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The Journal of Immunology, 2003, 170: 2564-2572.
Copyright © 2003 by The American Association of Immunologists

C/EBP{alpha} and Ets Protein Family Members Regulate the Human Myeloid IgA Fc Receptor (Fc{alpha}R, CD89) Promoter1

Toshibumi Shimokawa and Chisei Ra2

Allergy Research Center and Department of Immunology, Juntendo University School of Medicine, Tokyo, Japan; CREST, Japan Science and Technology Corp., Kawaguchi City, Japan; and Department of Molecular Cell Immunology and Allergology, Advanced Medical Research Center, Nihon University School of Medicine, Tokyo, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Fc{alpha}R (CD89), the FcR for IgA, is expressed exclusively in myeloid cells, including monocytes/macrophages, neutrophils, and eosinophils, and is thought to mediate IgA-triggered cellular functions in immunity. Here we demonstrate that the Fc{alpha}R 5'-flanking region from -102 to -64 relative to the ATG translation initiation codon is essential for promoter activity and contains two functional binding motifs for C/EBP and Ets family members at -74 and -92, respectively. EMSAs and cotransfection experiments show that C/EBP{alpha} acts as a major activator of the Fc{alpha}R promoter at least in immature myeloid cells. In addition, we found two additional functional targets of C/EBP{alpha} at -139 and -127. On the other hand, the Fc{alpha}R Ets binding motif could bind Elf-1 and mediate the trans-activation by cotransfected Elf-1, but a major component of the complex forming on this site appears to be an unidentified Ets-like nuclear protein that is preferentially detected in cells of hemopoietic origin. Furthermore, separation of the C/EBP and Ets binding sites reduces Fc{alpha}R promoter activity, suggesting some functional interaction between these factors. As the in vivo role of Fc{alpha}R is still incompletely defined, these findings reveal the features controlling the Fc{alpha}R promoter in myeloid lineage and provide a foundation for clarifying regulatory mechanisms of Fc{alpha}R gene expression associated with its potential roles.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Immunoglobulin A is the prominent Ig in secretions. While one of the protection mechanisms involving IgA is implicated in the neutralization and removal of environmental Ags from mucosal sites without inflammatory processes (1), an immune response through the specific FcR on effector cells has also been proposed as an active role for IgA in immunity. Indeed, the gene encoding a human myeloid Fc{alpha}R has been isolated and characterized (2). However, because no homologue of the human Fc{alpha}R gene has been isolated from any other species including the mouse to date, previous data regarding Fc{alpha}R functions are based on in vitro studies, except for more recent data obtained from transgenic mice expressing human Fc{alpha}R (3, 4).

Fc{alpha}R (CD89) is expressed exclusively in myeloid cell lineage associated with mucosal surfaces, including monocytes/macrophages (2, 5), neutrophils (2, 5), and eosinophils (6). Through Fc{alpha}R, IgA immune complexes are capable of triggering the effector functions of these cells, including phagocytosis, Ab-dependent cell-mediated cytotoxicity, superoxide production, and the release of inflammatory cytokines (reviewed in Ref. 7). Although the in vivo role of Fc{alpha}R is still incompletely defined, a recent report has demonstrated that Fc{alpha}R is expressed not only on circulating myeloid cells but also on liver Kupffer cells, and an active role for IgA was proposed as a second line of defense against invasive pathogens that have eluded destruction at the mucosal surface (3). In addition, altered Fc{alpha}R expression has been reported in allergic diseases (6), liver cirrhosis (8), HIV infection (9), and IgA nephropathy (IgAN)3 (10), suggesting a role for Fc{alpha}R in the pathogenesis of these diseases. In this regard Launay et al. (4) have recently shown that IgAN spontaneously develops in transgenic mice expressing human Fc{alpha}R on monocytes. Moreover, we have more recently identified functional promoter polymorphisms of the Fc{alpha}R gene (11) and suggested their association with IgAN (12). These observations raise the possibility that mechanisms causing certain inflammatory diseases might involve a regulatory factor of the Fc{alpha}R promoter, but mechanisms controlling Fc{alpha}R transcription are not well understood, and no transcription factors regulating this gene have been identified to date.

Using a transient transfection assay, we previously identified the promoter for the Fc{alpha}R gene that directs the expression of a reporter gene in the Fc{alpha}R-positive myeloid cell line U937 (11). In the present study we describe the identification of functional C/EBP and Ets sites within the Fc{alpha}R essential promoter region. In vitro assays demonstrate that the C/EBP site is bound by predominantly C/EBP{alpha}, and the Ets site is bound by Elf-1 and an unidentified Ets-like nuclear factor (HEL-NF1). Furthermore, cotransfection experiments show that C/EBP{alpha} and Elf-1 activate the Fc{alpha}R promoter.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture

The human promonocytic U937 and THP-1, premyelocytic HL60, T cell leukemia Jurkat, Burkitt’s lymphoma B cell Daudi, and epithelioma HeLa cell lines were cultured at 37°C in 5% CO2 and maintained in RPMI 1640 (Nissui Pharmaceutical, Tokyo, Japan) supplemented with 10% (v/v) heat-inactivated FBS (Life Technologies, Gaithersburg, MD), 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. The human hepatoma HepG2 cell line was maintained in DMEM (Life Technologies) with the supplements listed above.

