The Role of IFN-Γ in Regulating Nfkbiz Expression in Epidermal Keratinocytes

Biomedical Research (Tokyo) 36 (2) 103-107, 2015 The role of IFN-γ in regulating Nfkbiz expression in epidermal keratinocytes 1 2 3 4 Toshina ISHIGURO-OONUMA , Kazuhiko OCHIAI , Kazuyoshi HASHIZUME , and Masami MORIMATSU 1 Department of Biological Resources, Integrated Center for Sciences, Ehime University, Ehime 791-0295, Japan; 2 Department of Veter- inary Nursing and Technology, School of Veterinary Science, Nippon Veterinary and Life Science University, Tokyo 180-8602, Japan; 3 Laboratory of Veterinary Physiology, Co-Department of Veterinary Medicine, Faculty of Agriculture, Iwate University, Iwate 020-8550, Japan; and 4 Laboratory of Laboratory Animal Science and Medicine, Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan (Received 25 November 2014; and accepted 25 December 2014) ABSTRACT Nfkbiz is an inhibitor of nuclear factor κB (IκB) protein localized to the nucleus. We previously found that Nfkbiz gene-disrupted mice showed atopic dermatitis-like lesion, implying the important role of Nfkbiz in skin homeostasis. The purpose of this study was to examine the effect of interferon (IFN)-γ on Nfkbiz expression in keratinocytes. IFN-γ induced Nfkbiz expression at a comparable level to IL-1. Promoter analysis revealed that interferon-stimulated response element (ISRE) located in the Nfkbiz promoter region is important for responding to the stimulation. Inter- estingly, IFN-γ and IL-1 displayed synergism in terms of inducing Nfkbiz expression. By using selective inhibitors, we found that Janus activated kinase (JAK) 1 and nuclear factor (NF)-κB are important for Nfkbiz expression after IFN-γ stimulation and for synergism between IFN-γ and IL- 1. These findings indicate a possible important role of Nfkbiz in modulating the progression of inflammatory diseases in which IFN-γ and IL-1 are abundant. Nfkbiz is an inhibitor of nuclear factor κB (IκB) Interferon (IFN)-γ is a well-characterized, potent protein localized to the nucleus that is also termed inflammatory stimulus in a variety of biological sys- ‘molecule possessing ankyrin-repeats induced by li- tems (2). It exerts its cellular effects by interacting popolysaccharide (MAIL)’, ‘interleukin-1-inducible with its cell surface receptor, IFN-γR, which is com- nuclear ankyrin repeat protein (INAP)’, or IκBζ (5, posed of two subunits. The binding of IFN-γ to its 8, 20). Nfkbiz acts as a transcriptional regulator of receptor induces receptor oligomerization and acti- various genes on inflammatory stimuli such as LPS vation of the receptor-associated kinases called Ja- and IL-1 (19). We previously found that Nfkbiz nus activated kinase (JAK) 1 and JAK2 by trans- gene-disrupted mice showed atopic dermatitis-like phosphorylation (15). Activated JAKs phosphorylate lesion (16) and that Nfkbiz was constitutively ex- tyrosine residues within the cytoplasmic domains of pressed in epidermal keratinocytes (13). This consti- the receptor subunits, which act as docking sites for tutive expression was shown to be mediated by the STAT1 (2). Phosphorylation of STAT1 mediates di- activity of nuclear factor (NF)-κB (13). merization, which enables STAT1 dimers to translo- cate to the nucleus and bind to IFN-γ-activated sequence (GAS) or interferon-stimulated response Address correspondence to: Masami Morimatsu, DVM, element (ISRE) located within the promoters of Ph.D. IFN-γ-inducible genes (18). Keratinocytes express Laboratory of Laboratory Animal Science and Medi- cine, Department of Disease Control, Graduate School IFN-γ receptor and IFN-γ induces cytokine and che- of Veterinary Medicine, Hokkaido University, Sapporo mokine synthesis in these cells (4, 10). IFN-γ is also 060-0818, Japan known to promote exaggerated cytokine production Tel: +81-11-706-5106, Fax: +81-11-706-5106 in keratinocytes cultured from patients with atopic E-mail: [email protected] dermatitis, implying its important role in skin disor- 104 T. Ishiguro-Oonuma et al. ders (14). Recently, it was reported that several analyzed for the expression of Nfkbiz using an ABI ISREs are located at the Nfkbiz promoter region, al- PRISM 7700 with previously described primers and though there is no evidence that IFN-γ is involved probes (13). The amount of target gene expression in Nfkbiz expression (17). was calculated from the standard curves and was In the present study, we examined the effect of normalized using the expression of 18S ribosomal IFN-γ on Nfkbiz expression in epidermal keratino- RNA as an internal control. cytes and showed that IFN-γ is a strong inducer of Nfkbiz in keratinocytes. Luciferase assay. PAM212 cells were plated at 50% confluence in 12-well plates. After overnight cultiva- tion, the cells were transfected with plasmids (0.4 mg/ MATERIALS AND METHODS well) using FuGENE transfection reagent (Roche Animals. Wild-type pregnant C57BL/6 mice were Diagnostics, Indianapolis, IN, USA). The pRL-tk purchased from SLC Japan Inc. (Hamamatsu, Japan). vector was used as an internal control (Promega). The experimental procedure and care of animals Two days after transfection, IL-1 (20 ng/mL) and were