Different GATA Factors Dictate CCR3 Transcription in Allergic Inflammatory Cells in a Cell Type–Specific Manner

Su-Kang Kong, Byung Soo Kim, Tae Gi Uhm, Wonyong Lee, Gap Ryol Lee, Choon-Sik Park, Chul-Hoon Lee and Il Yup Chung
J Immunol June 1, 2013, 190 (11) 5747-5756; DOI: https://doi.org/10.4049/jimmunol.1203542

Abstract

The chemokine receptor CCR3 is expressed in prominent allergic inflammatory cells, including eosinophils, mast cells, and Th2 cells. We previously identified a functional GATA element within exon 1 of the CCR3 gene that is responsible for GATA-1–mediated CCR3 transcription. Because allergic inflammatory cells exhibit distinct expression patterns of different GATA factors, we investigated whether different GATA factors dictate CCR3 transcription in a cell type–specific manner. GATA-2 was expressed in EoL-1 eosinophilic cells, GATA-1 and GATA-2 were expressed in HMC-1 mast cells, and GATA-3 was preferentially expressed in Jurkat cells. Unlike a wild-type CCR3 reporter, reporters lacking the functional GATA element were not active in any of the three cell types, implying the involvement of different GATA factors in CCR3 transcription. RNA interference assays showed that small interfering RNAs specific for different GATA factors reduced CCR3 reporter activity in a cell type–specific fashion. Consistent with these findings, chromatin immunoprecipitation and EMSA analyses demonstrated cell type–specific binding of GATA factors to the functional GATA site. More importantly, specific inhibition of the CCR3 reporter activity by different GATA small interfering RNAs was well preserved in respective cell types differentiated from cord blood; in particular, GATA-3 was entirely responsible for reporter activity in Th2 cells and replaced the role predominantly played by GATA-1 and GATA-2. These results highlight a mechanistic role of GATA factors in which cell type–specific expression is the primary determinant of transcription of the CCR3 gene in major allergic inflammatory cells.

Introduction

The inflammatory infiltrates in allergic diseases such as asthma, allergic rhinitis, and atopic dermatitis consist primarily of Th2 cell subsets, mast cells, and eosinophils. CCR3 is a chemokine receptor initially thought to be specific to eosinophils (1, 2), but its cell surface expression was subsequently identified on Th2 cells (3) and mast cells (4). The mobilization of these cells may depend to a large extent on the expression of CCR3. The restricted expression pattern of CCR3 in these cell types leads to the notion that it plays an integral role in the pathogenesis of allergic diseases. Beyond the inflammatory pathways associated with CCR3 expression in these cell types, CCR3 has recently been demonstrated to be expressed in nonmotile cells, including airway epithelial cells (5, 6) and choroidal endothelial cells (7), where it is postulated to function in airway remodeling and neovascularization, respectively. These findings implicate CCR3 as a diagnostic marker and a therapeutic target of related inflammatory conditions. Understanding the molecular mechanism underlying CCR3 expression might provide a valuable approach for cell type–specific control of CCR3 expression.

The mammalian GATA transcription factor family consists of six members that share common features: they recognize the consensus sequence WGATAR and control gene expression, and they have two zinc finger motifs for DNA binding and protein–protein interactions. GATA-1, GATA-2, and GATA-3 are mainly expressed within hematopoietic lineages and play both distinct and overlapping roles in hematopoiesis by activating tissue-specific genes (8). Eosinophils and mast cells express both GATA-1 and GATA-2, consistent with the additional observation that these two GATA factors are often redundantly expressed in many hematopoietic cells including erythroid precursors and megakaryocytes (9–14).

GATA-1 is intimately associated with eosinophil development and eosinophil-specific gene expression. Ablation of a palindromic dblGATA enhancer binding site in the promoter of the mouse GATA-1 gene results in ablation of the eosinophil lineage in vivo (15), whereas GATA-1 expression reprograms an avian myeloblastic cell line into eosinophils (16). Ectopic expression of GATA-1 in human CD34+ hematopoietic progenitors gives rise to the eosinophil lineage in vitro, whereas GATA-1–deficient mice fail to develop eosinophil progenitors in the fetal liver (17). Functional GATA elements for GATA-1 binding are present in the promoters and/or regulatory regions of eosinophil-specific genes encoding major basic protein (MBP) (18), EOS47 (19), Charcot–Leyden crystal (CLC) protein (20), gp91phox (21), and CCR3 (22). With respect to mast cells, mice expressing a reduced level of GATA-1 display defective mast cell maturation and development (14, 23). Furthermore, ectopic GATA-1 expression activates the expression of mast cell–specific genes coding for carboxypeptidase A (12) and FcεRIα (24).

GATA-2–deficient mice lack mast cells, and the deletion of GATA-2 ablates mast cell differentiation of embryonic stem cells (25). GATA-2 functions cooperatively with PU.1 to specify the mast cell fate (26, 27). GATA-2 is necessary for early mast cell development (27, 28), whereas downregulation of GATA-2 is essential for maturation of bone marrow mast cells (29). GATA-2 also has instructive capacity in eosinophil development comparable to that of GATA-1 in vivo and in vitro, whereas a dominant negative form of GATA-2 prevents eosinophil formation as efficiently as a dominant negative GATA-1 (17). The timing of expression of GATA-2 is an important factor in the determination of eosinophil lineage. GATA-2 instructs C/EBPα-expressing granulocyte/monocyte progenitor to induce eosinophil generation while directing basophil and mast cell lineages upon suppression of C/EBPα at the granulocyte/monocyte progenitor stage (30). In addition, GATA-2 alone is able to activate transcription of the eosinophil-specific genes gp91phox (21) and EDN (31).

GATA-3 is highly expressed in Th2 cells and is critical for the commitment and differentiation of these cells; therefore, it is regarded as a Th2 cell master regulator (32). The GATA-3 transcript is also abundantly expressed in eosinophils and mast cells (12, 13, 17), although the functional role of GATA-3 in these cells has not been reported.

