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International Immunology Advance Access originally published online on December 22, 2005
International Immunology 2006 18(2):313-323; doi:10.1093/intimm/dxh370
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© The Japanese Society for Immunology. 2005. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

A role for Ets1, synergizing with AP-1 and GATA-3 in the regulation of IL-5 transcription in mouse Th2 lymphocytes

Jun Wang, M. Frances Shannon and Ian G. Young

Division of Molecular Bioscience, John Curtin School of Medical Research, Australian National University, Mills Road, Acton, ACT 0200 Australia

Correspondence to: I. G. Young; E-mail: ian.young{at}anu.edu.au


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
IL-5 is a key regulator of eosinophilic inflammation and is selectively expressed by antigen-activated Th2 lymphocytes. An important role for the proximal AP-1 and GATA sites in regulating IL-5 transcription is generally accepted but the significance of an adjacent Ets/NFAT site has remained unclear. We have investigated its role using the mouse Th2 clone D10.G4.1. Transcription of IL-5 reporter gene plasmids could be induced in D10 cells by phorbol myristate acetate/cyclic adenosine monophosphate (PMA/cAMP) stimulation and significantly further enhanced by activation of the mitogen-activated protein (MAP) kinase pathways. Strong induction of IL-5 mRNA was also induced by PMA/cAMP. Mutagenesis showed that the Ets/NFAT site is of critical importance along with the AP-1 and GATA sites in regulating IL-5 transcription stimulated by PMA/cAMP and MAP kinase activation. Transactivation was used to investigate the transcription factors which could function at the three sites and possible synergistic interactions. AP-1 (c-Fos/c-Jun) strongly induced IL-5 transcription and dominant negative AP-1 constructs confirmed that AP-1 plays an important role in regulating IL-5 expression. Ets1, unlike other members of the Ets/NFAT family, synergized strongly with AP-1 suggesting that Ets1 is the family member which functions at the Ets/NFAT site. AP-1/Ets1 transactivation also stimulated IL-5 mRNA expression. Ets1 binding to the proximal promoter region, demonstrated by chromatin immunoprecipitation, was stimulated by PMA/cAMP. The absolute dependence on the binding sites for Ets1, AP-1 and GATA-3 together with the strong synergy between Ets1 and AP-1 suggest close cooperative interactions between the three transcription factors in the regulation of IL-5 expression in mouse T cells.

Keywords: allergy, cytokines, gene regulation, T lymphocytes


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
IL-5 is one of the key cytokines produced by activated T lymphocytes that are involved in the regulation of immune and inflammatory responses. IL-5 appears to play a central role in the regulation of eosinophilia and contributes to several human diseases, including asthma (1–4). Understanding the regulation of this inducible cytokine may ultimately be valuable in treating various pathological conditions in humans involving eosinophilia. IL-5 is primarily produced by antigen-activated T lymphocytes of the Th2 subclass and is not produced by Th1 cells. Many of the studies to date of the IL-5 expression have been carried out using leukemias which do not show the cytokine expression characteristics of Th2 lymphocytes and therefore may have somewhat different regulatory mechanisms. In the present work, we have used the Th2 T cell clone D10.G4.1 to study the mechanisms of IL-5 expression in T cells and particularly the role of the Ets/NFAT site in the proximal promoter region.

Activation of T cells requires interaction of the TCR complex with antigens in association with the MHC and results in activation of protein kinase C (PKC) and an increase in intracellular calcium (5). A second co-stimulatory signal, which can be generated through the CD28 signaling pathway (6), is provided by antigen-presenting cells (7). Although T cell activation is usually mimicked in vitro by stimulation with phorbol myristate acetate (PMA) and ionomycin, IL-5 expression in mouse T cells is optimally stimulated by the combination of PMA and cyclic adenosine monophosphate (cAMP) (8, 9), consistent with a requirement for activation of PKC (stimulated by PMA) and activation of protein kinase K (PKA) (stimulated by cAMP) for optimal gene induction (10).

