ORIGINAL RESEARCH ARTICLE

Journal of

CCL27 Is Downregulated by Interferon Gamma via Epidermal Growth Factor Receptor in Normal Human Epidermal Keratinocytes

Cellular Physiology

MASARU KARAKAWA,1,2 MAYUMI KOMINE,1,2* YASUSHI HANAKAWA,3 HIDETOSHI TSUDA,2 KOJI SAYAMA,3 KUNIHIKO TAMAKI,1 AND MAMITARO OHTSUKI2 1

Department of Dermatology, University of Tokyo, Bunkyo-ku, Tokyo, Japan

2

Department of Dermatology, Jichi Medical University, Shimotsuke, Tochigi, Japan

3

Department of Dermatology, University of Ehime, Tou-on-shi, Ehime, Japan

The cutaneous T cell-attracting chemokine (CTACK)/CCL27 is indispensable in skin inflammation. CTACK/CCL27 is exclusively produced by epidermal keratinocytes to attract CCR10-expressing T lymphocytes to the skin. We investigated the mechanism of CTACK/ CCL27 production from normal human epidermal keratinocytes (NHEKs) by the proinflammatory cytokines TNFa and IFNg. CTACK/ CCL27 production was induced by TNFa via ERK, JNK, p38, and NFkB. The induction of CTACK/CCL27 by TNFa was suppressed by IFNg via a pathway dependent on JAK, STAT1, and STAT3. Our results also demonstrated that IFNg and TNFa induced the phosphorylation of EGFR and the following phosphorylation of ERK, which is partly responsible for the suppressive effect of IFNg on TNFa-induced production of CTACK/CCL27. Peri-lesional skin of psoriasis demonstrates early inflammatory changes as we have previously reported. CTACK/CCL27 expression was diffuse in the peri-lesional epidermis, while it was restricted to basal layer in lesional epidermis, suggesting that CTACK/CCL27 expression was induced in the early stage of psoriatic plaque formation, and IFNg could participate in the suppression of CTACK/CCL27 expression in the lesional epidermis, reflecting the later stage of psoriatic plaque formation. Our study suggests that CTACK/CCL27 may have a pivotal role in the early stage of psoriasis plaque formation, but should be downregulated in the later stage to induce inflammation characteristic for chronic psoriasis plaques. J. Cell. Physiol. 229: 1935–1945, 2014. © 2014 Wiley Periodicals, Inc.

Cutaneous T cell-attracting chemokine (CTACK)/CCL27 and thymus and activation-regulated chemokine (TARC)/CCL17 are chemokines indispensable in skin inflammation (Morales et al., 1999; Homey et al., 2002). CTACK/CCL27 is a skinspecific chemokine expressed in the epidermal keratinocytes, and it specifically binds to the CC chemokine receptor 10 (CCR10) (Homey et al., 2000), which contributes to a tissuerestricted leukocyte trafficking by exhibiting high receptor and tissue specificity. CTACK/CCL27 overexpression in the epidermis caused enhanced contact hypersensitivity to Th2 stimuli (Kagami et al., 2008). CTACK/CCL27 is critical in the development of atopic dermatitis in keratin14 promoterdriven IL-4 transgenic mice (Chen et al., 2006). Recent study showed decreased CTACK/CCL27 expression in psoriatic skin, no elevated expression in skin of atopic dermatitis or acute contact dermatitis (Riis et al., 2011a). Another study reported notable downregulation of CTACK/CCL27 in lesional skin of psoriasis (Gudjonsson et al., 2010), while the other study demonstrated that prominent decrease of CTACK/CCL27 expression after treatment with etanercept (Campanati et al., 2007). Although the precise role of CTACK/ CCL27 in inflammatory skin diseases is unknown, the mechanisms of its regulation are of interest when attempting to understand the molecular phathophysiology of inflammatory skin diseases. Tumor necrosis factor (TNF)a plays critical roles in several inflammatory disorders such as Crohn’s disease, rheumatoid arthritis, and psoriasis, and therefore, anti-TNF therapy is very effective in these disorders. IFNg is a Th1-type cytokine that has a characteristic expression pattern in certain Th1/Th17-type inflammatory disorders such as psoriasis

© 2 0 1 4 W I L E Y P E R I O D I C A L S , I N C .

Abbreviations: CTACK, cutaneous T cell attracting chemokine; NHEK, normal human epidermal keratinocytes; RT-PCR, reverse transcription polymerase chain reaction; TARC, thymus and activation-regulated chemokine; EGF, epidermal growth factor; ERK, extracellular signal-regulated kinase; JNK, jun-N-terminal kinase; TNF, tumor necrosis factor; IFN, interferon; STAT, signal transducer and activator of transcription; Th, T helper; TGF, transforming growth factor; NFkB, nuclear factor kappa B; JAK, janus kinase; IL, interleukin; MCP1, monocyte chemotactic protein 1; RANTES, regulated and normal T cell expressed and secreted; IP-10, interferon g-induced protein 10; GAS, interferon g activation sequence. The authors declared that they have no conflicts of interest. Contract grant sponsor: Ministry of Health, Labour and Welfare, and Ministry of Education, Culture, Sports, Science and Technology, Japan; Contract grant number: H23-028. *Correspondence to: Mayumi Komine, Department of Dermatology, Jichi Medical University, 3311-1 Yakushiji, Shimotsuke, Tochigi 329-0498, Japan. E-mail: [email protected] Manuscript Received: 6 August 2013 Manuscript Accepted: 2 April 2014 Accepted manuscript online in Wiley Online Library (wileyonlinelibrary.com): 8 April 2014. DOI: 10.1002/jcp.24643

