response in human skin. Acknowledgments We thank Karin Stenderup and Cecilia Rosada for providing cultures of primary keratinocytes. This work was made possible by support from the Lundbeck Foundation, the Novo Nordisk Foundation, The Danish Heart Foundation, Kgl. Hofbuntmager Aage Bangs Fond, Agnes og Poul Friis Fond, and Aase og Ejnar Danielsens Fond. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.cyto.2012.07.031.
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References [1] Bak RO, Mikkelsen JG. Regulation of cytokines by small RNAs during skin inflammation. J Biomed Sci 2010;17:53. [2] Jing Q, Huang S, Guth S, Zarubin T, Motoyama A, Chen J, et al. Involvement of microRNA in AU-rich element-mediated mRNA instability. Cell 2005;120:623–34. [3] Lagos-Quintana M, Rauhut R, Lendeckel W, Tuschl T. Identification of novel genes coding for small expressed RNAs. Science 2001;294:853–8. [4] Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 2004;116:281–97. [5] Lewis BP, Burge CB, Bartel DP. Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell 2005;120:15–20. [6] Seggerson K, Tang L, Moss EG. Two genetic circuits repress the Caenorhabditis elegans heterochronic gene lin-28 after translation initiation. Dev Biol 2002;243:215–25. [7] Olsen PH, Ambros V. The lin-4 regulatory RNA controls developmental timing in Caenorhabditis elegans by blocking LIN-14 protein synthesis after the initiation of translation. Dev Biol 1999;216:671–80. [8] Guo H, Ingolia NT, Weissman JS, Bartel DP. Mammalian microRNAs predominantly act to decrease target mRNA levels. Nature 2010;466:835–40. [9] Miyoshi K, Okada TN, Siomi H, Siomi MC. Characterization of the miRNA-RISC loading complex and miRNA-RISC formed in the Drosophila miRNA pathway. RNA 2009;15:1282–91. [10] Grimson A, Farh KK, Johnston WK, Garrett-Engele P, Lim LP, Bartel DP. MicroRNA targeting specificity in mammals: determinants beyond seed pairing. Mol Cell 2007;27:91–105. [11] Shin C, Nam JW, Farh KK, Chiang HR, Shkumatava A, Bartel DP. Expanding the microRNA targeting code: functional sites with centered pairing. Mol Cell 2010;38:789–802. [12] Krueger JG. The immunologic basis for the treatment of psoriasis with new biologic agents. J Am Acad Dermatol 2002;46:1–23. [quiz-6]. [13] Yi R, Poy MN, Stoffel M, Fuchs E. A skin microRNA promotes differentiation by repressing ‘stemness’. Nature 2008;452:225–9. [14] Lena AM, Shalom-Feuerstein R, di Val Cervo PR, Aberdam D, Knight RA, Melino G, et al. miR-203 represses ‘stemness’ by repressing DNp63. Cell Death Differ 2008;15:1187–95. [15] Sonkoly E, Wei T, Janson PC, Saaf A, Lundeberg L, Tengvall-Linder M, et al. MicroRNAs: novel regulators involved in the pathogenesis of psoriasis? PLoS ONE 2007;2:e610. [16] Wei T, Orfanidis K, Xu N, Janson P, Ståhle M, Pivarcsi A, et al. The expression of microRNA-203 during human skin morphogenesis. Exp Dermatol 2010;19:854–6. [17] Croker BA, Krebs DL, Zhang JG, Wormald S, Willson TA, Stanley EG, et al. SOCS3 negatively regulates IL-6 signaling in vivo. Nat Immunol 2003;4:540–5. [18] Sano S, Chan KS, DiGiovanni J. Impact of Stat3 activation upon skin biology: a dichotomy of its role between homeostasis and diseases. J Dermatol Sci 2008;50:1–14.
