Enhancer-Mediated Control of Macrophage-Specific Arginase I Expression Anne-Laure Pauleau, Robert Rutschman, Roland Lang, Alessandra Pernis, Stephanie S. Watowich and Peter J. This information is current as Murray of September 24, 2021. J Immunol 2004; 172:7565-7573; ; doi: 10.4049/jimmunol.172.12.7565 http://www.jimmunol.org/content/172/12/7565 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Enhancer-Mediated Control of Macrophage-Specific Arginase I Expression1

Anne-Laure Pauleau,2* Robert Rutschman,* Roland Lang,3* Alessandra Pernis,‡ Stephanie S. Watowich,† and Peter J. Murray4*

Arginase I expression in the liver must remain constant throughout life to eliminate excess nitrogen via the cycle. In contrast, arginase I expression in macrophages is silent until signals from Th2 cytokines such as IL-4 and IL-13 are received and the mRNA is then induced four to five orders of magnitude. Arginase I is hypothesized to play a regulatory and potentially pathogenic role in diseases such as asthma, parasitic, bacterial, and worm infections by modulating NO levels and promoting fibrosis. We show that Th2-inducible arginase I expression in mouse macrophages is controlled by an enhancer that lies ؊3 kb from the basal promoter. PU.1, IL-4-induced STAT6, and C/EBP␤ assemble at the enhancer and await the effect of another STAT6-regulated protein(s) that must be synthesized de novo. Identification of a powerful extrahepatic regulatory enhancer for arginase I provides potential to manipulate arginase I activity Downloaded from in immune cells while sparing liver function. The Journal of Immunology, 2004, 172: 7565Ð7573.

rginase I is a central metabolic of liver function, precursor of polyamines, essential molecules that have a plethora catalyzing the nitrogen elimination step of the Krebs of biological activities (1, 2). A urea cycle (1). In this context, arginase I expression must We were interested in the regulatory steps that lead to arginase remain constant throughout life. Genetic abnormalities in the genes I expression in macrophages. The rationale for this investigation is http://www.jimmunol.org/ that encode the urea cycle , including arginase I, are as- linked to the development of future agonists or antagonists of ar- sociated with a variety of pathological conditions related to the ginase I in diseases where the role of the enzyme is implicated in failure to eliminate nitrogen from the body (1). In contrast, argi- pathogenesis. It would be unlikely that direct agonists or antago- nase I expression is also found in nonhepatic cells (2–4) including nists of arginase I would have any useful pharmacological role cells of the hemopoietic system (2, 5), most significantly in mac- because of toxicity associated with inhibition or exacerbation of rophages (2, 5). The biological function of arginase I in macro- liver arginase I function. Therefore, we chose to investigate the phages is the subject of intense research, related predominantly to upstream regulatory steps that control arginase I gene expression its anticipated role in regulating NO production from activated in macrophages with the concept that these pathways could even- macrophages: being the common substrate for both argi- tually be regulated pharmacologically. by guest on September 24, 2021 nase I and the NO synthases (1, 6, 7). In vitro studies have estab- Unlike liver arginase I whose expression is constant throughout lished that arginase I can deplete arginine from macrophages and postnatal life (1, 11), macrophage arginase I expression is tightly from the milieu, leaving none available for NO synthases to gen- regulated. In resting murine macrophages, arginase I levels are erate NO (8, 9). This process is complex and not strictly dependent undetectable at the mRNA, protein, and enzymatic levels (5, 9). on substrate competition but also involves translational control of However, once exposed to cytokines that stimulate STAT6 activity NO synthase expression (10). The complexity of the competition (IL-4 and IL-13), arginase I mRNA, protein, and enzymatic levels between NO synthases and arginase I is speculated to have crucial are up-regulated four to five orders of magnitude (5, 9). The in vivo roles in diseases where NO levels must be tightly con- STAT6-mediated control of arginase I expression has also been trolled. Of further significance are the secondary metabolites of revealed in diseases dominated by Th2 responses including hel- arginase I, primarily , that can feed into pathways favor- minth, parasitic infections, and asthma (12–17). The tight control ing collagen biosynthesis, and hence fibrosis. Ornithine is also the over arginase I expression has led to the implication that it is crucially linked to pathologic sequelae in these diseases. We have previously shown that STAT6 is essential for IL-4/IL-13-mediated *Department of Infectious Diseases, St. Jude Children’s Research Hospital, Memphis, TN 38105; †Department of Immunology, M. D. Anderson Cancer Center, Houston, arginase I expression and that STAT6 regulates the expression of TX 77030; and ‡Department of Medicine, Columbia University, New York, NY another gene(s) that is required for expression (9). In this study, we 10032 define the regulatory mechanisms involved in this process. We show Received for publication September 26, 2003. Accepted for publication April that the regulation of arginase I in macrophages is controlled by a 13, 2004. complex enhancer element located 3-kb upstream of the transcription The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance start site. Surprisingly, the enhancer is regulated both directly and with 18 U.S.C. Section 1734 solely to indicate this fact. indirectly by STAT6 and a series of other transcription factors that 1 This work was supported by National Institutes of Health Cancer Center CORE P30 assemble in a temporal manner to induce arginase I gene expression. CA 21765, and by ALSAC. 2 Current address: Centre National de la Recherche Scientifique, UMR8125, Institute Materials and Methods Gustave Roussy, 39 rue Camille Desmoulins, Villejuif, France. Cells, Abs, and reagents 3 Institute fur Medizinische Mikrobiologie, Immunologie und Hygiene, Technical University, Munich, Germany. RAW macrophages were obtained from the American Type Culture Col- 4 Address correspondence and reprint requests to Dr. Peter J. Murray, Department of lection (Manassas, VA) and maintained in RPMI 1640 with 10% FBS, Infectious Diseases, St. Jude Children’s Research Hospital, 332 North Lauderdale, Room penicillin/streptomycin, and minimal nonessential amino acids (complete E8078, Mail Stop 320, Memphis, TN 38105. E-mail address: [email protected] RPMI). Mouse IL-4 and IL-10 were purchased from BD Biosciences/

Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00 7566 MECHANISM OF MACROPHAGE ARGINASE EXPRESSION

PharMingen (San Diego, CA) and resuspended to 1 ␮g/ml in complete were grown in complete RPMI on 10-cm plates until there were ϳ4 ϫ 106 RPMI before use. Final cytokine concentrations used are described in detail cells per plate. Two plates were assigned per time point and stimulation con- in Results specific for each experiment. Luciferase reporter constructs and dition. Cells attached to the plate were stimulated with IL-4 (10 ng/ml) for reagents for the analysis of luciferase expression were purchased from various times (typically 0, 1, 2, and 6 h). One plate was retained for accurate Promega (Madison, WI). Abs were purchased from Santa Cruz Biotech- counting of cell numbers. Cells were fixed by incubation in 1% formaldehyde nology (Santa Cruz, CA) and were used for chromatin immunoprecipita- for 10 min at room temperature and then washed twice with PBS containing tion or EMSA supershift reactions at a 1/100 dilution. The Abs are as PMSF as described in the Upstate Biotechnology protocol. Cells were scraped follows: anti-STAT6 (M200 rabbit, S20 rabbit, M20 goat), anti-IFN reg- from the plates and pelleted by centrifugation (200 ϫ g, 10 min) and then ulatory factor-4 (IRF-4)5 (M17 goat), anti-PU.1 (T21 rabbit), anti-C/EBP␤ resuspended in SDS lysis buffer (Upstate Biotechnology) and protein-DNA (C19, rabbit), anti-CBP (A22, rabbit). Anti-acetylated H3, anti-acetylated complexes fragmented by sonication using a Misonix Sonicator 3000 (Farm- H4, and anti-hyperacetylated H4 rabbit polyclonal Abs were from Upstate ingdale, NY) set at 80% power, 6°C constant temperature for 8 ϫ 30 s son- Biotechnology (Waltham, MA). An additional anti-STAT6 rabbit poly- ication cycles. These conditions were empirically established to give fragmen- clonal Ab (18) was a gift of Dr. J. Ihle (Department of Biochemistry, St. tation of DNA with a mean length of 500–800 bp. Insoluble material was Jude Children’s Research Hospital). For immunoprecipitations with goat removed by centrifugation at 15,000 ϫ g for 10 min. The lysate was then Abs, a rabbit anti-goat Ab (Pierce, Rockford, IL) was added at a final precleared with salmon sperm-saturated protein A-agarose slurry (Upstate Bio- dilution of 1/1000 before capture of Ig conjugates with protein A. technology) at a ratio of 50 ␮l per 2 ϫ 106 cell equivalents for2hat4°C. The agarose conjugates were removed by centrifugation and supernatants were RNA isolation, Northern blotting, and real-time RT-PCR incubated with polyclonal Abs to transcription factors as detailed in Materials and Methods. For anti-STAT6 immunoprecipitations, each Ab listed in the Total RNA was isolated from macrophages using TRIzol (Invitrogen, Materials and Methods was tested and the M200 Ab was found to be superior Carlsbad, CA) as described (19, 20). RNA was separated on formaldehyde in these assays. Accordingly, the M200 Ab was used for all subsequent ex- agarose gels and blotted to Hybond N membranes (Amersham Pharmacia periments. Following overnight incubation at 4°C, salmon sperm-saturated