The most critical regulatory sequences for CCR3 gene transcription reside in exon 1, which is 161 bp long (33) and includes five GATA binding sites. Our previous study, using reporters with a series of point mutations, EMSA, and chromatin immunoprecipitation (ChIP) assays, demonstrated that the first GATA site in the nontranslated first exon is solely responsible for GATA-1–mediated transactivation of CCR3 and exhibits high-affinity binding for the GATA factor in several cell types, including myeloid and epithelial cells (22), thus establishing GATA-1 as a critical activator of the CCR3 gene. However, as CCR3 is expressed at substantial levels in mast cells and Th2 cells that predominantly and/or exclusively express GATA-2 and GATA-3, respectively, we hypothesized that these two GATA factors might actively participate in the regulation of CCR3 expression in these allergic inflammatory cells. GATA-1 and GATA-2 are often functionally redundant in the regulation of genes that are specifically expressed in erythrocytes, eosinophils, and mast cells. However, no studies have been done at the molecular level on whether GATA-3 shares the activity that is predominantly performed by GATA-1 and GATA-2 in eosinophils and mast cells. In the current study, we elucidate the molecular basis of the regulation of CCR3 expression by the different GATA factors in a cell type–specific manner, using cell lines representing these allergic cells and cord blood (CB)–derived cells that are more physiologically relevant to the three allergic inflammatory cell types.

Materials and Methods

Ethics statement

Written informed consent was obtained from all subjects. The protocols used in this study were approved by Hanyang University’s ethics committee (HYG-11-019-1).

Materials

IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, stem cell factor (SCF), GM-CSF, and FLT3 ligand were purchased from R&D Systems (Minneapolis, MN).

Cell culture

Jurkat cells were purchased from the Korean Cell Line Bank (Seoul, Korea), and K562 cells were purchased from American Type Culture Collection (Manassas, VA). HMC-1 cells and EoL-1 cells were kindly given by Drs. Jae Hong Kim (Korea University, Seoul, Korea) and Yun-Jae Jung (Gacheon University, Incheon, Korea), respectively. Jurkat, EoL-1, and K562 cells were maintained in RPMI 1640 medium (Welgene, Seoul, Korea), and HMC-1 cells were maintained in IMDM (Welgene). All growth media were supplemented with 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin. Eosinophils, mast cells, and Th2 cells were induced from human umbilical CB mononuclear cells. Briefly, CD34+ cells were purified by density-gradient centrifugation over Ficoll-Paque PREMIUM 1.073 (density, 1.077 g/ml; GE Healthcare, Uppsala, Sweden), followed by positive selection using a MACS CD34+ MicroBead Kit (Miltenyi Biotec, Auburn, CA). Eosinophils were induced from CD34+ cells, as previously described (34), and confirmed by intracellular staining with anti-human MBP Ab (BD Pharmingen, San Diego, CA), which indicated > 90% purity. Mast cells were induced as previously reported (35). CD34+ cells were cultured in IMDM containing 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin and supplemented with a cytokine mixture of SCF (50 ng/ml), IL-6 (5 ng/ml), and IL-10 (5 ng/ml) for 7 d. Cells were then plated in 12-well plates at 2 × 106 cells per well in medium supplemented with SCF, IL-6, and IL-10, and incubated for up to 5 additional weeks, with medium changes every week. Purity of the CB mast cells was > 90%, as determined by staining with anti-human FcεRIα (Millipore, Bedford, MA) or anti–c-Kit Abs. Th2 cells were prepared from CB CD4+ cells, as previously described (36). CB CD4+ cells were purified using the CD4+ T Cell Isolation Kit (Miltenyi Biotec) according to the manufacturer's protocol and seeded in 12-well plates at 1 × 106 cells per well. The cells were cultured in RPMI 1640 containing 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin and supplemented with a cytokine mixture of IL-2 (10 ng/ml), IL-4 (10 ng/ml), and anti–IL-12 Ab (2 μg/ml) for 10 d. The CB Th2 cells were stimulated with plate-bound anti-CD3 Ab for 1 d. The purity of Th2 cells was confirmed by the presence of intracellular IL-4 and absence of intracellular IFN-γ.

GATA factor mRNA expression

Total mRNAs were extracted from cell lines and CB-derived cells, using TRI reagent (Molecular Research Center, Cincinnati, OH). First-strand cDNA was synthesized from 4 μg total RNA, using SuperScript II Reverse Transcriptase (Invitrogen Life Technologies) in a 20-μl reaction containing random primers, deoxynucleotide triphosphates (0.5 mM), MgCl2 (2.5 mM), and DTT (5 mM). Reverse transcription was performed at 42°C for 1 h, followed by heat inactivation at 70°C for 15 min. The synthesized cDNA was amplified for 30 cycles with Ex Taq DNA polymerase (TaKaRa, Shiga, Japan). The following primers were used in the amplification: GATA-1 forward, 5′-GCCCTGACTTTTCCAGTACC-3′, and reverse, 5′-CGAGTCTGAATACCATCCTTCC-3′; GATA-2 forward, 5′-TGTCACTGACGGAGAGCATG-3′, and reverse, 5′-CGTCTGACAATTTGCACAACAG-3′; GATA-3 forward, 5′-CTCATTAAGCCCAAGCGAAGG-3′, and reverse, 5′-GGGTTAAACGAGCTGTTCTTG-3′; GAPDH forward, 5′-CGTCTTCACCACCATGGAGA-3′, and reverse, 5′-CGGCCATCACGCCACAGTTT-3′.

Real-time PCR

Quantitative real-time PCR (qRT-PCR) analysis of CCR3 mRNA expression was performed in a 20-μl reaction with 1 μl cDNA, 1 μl of each primer (10 pM), and 10 μl SYBR Green Master Mix (Applied Biosystems, Foster City, CA), using the Applied Biosystems Prism 7900 Sequence Detection System (Applied Biosystems). PCR conditions were as follows: 2 min at 50°C and 10 min at 95°C, followed by 40 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 30 s. PCR was performed in triplicate in 96-well plates. CCR3 mRNA levels were normalized to PP1A mRNA level. The specificity of amplification was confirmed by melting curve analysis and gel electrophoresis. Relative expression was evaluated using the comparative cycle threshold (2−ΔΔCt) method and expressed as mean ± SEM. The following primers were used for amplification: CCR3 forward, 5′-ATGCTGGTGACAGAGGTGAT-3′, and reverse, 5′-AGGTGAGTGTGGAAGGCTTA-3′; PP1A forward, 5′-TCCTGGCATCTTGTCCATG-3′, and reverse 5′-CCATCCAACCACTCAGTCTTG-3′.