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vulgaris. TNFa has been revealed to induce CTACK/CCL27 through NFkB-dependent pathway (Vestergaard et al., 2005), IL-1b has been reported to induce CTACK/CCL27 via p38 and NFkB (Riis et al., 2011b), and IL-17 has been shown to downregulate CTACK/CCL27 through cyclooxygenase (COX) 2-dependent manner (Kanda et al., 2005); however, there has been no report on the regulatory role of IFNg, another essential molecule in inflammatory skin diseases, such as psoriasis and contact dermatitis, on CTACK/CCL27 expression. We investigated the regulation of CTACK/CCL27 by TNFa and IFNg, and identified novel regulatory mechanisms that are potentially involved in downregulation of CTACK/ CCL27 in the lesion of psoriatic plaques. Materials and Methods Antibodies and cytokines Anti-signal transducers and activators of transcription (STAT) 1, anti-STAT3, anti-ERK, anti-EGFR, anti-p38, anti-JNK, antiphospho-STAT1, anti-phospho-STAT3, anti-phospho-ERK, antiphospho-EGFR, anti-phospho-p38, anti-phospho-JNK, anti-rabbit IgG horseradish peroxidase (HRP) conjugate, and anti-mouse IgG HRP conjugate were purchased from Cell Signaling Technology Japan K.K. (Tokyo, Japan). Recombinant human TNFa, recombinant human interferon (IFN)g, recombinant human oncostatin M (OSM), and anti-cutaneous T cell-attracting chemokine (CTACK)/CCL27 antibody were from R&D Systems (Minneapolis, MN). Anti-HLA-DR antibody was from Dako Japan (Tokyo, Japan). Inhibitors for signal transduction Several inhibitors of signal transduction were added 30 min prior to stimulation with TNFa and IFNg. Parthenolide, SB202190, PD98059, PD153035, PD168393, c-jun N-terminal kinase (JNK) inhibitor 2, Janus kinase (JAK) inhibitor 1, and JAK3 inhibitor were purchased from Calbiochem (Merck, Darmstadt, Germany). The following concentrations were used: PD98059, 50 mM; PD153035, 5 mM; PD168393, 5 mM; SB202190, 5 mM; JNK inhibitor 2, 50 nM; JAK inhibitor 1, 0.1–1 ng/ml; JAK3 inhibitor, 2.5–10 ng/ml; parthenolide, 5 mM. Cell culture and treatment Normal neonatal foreskin human keratinocytes (NHEKs) were purchased from Iwaki Glass Co., Ltd (Tokyo, Japan), and cultured in Keratinocyte-Serum Free Medium from Invitrogen Corp. (Carlsbad, CA), supplemented with bovine pituitary extract (BPE) (Kyokuto Seiyaku, Tokyo, Japan) and epidermal growth factor (EGF) (R&D Systems) (keratinocyte growth medium, KGM). The NHEKs were trypsinized into 6-well plates. When they reached subconfluency (70–80% confluent), the medium was changed to Keratinocyte-Serum Free Medium without BPE or EGF (keratinocyte basal medium; KBM). After incubation in nonsupplemented medium for 24 h, IFNg at a concentration of 0– 100 U/ml was simultaneously added with or without TNFa (30 ng/ml). Enzyme-linked immunosorbent assay (ELISA) The culture supernatants were harvested at 24–72 h after stimulation and subjected to ELISA by using 96-well plates coated with a murine monoclonal antibody against human CTACK/CCL27 (R&D Systems). The ELISA was performed according to the manufacturer’s directions. The optical density of each well was determined using a microplate reader (Model 550; Bio-Rad, Tokyo, Japan) set to 450 nm. In addition, ELISA for TGFa and amphiregulin was performed using the Quantikine1 JOURNAL OF CELLULAR PHYSIOLOGY

