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. Thus, we identified Nfkbiz as one of the interferon-stimulated genes (ISGs). Our finding sug- gests that there may be a positive-feedback mechanism between IFN-γ and Nfkbiz because it is known that Nfkbiz enhances IFN-γ expression in response to certain kinds of inflammatory cytokine (7). In the following experiments, we examined the role of ISREs of the Nfkbiz promoter in Nfkbiz expression in keratinocytes. In general, ISRE exists in ISGs with the consensus sequence of AGTTCNNTTCC (where N is any nucleotide) (18). The IFN-γ/IL1- stimulated activation of Nfkbiz promoter was significantly lowered by the deletion of ISREs, indicating that ISREs in the Nfkbiz promoter are functional and important for responding to stimulation. IFN-γ synergistically induced Nfkbiz expression in concurrent stimulation with IL-1. It is known that IFN-γ plus IL-1 synergistically induce inflammatory cytokine expression in certain types of cell (3, 21). Both NF-κB binding sites and ISREs in the promoter region of target genes are important for this synergism (3). Although the biological significance of this synergism is still to be determined, it is possible that stimulus-induced Nfkbiz suppresses the progression of inflammatory conditions such as atopic dermatitis in a different way from constitutively expressed Nfkbiz. The JAK/STAT pathway plays a central role downstream of IFN signaling (1). JAK associates with the Fig. 1 Nfkbiz expression after IFN-γ stimulation. Nfkbiz IFN receptor, which transmits the signal to STAT mRNA of primary keratinocytes (a) and keratinocyte cell line family molecules. There are four kinds of JAK fam- PAM212 (b) was measured after stimulation with IFN-γ and/ or IL-1 for one hour using qRT-PCR. The values are shown ily molecule, JAK1, JAK2, JAK3, and Tyk2 (1). as fold change relative to the control. Among them, JAK1 and JAK2 are important for the 106 T. Ishiguro-Oonuma et al.

IFN-γ signaling pathway (18). We used two kinds of Bay11-7082 as an NF-κB inhibitor to examine the inhibitor of JAKs. One was a specific JAK2 inhibi- role of NF-κB in Nfkbiz expression after IFN-γ stim- tor, AG490, and the other was a broad inhibitor of ulation because NF-κB is involved in the constitutive the JAK family, pan-JAK inhibitor. We also used expression of Nfkbiz and is currently the only known

Fig. 2 Stimulus induced Nfkbiz promoter activity in deletion mutants. PAM212 cells were transfected with two kinds of deleted Nfkbiz promoter fragment cloned into pGL-3 basic vector. +1 indicates the transcription start site demonstrated using the primer extension method. These data represent the means ± S.E.M. of three samples. *P < 0.05 comparison of promoter responsiveness (ratio of the activities with and without stimulation) between fragments.