MAP kinase pathways are known to play a critical role in the activation of T cells during the immune response. Three parallel MAP kinase signal transduction pathways have been identified in mammalian cells. These are the extracellular signal-related kinase (ERK; 11), c-Jun N-terminal kinase (JNK; 12) and p38 MAP kinase (13, 14). The ERK pathway is involved in the positive selection of T cells in the thymus and in T cell activation (15, 16). JNK is also activated during T cell activation, and appears to integrate signals initiated at the TCR complex and the co-stimulatory molecule CD28 (17). TCR ligation or PMA treatment is sufficient to maximally induce ERK activity, whereas JNK and p38 activation requires a co-stimulatory signal such as CD28 ligand binding or ionomycin treatment, respectively (17–19).

Inhibition of IL-5 expression by the inhibitor SB203580 has been reported for human T cells (20) and for the mouse T cell clone D10.G4.1 (21), suggesting a role for p38 MAP kinase in IL-5 gene regulation. Similarly, inhibition by PD98059 suggests involvement of the ERK pathway in IL-5 expression in mouse T cells (22). Potentially, MAP kinase activation could influence IL-5 expression by increasing the levels and/or activation of AP-1 (23) and GATA-3 (21) as both transcription factors are implicated in regulating IL-5 expression.

A number of studies have been carried out on the transcriptional elements controlling mouse IL-5 expression. Evidence has been obtained supporting a key role for elements in the proximal promoter region in the induction of IL-5 transcription. Conserved lymphokine element 0 (CLE0) (24) is located at –53 to –39 in the mouse IL-5 promoter. It has been shown to be critical for IL-5 expression in EL-4 cells (25, 26) and in D10 cells (27), although Stranick et al., (28) found CLE0 not to be essential for IL-5 expression in antigen-stimulated D10 cells. CLE0 consists of adjacent putative AP-1 and Ets/NFAT binding sites. The major inducible proteins binding to the AP-1 region of mouse IL-5 CLE0 have been reported to be JunB and JunD in D10 cells (29), c-Fos, JunB and JunD in EL-4 cells (30) and c-Fos, JunB and NFAT in EL-4 cells (31). No binding to the Ets/NFAT site has been demonstrated in mouse T cell lines and it has not been clear whether this site is critical and whether an Ets or NFAT family member functions at this site. A recent study using human IL-5 promoter constructs in Jurkat cells (32) supports the possibility that a member of the Ets family may be involved in IL-5 gene regulation although further studies are desirable since Jurkat cells do not express IL-5 either constitutively or after stimulation. It has also been reported that the 5' end of the CLE0 element carries an Oct site which overlaps with the AP-1 site (33). However, recent work on human IL-5 transcription indicates that IL-5 expression can occur in the absence of Oct-2 (34).

There are several reports suggesting the importance of a GATA site (–71 to –66) located upstream of the CLE0 element in regulating IL-5 expression. These include studies in EL-4 cells (25, 30) and in D10.G4.1 (29, 35, 36). Siegel et al., (30) and Zhang et al., (29) showed that GATA-3 rather than GATA-4 bound to the GATA element. GATA-3 has been shown to be specifically expressed in Th2 cells and to be important in the expression of Th2-specific cytokines (37, 38, 57). In contrast to these findings, selectivity for GATA-4 has been reported for constitutive human IL-5 expression by the ATL-16T cell line (39, 40) leaving some uncertainty as to which GATA factor functions in IL-5 regulation.

In the present work, studies were carried out in the mouse Th2 clone D10.G4.1 to investigate the significance of the proximal Ets/NFAT site and to investigate possible synergistic interactions between Ets/NFAT family members and AP-1 and GATA-3. Evidence was obtained that the Ets/NFAT site is of equal importance to the adjacent AP-1 and GATA sites and that each of the three sites is required for IL-5 expression induced by a variety of T cell activation signals. The results also suggest that during T cell stimulation, elevation of AP-1 levels may be important in stimulating IL-5 expression and that Ets1 can strongly synergize with AP-1.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Cell culture
A subline of mouse Th2-type D10.G4.1 cells was used in these studies and was maintained in RPMI-1640 medium with the following additives: 10% FCS, 10 mM NaCl, 1 mM sodium pyruvate, 56.2 mM monothioglycerol, 60 µg ml–1 benzylpenicillin, 100 µg ml–1 streptomycin, 2 mM L-glutamine, 0.05 mM ß-mercaptoethanol and 25 units ml of IL-2. The IL-2 was prepared from Sf9 cells infected with a baculovirus expressing mouse IL-2 (26). The medium was changed every three days.