Human TNFa ELISA kit and the Amphiregulin Human ELISA kit (R&D Systems). RT-PCR and quantitative real-time (qRT)-PCR The total RNA samples were isolated using RNeasy Mini Kit (Quiagen, Valencia, CA). Reverse transcriptase-PCR was performed using a High-Capacity cDNA Reverse Transcription kit (Applied Biosystems, Norwalk, CT). The primers and probes for CTACK/CCL27, TGFa, amphiregulin, and glyceraldehyde-3phosphate dehydrogenase (GAPDH) were obtained from Applied Biosystems. The real-time PCR and RNA analysis were carried out in an ABI PRISM 7000 sequence detector (Applied Biosystems) by using a TaqMan RT-PCR Master Mix Reagents kit (Applied Biosystems), according to the manufacturer’s directions. Recombinant adenovirus Adenovirus vectors containing the genes for HA-tagged wild-type (WT) STAT1 and STAT3 (AxCAwtSTAT1; STAT1WT, and AxCAwtSTAT3; STAT3WT) and HA-tagged dominant-negative (dn) STAT1 and STAT3 (AxCAdnSTAT1; STAT1F, and AxCAdnSTAT3; STAT3F) (Nakajima et al., 1996), which contain a CAG promoter (chicken b-actin promoter with cytomegalovirus enhancer), were prepared by homologous recombination in 293 cells, as previously described (Hanakawa et al., 2000). DNA encoding wtSTAT1, 3 and dnSTAT1, 3 were kind gifts from Dr. Nakajima of Osaka University. NHEKs were infected with AxCAwtSTAT1, AxCAdnSTAT1, AxCAwtSTAT3, AxCAdnSTAT3, or AxCALacZ as a negative control, at a multiplicity of infection (MOI) of 10 in KGM. They were starved of EGF and BPE for 24 h, and incubated with or without TNFa (30 ng/ml) and IFNg (100 U/ml) for 48 h. Western blotting NHEKs were stimulated with 100 U/ml IFNg and disrupted in RIPA lysis buffer (Santa Cruz Biotechnology, Santa Cruz, CA). The concentrations of the extracted proteins were measured using a BCA Protein Assay kit (Pierce, Rockford, IL). The samples were boiled in sample buffer (50 mM Tris (pH 7.4), 0.14% SDS, 1% bmercaptoethanol (v/v)), and separated by 12.5% SDS–PAGE (20 mg ofproteinperlane).TheproteinsweretransferredtoanImmobilon-P transfer membrane (Millipore Corp. Merck, Billerica, MA) and incubated with the primary antibody overnight at 4°C. The signals were visualized using a chemiluminescence method (PhototopeHRP Western Blot Detection kit; GE Healthcare, Tokyo, Japan). Luciferase assays The pkB-luc, pGAS-luc, and pSTAT3-luc plasmids were purchased from Promega Corporation (Madison, WI), and the TK-Renilla-luc vector, from Clontech Laboratories (Takara Bio, Shiga, Japan). The transfection reagent FuGENE6 was purchased from Roche Applied Science (Mannheim, Germany). The plasmid transfection and assays were performed according to the manufacturers’ directions. NHEKs were cultured in 10-cm tissue culture dishes with 10 ml of KGM until 60–80% confluence. Five hours after the transfection procedure, the NHEKs were trypsinized into 6-well plates, and incubated in KGM overnight. The next day, medium was changed into KBM and incubated overnight. On the third day, the NHEKs were stimulated and incubated for 24 h. The firefly luminescence activity and the renilla luminescence activity were measured. The sample activity was calculated by dividing the firefly luminescence activity by the renilla luminescence activity.

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Immunohistochemistry Samples from seven cases of psoriasis embedded and frozen in OCT compound and surrounding normal skin of benign tumors from two cases as normal control were retrieved from the archives of the Department of Dermatology, Jichi Medical University. Immunohistochemical staining was performed using the avidin–biotin–horseradish peroxidase method (ABC standard; Vector Laboratories, Burlingame, CA) as previously described. Antibody for human CTACK/CCL27 at a concentration of 1 mg/ml, anti-HLADR antibody at a concentration of 1/100 were used as primary antibodies. Color was developed using diaminobenzidine as a substrate. Slides were examined and images were acquired using a microscope (BZ8000; KEYENCE, Osaka, Japan). Results TNFa-induced production of CTACK/CCL27 and inhibition by IFNg

To investigate the effect of TNFa and IFNg on CTACK/CCL27 production by keratinocytes, we stimulated normal human keratinocytes (NHEKs) with TNFa in the presence or absence of IFNg, and evaluated the supernatant CTACK/CCL27 concentration. We set the concentration of TNFa at 30 ng/ml, which is the minimal essential concentration for the complete induction of CTACK/CCL27, as seen in our previous experiments (Karakawa et al., 2010). At 24 h after stimulation, TNFa induced CTACK/CCL27 production. IFNg alone did not alter the production of CTACK/CCL27; however, when applied together, IFNg completely inhibited the production of CTACK/CCL27 induced by TNFa (Fig. 1a). The induction of CTACK/CCL27 by TNFa increased up to 72 h, and IFNg completely inhibited the induction at all the time points investigated (Fig. 1a). Next, IFNg concentrations ranging from 0 to 100 U/ml were applied to the cells, and Figure 1b shows that IFNg inhibited the production of CTACK/CCL27 induced by TNFa in a dosedependent manner. We evaluated this regulation at the mRNA level. Figure 1c demonstrates that TNFa induced CTACK/CCL27 mRNA expression, which was suppressed by IFNg. TNFa-induced production of CTACK/CCL27 was suppressed by inhibition of NFkB, p38MAPK, ERK, and JNK, and IFNg-mediated suppression of CTACK/CCL27 induced by TNFa was abolished by inhibition of ERK

NFkB is one of the transcription factors that play a major role in signaling pathways controlled by TNFa. In addition, ERK, p38, and JNK are mitogen-activated protein kinases (MAPKs) that are also involved in the signaling pathways stimulated by TNFa. We examined the involvement of those factors by utilizing the NFkB inhibitor parthenolide, the p38 inhibitor SB202190, the ERK inhibitor PD98059, and the JNK inhibitor 2. Parthenolide inhibited the production of CTACK/CCL27 induced by TNFa approximately by 45% in the NHEKs. Comparatively, SB202190 inhibited CTACK/CCL27 production by 70% (Fig. 2a). IFNg almost completely suppressed the production of CTACK/CCL27, irrespective of the presence of the inhibitors. Both PD98059 and the JNK inhibitor 2 inhibited the induction of CTACK/CCL27 by TNFa. Interestingly, PD98059 completely reversed the suppressive effect of IFNg on the production of CTACK/CCL27 induced by TNFa (Fig. 2b). We also tested ibuprofen, the COX2 inhibitor, because IL17 has been reported to suppress CTACK/CCL27 expression via COX2-dependent pathway (Kanda et al., 2005). The JOURNAL OF CELLULAR PHYSIOLOGY