[19] Moffatt CE, Lamont RJ. Porphyromonas gingivalis induction of microRNA-203 expression controls suppressor of cytokine signaling 3 in gingival epithelial cells. Infect Immun 2011;79:2632–7. [20] Asirvatham AJ, Gregorie CJ, Hu Z, Magner WJ, Tomasi TB. MicroRNA targets in immune genes and the Dicer/Argonaute and ARE machinery components. Mol Immunol 2008;45:1995–2006. [21] Sonkoly E, Ståhle M, Pivarcsi A. MicroRNAs: novel regulators in skin inflammation. Clin Exp Dermatol 2008;33:312–5. [22] Ivics Z, Hackett PB, Plasterk RH, Izsvak Z. Molecular reconstruction of Sleeping Beauty, a Tc1-like transposon from fish, and its transposition in human cells. Cell 1997;91:501–10. [23] Jakobsen M, Stenderup K, Rosada C, Moldt B, Kamp S, Dam TN, et al. Amelioration of psoriasis by Anti-TNF-a RNAi in the xenograft transplantation model. Mol Ther 2009;17:1743–53. [24] Bak RO, Stenderup K, Rosada C, Petersen LB, Moldt B, Dagnæs-Hansen F, et al. Targeting of human interleukin-12B by small hairpin RNAs in xenografted psoriatic skin. BMC Dermatology 2011;11:5. [25] Yi R, O’Carroll D, Pasolli HA, Zhang Z, Dietrich FS, Tarakhovsky A, et al. Morphogenesis in skin is governed by discrete sets of differentially expressed microRNAs. Nat Genet 2006;38:356–62. [26] Andl T, Murchison EP, Liu F, Zhang Y, Yunta-Gonzalez M, Tobias JW, et al. The miRNA-processing enzyme dicer is essential for the morphogenesis and maintenance of hair follicles. Curr Biol 2006;16:1041–9. [27] Nissan X, Denis JA, Saidani M, Lemaitre G, Peschanski M, Baldeschi C. miR-203 modulates epithelial differentiation of human embryonic stem cells towards epidermal stratification. Dev Biol 2011. [28] Zibert JR, Løvendorf MB, Litman T, Olsen J, Kaczkowski B, Skov L. MicroRNAs and potential target interactions in psoriasis. J Dermatol Sci 2010;58:177–85. [29] Anolik JH, Ravikumar R, Barnard J, Owen T, Almudevar A, Milner EC, et al. Cutting edge: anti-tumor necrosis factor therapy in rheumatoid arthritis inhibits memory B lymphocytes via effects on lymphoid germinal centers and follicular dendritic cell networks. J Immunol 2008;180:688–92. [30] Chong BF, Wong HK. Immunobiologics in the treatment of psoriasis. Clin Immunol 2007;123:129–38. [31] Johansen C, Funding AT, Otkjaer K, Kragballe K, Jensen UB, Madsen M, et al. Protein expression of TNF-alpha in psoriatic skin is regulated at a posttranscriptional level by MAPK-activated protein kinase 2. J Immunol 2006;176:1431–8. [32] Kunz S, Wolk K, Witte E, Witte K, Doecke WD, Volk HD, et al. Interleukin (IL)19, IL-20 and IL-24 are produced by and act on keratinocytes and are distinct from classical ILs. Exp Dermatol 2006;15:991–1004. [33] Sa SM, Valdez PA, Wu J, Jung K, Zhong F, Hall L, et al. The effects of IL-20 subfamily cytokines on reconstituted human epidermis suggest potential roles in cutaneous innate defense and pathogenic adaptive immunity in psoriasis. J Immunol 2007;178:2229–40. [34] He M, Liang P. IL-24 transgenic mice. in vivo evidence of overlapping functions for IL-20, IL-22, and IL-24 in the epidermis. J Immunol 2010;184:1793–8.
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Regulation of pro-inflammatory cytokines TNFa and IL24 by microRNA-203 in primary keratinocytes Maria Nascimento Primo, Rasmus O. Bak, Beatrice Schibler, Jacob Giehm Mikkelsen ⇑ Department of Biomedicine, Aarhus University, DK-8000 Aarhus C, Denmark
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Article history: Received 9 March 2012 Received in revised form 20 June 2012 Accepted 26 July 2012 Available online 20 August 2012 Keywords: MicroRNA Cytokine Inflammation Skin
a b s t r a c t Cutaneous homeostasis and innate immunity is procured by a complex circuitry of intercellular cytokine signaling. MicroRNAs are important posttranscriptional regulators of keratinocyte gene expression and assist in modulating the fine balance between cell proliferation and differentiation in skin. A characteristic microRNA profile in inflammatory skin suggests putative functions of microRNAs in perturbed cytokine production and signaling during chronic inflammatory skin conditions such as psoriasis. It remains unclear, however, why certain microRNAs are aberrantly expressed during skin inflammation and if they serve pro- and/or anti-inflammatory functions. In this report, we focus on cytokine regulation by microRNA-203 (miR-203), which is highly abundant in keratinocytes and upregulated in psoriatic lesions. By screening a panel of cytokines that are upregulated in psoriatic skin for regulation by miR-203, we identify the genes encoding the pro-inflammatory cytokines TNFa and IL24 as direct targets of miR-203. Studies of miR-203 overexpression, inhibition, and mutagenesis validate posttranscriptional regulation of TNFa and IL24 by miR-203 in cell lines and primary keratinocytes. Our findings suggest that miR-203 serves to fine-tune cytokine signaling and may dampen skin immune responses by repressing key proinflammatory cytokines. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction The alertness of the immune system relies on sophisticated and highly controlled intercellular signaling among immune cells and between immune cells and differentiated somatic cells. Cytokines are key modulators of the innate immune response and play a prominent role during inflammation. Given the sensitivity and cellular impact of signal transmission, the production of cytokines needs to be highly flexible and is thus strongly regulated at the transcriptional level. Moreover, a tight regulation is conferred by regulatory mechanisms that operate during RNA processing and translation. Cytokine-encoding mRNAs are typically short-lived transcripts due to the presence of AU-rich elements located in the 30 untranslated regions (UTRs) of mRNA transcripts, which under normal cellular conditions promote rapid mRNA decay by attracting AU-rich elements-binding proteins and RNA degradation enzymes [1]. Moreover, components of AU-rich elements-mediated decay pathways may act together with the microRNA machinery to regulate the stability and translation of cytokine-encoding mRNAs [2]. ⇑ Corresponding author. Address: Department of Biomedicine, University of Aarhus, Wilh. Meyers Allé, Bldg. 1240, DK-8000 Aarhus C, Denmark. Tel.: +45 89421671. E-mail address: [email protected] (J.G. Mikkelsen). 1043-4666/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.cyto.2012.07.031
MicroRNAs (miRNAs) constitute a family of small (21nt), noncoding RNA molecules which regulate up to one-third of human genes by triggering either translational repression or mRNA degradation [3–5]. MicroRNA-directed gene suppression is accomplished in association with RNAi-induced silencing complex which recruits factors that facilitate mRNA destabilization or repression of the translation process [4,6–9]. Base-pairing at position 2–8 relative to the 50 end of the miRNA, termed the ‘seed region’, is of particular importance for gene suppression by miRNAs [10,11] and is in many cases sufficient to regulate gene expression. Chronic inflammatory diseases are characterized by aberrant cytokine signaling in the afflicted tissue. Such variations could potentially reflect abnormal expression of immune-regulatory miRNAs since a growing list of miRNAs has been associated with the pathogenesis of immune diseases. In psoriatic skin lesions, keratinocytes contribute to a chronic inflammatory state by responding to and producing cytokines that affect cell proliferation and differentiation, promote angiogenesis, and attract skin-homing immune cells [12]. MicroRNA-203 is highly expressed in keratinocytes and plays a role in skin morphogenesis, as supported by its suppression of the transcription factor p63 [13,14]. This miRNA is upregulated in psoriatic lesions and is thought to play a potential part in disease progression through its repression of suppressor of cytokine signaling 3 (SOCS3) [15,16], which is a negative regulator of STAT3 [17,18]. In addition, miR-203 was recently found to
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target SOCS6 [19]. Together, these findings indicate that upregulation of miR-203 facilitates repression of immunosuppressive genes and plays a potential part in disease progression. However, there is no formal proof of this assumption, and miR-203 could have additional targets in a complex network of immune-modulatory interactions in which the miRNA under certain conditions acts as an activator and under other circumstances as a suppressor of immune signaling. In this report, we address the hypothesis that miR-203 is a modulator of cytokine signaling with the ability to both accelerate and suppress an innate immune response. To do so, we challenged the notion that cytokine-encoding mRNAs are rare primary miRNA targets [20] and screened a panel of 30 UTRs from mRNAs, all encoding cytokines upregulated in human psoriatic skin, for sensitivity to miR-203-directed regulation. We demonstrate that the pro-inflammatory cytokines tumor necrosis factor a (TNFa) and interleukin24 (IL24) are direct targets of miR-203 in a keratinocyte cell line and primary keratinocytes. Our findings lend support to the notion that miR-203 serves to fine-tune, or balance, cytokine signaling by down-regulating members of the SOCS gene family as well as proinflammatory cytokines, possibly indicating that enhanced production of miR-203 in psoriatic skin could be part of an anti-inflammatory response. 2. Materials and methods
protocol. Primary keratinocytes were seeded in 6-well plates (6 104 cells/well) 1 day before transduction performed with an MOI of 10. Seventy-two hours post-transduction, primary keratinocyte culture supernatants were harvested for quantification of secreted protein, and total RNA was extracted using the SV Total RNA Isolation System (Promega, Madison, WI, USA) according to manufacturer’s protocol. 2.3. Bioinformatics miR-203 target sites in SOCS6, TNFa and IL24 mRNAs were predicted by PicTar 5, RNAhybrid and TargetScan 5.2 softwares available at http://pictar.mdc-berlin.de/, http://bibiserv.techfak. uni-bielefeld.de/rnahybrid/ and http://www.targetscan.org/, respectively. 2.4. Statistical analyses All experiments were performed in triplicates and data are depicted as mean + SEM. All p-values were calculated by a two-tailed Student’s T-test to test the null hypothesis of no difference between two compared groups. The assumption of equal variances was tested by the F-test. In all statistical analyses, p-values <0.05 were considered significant. Further details related to materials and methods are provided in the Supplementary Materials and Methods.