Plasmid constructs

The promoterless firefly luciferase reporter plasmid pGL3-dBH, and the 929- and 259-bp Fc{alpha}R 5'-flanking region-luciferase constructs pGL-929 and pGL-259 (hereafter we refer to original pGL-732 and pGL-62 as pGL-929 and pGL-259), respectively, were previously described (11). To construct Fc{alpha}R promoter deletion mutants with various 3' end points, PCR-amplified DNA fragments were cloned into MluI/XhoI-digested pGL3-dBH. Constructs with a mutated Ets site on antisense strand at -87 to -90 from GGAA to TTAA, a mutated C/EBP site at -74 to -66 from TGTCGTAAG to TGTCCACCA, or mutated C/EBP sites at -139 to -131 from TGAGGCAAT to TGAGCACCA and at -127 to -119 from TGTGGAAAT to TGTGCACCA, and constructs with an extra 4 bp (GTAC) or 10 bp (GTACCTTAGA) between the C/EBP and Ets binding sites (at -80/-81) were generated by two-step PCR mutagenesis; underlining indicates mutated sequences. The human C/EBP{alpha} expression vector (hCMV-C/EBP{alpha}) (13) was provided by Dr. G. J. Darlington (Baylor College of Medicine, Houston, TX), and the corresponding empty vector (pCMV-Empt) was constructed by digestion with BamHI, followed by self-ligation. The human Elf-1 expression vector (pcDNAf-Elf-1) (14) was provided by Dr. J. M. Leiden (Harvard School of Public Health, Houston, TX), and the corresponding empty vector (pcDNA-Empt) was constructed by digestion with EcoRV and XbaI, followed by blunting and self-ligation.

Transfection

Plasmids were purified using a Maxiprep kit (Qiagen, Hilden, Germany) and transfected into cells using Superfect transfection reagent (Qiagen) in accordance with the manufacturer’s instructions. Briefly, U937 and THP-1 cells were plated at 2.5 x 106 cells in a 60-mm dish and transfected with 5 µg of DNA using 20 µl of Superfect transfection reagent. Jurkat cells were plated at 5 x 105 cells/well of a 24-well plate and transfected with 1 µg of DNA using 4 µl of the transfection reagent. HeLa cells were plated at 1.7 x 105 cells/well of a six-well plate the day before transfection and transfected with 2 µg of DNA using 10 µl of the transfection reagent. To normalize for transfection efficiency, the plasmid pRL-CMV (Promega, Madison, WI), which contains the Renilla luciferase gene driven by the CMV immediate early promoter, was included in transfections as 10% of the total DNA, except for HeLa and Jurkat cells in which it was included as 1 and 2%, respectively. All transfections were performed in duplicate. Cells were harvested 24 h after transfection and assayed for firefly and Renilla luciferases in a luminometer (Lumat LB9507, Berthold, Wildbad, Germany) using the Dual-Luciferase reporter assay system (Promega).

Transactivation of the Fc{alpha}R promoter

For trans-activation experiments for C/EBP{alpha}, pRL-SV40 (Promega), which contains the Renilla luciferase gene driven by the SV40 early promoter, was used to normalize for transfection efficiency. Jurkat cells were transfected as described above with 0.7 µg of reporter plasmid, 0.1 µg of the hCMV-C/EBP{alpha} expression plasmid, 0.03 µg of pRL-SV40, and sheared empty vector pCMV-Empt to a total of 1 µg of DNA/transfection. Control plates received 0.3 µg of the empty vector to allow a precise comparison. For trans-activation experiments for Elf-1, HeLa cells were transfected as described above with 1.8 µg of reporter plasmid, 0.2 µg of the pcDNAf-Elf-1 expression plasmid or empty vector (pcDNA-Empt), and 0.02 µg of pRL-CMV. The luciferase activities were measured 24 h after transfection.

Preparation of nuclear extracts

Nuclear extracts were prepared according to the method described by Schreiber et al. (15). Buffers A and C were supplemented with freshly added antipain, aprotinin, leupeptin, soybean trypsin-chymotrypsin inhibitor, pepstatin A (all 5 µg/ml), 1 mM benzamidine, 0.5 and 1 mM PMSF (all from Sigma-Aldrich, St. Louis, MO) for buffers A and C, respectively, and 0.5 mM DTT. Extracts were stored at -80°C, and protein concentrations were determined by the Bradford method (Bio-Rad, Hercules, CA) using {gamma}-globulin as a standard.

