CCL27 expression is regulated by both p38 MAPK and IKKβ signalling pathways

Cytokine 56 (2011) 699–707

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Cytokine journal homepage: www.elsevier.com/locate/issn/10434666

CCL27 expression is regulated by both p38 MAPK and IKKb signalling pathways Jette Lindorff Riis ⇑, Claus Johansen, Christian Vestergaard, Kristian Otkjaer, Knud Kragballe, Lars Iversen Department of Dermatology, Aarhus Sygehus, Aarhus University Hospital, Aarhus C, Denmark

a r t i c l e

i n f o

Article history: Received 23 June 2010 Received in revised form 31 August 2011 Accepted 10 September 2011 Available online 10 October 2011 Keywords: CCL27 IKKb p38 MAPK MSK1 Mnk

a b s t r a c t The skin-specific chemokine CCL27 is believed to play a pivotal role in establishing the inflammatory infiltrate characteristic for common inflammatory skin diseases. Through binding to the chemokine receptor 10 (CCR10), CCL27 mediates inflammation by promoting lymphocyte migration into the skin. Little is known about the regulation of CCL27 gene expression. The purpose of our study was to investigate the regulation of the IL-1b-induced CCL27 gene expression in normal human keratinocytes (NHEK). Preincubation of NHEK with the inhibitory jB (IjB) kinase (IKK) inhibitor, SC-514, or the p38 mitogenactivated protein kinase (MAPK) inhibitor, SB202190, revealed a profound reduction in both CCL27 mRNA and CCL27 protein expression indicating the significance of these pathways in the regulation of CCL27 expression. Furthermore, the impact of inhibitors of mitogen- and stress-activated kinase 1 (MSK1) or the mitogen-activated protein kinase-interacting kinases (Mnk1 + 2), downstream kinases of p38 MAPK, on IL-1b-induced CCL27 expression in NHEK were investigated. We identified seven NF-jB binding elements upstream from the CCL27 gene start codon using electrophoretic mobility shift assay (EMSA). Supershift analyses demonstrated the involvement of the p50/p65 NF-jB heterodimer. We conclude that IL-1b-induced CCL27 gene expression in NHEK is regulated through the p38 MAPK/MSK1/Mnk1 + 2 as well as the IKKb/NF-jB signalling pathways. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction Common inflammatory skin diseases, such as psoriasis, atopic dermatitis and contact dermatitis, are characterised by a distinct dermal inflammatory infiltrate primarily consisting of lymphocytes. The recruitment of T cells is a critical step in the pathogenesis of such skin disorders and is directed by the gradients of numerous chemokines produced by resident or immigrated cells [1]. The lymphocytes responsible for dermal immunity home from the blood to the skin through a multistep process involving vascular endothelial cell recognition and extravasation [2]. CCL27/CTACK (cutaneous T-cell-attracting chemokine) is a skinspecific CC-chemokine continuously expressed by epidermal keratinocytes [3]. CCL27 specifically binds the chemokine receptor 10 (CCR10) [4], which is expressed almost exclusively on skin homing CLA+ lymphocytes [3,5]. Thus, CCL27 contributes to a tissuerestricted leucocyte trafficking by exhibiting high receptor and

Abbreviations: MAPK, mitogen-activated protein kinase; MSK, mitogen- and stress-activated protein kinase; Mnk, mitogen-activated protein kinase (MAPK)interacting kinase; IjB, inhibitory jB; IKK, IjB kinase; NF-jB, nuclear factor-jB; NHEK, normal human epidermal keratinocytes. ⇑ Corresponding author. Address: Department of Dermatology, Aarhus Sygehus, PP Oerumsgade 11, DK-8000 Aarhus C, Denmark. Tel.: +45 89491848; fax: +45 89491850. E-mail address: [email protected] (J.L. Riis). 1043-4666/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.cyto.2011.09.007

tissue specificity and induces inflammation by promoting lymphocyte migration into the skin [6]. Immunohistochemically CCL27 has been found highly upregulated in inflammatory skin diseases, such as atopic dermatitis, psoriasis, and contact dermatitis [5]. Furthermore, increased serum levels of CCL27 in patients suffering from atopic dermatitis and psoriasis vulgaris have been demonstrated [7]. The expression of CCL27 is inducible in cultured normal human keratinocytes (NHEK) by the pro-inflammatory cytokines TNFa and IL-1 [3]. However, little is known about the signalling pathways involved in the regulation of CCL27 gene expression. The mitogen-activated protein kinases (MAPKs) represent a family of protein kinases, which regulates different cellular responses including inflammation through phosphorylation of specific kinases [8]. The MAPK family consists of extracellular signalregulated protein kinases (ERK), c-Jun NH2-terminal kinases (JNK), ERK5, and the p38 MAP kinases (p38 MAPK). The p38 MAPK indirectly affects gene transcription or translation of specific proteins during an inflammatory response by interacting with transcription factors and kinases. For example, the IL-1b-induced IL-20 gene expression has recently been demonstrated to act through p38 MAPK [9] and p38 MAPK has recently been reported as a regulator of IL-1b-induced IL-24 expression [10]. Thus, the p38 MAPK is a pivotal factor in inflammatory cellular responses. Mitogen- and stress-activated protein kinase 1 (MSK1) is a downstream kinase of the p38 MAPK. The p38 MAPK/MSK