Flow cytometry

Cells were fixed in 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO) for 15 min at 4°C, washed with 2% BSA in PBS, and then stained with PE-conjugated anti-human CCR3 Ab (R&D Systems) or isotype-matched Ab (BD Pharmingen, San Diego, CA). To determine expression of intracellular IL-4 and IFN-γ, cells were stained with an FITC-conjugated anti-human IL-4 (BioLegend, San Diego, CA) and PE-conjugated anti-human IFN-γ Ab (BioLegend). Isotype-matched Ab was also used to stain the cells. Stained cells were analyzed using a flow cytometer, and data analysis was performed using CellQuest Software (Becton Dickinson, San Jose, CA).

Western blot analysis

Cells were lysed in RIPA buffer (50 mM Tris-Cl [pH 7.4], 0.1% NaN3, 1% Nonidet P-40, 0.25% sodium deoxycholate, 1 mM EDTA, 1 mM Na3VO4, 1 mM NaF, and protease inhibitor mixture) supplemented with 0.4 M NaCl. Lysates were centrifuged, and the resulting supernatants were subjected to Western blot analysis. A total of 30 μg of the cell lysate was resolved by SDS-PAGE and then transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% nonfat dry milk and then probed with anti–GATA-1 (M-20; Santa Cruz Biotechnology, Santa Cruz, CA), anti–GATA-2 (H-116; Santa Cruz Biotechnology), anti–GATA-3 (HG3-35; Santa Cruz Biotechnology), anti–friend of GATA-1 (FOG-1) (M-20; Santa Cruz Biotechnology), and anti-GAPDH Abs (Santa Cruz Biotechnology). The membranes were incubated with anti-goat HRP-conjugated Ab (Santa Cruz Biotechnology) for GATA-1, anti-rabbit HRP-conjugated Ab (Cell Signaling Technology, Beverly, MA) for GATA-2, and anti-mouse HRP-conjugated Ab (Cell Signaling Technology) for GATA-3. Immunostained proteins were visualized using an ECL detection system (Amersham Biosciences, Buckinghamshire, U.K.).

ChIP analysis

Physical associations between GATA factors and the CCR3 regulatory region in K562 and HMC-1 cells were analyzed using a ChIP assay kit (Millipore) according to the manufacturer’s instructions. Cells were treated with formaldehyde for 10 min at 37°C, incubated in lysis buffer, and sonicated to shear the chromatin. The cross-linked protein–DNA complexes were immunoprecipitated with the Abs used for Western blot analysis. Goat IgG (Sigma-Aldrich), rabbit IgG (Sigma-Aldrich), and mouse IgG (Abcam) were used as control Abs for GATA-1, GATA-2, and GATA-3, respectively. Anti-histone 3 Ab (Abcam, Cambridge, U.K.) and water were used as positive and negative controls, respectively. The Ab-bound complexes were pulled down with protein A-agarose/salmon sperm DNA, and the cross-links were reversed. DNA was recovered through phenol/chloroform extraction and ethanol precipitation and used as a template for PCR amplification, with PCR primers targeted to regions spanning the functional GATA element in exon 1 of the CCR3 gene (nucleotides −65 to +197, with numbering according to the longest exon 1), as previously described (22). The following primers were used in the amplification: forward primer, 5′-GCTAGTCTGTTTAAAACAGGAAG-3′, and reverse primer, 5′-TGGAAAAGCGACACCTACCT-3′.

Luciferase assay for reporter plasmids

CCR3 reporter plasmids were previously described (22). Four cell lines and CB-derived cells were cotransfected with reporter plasmids and pRL-TK vector (Promega, Madison, WI), using Lipofectamine 2000 Reagent (Invitrogen Life Technologies) or Amaxa 4D-Nucleofector (Lonza, Koln, Germany). Luciferase activity was measured using the Dual-Luciferase Reporter System (Promega), and transfection efficiency was normalized to Renilla luciferase activity.

RNA interference

Small interfering RNA (siRNA)–dependent knockdown experiments were conducted using GATA-2 siRNA (Santa Cruz Biotechnology) and ON-TARGETplus SMARTpool siRNA for GATA-1, GATA-3, and nontargeting (scrambled) control pools (Dharmacon, Chicago, IL). The sequence of FOG-1 double-stranded siRNA was previously described (37). Briefly, cells were cotransfected with siRNA, and the CCR3 reporter using Amaxa 4D-Nucleofector and luciferase activities was measured. An aliquot of the transfected cells was taken for Western blot analysis of GATA factor protein expression.

Nuclear extract preparation and EMSA

Cells were lysed with RIPA buffer containing 0.15 M NaCl and resuspended in RIPA buffer containing 0.4 M NaCl. Lysates were centrifuged, and the resulting supernatants (nuclear extracts) were analyzed by EMSA. A total of 4 μg nuclear extract was incubated with the [32P]-labeled probe for 20 min in binding buffer [50 mM Tris-Cl (pH 7.5), 20% glycerol, 5 mM MgCl2, 2.5 mM EDTA, 2.5 mM DTT, 250 mM NaCl, and 0.25 mg/ml poly(dI-dC) • poly(dI-dC)]. For supershift analysis, extracts were incubated with the anti-GATA Abs used for Western blot analysis and control Abs (Sigma-Aldrich), followed by radiolabeled probe.

Statistical analysis

All statistical analyses were performed using the Microsoft Excel data analysis program or SPSS 10.0 software (SPSS, Chicago, IL). An independent t test was used to compare relative reporter activities between two groups. Comparisons of the activities in three or more independent groups were performed using one-way ANOVA followed by post hoc analysis. Differences with a p value < 0.05 were considered statistically significant. The results are expressed as mean ± SEM.