EMSA

Equimolar concentrations of each complementary single-stranded oligodeoxynucleotide (ODN) were annealed in 50 mM NaCl, 10 mM Tris-HCl (pH 7.6), and 10 mM MgCl2 and were 5'-end-labeled with T4 polynucleotide kinase and [{gamma}-32P]ATP (NEN, Boston, MA). Protein-DNA binding reactions were performed in 20-µl reaction volumes containing radiolabeled probe (25 fmol), 6 µg of nuclear extract, 10% buffer C (15), 0.5–1 µg of poly(dI-dC)·poly(dI-dC), 50 mM KCl, 20 mM Tris-HCl (pH 7.6), 0.2 mM EDTA, 1 mM DTT, 1 mM MgCl2, 0.5 mM spermidine, 0.01% Triton X-100, and 5% glycerol for 30 min on ice. Specific rabbit polyclonal antisera against human C/EBP{alpha}, C/EBP{beta}, C/EBP{delta}, C/EBP{epsilon}, PU.1, Elf-1, Fli-1, Ets-1/Ets-2, and Ets-2 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA), and control rabbit IgG was purchased from Sigma-Aldrich. Particular Ab (2 µg) or unlabeled competitor ODN were added to binding buffer and incubated on ice for 30 min before the addition of radioactive probe. Protein-DNA complexes were separated at 150 V on 4% nondenaturing polyacrylamide gels in 0.25 x TBE buffer (1x TBE: 89 mM Tris-Hcl; 89 mM boric acid, and 2 mM EDTA) at room temperature.

ODNs used in EMSA

ODNs used as double-stranded probes and competitors are as follows: Fc{alpha}R wild-type -82 to -52, wtC/EBP (5'-GAGCTTATTGTCGTAAGAATATCTGTCATCC-3'); Fc{alpha}R C/EBP site mutation -82 to -52, mutC/EBP (5'-GAGCTTATTGTCCACCAAATATCTGTCATCC-3'); high affinity consensus-binding site for C/EBP proteins, CEBP (5'-TGCAGATTGCGCAATCTGCA-3') (16), Fc{alpha}R wild-type -103 to -74, wtETS, (5'-TCTGTCCTCATACTTCCTGCGGAGCTTATT-3'); Fc{alpha}R Ets site mutation -103 to -74, mutETS (5'-TCTGTCCTCATACTTAATGCGGAGCTTATT-3'); consensus-binding site for Drosophila E74, E74 (5'-AATAACCGGAAGTAACTC-3') (17); consensus-binding site for Ets-1 derived from TCR{alpha} enhancer T{alpha}2, TCR{alpha} T{alpha}2 (5'-TTCCAGAGGATGTGGCTTCTGCGGGA-3') (18); consensus-binding site for Elf-1 derived from the IL-2 enhancer NFAT-1, IL-2 NFAT-1 (5'-AGAAAGGAGGAAAAACTGTTTCATACAGAAGGC-3') (14); and consensus-binding site for PU.1 derived from the SV40 enhancer, SV40 PU box (5'-TGAAATAACCTCTGAAAGAGGAACTTGGTTAGGTA-3') (19). Underlining indicates mutated sequences.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The Fc{alpha}R promoter contains a myeloid-specific essential regulatory region

Previously, we identified the Fc{alpha}R promoter in 929 bp of the Fc{alpha}R 5'-flanking region, which directs the expression of a reporter gene in myeloid cell line U937, but ~100-fold less activity in the cervical carcinoma cell line HeLa (11). To analyze in detail the cell type specificity of this region, 5' and 3' truncation mutants were fused to the firefly luciferase gene and transfected into various cell lines. In our previous papers (11, 12), we numbered nucleotide positions from the major transcription site that had been identified by de Wit et al. (20). However, since the Fc{alpha}R promoter appears to contain multiple transcription start sites (20) (our unpublished observations), we hereafter designate the ATG translation initiation codon as +1. In this numbering system, the previously identified major transcription start site corresponds to nucleotide -197.

As shown in Fig. 1, 929 bp of the 5'-flanking region (pGL-929) showed high levels of promoter activity in the Fc{alpha}R-positive myeloid cell lines U937 and THP-1, but ~2 orders of magnitude less activity in Fc{alpha}R-negative Jurkat T and nonhemopoietic HeLa cell lines. Similar results were obtained with the 5'-deleted DNA fragment (pGL-259). In contrast, the 3' deletion to -140 (pGL-259/-140) resulted in 200- and 70-fold reductions of luciferase activity in U937 and THP-1 cells, respectively (Fig. 1). With this construct, luciferase activity in Jurkat and HeLa cells reduced by only 2- and 7 fold, respectively, suggesting a strong tissue-specific positive regulatory element(s) within the 139-bp proximal flanking region.



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  		FIGURE 1. Cell type specificity of the Fc{alpha}R promoter. The Fc{alpha}R genomic organization is displayed at the top. The black boxes denote the protein-coding sequences: S1 and S2, signal sequences; EC1 and EC2, extracellular Ig-like domains; TM/C, transmembrane and cytoplasmic domains. The numbers indicate nucleotide positions relative to the ATG translation initiation codon at +1. The bent arrows indicate the major transcription start site (-197) identified by de Wit et al. (20 ). The indicated cells were transfected with each construct as described in Materials and Methods. Firefly luciferase activity was normalized to Renilla luciferase activity. Values represent fold activity over the level of the promoterless construct (pGL3-dBH) and the SEM in each cell line. The numbers of independent experiments employed to calculate these data are shown in parentheses. The means of the background values (firefly luciferase/Renilla luciferase of pGL3-dBH) were: U937, 2,587 relative light units (RLU)/597,560 RLU; THP-1, 2,117 RLU/25,571,152 RLU; Jurkat, 74,644 RLU/3,081,873 RLU; HeLa, 21,790 RLU/84,676,505 RLU.

