Disrupting the DNA Binding of EGR-1 with a Small-Molecule Inhibitor Ameliorates 2,4-Dinitrochlorobenzene-Induced Skin Inflammation

EGR1 encodes a transcription factor containing three zinc-finger (ZF) DNA-binding domains of the Cys2-His2 type. It is rapidly induced by variety extracellular stimuli and involved in multiple physiological processes, including apoptosis, cell proliferation, inflammation (Li et al., 2019Li T.T. Liu M.R. Pei D.S. Friend or foe, role EGR-1 cancer.Med Oncol. 2019; 37: 7Crossref PubMed Scopus (24) Google Scholar). mediates inflammatory responses various tissues (Cho 2006Cho S.J. Kang M.J. Homer R.J. H.R. Zhang X. Lee P.J. al.Role early growth response-1 (Egr-1) interleukin-13-induced remodeling.J Biol Chem. 2006; 281: 8161-8168Abstract Full Text PDF (85) Scholar; Shin 2013Shin S.Y. J.M. Lim Y. Y.H. Transcriptional regulation growth-regulated oncogene alpha gene response protein-1 to tumor necrosis stimulation.Biochim Biophys Acta. 2013; 1829: 1066-1074Crossref (21) Son 2008Son S.W. Min B.W. Regulatory mechanism TNFalpha autoregulation HaCaT cells: EGR-1.Biochem Res Commun. 2008; 374: 777-782Crossref Yan 2000Yan S.F. Fujita T. Lu J. Okada K. Shan Zou Mackman N. al.Egr-1, master switch coordinating upregulation divergent families underlying ischemic stress.Nat Med. 2000; 6: 1355-1361Crossref (388) In skin, expression upregulated dermal wound area (Bryant 2000Bryant M. Drew G.M. Houston P. Hissey Campbell C.J. Braddock Tissue repair with therapeutic factor.Hum Gene Ther. 11: 2143-2158Crossref (58) Scholar) psoriasis (Fang 2007Fang Wee S.A. Ronski Fan H. Tao S. Lin Q. Evidence as differentially expressed among proliferative skin diseases.Genomic 2007; 1: 75-85Crossref (15) regulates IL-33–induced TSLP (Ryu 2015Ryu W.I. Kim J.H. Bae H.C. Ryu H.J. IL-33 induces Egr-1-dependent via MAPK pathways human keratinocytes.Exp Dermatol. 2015; 24: 857-863Crossref (38) IL-17–induced psoriasin (S100A7) (Jeong 2014Jeong S.H. Jang al.Egr-1 key regulator IL-17A-induced psoriasis.Exp 2014; 23: 890-895Crossref (16) keratinocytes, suggesting that might play mediating response. However, functional chronic conditions, such atopic dermatitis (AD), has not been assessed. Therefore, we investigated hypothesis crucial for evaluated impact targeting domain small-molecule drug on amelioration inflammation. TNF-α major proinflammatory cytokine elevated AD-like lesions mouse model 2,4-dinitrochlorobenzene (DNCB) (Schottelius 2010Schottelius A.J. Zügel U. Döcke W.D. Zollner T.M. Röse L. Mengel A. al.The mitogen-activated protein kinase-activated kinase 2 p38/TNF-alpha pathway systemic cutaneous inflammation.J Invest 2010; 130: 481-491Abstract (32) We also observed increased cytokines chemokines (Supplementary Figure S1a). Because known be S1b), reasoned loss function could modulate decreasing chemokine expression. To test this possibility, knocked down keratinocytes short hairpin (sh) RNA confirmed silencing inhibited TNF-α‒induced EGR-1–regulated cytokines, TSLP, IL-17, CCL5, CXCL1 (Figure 1a Supplementary S1c). Quantitative real-time PCR showed suppression TNF-α–induced mRNA shRNA transfection compared scrambled control (Figures 1b S1d), contributes DNCB stimulated 1c, upper blots), DNCB-induced using Egr1-null mice. All experiments were conducted according national guidelines approved Institutional Animal Care Use Committee Konkuk University (Seoul, South Korea). Wild-type (Egr1+/+) (Egr1−/−) mice treated dorsal S2a). The Egr1+/+ subjected applications developed severe lesions, whereas Egr1−/− only mild 1c). Histological analysis shows epidermal thicknesses 1d S2b) population toluidine blue stain‒positive cells 1e S2c) significantly reduced those Immunohistochemistry infiltrations myeloperoxidase-positive neutrophils, F4/80-positive macrophages, CD3ε-positive T cells, IL-17‒positive substantially S3a). Reduced myeloperoxidase, F4/80, CD3ε levels S3b). Immunofluorescence staining was markedly DNCB-treated 1f). These vitro vivo results suggest microenvironment through chemokines. next wished determine whether potential target AD. investigate benefit targeting, new