2.1. Cell lines and primary keratinocytes HaCaT, HeLa, HEK293 and 293T cells were cultured at 37 °C in 5% (v/v) CO2 and maintained in Dulbeccoo’s modified Eagle’s medium (Cambrex, Verviers, Belgium) supplemented with 10% fetal calf serum, penicillin (100 U/mL), streptomycin (0.1 mg/mL), and L-glutamine (265 mg/L). Primary keratinocytes were cultured at 37 °C in 5% (v/v) CO2 and maintained in Serum-free Keratinocyte medium (Gibco BRL-Life Technologies, Eggenstein, Germany) supplemented with bovine pituitary extract (25 lg/mL) and recombinant epidermal growth factor (0.1–0.2 ng/mL). 2.2. Lentiviral vector production and transduction For production of lentiviral vectors, 293T cells were seeded in 10-cm dishes 1 day before transfection (4 106 cells/dish). Cells were transfected by the calcium phosphate method with 3.75 lg pMD.2G, 3 lg pRSV-Rev, 13 lg pMDLg/pRRE, and 13 lg lentiviral transfer vector. Forty-eight hours after transfection the viral supernatants were harvested and passed through 0.45 lm filters (Sarstedt, Nümbrecht, Germany). Titer assays based on colony formation were performed in HaCaT cells seeded in 6-well plates (5 104 cells/well) 1 day before transduction. Lentiviral supernatants for transduction of naïve HaCaT and HaCaT-203 cells were serially diluted and supplemented with polybrene (8 lg/mL; Sigma–Aldrich, Milwaukee, WI, USA). Transduced HaCaT cells were grown in 1 lg/mL puromycin-containing medium (Sigma–Aldrich, Milwaukee, WI, USA) for 10 days after which the number of colonies was counted and the titer was calculated. HaCaT-203 and naïve HaCaT cells were seeded in 6-well plates (6 104 cells/well) 1 day before transduction. In high-dose experiments, transductions were performed at an MOI of 50, and total RNA was extracted 72 h post-transduction using the SV Total RNA Isolation System (Promega, Madison, WI, USA) according to manufacturer’s protocol. For stable transduction experiments, transductions were performed at an MOI of 5, followed by puromycin selection (final concentration of 1 lg/mL; Sigma–Aldrich, Milwaukee, WI, USA) for 14 days. Total RNA was extracted using the SV Total RNA Isolation System (Promega, Madison, WI, USA) according to manufacturer’s
3. Results 3.1. Identification of cytokine-encoding genes as potential targets of miR-203 Skin inflammation is characterized by deregulated expression of a wealth of cytokines and several miRNAs. The abundance of miR203 in lesional psoriatic skin could suggest that this particular miRNA contributes to the progression of the disease and/or is aberrantly expressed as part of a protective response. To study the regulatory properties of miR-203 in cytokine production, we set out to screen a panel of genes associated with the cytokine network in psoriasis as possible targets of miR-203 using a dual luciferase reporter assay. Candidate target sequences were fused with the Renilla luciferase (Rluc) reporter gene in context of the psiCHECK-2 reporter plasmid, allowing the use of Rluc expression as a measure for potential miRNA targeting of the fusion mRNA. Expression of firefly luciferase from a separate expression cassette in the same plasmid allowed for normalization in case of transfection differences. Different reporter constructs were generated containing 30 UTRs from six genes encoding cytokines that are upregulated in psoriatic skin (TNFa, IL12B, IL15, IL17, IL20 and IL24) and from the gene encoding SOCS6 (which was identified as a miR-203 target gene during the course of these studies; [19]). In addition, we included three positive controls, a single perfect target site for miR-203 (psiCHECK.miR203target) and the 30 UTRs derived from the genes encoding transcription factor p63 and SOCS3 which are both validated targets of miR-203 [14,21]. MicroRNA-203 was expressed from a 335-bp human genomic DNA segment encoding the miR-203 precursor transcribed by a CMV promoter contained within a Sleeping Beauty (SB) DNA transposon plasmid (pT2/CMV-pri-miR-203.SV40-neo; Fig. 1a), allowing studies of both transiently and stably expressed miR-203. The reporter plasmids were co-transfected into HEK293 cells that do not have endogenous expression of miR-203, along with either pT2/CMV-pri-miR-203.SV40-neo or a negative control plasmid, pUC19. As shown in Fig. 1b, the functionality of plasmid-encoded miR-203 was verified as it was found to potently
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Fig. 1. Functional screening of potential miR-203 target genes. (a) Schematic representation of an SB DNA transposon-based vector expressing the pri-miR-203 sequence. The processing of pri-miR-203 to pre-miR-203, and the RNA-induced silencing complex (RISC) loaded with mature miR-203 is schematically shown below the vector. (b) Identification of SOCS6, TNFa, and IL24 as potential target genes of miR-203. HEK293 cells were co-transfected with either pUC19 (negative control) or the pri-miR-203encoding vector and the psiCHECK reporter plasmid containing a luciferase gene fused to a fragment of the indicated gene. ‘Control’ indicates a psiCHECK plasmid without a target sequence, whereas a psiCHECK plasmid with a perfect target sequence for miR-203 was included in transfections labeled ‘miR-203 target’. p < 0.01; p < 0.001. Experiments were performed in triplicates, and the data are depicted as mean + SEM.