Biotech, Piscataway, NJ) for Northern analysis as described (9). Real time Downloaded from protein A-agarose was added at a ratio of 30 ␮l/ml lysate and incubated at 4°C RT-PCR was performed as described in detail using Superscript II (In- with gentle rocking for 2 h. Immunoprecipitated material was washed and vitrogen) for reverse transcription and FAM-labeled probes (20) listed in eluted exactly according to the Upstate Biotechnology protocol. DNA cross- Table I. links were reversed by incubation with 20 ␮l of 5 M NaCl/500 ␮l eluted PAC isolation, promoter cloning, oligonucleotide definition and material for4hat65°C. DNA was extracted with phenol/chloroform and then precipitated. DNA from each immunoprecipitation was resuspended in 50 ␮l location, site-directed mutagenesis of 10 mM Tris, pH 8.0, 1 mM EDTA and subjected to PCR analysis for the arginase I enhancer using primers 391 and 379 (Table I) using conditions Probes from the proximal region of the arginase I promoter were used to http://www.jimmunol.org/ screen a mouse 129 PAC library supplied by the Human Genome Mapping empirically determined to amplify the amplicons before reaching the plateau Project Resource Centre (Hinxton, U.K.). Four clones (510-F22, 513-G17, (generally 25–26 cycles). Negative control reactions for background were per- 522-M2, and 526-J2) were identified. Bacteria were grown in 25 ␮g/ml formed with primers specific for the IL-12p40 and KC promoters that do not kanamycin according to the supplier’s instructions. Plasmid DNA was iso- recruit STAT6 (Table I). Positive control reactions were performed using the lated according to the supplier’s instructions. The presence of the arginase “input” samples to the immunoprecipitation reactions that had their cross-links I promoter was confirmed by PCR using oligonucleotides specific for re- reversed according to the Upstate Biotechnology protocol. gions within the promoter. Oligonucleotides to amplify the promoter frag- EMSAs ments, introduce mutations, and perform EMSA are detailed in Table I including the position of each oligonucleotide relative to the A residue in EMSAs reactions were performed as described in detail (21, 22) using 5 ␮g the initiation codon of the arginase I gene. The detailed description of the of nuclear extract and oligonucleotide probes detailed in Table I and the murine arginase I locus can be found at: www.ensembl.org/Mus_muscu- Fig. 5 legend. Supershift reactions were performed using 1 ␮g of each Ab by guest on September 24, 2021 lus/geneview?gene ϭ ENSMUSG00000019987. as detailed in Materials and Methods. Complexes were resolved on 1% Promoter fragments were cloned into the pGL3-basic vector (Promega) Tris-borate-EDTA acrylamide gels. using the restriction sites detailed in Table I. Site-directed mutagenesis was performed using the Stratagene (La Jolla, CA) Quik-Change procedure DNase I hypersensitivity according to the manufacturer’s instructions. All mutations were made in Ϫ Ϫ RAW cells were stimulated for 2 or 4 h with 10 ng/ml IL-4. Nuclei were the 31/ 3810 construct. In all cases, each mutation was constructed and isolated from ϳ250 ϫ 106 cells by resuspension in lysis buffer (10 mM analyzed completely independently twice. Tris, pH 7.5, 10 mM NaCl, 3 mM MgCl2, 1 mM PMSF, 0.05% Nonidet Transfection of RAW cells and reporter assays P-40). Additional Nonidet P-40 was added to the lysis buffer to a final concentration of 0.1% to achieve cell membrane lysis. The nuclei were RAW cells were grown in complete RPMI. Cells were harvested by gentle then gently resuspended in DNase I digestion buffer (40 mM Tris, pH 7.9, ϳ ϫ 6 scraping, centrifuged (200 ϫ g, 8 min) and washed once with PBS. Cells 10 mM NaCl, 6 mM MgCl2, 10 mM CaCl2)to 20 10 nuclei/ml. were resuspended in Optimem (Invitrogen) at a density of 1 ϫ 107 cells per Aliquots of nuclei were incubated with a dilution range of highly purified ml. Cells (0.5 ml) were gently mixed with 10 ␮g of plasmid DNA and DNase I (Amersham Pharmacia Biotech) over 0–2000 U/ml for 20 min at electroporated at 250 V, 975 ␮F in 0.4-cm cuvettes. Each transfection was 37°C. DNA was isolated by first digesting nuclei and associated proteins made up to 6 ml with complete RPMI and plated at 1 ml/well in 12-well with proteinase K (100 ␮g/ml) in SDS lysis buffer (100 mM Tris, pH 8.5, plates. Following overnight incubation at 37°C, the medium was replaced 5 mM EDTA, 0.2% SDS, 200 mM NaCl) at 55°C for 2 h. DNA was then (1 ml) and the cells were allowed to rest for 2–3 h. Cells (duplicate wells) precipitated with isopropanol and resuspended in 10 mM Tris, pH 8.0, 1 were left untreated or stimulated with IL-4 or IL-4 and IL-10 for 16–20 h. mM EDTA to a final concentration of 1 ␮g/␮l. Ten micrograms were Reporter activity was performed with the Promega reagents according to digested to completion overnight with EcoRI and EcoRV. Samples were ϩ the manufacturer’s instructions and luciferase activity was measured using resolved on a 0.7% agarose gel, transferred to Hybond N membranes, and a luminometer set to a 10-s measurement time. hybridized with a 1.8-kb probe from the arginase I promoter encompassing Stable RAW cell lines containing luciferase reporter constructs were the Ϫ2890/Ϫ3386 region that contains the enhancer element. Southern derived by linearizing each plasmid with Mlu I immediately 5Ј to the be- blots were washed at high stringency (0.1% SDS, 0.1ϫ SSC, 65°C) for 3 h ginning of the arginase I 5Ј genomic fragments. These plasmids were co- and exposed to film. transfected by electroporation as described above with XhoI-linearized pCDNA1 at a 10:1 ratio. After selection in 250 ␮g/ml G418 over a 2-wk Results period, lines were plated and stimulated with IL-4 or IL-4 and IL-10 and Characterization of a transfectable system to study arginase I assayed for luciferase activity 14 h later. regulation Chromatin immunoprecipitations (ChIP) To study the regulation of arginase I gene expression in macro- ChIP was performed as described by the manufacturer of the ChIP reagents phages through techniques such as promoter dissection, a trans- (Upstate Biotechnology), with the following modifications. RAW cells fectable system was essential. Primary macrophages are unsuitable for this purpose because they cannot be readily transfected. There- 5 Abbreviations used in this paper: IRF, IFN regulatory factor; ChIP, chromatin im- fore, we turned to the RAW macrophage cell line as a model sys- munoprecipitation; CBP, CREB binding protein. tem. Using Northern blotting and real-time RT-PCR, we first asked The Journal of Immunology 7567