Plasmid construction
The –1170mIL5Luc reporter construct contains ~1170 bp of sequence preceding the transcription initiation site of the mouse IL-5 promoter. A SacI fragment from –1170SynCATmIL5 construct which encompasses the –1174 to +26 of the 5'-unstranslated region of the mouse IL-5 gene was cloned into the SacI site of the pXPG luciferase reporter construct (41). The mutations of AP-1, Ets/NFAT and GATA binding sites of IL-5 promoter were made in this construct using QuickChangeTM site-directed mutagenesis system (Stratagene). The –88mIL5Luc construct which carries the proximal 88 bp of the mIL-5 promoter was generated by PCR (forward 5'-CCTTTATTAGGTGTCCTCTATC-3', reverse 5'-AAGGAAACGACTTCCGGTCGCGA-3'). Addition of a BamHI site on the sense primer and a KpnI site on the anti-sense primer allowed the fragment to be subcloned into the pXPG luciferase reporter plasmid. The constructs were verified by sequencing.

To construct the GATA-3 expression plasmid, a 2056-bp cDNA encoding GATA-3 was released by EcoRI from pGMmc5bB (42). This fragment was cloned into the EcoRI site of pEFIRES-P plasmid (43) under the control of the EF-1{alpha} promoter. The GATA-4 expression plasmid was similarly constructed using a 1799-bp EcoRI fragment encoding GATA-4 from pMT2mGATA-4 (44). The c-Jun, c-Fos, Ets1 and Elf1 expression constructs in the pEFBOS expression vector under the control of the EF-1{alpha} promoter were described previously (45) (a gift from I. Kola). The other constructs used have been described elsewhere: NFATp and NFATc expression plasmids, (46) dominant negative c-Jun (A-Jun) and c-Fos (A-Fos), (47, 48) and MAP kinase expression constructs Raf-BXB-CX, MLK3 and MKK6E, (19) (provided by U. R. Rapp).

Optimization of transfection efficiency
An expression plasmid containing the gene encoding green fluorescent protein (GFP) was used to optimize transfection efficiency in D10 cells. A total of 5 x 106 cells were mixed with 5 µg of GFP plasmid in 300 µl of RPMI-1640 supplemented with 20% FCS in electroporation cuvettes for 15 min at room temperature and subjected to voltage pulses ranging from 230 to 310 V at 975 µF capacitance in a GenePulser apparatus (Bio-Rad). Transfected cells were cultured in 5 ml of complete RPMI-1640 medium for 20 h. One millilitre of cells were transferred into FACS analysis tubes and stained with propidium iodide at 10 µg ml–1 for 30 min and then subjected to FACS assay. The highest level of viable cells expressing GFP was obtained with 270 V and 975 µF capacitance.

Transfection assay
D10 cells were grown in RPMI-1640 complete medium and subcultured at 5 x 105 cells ml–1 the day before transfection. The cells were harvested and re-suspended in the same medium. A total of 5 x106 cells were incubated with 20 µg of DNA (5 µg of IL-5 reporter plasmids, 5 µg of different expression plasmid and empty plasmid to make up to 20 µg of total DNA) in 300 µl of growth medium supplemented with 20% FCS for 15 min at room temperature, and subjected to electroporation at 270 V and 975 µF capacitance using GenePulser (Bio-Rad). The cells were transfered into 5 ml of fresh medium and incubated at 37°C for 20 h. Transfected cells were treated with or without PMA and dibutyryl cAMP (cAMP) for 9 h (25 ng ml–1 PMA and 1 mM cAMP).

For transactivation effects on endogenous IL-5 mRNA expression, D10 cells were co-transfected with plasmid containing relevant transcription factors (AP-1 and Ets1) and GFP construct at 5 : 1 ratio. After 20 h recovery, transfected cells were sorted by FACS for GFP-positive cells. Expression of IL-5 mRNA was analyzed by real-time PCR.