in accordance with the National University IFN-γ (20 ng/mL) were added. After an additional Corporation Hokkaido University Regulations on one hour of incubation, the cells were washed once Animal Experimentation. in PBS and lysed in Passive Lysis Buffer (Promega). Luciferase activity was measured using the Dual- Cell culture. Primary keratinocytes were isolated Luciferase reporter assay system (Promega) as pre- from neonatal mice as previously reported (13). In viously described (6). brief, neonatal mice were sacrificed within 3 days of birth, and the skin was excised and sterilized using RESULTS 70% ethanol. After overnight dispase treatment to separate the epidermis and dermis, the epidermis was Expression analysis of Nfkbiz after stimulation with trypsinized for 10 min at 37°C to disaggregate the IFN-γ in primary keratinocytes and keratinocyte cell keratinocytes. The trypsin was neutralized with PBS line PAM212 containing 5% chelex-treated fetal calf serum (FCS). To determine the effect of IFN-γ on Nfkbiz expres- Then, cells were washed with serum-free PBS twice sion in keratinocytes, we carried out qRT-PCR. As a and suspended in serum-free keratinocyte growth positive control, we used IL-1 because this cytokine medium (KGM2 Bullet kit; Cambrex, Walkersville, is known to induce Nfkbiz expression strongly in MD, USA) containing 20 μM CaCl2, 1 × human ke- keratinocytes and macrophages. In primary keratino- ratinocyte growth supplement, 100 U/mL penicillin, cytes, IFN-γ increased Nfkbiz expression by 7.7-fold and 100 μg/mL streptomycin. The mouse keratino- (Fig. 1a). The magnitude of Nfkbiz expression by cyte cell line PAM212 was a kind gift from Dr. IFN-γ was comparable to that by IL-1 (Fig. 1a). Hajime Nakano (Hirosaki University). The cells were IFN-γ also increased Nfkbiz expression by 1.8-fold in maintained at 37°C in 5% CO2 in Eagle’s minimum the keratinocyte cell line PAM212 (Fig. 1b). Interest- essential medium supplemented with 10% heat-inac- ingly, co-stimulation with IFN-γ plus IL-1 synergis- tivated FCS, 100 U/mL penicillin, and 100 μg/mL tically enhanced Nfkbiz expression in both primary streptomycin. keratinocytes and PAM212 (Fig. 1). Reagents. IL-1β was obtained from Wako (Osaka, Promoter analysis of Nfkbiz Japan). Bay11-7082 was obtained from Biomol Re- To determine whether ISREs in the Nfkbiz promoter search Laboratories (Plymouth Meeting, PA, USA). are important for Nfkbiz expression, we performed AG490 and pan-JAK inhibitor were obtained from truncation analysis of this promoter. We used two Calbiochem (San Diego, CA, USA). fragments. One was a −723 to +18-bp Nfkbiz promoter fragment that contains three ISREs and the Quantitative reverse transcription-polymerase chain other was a −357 to +18-bp Nfkbiz promoter frag- reaction (qRT-PCR). Total RNA was prepared from ment. We transfected these constructs into PAM212 cells using TRIzol reagent (Invitrogen Corp., Carls- cells and measured luciferase activity in the presence bad, CA, USA). After RNA extraction, reverse tran- or absence of IFN-γ plus IL-1. When the ISRE con- scription reaction was carried out using SuperScript taining region (−723 to −358) was deleted, promoter II (Invitrogen) according to the manufacturer’s in- responsiveness significantly decreased (Fig. 2). The structions. Complementary DNA was quantitatively promoter activities of unstimulated controls were IFN-γ induces Nfkbiz expression 105 also decreased by the deletion. combined stimulation, pan-JAK inhibitor and Bay11- 7082 showed significant effects on Nfkbiz expres- Effects of selective inhibitors on Nfkbiz expression sion in PAM212 cells treated with IL-1 alone or in keratinocytes IFN-γ alone (Fig. 3c). We used selective inhibitors to investigate which signaling pathway is involved in Nfkbiz expression DISCUSSION after stimulation with IFN-γ plus IL-1 in keratinocytes. We used three compounds; AG490 as a JAK2 Nfkbiz was originally identified in macrophages (8). inhibitor, pan-JAK inhibitor as a broad inhibitor of Interestingly, Nfkbiz gene-disrupted mice showed JAK 1 to 3, and Bay11-7082 as an inhibitor of NF- atopic dermatitis-like lesion, indicating that Nfkbiz κB. In primary keratinocytes, pan-JAK inhibitor and has an important role in the skin (16). However, the Bay11-7082 significantly reduced Nfkbiz expression, role of Nfkbiz in keratinocytes is largely unknown. although AG490 did not show any effect after stim- We previously reported that Nfkbiz is constitutively ulation (Fig. 3a). Similar effects were observed when expressed in keratinocytes and its expression is me- PAM212 cells were analyzed (Fig. 3b). Next, to an- diated by NF-κB (13). In this study we examined alyze the signaling mechanism in detail, we stimu- the role of IFN-γ in regulating Nfkbiz expression in lated PAM212 cells with a single stimulus, IL-1 or keratinocytes. IFN-γ induced Nfkbiz expression at a IFN-γ, after treatment with inhibitors. Similar to comparable level to IL-1, a strong inducer of Nfkbiz in keratinocytes, indicating that IFN-γ plays an important role in regulating Nfkbiz expression in keratinocytes.

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