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signalling cascade is activated by stress and inflammatory cytokines [11]. The mitogen-activated protein kinase-interacting kinases (Mnk1 + 2), substrates of ERK and p38 MAPK, phosphorylate eukaryotic initiation factor 4E (eIF4E), which regulates translation [12,13]. Other less well-characterised substrates of Mnk1 + 2 are a number of proteins involved in the translational machinery which, e.g. through binding to AU-rich elements modulate the stability or translation of specific mRNAs [14]. The transcription factor nuclear factor jB (NF-jB) is involved in regulation of the expression of many genes involved in the inflammatory process. NF-jB is a hetero- or homodimer composed of the ‘rel’ family of proteins including the five members; RelA/p65, RelB, c-Rel, p52, and p50 [15]. In quiescent cells, NF-jB is held inactive in the cytoplasm by the NF-jB inhibitory proteins, the IjBs. On cellular activation, the IjB kinase (IKK) becomes activated leading to phosphorylation of the IjBs, allowing nuclear NF-jB entry [16]. Once activated NF-jB initiates gene transcription of various target genes. NF-jB binding sites are represented in promoter regions of many cytokine and chemokine genes related to immune and inflammatory responses [17]. The promoter region of the CCL27 gene contains NF-jB binding sites which may regulate transcription of the gene [18]. NF-jB is involved in the regulation of CCL27, inasmuch as non-specific NF-jB-inhibitors repress TNFa-induced CCL27 production, and the NF-jB p50/p65 heterodimer is proposed to be the predominant dimer [18]. Another study found TNFa-induced CCL27 production suppressed by inhibited NF-jB activity, and the p50 and p65 NF-jB subunits were suggested to be responsible for this TNFainduced CCL27 production [19]. Besides, in the human keratinocyte cell line, HaCaT cells, the combined TNFa- and IL-1b-induced production of CCL27 is downregulated by inhibitors of both p38 MAPK and NF-jB [20]. The purpose of this in vitro study was to investigate the signal transduction pathways regulating CCL27 expression in further details. We determined both the p38 MAPK signalling pathway along with its substrates MSK1 and Mnk1 + 2, and the IKK/NF-jB signal-transduction cascade as key players in the regulation of IL-1b-induced CCL27 production. Furthermore, we identified several NF-jB DNA binding sites in the CCL27 promoter region.

2. Materials and methods 2.1. Cell cultures Normal human epidermal keratinocytes (NHEK) were obtained by trypsinization of skin samples from adult patients undergoing plastic surgery as previously described [21]. Second passage keratinocytes were grown in a 37 °C, humidified, 5% CO2 atmosphere in keratinocyte serum-free medium (Invitrogen, Carlsbad, CA) containing 5 ng/ml human recombinant epidermal growth factor and 50 lg/ml bovine pituitary extract, and with gentamicin 5 lg/ml added. 24 h prior to stimulation the medium was changed to keratinocyte basal medium (keratinocyte serum-free medium without growth factors). Cells were grown to about 70% confluence. Cells were stimulated with IL-1b (10 ng/ml; R&D Systems, Oxon, UK). IL-1b was diluted in PBS containing bovine serum albumin. In some experiments, cells were pre-incubated with the p38a/b inhibitor SB202190 (10 lM), the IKKb inhibitor SC-514 (50 lM), the MSK1 inhibitor Ro-31-8220 (5 lM), the IKK inhibitor III BMS-345541 (10 lM), the PKC inhibitor Gö6983 (60 nM), or the Mnk inhibitor CGP57380 (50 lM) (Calbiochem, San Diego, CA). The inhibitors were added 30 min before stimulation of the cells, except SC-514 and CGP57380, which were added 1 h prior to stimulation. In all experiments vehicle (PBS with BSA and DMSO) was added to the controls. All incubations were carried out in duplicates.

2.2. RNA extraction Keratinocytes were washed once in ice-cold sterile phosphatebuffered saline (Gibco, Invitrogen, Carlsbad, CA). RNA was purified with SV Total RNA Isolation System (Promega, Madison, WI). 175 ll of SV RNA lysis buffer was added to the cells before dishes were scraped. The lysates were transferred to Eppendorf tubes and kept at 80 °C until RNA isolation. The lysates were thawed on ice. A 350 ll of SV RNA Dilution Buffer was added and tubes inverted 10 times. Samples were heated for 3 min at 70 °C and afterwards centrifuged at 13,000g at room temperature for 10 min. Supernatants were transferred to new Eppendorf tubes. 200 ll of 96% ethanol was added to the supernatant and gently mixed. The mixture was transferred to spin baskets attached to a Vac-Man with MiniPrep Vacuum Adapters (Promega, Madison, WI). Total RNA was purified according to the vacuum protocol, as described by the manufacturer. Ultimately, RNA was dissolved in RNase/DNase-free water and stored at 80 °C until further use. 2.3. Quantitative real-time RT-PCR For reverse transcription (RT), Taqman Reverse Transcription reagents (Applied Biosystems, Foster City, CA) were used according to the manufacturer’s instructions including the use of random hexamers as primers. RT thermal cycling was carried out using a Peltier Thermal Cycler-200 (MJ Research, Inc., Waltham, MA). A 10 min 25 °C incubation step, followed by a 30 min 48 °C RT step and finally a 5 min 95 °C RT inactivation step. The cDNA was stored Ò at 80 °C. CCL27 mRNA expression was determined with TaqMan Gene Expression Assay (assay ID: Hs00171157_m1, Applied Biosystems). The housekeeping gene used for normalisation was 18 s rRNA (assay ID: Hs99999901_s1, Applied Biosystems). PCR mastermix was TaqMan Universal PCR Master Mix (Applied Biosystems). The expression of each gene was analysed in triplicates at a reaction volume of 25 ll. PCR conditions were 2 min at 50 °C, 10 min at 95 °C followed by 50 cycles of 15 s at 95 °C, and 60 s at 60 °C. Real-time PCR machine was a Rotorgene-3000 (Corbett Research, Sydney, Australia). Relative gene expression levels were determined by using the relative standard curve method as outlined in User Bulletin No. 2 (ABI Prism 7700 sequencing detection system, Applied Biosystems). Briefly, a standard curve for each gene was made of 4-fold serial dilutions of total RNA from IL-1b-stimulated keratinocytes. The curve was then used to calculate relative amounts of target mRNA. 2.4. ELISA The supernatant was transferred to Eppendorf tubes and snapfrozen in liquid nitrogen. CCL27 expression was measured by ELISA (Duo-SetÒ ELISA Development System, R&D Systems, Oxon, UK). Wells of a 96-well Maxisorp (Invitrogen, Carlsbad, CA) plate were incubated overnight at room temperature with 100 ll of capture antibody. The wells were washed three times in wash buffer (PBS containing 0.05% TweenÒ 20 (PBST). The wells were then blocked with 300 ll of PBS with 1% bovine serum albumin (BSA) for 1 h and washed three times with wash buffer. The samples and the standards were diluted in PBS containing 1% BSA and 100 ll were added. The samples were diluted 1:1 and measured in doublets. Wells were incubated 1.5 h at room temperature and then left overnight at 4 °C. On the third day wells were washed three times with PBST. 100 ll of the biotinylated detection antibody (50 ng/ml) was added before 2 h of incubation. After three times wash in PBST, 100 ll of the HRP conjugated streptavidin was added in a 1:200 dilution and incubated at room temperature for 20 min. The wells were washed again times three in PBST and visualised by adding 100 ll of substrate solution (R&D Systems, Oxon, UK) and incubated