Table I. Oligonucleotides used in this study

Direction or Oligo Site Purpose Partner/Target Sequence Location

375 Mlu I 4 031 CGCACGCGTAAGCGCTCCTTGTATGGGTG Ϫ3741/Ϫ3760 376 Mlu I 3 377 TCACGCGTATTGCCAGGAATATACCAGA Ϫ3789/Ϫ3808 377 Mlu I 4 376 CGCACGCGTTGGCCTCAGAACATCTAAG Ϫ3307/Ϫ3325 378 Mlu I 3 379 TCACGCGTCGCTGTGAAAGGATCTATCA Ϫ3367/Ϫ3386 379 Mlu I 4 378 CGCACGCGTAAAGTGGCACAACTCACGTA Ϫ2890/Ϫ2909 380 Mlu I 3 032 TCACGCGTGGGCCATGGTATGTGT Ϫ3168/Ϫ3183 387 Mlu I 4 378 CGCACGCGTGAGTCAGACTGGGGTGTCAG Ϫ3216/Ϫ3235 388 Mlu I 3 389 TCACGCGTGACAGTCCTTTGTGAAGACT Ϫ3268/Ϫ3287 389 Mlu I 4 388 CGCACGCGTCCCTTTACTCTGTGTGATT Ϫ3127/Ϫ3145 390 Mlu I 4 380 CGCACGCGTGCTCTCTGACTTCCTTATTG Ϫ3020/Ϫ3039 391 Mlu I 3 392 or 379 TCACGCGTGGTAGCCGACGAGAG Ϫ3053/Ϫ3067 392 Mlu I 4 391 CGCACGCGTAGTGGCACAACTCACGTACA Ϫ2861/Ϫ2880 393 Mlu I 3 394 TCACGCGTTGTACGTGAGTTGTGCC Ϫ2895/Ϫ2911 394 Mlu I 4 393 CGCACGCGTTCAGTGCACAAGTCCAGTTG Ϫ2766/Ϫ2765 395 Mlu I 3 032 TCACGCGTGAACAGGCAAACAATACGAT Ϫ2799/Ϫ2818 402 Mlu I Linker CGCGCTTTGTTAGGAAGTGAGGCATTGTTCAGACTTCCTTATGC Ϫ2950/Ϫ2898 403 Mlu I Linker CGCGGCATAAGGAAGTCTGAACAATGCCTCACTTCCTAACAAAG Ϫ2950/Ϫ2898 404 Mlu I Linker CGCGCTTTGTTATTTTGTGAGGCATTGTTCAGACAAAATTATGC Ϫ2950/Ϫ2989 405 Mlu I Linker CGCGGCATAATTTTGTCTGAACAATGCCTCACAAAATAACAAAG Ϫ2950/Ϫ2989

408 Mlu I Linker CGCGCAGAAGGCTTTGTCAGCAGGGCAAGACTATACTTTG Ϫ2985/Ϫ3020 Downloaded from 409 Mlu I Linker CGCGCAAAGTATAGTCTTGCCCTGCTGACAAAGCCTTCTG Ϫ2985/Ϫ3020 410 Mlu I Linker CGCGATGCTTTCTTATGAACAGGCTGTATTAGCCAACAGTCCTGTC Ϫ2912/Ϫ2954 411 Mlu I Linker CGCGGACAGGACTGTTGGCTAATACAGCCTGTTCATAAGAAAGCAT Ϫ2912/Ϫ2954 419 Mlu I 3 392, 422 TCACGCGTCAGAAGGCTTTGTCAGCAG Ϫ3002/Ϫ3020 420 Mlu I 3 422, 392 TCACGCGTCTATACTTTGTTAGGAAGTG Ϫ2975/Ϫ2994 421 Mlu I 3 392 TCACGCGTGTTCAGACTTCCTTATGC Ϫ2950/Ϫ2967 422 Mlu I 4 391, 419, 420 TCACGCGTGTTGGCTAATACAGCCTG Ϫ2924/Ϫ2938 423 Mutant GGCAAGACTATACTTTGTTACTCAGTGAGGCATTGT Ϫ2966/Ϫ3001 424 Mutant ACAATGCCTCACTGAGTAACAAAGTATAGTCTTGCC Ϫ2966/Ϫ3001 http://www.jimmunol.org/ 425 Mutant AGGCATTGTTCAGACCTCATTATGCTTTCTTAT Ϫ2942/Ϫ2974 426 Mutant ATAAGAAAGCATAATGAGGTCTGAACAATGCCT Ϫ2942/Ϫ2974 427 Mutant ACTTCCTTATGCTGCGCTATGAACAGGCTGTATTA Ϫ2927/Ϫ2961 428 Mutant TAATACAGCCTGTTCATAGCGCAGCATAAGGAAGT Ϫ2927/Ϫ2961 447 Mutant CATTGTTCAGACTTCCGGATGCTTTCTTATGAACAG Ϫ2936/Ϫ2971 448 Mutant CTGTTCATAAGAAAGCATCCGGAAGTCTGAACAATG Ϫ2936/Ϫ2971 449 Mutant AGCTCATCTTCAATAACTCAGTCAGAGAGCAGAAGG Ϫ3014/Ϫ3049 450 Mutant CCTTCTGCTCTCTGACTGAGTTATTGAAGATGAGCT Ϫ3014/Ϫ3049 451 Mutant AGAGACCAGCTCATCTTCCCTCCGGAAGTCAGAGAGCAGAAG Ϫ3015/Ϫ3056 452 Mutant CTTCTGCTCTCTGACTTCCGGAGGGAAGATGAGCTGGTCTCT Ϫ3015/Ϫ3056 021 Xho I 4 CGCCTCGAGGCTGCATGTGCTCGG Ϫ31/Ϫ45 022 Mlu I 3 021 CGCACGCGTAGAACTGCTTTGGGTTGTCA Ϫ639/Ϫ657 by guest on September 24, 2021 023 Mlu I 3 021 CGCACGCGTAAATGGGTTCTTCGGGTCA Ϫ1045/Ϫ1063 024 Mlu I 3 021 CGCACGCGTAATGTAAGGTCAAGCGATTT Ϫ2346/Ϫ2365 025 Mlu I 3 021 CGCACGCGTAGATTGCCAGGAATATACCA Ϫ3791/Ϫ3810 026 Mlu I 3 021 CGCACGCGTCATAAGGGTATGCGTTAATC Ϫ6416/Ϫ6435 027 Mlu I 3 021 CGCACGCGTCCCAATGAAGAAGCTAGAGA Ϫ8235/Ϫ8254 028 Mlu I 3 021 CGCACGCGTCTGATACCCAAATAGTTCCT Ϫ10672/Ϫ10691 031 Mlu I 3 032 TCACGCGTGCGAGCCTTCCCGTAG Ϫ4277/Ϫ4292 032 Mlu I 4 031 CGCACGCGTCCATACACACGACGGTTCCA Ϫ2640/Ϫ2659 033 Mlu I 3 034 CGCACGCGTGGTAAGGGCCACTAGGACTT Ϫ7460/Ϫ7479 034 Mlu I 4 033 CGCACGCGTACAGAGTFFFCAGCTACGG Ϫ4264/Ϫ4282 035 Mlu I 3 036 CGCACGCGTCCCACCACAGAGAACCCTA Ϫ10094/Ϫ10113 036 Mlu I 4 035 TGCACGCGTATGGTGGTCATGTCAACTGC Ϫ7575/Ϫ7594 037 Mlu I 3 038 CGCACGCGTCCATCGGCTCACCTCTATC Outside contig 038 Mlu I 4 037 CGCACGCGTTGAAGGGATTTGGGTATGGA Ϫ10283/Ϫ10322 039 Mlu I 3 040 CGCACGCGTGTTTGGCTGAGAACTATGTT Outside contig 040 Mlu I 4 039 CGCACGCGTGTCTVCTCATTGGCTAGGA Outside contig 396 EMSA CAGAAGGCTTTGTCAGCAGGGCAAGACTATACTTTG 397 EMSA CAAAGTATAGTCTTGCCCTGCTGACAAAGCCTTCTG 398 EMSA CTTTGTTAGGAAGTGAGGCATTGTTCAGACTTCCTTATGC 399 EMSA GCATAAGGAAGTCTGAACAATGCCTCACTTCCTAACAAAG 400 EMSA ATGCTTTCTTATGAACAGGCTGTATTAGCCAACAGTCCTGTC 401 EMSA GACAGGACTGTTGGCTAATACAGCCTGTTCATAAGAAAGCAT 470 EMSA CAGAAGGCggTGTCAGCAGGGCAAGACTATACTTTG 471 EMSA CAAAGTATAGTCTTGCCCTGCTGACAccGCCTTCTG 472 EMSA CTTTGTTAGttAGTGAGGCATTGTTCAGACTTCCTTATGC 473 EMSA GCATAAGGAAGTCTGAACAATGCCTCACTaaCTAACAAAG 474 EMSA CTTTGTTAGGAAGTGAGGCATTGTTCAGACTaaCTTATGC 475 EMSA GCATAAGttAGTCTGAACAATGCCTCACTTCCTAACAAAG 476 EMSA ATGCTaatTTATGAACAGGCTGTATTAGCCAACAGTCCTGTC 477 EMSA GACAGGACTGTTGGCTAATACAGCCTGTTCATAAattAGCAT 478 EMSA ATGCTTTCTTATGAACAGGCTGTATTAtttAACAGTCCTGTC 479 EMSA GACAGGACTGTTaaaTAATACAGCCTGTTCATAAGAAAGCAT 319 ChIP KC GGAGTTCGGACTTTCGGGAAG 320 ChIP KC GTGCTCAGGGCGAGGTG 321 ChIP IL-12p40 GGAAAGGTGGCCCAGATACAC 322 ChIP IL-12p40 TGAATAGAGGCGGCAATG RL75 RT-PCR Arginase I ACAGTCTGGCAGTTGGAAGCATC RL76 RT-PCR Arginase I GGGAGTCCCCAGGAGAATCCT RL83 RT-PCR Actin ACCCACACTGTGCCCATCTAC RL84 RT-PCR Actin AGCCAAGTCCAGACGCAGG RL85 Probe AGGGCTATGCTCTCCCTCACGCCA 7568 MECHANISM OF MACROPHAGE ARGINASE EXPRESSION