inhibitor (named AB1711) selectively disrupts DNA‒binding activity without affecting upstream signaling regulating (see Materials Methods Figures S4 S5) had no cytotoxic effect even when exposed up 80 μM concentration S6). By electrophoretic mobility shift assay, AB1711 prevented DNA binding dose-dependent manner 2a) but did affect activator S7), may have unwarranted off-target effects. better understand how interferes activity, performed molecular docking study predict possible mode EGR-1. harbors Cys2-His2‒type ZF (ZF1, ZF2, ZF3) (Pavletich Pabo, 1991Pavletich N.P. Pabo C.O. Zinc finger-DNA recognition: crystal structure Zif268-DNA complex at 2.1 A.Science. 1991; 252: 809-817Crossref (1703) Docking simulation interact ZF1 ZF2 2b). Five residues (Arg357, His358, Arg360, Ile361, Gly364) six (Lys366, Arg375, Phe377, Ser378, Arg379, His382) predicted S8). On basis simulation, expected AB1771 would interfere interacting specific amino acids ZF2. Next, determined alters EGR-1‒regulated activation quantitative PCR. Pretreatment genes 2c). genes, except IL1B, umbilical vein endothelial monocytic THP1 S9). efficacy NC/Nga mice, an inbred strain develops AD‒like under exposure environmental factors (Matsuda 1997Matsuda Watanabe Geba G.P. Sperl Tsudzuki Hiroi al.Development dermatitis-like lesion IgE hyperproduction mice.Int Immunol. 1997; 9: 461-466Crossref (668) ear after being challenged 14 days 2d S10a). Repeated topical application effectively ameliorated DNCB-elicited 2d) thickenings 2e S10b). Dexamethasone used reference drug. infiltration blue‒stained into dermis 2f S10c). Itching prominent clinical symptom Various IL-33, IL-13, IL-31, directly sensitize sensory neurons, causing itching (Wang Kim, 2020Wang F. B.S. Itch: A paradigm neuroimmune crosstalk.Immunity. 2020; 52: 753-766Abstract Inflammasome-activated IL-1β (Fan 2020Fan J.J. Gao B. Song A.Q. Zhu Y.J. Zhou Li W.Z. al.Spinal cord NLRP1 inflammasome dry itch mice.J Neuroinflammation. 17: 122Crossref (4) Scholar), IL-17 enhances secretion 2012Cho K.A. Suh J.W. K.H. J.L. Woo IL-22 enhance stimulating ROS-NLRP3-caspase-1 pathway.Int 2012; 147-158Crossref (100) pruritogenic (e.g., IL-1β), antipruritic DNCB-challenged ears Scratching behaviors recorded 1 hour 7, 14, 21 challenge presence dexamethasone Video S1-S3), total scratching bouts counted. Notably, repeated number elicited 2g). Collectively, our can lead enhanced reducing summary, demonstrated Egr1 deficiency knockdown downregulated identified synthetic compound AB1711, which associates EGR-1, monocytes, alleviated plays disrupting feasible approach. propose selective promising strategy improve Further detailed studies will necessary clarify roles AD pathogenesis. No datasets generated analyzed during study. Hyunjin Yeo: http://orcid.org/0000-0001-9978-8472 Sung Ahn: http://orcid.org/0000-0002-1236-2143 Jeong Yeon Lee: http://orcid.org/0000-0003-1298-7466 Euitaek Jung: http://orcid.org/0000-0003-3091-0649 Munki Jeong: http://orcid.org/0000-0002-4945-4914 Gi Sue Kang: http://orcid.org/0000-0002-2237-9088 Seunghyun http://orcid.org/0000-0002-5069-9513 Youngshim http://orcid.org/0000-0003-1206-0556 Dongsoo Koh: http://orcid.org/0000-0001-8984-0273 Young Han http://orcid.org/0000-0003-4060-6587 Yoongho Lim: http://orcid.org/0000-0002-8945-3027 Soon Shin: http://orcid.org/0000-0002-1075-0011 authors state conflict interest. This supported Basic Science Research Program National Foundation Korea funded Ministry Science, ICT Future Planning Republic (grant Korea-2020R1A2C1005845). paper Professor Conceptualization: SYS, YHL, DK, YLim; Data Curation: Formal Analysis: HY, SSA, YLee; Funding Acquisition: SYS; Investigation: SSA; Methodology: JYL, EJ, MJ, GSK, SA, YLee, DK; Supervision: Writing – Review Editing: YLim, DK Download .pdf (5.03 MB) Help pdf files Materialshttps://www.jidonline.org/cms/asset/b180d8e9-81ea-4ccf-9f9a-ea63fa57272d/mmc2.mp4Loading ... .mp4 (495.52 S1https://www.jidonline.org/cms/asset/ee427895-7a49-43c2-b30a-2f5ff9fe95b5/mmc3.mp4Loading (494.98 S2https://www.jidonline.org/cms/asset/a848d676-0a20-4c48-b8ee-743ce703b073/mmc4.mp4Loading (241.62 S3