 
Essential regulatory region located within -102/-64 bp of the Fc{alpha}R translation initiation site contains functional C/EBP and Ets binding motifs

To determine the functional cis elements of the Fc{alpha}R promoter, we constructed successive 3' truncation mutants and transfected them into U937 cells. As shown in Fig. 2, deletion to -30 (pGL-259/-30) resulted in a 2-fold decrease in promoter activity, suggesting the presence of a positive regulatory element. Further deletion to -64 (pGL-259/-64) did not affect promoter activity. However, deletion to -103 (pGL-259/-103) abolished almost all promoter activity, indicating that the promoter region between -102 and -64 is essential. Therefore, we analyzed this region in greater detail.



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  		FIGURE 2. Deletion analysis of the Fc{alpha}R promoter region. U937 cells were transfected with the indicated constructs as described in Materials and Methods. Values are corrected for transfection efficiency and represent activity relative to pGL-259 set as 100%. Thin bars represent SEM from 6–10 transfections. *, p < 0.002 compared with pGL-259 by paired two-tailed t test; **, p < 0.001 compared by pGL-259/-64 by two-tailed t test. The means of the control values (firefly luciferase/Renilla luciferase of pGL-259) were 677,298 RLU/894,724 RLU.

 
As shown in Fig. 3, we found within the essential region two potential binding motifs for the Ets family at -92 to -85 on the lower strand (5'-CAGGAAGT-3') (21) and for the C/EBP family at -74 to -66 (5'-TGTCGTAAG-3') (22). As shown in Fig. 4, when the potential C/EBP site was mutated in the context of the -259/-64 promoter (pGLmCE3–259/-64), the promoter activity in U937 cells was reduced by 3-fold and was almost identical with that seen in the deletion mutant that removed the site (pGL-259/-81). When the potential Ets site was mutated (pGLmETS-259/-64), promoter activity was reduced by 4-fold. Also, mutation of both sites (pGLmETSmCE3–259/-64) resulted in a further reduction in promoter activity, although the reduced activity was higher than that seen in the deletion mutant that removed both sites (compare with pGL-259/-103). A similar deleterious effect of these mutations was also observed in the context of the 259-bp promoter with U937 and THP-1 cells (Fig. 4 and data not shown). These data demonstrate that both the Ets and C/EBP sites in the essential region are functionally critical.



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  		FIGURE 3. Transcription factor binding motifs in the Fc{alpha}R promoter region and ODN employed for EMSA. The numbers indicate nucleotide positions relative to the ATG translation initiation codon at +1. Potential binding sequences for Ets and C/EBP families are boxed. In addition to the Ets site at -92 and the C/EBP site at -74 in the essential regulatory region, two potential C/EBP-binding sites located at -139 and -127 are also shown. Mutated sequences are indicated in bold.

 


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  		FIGURE 4. Deletion and mutation analyses of the Ets and C/EBP sites in the Fc{alpha}R essential promoter region. U937 cells were transfected with the indicated constructs as described in Materials and Methods. Thin bars represent the SEM from three to nine transfections. *, p < 0.001 compared with pGL-259/-64 by two-tailed t test; **, p < 0.001, p < 0.002, and p < 0.05 compared with pGLmCE3–259/-64, pGLmETS-259/-64, and pGL-259/-103, respectively, by two-tailed t test; ***, p < 0.005 compared with pGL-259 by paired two-tailed t test; ****, p < 0.001 and p < 0.01 compared with pGLmCE3–259 and pGLmETS-259, respectively, by two-tailed t test.

 
C/EBP{alpha} predominantly binds to the functional C/EBP site in the Fc{alpha}R promoter region

To identify nuclear protein(s) binding to the functional C/EBP site, an ODN spanning from -82 to -52 (wtC/EBP; Fig. 3) was subjected to EMSA. As shown in Fig. 5A, U937 nuclear extracts produced complexes (lane 2) whose formation was prevented by unlabeled self ODN (lanes 3 and 4), but only partially by the mutated C/EBP site (mutC/EBP; Figs. 3 and 5A, lanes 5 and 6). In addition, the previously described high affinity C/EBP-binding sequence (CEBP) (16) considerably prevented the complex formation (lanes 7 and 8), strongly suggesting that the factors specifically recognizing the functional C/EBP site belong to the C/EBP family.



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  		FIGURE 5. EMSA of the Fc{alpha}R promoter C/EBP site. A, U937 nuclear extracts were incubated with the labeled wtC/EBP probe in the absence (lane 2) or the presence of 20- and 100-fold molar excesses of cold specific competitor (wtC/EBP; lanes 3 and 4), mutated C/EBP site competitor (mutC/EBP; lane 5 and 6), and high affinity C/EBP site competitor (16 ) (CEBP; lane 7 and 8). Lane 1 contains probe alone. The specifically formed C/EBP complexes are indicated. B, U937 nuclear extracts were incubated with the labeled wtC/EBP probe in the presence of rabbit control IgG (lane 1) or antisera against C/EBP{alpha} (lane 2), C/EBP{beta} (lane 3), C/EBP{alpha} and C/EBP{beta} (lane 4), C/EBP{delta} (lane 5), and C/EBP{epsilon} (lane 6). {circ}, Supershifted complexes. C, U937 nuclear extracts were incubated with the labeled CEBP probe in the absence (lane 1) or the presence of a 100-fold molar excess of cold Fc{alpha}R C/EBP site competitor (wtC/EBP; lane 2), rabbit control IgG (lane 3), antisera against C/EBP{alpha} (lane 4), C/EBP{beta} (lane 5), and C/EBP{alpha} and C/EBP{beta} (lane 6). Lane 1 contains probe alone. {circ}, Supershifted complexes. The numbers below each lane number indicate the relative intensity of specific complexes as determined by NIH Image software.