Luciferase assays
After 9 h stimulation, the cells were harvested by centrifugation, washed twice with PBS and then lysed with three rounds of freeze-thaw lysis in liquid nitrogen in a lysis buffer containing 10 mM K2HPO4, 1 mM EDTA and 0.2 mM dithiothreitol (DTT). The debris was removed by centrifugation. Protein concentrations of lysates were determined using Bradford protein assay (Bio-Rad). In the luciferase assays, 15 µg of protein extracts were dispensed into 96-well plates and mixed with 200 µl of assay buffer containing 100 mM potassium phosphate buffer (4 parts 100 mM K2HPO4 : 1 part 100 mM KH2PO4), 8 mM MgSO4, 2 mM DTT, 0.75 mM ATP and 0.175 mM co-enzyme A. The reactions were initiated by adding 40 µl of 1 mM D-luciferin. The light signal was measured using a TopCounter (Packard). The linearity of the assay was confirmed for high activity samples by measuring the activity of 2-fold dilutions of the protein samples. The background of assay was very low and taken away from all values. Mean and standard derivations of at least three independent experiments are shown in the figures.

Real-time PCR analysis
Total RNA was prepared from stimulated and unstimulated T cells using TriReagent (Sigma–Aldrich) according to manufacturer's instructions. RNA was treated with DNase I and reverse transcribed using Superscript II reverse transcriptase (Invitrogen) as detailed in the manufacturer's instructions. SYBR Green real-time PCRs were performed with 50 ng of cDNA in a total volume of 25 µl on an ABI 7500 sequence detector (Applied Biosystems) with primer for the IL-5 cDNA (forward 5'-AGCACAGTGGTGAAAGAGACCTT-3', reverse 5'-TCCAATGCATAGCTGGTGATTT-3'). The Ct values for IL-5 were normalized to the housekeeper gene ubiquitin-conjugating enzyme E2D.

The following PCR conditions were used: stage1, 50°C for 2 min for one cycle; stage 2, 95°C for 10 min for one cycle and stage 3, 95°C for 15 s and 60°C for 1 min for 40 cycles. Since PCR amplification was monitored by SYBR green, the absence of non-specific amplification was determined by analyzing the dissociation curve of the PCR amplification products.

Chromatin immunoprecipitation analysis
Chromatin immunoprecipitation (ChIP) analysis was performed using a ChIP assay kit from Upstate Biotechnology according to the manufacturer's instructions with slight modifications. In brief, 2 x 107 D10 T cells were treated with formaldehyde (1%) for 15 min and the reaction was terminated by the addition of 0.25 M glycine. Cells were washed twice with PBS and re-suspended in SDS lysis buffer (Upstate Biotechnology) and sonicated to shear chromatin. The samples were pre-cleared with 80 µl of salmon sperm DNA–protein A Agarose (Upstate Biotechnology) following by addition of Ets1 antibody (Santa Cruz Biotechnology) or without antibody as a control, overnight with rotation at 4°C. After wash with low-salt buffer, high-salt buffer, LiCl wash buffer and Tris–EDTA buffer (Upstate Biotechnology), the cross-link between DNA and protein was reversed at 65°C overnight. Samples were recovered by phenol–chloroform extraction and re-suspended in MilliQ water for real-time PCR analysis. Using the conditions described above, SYBR Green real-time PCRs were performed with IL-5setA primer flanking Ets binding site of the IL-5 proximal promoter region (forward 5'-ACCCTGAGTTTCAGGACTCG-3', reverse 5'-TCCCCAAGCAATTTATTCTCTC-3') and IL-5setB primer located at –3-kb region of the IL-5 promoter as a control (forward 5'-ATAGGCGTTAGGCACCATGT-3', reverse 5'-TGGGGTGCATGATGTGAA-3'). The amount of precipitated target sequence was determined by normalization with the total input.