Fig. 3 Effects of selective inhibitors on Nfkbiz expression in keratinocytes. Nfkbiz mRNA of primary keratinocytes (a) and keratinocyte cell line PAM212 (b, c) pre-treated with selective inhibitors was measured after combined stimulation with IFN-γ plus IL-1 (a, b) or single stimulation with IFN-γ or IL-1 alone (c) for one hour using qRT-PCR. The values are shown as fold change relative to the control. *P < 0.05 compared with no inhibitor. IFN-γ induces Nfkbiz expression 107 transcriptional factor of Nfkbiz in keratinocytes (13). motifs. J Biol Chem 276, 12485–12488. Pan-JAK inhibitor and Bay11-7082 blocked the ef- 6. Ito T, Morimatsu M, Oonuma T, Shiina T, Kitamura H and Syuto B (2004) Transcriptional regulation of the MAIL gene fect of stimulation. These results suggest that JAK1 in LPS-stimulated RAW264 mouse macrophages. Gene 342, but not JAK2 is critical for Nfkbiz expression in the 137–143. JAK/STAT pathway and that NF-κB is an important 7. Kannan Y, Yu J, Raices RM, Seshadri S, Wei M, Caligiuri transcriptional factor to respond to an inflammatory MA and Wewers MD (2011) IkappaBzeta augments IL-12- stimulus such as IFN-γ as well as the constitutive and IL-18-mediated IFN-gamma production in human NK cells. Blood 117, 2855–2863. expression of Nfkbiz in the resting keratinocytes. Our 8. Kitamura H, Kanehira K, Okita K, Morimatsu M and Saito M findings that both JAK/STAT and NF-κB pathways (2000) MAIL, a novel nuclear I kappa B protein that potenti- are important for the synergism are consistent with ates LPS-induced IL-6 production. FEBS Lett 485, 53–56. a previous report on CXCL10 (3). Although STAT3 9. Krappmann D (2013) Shaping oncogenic NF-kappaB activity in the nucleus. Blood 122, 2146–2147. is known to play a very important role in inducing 10. Nickoloff BJ (1987) Binding of 125I-gamma interferon to cul- Nfkbiz expression in immune cells among the STAT tured human keratinocytes. J Invest Dermatol 89, 132–135. family (9, 11, 12), the central STAT molecule acting 11. Okamoto K, Iwai Y, Oh-Hora M, Yamamoto M, Morio T, downstream of IFN-γ signaling is STAT1 or STAT2 Aoki K, Ohya K, Jetten AM, Akira S, Muta T and Takayanagi rather than STAT3 (1, 15). The precise role of STATs H (2010) IkappaBzeta regulates T(H)17 development by co- operating with ROR nuclear receptors. Nature 464, 1381– in the regulation of Nfkbiz expression seems to be 1385. complicated and further analysis is needed. 12. Okuma A, Hoshino K, Ohba T, Fukushi S, Aiba S, Akira S, In conclusion, we showed that IFN-γ induces Ono M, Kaisho T and Muta T (2013) Enhanced apoptosis by Nfkbiz expression in epidermal keratinocytes. The disruption of the STAT3-IkappaB-zeta signaling pathway in effect of IFN-γ on Nfkbiz induction is synergistical- epithelial cells induces Sjögren’s syndrome-like autoimmune disease. Immunity 38, 450–460. ly enhanced by concurrent stimulation with IL-1. 13. Oonuma T, Morimatsu M, Ochiai K, Iwanaga T and Hashizume ISREs in the Nfkbiz promoter region are important K (2007) Role of NF-kappaB in constitutive expression of for the response to IFN-γ stimulation. JAK1, but not MAIL in epidermal keratinocytes. J Vet Med Sci 69, 279–284. JAK2, and the NF-κB pathway are important for 14. Pastore S, Corinti S, La Placa M, Didona B and Girolomoni G (1998) Interferon-gamma promotes exaggerated cytokine Nfkbiz expression in keratinocytes. production in keratinocytes cultured from patients with atopic dermatitis. J Allergy Clin Immunol 101, 538–544. 15. Ramana CV, Gil MP, Schreiber RD and Stark GR (2002) Acknowledgments Stat1-dependent and -independent pathways in IFN-gamma- This work was supported by Research Fellowships of dependent signaling. Trends Immunol 23, 96–101. 16. Shiina T, Konno A, Oonuma T, Kitamura H, Imaoka K, Japan Society for the Promotion of Science for Young Takeda N, Todokoro K and Morimatsu M (2004) Targeted Scientists and KAKENHI scientific research grants disruption of MAIL, a nuclear IkappaB protein, leads to se- from the Ministry of Education, Culture, Sports, vere atopic dermatitis-like disease. J Biol Chem 279, 55493– Science and Technology of Japan (No. 19380164 55498. and 26450400). 17. Takaoka A, Yanai H, Kondo S, Duncan G, Negishi H, Mizutani T, Kano S, Honda K, Ohba Y, Mak TW and Taniguchi T (2005) Integral role of IRF-5 in the gene induction programme REFERENCES activated by Toll-like receptors. Nature 434, 243–249. 18. Wesoly J, Szweykowska-Kulinska Z and Bluyssen HA (2007) 1. Au-Yeung N, Mandhana R and Horvath CM (2013) Tran- STAT activation and differential complex formation dictate se- scriptional regulation by STAT1 and STAT2 in the interferon lectivity of interferon responses. Acta Biochim Pol 54, 27–38. JAK-STAT pathway. JAKSTAT 2, e23931. 19. Yamamoto M, Yamazaki S, Uematsu S, Sato S, Hemmi H, 2. Boehm U, Klamp T, Groot M and Howard JC (1997) Cellu- Hoshino K, Kaisho T, Kuwata H, Takeuchi O, Takeshige K, lar responses to interferon-gamma. Annu Rev Immunol 15, Saitoh T, Yamaoka S, Yamamoto N, Yamamoto S, Muta T, 749–795. Takeda K and Akira S (2004) Regulation of Toll/IL-1-recep- 3. Burke SJ, Goff MR, Lu D, Proud D, Karlstad MD and Collier tor-mediated gene expression by the inducible nuclear protein JJ (2013) Synergistic expression of the CXCL10 gene in re- IkappaBzeta. Nature 430, 218–222. sponse to IL-1beta and IFN-gamma involves NF-kappaB, 20. Yamazaki S, Muta T and Takeshige K (2001) A novel IkappaB phosphorylation of STAT1 at Tyr701, and acetylation of his- protein, IkappaB-zeta, induced by proinflammatory stimuli, tones H3 and H4. J Immunol 191, 323–336. negatively regulates nuclear factor-kappaB in the nuclei. J 4. Fujisawa H, Wang B, Sauder DN and Kondo S (1997) Ef- Biol Chem 276, 27657–27662. fects of interferons on the production of interleukin-6 and in- 21. Yeruva S, Ramadori G and Raddatz D (2008) NF-kappaB- terleukin-8 in human keratinocytes. J Interferon Cytokine Res dependent synergistic regulation of CXCL10 gene expression 17, 347–353. by IL-1beta and IFN-gamma in human intestinal epithelial 5. Haruta H, Kato A and Todokoro K (2001) Isolation of a novel cell lines. Int J Colorectal Dis 23, 305–317. interleukin-1-inducible nuclear protein bearing ankyrin-repeat