 
The C/EBP family belongs to the basic leucine zipper transcription factors and includes at least six members that homodimerize or heterodimerize with each other in a tissue-specific manner (reviewed in Ref. 23). In the hemopoietic system, C/EBP{alpha}, C/EBP{beta}, C/EBP{delta}, and C/EBP{epsilon} are present predominantly in myeloid cells, and their expression is differentially regulated during myeloid differentiation (24, 25). Supershift EMSA showed that the specific complexes were eliminated and supershifted by anti-C/EBP{alpha} Ab (Fig. 5B, lane 2) and weakly affected by anti-C/EBP{beta} Ab (lane 3). As well, a further modest reduction in complex formation was observed with anti-C/EBP{beta} Ab in addition to anti-C/EBP{alpha} Ab (lane 4). In contrast, control rabbit IgG (lane 1) and antisera against C/EBP{delta} and C/EBP{epsilon} had no effect (lanes 5 and 6). A reverse EMSA clearly demonstrated that complexes with the high affinity C/EBP consensus binding sequence were completely competed by the Fc{alpha}R C/EBP site (lane 2 in Fig. 5C) and were supershifted predominantly by anti-C/EBP{alpha} Ab and partially by anti-C/EBP{beta} Ab (lanes 4 and 5, respectively). Moreover, the combination of antisera against C/EBP{alpha} and C/EBP{beta} resulted in a further reduction in complex formation (lane 6). Taken together, we concluded that the complexes recognizing the functional C/EBP site in the Fc{alpha}R promoter contain predominantly C/EBP{alpha} homodimers and in part C/EBP{beta}-containing dimers.

C/EBP{alpha} trans-activates the Fc{alpha}R promoter

To examine whether C/EBP{alpha} functions as a regulator of the Fc{alpha}R promoter, trans-activation experiments were performed in Jurkat cells. As shown in Fig. 6A, Fc{alpha}R promoter activity was considerably increased (26-fold) by cotransfected C/EBP{alpha}. A mutation in the C/EBP site resulted in a strong decrease in the level of absolute promoter activity in the presence of C/EBP{alpha} (Fig. 6A, pGLmCE3–259). However, the trans-activation by C/EBP{alpha} was decreased only slightly, and the pGLmCE3–259 construct was still trans-activated to >20-fold. Because the promoterless plasmid was no longer trans-activated by C/EBP{alpha} (pGL3-dBH; Fig. 6A), this indicates that the Fc{alpha}R promoter region contains an additional C/EBP{alpha} target site(s). In this regard we found two potential C/EBP binding sites (-139 to -131 and -127 to -119) upstream of the C/EBP site at -74 (Fig. 3). To determine whether these additional C/EBP sites compensate for the mutated -74 C/EBP site, we constructed the plasmid pGLmCE12–259 in which both upstream C/EBP sites are mutated. As shown in Fig. 6B, when this plasmid was cotransfected with C/EBP{alpha}, we observed an ~10-fold decrease in trans-activation by C/EBP{alpha} (compared with pGL-259 in Fig. 6A), but Fc{alpha}R promoter activity was still increased 2.6-fold (pGLmCE12–259; Fig. 6B). This trans-activation depends on the functional C/EBP site at -74, since the mutant promoter containing the further mutation in the -74 C/EBP site (pGLmCE123–259) was no longer trans-activated by C/EBP{alpha}. Taken together, these results indicate that C/EBP{alpha} functions as a major activator of the Fc{alpha}R promoter and both the -74 C/EBP site and the additional upstream target(s) are required for full activation.



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  		FIGURE 6. Trans-activation of the Fc{alpha}R promoter by C/EBP{alpha}. Jurkat cells were transfected with the indicated constructs in the absence or the presence of hCMV-C/EBP{alpha} as described in Materials and Methods. Two potential C/EBP binding sites located at -139 and -127 are indicated in addition to the Ets site at -92 and C/EBP site at -74 in the essential regulatory region. Values are corrected for transfection efficiency and are presented as the fold activity over the level of the promoterless construct (pGL3-dBH) in the absence of C/EBP{alpha}. Fold stimulation by C/EBP{alpha} represents promoter activity with cotransfected CEBP{alpha} divided by that obtained without the expression vector. The data represent the mean and SEM from three or four transfections. *, Significant decrease (p < 0.05) compared with pGL-259 by one-tailed t test for matched pairs.