Western blot analysis
Nuclear extracts were prepared according to the method by Schreiber et al. (49). Protein concentrations were determined by the Bradford assay (Bio-Rad). Proteins (15 µg) were resolved by SDS-PAGE, transferred to nitrocellulose and subjected to western blot analysis using anti-Ets1 antibody (Santa Cruz Biotechnology). Chemiluminescence was detected using ECL reagents (Amersham Biosciences) according to the manufacturer's instructions.

Electrophoretic mobility shift assays
Recombinant c-Jun and c-Fos (50) (a gift from D. Tremethick) and recombinant Ets1 (45) (supplied by I. Kola) were prepared as previously described. Double-stranded oligonucleotides were end labeled with [{gamma}-32p]dATP by T4 polynucleotide kinase (51). The 15 ng of recombinant proteins (AP-1 and Ets1) and 0.1 ng radiolabeled oligonucleotides were added to the binding buffer containing 1 µg of poly (dI.dC), 25 mM HEPES, 25 mM NaCl, 10% glycerol, 1 mM EDTA, 1 mM DTT and 0.25% Nonidet P-40 in a final volume of 25 µl. Protein–DNA complex was resolved on a 4.5% non-denaturing polyacrylamide gel containing 0.5x TBE (0.5 mM Tris, 42 mM boric acid, 1 mM EDTA, pH 8.3). The gels were dried and exposed to X-ray film (Kodak) at room temperature overnight. In competition experiments, 100-fold excess of unlabeled oligonucleotides which correspond to the same binding site or other unrelated binding sequences were added to the reactions.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Reporter gene assay for IL-5 expression in D10 cells
A transient transfection system was established using a derivative of the mouse Th2 clone D10.G4.1 to facilitate studies of the transcriptional elements involved in IL-5 gene induction and for transactivation experiments. Optimum conditions for electroporation were obtained with 270 V and 975 µF capacitance in which 27% of transfected cells expressed GFP. Using these conditions, transient transfection of a luciferase reporter construct (–1170mIL5Luc) carrying 1170 bases of the upstream region of the mouse IL-5 gene into D10 cells, and subsequent stimulation of the cells with PMA and dibutyryl cAMP (cAMP) gave significant induction of luciferase expression. It was found that maximal stimulation was achieved after 9–12 h (data not shown). Using optimal conditions for electroporation and stimulation, 36-fold induction of luciferase expression was achieved with PMA/cAMP (Fig. 1B). PMA alone gave little stimulation, whereas the stimulation with cAMP alone was 10-fold. In addition to induction of luciferase, strong induction of IL-5 mRNA by PMA/cAMP was shown by real-time PCR (Fig. 1C). The synergistic activation of IL-5 expression by PMA and cAMP in D10 cells is in agreement with our previous studies (26, 52).


Figure 1
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  		Fig. 1. Role of transcription factor binding sites in the proximal promoter region. (A) Proximal promoter region of the mouse IL-5 gene. The putative binding sites for AP-1, Ets/NFAT and GATA and the mutations introduced into these sites to test their functional involvement in IL-5 gene expression. (B) Effect of mutation of binding sites on promoter activity. Mutations in putative elements were introduced into the –1170mIL5Luc reporter construct. The –88mIL5Luc carries a deletion of –89 to –1170. Wild-type and mutant constructs were introduced into D10 cells and luciferase was measured after stimulation with PMA and cAMP as indicated. Results represent the average of at least three independent experiments. (C) Effect of PMA/cAMP stimulation on induction of IL-5 mRNA levels determined by real-time PCR analysis.

 
Effect of MAP kinase activation
As MAP kinases are known to play a critical role in the activation of T cells during immune responses, we tested the effect of specific stimulation of the individual MAP kinase pathways on IL-5 transcription. The approach used expression plasmids encoding constitutively active versions of the corresponding upstream kinases (19). The constructs used were a constitutively active kinase mutant of Raf (Raf-BXB-CX) which serves as a specific ERK activator; MLK3, which when over-expressed results in a strong activation of JNK without affecting ERK and p38 MAP kinase activities and an activated mutant of MKK6 (MKK6E) which serves as a specific activator of p38 (19).