down-regulate the Rluc fusion transcript carrying a perfect miR203 target site (about 90% knockdown). Also, the two previously reported targets of miR-203, p63 and SOCS3, were targeted by transiently expressed miR-203, resulting in both cases in approximately 50% knockdown. Among the remaining target sequences, transient expression of miR-203 resulted in targeting of Rluc transcripts fused to sequences derived from SOCS6, TNFa and IL24, resulting in 25, 45, and 20% knockdown, respectively, compared to the negative control (p < 0.01). These findings suggest that mRNAs encoding SOCS6, TNFa and IL24 are targeted by miR-203. 3.2. SOCS6, TNFa, and IL24 mRNA transcripts are direct targets of miR203 Human miRNAs mainly target the 30 UTRs of mRNAs, predominantly by complete sequence complementarity within the ‘seed region’. Thus, to identify potential miR-203 target sites within mRNAs encoding SOCS6, TNFa, and IL24, we performed a bioinformatic search for miR-203 seed match sequences using PicTar 5, RNAhybrid and TargetScan 5.2 softwares. As a result, we identified four potential miR-203 target sites within each of the 30 UTRs of SOCS6 and IL24 mRNA transcripts and two within the 30 UTR of TNFa mRNA (Fig. 2a, upper panel). To decipher the importance of these potential miR-203 target sites, we generated mutated 30 UTR sequences for each of the three genes in which all the predicted miR-203 seed binding sites were abolished by introducing a 4-bp mutation (Fig. 2a, bottom panel). The seed match mutation sequences were designed strictly on the basis of computational prediction of sequences that would have limited complementarity to the miR-203 sequence and at the same time would not match the seed region
of any other human miRNA. The mutated 30 UTR sequences were fused to the Rluc gene in the psiCHECK reporter plasmid, and the susceptibility of the fusion mRNAs to miR-203 regulation was compared with the corresponding mRNAs containing the native SOCS6, TNFa, and IL24 30 UTRs. As shown in Fig. 2b–d, the mutated seed matches completely abolished miR-203-mediated repression of expression from mRNA transcripts containing the SOCS6, TNFa, and IL24 sequences, suggesting that miR-203 interacted directly with one or more of the computationally predicted target sites within the 30 UTRs of SOCS6-, TNFa-, and IL24-encoding mRNAs. To further confirm that the regulation of the Rluc fusion transcripts by miR-203 was mediated by the predicted target sites in the mutated 3’ UTRs, we generated a mutant miR-203 (miR-203mut) with ‘seed region’-complementarity to the mutated 30 UTR target sites carrying the 4-bp mutations. Due to the presence of computationally predicted target sites for miR-203-mut within the Rluc gene, we replaced the Rluc reporter gene within the psiCHECK.IL24-mut plasmid with the eGFP gene. HEK293 cells were co-transfected with the modified reporter vector (psiCHECK.eGFP-IL24-mut) and with plasmids expressing either primiR-203 or pri-miR-203-mut. As shown in Fig. 2e, over-expression of pri-miR-203-mut led to repression of eGFP expression from an mRNA fusion transcript containing the mutant IL24 sequence, resulting in approximately 20% knockdown compared to co-transfections with plasmids encoding either the unmodified pri-miR203 or pri-miR-125b (the latter included as an additional negative control). Taken together, these findings validated the importance of mRNA-miRNA complementarity, demonstrating that the generegulatory effects of miR-203 were achieved by direct interaction with target sequences within the 30 UTRs of the affected genes.
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Fig. 2. Site-directed mutagenesis validates SOCS6, TNFa and IL24 mRNAs as direct targets of miR-203. (a, top panel) Schematic representation of the 30 UTRs of SOCS6, TNFa and IL24 mRNA transcripts (grey bars) with indication of the predicted target sites for miR-203 (black boxes). Numbers underlying the black boxes represent the first and the last basepair position within the NCBI reference. (a, bottom panel) Schematic representation of the miR-203 seed match mutations and miR-203 mutations (bold nucleotides are written in bold). (b–e) Disruption of miR203-directed gene regulation by introduced mutations. In panels (b–d) HEK293 cells were co-transfected with the indicated psiCHECK vectors and either pUC19 (negative control) or the pri-miR-203-encoding vector; in panel (e), HEK293 cells were transfected with psiCHECK.eGFP-IL24-mut and the vectors indicated. p < 0.001; p < 0.0001. Experiments were performed in triplicates, and the data are depicted as mean + SEM.
3.3. Potent knockdown of endogenously expressed SOCS6, TNFa, and IL24 mRNAs in HaCaT cells with engineered over-expression of miR203 To investigate miR-203 suppression of endogenously expressed full-length mRNA transcripts encoding SOCS6, TNFa, and IL24, we utilized the bipartite SB DNA transposon system to establish HaCaT keratinocyte cell lines with engineered over-expression of miR203. HaCaT cells were co-transfected with pT2/CMV-pri-miR203.SV40-neo (Fig. 1a) and either a plasmid encoding an inactive mutated SB transposase or the hyperactive SB100X transposase [22]. The number of neomycin-resistant colonies was significantly increased in the presence of SB100X relative to the mutated transposase (Supplementary Fig. S1a), indicating that genomic integration of the bicistronic vector was efficiently accomplished by a transposase-directed mechanism. We next isolated and expanded three individual clones containing the transposon (HaCaT-203 clones A, B, and C) and detected increased expression of miR-203 in all three HaCaT clones, when compared to the naïve HaCaT cell line, ranging from 8.7 to 12.4-fold increase (Supplementary Fig. S1b). MicroRNA-203-directed regulation of endogenously expressed SOCS6, TNFa, and IL24 were evaluated by qRT-PCR on naïve HaCaT cells and on the HaCaT-203 clone with highest miR-203 expression (clone C, from now on designated HaCaT-203). For all three target genes, over-expression of miR-203 repressed the mRNA levels, resulting in levels that were reduced to 40%, 5%, and 10% of the levels of mRNA transcripts present in naïve HaCaT cells for SOCS6, TNFa, and IL24 mRNA, respectively (Fig. 3a–c, grey bars). To validate that post-transcriptional suppression by miR-203 resulted in reduced levels of protein in keratinocytes, we investigated TNFa
