The AP-1 Transcription Factor c-Jun Promotes Arthritis by Regulating Cyclooxygenase-2 and Arginase-1 Expression in Macrophages This information is current as of September 28, 2021. Nicole Hannemann, Jutta Jordan, Sushmita Paul, Stephen Reid, Hanns-Wolf Baenkler, Sophia Sonnewald, Tobias Bäuerle, Julio Vera, Georg Schett and Aline Bozec J Immunol published online 15 March 2017 http://www.jimmunol.org/content/early/2017/03/15/jimmun Downloaded from ol.1601330 Supplementary http://www.jimmunol.org/content/suppl/2017/03/15/jimmunol.160133 http://www.jimmunol.org/ Material 0.DCSupplemental Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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Published March 15, 2017, doi:10.4049/jimmunol.1601330 The Journal of Immunology The AP-1 Transcription Factor c-Jun Promotes Arthritis by Regulating Cyclooxygenase-2 and Arginase-1 Expression in Macrophages Nicole Hannemann,* Jutta Jordan,† Sushmita Paul,‡ Stephen Reid,x Hanns-Wolf Baenkler,* Sophia Sonnewald,x Tobias Ba¨uerle,† Julio Vera,‡ Georg Schett,* and Aline Bozec* Activation of proinflammatory macrophages is associated with the inflammatory state of rheumatoid arthritis. Their polarization and activation are controlled by transcription factors such as NF-kB and the AP-1 transcription factor member c-Fos. Surprisingly, little is known about the role of the AP-1 transcription factor c-Jun in macrophage activation. In this study, we show that mRNA and protein levels of c-Jun are increased in macrophages following pro- or anti-inflammatory stimulations. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment cluster analyses of microarray data using wild-type and c-Jun– Downloaded from deleted macrophages highlight the central function of c-Jun in macrophages, in particular for immune responses, IL production, and hypoxia pathways. Mice deficient for c-Jun in macrophages show an amelioration of inflammation and bone destruction in the serum-induced arthritis model. In vivo and in vitro gene profiling, together with chromatin immunoprecipitation analysis of macrophages, revealed direct activation of the proinflammatory factor cyclooxygenase-2 and indirect inhibition of the anti- inflammatory factor arginase-1 by c-Jun. Thus, c-Jun regulates the activation state of macrophages and promotes arthritis via differentially regulating cyclooxygenase-2 and arginase-1 levels. The Journal of Immunology, 2017, 198: 000–000. http://www.jimmunol.org/ he transcription factor family AP-1 is composed of homo- Whereas resident macrophages regulate tissue homeostasis, and heterodimeric complexes, which consist of Jun, Fos, macrophages recruited from the bloodstream activate specific T activating transcription factor, and musculoaponeurotic transcriptional programs, which allow them to polarize into pro- or fibrosarcoma proteins. These dimers are involved in different anti-inflammatory macrophages (4). AP-1 transcription factors cellular processes, such as proliferation, apoptosis, and differen- have been described to orchestrate inflammatory responses in tiation (1). In macrophages, AP-1 proteins can regulate inflam- macrophages, in particular by the expression of inflammatory matory processes through activation of cytokine production. For mediators (5). JunD for instance was recently shown to induce instance, the activation of IL and TLR leads to the initiation of an macrophage activation and cytokine secretion, by modulating their by guest on September 28, 2021 MAPK signaling cascade through MyD88, resulting in the acti- gene expression levels (6, 7). Furthermore, activation of JNK in vation of AP-1 in macrophages (2, 3). macrophages is required for the establishment of obesity-induced Macrophages perform diverse homeostatic functions in the de- insulin resistance and inflammation. JNK-deficient macrophages velopment, tissue repair, and immune responses against pathogens. expressed fewer proinflammatory chemokines and cytokines, such as Ccl2, Ccl5, IL-6, IL-12, and TNF (8). Additionally, JNK *Department of Internal Medicine 3—Rheumatology and Immunology, University blockade suppresses matrix metalloproteinases and thus inhibits ar- Hospital Erlangen, Friedrich Alexander University Erlangen–Nuremberg, 91054 thritic joint destruction in a rat adjuvant-induced arthritis model (9). Erlangen, Germany; †Preclinical Imaging Platform Erlangen, Institute of Radiology, University Hospital Erlangen, 91054 Erlangen, Germany; ‡Laboratory of Systems One JNK target is c-Jun, suggesting that c-Jun could also represent a Tumor Immunology, Department of Dermatology, University Hospital Erlangen, potent regulator of macrophage activation during inflammation. 