The –1170mIL5Luc construct was co-transfected with each of the MAP-kinase-stimulating expression constructs into D10 cells. Expression of activated Raf gave 6-fold induction of IL-5 promoter activity, MLK3 gave 2-fold induction but MKK6E had no effect (Fig. 2A). The stimulation of all three MAP kinase pathways simultaneously gave 9-fold induction (Fig. 2A). In contrast to these relatively small effects, activation of each MAP kinase pathway in D10 cells gave strong synergistic stimulation with PMA/cAMP (Fig. 2B). The stimulations achieved with MKK6E, Raf and MLK3 were, respectively, 34-fold, 27-fold and 21-fold the additive levels obtained with cells stimulated with PMA/cAMP or constitutively active MAP kinase expression construct alone. Thus, activation of each MAP kinase pathway can strongly enhance IL-5 expression in cells stimulated with PMA/cAMP. T cell expression of TNF{alpha} is similar in that it is also induced by activation of each MAP kinase pathway (53).


Figure 2
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  		Fig. 2. Effect of MAP kinase pathway activation on IL-5 expression. (A) MAP kinase activation alone. The indicated expression plasmids were co-transfected into D10 cells together with –1170mIL5Luc and luciferase activity was determined. (B) Effect of additional stimulation with PMA/cAMP. Results represent the average of at least three independent experiments.

 
Mutation of the transcription factor binding sites in the proximal promoter region
The reporter construct used in the experiments described above had 1170 bases of upstream sequence to try and ensure that any relevant upstream regulatory sites were included. However, for stimulation of IL-5 expression by PMA/cAMP, it was shown that a truncated construct (–88mIL5Luc), carrying just the proximal promoter region of the gene (Fig. 1B), gave similar levels of induction to the –1170mIL5Luc and exhibited a similar pattern as –1170mIL5Luc in response to different stimuli (data not shown). This construct was also very strongly stimulated by MAP kinase activation (Fig. 3), indicating that the major positive control elements involved in the stimulations observed were in the proximal promoter region. Within the proximal promoter region there are three putative transcription factor binding sites: Ets/NFAT (–41 to –47), AP-1 (–48 to –54) and GATA (–67 to –72) (Fig. 1A). Although the importance of the AP-1 and GATA sites are reasonably well accepted, we wished to determine the relative importance of the Ets/NFAT site in comparison to these sites. Mutation of the Ets/NFAT site abolished inducible expression of the –1170mIL5Luc expression construct (Fig. 1B) as did mutation of the other two sites indicating a critical role for each site in gene expression induced by PMA/cAMP. To determine whether the stimulation of IL-5 expression mediated by activation of the MAP kinase pathways was also dependent on the Ets/NFAT, AP-1 and GATA elements, wild-type and mutant –1170mIL5Luc constructs were transfected together with expression vectors for Raf-BXB-CX, MLK3 or MKK6E into D10 cells. Mutation of each of the three transcription factor binding sites greatly reduced the induction of IL-5 expression mediated by activation of each MAP kinase pathway together with PMA/cAMP stimulation (Fig. 3).


Figure 3
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  		Fig. 3. Effect of mutation of transcription factor binding sites on IL-5 induction by activation of MAP kinase pathways. The indicated expression plasmids were co-transfected into D10 cells together with wild-type –1170mIL5Luc or mutated reporter constructs and luciferase activity was determined after stimulation with PMA/cAMP. The mutations carried by the different constructs are indicated and described in detail in Fig. 1(A). Results represent the average of at least three independent experiments.

 
Investigation of the transcription factors active at the AP-1, Ets/NFAT and GATA sites by transactivation
Transactivation experiments were carried out using expression constructs encoding relevant transcription factors to identify those that could stimulate expression of the IL-5 gene and to test for synergistic interactions. The transcription factors used were c-Fos and c-Jun (AP-1 site) Ets1, Elf1, NFATp or NFATc (Ets/NFAT site) and GATA-3 or GATA-4 (GATA site). Expression constructs for these transcription factors were transiently co-transfected together with the –1170mIL5Luc reporter construct and expression was measured with and without stimulation by PMA/cAMP.