Fig. 1. The induction of CTACK/CCL27 expression by TNFa and its inhibition by IFNg. a: The NHEKs were stimulated by TNFa (30 ng/ml) and IFNg (100 U/ml). The supernatants were harvested at the indicated time points and subjected to ELISA. b: The NHEKs were stimulated by 30 ng/ml of TNFa and the indicated concentrations of IFNg. The supernatants were harvested after 48 h and subjected to ELISA. c: The NHEKs were stimulated by TNFa and IFNg. The cells were harvested at the indicated time points, and the RNA was extracted. They were reverse transcribed into cDNAs that were subjected to real-time PCR analysis. Results are expressed as mean  SD of three independent experiments.

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blot analysis demonstrated that STAT1F overexpressed in the NHEKs inhibited the phosphorylation of STAT1 induced by IFNg (Fig. 3b, upper part). We investigated whether STAT1 phosphorylation is involved in the suppression of CTACK/CCL27 induction by IFNg. To this end, we introduced STAT1WT and STAT1F into the NHEKs that were stimulated with TNFa and IFNg, and evaluated the supernatant concentration of CTACK/CCL27. IFNg inhibited the production of CTACK/CCL27 induced by TNFa by almost 90% in the mock-transfected control (data not shown) and STAT1WT-transfected cells. Comparatively, it inhibited production of CTACK/CCL27 only by 60% in the STAT1F-transfected cells (Fig. 3b, lower part). The STAT3 wild-type expression vector inhibited TNFainduced CTACK/CCL27 production

STAT3 is a member of the STAT family that is widely activated in a variety of cellular contexts. In our system, either TNFa or IFNg induced STAT3-luciferase activity, indicating that both TNFa and IFNg alone can activate STAT3 (Fig. 3c). STAT3F is a dominant-negative mutant of STAT3 that has the tyrosine residue at 705 replaced by a phenylalanine residue. Following overexpression in the NHEKs, STAT3F attenuated the phosphorylation of STAT3 (Fig. 3d, upper part). Subsequent to introducing STAT3WT and STAT3F into NHEKs, we stimulated them with TNFa and/or IFNg. TNFa-induced production of CTACK/CCL27 was completely inhibited by introduction of STAT3WT (Fig. 3d, lower part). Oncostatin M (OSM), an activator of STAT3, suppressed CTACK/CCL27 expression in NHEKs

Fig. 2. TNFa-induced production of CTACK/CCL27 was partially inhibited by the NFkB inhibitor (parthenolide), the p38 inhibitor (SB202190), and the ERK inhibitor (PD98059). a: The NHEKs were pretreated with signal inhibitors parthenolide (5 mM) and SB202190 (5 mM), and stimulated by TNFa (30 ng/ml) and IFNg (100 U/ml). The supernatants were harvested after 48 h of incubation and subjected to ELISA. b: The NHEKs were pretreated with the ERK inhibitor PD98059 (50 mM) or JNK inhibitor 2 (50 nM), and stimulated by TNFa (30 ng/ml) and IFNg (100 U/ml). The supernatants were harvested after 48 h and subjected to ELISA. Results are expressed as mean  SD of three independent experiments.

suppressive effect of IFNg on the induction of CTACK/CCL27 by TNFa was not affected by the addition of ibuprofen (data not shown), suggesting that the suppressive effect of IFNg on CTACK/CCL27 production was not dependent on COX2. IFNg did not inhibit the activation of NFkB by TNFa

To determine whether IFNg inhibits TNFa-induced CTACK/ CCL27 production by reducing NFkB activity, we examined the effect of IFNg on TNFa-induced NFkB activity. Our results demonstrated that the induction of NFkB activity by TNFa was not reduced by the addition of IFNg (Fig. 3a). The STAT1 dominant-negative mutant partially reversed the inhibitory effect of IFNg on the TNFa-induced CTACK/CCL27 production

STAT 1F is a dominant-negative mutant of STAT1 that has the tyrosine residue at 701 replaced by a phenylalanine. Western JOURNAL OF CELLULAR PHYSIOLOGY

IL-17 has been reported to suppress CTACK/CCL27 expression (Kanda et al., 2005), and activate STAT3 in keratinocytes (Shi et al., 2011). These facts, together with our current data, suggest that activated STAT3 interferes with the expression of CTACK/CCL27. We checked whether OSM, another STAT3-activating cytokine, suppresses CTACK/ CCL27 expression in a manner similar to that by IFNg and IL17. NHEKs were separately stimulated by TNFa with and without OSM, the culture supernatant collected, and were analyzed using ELISA. As shown in Figure 3e, OSM suppressed the TNFa-induced CTACK/CCL27 expression. Moreover, OSM did not show enhanced suppression of CTACK/CCL27 when used together with IFNg. JAK inhibitor 1 and JAK3 inhibitor inhibited the suppression of TNFa-induced CTACK/CCL27 production by IFNg

The JAK-STAT pathway is a major pathway in IFNg signaling. The JAK inhibitor 1 inhibits JAK1, JAK2, and Tyk2 at low doses, and also inhibits JAK3 at high doses. The JAK3 inhibitor is a specific inhibitor for JAK3. The JAK inhibitor 1 (Fig. 4a) and JAK3 inhibitor (Fig. 4b) inhibited the suppressive effect of IFNg on TNFa-induced CTACK/CCL27 production. These results indicate that JAKs, including JAK3, are required for the suppression of CTACK/CCL27 by IFNg.