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for 20 min. The reactions were stopped by adding 2 N H2SO4. The results were measured using an ELISA reader (Lab Systems iEMS Reader MF, Copenhagen, Denmark) at 450 nm. Readings at 540 nm were subtracted to correct for optical imperfections in the plate. The mean minimum detectable dose (MDD) of CCL27 is 1.55 pg/ml. 2.5. Western blot analysis Whole cell protein extracts from cultured keratinocytes were isolated as previously described [22]. Equal protein amounts as determined by Bradford assay [23] were separated on SDS–PAGE 4–12% gels and blotted onto nitrocellulose membranes. Membranes were incubated with the p-p65(Ser276)(Catalogue No. 3037; Cell Signaling Technology) or b-actin (Catalogue No. A-1978; Sigma). The antibodies were detected with anti-rabbit IgG-HRP (Catalogue No. 7074; Cell Signaling Technology), or with anti-mouse (Catalogue No. p0447; Dako) in a standard ECL reaction (Amersham Biosciences) according to the manufacturer’s instructions. 2.6. Nuclear extraction Nuclear extracts were prepared as previously described [24]. Briefly a hypoton buffer (10 mM Hepes, 10 mM KCl, 0.2 mM EDTA, 0.1 mM EGTA, 1 mM DTT, pH 7.9) containing 0.5% detergent was added to the cultured cells. The cells were released from the Petri dish by a rubber policeman. The suspension was passed through a 27-gauge needle six times and centrifuged at 13,600g at 4 °C for 1 min. The supernatant were considered the cytosolic extract. The pelleted nuclei were resuspended in a hyperton buffer (20 mM Hepes, 0.4 mM NaCl, 1 mM EDTA and 1 mM DTT, pH 7.9) and rotated at 4 °C for 30 min before centrifuged at 13,600g for 5 min. The supernatant was considered the nuclear extract. Protein concentration was determined by Bradford [23] with BSA as standard. 2.7. Electrophoretic mobility shift assay (EMSA) The promoter region of CCL27 (Entrez Nucleotide, Accession No. NT008413) was searched for potential NF-jB binding sites (TFSEARCH version 1.3) [25] resembling the consensus sequence. We identified seven potential NF-jB binding sites (bold) and matching oligonucleotides were designed. Furthermore, seven corresponding oligonucleotides with mutated (underlined) NF-jB binding site sequence (bold) were synthesized (DNA Technology A/S, Aarhus, Denmark).

hCCL27(S1): hCCL27(S1M): hCCL27(S2): hCCL27(S2M): hCCL27(S3): hCCL27(S3M1): hCCL27(S3M2): hCCL27(S4): hCCL27(S4M): hCCL27(S5): hCCL27(S5M1): hCCL27(S5M2): hCCL27(S6): hCCL27(S6M): hCCL27(S7): hCCL27(S7M):