 
Characterization of the DNA-protein complexes associated with the functional Ets site in the Fc{alpha}R promoter region

As shown in Fig. 7A, EMSA using the functional Ets site of the Fc{alpha}R promoter showed four complexes in U937 nuclear extracts (E1, E2, E3, and E4; lane 2). Complexes E1 and E2 were competed by self ODN (compare lanes 3 and 4 with lane 2), but not by the mutated Ets site (mutETS; lanes 5 and 6), indicating that they recognize the functional Ets site. Furthermore, they were also competed by consensus binding motifs for a Drosophila Ets protein E74 (17) (lanes 7 and 8) and other known Ets family members, although their competition efficiencies varied (see Fig. 8B). These results strongly suggest that complexes E1 and E2 contain Ets family members. On the other hand, complexes E3 and E4 were nonspecific, because they were competed by neither self ODN (Fig. 7A, lanes 3 and 4) nor binding sequences for other Ets proteins (Fig. 7A, lanes 7 and 8, and see Fig. 8B).



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  		FIGURE 7. EMSA of the Fc{alpha}R promoter Ets site. A, The labeled wtETS probe was incubated without (lane 1) or with U937 nuclear extracts in the absence (lanes 2) or the presence of 20- and 100-fold molar excesses of cold specific competitor (wtETS; lanes 3 and 4), mutated Ets site competitor (mutETS; lanes 5 and 6), and Drosophila E74-binding site (17 ) (E74; lanes 7 and 8). At least four complexes, E1, E2, E3, and E4, can be detected and are indicated. B, Cell type specificity of DNA-binding proteins that interact with wtETS. Lane 1, U937 cells (monocytic cell line); lane 2, THP-1 cells (monocytic cell line); lane 3, HL60 cells (myelocytic cell line); lane 4, Jurkat cells (T cell line); lane 5, Daudi cells (B cell line); lane 6, HeLa cells (epithelial carcinoma); lane 7, HepG2 cells (hepatocellular carcinoma). The numbers below each lane number indicate the relative intensity of complex E1.

 


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  		FIGURE 8. Characterization of HEL-NF1 DNA-binding properties. U937 nuclear extracts were incubated with the labeled wtETS probe. A, Supershift EMSA using rabbit control IgG (lane 1) or rabbit antisera against PU.1 (lane 2), Ets-2 (lane 3), Elf-1 (lane 4), Fli-1 (lane 5), and Ets-1/Ets2 (lane 6). The specifically formed complexes HEL-NF1 and E2 are indicated. {circ}, Supershifted complexes. B, Supershift EMSA without (lane 1) or with a 100-fold molar excess of the indicated competitor ODN in the absence (lanes 1, 2, 4, and 6) or the presence of antisera against Elf-1 (lane 3), Ets-2 (lane 5), and Fli-1 (lane 7). The numbers below each lane number indicate the relative intensity of HEL-NF1.

 
We next examined the cell type specificity of the Ets-binding factors. Fig. 7B demonstrates that complex E1 is present in a wide variety of hemopoietic cells and is much less abundant in nonhemopoietic HeLa and HepG2 extracts. On the other hand, complex E2 was detected exclusively in U937 cells and was absent in other cell lines, including THP-1 (Fig. 7B). Because the deleterious effect of the mutated Ets site on Fc{alpha}R promoter activity was also observed in THP-1 cells to a similar extent as in U937 cells (data not shown), complex E2 is unlikely to mediate Fc{alpha}R promoter activity through the functional Ets site. Taken together, we conclude that complex E1, which is predominantly restricted to cells of hemopoietic origin, is responsible for Fc{alpha}R promoter activity. Hereafter this complex denotes hemopoietic Ets-like nuclear factor 1 (HEL-NF1).

To attempt to identify the Ets family member representing HEL-NF1, a supershift EMSA was performed. As shown in Fig. 8A, no considerable reduction in complex formation was observed with Abs against PU.1, Ets-2, Elf-1, Fli-1, and Ets-1/Ets-2, but supershifted bands were seen exclusively after addition of anti-Fli-1 Ab (lane 5), suggesting that a very minor proportion of the complexes forming on the Ets site contains Fli-1. Again we observed neither elimination nor reduction of HEL-NF1 or complex E2 after adding anti-Fli-1 Ab.

To examine whether a minor portion of HEL-NF1 contains one of the Ets proteins that we tested, we performed supershift EMSA in combination with competitor ODN containing Ets-binding motifs that influence the DNA binding affinity of different Ets proteins. The TCR{alpha} enhancer T{alpha}2 site has previously been shown to bind Ets-1, but not Elf-1 (14). When used as a competitor, the T{alpha}2 site greatly reduced complex formation on the Fc{alpha}R Ets site, but a minor part comigrating with HEL-NF1 was still detected (Fig. 8B, compare lane 2 with lane 1). This remaining species was totally abolished by the anti-Elf-1 Ab (lane 3), indicating that Elf-1 can weakly bind the Fc{alpha}R Ets site. In contrast, complex resistant to the IL-2 promoter NFAT-1 competitor, which interacts with Elf-1, but not with Ets-1 (14), was not affected by the anti-Ets-2 Ab (Fig. 8B, compare lane 5 with lane 4). This suggests that Ets-2 is not a component of HEL-NF1, because Ets-1 and closely related Ets-2 have been shown to recognize similar binding sequences (26). A similar analysis was performed for the anti-Fli-1 Ab that produced supershifted bands in Fig. 8A. The SV40 PU box has been reported to bind PU.1, but not Fli-1 (27). However, a minor complex resistant to this competitor was not affected by the anti-Fli-1 Ab while the supershifted bands were still detected (Fig. 8B, lane 7). This result suggests that the supershifted complexes might result from Ab-triggered induction or stabilization of Fli-1 binding to the Ets site rather than direct DNA binding, as reported for several nuclear factors (28). Taken together, we concluded that although a major component of HEL-NF1 is an unidentified Ets-like factor, Elf-1 can also bind to the Fc{alpha}R Ets site with a low affinity.