Inhibitors of EGF receptor tyrosine phosphorylation inhibited the suppression of CTACK/CCL27 by IFNg

EGF receptor (EGFR) is constitutively activated in NHEKs cultured in growth medium supplemented with EGF, and also in basal medium in which ligands of EGFR, such as TGFa and

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Fig. 3. The role of STAT1 and STAT3 in IFNg- and TNFa-regulated CTACK/CCL27 production. The subconfluent NHEKs were transfected with the pkB-luc construct (a), or STAT3-luc (c) and the TK-Renilla-luc construct. The cells were trypsinized at 5 h after transfection, incubated overnight, stimulated with TNFa and IFNg, and subjected to the dual luciferase assay. The AxCAwtSTAT1; STAT1WT or AxCAdnSTAT1; STAT1F-containing adenovirus vectors (b) or the AxCAwtSTAT3; STAT3WT or AxCAdnSTAT3; STAT3F-containing adenovirus vectors (d) were introduced into the NHEKs and incubated for 5 h. The cells were trypsinized into 6-well plates, incubated overnight, stimulated with IFNg for the indicated time period, harvested, and subjected to Western blot analysis (upper parts (b), (d)). The transfected NHEKs were stimulated and incubated for 48 h. The supernatants were harvested and subjected to ELISA (lower parts). Results are expressed as mean  SD of three independent experiments. (e) Oncostatin M (OSM) and IFNg were added separately to the NHEKs stimulated with TNFa, the cells were centrifuged, and the supernatant was analyzed using ELISA. The results are expressed as mean  SD of three independent experiments.

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HB-EGF, are produced from the growing NHEKs. The inhibitors of EGFR tyrosine phosphorylation, PD153035 and PD168393, were added 30 min before the stimulation with TNFa, cultured for 48 h, and the culture supernatant was subjected to ELISA. Inhibition of EGFR tyrosine phosphorylation by these inhibitors reduced the suppressive effect of IFNg on the production of CTACK/CCL27. This result suggests that IFNg suppressed CTACK/CCL27 production through EGFR phosphorylation (Fig. 4c). ERK and EGFR were phosphorylated following stimulation with TNFa and IFNg

Fig. 4. Inhibitors of tyrosine kinase of JAK and EGFR inhibited the suppressive effect of IFNg on CTACK/CCL27 production induced by TNFa. The NHEKs were incubated with JAK inhibitor 1 (a) or JAK3 inhibitor (b) 30 min prior to stimulation with TNFa (30 ng/ml) or IFNg (100 U/ml). The cells were incubated for 48 h, and the supernatants were harvested and subjected to ELISA. The NHEKs were treated with inhibitors for EGFR phosphorylation, PD153035 (5 mM) or PD168393 (5 mM), 30 min prior to stimulation with TNFa and/or IFNg, incubated for 48 h, and the supernatants were harvested and subjected to ELISA (c). Results are expressed as mean  SD of three independent experiments.

We next investigated if ERK is phosphorylated following stimulation by IFNg. We focused on ERK phosphorylation at the later time points to assess whether it is activated following EGFR activation. The results demonstrated that IFNg induced phosphorylation of ERK in a time-dependent manner. Especially, IFNg induced weak phosphorylation of ERK that began 4 h after stimulation and subsided after 48 h (Fig. 5a). We next investigated whether EGFR was phosphorylated following stimulation by IFNg. In the first 30 min, IFNg alone did not induce EGFR phosphorylation, whereas at 30 and 120 min, weak phosphorylated EGFR bands were observed (Fig. 5b). The longer time points demonstrated that IFNg alone induced phosphorylation of EGFR at 2 and 4 h (Fig. 5c). These results suggest that EGFR phosphorylation is indirectly induced by IFNg, perhaps through de novo protein synthesis. We further confirmed this by adding cycloheximide (CHX), the protein synthesis inhibitor, in the culture. Addition of CHX suppressed the phosphorylation of EGFR by IFNg at late time points (Fig. 5d). These results indicate that IFNg induces ERK phosphorylation in a time-dependent manner, which possibly follows the IFNg-induced activation of EGFR. Therefore, we examined whether EGFR is located upstream of the ERK phosphorylation signal. EGFR inhibitor PD168393, when added to the medium right before the stimulation with IFNg, suppressed the phosphorylation of ERK during the late time points (4–48 h), indicating that the later time-point phosphorylation of ERK occurred through EGFR phosphorylation (Fig. 5e). Our results demonstrating that the EGFR inhibitor reduced the suppressive effect of IFNg motivated us to assess whether EGFR ligands are produced from keratinocytes stimulated by IFNg. In the culture supernatant from keratinocytes stimulated by IFNg, EGF was not detected, but substantial concentrations of the known EGFR ligands, TGFa and amphiregulin, were identified (Fig. 5f, upper part), while at the mRNA level, TGFa was induced, but amphiregulin was not (Fig. 5f, lower part). We tested whether EGF ligands could suppress TNFa-induced CTACK/CCL27 production. We added each of EGF, HB-EGF or amphiregulin in the culture together with TNFa, collected the culture supernatant 48 h after stimulation and performed ELISA. On the contrary to our anticipation, none of these EGFR ligands suppressed the CTACK/CCL27 levels induced by TNFa (data not shown). We speculated that a combined stimulation by EGFR ligands and other signaling molecules might be involved in suppressing the CTACK/CCL27 production. Inhibition of ERK phosphorylation augmented phosphorylation of p38, while it suppressed phosphorylation of JNK induced by TNFa and/or IFNg