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Gel shift assays were performed as previously described [26]. Oligonucleotides were labelled by T4 polynucleotide kinase (Promega, Madison, WI) and purified on a Nick Spin column (Sephadex G-50, Pharmacia, Uppsala, Sweden). Nuclear protein (3 lg) was preincubated for 10 min in binding buffer (Promega, Madison, WI) and hypoton buffer (10 mM Hepes, 10 mM KCl, 0.2 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 1 protease inhibitors and 0.5 mM PMSF). A 1 ll of 32P-labelled NF-jB probe was added and the suspension was incubated for additional 20 min. Samples were separated on a 6% NovexÒ DNA retardation gel (Invitrogen, Carlsbad, CA) run at 100 V for 2 h at 4 °C in 0.5  TBE buffer (22.5 mM Tris-base, 4 mM boric acid and 1 mM EDTA) and visualised after exposure to X-ray film. Supershifts were performed by adding 2 ll of the corresponding commercially available antibodies specific for the individual NF-jB proteins (NF-jB p50 (C-19) cat# sc1190 goat polyclonal IgG and NF-jB p65 (H-286) cat# sc-7151 rabbit polyclonal IgG, Santa Cruz Biotechnology, Santa Cruz, CA) to the binding reactions. After 15 min of incubation the 32P-labelled NF-jB probe was added. Incubation was then allowed to proceed for additional 30 min. In control experiments a specific competitor (unlabelled hCCL27(S3) oligo) or a non-specific competitor (unlabelled SP-1 oligo) (E323B, Promega, Madison, WI) was added 10 min before addition of labelled hCCL27(S3) oligo. 2.8. Statistics Results were expressed as mean ± standard deviation (SD). Statistical significance (p < 0.05) was assessed by one sample t-test. To test for normal distribution, a probability test was conducted. 3. Results 3.1. CCL27 expression is induced by IL-1b in NHEK To study the dynamics of CCL27 mRNA (Fig. 1A) and protein (Fig. 1B) expression in IL-1b-stimulated NHEK, time course studies were conducted. Total RNA and protein were extracted and subsequently, CCL27 mRNA (relative to housekeeping gene 18s rRNA) and CCL27 protein expression levels were determined by quantitative real-time RT-PCR and ELISA techniques, respectively. After 4 h of IL-1b-stimulation, a significant upregulation of CCL27 mRNA expression was found, and maximal CCL27 mRNA expression (6-fold induction) was detected after 6 h of stimulation. A significant increase in CCL27 protein level was measured after 6 h of stimulation, while maximal protein level (1.7-fold induction) was observed 36 h after stimulation with IL-1b.

50 -GGGCTAGGGAATCCCTGATTCA-30 50 -GGGCTAGCCCAAAACTGATTCA-30 50 -CATCGGGGGGAATCCGGTAGCT-30 50 -CATCGGGGCCCAAAAGGTAGCT-30 50 -AAGAGGGGGAGATTCCTAGAAG-30 0

0

5 -AAGAGGGCCCGATTCCTAGAAG-3

50 -AAGAGGGCCCGAAAACTAGAAG-30 50 -GAACCTGGGAGATTTCCTTGGG-30 50 -GAACCTGCCCGAAAACCTTGGG-30 50 -TTTCCTTGGGATTTCCTCAGTG-30 50 -TTTCCTTGCCCTTTCCTCAGTG-30 50 -TTTCCTTGCCCTAAACTCAGTG-30 50 -GGAGAGGGGATGTTCCTCCAGG-30 50 -GGAGAGGCCCTGAAACTCCAGG-30 50 -CATAAAGGGCCTTCCCCCAGCT-30 50 -CATAAACCCCCAAACCCCAGCT-30

3.2. The p38 MAPK signalling pathway regulates CCL27 gene expression To characterise the role of the p38 MAPK signalling cascade in the regulation of CCL27 expression cultured NHEK were preincubated with the p38 MAPK inhibitor SB202190. This inhibitor was added to the cells 30 min prior to IL-1b-stimulation. NHEK were stimulated with IL-1b for 6 h before CCL27 mRNA expression was measured. A stimulation period of 24 h was chosen for analyzing the CCL27 protein level. Incubation with the p38 MAPK inhibitor prior to stimulation resulted in a marked reduction in the expression of CCL27 mRNA (approximately 37%) as well as CCL27 protein (approximately 35%) (Fig. 2A and B). These findings indicate that IL-1b-induced CCL27 gene expression is regulated through the p38 MAPK signalling pathway. When SB202190 alone was added to NHEK no inhibition of CCL27 mRNA expression was found, whereas the CCL27 protein level was slightly inhibited by SB202190 compared with NHEK treated with vehicle (DMSO)

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Fig. 1. IL-1b-induced CCL27 gene expression. Time study of IL-1b-induced CCL27 expression. (A) CCL27 mRNA expression. (B) CCL27 protein level. Normal human keratinocytes were cultured until a confluency of 50–70% and incubated with either vehicle (controls) or IL-1b (10 ng/ml) for different time periods. Controls were incubated with vehicle for 24 h and for 60 h for RNA and protein purposes, respectively. The mean value of controls for each culture has been set to index 100. Each bar represents mean ± SD. ⁄p < 0.05 compared with controls. n = 3.

alone. To ensure these results were not due to unspecific inhibition, NHEK were also preincubated with the protein kinase C (PKC) inhibitor Gö6983. The PKC family of enzymes is involved in controlling the function of other proteins through phosphorylation. PKC enzymes play important roles in several signal-transduction cascades, but inhibition of PKC did not change either CCL27 mRNA or CCL27 protein expression (Fig. 2A and B). 3.3. Downstream kinases of p38 MAPK play a pivotal role in CCL27 regulation Next, we investigated the role of p38 MAPK downstream kinases in the regulation of CCL27 gene expression. The p38 MAPKactivated kinase: mitogen- and stress-activated kinase 1 (MSK1) has been suggested to increase the transcriptional activity of NF-jB [27]. NHEK were preincubated with an inhibitor of MSK1, Ro-31-8220, followed by stimulation with IL-1b. Preincubation with Ro-31-8220 resulted in a 74% reduction in mean CCL27 expression at mRNA level (Fig. 3A) and a 37% reduction in CCL27 expression at protein level (Fig. 3B). Ro-31-8220 inhibited CCL27 mRNA and protein expression to levels equivalent to those observed in vehicle-treated NHEK. Thus, preincubation with

Fig. 2. The p38 MAPK inhibitor SB202190 inhibits IL-1b-induced CCL27 expression. (A) CCL27 mRNA and (B) CCL27 protein level in NHEK preincubated for 1 h with DMSO, the p38 MAPK inhibitor SB202190 (10 lM) or the PKC inhibitor Gö6983 (60 nM) before stimulation with vehicle or IL-1b (10 ng/ml) for 6 h for mRNA purposes or for 24 h for protein determination. All experiments were carried out in doublets. Each bar represents mean ± SD. ⁄p < 0.05 compared with IL-1b-stimulated cells. ⁄⁄p < 0.05 compared with vehicle-stimulated cells. (A) n P 5. (B) n P 3.

