The Transcription Factor p63 Is a Direct Effector of IL-4– and IL-13–Mediated Repression of Keratinocyte Differentiation

Atopic Dermatitis is an inflammatory skin disease associated with broad defects in barrier function caused by increased levels of type-2 cytokines (IL-4 and IL-13) that repress keratinocyte (KC) differentiation. Although crucial mediating allergic disease, the mechanisms for gene repression induced remain unclear. In this study, we determined requires master regulator epidermal differentiation program, p63. We found cytokine–mediated inhibition expression genes involved early KC differentiation, including keratin 1, 10, DSC-1, reversed p63 blockade. Type-2 cytokines, through p63, also regulate additional CHAC-1, STC2, CALML5. The regulation these ablated small interfering RNA as well. addition, IL-4 IL-13 Staphylococcus aureus lipoteichoic acid work combination to further suppress program. Finally, inhibit activity Notch, a transcription factor required induce conclusion, blockade are multifactorial, involving pathways converge on factors critical development, Notch. 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Real-time PCR analysis shows two represses K1, K10, U D Supplementary S1b). Scholar), can Transcription proliferating cells, downregulation concomitant 2a, show there multiple p63-binding sites upstream downstream promoter regions Gene Regulation Database (GTRD), database factor–binding identified ChIP-sequencing experiments (Yevshin 2017Yevshin I. Sharipov Valeev Kel Kolpakov GTRD: binding ChIP-seq experiments.Nucleic Acids Res. 2017; 45: D61-D67Crossref (120) To explore transfected control (siRNA). Transfected hours IL-13. Cells differentiate days. was 2b). 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S3, siRNA, dependence expression. contrast, HSD3B1 long IL-4– IL-13–regulated (Gingras Simard, 1999Gingras Simard Induction 3beta-hydroxysteroid dehydrogenase/isomerase type interleukin-4 normal prostate immortalized keratinocytes, colon, cervix cancer lines.Endocrinology. 140: 4573-4584Crossref contains no observed 3c). requirement targets. occur absence consistent report showing transducer 6 (STAT6) activated repressed (Czimmerer 2018Czimmerer Daniel Horvath Rückerl Nagy Kiss al.The STAT6 mediates enhancers limits alternatively polarized macrophages.Immunity. 48: 75-90.e6Abstract (99) During signaling, translocated nucleus (Ohmori Hamilton, 2000Ohmori Hamilton T.A. Interleukin-4/STAT6 STAT1 NF-kB dependent mechanisms.J Biol Chem. 2000; 275: 38095-38103Abstract (87) neither nor alter constitutive nuclear localization S4). did find form lacking transactivation domain functioned dominant-negative inhibitor block IL-13–mediated S5), suggesting rather than DNA responsible p63-mediated transcription. previously specific changes Notch-regulated (Brauweiler 2017Brauweiler Hall C.F. Goleva D.Y.M. inhibits pathway.J 137: 2030-2033Abstract (7) NRARP HES, Notch-mediated HK DAPT, molecule inhibitor, included control. showed HES mRNA downregulated 4a). still 4b). occurred presence LTA. A DAPT treatment significant decreases 4a b). mechanism next examined protein. Primary HKs DAPT. After treatment, analyzed blot intracellular domain. 4c generation domain, especially increased; therefore, increases appeared additional, lower molecular weight band indicate change covalent modification transcriptionally phosphorylated (Carrieri Dale, 2016Carrieri F.A. Dale J.K. Turn down notch.Front Dev 4: 151PubMed quantitated upper bands correlated band. (lanes 5 10) completely induction correlates DAPT-mediated reductions HES. modulates (Nguyen 2006Nguyen B.C. Lefort Mandinova Antonini Devgan Della Gatta al.Cross-regulation between commitment 2006; 1028-1042Crossref (300) Rangarajan directly test DMSO calcium. indicated (K1, DSC-1) performed. Significant inhibition, particularly 5a). partially resistant 5b, DMSO- DAPT-treated previously. Further cytokines. depends part inductive generated demonstrate knockdown reverses our knowledge, pathway converging propose model, 6, mediated fate. suppression schematic illustrating gradient versus layers points (Omori-Miyake 2014Omori-Miyake Tsunemi Kawashima Yagi assessment IL-4- IL-13-mediated structural components keratinocytes mice humans.J 2014; 134: 1342-1350Abstract (62) although recent large set enhancer chromatin modifications, accessibility, histone acetylase deacetylase even promoter. Consistent STAT6-binding sites, GTRD Interestingly, provided insight relationship (Pattison 2018Pattison J.M. Melo S.P. Piekos S.N. Torkelson J.L. Bashkirova Mumbach M.R. al.Retinoic BMP4 cooperate dynamics during surface commitment.Nat Genet. 