whether IL-4 and IL-13 induced arginase I mRNA expression as we had found previously in primary macrophages (9). The results show that arginase I mRNA was strongly induced in a time-de- pendent manner (Fig. 1). Neither IL-10 nor LPS induced arginase I mRNA alone but IL-10 was synergistic with both IL-4 and IL-13 in inducing the mRNA, precisely as reported in primary macro- phages (5, 9). Importantly, IL-4 or IL-13 induced a four to five orders-of-magnitude increase in arginase I mRNA levels with the same kinetics as in primary macrophages (5, 9). Taken together, the results argue that RAW cells were a suitable model to search for STAT6-responsive regulatory regions within the arginase I promoter.

Isolation of the IL-4-responsive region of the arginase I locus Extensive studies have characterized the rat and human arginase I promoters to define the regulatory elements that control the pre- cise, coordinate expression of the urea cycle encoding genes (11, 23–26). However, little is known about the mouse arginase I pro-

moter. Preliminary studies established that regions immediately up- Downloaded from stream of the anticipated transcriptional start site were constitutively active in luciferase reporter assays in HepG2 cells, a human hepatoma cell line (A.-L. Pauleau, unpublished data). However, none of these constructs was responsive to IL-4 when transfected into RAW cells suggesting that the IL-4-responsive region was not located in the

proximal promoter region (data not shown). http://www.jimmunol.org/ We next cloned the mouse arginase I promoter region from FIGURE 1. Induction of arginase I expression in RAW macrophages. a, mouse PAC clones containing the entire arginase I locus. Frag- RAW cells were treated with the stimulants shown at the top of the figure ϳ for 3, 10, or 20 h. RNA was extracted and separated by denaturing gel ments encompassing 6 kb relative to the initiation codon were electrophoresis. Arginase I mRNA levels were detected by Northern blot- cloned upstream of a luciferase reporter vector and transfected into ting using a mouse arginase I cDNA probe. b, RNA was extracted from RAW cells to test their response to IL-4, and, synergistically with RAW cells treated as shown in a. Arginase I mRNA levels were quantified IL-1 Constructs containing ϳ4orϳ6 kb regions upstream of the using real-time RT-PCR and normalized to 10,000 copies of actin mRNA. transcriptional start site were strongly responsive to IL-4 or IL-4 Data are representative of three independent experiments. and IL-10 (Fig. 2). The Ϫ31/Ϫ3810 construct induced a 42-fold increase above background luciferase activity that was further aug- by guest on September 24, 2021 mented by IL-10. These results suggested that the IL-4 responsive element was present within this sequence. Systematic dissection of