Ro-31-8220 completely abolished the IL-1b-induced CCL27 production at both mRNA and protein levels. These results indicate that MSK1 regulates CCL27 gene expression. Secondly, the p38 MAPK-activated kinases, mitogen-activated protein kinase (MAPK)-interacting kinases 1 and 2 (Mnk1 + 2) were studied. Mnk1 and Mnk2 phosphorylate eukaryotic initiation factor 4E (eIF4E), a protein playing an essential role in initiating the process of translation [12,13]. Previously, pretreatment of NHEK with the Mnk inhibitor CGP57380 prior to Anisomycin-stimulation, a known p38 MAPK inducer, has abolished eIF4E phosphorylation [28]. These data suggest the Mnks as major phosphorylators of eIF4E. The same article shows an IL-1b-induced eIF4E phosphorylation. We preincubated NHEK with the Mnk1 + 2 inhibitor CGP57380 [29] and subsequently stimulated with IL-1b for 6 h. Preincubation with CGP57380 prior to IL-1b-stimulation resulted in a 35% reduction in the CCL27 mRNA level and a 37% reduction in CCL27 protein level when compared with IL-1b-stimulated NHEK (Fig. 3C and D). Inhibition of Mnk1 and Mnk2 resulted in CCL27 protein levels similar to vehicle levels, thus obliterating the IL-1b-induced CCL27 protein production. These data suggest that the Mnks are part of the regulatory mechanisms involved in CCL27 gene expression. Because both MSK1 and the Mnks are activated downstream of ERK as well as of p38 MAPK, we investigated the influence of MEK

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Fig. 3. The p38 MAPK regulates CCL27 gene expression through MSK1 and the Mnks. (A and C) CCL27 mRNA expression. (B, D and E) CCL27 protein level. NHEK were grown in keratinocyte serum-free medium with growth factors. 24 h prior to preincubation with inhibitors, the medium was changed to keratinocyte serum-free medium without growth factors. The MSK1 inhibitor Ro-31-8220 (5 lM), the Mnk1 + 2 inhibitor CGP57380 (50 lM), or the MEK inhibitor PD98059 (50 lM) was added to cells half an hour before IL-1b-stimulation (10 ng/ml). Each bar represents mean ± SD. ⁄p < 0.05 compared with IL-1b-stimulated NHEK. ⁄⁄p < 0.05 compared with vehicle-stimulated cells. (A, B and E): n = 4. (C and D): n = 3.

inhibition (an upstream kinase of ERK) on CCL27 production. We found no significant alterations in CCL27 protein level after pretreating NHEK with the MEK inhibitor, PD98059, prior to IL-1b-stimulation (Fig. 3E). 3.4. CCL27 expression is regulated by the IKKb signalling pathway Former studies have indicated an important role of NF-jB in CCL27 gene expression. Therefore, NHEK were incubated with inhibitors of the IKK/NF-jB signalling pathway (Fig. 4A and B). We added the specific IKKb inhibitors SC-514 [30] or BMS345541 [31] to NHEK prior to IL-1b-stimulation. SC-514-incubation alone resulted in a minor downregulation of CCL27 protein level (p = 0.0092) compared with DMSO-incubated vehicle-treated controls. Inhibition of IKKb with SC-514 downregulated the mean

CCL27 mRNA expression by 53% whereas mean CCL27 protein level was decreased by 25%. Preincubation of NHEK with BMS-345541 resulted in a complete abrogation of the IL-1b-induced CCL27 mRNA as well as protein expression. Together these results indicate an IKK/NF-jB mediated pathway of CCL27 transcriptional activation. 3.5. Identification of seven NF-jB binding sites in the CCL27 promoter region NF-jB initiates gene transcription of target genes containing the classic 9-10 base pair long jB consensus sequence (Table 1). Therefore, we searched the CCL27 gene sequence on human chromosome 9 and identified seven putative NF-jB binding sites (named CCL27(S1-S7) in order of distoproximal location) within

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a 3.2 kb region 50 of the CCL27 coding sequence (Table 1). The seven potential NF-jB binding sites all express high homology with the classic jB consensus sequence. We investigated the NF-jB DNA binding activity to the potential NF-jB binding sites in the suspected CCL27 gene promoter region by EMSA. Along with oligonucleotides (hCCL27(S1-S7)) matching the promoter sequences holding the NF-jB consensus sequence corresponding oligonucleotides mutated in either 3 or 6 of their potential 9-10 NF-jB binding base pairs (hCCL27(S1M-S7M) were employed (Table 1). NHEK were stimulated with IL-1b or incubated with vehicle and the nuclear extracts were incubated with the oligonucleotides. We found that NF-jB binds to all the seven sequences shown in Table 1 and observed a clear increase in NF-jB DNA binding activity when comparing controls with IL-1b-stimulated NHEK (Fig. 5A, Supplementary Fig. 1). When nuclear extract was incubated with one of the mutated oligonucleotides (hCCL27(S2M-S7M)) no binding was seen, regardless of whether six or just three base pairs were changed (Fig. 5A, Supplementary Fig. 1). Although, NF-jB DNA binding was observed when nuclear extract were incubated with the mutated hCCL27(S1M) oligo the binding activity was clearly decreased compared with incubation with the non-mutated hCCL27(S1) oligo (Supplementary Fig. 1). 3.6. IL-1b-induced NF-jB DNA binding is inhibited by SC-514

Fig. 4. IKK inhibitors inhibit IL-1b-induced CCL27 expression. (A) CCL27 mRNA and (B) CCL27 protein level in NHEK preincubated with either SC-514 or BMS-345541, both inhibitors of the IjB kinase (IKK), or DMSO (controls). BMS-345541 (10 lM) was added to the cells half an hour before stimulation whereas SC-514 (50 lM) was added 1 h prior to IL-1b-stimulation (10 ng/ml). The mean value of controls for each culture has been set to index 100. Each bar represents mean ± SD. ⁄p < 0.05 compared with IL-1b-stimulated cells. ⁄⁄p < 0.05 compared with vehicle-stimulated cells. n P 4.