Results

Expression of CCR3 and GATA factors in representative allergic inflammatory cells

To investigate the regulation of CCR3 expression by GATA-1, GATA-2, and GATA-3 in allergic inflammatory cells, we first examined the expression of these molecules in three different cell lines: Jurkat T cells, EoL-1 eosinophilic cells, and HMC-1 mast cells. These three representative cell lines constitutively expressed appreciable levels of CCR3 mRNA expression relative to K562 cells (Fig. 1A). Cell surface CCR3 expression was largely consistent with CCR3 mRNA expression (Fig. 1A). EoL-1 cells expressed GATA-2 mRNA and protein, and HMC-1 cells expressed both GATA-1 and GATA-2 transcripts and proteins at comparable levels. With respect to expression of GATA-3, although GATA-3 mRNA was previously shown to be relatively abundantly expressed in eosinophils and mast cells (12, 13, 17), expression of GATA-3 protein has not been reported in these cell types. In our study, EoL-1 and HMC-1 cells expressed GATA-3 mRNA, but not protein (Fig. 1B), and Jurkat cells expressed GATA-3 protein at a high level and GATA-2 protein at a lower but detectable level (Fig. 1B). Thus, GATA-3 protein expression did not necessarily match its mRNA expression, in line with a previous study that showed a significant positive correlation between mRNA and protein levels only for a subset of the genes (38). By contrast, K562 cells expressed a low level of CCR3 mRNA and protein (Fig. 1A). This cell line was used as a reference for regulation of CCR3 transcription mediated by GATA factors because we have previously used this cell line and various CCR3 reporter plasmids to dissect GATA-1–dependent regulation of the CCR3 gene at the molecular level (22). K562 cells are known to abundantly express GATA-1 mRNA and protein and GATA-2 mRNA (14, 39, 40), but expression of GATA-2 protein has not been reported. Our new data showed that GATA-2 protein was also abundantly expressed in K562 cells (Fig. 1B). In addition, like the eosinophilic and mast cell lines examined in this study, K562 cells did not express GATA-3 protein. Overall, the four cell types displayed distinct expression profiles of GATA factor family members at the protein level. The concomitant expression of GATA-2 and/or GATA-3 together with CCR3 raised the possibility that these two GATA factors could modulate CCR3 transcription individually or in combination in addition to GATA-1, which is known to be necessary for CCR3 transcription (22).

FIGURE 1.
FIGURE 1.

Four different cell types expressing CCR3 display distinct expression patterns of different GATA factors. (A) Expression of CCR3 mRNA and protein was analyzed by qRT-PCR and FACS, respectively. The qRT-PCR results shown were representative of three or more independent experiments and expressed as mean ± SEM relative to that of K562 cells. (B) Expression of mRNA and protein for three GATA factors was assessed by RT-PCR and Western blotting, respectively. GAPDH mRNA and protein were used as loading controls.

Involvement of different GATA factors in CCR3 transcription in allergic inflammatory cells

We previously demonstrated GATA-1–mediated regulation of CCR3 gene expression. Introduction of a dominant negative form of GATA-1 or GATA-1 siRNA into K562 cells resulted in a marked decrease in transcriptional activity of the CCR3 reporter (22). The cis-acting element responsible for GATA-1–mediated transcription maps to the functional GATA site within exon 1 of the gene (Fig. 2A). A point mutation or deletion of this site leads to severe reduction of transcription in K562 cells (22). Although the first GATA site, AGATAC, is different from the 3′ end of the consensus GATA binding motif (A/T)GATA(A/G), it exhibited high-affinity GATA-1 binding in vitro and was occupied by GATA-1 in chromatin. We next determined reporter activities in the three types of allergic inflammatory cells. The wild-type reporter was active in all three cell types, as in K562 cells, but the two reporters lacking the functional GATA site had virtually no activity (Fig. 2B), indicating that different GATA factors may transactivate the CCR3 reporter through binding to the functional GATA site. Because HMC-1 mast cells redundantly express both GATA-1 and GATA-2 proteins (Fig. 1), the CCR3 reporter activities seen in HMC-1 cells could result from the integrated effect of GATA-1 and GATA-2. In contrast, as EoL-1 cells expressed GATA-2 protein exclusively and Jurkat cells predominantly expressed GATA-3 protein, GATA-2 and GATA-3 were likely to be responsible for activation of the reporter in these respective cell types, raising the possibility that GATA-2 and GATA-3 can also activate CCR3 transcription.

FIGURE 2.
FIGURE 2.

GATA factor–dependent transactivation of CCR3 in the four different cell types. (A) Three CCR3 constructs were previously described (22). The wild-type construct of the CCR3 gene spanned exon 1 and the proximal intron 1 sequence and contained the first GATA element, which was shown to be solely responsible for GATA-1–mediated transactivation. Two additional constructs lacking the functional GATA site were included to confirm GATA-specific regulation of the constructs. Open and closed ovals indicate wild-type and mutant GATA sites, respectively. (B) The three reporters were transfected into the four cell lines with Lipofectamine 2000, and the reporter activities were measured 48 h after transfection. Transfection efficiency was normalized to cotransfected Renilla luciferase activity. The transcription activities were determined as relative values (%) compared with the transcription activity of pGL3 vector. The data were expressed as mean ± SEM of three independent experiments performed in triplicate.