FIGURE 2. Identification of the IL-4-responsive element of the arginase I promoter. a, Initial constructs were made from PAC clones containing the entire arginase I locus. Constructs are numbered according to their position relative to the A residue in the initiation codon of mouse arginase I. aÐc, Progressive reduction to isolate the arginase I enhancer. For clarity, a selection of plasmids used to isolate the enhancer is shown. In each case, black shading indicates plasmids that were IL-4-responsive in transfection assays. Numbering to the right indicates that distal elements of the promoter (e.g., ϩ Ϫ2640/Ϫ4292) were fused to the Ϫ31/Ϫ657 (data not shown) or Ϫ31/Ϫ2365 plasmids which provide the information necessary for basal transcription, including the TATA element and transcriptional start site. Hatched lines indicate that a region was directly cloned 5Ј to the Ϫ31/Ϫ2365 fragment and are shown aligned to the larger fragments depicted in a. RAW cells were transiently transfected and then stimulated with IL-4 or IL-4 and IL-10 for 16–20 h and assayed for luciferase activity as described in Materials and Methods. Fold induction relative to transfected cells left untreated is shown on the ordinate. Data are representative of multiple experiments used to define the IL-4-responsive region where fragments were cloned into the Ϫ31/Ϫ657 or Ϫ31/Ϫ2365 plasmids in either orientation. The Journal of Immunology 7569

FIGURE 3. The IL-4-responsive region of the argi- nase I promoter is an enhancer. The IL-4-responsive fragment Ϫ2890/Ϫ3067 was cloned downstream of the polyadenylation signal in each plasmid as shown in aÐc as a test of enhancer activity. RAW cells were transiently transfected and then stimulated with IL-4 or IL-4 and IL-10 for 16–20 h and assayed for luciferase activity as described in Materials and Methods. Fold induction relative to transfected cells left untreated is shown on the ordinate. Note that enhancer activity in the pGL3-promoter vector (a) is lower because the basal activity of this plasmid is extremely high as a

result of the strong SV40 promoter element. Downloaded from http://www.jimmunol.org/

this region led to a 159-bp sequence sufficient to induce reporter RAW cell lines that were generated by cotransfecting linearized activity in response to IL-4 (Fig. 2). In these constructs, putative versions of these plasmids and then stimulating with IL-4 or IL-4 IL-4-responsive regions were fused to basal arginase I promoter and IL-10 and assaying for luciferase activity (data not shown). fragments represented by the Ϫ31/Ϫ657 and Ϫ31/Ϫ2356 regions We also mapped the transcription initiation site of the macroph- of the promoter. These constructs by themselves were unrespon- age-induced arginase I mRNA relative to the liver mRNA. Using sive to IL-4 until fused to fragments containing the 159-bp se- Northern blotting, primer extension and 5Ј RACE, we found that quence. Qualitatively similar results were obtained using stable the arginase I mRNA begins in a region within 10 bp of the rat liver by guest on September 24, 2021

FIGURE 4. Mutagenesis analysis of the arginase I enhancer. Mutations were introduced into the Ϫ31/Ϫ3810 plasmid using site-directed mutagen- esis. Mutations, labeled A through G, were constructed as shown (a). Each mutant was made twice, indepen- dently, and the results from one series of experiments are shown. Mutants were transfected into RAW cells, stim- ulated with IL-4 or IL-4 ϩ IL-10 and assayed for luciferase activity (b). Fold induction relative to transfected cells left untreated is shown on the ordinate. The position of the EMSA probes used in Fig. 5 are shown underlined. 7570 MECHANISM OF MACROPHAGE ARGINASE EXPRESSION Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 5. (Legend continues) The Journal of Immunology 7571

In silico analysis of the enhancer using programs designed to predict transcription factor binding sites (e.g., TransFac) suggested the presence of multiple putative binding sites for transcription factors including C/EBP␤, PU.1, and STAT6 (Fig. 4a). To ascer- tain the importance of these sites, we initially attempted to further reduce the size of the enhancer by creating fragments progres- sively reduced in size from either end. Surprisingly, none of these fragments displayed IL-4 responsiveness suggesting that the en- hancer could not be minimized in this manner (data not shown). We then created plasmids containing mutations in each putative transcription factor or adjacent sequences in the case of the putative PU.1 sites with the rationale that PU.1 forms tight complexes with proteins such as IRF-4 that we had identified in microarray screens and considered potentially important in regu- lation of the enhancer (see Discussion). All mutations were made in the Ϫ31/Ϫ3810 plasmid that contains the enhancer, the basal FIGURE 6. Model for the macrophage-specific regulation of arginase I promoter, and the transcription start site. Each mutant lost its abil- gene expression. In this model, STAT6 has both direct and indirect roles. ity to induce reporter expression in response to IL-4 or IL-4 and

