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* Department of Infectious Diseases, St. Jude Children’s Research Hospital, Memphis, TN 38105;
Department of Immunology, M. D. Anderson Cancer Center, Houston, TX 77030; and
Department of Medicine, Columbia University, New York, NY 10032
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Abstract |
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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 in immune cells while sparing liver urea cycle function. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We were interested in the regulatory steps that lead to arginase I expression in macrophages. The rationale for this investigation is linked to the development of future agonists or antagonists of arginase I in diseases where the role of the enzyme is implicated in pathogenesis. It would be unlikely that direct agonists or antagonists of arginase I would have any useful pharmacological role because of toxicity associated with inhibition or exacerbation of liver arginase I function. Therefore, we chose to investigate the upstream regulatory steps that control arginase I gene expression in macrophages with the concept that these pathways could eventually be regulated pharmacologically.
Unlike liver arginase I whose expression is constant throughout postnatal life (1, 11), macrophage arginase I expression is tightly regulated. In resting murine macrophages, arginase I levels are undetectable at the mRNA, protein, and enzymatic levels (5, 9). However, once exposed to cytokines that stimulate STAT6 activity (IL-4 and IL-13), arginase I mRNA, protein, and enzymatic levels are up-regulated four to five orders of magnitude (5, 9). The STAT6-mediated control of arginase I expression has also been revealed in diseases dominated by Th2 responses including helminth, parasitic infections, and asthma (12, 13, 14, 15, 16, 17). The tight control 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 arginase I expression and that STAT6 regulates the expression of another gene(s) that is required for expression (9). In this study, we define the regulatory mechanisms involved in this process. We show that the regulation of arginase I in macrophages is controlled by a complex enhancer element located 3-kb upstream of the transcription start site. Surprisingly, the enhancer is regulated both directly and indirectly by STAT6 and a series of other transcription factors that assemble in a temporal manner to induce arginase I gene expression.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RAW macrophages were obtained from the American Type Culture Collection (Manassas, VA) and maintained in RPMI 1640 with 10% FBS, penicillin/streptomycin, and minimal nonessential amino acids (complete RPMI). Mouse IL-4 and IL-10 were purchased from BD Biosciences/PharMingen (San Diego, CA) and resuspended to 1 µg/ml in complete RPMI before use. Final cytokine concentrations used are described in detail in Results specific for each experiment. Luciferase reporter constructs and reagents for the analysis of luciferase expression were purchased from Promega (Madison, WI). Abs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and were used for chromatin immunoprecipitation or EMSA supershift reactions at a 1/100 dilution. The Abs are as follows: anti-STAT6 (M200 rabbit, S20 rabbit, M20 goat), anti-IFN regulatory factor-4 (IRF-4)5 (M17 goat), anti-PU.1 (T21 rabbit), anti-C/EBP (C19, rabbit), anti-CBP (A22, rabbit). Anti-acetylated H3, anti-acetylated H4, and anti-hyperacetylated H4 rabbit polyclonal Abs were from Upstate Biotechnology (Waltham, MA). An additional anti-STAT6 rabbit polyclonal Ab (18) was a gift of Dr. J. Ihle (Department of Biochemistry, St. Jude Children’s Research Hospital). For immunoprecipitations with goat Abs, a rabbit anti-goat Ab (Pierce, Rockford, IL) was added at a final dilution of 1/1000 before capture of Ig conjugates with protein A.
RNA isolation, Northern blotting, and real-time RT-PCR
Total RNA was isolated from macrophages using TRIzol (Invitrogen, Carlsbad, CA) as described (19, 20). RNA was separated on formaldehyde agarose gels and blotted to Hybond N membranes (Amersham Pharmacia Biotech, Piscataway, NJ) for Northern analysis as described (9). Real time RT-PCR was performed as described in detail using Superscript II (Invitrogen) for reverse transcription and FAM-labeled probes (20) listed in Table I.
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Probes from the proximal region of the arginase I promoter were used to screen a mouse 129 PAC library supplied by the Human Genome Mapping Project Resource Centre (Hinxton, U.K.). Four clones (510-F22, 513-G17, 522-M2, and 526-J2) were identified. Bacteria were grown in 25 µg/ml kanamycin according to the supplier’s instructions. Plasmid DNA was isolated according to the supplier’s instructions. The presence of the arginase I promoter was confirmed by PCR using oligonucleotides specific for regions within the promoter. Oligonucleotides to amplify the promoter fragments, introduce mutations, and perform EMSA are detailed in Table I including the position of each oligonucleotide relative to the A residue in the initiation codon of the arginase I gene. The detailed description of the murine arginase I locus can be found at: www.ensembl.org/Mus_musculus/geneview?gene = ENSMUSG00000019987.