Table 1 Potential NF-jB binding sites in the promoter region of the CCL27 gene. 50 -GGGRNNYYCC-30

Consensus

50 -GGGAATCCC-30

CCL27(S1) CCL27(S1 M)

bp-3072–3080

CCL27(S2) CCL27(S2 M)

bp-2603–2611

5 -GCCCAAAAC-3 50 -GGGGAATCC-30

CCL27(S3) CCL27(S3M1)

bp-2025–2034

50 -GGCCCAAAA-30 50 -GGGAGATTCC-30

0

0

50 -GCCCGATTCC-30

CCL27(S3M2) CCL27(S4) CCL27(S4M)

bp-1494–1503

50 -GCCCGAAAAC-30 50 -GGGAGATTTC-30

CCL27(S5) CCL27(S5M1)

bp-1482–1491

50 -GCCCGAAAAC-30 50 -TGGGATTTCC-30

Next, we analysed the impact of SC-514 on NF-jB DNA binding to the hCCL27(S1-S7) oligonucleotides. Preincubation of NHEK with SC-514 prior to IL-1b-stimulation resulted in a reduction of the NF-jB DNA binding activity to all seven oligonucleotides (Fig. 5A, Supplementary Fig. 1). These results are parallel to the previously shown reduction in CCL27 mRNA expression after preincubation with SC-514 and suggest the IKK/NF-jB signalling cascade to be part of the regulatory mechanisms responsible for the CCL27 gene expression induced by IL-1b. 3.7. Involvement of the p50/p65 NF-jB dimer in IL-1b-triggered CCL27 expression To characterise the NF-jB subunits responsible for the DNA binding supershift analysis was performed. Incubation of nuclear extract with antibodies against the p50 and/or the p65 NF-jB subunits both revealed a complete supershift of the NF-jB band (Fig. 5) indicating involvement of the p50/p65 dimer in the regulation of IL-1b-triggered CCL27 expression (Fig. 5B). 3.8. IL-1b-induced NF-jB DNA binding involves p38 MAPK Finally, the effect of inhibition of p38 MAPK, MSK1 and Mnk on NF-jB DNA binding to the hCCL27(S5) oligonucleotide was investigated. We found that preincubation of keratinocytes with the p38 MAPK inhibitor SB202190 (Fig. 5C, lane 5) resulted in a reduced NF-jB DNA binding activity while preincubation using either the MSK1 inhibitor Ro31-8220 (lane 4) or the Mnk inhibitor CGP57380 (lane 3) revealed no statistically significantly altered DNA binding of NF-jB.

50 -TGCCCTTTCC-30

CCL27(S5M2) CCL27(S6) CCL27(S6M)

bp-1030–1039

50 -TGCCCTAAAC-30 50 -GGGATGTTCC-30

CCL27(S7) CCL27(S7M)

bp-697–706

50 -GCCCTGAAAC-30 50 -GGGCCTTCCC-30 50 -CCCCCAAACC-30

The consensus sequence of the classic NF-jB binding site is shown in the top row (G, guanine, A, adenine, T, thymine, C, cytosine, R, purine, Y, pyrimidine, and N, any nucleotide). Below are seven putative NF-jB binding sites identified in the CCL27 gene along with their base pair (bp) numbers (Entrez Nucleotide, NT008413). Below each original sequence the mutated sequence(s) is(are) listed (mutations are underscored).

3.9. The role of p38 MAPK, Mnk, and MSK1 in NF-jB activation To address whether p38 MAPK, Mnk, and MSK1 are required for the activation of NF-jB in IL-1b-stimulated NHEK, we preincubated NHEK with SB202190, CGP57380, or Ro-31-8220, respectively, prior to IL-1b-stimulation. Whole cell extracts were analysed by Western blotting using antibodies against the phosphorylated p65 NF-jB subunit. Each inhibitor revealed involvement in the activation of NF-jB through phosphorylation of the p65 subunit (Fig. 6).

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Fig. 6. The role of p38 MAPK, Mnk, and MSK1 in NF-jB activation. The Mnk inhibitor CGP57380 (50 lM), MSK1 inhibitor Ro-31-8220 (5 lM), or the p38 MAPK inhibitor SB202190 (10 lM) was added to NHEK half an hour before IL-1b-stimulation (10 ng/ml) (30 min). Western blottings were performed using whole cell protein extracts separated on SDS–PAGE 4–12% gels. Primary antibodies against the phosphorylated p65(Ser276) NF-jB subunit (p-p65) or against b-actin, ensuring equal loading, were used. The gel shown is representative for three different experiments.