ERK is involved in the TNFa-induced expression of CTACK/CCL27, but it assists IFNg in inhibiting CTACK/ CCL27 expression. Such an intricate involvement of ERK in JOURNAL OF CELLULAR PHYSIOLOGY

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Fig. 5. TNFa and IFNg induced phosphorylation of ERK and EGFR. IFNg induced ERK phosphorylation during the late time points via EGFR phosphorylation, which required protein synthesis. TNFa and IFNg caused the release of EGFR ligands in the supernatant. The inhibitor of ERK phosphorylation suppressed JNK phosphorylation, but prolonged p38 phosphorylation. a: After incubation for the indicated time period, the cells were harvested, and cell lysates were subjected to Western blotting with anti-phospho ERK, anti-ERK2, and anti-a tubulin antibodies. The experiment was repeated three times, and representative blots are shown. b: The NHEKs were harvested at the indicated time points. The cell lysates were subjected to Western blotting with anti-tyrosine phosphorylated EGFR, anti-EGFR, and anti-a tubulin antibodies. c: The cells were harvested at the indicated time points, and the cell lysates were subjected to Western blot analysis. d: Cycloheximide was added to the medium when the cells were stimulated with IFNg, the cells were harvested at indicated time points, and cell lysates were subjected to Western blot with anti-tyrosine phosphorylated EGFR, anti-EGFR, and anti-GAPDH antibodies. The experiment was repeated three times, and representative blots are shown. e: NHEKs were incubated with IFNg for the indicated hours with or without PD168393, the inhibitor of EGFR phosphorylation. The cells were harvested and analyzed with Western blot using anti-phospho ERK, anti-ERK, and anti-GAPDH antibodies. f: NHEKs were separately incubated with TNFa and IFNg, and the supernatant and cells were harvested at the indicated time points, and were analyzed using ELISA and realtime PCR, respectively, to detect the levels of TGFa and amphiregulin. g: NHEKs were separately stimulated with IFNg and TNFa with or without PD98059, were harvested, and were analyzes using Western blot with anti-phospho-p38, anti-phospho-JNK, anti-p38, and anti-JNK antibodies.

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CTACK/CCL27 expression led us to investigate the interaction of ERK with other MAPK members. When the phosphorylation of ERK was inhibited by PD98059, a prolonged phosphorylation of p38 was observed following stimulation by TNFa and/or IFNg, while the phosphorylation of JNK was suppressed (Fig. 5g). Following stimulation by TNFa alone, phosphorylation of p38 was observed at 5, 15, 60 min, which disappeared between 120 and 360 min. A combination of TNFa and IFNg induced p38 phosphorylation between 5 and 15 min, disappearing at 60, 120, and 360 min. When PD98059 was added, phosphorylation of p38 continued for 360 min for both the above conditions (Fig. 5g). The late-phase phosphorylation of ERK induced by IFNg may cause a late-phase suppression of CTACK/CCL27 through the inhibition of p38 activity. Immunohistochemical study of expression of CTACK and HLA-DR in psoriatic lesions and peri-lesional epidermis

In order to explore the clinical meaning of regulation of CTACK expression, we performed immunohistochemical

study using lesional and peri-lesional skin from patients with psoriasis. In our previous study, we have demonstrated the early inflammatory changes in the peri-lesional epidermis of psoriatic lesions (Komine et al., 2007). We, and several previously published reports, assume that peri-lesional skin shows early inflammatory changes in the formation of psoriatic lesion, while the lesional skin shows the later stage (Van de Kerkhof et al., 1991; Komine et al., 2007). Epidermal keratinocytes express HLA-DR in response to IFNg (Albanesi et al., 1998), which makes it the marker for IFNg influence in the epidermis. CTACK/CCL27 was stained diffusely in the whole epidermis in peri-lesional skin, while it was stained mainly in the basal layer in the lesional epidermis similar to normal healthy skin (Fig. 6a–c). HLA-DR was stained on epidermal keratinocytes in intercellular pattern as well as in infiltrating cells in lesional skin, while it was stained mainly in epidermal Langerhans cells in the peri-lesional epidermis without staining on epidermal keratinocytes (Fig. 6d–g), suggesting that keratinocytes in the lesional epidermis are influenced by IFNg, while keratinocytes in the peri-lesional epidermis are not.