and IL24 protein levels in both naïve HaCaT and HaCaT-203 cells. Indeed, over-expression of miR-203 led to a marked reduction of TNFa, resulting in 80% knockdown of both TNFa and IL24 protein levels (Fig. 3b and c, black bars). These findings confirmed the potential of miR-203 to alter expression levels of cytokines and possibly interact directly with distinct components of the inflammatory response in keratinocytes. To investigate whether expression of the potential miR-203 target genes varied with the miR-203 expression level, we analyzed the IL24 mRNA levels in the other two HaCaT-203 clones (clones A and B). As illustrated in Fig. 3d, IL24 mRNA levels inversely correlated with miR-203 levels in the four HaCaT keratinocyte cell lines, indicating that suppression of IL24 mRNA transcripts was directly dependent on miR-203 levels. 3.4. Establishment of miR-203 inhibition by lentiviral vector-encoded ‘antagomiRs’ We have previously provided evidence of potent and sustained in vivo knockdown of cytokine-encoding genes by the use of lentiviral vectors encoding small interfering RNAs [23,24]. Hence, we decided to explore the same lentiviral platform for construction of viral vectors expressing short, non-coding RNAs that were complementary to miRNAs as a method for persistently antagonizing miRNA function in vitro. A cassette expressing such an RNA ‘antagomiR’ directed against miR-203 (antagomiR-203) from an H1 promoter was cloned into a lentiviral vector plasmid (previously described in [24]) and analyzed for its ability to target miR-203. HaCaT-203, HaCaT, and HeLa cells were co-transfected with the Rluc reporter plasmid encoding the Rluc gene fused with the perfect miR-203 target site (psiCHECK.miR203target) and with either
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Fig. 3. Stable over-expression of miR-203 leads to marked reduction of endogenous SOCS6, TNFa, and IL24 in HaCaT cells. (a–c) Reduced levels of SOCS6, TNFa, and IL24 in HaCaT-203 cells. mRNA levels were evaluated by qRT-PCR in naïve HaCaT cells and a HaCaT-203 clone stably over-expressing miR-203. TNFa and IL24 protein levels (panels (b) and (c), respectively) were measured by ELISA from naïve HaCaT and HaCaT-203 cell supernatants. (d) IL24 mRNA levels (black line) and miR-203 levels (light-grey columns) were evaluated by qRT-PCR in all HaCaT cell lines. For all experiments naïve HaCaT cells and HaCaT-203 cells were incubated with anisomycin (500 ng/mL) for 18 h before both RNA and protein collection. p < 0.001; p < 0.0001. Experiments were performed in triplicates, and the data depicted as mean + SEM.
the lentiviral vector plasmid encoding antagomiR-203 (pLV/antagomiR-203) or pUC19 plasmid (negative control). The Rluc fusion transcript was thus targeted by endogenously expressed miR203, and transient expression of antagomiR-203 was suspected to relieve this suppression. As shown in Fig. 4a, the presence of antagomiR-203 led to increased luciferase activities in all three cell lines. We detected a 4.6-fold increase in HaCaT-203 cells, a 3.3-fold increase in HaCaT cells and a 2.1-fold increase in HeLa cells. We also evaluated miR-203 levels by qRT-PCR analysis (Fig. 4a, insert) and found that the miR-203 expression pattern in the three cell lines correlated directly with the degree of miR-203 inhibition. Hence, the impact of antagomiR-203 was most pronounced in the cells with highest levels of miR-203. We also evaluated the dose-dependency of antagomiR-203 by co-transfecting the three cell lines with psiCHECK.miR203target and increasing amounts of the antagomiR-203-encoding lentiviral plasmid. A dose–response relationship was obtained in the three cell lines, as increased amounts of antagomiR-203-encoding plasmid resulted in increased Rluc expression (Fig. 4b). Taken together,
our results showed robust miR-203 inhibition by introduction of antagomiR-203 and demonstrated that the level of inhibition was dependent on both the endogenous miR-203 expression level and the antagomiR dose. To evaluate the potency of lentiviral vector-delivered antagomiRs, HaCaT cells were transduced with lentiviral vectors encoding antagomiR-203 (LV/antagomiR-203). As negative controls, we included a vector which did not carry an antagomiR (LV/vehicle) and a vector encoding an antagomiR predicted not to target any known miRNA (LV/antagomiR-neg). For all three vectors, cells were transduced at a multiplicity of infection (MOI) of approximately 120. On the following day, cells were transfected either with psiCHECK.miR203target or pUC19 (negative control), and luciferase activities were measured 48 h post-transfection. As shown in Fig. 4c, expression of antagomiR-203 from lentiviral vectors resulted in almost complete relief of miR-203-mediated suppression of the reporter (p < 0.01). This demonstrated that lentiviral delivery of antagomiR-203 efficiently inhibited the functional properties of miR-203.