Inhibition of CCR3 transcription by siRNAs specific for different GATA factors

To determine the functional involvement of the three different GATA factors in the activation of CCR3 transcription, we examined whether siRNAs specific for the different GATA factors could inhibit CCR3 reporter activities. When Jurkat cells, which expressed a high level of GATA-3 protein and a modest level of GATA-2 protein, were transfected with GATA-3 siRNA, both the reporter activity and the GATA-3 protein level were decreased, whereas GATA-1 and GATA-2 siRNAs had little effect on the CCR3 reporter activity (Fig. 3A, 3B). In HMC-1 cells, which expressed both GATA-1 and GATA-2 proteins at comparable levels, either GATA-1 or GATA-2 siRNA considerably reduced reporter activity, and a combination of both siRNAs further inhibited the reporter activity with a decrease in the levels of GATA-1 and GATA-2 proteins. In contrast, GATA-3 siRNA had little effect on the reporter activity in this cell line (Fig. 3B). These results indicate that GATA-1 and GATA-2 individually transactivate the CCR3 reporter in HMC-1 cells. In EoL-1 cells, which exclusively expressed GATA-2 protein, GATA-2 siRNA inhibited reporter activity accompanied by a decrease in the GATA-2 protein level. Thus, availability of GATA factors and their expression level serve as the primary determinant of GATA factor–mediated CCR3 transactivation. Intriguingly, although transfection of K562 cells, which abundantly expressed both GATA-1 and GATA-2 proteins, with GATA-1 or GATA-2 siRNA led to significant decreases in the respective protein levels, only GATA-1 siRNA inhibited the CCR3 reporter activity (Fig. 3A, 3B), whereas GATA-2 siRNA augmented the CCR3 reporter activity. Further experiments showed that the CCR3 reporter activity was upregulated by GATA-2 siRNA in a dose-dependent manner (Supplemental Fig. 1). At this time, we do not know the mechanisms by which GATA-2 regulates the CCR3 reporter activity in this cell type. In addition, because FOG-1 activates or represses numerous GATA-1 target genes (41), we tested the effect of FOG-1 siRNA on CCR3 reporter activity in K562 cells. FOG-1 protein was highly expressed in K562 cells, and its level was reduced by a FOG-1 siRNA (Fig. 4A). The FOG-1 siRNA inhibited CCR3 reporter activity in a dose-dependent manner (Fig. 4B). When equal concentrations (50 nM) of GATA-1, GATA-2, and FOG-1 siRNAs were introduced into K562 cells, a simultaneous knockdown of FOG-1 and GATA-1 further reduced the CCR3 reporter activity in an additive manner (Fig. 4C). FOG-1 siRNA also reduced the effect of GATA-2 siRNA. When a nuclear extract of K562 cells that had been transfected with FOG-1 siRNA was incubated with the EMSA probe harboring the functional GATA site, GATA-1 binding was nearly eliminated (Fig. 4D). This result suggests that the GATA-1 binding is FOG-1 dependent in this cell type.

FIGURE 3.
FIGURE 3.

Different GATA factors transactivate the CCR3 reporter in a cell type–specific manner. siRNAs directed against different GATA factors were introduced together with the CCR3 reporter into the four cell lines. K562 and HMC-1 cells were transfected with 50 nM siRNA, using Lipofectamine 2000, whereas Jurkat and EoL-1 cells were transfected with 100 nM siRNAs, using Amaxa 4D-Nucleofector. (A) An aliquot of the transfectants was subjected to Western blotting to confirm knockdown efficiency. Two cell lines that express both GATA-1 and GATA-2, K562 and HMC-1, were tested with GATA-1 and GATA-2 siRNAs, whereas EoL-1 and Jurkat cells were tested with GATA-2 and GATA-3 siRNAs, respectively. The results are representative of two to three independent experiments. (B) Activity of the CCR3 reporters was measured. Transfection efficiency was normalized to cotransfected Renilla luciferase activity. The transcription activities were determined as relative values (%) compared with the transcription activity of pGL3 vector. The data are expressed as mean ± SEM of three independent experiments performed in triplicate. Statistical significance was obtained using ANOVA, compared with scrambled siRNA. *p < 0.05, **p < 0.01.

FIGURE 4.
FIGURE 4.

FOG-1 knockdown leads to downregulation of the CCR3 reporter activity in K562 cells. (A) K562 cells were transfected with 50–200 nM FOG-1 siRNA or scrambled siRNA (200 nM), and were examined for knockdown efficiency with anti-FOG Ab. (B) The CCR3 reporter was cotransfected with increasing concentrations of FOG-1 siRNA or scrambled siRNA (0, 10, 25, 50, and 100 nM) into K562 cells. Data represent the mean ± SEM of three independent experiments, performed in triplicate. Statistical significance was obtained using an independent t test, compared with scrambled siRNA. *p < 0.05, **p < 0.01. (C) Equal concentrations (50 nM) of FOG-1, GATA-1, and GATA-2 siRNAs individually or in combination were introduced into K562 cells with the CCR3 reporter. Data represent means ± SEM of three independent experiments, performed in triplicate. The data are expressed as mean SEM of three independent experiments performed in triplicate. Statistical significance was obtained using ANOVA, compared with scrambled siRNA. *p < 0.05, **p < 0.01. (D) K562 cells were transfected with FOG-1 (200 nM) or scrambled siRNAs (200 nM) into K562 cells, and cultured for 36 h. The nuclear extract was then preincubated with anti–GATA-1 (S) or control Abs (C) before being mixed with the EMSA probe.

In vitro and in vivo binding of different GATA factors to the functional GATA site

We examined whether CCR3 transactivation by the GATA factors paralleled their ability to bind to the functional GATA site. When nuclear extract from Jurkat cells was incubated with Abs against different anti-GATA factors, the DNA–protein complex was supershifted strongly with anti–GATA-3 and weakly with anti–GATA-2, but not with anti–GATA-1. In EoL-1 cells, which expressed only GATA-2 protein, only anti–GATA-2 induced a supershift of the complex. In HMC-1 cells, where GATA-1 and GATA-2 siRNAs almost equally efficiently inhibited the CCR3 reporter activity, both GATA-1 and GATA-2 Abs induced the supershifted complex, although anti–GATA-1 had a greater effect than did anti–GATA-2 (Fig. 5A). Thus, the DNA binding activities of the GATA factors appeared to strictly correlate with their expression levels and their abilities to transactivate the CCR3 reporter. Furthermore, in agreement with the inability of GATA-2 siRNA to inhibit CCR3 reporter activity in K562 cells, anti–GATA-2 failed to form the supershifted complex and slightly inhibited formation of the DNA–protein complex that presumably harbored GATA-1 (Fig. 5A). To investigate the interaction of GATA factors with the functional GATA site in the CCR3 regulatory region in vivo, a ChIP assay was performed. Cross-linked chromatin was sonicated and immunoprecipiated with anti–GATA factor Abs, the respective control Abs, or anti-histone 3 Ab, the latter two being controls to determine the specificity and efficiency of ChIP assay, respectively. The resulting immunoprecipitates were analyzed with PCR primers targeted to region −65 to +197, which spans the functional GATA element in exon 1 of the CCR3 gene. Specific DNA bands were observed for anti–GATA-2 immunocomplexes from EoL-1 cells (Fig. 5B). Both GATA-3 and GATA-2, although to a lesser extent, bound to the region containing a functional GATA element in Jurkat cells. In HMC-1 cells, both GATA-1 and GATA-2 proteins occupied this region of the CCR3 gene. Consistent with the DNA binding pattern of GATA factors in nuclear extract of K562 cells in the EMSA analysis, only anti–GATA-1 was able to pull down GATA-1–DNA complexes. These results indicate that the three GATA factors associate with chromatin at the sequence spanning the functional GATA element within the CCR3 regulatory region in a cell type–specific manner.