Promoter fragments were cloned into the pGL3-basic vector (Promega) using the restriction sites detailed in Table I. Site-directed mutagenesis was performed using the Stratagene (La Jolla, CA) Quik-Change procedure according to the manufacturer’s instructions. All mutations were made in the –31/–3810 construct. In all cases, each mutation was constructed and analyzed completely independently twice.
Transfection of RAW cells and reporter assays
RAW cells were grown in complete RPMI. Cells were harvested by gentle scraping, centrifuged (200 x g, 8 min) and washed once with PBS. Cells were resuspended in Optimem (Invitrogen) at a density of 1 x 107 cells per ml. Cells (0.5 ml) were gently mixed with 10 µg of plasmid DNA and electroporated at 250 V, 975 µF in 0.4-cm cuvettes. Each transfection was made up to 6 ml with complete RPMI and plated at 1 ml/well in 12-well plates. Following overnight incubation at 37°C, the medium was replaced (1 ml) and the cells were allowed to rest for 2–3 h. Cells (duplicate wells) were left untreated or stimulated with IL-4 or IL-4 and IL-10 for 16–20 h. Reporter activity was performed with the Promega reagents according to the manufacturer’s instructions and luciferase activity was measured using a luminometer set to a 10-s measurement time.
Stable RAW cell lines containing luciferase reporter constructs were derived by linearizing each plasmid with Mlu I immediately 5' to the beginning of the arginase I 5' genomic fragments. These plasmids were cotransfected by electroporation as described above with XhoI-linearized pCDNA1 at a 10:1 ratio. After selection in 250 µg/ml G418 over a 2-wk period, lines were plated and stimulated with IL-4 or IL-4 and IL-10 and assayed for luciferase activity 14 h later.
Chromatin immunoprecipitations (ChIP)
ChIP was performed as described by the manufacturer of the ChIP reagents (Upstate Biotechnology), with the following modifications. RAW cells were grown in complete RPMI on 10-cm plates until there were 
4 x 106 cells per plate. Two plates were assigned per time point and stimulation condition. Cells attached to the plate were stimulated with IL-4 (10 ng/ml) for various times (typically 0, 1, 2, and 6 h). One plate was retained for accurate counting of cell numbers. Cells were fixed by incubation in 1% formaldehyde for 10 min at room temperature and then washed twice with PBS containing PMSF as described in the Upstate Biotechnology protocol. Cells were scraped from the plates and pelleted by centrifugation (200 x g, 10 min) and then resuspended in SDS lysis buffer (Upstate Biotechnology) and protein-DNA complexes fragmented by sonication using a Misonix Sonicator 3000 (Farmingdale, NY) set at 80% power, 6°C constant temperature for 8 x 30 s sonication cycles. These conditions were empirically established to give fragmentation of DNA with a mean length of 500–800 bp. Insoluble material was removed by centrifugation at 15,000 x g for 10 min. The lysate was then precleared with salmon sperm-saturated protein A-agarose slurry (Upstate Biotechnology) at a ratio of 50 µl per 2 x 106 cell equivalents for 2 h at 4°C. The agarose conjugates were removed by centrifugation and supernatants were incubated with polyclonal Abs to transcription factors as detailed in Materials and Methods. For anti-STAT6 immunoprecipitations, each Ab listed in the Materials and Methods was tested and the M200 Ab was found to be superior in these assays. Accordingly, the M200 Ab was used for all subsequent experiments. Following overnight incubation at 4°C, salmon sperm-saturated protein A-agarose was added at a ratio of 30 µl/ml lysate and incubated at 4°C with gentle rocking for 2 h. Immunoprecipitated material was washed and eluted exactly according to the Upstate Biotechnology protocol. DNA cross-links were reversed by incubation with 20 µl of 5 M NaCl/500 µl eluted material for 4 h at 65°C. DNA was extracted with phenol/chloroform and then precipitated. DNA from each immunoprecipitation was resuspended in 50 µl 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 empirically determined to amplify the amplicons before reaching the plateau (generally 25–26 cycles). Negative control reactions for background were performed with primers specific for the IL-12p40 and KC promoters that do not recruit STAT6 (Table I). Positive control reactions were performed using the "input" samples to the immunoprecipitation reactions that had their cross-links reversed according to the Upstate Biotechnology protocol.