Trans-activation of the Fc{alpha}R promoter by Elf-1

Because our EMSA indicates that Elf-1 can bind to the Fc{alpha}R functional Ets site, trans-activation experiment was performed in HeLa cells. As shown in Fig. 9, Fc{alpha}R promoter activity was increased to 11-fold by cotransfected Elf-1 (pGL-259). This trans-activation was decreased (3-fold reduction) by mutation at the Ets site (pGLmETS-259), but promoter activity was still increased 4-fold. This is probably due to a cryptic Elf-1 binding sequence in the reporter plasmid backbone, because background values of the promoterless plasmid were also activated by Elf-1 (4-fold increase) identical with that seen in pGLmETS-259 with Elf-1 (Fig. 9). These results suggest that regulation of the Fc{alpha}R promoter through the functional Ets site could be in part mediated by Elf-1.



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  		FIGURE 9. Trans-activation of the Fc{alpha}R promoter by Elf-1. The indicated constructs were cotransfected into HeLa cells with or without pcDNAf-Elf-1 as described in Materials and Methods. Values are corrected for transfection efficiency and are presented as the fold activity over the level of the promoterless construct (pGL3-dBH) in the absence of Elf-1. Fold stimulation by Elf-1 represents promoter activity with cotransfected Elf-1 divided by that obtained without the expression vector. The data represent the mean and SEM from three transfections. *, Significant decrease (p < 0.02) compared with pGL-259 by one-tailed t test for matched pairs.

 
Extending the distance between the adjacent C/EBP and Ets binding sites reduced Fc{alpha}R promoter activity

The binding sites for C/EBP and HEL-NF1 in the essential regulatory region of the Fc{alpha}R promoter are in close proximity (only 10 bp between them; see Fig. 3). To investigate the possibility that C/EBP and HEL-NF1 cooperate with each other, we constructed 4- and 10-bp insertion mutants between these binding sites to alter the orientation on the helix and the distance between these factors. As displayed in Fig. 10, both insertions reduced Fc{alpha}R promoter activity, suggesting that interaction of C/EBP and HEL-NF1 might contribute to expression of the Fc{alpha}R gene.



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  		FIGURE 10. Effect of the distance between the Ets and C/EBP sites on Fc{alpha}R promoter activity. U937 cells were transfected with the indicated constructs as described in Materials and Methods. Indicated are only the Ets and C/EBP sites in the essential regulatory region at -92 and -74, respectively. Firefly luciferase activity was determined as described in Fig. 2. Thin bars represent the SEM from three transfections. *, Significant decrease (p < 0.05) compared with pGL-259 by two-tailed t test for matched pairs.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
While the in vivo role of Fc{alpha}R is still incompletely clarified, uncovering the mechanisms controlling Fc{alpha}R gene expression will elucidate mechanisms underlying altered Fc{alpha}R expression in response to environmental factors in normal and various pathological situations, which leads to understanding of IgA-mediated immune defense through Fc{alpha}R. This is the first report describing the identification of transcription factors involved in Fc{alpha}R gene expression.

In this study we identified C/EBP{alpha} as a major activator of the Fc{alpha}R promoter using immature myeloid cell lines. Myeloid precursors can develop into either granulocytes or monocytes/macrophages, and Fc{alpha}R is expressed on both cell lineages (2, 5, 6). However, targeted disruption in mice (29) and enforced expression in immature myeloid cell lines (30) have recently demonstrated that C/EBP{alpha} is a critical factor for granulocytic, but not monocytic, differentiation. Moreover, it has been shown that C/EBP{alpha} is rapidly down-regulated during the monocytic differentiation and is undetectable in human peripheral blood monocytes, in contrast to the observation of its high level expression in blood neutrophils as well as its rapid up-regulation during granulocytic differentiation (30, 31). These observations indicate that while C/EBP{alpha} is implicated as a granulocyte regulator of Fc{alpha}R expression, the mechanism regulating monocytic expression involves another transcription factor(s). One such candidate is a distinct member of the C/EBP family. For example, the expression of C/EBP{beta} has been shown to increase during monocytic differentiation (32). Furthermore, targeted disruption of C/EBP{beta} in mice demonstrated monocyte/macrophage dysfunction resulting in high susceptibility to bacterial infection (33, 34). In our preliminary experiments using nuclear extracts from primary monocytes, complexes with the functional C/EBP site at -74 were supershifted by anti-C/EBP{beta} Ab, but not anti-C/EBP{alpha} Ab (our unpublished observations). Although it remains to be determined whether the Fc{alpha}R promoter is activated by C/EBP{beta}, C/EBP{beta} may regulate certain monocyte functions through controlling Fc{alpha}R expression levels.