Fig. 6. In vivo expression of CTACK/CCL27 in lesional and peri-lesional epidermis of psoriasis. Skin samples from seven patients of plaque type psoriasis vulgaris were stained with anti-CTACK/CCL27 antibody (a,b), or with anti-HLA-DR antibody (d,e,g,h). Skin samples from normal control individuals were stained with anti-CTACK/CCL27 antibody (c), and anti-HLA-DR antibody (f,i). The representative images were shown in the figure. a: Basal layer of epidermis was stained with anti-CTACK/CCL27 antibody in lesional skin, while (b) the whole epidermis was stained in peri-lesional skin of psoriasis patients by anti-CTACK/CCL27 antibody. c: Normal healthy control samples showed staining of CTACK/CCL27 in the basal layer of the epidermis. d: HLA-DR was stained in the epidermal keratinocytes in lesional skin as well as dendritic cells lymphoid cells and endothelial cells, while (e,f) only Langerhans cells were stained in the peri-lesional and normal epidermis. Dermal infiltrating cells were also positive for HLA-DR both in lesional and peri-lesional skin. g: High magnification of the epidermis of d) shows cell surface staining of HLA-DR on epidermal keratinocytes. h,i: High magnification of (e) and (f) shows HLA-DR staining in the epidermal dendritic cells in epidermis of peri-lesion and normal healthy controls.

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Discussion

We clearly demonstrated in this study that the expression of CTACK/CCL27 in the lesional epidermis of psoriasis patients is restricted to basal layer of epidermal keratinocytes, while it was diffusely distributed through the whole epidermis in perilesional skin, and that the production of CTACK/CCL27 induced by TNFa is suppressed by the addition of IFNg at both the protein and mRNA level. The similar suppressive effect of IFNg on inflammatory molecules has been reported only in endothelial cells, in which attachment of Th1 cells to endothelial cells is suppressed by IFNg (Norman et al., 2008). IL-1b has been reported to induce CTACK/CCL27 expression (Riis et al., 2011b), and IL-17, one of the most pathognomonic cytokines in psoriasis, has been reported to suppress CTACK/ CCL27 expression in NHEKs (Kanda et al., 2005). We assume that CTACK/CCL27 is induced by early inflammatory cytokines, such as TNFa and IL-1b, in the peri-lesional epidermis (i.e., the early stage of plaque formation) and then it is suppressed by pathognomonic cytokines, such as IL-17 and IFNg in the central lesion of the plaque (i.e., the late stage of plaque formation). The induction of CTACK/CCL27 was dependent on ERK, JNK, p38, and NFkB, which is compatible with the previous reports (Vestergaard et al., 2005; Kagami et al., 2006; Riis et al., 2011b). The suppressive effect of IFNg on the production of CTACK/CCL27 and the mechanism of

its action have been investigated for the first time in this study. IFNg signals via STAT1 (O‘Shea, 1997) and phosphorylates STAT3 in certain cell types (Caldenhoven et al., 1999; Kaur et al., 2003). We demonstrated that IFNg phosphorylated STAT3 as well as STAT1, and that activation of STAT3 suppressed TNFa-induced CTACK/CCL27 production. In addition, we revealed that STAT1 was partially involved in the suppressive effect of IFNg on CTACK/CCL27 production. The precise mechanism underlying CTACK/CCL27 suppression via STAT3 is unknown. Although STATs are transcriptional activators, they also appear to act as transcriptional repressors (Zhou and Waxman, 1999; Chew et al., 2014). Previous reports indicated that STAT3 acted as a transcriptional repressor to PPARa. We did not examine the mechanism underlying STAT3-induced inhibition of CTACK/CCL27 transcription, but we can speculate that it may take place directly or through the involvement of other transcription factors such as PPARa. JAKs are key molecules in the signaling pathway of IFNg and are recruited to the IFN receptor, and phosphorylate STATs (Hu and Ivashkiv, 2009). Our study demonstrated for the first time that JAKs are involved in the suppressive effect of IFNg on CTACK/CCL27 production, probably by activating STAT1 and STAT3. EGFR is deeply involved in chemokine production. Indeed, production of MCP1/CCL2, RANTES/CCL5, and IP-10/

Fig. 7. Proposed schematic representation of the regulation of CTACK/CCL27 production by NHEKs. This figure shows our hypothetic scheme on the regulation of CTACK/CCL27 expression by TNFa and IFNg; TNFa induces CTACK/CCL27 via ERK, p38, JNK, and NFkB pathway. TNFa also activates STAT3, which has suppressive effect on CTACK/CCL27 expression. IFNg suppresses TNFa-induced CTACK/ CCL27 expression through JAK/STAT1, probably via EGFR and subsequent ERK activation through de novo protein synthesis, as well as through JAK/STAT3 pathway.