EMSAs
EMSAs reactions were performed as described in detail (21, 22) using 5 µg of nuclear extract and oligonucleotide probes detailed in Table I and the Fig. 5 legend. Supershift reactions were performed using 1 µg of each Ab as detailed in Materials and Methods. Complexes were resolved on 1% Tris-borate-EDTA acrylamide gels.
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RAW cells were stimulated for 2 or 4 h with 10 ng/ml IL-4. Nuclei were isolated from 250 x 106 cells by resuspension in lysis buffer (10 mM Tris, pH 7.5, 10 mM NaCl, 3 mM MgCl2, 1 mM PMSF, 0.05% Nonidet 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 then gently resuspended in DNase I digestion buffer (40 mM Tris, pH 7.9, 10 mM NaCl, 6 mM MgCl2, 10 mM CaCl2) to 20 x 106 nuclei/ml. Aliquots of nuclei were incubated with a dilution range of highly purified DNase I (Amersham Pharmacia Biotech) over 0–2000 U/ml for 20 min at 37°C. DNA was isolated by first digesting nuclei and associated proteins with proteinase K (100 µg/ml) in SDS lysis buffer (100 mM Tris, pH 8.5, 5 mM EDTA, 0.2% SDS, 200 mM NaCl) at 55°C for 2 h. DNA was then precipitated with isopropanol and resuspended in 10 mM Tris, pH 8.0, 1 mM EDTA to a final concentration of 1 µg/µl. Ten micrograms were digested to completion overnight with EcoRI and EcoRV. Samples were resolved on a 0.7% agarose gel, transferred to Hybond N+ membranes, and hybridized with a 1.8-kb probe from the arginase I promoter encompassing the –2890/–3386 region that contains the enhancer element. Southern blots were washed at high stringency (0.1% SDS, 0.1x SSC, 65°C) for 3 h and exposed to film.
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Results |
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To study the regulation of arginase I gene expression in macrophages through techniques such as promoter dissection, a transfectable system was essential. Primary macrophages are unsuitable for this purpose because they cannot be readily transfected. Therefore, we turned to the RAW macrophage cell line as a model system. Using Northern blotting and real-time RT-PCR, we first asked 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-dependent 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 macrophages (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.
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Extensive studies have characterized the rat and human arginase I promoters to define the regulatory elements that control the precise, coordinate expression of the urea cycle encoding genes (11, 23, 24, 25, 26). However, little is known about the mouse arginase I promoter. Preliminary studies established that regions immediately upstream 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).
We next cloned the mouse arginase I promoter region from mouse PAC clones containing the entire arginase I locus. Fragments encompassing 6 kb relative to the initiation codon were cloned upstream of a luciferase reporter vector and transfected into RAW cells to test their response to IL-4, and, synergistically with IL-1 Constructs containing 4 or 6 kb regions upstream of the transcriptional start site were strongly responsive to IL-4 or IL-4 and IL-10 (Fig. 2). The –31/–3810 construct induced a 42-fold increase above background luciferase activity that was further augmented by IL-10. These results suggested that the IL-4 responsive element was present within this sequence. Systematic dissection of this region led to a 159-bp sequence sufficient to induce reporter activity in response to IL-4 (Fig. 2). In these constructs, putative IL-4-responsive regions were fused to basal arginase I promoter fragments represented by the –31/–657 and –31/–2356 regions of the promoter. These constructs by themselves were unresponsive to IL-4 until fused to fragments containing the 159-bp sequence. Qualitatively similar results were obtained using stable RAW cell lines that were generated by cotransfecting linearized versions of these plasmids and then stimulating with IL-4 or IL-4 and IL-10 and assaying for luciferase activity (data not shown).
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The IL-4-responsive region behaves as a classical enhancer
While creating the constructs to characterize the IL-4-responsive region, we noticed that the element could be cloned into reporter constructs in either orientation or independent of the location from the start site of the reporter. This suggested that the 159-bp fragment may act as a classically defined enhancer element. To experimentally test this possibility, we cloned the 159-bp fragment downstream of the polyadenylation site in luciferase constructs driven by the strong SV40 promoter, or two non-IL-4-responsive arginase I proximal promoter fragments (Fig. 3). When these constructs were transfected into RAW cells and stimulated with IL-4, the 159-bp fragment increased IL-4 responsiveness. This was also true for enhancing the activity of the SV40 promoter, whose basal activity is extremely high, 3- to 6-fold (Fig. 3a). For the –31/–657 or –31/–2365 constructs, basal activity of these promoters is low and augmented slightly by the addition of IL-4 but robustly induced when the 159-bp fragment is cloned downstream. These results define the IL-4-responsive element of the arginase I promoter as an enhancer.