AP-1 (c-Fos/c-Jun) gave dramatic transactivation of 400-fold over normal unstimulated levels of expression (Fig. 4A), indicating that IL-5 gene induction can be achieved by raising the levels of AP-1, without the need for stimulation. Transactivation by Ets, NFAT and GATA family members gave much smaller increases in expression of up to 4-fold in the absence of stimulation. The effect of stimulation by PMA/cAMP on the level of transactivation achieved was also tested. For AP-1, the values after transactivation and stimulation were 250-fold higher than with –1170mIL5Luc alone (Fig. 4B). Transactivation by Ets1, Elf1, NFATp and NFATc was also tested following stimulation with PMA/cAMP. Elf1 and NFATp gave lower levels of transactivation than Ets1 and NFATc which gave levels of transactivation of ~25-fold (Fig. 4B). Although these experiments did not clarify whether Ets1 or NFATc was involved at the Ets/NFAT site, the involvement of Ets1 at this site is supported by its unique ability to synergize with AP-1 (see below). GATA-3 gave slightly less transactivation than Ets1 (14-fold) and was consistently more effective than GATA-4 (Fig. 4B). Our data suggest that GATA-4 can function at the GATA site although less effectively than GATA-3. However, in Th2 cells, GATA-3 is more likely than GATA-4 to function at the GATA site since it is specifically expressed in these cells.


Figure 4
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  		Fig. 4. Transactivation of expression of –1170mIL5Luc using expression constructs for relevant transcription factors. The reporter construct was co-transfected with indicated expression constructs for relevant transcription factors into D10 cells and luciferase activity measured. (A) Transactivation using unstimulated cells. (B) Transactivation using cells stimulated with PMA/cAMP. (C) Transactivation using pairwise combinations of AP-1 and the indicated transcription factors. Results represent the average of at least three independent experiments. (D) Effect of AP-1/Ets1 transactivation on mRNA levels of IL-5 determined by real-time PCR.

 
Synergy of Ets1 with AP-1
Since the Ets and AP-1 binding sites are adjacent in the proximal region of the mouse IL-5 promoter and Ets1 and AP-1 have been reported to form a complex in T lymphocytes (54), it was of interest to determine if AP-1 could synergize with Ets1 or any of the other Ets/NFAT family members potentially able to bind to the Ets/NFAT site. Therefore, expression constructs for c-Fos and c-Jun (AP-1) were transiently co-transfected in combination with expression constructs for the other relevant transcription factors into D10 cells. Without stimulation, AP-1 and Ets1 gave strong synergistic transactivation of the –1170mIL5Luc reporter construct elevating expression levels to 10-fold over the additive stimulation of AP-1 and Ets1 alone. Over-expression of the other transcription factors tested, including GATA-3 and GATA-4, did not result in strong synergistic stimulation with AP-1 (Fig. 4C). The demonstrated strong synergy between AP-1 and Ets1 provides evidence for a role for Ets1 in regulating IL-5 expression at the Ets/NFAT site in Th2 lymphocytes. Real-time PCR was also used to demonstrate that AP-1/Ets1 transactivation also induced an elevation of IL-5 mRNA levels (Fig. 4D).

Activation pathways affecting transactivation
The effect of PMA and cAMP, alone and in combination, on transactivation was tested in more detail. In each case, PMA stimulation resulted only in a minor increase in transactivation (Fig. 5). cAMP was more effective, particularly with AP-1, suggesting that PKA activation is important in AP-1 activation in D10 cells. Strong synergistic stimulation was observed when PMA and cAMP were used together (Fig. 5). The responses to stimulation by PMA, cAMP or both agents in the transactivation experiments were similar to the responses observed with the –1170mIL5Luc plasmid, although significantly higher values of IL-5 expression were obtained by transactivation.


Figure 5
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  		Fig. 5. Effect of stimulation on transactivation by relevant transcription factors. Expression constructs for the indicated transcription factors were introduced into D10 cells together with the –1170mIL5Luc reporter and luciferase activity was measured after stimulation by PMA, cAMP or both. Results represent the average of at least three independent experiments.