Additional control is exerted by an Ets binding site. At present, the identity of HEL-NF1 is unknown. In our characterization Elf-1 is capable of binding to the Fc{alpha}R Ets site with low affinity. Although it is unlikely to represent a major component of HEL-NF1 (Fig. 8), cotransfection experiments demonstrated that Elf-1 is able to induce some degree of trans-activation of the Fc{alpha}R promoter through the functional Ets site (Fig. 9), indicating that it acts as a transcriptional activator of the Fc{alpha}R gene in this context. A regulatory role for the Fc{alpha}R Ets site may not be a consequence of binding of the HEL-NF1 major component alone, but rather a result of combinatorial interactions among coexpressed Ets proteins such as Elf-1.

Although C/EBP{alpha} and Ets family members including Elf-1 have to date been shown to regulate myeloid-specific gene expression, they are also expressed outside the myeloid lineage. Also, we detected HEL-NF1 in a wide variety of hemopoietic cell lines, including Fc{alpha}R-negative cells. Considering previously reported promoters that are regulated by multiple transcription factors, it is suggested that cell type-specific expression of the Fc{alpha}R gene is regulated by some combination of C/EBP{alpha} and other transcription factors, including HEL-NF1. In fact, C/EBP{alpha} has recently been reported to physically interact with several Ets family members, such as Ets-1 and Fli-1 (35). In our first approach, artificial spacing introduced between the functional Ets and C/EBP binding sites in the essential region significantly decreased promoter activity (Fig. 10), suggesting that HEL-NF1 and C/EBP{alpha} interact and cooperatively regulate Fc{alpha}R gene expression. Understanding the mechanism of this regulation will require further studies, including a study to determine the identity of HEL-NF1.

In addition to the C/EBP site at -74, we found two targets of C/EBP{alpha} at -139 and -127. In the cotransfection experiments with Jurkat cells, mutation in both sites at -139 and -127 showed a 10-fold decrease (from 26- to 2.6-fold) in trans-activation by C/EBP{alpha}, whereas mutation in the -74 C/EBP site resulted in a significant, but modest, decrease (from 26- to 22-fold), suggesting that the two additional C/EBP sites appear to be more critical than the -74 site. Alternatively, the -74 C/EBP site might function critically in combination with the Ets site as discussed above, because mutation in the C/EBP site at -74 showed a considerable reduction in the level of Fc{alpha}R promoter activity in Jurkat cells cotransfected with C/EBP{alpha} (Fig. 6A) as well as U937 cells (Fig. 4). In addition, because the effects of C/EBP{alpha} through the -74 site and the -139 and -127 sites appear to be additive, it is possible that the two upstream C/EBP sites act independently of the -74 site. In this context, the Fc{alpha}R promoter might be regulated through two independent mechanisms involving C/EBP factors.

While the deletion from -102 to -64 (pGL-259/-103) resulted in the loss of almost all promoter activity (Figs. 2 and 4), the construct with double mutation of the Ets and C/EBP sites in the context of the -259/-64 promoter (pGLmETSmCE3–259/-64) still retained low, but significant, activity (Fig. 4), strongly suggesting an additional cis element within the essential region. Such a sequence is likely to be located around the Ets site, because the C/EBP mutation in the context of the -259/-64 promoter (pGLmCE3–259/-64) reduced promoter activity to a level almost identical with that seen in the mutant that deleted the C/EBP, but not the Ets, site (pGL-259/-81; Fig. 4). It has been emphasized that transcriptional regulation mediated by many Ets proteins requires or is in concert with additional transcription factors (36). Further analyses with point mutations will identify novel important sequences.

In summary, the human Fc{alpha}R promoter contains cis elements that appear to play a critical role in the regulation mediated by C/EBP{alpha} and Ets family members. Further examination of the interplay between these factors and additional factors should provide the foundation for understanding the regulatory mechanism of Fc{alpha}R in terminal differentiation into functionally specialized immune cells and for resolving how the Fc{alpha}R promoter is regulated in a reflection of its potential roles.


   Acknowledgments
 
We thank Dr. Gretchen J. Darlington (Baylor College of Medicine, Houston, TX) for the C/EBP{alpha} expression vector, and Dr. Jeffrey M. Leiden (Harvard School of Public Health, Houston, TX) for the Elf-1 expression vector. We also thank Dr. Shinsaku Togo for experimental help, and Drs. Takafumi Uchida and Yasuhiro Gon for preparing the figures for this manuscript.


   Footnotes
 
1 This work was supported in part by grants in aid from the Japanese Ministries of Education, Health, and Welfare and the Environment Agency. Back

2 Address correspondence and reprint requests to Dr. Chisei Ra, Department of Molecular Cell Immunology and Allergology, Advanced Medical Research Center, Nihon University School of Medicine, 30-1 Oyaguchi-kamimachi, Itabashi-ku, Tokyo 173-8610, Japan. E-mail address: fcericra{at}med.nihon-u.ac.jp Back

3 Abbreviations used in this paper: IgAN, IgA nephropathy; CEBP, high affinity C/EBP-binding sequence; ODN, oligodeoxynucleotide; RLU, relative light units. Back

Received for publication July 22, 2002. Accepted for publication December 20, 2002.


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 Results
 Discussion
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