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CXCL10 induced by TNFa is suppressed by EGFR signals (Mascia et al., 2003). TARC/CCL17 induced by TNFa and IFNg was upregulated by the addition of the EGFR tyrosine kinase inhibitor (Komine et al., 2005). Production of CTACK/CCL27 stimulated by TNFa and IL-1b was enhanced by the EGFR inhibitors, and that introduction of constitutively active Ras, the downstream molecule of EGFR, suppressed the production of CTACK/CCL27 (Pivarcsi et al., 2007). Our study revealed that the EGFR tyrosine kinase inhibitors and the inhibitor of ERK phosphorylation, the downstream molecule of EGFR signaling, inhibited the suppressive effect of IFNg on CTACK/ CCL27 production, which newly demonstrated that suppressive effect of IFNg was dependent on activation of EGFR and ERK. IFNg has been reported to activate EGFR through the production of TGFa (Matsuura et al., 1999), and EGF, HB-EGF, TGFa, and amphiregulin are natural ligands of EGFR (Chan et al., 2004). We speculated that these EGFR ligands suppressed the production of CTACK/CCL27 via an autocrine or paracrine pathway in keratinocytes, however, each of these EGFR ligands did not suppress the CTACK/CCL27 production induced by TNFa. We do not know the mechanism of EGFR activation by IFNg, but we speculated that other signaling pathways activated by IFNg, such as STAT1, may be needed in the suppressive effect through EGFR activation. TNFa can transactivate EGFR (Izumi et al., 1994; Hirota et al., 2001; Sakamoto et al., 2005). Several stimuli, including those by phorbol esters and cytokines such as TNFa, have been reported to activate EGFR by inducing the shedding of EGFR ligands by tumor necrosis factor alpha converting enzyme (TACE) or by disintegrin and metalloprotease (ADAM) 17 (Argast et al., 2004). TACE was first recognized as an enzyme that cleaves soluble TNFa from its membranebound form, and it is now known to release EGFR ligands from their membrane-bound forms. We assumed that TNFa activated EGFR through a similar mechanism in our experiment. STAT3 is known to function downstream of EGFR in many cell types, and it has been reported that EGFR signals induce STAT3 activation in keratinocytes (Sano et al., 2008). Our results indicated that TNFa not only induced CTACK/ CCL27 production through the NFkB and p38 pathways, but also has a suppressive effect on the production of CTACK/ CCL27 through activation of STAT3 and ERK via EGFR activation. The effect of ERK on the production of CTACK/ CCL27 is complex, mainly because ERK is involved in the induction of CTACK/CCL27 expression by TNFa, and also in the suppression of CTACK/CCL27 by IFNg. We do not know the precise underlying mechanism of action, but our results showed that IFNg resulted in late-phase activation of ERK, that ERK inhibition enhanced p38 phosphorylation, and that p38 was positively involved in CTACK/CCL27 expression, which may explain the late-phase suppression of CTACK/CCL27 through the IFNg-ERK-p38 pathway. The crosstalk between MAP kinases has been reported (Gazel et al., 2008), but the ERK inhibition resulted in prolonged activation of p38 is a novel finding. We assumed that ERK and p38 may induce set of transcription factors, such as Elk-1 and SAP-1, or c-Fos and nMyc, respectively, which could work directly on the promoter region of CTACK/CCL27, or through other transcription factors or co-factors as previously described (Lo et al., 2006; Gazel et al., 2008; Riis et al., 2011b; Benbernou et al., 2013). Figure 7 demonstrates a schematic view of the regulatory mechanisms of CTACK/CCL27 production revealed by our study. We suggest the biological connotations of our findings are that CTACK/CCL27 is induced by TNFa in the early stage of inflammation. The major sources of TNFa in psoriasis have been suggested to be dendritic cells (DCs), such as TNF- and iNOS-producing DCs (TipDCs), or other inflammatory DCs, during the initiation phase of psoriasis pathogenesis (Lowes JOURNAL OF CELLULAR PHYSIOLOGY

et al., 2005). In the peri-lesional skin of psoriasis—which is considered to be the early stage of psoriatic lesion formation— there are many activated DCs along the epidermal-dermal junction, but the infiltration of lymphocytes is low (Komine et al., 2007). This early inflammation plays an important role in the initiation of skin inflammation and induction of CTACK/ CCL27. CTACK/CCL27 induced in the epidermis in the early stage of psoriatic plaque formation may attract cells which express CCR10, such as Th22, into the epidermis, which in turn play essential part in establishing psoriatic plaques in the subsequent stage by producing IL-22 (Duhen et al., 2009; Eyerich et al., 2009; Fujita, 2013). CTACK/CCL27 may be suppressed at certain stages of inflammation when copious amounts of IFNg and IL-17 are produced from infiltrating T cells, such as Th1 and Th17 cells, in the milieu. Th17 cells, which produce IL-17 and IFNg, have been reported to be increased in the psoriatic lesions, and they are believed to be mainly involved in the psoriatic lesion formation during the late stage (Nestle et al., 2009). Subsequent to this suppression, disease-specific changes such as neutrophil infiltration in psoriasis may be induced by disease-specific cytokines and chemokines. The expression of IFNg by infiltrating T lymphocytes is enhanced in inflammatory skin diseases such as atopic dermatitis and psoriasis, especially in the chronic stages (Horikawa et al., 2002). In the chronic stages of many inflammatory skin diseases, thickening of the epidermis is a common finding, which may imply an abundance of EGFR ligands. Activation of EGFR during inflammation could serve as a switch that signals the end of the general initiation stage and the beginning of the disease-specific chronic stage. Activation of EGFR has been reported to suppress production of TARC/CCL17 (Komine et al., 2005), one of general initiators of skin inflammation, MCP-1/CCL2, RANTES/ CCL5, and IP-10/CXCL10, and induce the production of IL-8/ CXCL8 and GM-CSF, the neutrophil attractant and growth factor (Mascia et al., 2003; Pastore et al., 2005), which induce secondary neutrophil infiltration. These previously reported facts strongly support our hypothesis. Acknowledgments

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