The STAT6-mediated induction of factor X that assembles at the promoter IL-10 although mutations in the putative STAT6 binding site com- Downloaded from is speculative. pletely abrogated reporter activity while mutations at other sites (e.g., mutants B and E) retained some inducibility (Fig. 4b). Sim- ilar results were found using stable RAW cell lines generated by transcription start site previously mapped by Mori and colleagues transfecting linearized versions of these plasmids. This data, com- (data not shown, and Ref. 25). This suggests that the IL-4 responsive bined with the inability to reduce the overall size of the enhancer region was unlikely to act as an alternative promoter in macrophages. suggested that IL-4-mediated regulation of arginase I gene regu- http://www.jimmunol.org/ lation was more complex than expected and required the assembly The IL-4-responsive region behaves as a classical enhancer of a variety of transcription factors to the enhancer. While creating the constructs to characterize the IL-4-responsive region, we noticed that the element could be cloned into reporter Factors that assemble at the enhancer constructs in either orientation or independent of the location from ChIP and EMSA reactions were performed to characterize the fac- the start site of the reporter. This suggested that the 159-bp frag- tors that can bind to the enhancer. In untreated RAW cells, STAT6, ment may act as a classically defined enhancer element. To exper- and C/EBP␤ were weakly detected at the enhancer (Fig. 5a). Fol- ␤ imentally test this possibility, we cloned the 159-bp fragment lowing IL-4 treatment, however, STAT6, C/EBP , and the coac- by guest on September 24, 2021 downstream of the polyadenylation site in luciferase constructs tivator CREB binding protein were recruited to the enhancer in a driven by the strong SV40 promoter, or two non-IL-4-responsive time-dependent manner. Correlative data from EMSA binding re- arginase I proximal promoter fragments (Fig. 3). When these con- actions also showed that IL-4-inducible complexes bound to oli- structs were transfected into RAW cells and stimulated with IL-4, gonucleotide probes encompassing the putative STAT6 and the 159-bp fragment increased IL-4 responsiveness. This was also C/EBP␤ sites (Fig. 5f). Most interestingly, ChIP experiments true for enhancing the activity of the SV40 promoter, whose basal showed that STAT6 itself is recruited to the enhancer which sug- activity is extremely high, 3- to 6-fold (Fig. 3a). For the Ϫ31/ gests that this factor plays two distinct functions in arginase I reg- Ϫ657 or Ϫ31/Ϫ2365 constructs, basal activity of these promoters ulation: STAT6 directly binds the enhancer as shown here, but also is low and augmented slightly by the addition of IL-4 but robustly directs the regulation of one or more other genes that are crucial induced when the 159-bp fragment is cloned downstream. These for arginase expression (9). This indirect role of STAT6 was re- results define the IL-4-responsive element of the arginase I pro- vealed in our previous work showing that cycloheximide blocks moter as an enhancer. arginase I expression induced by IL-4. ChIP analysis also showed

FIGURE 5. Factors that bind to the arginase I enhancer. a and b, ChIP experiments were performed with RAW cells treated with IL-4 for the times indicated (0–4 h). Lysates from fixed and sonicated cells were immunoprecipitated with Abs to IRF-4, STAT6, C/EBP␤, and PU.1. Immunoprecipitated material was assayed by PCR for the arginase I enhancer using primers 379 and 391. Data are representative of seven independent experiments. c and d, ChIP experiments were performed to detect acetylated histones at the enhancer. Abs to acetylated histones H4 (Ac-H4), hyperacetylated H4 (Hyp-Ac-H4), and H3 (Ac-H3) were used (c) along with control immunoprecipitations for STAT6 (d) to show the IL-4-mediated recruitment of this factor for a given experiment. Data are representative of two independent experiments. e and f, EMSA analysis of complexes that can bind to regions of the arginase I enhancer. Labeled duplex probes spanning the enhancer (e, oligonucleotides 398 and 399; f, oligonucleotides 400 and 401) were incubated with nuclear extracts from IL-4-stimulated RAW cells and subjected to EMSA analysis. Preliminary experiments established the identity of nonspecific (NS) bands in these reactions. Polyclonal Abs were used to supershift PU.1 (PU.1 SS) in e. In some reactions, a 100-fold excess of cold competitor oligonucleotide duplex was added (Comp.). EMSA data is representative of four independent experiments. gÐi, DNase I hypersensitivity mapping in the region of the enhancer. g, Map of the experimental design showing the location of the enhancer and probe. Complete digestion with EcoRI and EcoRV generates a 5.4-kb fragment. h, Control total RNA isolation and Northern blotting to confirm the induction of the arginase I mRNA in the same cells used for hypersensitivity mapping. i, DNase I hypersensitivity mapping following IL-4 treatment for 2 or 4 h. Nuclei were isolated from stimulated RAW cells and exposed to dilutions of DNase I for 20 min. DNA was isolated and digested to completion with EcoRI and EcoRV and analyzed by Southern blotting. Note the presence of the Arrows indicate the presence of potential fragments hypersensitive .(ء) expected 5.4-kb fragment detected by the probe along with background hydridization to DNase I digestion at ϳ3, 1.9, and 0.8 kb. The location of the enhancer is Ϫ2890/Ϫ3067 relative to the initiation codon. Note that IL-4 treatment does not affect the appearance of any hypersensitive sites in this region. 7572 MECHANISM OF MACROPHAGE ARGINASE EXPRESSION that PU.1 was constitutively bound to the enhancer (Fig. 5b). colleagues (2) who reported that they identified a STAT6 site in a Within the enhancer, the most likely binding sites for PU.1 are the similar locale. ChIP experiments and EMSA analysis showed that GGAA motifs (Fig. 4). Site-directed mutagenesis of these sites STAT6 was directly recruited to the enhancer. This finding was substantially reduced enhancer activity (Fig. 4). Thus, PU.1 could surprising considering that our previous data suggested that serve as a factor that coordinates factor assembly at the enhancer. STAT6 functioned by directing the expression of another gene(s) The ChIP data also correlated with supershift EMSA results de- whose activity was required for arginase I mRNA expression. finitively showing that PU.1 bound to an oligonucleotide probe Therefore, STAT6 appears to have two functions, both direct and encompassing the putative PU.1 binding sites (Fig. 5e). Finally, we indirect (Fig. 6). This scenario is reminiscent of the IL-4 regulation performed additional ChIP experiments to ask whether chromatin of the polymeric Ig receptor gene where STAT6 also appears to at the enhancer was in a closed or open configuration based on the have a dual function (28). Other factors that bind to the enhancer levels of acetylated histones (Fig. 5c). The results showed that include PU.1, a crucial transcription factor in macrophage devel- acetylated and hyperacetylated histone H4 and acetylated H3 were opment and biology, C/EBP␤ and the coactivator CBP. Mutations readily immunoprecipitated from unstimulated cells, indicating in the putative binding sites for PU.1 and C/EBP␤ also reduced the that the arginase I enhancer is most likely in an open configuration activity of the enhancer. Overall, it appears that multiple factors awaiting signals for rapid transcriptional activation. These results form a complex at the enhancer and likely assemble in the correct were correlated with DNase I hypersensitivity assays (Fig. 5, gÐi) temporal order. Thus, STAT6 directly binds, along with PU.1, that showed that IL-4 does not regulate the inducible formation of CBP, C/EBP␤, and possibly other components, and this complex hypersensitive sites around the region of the enhancer. There were awaits the STAT6-regulated production of another factor that es- three hypersensitive sites detected within a 5.4-kb region of the tablishes transcriptional regulation of the locus. Downloaded from enhancer (Fig. 5, g and i) that potentially represent regions in and To search for the STAT6-regulated factor(s), we have used two around the enhancer that are more accessible to DNase I digestion. types of microarray approaches. The first, an Affymetrix screen Therefore, the results support the notion that the DNA in this re- (Santa Clara, CA), was designed to focus on the identification of gion is likely in a configuration readily accessible to the IL-4- IL-4-regulated genes. In this pool of genes, we expected to identify regulated factors that stimulate arginase I transcription. transcription factors that could then be tested as candidate factors for regulating the enhancer. In this group, two mRNAs were iden- http://www.jimmunol.org/ Discussion tified, Krox-20 and IRF-4, whose induction was confirmed by We have identified the mechanism of macrophage-specific argi- Northern and immunoblotting analysis. Krox-20 had previously nase I expression. The results show that an enhancer located ϳ3kb been identified as IL-4 regulated in B cells (29). However, trans- from the transcriptional start site is responsive to signals delivered fection experiments failed to confirm any role for this factor in from the Th2 cytokine IL-4, via STAT6. However, the regulation arginase I regulation (data not shown). IRF-4 was appealing be- of the enhancer is complex, because STAT6 both directly binds to cause it forms a complex with PU.1 that is understood in atomic the enhancer, and directs the synthesis of other genes required for detail (30), and plays an important role in regulating IL-4 re- arginase I expression. Furthermore, we found that multiple factors Ϫ/Ϫ