91054 Erlangen, Germany; and xDivision of Biochemistry, Friedrich Alexander Uni- versity Erlangen–Nuremberg, 91054 Erlangen, Germany About 1% of the population worldwide is afflicted by rheumatoid arthritis, a chronic inflammatory disease of the joints, which is ORCIDs: 0000-0002-9185-6255 (S.S.); 0000-0002-3076-5122 (J.V.); 0000-0001- 8174-2118 (A.B.). characterized by synovial hyperplasia, cartilage degradation, and Received for publication August 2, 2016. Accepted for publication February 17, bone destruction (10). Macrophages are centrally involved in the 2017. process of arthritis (11). Their numbers in the synovial membrane This work was supported by funding from the Staedtler Foundation, Deutsche are increased during arthritis and exhibit a highly activated phe- Forschungsgemeinschaft Grants A01-CRC1181, Z-CRC1181, SPP1468 (IMMUNO- BONE program), and BO3811/1-1 (Emmy Noether program), and by Interdis- notype with increased expression of proinflammatory cytokines ziplina¨re Zentrum fur€ Klinische Forschung Project D23. (12). In addition to their proinflammatory properties, they contrib- The sequences presented in this article have been deposited in the National Center for ute to the destruction of bone and cartilage in the acute and chronic Biotechnology Information’s Gene Expression Omnibus database (https://www.ncbi. phases of arthritis (12). However, whether AP-1 proteins in mac- nlm.nih.gov/geo) under accession number GSE88898. rophages influence inflammatory arthritis still remains unknown. Address correspondence and reprint requests to Dr. Aline Bozec, Department of Internal Medicine 3—Rheumatology and Immunology, University Hospital Erlangen, In this study, we show that c-Jun participates in macrophage Ulmenweg 18, 91054 Erlangen, Germany. E-mail address: [email protected] activation by regulating the expression pattern of pro- and anti- The online version of this article contains supplemental material. inflammatory genes upon the stimulation with diverse Th1 or Abbreviations used in this article: AC, apoptotic cell; Arg1, arginase-1; ChIP, chro- Th2 cytokines, pro- or anti-inflammatory stimulations, and mi- matin immunoprecipitation; Cox2, cyclooxygenase-2; GO, Gene Ontology; KEGG, crobial products. Gene Ontology (GO) cluster analysis unravels the Kyoto Encyclopedia of Genes and Genomes; MRI, magnetic resonance imaging. important role of c-Jun in immune pathways and IL production. Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$30.00 Two targets of c-Jun identified were the proinflammatory enzyme www.jimmunol.org/cgi/doi/10.4049/jimmunol.1601330 2 c-Jun PROMOTES INFLAMMATORY MACROPHAGE SIGNALING cyclooxygenase-2 (Cox2, also known as PG-endoperoxide syn- Western blot analysis thase 2) and the anti-inflammatory enzyme arginase-1 (Arg1). Cells were lysed using Frackelton buffer (10 mM Tris, 50 mM NaCl, 30 mM Using two conditionally deleted c-Jun mice, controlled under the NaPPi, 50 mM NaF, 1% Triton X-100) supplemented with protease inhibitor Mx-1 or the lysozyme 2 promoter, named c-JunDMx or c-JunDLysM, mixture (cOmplete ULTRA; Roche, Basel) followed by three freeze and respectively, we confirmed the direct activation of Cox2 and the thaw cycles in liquid nitrogen. Afterward, lysates were boiled for 5 min in 3 3 6 indirect inhibition of Arg1 by c-Jun. Moreover, using the K/BxN 1 Laemmli buffer and centrifuged. Cell extracts from 0.25 10 mac- rophages were separated by NaDodSO4-PAGE on a 10% acrylamide gel. arthritis model, we showed reduced joint inflammation in c-Jun– Semidry immunoblotting was carried out on polyvinylidene difluoride deficient mice by histological analyses and in vivo magnetic res- membrane, which was further incubated overnight at 4˚C with rabbit anti- onance imaging (MRI). Finally, we confirmed the direct binding mouse c-Jun (1:1000; Cell Signaling Technology, Leiden, the Nether- of c-Jun on the Cox2 promoter in macrophages through chromatin lands), rabbit anti-mouse GAPDH (1:5000; Cell Signaling Technology), and mouse anti-mouse b-actin Ab (1:5000; Sigma-Aldrich, St. Louis, MO)