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Fig. 4. Inhibition of miR-203 function by plasmid or lentiviral delivery of DNA-encoded antagomiR-203. (a) Impact of antagomiR-203 in cells with variable levels of miR-203. Cell lines were co-transfected with the psiCHECK.miR203target vector and either pUC19 (negative control) or the lentiviral plasmid expressing the antagomiR-203 (pLV/ antagomiR-203). miR-203 Ct values from qRT-PCR are shown in the small insert (all cell lines showed similar Ct values of the reference small RNA, RU48). (b) Suppression of miR-203 activity is increased with enhanced dosages of antagomiR-203. Cell lines were co-transfected with the psiCHECK.miR203target vector and with increasing amounts of pLV/antagomiR-203. (c) Repression of miR-203 activity by lentivirally delivered antagomiR-203. HaCaT cells were transduced at an MOI of 120 with LV/antagomiR-203, LV/ antagomiR-neg, or LV/vehicle and transfected with either pUC19 (negative control) or psiCHECK.miR203target vector (miR-203 target). p < 0.01; p < 0.001; p < 0.0001. Experiments were performed at least in triplicates, and the data are depicted as mean + SEM.
3.5. miR-203 regulates TNFa and IL24 protein levels in primary keratinocytes During the course of our studies, SOCS6 was reported to be a target for miR-203 [19]. Here, we chose to focus our analyses on the pro-inflammatory cytokines IL24 and TNFa as targets of miR203 in a potential cytokine-regulating response. We analyzed the levels of IL24 mRNA in naïve HaCaT and HaCaT-203 cells after exposure of the cells to LV/antagomir-203 and measured a significant increase in the levels of IL24 mRNA in both cell lines (Supplementary Fig. S2). Notably, the effect of antagomiR-203 was persistent, allowing long-term repression of miR-203 function by antagomiR-203 expressed from genomically integrated lentiviral vectors (Supplementary Fig. S2). We also observed that the levels of detectable miR-203 in HaCaT-203 cells were unaffected by the treatment with antagomiR-203, lending support to the notion that the miRNA was antagonized, and not degraded, by the antagomiR (Supplementary Fig. S2).
To verify this type of regulation in primary cells, we transferred antagomiR-203-encoding lentiviral vectors to primary keratinocytes at an MOI of 10 and subsequently, 3 days after transduction, measured levels of mRNA and protein by qRT-PCR and ELISA, respectively. As depicted in Fig. 5a, we did not detect a statistically significant decrease in the levels of miR-203 in LV/antagomirR203-treated primary keratinocytes, when compared to cells transduced with LV/antagomiR-neg, in accordance with our observations in HaCaT and HaCaT-203 cells (Supplementary Fig. S2). Nevertheless, we measured a 1.4-fold increase of both TNFa and IL24 mRNA levels in the presence of vector-encoded antagomiR203 in primary keratinocytes (p < 0.0001) (grey bars, Fig. 5b and c). In agreement with the detection of enhanced mRNA levels for both cytokines, we measured increased levels of both proteins in the presence of miR-203 (1.8 and 1.5-fold for TNFa and IL24, respectively) (black bars, Fig. 5b and c). These data demonstrated that the antagomiRs did not facilitate miRNA degradation, but that suppression of miR-203 activity correlated with increased levels of
Fig. 5. miR-203 is a regulator of TNFa and IL24 in primary keratinocytes. Primary keratinocytes were transduced at an MOI of 10 with LV/vehicle, LV/antagomiR-neg or LV/ antagomiR-203 and RNA and protein samples were collected 3 days after transduction. (a) Levels of miR-203 in lentiviral vector-treated keratinocytes. miR-203 levels were evaluated by qRT-PCR. (b) Increased expression of TNFa in primary keratinocytes treated with antagomirR-203. TNFa mRNA and protein levels were evaluated by qRT-PCR and ELISA, respectively. (c) Enhanced expression of IL24 in primary keratinocytes treated with antagomirR-203. IL24 mRNA and protein levels were evaluated by qRT-PCR and ELISA, respectively. For all experiments primary keratinocytes were incubated with anisomycin (500 ng/mL) for 18 h before RNA and protein collection. p < 0.05; p < 0.001; p < 0.0001. All experiments were performed in triplicates and data depicted as mean + SEM.
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both TNFa and IL24 mRNA transcripts and protein. In summary, our data demonstrate that expression of TNFa and IL24 is directly regulated by miR-203 suggesting that miR-203 may act as a suppressor as well as an activator of immune responses in skin through regulation of SOCS3 and SOCS6 as well as the pro-inflammatory cytokines TNFa and IL24.