sponses (31–34). However, macrophages from IRF-4 mice had by guest on September 24, 2021 assemble at the enhancer, a property in keeping with other en- completely normal IL-4-mediated induction of arginase I (data not hancer elements. shown). We also designed a microarray screen taking advantage of Arginase I expression in the liver is constitutive and must be the fact that cycloheximide blocks induction of arginase I mRNA maintained constantly, and coordinately, with the other enzymes that make up the urea cycle (1, 2). The basal, hepatocyte regulation when macrophages are stimulated with IL-4. Macrophages were of the genes encoding the urea cycle enzymes have been exten- treated with IL-4 with or without cycloheximide for various times sively dissected (11, 23–27). In contrast, the expression of the and mRNA expression was measured on M20 arrays that contain ϳ arginase I gene in macrophages is silent until the cells are stim- 20,000 cDNAs. Here, we were searching for genes that were ulated with IL-4 or IL-13. Both of these cytokines activate STAT6 induced by IL-4 in the presence or absence of cycloheximide. that is essential for many of the signal transduction events down- Genes that require new protein synthesis to be induced (e.g., ar- stream of the IL-4 or IL-13 receptors. Although previous studies ginase I) would not be present in the samples treated with cyclo- have shown that a variety of signals can regulate macrophage ar- heximide. In this screen, we identified a candidate transcription ginase I expression, IL-4 and IL-13 are by far the most potent. We factor, ATF1. However, the induction of this protein was weak and began to search for the IL-4-regulated region of the arginase I not dependent on STAT6 (data not shown). At this stage, it is not promoter using the studies of Mori and colleagues (24, 25) as a possible to definitively determine whether STAT6 induces a tran- guide. These investigators have dissected the regulatory regions scription factor. It is possible that STAT6 regulates other processes involved in the hepatocyte-specific expression of the arginase I required for transcription of the arginase I gene, including chro- gene in rats and humans. When fused to reporter constructs and matin-dependent factors. In addition, we cannot definitively ex- transfected into macrophages, arginase I promoter constructs con- clude the possibility that cycloheximide regulates the degradation taining the previously identified hepatocyte-specific regulatory re- of a factor(s) that assembles at the enhancer. This effect would gions were not responsive to IL-4. Therefore, we searched up- preclude indirect effects of STAT6 on other genes. stream of the transcriptional start site and eventually identified a Direct pharmacological manipulation of arginase I in diseases small region, ϳ3 kb from the start site that conferred IL-4-respon- where macrophage activity is implicated will most likely be im- siveness upon arginase I basal promoter fragments. This region possible because of liver toxicity associated with the disruption of acted as a true enhancer element: it was functional independent of the urea cycle. For example, if arginase I activity promotes fibrosis distance from the start site and was active in either orientation. The in schistosomiasis (12, 14, 35) or accentuates asthmatic reactions arginase I enhancer could not be reduced in size, and mutations (13), then our ability to regulate arginase I activity only in mac- introduced into putative transcription factor binding sites all rophages would be an appealing target. This report defines the affected activity. The activity of the enhancer was completely ab- basis for macrophage specificity and suggests that the control of rogated when a mutation in the putative STAT6 binding site was the regulation of the arginase I enhancer could be targeted while introduced, data that is consistent with the findings of Morris and sparing constitutive expression of arginase I in the liver. The Journal of Immunology 7573

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