Fig. 5. Identification of NF-jB DNA binding sites. (A) The NF-jB DNA binding activity to the seven putative NF-jB binding sites located upstream from the start codon of the CCL27 gene was analysed by electrophoretic mobility shift assay (EMSA). NHEK were incubated with vehicle (controls) or IL-1b (10 ng/ml) or preincubated with the IKKb inhibitor SC-514 for an hour and stimulated with vehicle or IL-1b (10 ng/ml) for 75 min. The nuclear fraction was isolated and incubated with the synthetic NF-jB oligonucleotides. NF-jB DNA binding to the oligonucleotide hCCL27(S5) and to its two corresponding mutated oligonucleotides hCCL27(S5M1) and hCCL27(S5M2) is shown. In the hCCL27(S5M1) oligonucleotide three nucleotides in the NF-jB binding site have been mutated (underscored) (TGCCCTTTCC) whereas in the hCCL27(S5M2) oligonucleotide six nucleotides were mutated (underscored) (TGCCCTAAAC). Representative gel of three different experiments is shown. (B) EMSA on NHEK incubated with vehicle (controls) or IL-1b (10 ng/ml) including supershift analysis was carried out after incubation of nuclear extracts with p50 and p65 antibodies separately and together. NF-jB binding activity to the oligonuleotide hCCL27(S5) was present in IL-1b-stimulated keratinocytes whereas no binding activity was observed in controls. (C) NHEK were incubated with vehicle (controls) or IL-1b (10 ng/ml) or preincubated with the p38 MAPK inhibitor SB202190, the MSK1 inhibitor Ro-31-8220 or the Mnk inhibitor CGP57380 for an hour and stimulated with IL-1b (10 ng/ml) for 75 min. NS = non-specific bands.

4. Discussion Chemokines are major promoters of recruiting lymphocytes into sites of inflammation. CCL27 mediates its function by selec-

tively attracting Th1 and Th2 T cells expressing CCR10. This skinspecific chemokine is selectively and continuously produced by keratinocytes, which play an important role in immune responses in inflammatory skin disorders. Moreover, the CCL27 expression is believed to be enhanced in inflammatory skin diseases, such as atopic dermatitis [5,7], and recent studies have proposed CCL27 as a key player in skin cancer [32,33]. In this study, focusing on the regulatory mechanisms of CCL27 gene expression, we confirmed that CCL27 gene expression is inducible by the proinflammatory cytokine IL-1b. By the use of inhibitors of signal transduction pathways, we determined that p38 MAPK/MSK1/Mnk1 + 2-dependent signalling as well as NF-jB regulate this IL-1b-induced CCL27 mRNA and protein expression. The MAPK signalling pathways are known to be involved in various cellular processes such as proliferation, differentiation, apoptosis and inflammation. Through preincubation of NHEK with the p38 MAPK inhibitor SB202190, we found a pronounced reduction in both CCL27 mRNA and protein expression indicating significance of the p38 MAPK signalling pathway in CCL27 regulation. Kagami et al. have reported TNFa- plus IL-1b- induced CCL27 protein expression inhibited by SB202190 and NF-jB inhibitors (parthenolide) in HaCaT cells, while the IL-1b-induced CCL27 protein level was unaffected [20]. However, in this work, we used NHEK as a model to study CCL27 expression. To examine the role of IKKb in the activation of NF-jB in IL-1b-induced CCL27 production we used SC-514, a selective inhibitor of IKKb with no effect on other IKK isoforms or serine-threonine and tyrosine kinases [30]. We found the IL-1b-induced CCL27 mRNA and protein expression inhibited by both SB202190 and IKK inhibitors. These different findings may be explained by the use of different concentration of inhibitors, incubation periods, cell type, and/or culturing conditions. Chaturvedi et al. have demonstrated abnormalities in the NF-jB signalling pathway in HaCaT cells including markedly diminished levels of IKKb and lack of induction of kinase activity in response to inflammatory stimuli [34]. These studies suggest caution in using HaCaT cells as a substitute for normal keratinocytes when studying signal transduction in the skin. To our knowledge, MSK1 has never been investigated regarding CCL27 regulation. MSK1 has been found activated in lesional psoriatic skin and regulates expression of proinflammatory cytokines [35] suggesting MSK1 to play an important role in the regulation of inflammatory skin diseases. In the present study, we found IL1b-induced CCL27 expression robustly suppressed by inhibited

