A TGF-β− and p63-Responsive Enhancer Regulates IFN- κ Expression in Human Keratinocytes This information is current as Katrin Klein, Christina Habiger, Thomas Iftner and Frank of September 24, 2021. Stubenrauch J Immunol published online 14 February 2020 http://www.jimmunol.org/content/early/2020/02/13/jimmun ol.1901178 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2020/02/13/jimmunol.190117 Material 8.DCSupplemental http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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Published February 14, 2020, doi:10.4049/jimmunol.1901178 The Journal of Immunology A TGF-b– and p63-Responsive Enhancer Regulates IFN-k Expression in Human Keratinocytes Katrin Klein, Christina Habiger, Thomas Iftner, and Frank Stubenrauch Type I IFNs have antiviral and immune-modulating activities. IFN-a/-b have very low basal expression levels but are strongly induced upon activation of pattern recognition receptors. In contrast, IFN-k is constitutively expressed in uninfected keratinocytes and responds only weakly to pattern recognition receptor activation. IFN-k expression has been implicated in the pathogenesis of inflammatory skin diseases and in limiting human papillomavirus replication in human keratinocytes. We have identified an enhancer ∼5 kb upstream of the IFNK gene driving its expression in keratinocytes. The enhancer consists of binding sites for the transcription factors jun-B, SMAD3/4, AP-2a/g, and p63, of which the latter two are key regulators of keratinocyte biology. The jun-B and SMAD3/4 elements confer activation by the TGF-b pathway. Furthermore, inhibition of ERK1/2 kinases activates IFN-k expression. Our study provides a framework for the cell type–specific, constitutive expression of IFN-k and its modulation Downloaded from by signal transduction pathways in human keratinocytes. The Journal of Immunology, 2020, 204: 000–000. nterferons are secreted cytokines that bind to membrane re- activated by virus infection or PRR signaling (5–10). IFN-ε is ceptors and initiate signal transduction cascades that result in constitutively expressed in the reproductive organs and can be I the increased transcription of several hundred IFN-stimulated induced by estrogen but not by PRR signaling or overexpression genes (ISG) (1, 2). ISG proteins inhibit a variety of steps during the of IRF3 or 7 (6). IFN-k is constitutively expressed in normal http://www.jimmunol.org/ infection and replication of many viruses, modulate the immune human keratinocytes (NHK), peripheral blood monocytes, and response, and have antitumor activities (1, 2). IFNs are classified monocyte-derived dendritic cells (7, 11). LaFleur et al. (7) according to the membrane receptor used: type I IFN bind to the reported a 3- to 4-fold induction of IFNK upon infection with IFNAR1/2 receptor, type II IFN bind to the IFNGR1/2 receptor, encephalomyocarditis virus 5 h postinfection, which increased and type III IFN bind to the IFNLR1/IL-10RB receptor (1, 2). The up to 10-fold at 15 h postinfection. Stimulation of NHK with type I IFN family in humans consists of more than 10 subtypes of poly(I:C), an activator of RIG-I, MDA5, and TLR3 pathways, for IFNA and single IFNB1,-E,-K, and -W1 genes. Type I IFN-a and 6 h resulted in only a 1.5-fold induction of IFNK, whereas IFNB1 -b and type III IFN-l are present at very low levels in many cell and -L1 were activated 1500- and 3000-fold, respectively (8). types but are strongly induced upon the activation of pattern Similarly, poly(dA:dT), which activates several cytoplasmic DNA by guest on September 24, 2021 recognition receptors (PRR), such as TLR and RIG-I–like recep- sensors and RIG-I, induced IFNK 3- to 4-fold in the human tors, and DNA sensors such as IFI16, cGAS, and RNA polymerase keratinocyte NIKS cell line, whereas IFNA,-B1,and-L1 were III (1, 2). PRR signaling cascades activate IFN regulatory factor activated 110-, 15,000- and 40,000-fold, respectively (12, 13). (IRF) 3 and 7, NF-kB family members p50 and p65, and a het- Semiquantitative analyses also suggested a strong induction of erodimeric complex of ATF-2 and Jun (1, 2). Different combi- IFN-b, but not of IFN-k, by Sendai virus infection in human nations of these transcription factors (TF) are required for the keratinocytes expressing the human papillomavirus (HPV) 16 E7 induction of IFNB1 (IRF3, p50/p65, ATF-2/Jun), IFNA (IRF7), protein (9). Furthermore, inhibitors of the epidermal growth factor and IFNL (IRF3, IRF7, p50/p65) (3, 