4. Discussion Damaged epidermal tissue is continuously repaired by mechanisms that govern homeostasis of the skin. Self-renewing epidermal stem cells serve as a source of keratinocytes replacing injured keratinocytes as well as keratinocytes that are lost during epidermal differentiation and the constant turnover of skin tissue. Skin homeostasis is maintained through complex interactive processes that involve intercellular communication and intracellular signaling facilitated by a complex circuitry of activities by chemokines and cytokines as well as growth factors and activated transcription factors. Together, these factors control the balance between proliferation, differentiation, and apoptosis of keratinocytes. Due to the potential of pro-inflammatory cytokines to induce continuous tissue inflammation and damage, it is vital that the production of cytokines by keratinocytes and skin-homing lymphocytes is tightly regulated during skin homeostasis as well as during inflammatory responses toward infections and injury. MicroRNAs are considered to have a significant role during epidermal remodeling and skin development, as specific gene ablation of the Dicer enzyme, which is essential for processing of miRNAs, leads to loss of miRNA expression, abnormal hair follicle development, and impaired epidermal differentiation [25,26]. Several studies have identified miR-203 as a skin-specific miRNA expressed in differentiated murine and human epidermal keratinocytes [13,27], and global miRNA analyses have shown that miR-203 is upregulated in lesional psoriatic skin [15,28]. In this report, we investigated putative anti-inflammatory functions of miR-203 mediated through direct regulation of the expression of pro-inflammatory cytokines in keratinocytes. Among a panel of cytokines that are central factors in chronic skin inflammation leading to psoriasis, we identified TNFa- and IL24-encoding mRNAs as specific targets of miR-203. By managing the levels of miR-203 in keratinocytes, either by over-expressing miR-203 or by inhibiting miR-203 function, we found that miR-203 plays an active role in modulating inflammatory cytokines in skin cells. Hence, together with the findings of other laboratories, our data suggest that miR-203 possesses properties of both anti- and pro-inflammatory character and, thus, may serve a buffering function in skin homeostasis. TNFa is a central cytokine in innate and adaptive immunity, and its vital role during chronic inflammation has been supported by the fact that systemically administered TNFa inhibitors are currently successfully employed for treatment of rheumatoid arthritis and severe psoriasis [29,30]. In accordance, we recently demonstrated amelioration of psoriatic lesions in a skin xenotransplantation model by local, lentiviral administration of small hairpin RNAs targeting TNFa-encoding mRNAs through the RNA interference pathway [23]. Given the regulatory actions of miR-203 on TNFa mRNA in primary keratinocytes, it may seem contradictory that the levels of both TNFa protein and miR-203 are increased in lesional psoriatic skin. However, our findings lend support to the hypothesis that the upregulation of miR-203 in psoriasis is a probable consequence of the disease development rather than a causative factor in the development or progression of the disease. Notably, increased levels of TNFa protein in lesional psoriatic skin are not the result of increased mRNA levels but rather of modulated posttranscriptional regulation of expression [31]. Whether such balanced levels of TNFa mRNA in psoriatic skin involve the
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potential degradation of TNFa-encoding mRNA by increased levels of miR-203 is not clear at the present stage. However, together with well-defined signaling cascades, such as the p38 MAPK and NF-rB pathways, miR-203 may serve to modulate the expression of TNFa. With our current knowledge it is not clear whether the increase of miR-203 observed in psoriatic skin is sufficient to influence or control the production of TNFa and IL24. However, by antagonizing miR-203 function in primary keratinocytes by lentivirus-encoded antagomiRs, we observed a significant influence on the expression of both TNFa and IL24, indicating a direct correlation with miR-203 activity. Future studies based on managing of miR-203 in the xenotransplantation skin model will reveal whether pro-inflammatory cytokines in a similar fashion can be regulated through lentiviral delivery of expression cassettes expressing miR-203 or inhibitors of miR-203 function. Such experiments will address also to which extent small changes in the cellular content of active miR-203 have an impact on the expression on TNFa and IL24. IL24 is one of several cytokines which is known to directly mediate the cross-talk between skin-infiltrating T cells and resident cells in skin [32]. This cytokine is constitutively expressed by proliferating keratinocytes in vitro, although it is preferentially expressed in inflamed skin by Th2 cells and activated monocytes, resulting in persistent STAT3 activation in keratinocytes, which leads to expression of inflammatory mediators in skin [32]. Moreover, the gene expression profile in IL24-induced keratinocytes is consistent with the profile that is evident during the re-epithelialization phase in wound healing, establishing IL24 as an important modulator in epidermal remodeling [33]. In accordance, it has been demonstrated recently that IL24-transgenic mice die neonatally, with increased epidermal thickness and significant macrophage infiltration in the dermis [34]. Interestingly, transgenic mice over-expressing miR-203 also frequently die shortly after birth, but are shown by histological evaluation to present with a thinner epidermis relative to wild-type mice [13]. These findings suggest that miR-203 and IL24 have antagonistic roles in skin homeostasis, lending support to our finding of IL24-encoding mRNA as a direct target of miR-203 in primary keratinocytes. Taken together, our results demonstrate that the inflammatory cytokines TNFa and IL24 are direct targets of miR-203 in keratinocytes, suggesting a new level of complexity in epidermal remodeling and skin homeostasis. Even though the physiological function of miR-203 and the reason for its enhanced expression during inflammation in skin is still debatable, our findings propose that miR-203 plays a vital role in epidermal remodeling and cytokine signaling. MicroRNA-203 is known to promote epidermal differentiation by repressing expression of p63 [14]. Combined with evidence of miR-203-directed regulation of members of the SOCS gene family [16,19], our findings of pro-inflammatory cytokines as additional targets define both pro- and anti-inflammatory functions of miR-203, suggesting that miR-203 is a potential key player in fine-tuning the innate immune response in human skin. Acknowledgments We thank Karin Stenderup and Cecilia Rosada for providing cultures of primary keratinocytes. This work was made possible by support from the Lundbeck Foundation, the Novo Nordisk Foundation, The Danish Heart Foundation, Kgl. Hofbuntmager Aage Bangs Fond, Agnes og Poul Friis Fond, and Aase og Ejnar Danielsens Fond. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.cyto.2012.07.031.
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