FIGURE 5.
FIGURE 5.

Different GATA factors bind the functional GATA element in the regulatory region of the CCR3 gene in a cell type–specific manner. Cell type–specific binding of GATA factors was observed: GATA-1 for K562; GATA-2 and GATA-3 for Jurkat; GATA-2 for EoL-1; and GATA-1 and GATA-2 for HMC-1 cells. (A) EMSA was performed with nuclear extract of the four cell lines and a [32P]-labeled probe including the functional GATA element. For supershift assays, specific (S) and control (C) Abs for the different GATA factors were used as follows: GATA-1, goat anti-human GATA-1 Ab (M-20) and goat IgG (Sigma-Aldrich); GATA-2, rabbit anti-human GATA-2 Ab (H-116) and rabbit IgG (Sigma-Aldrich); GATA-3, mouse anti-human GATA-3 Ab (HG3-35) and mouse IgG (Abcam), respectively. All Abs were purchased from Santa Cruz Biotechnology, unless otherwise mentioned. (B) ChIP assay. Specific (S) and control (C) Abs for the GATA factors were used to pull down DNA fragments bound to GATA factors. Anti-histone 3 Ab (Abcam) was used as a positive control for ChIP efficiency. DW indicates no DNA template and was used to show lack of nonspecific binding in the ChIP assays. The pulled-down DNA fragments were amplified by conventional PCR using a specific primer pair. The results are representative of three independent experiments.

Expression of GATA factors and CCR3 reporter activity in eosinophils, mast cells, and Th2 cells differentiated from CB

To investigate the physiologic relevance of transcriptional activation of the CCR3 gene by GATA factors, we induced in vitro differentiation of the above three cell types by culturing CB CD34+ cells for eosinophils and mast cells and by in vitro polarization of CB CD4+ cells for Th2 cells. The identity of CB-derived eosinophils and mast cells was confirmed by demonstrating that the majority of eosinophils expressed MBP at day 24 (95.2 ± 2.1%; n = 3) and mast cells expressed FcεR1α at day 42 (90.1 ± 3.5%; n = 3). The differentiated CB Th2 cells had a typically skewed Th2 cytokine profile of intracellular IL-4high and IFN-γ−. Thus, the CB-derived cell types expressed their respective signature molecules (Fig. 6A). The eosinophils derived from CB CD34+ cells highly expressed CCR3 mRNA and protein, whereas mast cells expressed CCR3 mRNA and protein at low levels, as previously reported (35, 42). The in vitro polarized Th2 cells derived from CB CD4+ cells expressed CCR3 at intermediate level (Fig. 6B). In addition, the CB CD34+ cell–derived eosinophils and mast cells expressed both GATA-1 and GATA-2 proteins, but not GATA-3, whereas Th2 cells exclusively expressed GATA-3 mRNA and protein (Fig. 6C). When CCR3 reporters driven by either wild-type or mutant GATA elements were introduced into the three cell types, the wild-type reporter was activated in all three cell types with the greatest activity in eosinophils, whereas the mutant reporter had virtually no activity in any of the three cell types (Fig. 6D). This result indicates that the functional GATA site is critical for GATA factor–mediated transactivation of the CCR3 reporter in the three different cell types, as in the representative cell lines. Introduction of individual GATA-1 or GATA-2 siRNAs resulted in a decrease in reporter activity in eosinophils and mast cells, and combined treatment with GATA-1 and GATA-2 siRNAs had an additive inhibitory effect on CCR3 reporter activity. In contrast, only GATA-3 siRNA reduced reporter activity in Th2 cells. These results suggest that regulation of CCR3 reporter activity by different GATA factors is almost exactly phenocopied in CB-derived eosinophils, mast cells, and Th2 cells in a cell type–specific manner (Fig. 6D).

FIGURE 6.
FIGURE 6.

Transactivation of the CCR3 reporter by the different GATA factors is almost exactly duplicated in the three cell types derived from CB. (A) Eosinophils and mast cells were derived from CB CD34+ cells, and Th2 cells were generated by in vitro polarization of CB CD4+ cells, as described in Materials and Methods. Identities of eosinophils, mast cells, and Th2 cells were confirmed by expression of their signature molecules: MBP, FcεRIα, and IL-4highIFN-γ−, respectively. (B) CCR3 mRNA and protein were analyzed in the three cell types. The qRT-PCR result is representative of three independent experiments and expressed as mean ± SEM relative to that of mast cells. (C) mRNAs and proteins of the three GATA factors were analyzed. GAPDH mRNA and protein were used as loading controls. (D) CCR3 reporter activities. GATA factor siRNAs were transfected into the three CB-derived cell types using Amaxa 4D-Nucleofector. Transfection efficiency was normalized to cotransfected Renilla luciferase activity. The transcription activities were determined as relative values (%) compared with the transcription activity of pGL3 vector. The data were expressed as mean ± SEM of three independent experiments performed in triplicate. Statistical significance was obtained using ANOVA, compared with scrambled siRNA. *p < 0.05, **p < 0.01.