4). (EGF) receptor (EGFR) and of MEK activate IFN-k and ISG In contrast to IFN-a,-b, and -l, IFN-ε and -k are expressed in expression in NHK (14, 15). a cell type–dependent manner and are not (or only very weakly) Increased expression of IFN-k has been implicated in the pa- thology of cutaneous lupus erythematosus and allergic contact dermatitis, whereas decreased expression has been observed in Institute for Medical Virology and Epidemiology of Viral Diseases, University psoriasis, atopic dermatitis, and high-risk (HR) HPV–infected Hospital Tuebingen, University of Tuebingen, D72076 Tuebingen, Germany lesions (9, 12, 16, 17). HR-HPV have also been shown to diminish ORCIDs: 0000-0002-5929-7078 (T.I.); 0000-0002-6685-299X (F.S.). IFN-k expression in cultivated keratinocytes (8, 9, 13, 18, 19). Received for publication September 26, 2019. Accepted for publication January 19, 2020. IFN-k repression is mainly caused by the HR-HPV E6 protein and involves an increase in DNA methylation at CpG islands in the This work was supported by a grant from the Wilhelm-Sander-Stiftung (2007.007.3 to F.S.). IFNK promoter (9, 18). Transcriptome analyses upon inducible Address correspondence and reprint requests to Prof. Frank Stubenrauch, University expression of IFN-k in HPV31-positive keratinocytes have Hospital Tuebingen, Elfriede-Aulhorn-Straße 6, 72076 Tuebingen, Germany. E-mail revealed that 71% of IFN-k target genes are known ISG, indi- address: [email protected] cating that its ability to regulate gene expression is similar to The online version of this article contains supplemental material. IFN-a and -b (20). IFN-k has anti-HPV activity, and the ISG Abbreviations used in this article: BS, binding site; ChIP, chromatin immunoprecip- Sp100 and IFI16 have been shown to limit HR-HPV replication itation; EGF, epidermal growth factor; EGFR, EGF receptor; FLuc, firefly luciferase; GLuc, Gaussia luciferase; HPV, human papillomavirus; HR, high-risk; HS, hyper- (20–23). Reconstitution of IFN-k expression in HR-HPV–positive sensitive; ISG, IFN-stimulated gene; mt, mutated; NHK, normal human keratinocyte; cells might therefore have therapeutic potential, and in line with PRR, pattern recognition receptor; qPCR, quantitative PCR; siRNA, small interfering this, a recent report suggested that TGF-b1 specifically induces RNA; TF, transcription factor; TFBS, TF binding site; wt, wild-type. IFN-k by demethylation of the IFNK promoter in HPV16-positive Copyright Ó 2020 by The American Association of Immunologists, Inc. 0022-1767/20/$37.50 cells but not in normal keratinocytes (18). www.jimmunol.org/cgi/doi/10.4049/jimmunol.1901178 2 REGULATION OF IFN-k EXPRESSION IN KERATINOCYTES The cell type–specific expression of IFN-k and its deregulated poly(dA:dT) or poly(dI:dC)] and incubated at room temperature for 5 min. expression in pathologic conditions prompted us to investigate its Then, double-stranded 59-DY681–labeled oligonucleotides (40 fmol) transcriptional regulation. We have identified an enhancer element were added and incubated for 15 min at room temperature. Complexes were separated in 3 or 5% polyacrylamide-0.53 Tris-borate-EDTA gels. that controls IFNK expression in human keratinocytes. IFNK ex- Fluorescent signals were recorded with an Odyssey Fc Infrared Imaging pression requires binding sites (BS) for AP-2, SMAD3/4, jun-B, System (LI-COR Biosciences). and p63 proteins in this enhancer. AP-2 and p63 proteins are Compound treatments highly expressed in keratinocytes and control important aspects of keratinocyte biology and thus most likely contribute to the Cells were seeded in six-well plates and treated the next day with the keratinocyte-specific expression of IFN-k. SMAD3/4 proteins can respective substances or vehicle controls as listed in Supplemental Table I. be activated by the TGF-b signal transduction cascade (24), and After 24 h, RNA was isolated using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions and reverse-transcribed using consistent with this, the TGF-b pathway activates IFN-k expres- the QuantiTect Reverse Transcription Kit (Qiagen). Twenty-five nano- sion, which is dependent on both the SMAD3/4 and jun-B BS. grams of cDNA per well were used for quantitative RT-PCR using the Furthermore, inhibition of ERK1/2 kinases activates IFN-k ex- SYBR Green I Master Mix (Roche) and analyzed with the Light Cycler pression. Interestingly, the simultaneous activation by TGF-b1 480 Instrument (Roche). The primers are listed in Supplemental Table I.