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, PU.1, and STAT6 (Fig. 4a). To ascertain the importance of these sites, we initially attempted to further reduce the size of the enhancer by creating fragments progressively reduced in size from either end. Surprisingly, none of these fragments displayed IL-4 responsiveness suggesting that the enhancer could not be minimized in this manner (data not shown). We then created plasmids containing mutations in each putative transcription factor binding site 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 regulation of the enhancer (see Discussion). All mutations were made in the –31/–3810 plasmid that contains the enhancer, the basal promoter, and the transcription start site. Each mutant lost its ability to induce reporter expression in response to IL-4 or IL-4 and IL-10 although mutations in the putative STAT6 binding site completely abrogated reporter activity while mutations at other sites (e.g., mutants B and E) retained some inducibility (Fig. 4b). Similar results were found using stable RAW cell lines generated by transfecting linearized versions of these plasmids. This data, combined with the inability to reduce the overall size of the enhancer suggested that IL-4-mediated regulation of arginase I gene regulation was more complex than expected and required the assembly of a variety of transcription factors to the enhancer.
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ChIP and EMSA reactions were performed to characterize the factors that can bind to the enhancer. In untreated RAW cells, STAT6, and C/EBP were weakly detected at the enhancer (Fig. 5a). Following IL-4 treatment, however, STAT6, C/EBP, and the coactivator CREB binding protein were recruited to the enhancer in a time-dependent manner. Correlative data from EMSA binding reactions also showed that IL-4-inducible complexes bound to oligonucleotide probes encompassing the putative STAT6 and C/EBP sites (Fig. 5f). Most interestingly, ChIP experiments showed that STAT6 itself is recruited to the enhancer which suggests that this factor plays two distinct functions in arginase I regulation: STAT6 directly binds the enhancer as shown here, but also directs the regulation of one or more other genes that are crucial for arginase expression (9). This indirect role of STAT6 was revealed in our previous work showing that cycloheximide blocks arginase I expression induced by IL-4. ChIP analysis also showed that PU.1 was constitutively bound to the enhancer (Fig. 5b). Within the enhancer, the most likely binding sites for PU.1 are the GGAA motifs (Fig. 4). Site-directed mutagenesis of these sites substantially reduced enhancer activity (Fig. 4). Thus, PU.1 could serve as a factor that coordinates factor assembly at the enhancer. The ChIP data also correlated with supershift EMSA results definitively showing that PU.1 bound to an oligonucleotide probe encompassing the putative PU.1 binding sites (Fig. 5e). Finally, we performed additional ChIP experiments to ask whether chromatin at the enhancer was in a closed or open configuration based on the levels of acetylated histones (Fig. 5c). The results showed that acetylated and hyperacetylated histone H4 and acetylated H3 were readily immunoprecipitated from unstimulated cells, indicating that the arginase I enhancer is most likely in an open configuration awaiting signals for rapid transcriptional activation. These results were correlated with DNase I hypersensitivity assays (Fig. 5, g–i) that showed that IL-4 does not regulate the inducible formation of hypersensitive sites around the region of the enhancer. There were three hypersensitive sites detected within a 5.4-kb region of the enhancer (Fig. 5, g and i) that potentially represent regions in and around the enhancer that are more accessible to DNase I digestion. Therefore, the results support the notion that the DNA in this region is likely in a configuration readily accessible to the IL-4-regulated factors that stimulate arginase I transcription.
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Discussion |
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3 kb from the transcriptional start site is responsive to signals delivered from the Th2 cytokine IL-4, via STAT6. However, the regulation of the enhancer is complex, because STAT6 both directly binds to the enhancer, and directs the synthesis of other genes required for arginase I expression. Furthermore, we found that multiple factors assemble at the enhancer, a property in keeping with other enhancer elements.