Discussion

CCR3, which is commonly expressed in allergic inflammatory cells, including Th2 cells, eosinophils, and mast cells, is the major chemokine receptor responsible for mobilization of these cells to inflamed tissues. We have previously demonstrated that a cis-acting GATA binding motif in the untranslated first exon of CCR3 gene is critical for transactivation of the gene (22). Given that these allergic inflammatory cells express different GATA factors individually or in combination, and that GATA factors share an action mechanism and the majority of target sites in the cells (43, 44), we asked whether different GATA factors or their combination might be involved in the regulation of CCR3, depending on the cell type. Our results show the engagement of different GATA factors in CCR3 transcription in a cell type–specific manner: GATA-1 and GATA-2 function redundantly in eosinophils and mast cells in an additive manner, whereas GATA-3 is necessary for CCR3 transcription in Th2 cells. In particular, the positive regulation of CCR3 transcription by GATA-3 is unique to Th2 cells. Moreover, these three GATA factors appear to exert their functions through an identical GATA site in the chromatin. The primary determinant of which GATA factor is involved in CCR3 transcription is which GATA factor protein or proteins are predominantly expressed in the cell, as the availability of a particular GATA factor confers specific binding to the functional GATA site of the CCR3 regulatory region and subsequent transactivation. Furthermore, this study provides compelling evidence for the functional redundancy of GATA-3 in gene expression in which GATA-1 and GATA-2 predominantly act to activate transcription in allergic inflammatory cells. To our knowledge, this study is the first to elucidate how these three GATA factors redundantly regulate transcription of a particular gene at the molecular level in a cell type–specific manner.

Our results show that in HMC-1 cells, CB-derived eosinophils, and mast cells expressing comparable levels of endogenous GATA-1 and GATA-2, the CCR3 reporter activity was decreased upon RNA silencing of either GATA-1 or GATA-2 and was further reduced upon knockdown of both. Thus, GATA-1 and GATA-2 appear to have an additive effect on CCR3 transactivation. GATA-1 and GATA-2 are known to elicit transactivation of numerous eosinophil- and mast cell–specific genes (45). At a molecular level, GATA-1 stimulates not only transactivation of MBP (18), EOS47 (19), CLC protein (20), gp91phox (21), and CCR3 (22) genes that are specific for eosinophils, but also genes encoding carboxypeptidase A (12) and FcεRIα (24) that are specific for mast cells. GATA-2 stimulates transactivation of gp91phox (21) and EDN (31) in eosinophils. In most cases, these two GATA factors participate in transactivation of their target genes through direct binding to GATA sites in their regulatory or promoter regions. Because GATA-1 and GATA-2 are redundantly expressed in eosinophils and mast cells, at first glance it might be assumed that they play overlapping roles in regulation of their target genes and/or compensate for each other in these cell types. However, considerable gene-specific differences can be noted in their roles in the expression of eosinophil-specific genes. For example, GATA-1 and GATA-2 can equally effectively transactivate the gp91phox gene (21); GATA-1, but not GATA-2, transactivates the MBP gene (18), whereas GATA-2, but not GATA-1, strongly transactivates the EDN gene (31); and GATA-2 represses GATA-1–mediated transactivation of MBP and gp91phox genes (18, 21), but activates transactivation of the EDN gene, partly through upregulation of GATA-1 (31). Thus, the specific roles of these two GATA factors in transactivation of their target genes are divergent among eosinophil- and/or mast cell–specific genes. However, most of these observations were largely based on transfection of genes encoding GATA-1 and GATA-2. As overexpression studies risk off-target effects and provide the opportunity for proteins to aberrantly affect cellular regulatory networks, the effects of ectopically expressed GATA factors on their target genes remain uncertain with respect to physiologic contexts. In this sense, knockdown experiments that allow us to assess function by controlling endogenous expression levels have greater physiologic significance, and our knockdown data strongly support the redundant function of GATA-1 and GATA-2 in CCR3 expression.

It was previously demonstrated that cells expressing surface IL-5Rα within a human common myeloid progenitor population give rise exclusively to an eosinophil progenitor. These eosinophil progenitor cells express surface CCR3 at a low level, and CCR3 expression increases as the cells mature (46). CB-derived mast cells express a modest level of CCR3 at a relatively early stage during the differentiation period of 9 wk (35). It is therefore thought that CCR3 is expressed at a very early stage in the development of human eosinophils and mast cells. GATA-1 and GATA-2 mRNAs are equally coexpressed in eosinophil progenitors (46, 47) and human peripheral blood eosinophils (13, 17). GATA-1 and GATA-2 proteins coexist in primary cultures of bone marrow–derived mast cells, although GATA-2 is more abundantly expressed in immature mast cells than is GATA-1, and vice versa in mature mast cells (14). In addition, in our CB-derived eosinophils and mast cells, both GATA-1 and GATA-2 mRNAs were expressed during the entire development/differentiation period (data not shown). Because the expression profiles of these two GATA factors largely overlap in these two cell types, they may transactivate the CCR3 gene individually or in combination by acting on the same functional GATA site throughout cellular development/differentiation. Although CB provides an excellent cellular source for eosinophils and mast cells for studying the regulation of genes specific to these cell types, it is difficult to assess temporal expression profiles of GATA-1 and GATA-2 in a particular cell because the expression of specific GATA factors changes during development/differentiation and such changes are often measured in a cell population. Therefore, this model per se would not be suitable for examining the involvement of different GATA factors in CCR3 transactivation in relation to developmental stage. Nonetheless, monitoring the expression of GATA factors during developmental processes of these cells increases our understanding of the transcriptional networks that regulate lineage-specific gene expression.

Although GATA-3 is highly expressed in Th2 cells and is essential for the differentiation of these cells (32), GATA-3 expression and function are not limited to Th2 cells. GATA-3 is expressed in multiple tissues and cell types (48) and is required for T cell development (49) and the functions of NK cells (50) and regulatory T cells (51). Moreover, GATA-3 transcript is relatively abundantly expressed in eosinophils and mast cells (12, 13, 17). Thus, GATA-3 plays multifaceted roles in immune functions in diverse cell types. Our results demonstrated that GATA-3 binds to the functional GATA site of the CCR3 regulatory region in vitro and in vivo (Fig. 5) and, more importantly, is required for transactivation of the CCR3 reporter in Jurkat cells and in vitro polarized Th2 cells from CB (Figs. 3, 6). Therefore, GATA-3 serves as an important activator of CCR3 expression in Th2 cells that express GATA-3 protein, but not GATA-1 or GATA-2. This result emphasizes the important finding that GATA-3 can replace GATA-1 and GATA-2 in CCR3 expression, a function that GATA-1 and GATA-2 typically perform in a predominant manner at the molecular level. GATA-3 was previously demonstrated to successfully sustain embryonic erythropoiesis by rescuing GATA-1–deficient mice from embryonic lethality, although the underlying molecular mechanism is totally unknown (52). In addition, GATA-2 and GATA-3 function redundantly in early hematopoietic progenitors to sustain GATA-2 gene expression via multiple cis-acting GATA motifs upstream of the regulatory region (53). The results from these studies and our current data provide evidence for functional substitution of GATA-1 and/or GATA-2 with GATA-3.