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MSK1 activity, indicating MSK1 as essential to IL-1b-induced CCL27 expression. The specificity of chemical kinase inhibitors is essential in studies of signal-transduction pathways. Inhibitors of signalling pathways are generally referred to as specific, although this may not be the case [36]. In this study, MSK1 activity was inhibited using the protein kinase inhibitor Ro-318220, a kinase inhibitor commonly used. This inhibitor has been reported to inhibit but not to abolish the activity of PKC, p70 ribosomal protein S6 kinase, MAPK-1b and glycogen synthase kinase-3b with potency similar to that of MSK1 [36]. However, a recent study showed that Ro-318220 inhibits the MSK1 kinase activity without affecting MSK1 phosphorylation suggesting that p38 MAPK activity is unaffected [9]. Furthermore, previous reports have demonstrated that the activity of several other p38 MAPK activated kinases was almost unaffected by Ro-318220 [9,36]. Others have shown significant reduction in MSK1-phosphorylation in p38 MAPK but not ERK inhibited IL-1b-stimulated NHEK [9,35], indicating p38 MAPK as the main MSK1 activating kinase. Based on these considerations, we suggest that MSK1 is a significant regulator of CCL27 gene expression. Knowledge on the involvement of Mnk1 and Mnk2 in inflammatory skin diseases is sparse. A recent publication demonstrated that the Mnk inhibitor CGP57380 was capable of suppressing IL-1b-stimulated Mnk1 activation in NHEK [28], and overexpression of the Mnk1 substrate eIF4E has been demonstrated in psoriatic skin [37]. Furthermore, there is evidence that the Mnks regulate the production of the pro-inflammatory cytokine TNFa [14]. Thus, Mnk1 and Mnk2 may be important regulators of inflammatory responses in cutaneous diseases. In this study, we found IL-1b-induced CCL27 mRNA expression suppressed by inhibited Mnk1 + 2 activity suggesting that Mnk1 + 2 may affect mRNA stability. Furthermore, we demonstrated a complete abolishment of the IL-1b-induced CCL27 protein production following Mnk1 + 2 inhibition, which may reflect the inhibited Mnk1 + 2 activity on, e.g. eIF4E phosphorylation leading to a downsized translation of CCL27 mRNA into protein. Recent publications has shown that CCL27 is reduced in psoriasis while enhanced in atopic dermatitis [38,39]. Furthermore, several chemokines, e.g. CCL27 and the Th2-type chemokine CCL17/ TARC have been shown to be regulated through the p38 MAPK signalling pathway. We recently investigated the role of MSK1 in a murine chronic skin inflammation model [40]. However, to our knowledge, p38 MAPK activity in atopic skin as well as the role of the p38 MAPK down stream kinases MSK1 and Mnk in atopic dermatitis involved skin remains to be investigated. Upon stimulation, inactive NF-jB complexes kept in the cytoplasm become activated by phosphorylation leading to nuclear translocation of active NF-jB dimers. We identified seven putative NF-jB sites in the suspected CCL27 promoter region, three of which was pointed out earlier as potential NF-jB elements by Vestergaard et al. [18]. By in vitro binding assays we demonstrated that the NF-jB complex was capable of binding to all seven synthetic CCL27 NF-jB binding sites, that this NF-jB DNA binding was inducible by IL-1b, and that SC-514 was capable of inhibiting this binding. While the p38 MAPK inhibitor SB202190 likewise inhibited NF-jB DNA binding no effect was surprisingly found as a result of MSK1 or Mnk inhibition. A possible explanation of these findings could be that the effects of the kinases down stream of p38 MAPK act through transactivation of the NF-kB complex rather than through DNA binding activity directly. Although p38 MAPK is known to activate a number of kinases, searching the literature do not bring upon other specific candidates. Furthermore, the mechanisms of a potential Mnk or MSK1 promoted NF-jB transactivation remains to be investigated. Furthermore, EMSA revealed that the p50/p65 NF-jB heterodimer was the one responsible for the IL-1b-induced NF-jB binding

to the synthetic CCL27 DNA sequences. These findings are consistent with previous reports suggesting the p50/p65 NF-jB heterodimer responsible for TNFa-induced CCL27 production [18,19]. We also demonstrated that mutation of six of the seven CCL27 NF-jB binding elements completely abrogated the DNA binding of the NF-jB complex even if only three bases were changed, whereas NF-jB binding to the mutated hCCL27(S1M) oligo was clearly compromised. The induction of CCL27 gene expression by IL-1b may result from cooperative interactions between NF-jB binding sites within the CCL27 gene promoter. Our experiments strongly indicate that the mechanisms of CCL27 production in NHEK are dependent on NF-jB activation and therefore, that CCL27 gene expression is NF-jB driven. Because the binding activity of an oligonucleotide in vitro is not necessarily representative of its functional activity within cells there is a need for further studies, e.g. reporter assays, which will add to our understanding of the relative contribution of the various NF-jB elements in the CCL27 promoter region. Delineating the molecular mechanisms responsible for the IL-1b induction of CCL27 gene expression is important for a better understanding of the regulation of CCL27 expression in cutaneous immune responses and hereby in inflammatory disease. In this study, we demonstrated that IL-1b-induced CCL27 gene expression is regulated by both the p38 MAPK/MSK1/Mnk1 + 2 signalling pathway, the IKK signal transduction cascade, and through an NF-jB dependable mechanism. Therefore, the herein involved kinases as well as NF-jB are potential targets for future therapeutic approaches to inflammatory skin diseases. The p38 MAPK signalling cascade is attractive for pharmacological intervention because of its well-documented involvement in inflammatory processes. However, in the search for new effective therapeutic drugs for the treatment of inflammatory skin diseases side effects has to be taken into account. Former studies have reported serious side effects related to the use of p38 MAPK inhibitors, e.g. p38 inhibited transgenic mice developed cardiac hypertrophy [41]. Therefore, downstream kinases of p38 MAPK like MSK1 and Mnk with the likelihood of presenting a less pronounced side effect profile than p38 MAPK may be potential new targets of tomorrow’s biologicals. Conflict of interest The authors state no conflict of interest. Acknowledgements This work was supported by The Aage Bang Foundation, The Master Cabinetmaker Sophus Jacobsen and Wife Astrid Jacobsen Foundation, The Novo Nordic Foundation, The Lundbeck Foundation, and The Danish Research Agency. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.cyto.2011.09.007. References [1] Schon MP, Zollner TM, Boehncke WH. The molecular basis of lymphocyte recruitment to the skin: clues for pathogenesis and selective therapies of inflammatory disorders. J Invest Dermatol 2003;121:951–62. [2] Butcher EC, Williams M, Youngman K, Rott L, Briskin M. Lymphocyte trafficking and regional immunity. Adv Immunol 1999;72:209–53 [209–53]. [3] Morales J, Homey B, Vicari AP, Hudak S, Oldham E, Hedrick J, et al. CTACK, a skin-associated chemokine that preferentially attracts skin-homing memory T cells. Proc Natl Acad Sci USA 1999;96:14470–5. [4] Homey B, Wang W, Soto H, Buchanan ME, Wiesenborn A, Catron D, et al. Cutting edge: the orphan chemokine receptor G protein-coupled receptor-2

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