Arginase I expression in the liver is constitutive and must be maintained constantly, and coordinately, with the other enzymes that make up the urea cycle (1, 2). The basal, hepatocyte regulation of the genes encoding the urea cycle enzymes have been extensively dissected (11, 23, 24, 25, 26, 27). In contrast, the expression of the arginase I gene in macrophages is silent until the cells are stimulated with IL-4 or IL-13. Both of these cytokines activate STAT6 that is essential for many of the signal transduction events downstream of the IL-4 or IL-13 receptors. Although previous studies have shown that a variety of signals can regulate macrophage arginase I expression, IL-4 and IL-13 are by far the most potent. We began to search for the IL-4-regulated region of the arginase I promoter using the studies of Mori and colleagues (24, 25) as a guide. These investigators have dissected the regulatory regions involved in the hepatocyte-specific expression of the arginase I gene in rats and humans. When fused to reporter constructs and transfected into macrophages, arginase I promoter constructs containing the previously identified hepatocyte-specific regulatory regions were not responsive to IL-4. Therefore, we searched upstream of the transcriptional start site and eventually identified a small region, 3 kb from the start site that conferred IL-4-responsiveness upon arginase I basal promoter fragments. This region acted as a true enhancer element: it was functional independent of distance from the start site and was active in either orientation. The arginase I enhancer could not be reduced in size, and mutations introduced into putative transcription factor binding sites all affected activity. The activity of the enhancer was completely abrogated when a mutation in the putative STAT6 binding site was introduced, data that is consistent with the findings of Morris and colleagues (2) who reported that they identified a STAT6 site in a similar locale. ChIP experiments and EMSA analysis showed that STAT6 was directly recruited to the enhancer. This finding was surprising considering that our previous data suggested that STAT6 functioned by directing the expression of another gene(s) whose activity was required for arginase I mRNA expression. Therefore, STAT6 appears to have two functions, both direct and indirect (Fig. 6). This scenario is reminiscent of the IL-4 regulation of the polymeric Ig receptor gene where STAT6 also appears to have a dual function (28). Other factors that bind to the enhancer include PU.1, a crucial transcription factor in macrophage development and biology, C/EBP and the coactivator CBP. Mutations in the putative binding sites for PU.1 and C/EBP also reduced the activity of the enhancer. Overall, it appears that multiple factors form a complex at the enhancer and likely assemble in the correct temporal order. Thus, STAT6 directly binds, along with PU.1, CBP, C/EBP, and possibly other components, and this complex awaits the STAT6-regulated production of another factor that establishes transcriptional regulation of the locus.
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20,000 cDNAs. Here, we were searching for genes that were induced by IL-4 in the presence or absence of cycloheximide. Genes that require new protein synthesis to be induced (e.g., arginase I) would not be present in the samples treated with cycloheximide. In this screen, we identified a candidate transcription factor, ATF1. However, the induction of this protein was weak and not dependent on STAT6 (data not shown). At this stage, it is not possible to definitively determine whether STAT6 induces a transcription factor. It is possible that STAT6 regulates other processes required for transcription of the arginase I gene, including chromatin-dependent factors. In addition, we cannot definitively exclude the possibility that cycloheximide regulates the degradation of a factor(s) that assembles at the enhancer. This effect would preclude indirect effects of STAT6 on other genes. Direct pharmacological manipulation of arginase I in diseases where macrophage activity is implicated will most likely be impossible because of liver toxicity associated with the disruption of the urea cycle. For example, if arginase I activity promotes fibrosis in schistosomiasis (12, 14, 35) or accentuates asthmatic reactions (13), then our ability to regulate arginase I activity only in macrophages would be an appealing target. This report defines the basis for macrophage specificity and suggests that the control of the regulation of the arginase I enhancer could be targeted while sparing constitutive expression of arginase I in the liver.
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Acknowledgments |
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Footnotes |
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2 Current address: Centre National de la Recherche Scientifique, UMR8125, Institute Gustave Roussy, 39 rue Camille Desmoulins, Villejuif, France.
3 Institute fur Medizinische Mikrobiologie, Immunologie und Hygiene, Technical University, Munich, Germany.
4 Address correspondence and reprint requests to Dr. Peter J. Murray, Department of Infectious Diseases, St. Jude Children’s Research Hospital, 332 North Lauderdale, Room E8078, Mail Stop 320, Memphis, TN 38105. E-mail address: peter.murray{at}stjude.org
5 Abbreviations used in this paper: IRF, IFN regulatory factor; ChIP, chromatin immunoprecipitation; CBP, CREB binding protein.
Received for publication September 26, 2003. Accepted for publication April 13, 2004.
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References |
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)-hydroxy-L-arginine controls the growth of Leishmania inside macrophages. J. Exp. Med. 193:777.
M2 (CD11b/CD18) expression by cytokines is regulated by Stat3-dependent activation of PU. 1. J. Biol. Chem. 277:19001.This article has been cited by other articles:
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