Although the presence of GATA factor proteins is certainly a critical factor in transactivation of the CCR3 reporter through binding to the functional GATA site of the gene, this is not always the case. For example, despite the fact that K562 cells express both GATA-1 and GATA-2 proteins at comparable levels, GATA-2 failed to bind to the functional GATA site both in vitro and in vivo (Fig. 5) and did not promote CCR3 reporter activity (Fig. 3). Rather, GATA-2 appeared to inhibit CCR3 reporter activity in K562 cells, as GATA-2-specific siRNA dose-dependently augmented reporter activity (Supplemental Fig. 1). Sequence analysis of GATA-2 cDNA from K562 cells revealed that none of the amino acid residues in the GATA-2 C-finger and its flanking regions was altered (data not shown), ruling out the possibility that the failure of GATA-2 to bind the GATA site was due to altered structure of its DNA binding domain in K562 cells. Assuming that binding of GATA factors to their binding sites is a prerequisite for transactivation, we do not know what prevents binding of GATA-2 to the naked GATA motif in vitro (Fig. 5A) or to the chromatin DNA (Fig. 5B).

It is worth noting that FOG-1, a coregulator of GATA factors, appears to promote CCR3 reporter activity in K562 cells, as FOG-1 siRNA dose-dependently reduced reporter activity (Fig. 4). FOG-1 has been shown to facilitate chromatin occupancy of GATA-1 at WGATAR sites in the upstream region of the GATA-2 locus, replacing GATA-2 (54). On the basis of this suggested model, we speculate that a decrease in GATA-2 level induced by GATA-2 siRNA might release FOG-1 from its complex with GATA-2. As a result, increased interaction of FOG-1 and GATA-1 might enhance transactivation of the CCR3 reporter in K562 cells. However, as GATA-2 essentially acts as an activator of the CCR3 reporter in other cell types that express both GATA proteins, including HMC-1 cells, CB eosinophils, and mast cells, further studies are needed to investigate whether GATA-2 functions in a context-dependent manner and why it has a qualitatively different outcome at the same chromatin site. An interaction between FOG-1 and GATA-1 is critical for many GATA factor activities that are required for erythropoiesis and megakaryopoiesis (55), whereas FOG-1 antagonizes the fate choice of multipotential progenitor cells for the eosinophil (56) and mast cell lineages (57). These reports are apparently contradictory to our finding that FOG-1 upregulates the CCR3 gene, a type of gene that is preferentially expressed in eosinophils and mast cells. This observation is hardly explainable. However, it is worthwhile to note that K562 cells express high levels of endogenous FOG-1 as well as GATA-1 and thus show features of the erythroid lineage, in which FOG-1 acts as a cofactor for the upregulation of an erythroid-specific gene (55). Therefore, we speculate that downregulation of CCR3 by FOG-1 siRNA in K562 cells (which is equivalent to upregulation of CCR3 by FOG-1) might manifest itself as the result of the cellular context. It would be interesting to see whether the FOG-1 regulation of CCR3 occurs in different cell types of different levels of FOG-1 and GATA factors.

Th2 cells, mast cells, and eosinophils are the major cells in allergic inflammatory conditions, including asthma, rhinitis, and dermatitis. GATA factors, acting individually or in combination, not only play central roles in the development and differentiation of these cell types but also modulate a wide variety of their regulatory and effector functions, thus contributing to the pathophysiology of these diseases by regulating the expression of genes that are specific for Th2 cells (IL-4 and IL-13), eosinophils (MBP, EDN, and CLC protein), and mast cells (carboxypeptidase A and FcεRIα). The present study reveals that the CCR3 gene is commonly regulated by three different GATA factors in a cell type–specific manner. This idea heightens the possibility that anti-GATA therapy directed against all three GATA factors would be highly effective in intervention for diseases with complex pathophysiology, such as asthma. Antisense-based therapies that are directed against GATA-3 have been shown to be effective in the prevention and treatment of experimental allergic asthma (58, 59). In this light, a mixture of siRNAs specific for these three GATA factors might provide a more attractive therapy than targeting a single GATA factor, which is hardly sufficient to dampen the array of sophisticated inflammatory responses that manifest in heterogeneous diseases, both clinically and at the molecular level.

In summary, we show that multiple GATA factors participate in transactivation of the CCR3 gene in a cell type–specific manner through chromatin occupancy of the same GATA site in the gene’s regulatory region. The GATA factors expressed in a given cell type and their concentrations are the primary determinants for transactivation of the gene. The functional redundancy of GATA-1, GATA-2, and GATA-3 is demonstrated by transactivation of a gene previously thought to be eosinophil specific, especially the finding that positive regulation of the CCR3 gene by GATA-3 is a Th2 cell–specific mechanism. This study provides a mechanistic principle for the function of three GATA factors in the regulation of gene expression.

Disclosures

The authors have no financial conflicts of interests.

Acknowledgments

We thank Drs. Woo Sung Lim and Tae Wook Yoo (Woosung Hospital, Ansan, Republic of Korea) for providing CB, Prof. Philippe Martiat (University of Brussels, Brussels, Belgium) for technical assistance in preparing CB Th2 cells, and Prof. Jae Hong Kim and Yun-Jae Jung for provision of cell lines.

Footnotes

  • This work was supported by Grant 2011-0014580 (to I.Y.C.) from the National Research Foundation, Republic of Korea. S.-K.K. and B.S.K. were partially supported by the BrainKorea 21 project.

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    CB
    cord blood
    ChIP
    chromatin immunoprecipitation
    CLC
    Charcot–Leyden crystal
    FOG-1
    friend of GATA-1
    MBP
    major basic protein
    qRT-PCR
    quantitative real-time PCR
    SCF
    stem cell factor
    siRNA
    small interfering RNA.

  • Received December 28, 2012.
  • Accepted March 29, 2013.

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