50: 1658-1665Crossref (30) affects major establishing epigenetic landscape modifications assembly. Repression area mutations abolish regulation. manner, specify varied programs pathways. codependence hypothesize enhancer-binding protein, STAT6, initial marks. establish long-range topological interactions their occur, edit landscape. DNA-binding area. Repression, does gene; rather, distal location up downstream. operates program stage-dependently drive processes ranging progenitor expansion complex, encodes (Botchkarev, 2015Botchkarev V.A. Integration factor-regulated differentiation.J Investig Dermatol Symp Proc. 17: 30-32Abstract (9) Koster observation alternative based helps explain conflicting determination STAT6-mediated modulate fate decisions maturation. translocates KCs. Rather, full-length containing oligomerization inhibition. Our (TLR) ligand acts TLRs recognize conserved microbial structures activating innate immunity. Emerging evidence suggests global patterns inducing serving enhance (Foster 2007Foster S.L. Hargreaves D.C. Medzhitov Gene-specific TLR-induced modifications.Nature. 447: 972-978Crossref (862) vivo proliferation, recruitment 2019Brauweiler damages IL-1-mediated 1753-1761.e4Abstract (13) LTA–induced LTA-mediated likely enable fate, resulting heterogeneity play well found, types type-I suggest quantity more repression. signaling. Active juncture Spinous conditional null apparent could contribute pathway. Whereas restricts (Blanpain 2006Blanpain Lowry W.E. Pasolli H.A. Fuchs Canonical notch switch lineage.Genes 3022-3035Crossref (312) linked maintenance skin. postulate leading cytokine-p63 occurs TLR Additional efforts fully understand complexity regulators Understanding alterations ligands will help make progress novel approaches disease. (Cascade Biologics, Grand Island, NY) grown serum-free medium (EpiLife, Cascade Biologics), 1% supplement 0.06 mM chloride, antibiotics. plated 100,000 per 24-well plate, coated collagen matrix allowed adhere overnight before When indicated, (InvivoGen, San Diego, CA), (Research And Diagnostic Systems; Minneapolis, MN), ?M (Tocris Bioscience; Bristol, United Kingdom). Total isolated RNeasy Mini Kits (Qiagen, Valencia, CA) according manufacturer’s instructions. ?g reverse transcribed using Qiagen Quantiscript kit (Qiagen) dual-labeled fluorogenic probe method ABI Prism 7300 sequence detector (Applied Biosystems, Foster City, CA). Probes CALML5, FLG, HSD3B1, NRARP, HES-1, ?-actin purchased Applied Biosystems. Amplification reactions MicroAmp optical plates Biosystems) 25-?l volume. All normalized ?-actin. Second-passage 50–60% confluence instructions Lipofectamine 2000 (Invitrogen, 20 nontargeting (microarray minimal targeting genes) SMARTpool (Dharmacon-Thermo Fisher, Pittsburgh, PA). ensure viability silencing, trypsinized transfection. harvested RIPA lysis buffer protease (complete, Roche Holdings AG, Basel, Switzerland). Cellular debris pelleted centrifugation, clarified lysates resuspended Laemmli buffer, proteins resolved 4–15% gel (Bio-Rad Laboratories, Hercules, Transferred blotted antibodies directed against (BioLegend, Systems), (Cell Signaling, Cambridge, MA), (Sigma-Aldrich, St. Louis, MO) detected enhanced chemiluminescence (MilliporeSigma, Burlington, MA). media alone, washed cross formaldehyde. ChIP assay Agarose (Pierce/ThermoFisher, Pittsburg, PA) immunoprecipitated nonspecific IgG antibody Signaling). Primers designed basis forward: 5?-TGGGAAACTAAAGTACTGAC-3? reverse: 5?-CCCTCAAGGCCACCTAACTC-3?. amplification SYBR green Biosystems). (DNp63 alpha [Addgene, MA]) cloned lentiviral pUltra vector (Addgene), contained bicistronic concurrently EGFP. mutant cloned, confirmation cloning sequencing, lentivirus transfection plasmid constructs packaging plasmids (OriGene, Rockville, MD) 293 FT viral supernatant used infect KCs; 48 infection, 95% EGFP positive, reflecting retroviral transduction. transduction, statistical analyses conducted GraphPad (GraphPad Software, Inc., Comparisons ANOVA techniques Student’s t-tests appropriate. online Gtrd18-06.biouml.org, Anne Brauweiler: http://orcid.org/0000-0002-6527-8308 Donald Y.M. Leung: http://orcid.org/0000-0002-0177-3844 Elena Goleva: http://orcid.org/0000-0002-4685-9318 authors state conflicts interest. wish thank Cliff technical support. supported National Institutes Health Institute Infectious Diseases grant U19 AI117673, Arthritis Musculoskeletal Skin R01 AR41256, Edelstein Family Chair Pediatric Allergy-Immunology Jewish (Denver, CO). Conceptualization: AMB, EG, DYML; Data Curation: AMB; Methodology: Writing - Original Draft Preparation: Review Editing: DYML Download .pdf (.33 MB) Help pdf files figures