 
Requirement for the proximal transcription factor binding sites for transactivation
A proximal promoter construct (–88mIL5Luc) was used to determine what features of the transactivation observed with the –1170mIL5Luc construct were also observed with the –88mIL5Luc construct. The transactivation results were quite similar to those obtained with the –1170mIL5Luc construct. Dramatic transactivation by AP-1 (430-fold above unstimulated levels) was also observed with the –88mIL5Luc construct in the absence of stimulation and AP-1 and Ets1 gave 3-fold synergistic stimulation (data not shown). These results suggest that the transcription factor binding sites in the proximal promoter region are involved in the transactivations observed. This was verified using derivatives of the –1170mIL5Luc construct carrying mutations at the AP-1, Ets/NFAT or GATA sites (Fig. 6). In each case, the transactivation observed with AP-1, Ets1 or GATA-3 was reduced to 1% or less by mutation of the relevant transcription factor binding sites. Significantly, each transactivation was also reduced to a low level by mutating either of the other two binding sites. This indicates that the same mechanism of gene induction is involved in the transactivations observed as in induction by PMA/cAMP and MAP kinase activation. It indicates that each of the three binding sites is obligatorily required for expression of the IL-5 gene and suggests that close cooperation between AP-1, Ets1 and GATA-3 may be involved.


Figure 6
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  		Fig. 6. Effect of mutation of the indicated transcription factor binding sites on the transactivation of –1170mIL5Luc by AP-1, Ets1 or GATA-3. After co-transfection of the respective constructs, the D10 cells were stimulated with PMA/cAMP and luciferase activity was measured. The mutations carried by the different constructs are indicated and described in detail in Fig. 1(A). Results represent the average of at least three independent experiments.

 
Transactivation by wild type and dominant negative c-Fos and c-Jun
In view of the high levels of transactivation of the –1170mIL5Luc construct achieved with AP-1 (c-Fos/c-Jun), experiments were carried out to determine the level of transactivation by c-Fos and c-Jun alone. Expression constructs were transfected together with –1170mIL5Luc into D10 cells and the cells stimulated with PMA and cAMP. Transactivation by c-Jun was much more effective than by c-Fos but transactivation by a combination of the two was by far the most effective (Fig. 7A). The low level of transactivation by c-Fos is consistent with the inability of c-Fos to form transcriptionally active dimers. The moderate activity of c-Jun in comparison to c-Fos/c-Jun indicates that c-Jun homodimers are less active than c-Fos/c-Jun heterodimers in stimulating transcription from the IL-5 promoter.


Figure 7
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  		Fig. 7. Activity of wild-type and dominant negative c-Fos and c-Jun in IL-5 gene expression. (A) Transactivation. D10 cells were co-transfected with the reporter construct –1170mIL5Luc and either c-Fos or c-Jun or both and luciferase activity was measured after stimulation with PMA/cAMP. (B) Inhibition of –1170mIL5Luc expression by dominant negative c-Fos and c-Jun. Results represent the average of at least three independent experiments.

 
Experiments were also carried out to see if normal induction of IL-5 expression by PMA/cAMP could be inhibited by dominant negative AP-1 expression constructs. Dominant negative c-Fos (A-Fos) and c-Jun (A-Jun) have been made by fusing an acidic amphipathic extension to the N-terminus of the c-Jun and c-Fos leucine zipper domains. The acidic extension of c-Jun and c-Fos interacts with the basic region of wild-type c-Jun and c-Fos forming a coiled-coil extension of the leucine zipper and thus prevents the basic region of wild-type c-Jun and c-Fos binding to DNA (47, 48). To test for repression of IL-5 expression, dominant negative c-Fos and c-Jun expression plasmids were co-transfected together with –1170mIL5Luc into D10 cells and IL-5 expression induced with PMA and cAMP. Dose-dependent repression of the IL-5 promoter activity by a combination of dominant negative c-Fos and c-Jun was observed which was very significant at higher levels of transfected DNA (Fig. 7B). The dominant negative c-Fos construct alone also significantly inhibited the transcriptional activity of the mouse IL-5 promoter consistent with the other evidence obtained of strong involvement of an AP-1 Fos/Jun heterodimer in IL-5 gene expression.

Functional independence of the AP-1 and Ets sites
The putative AP-1 and Ets1 sites are adjacent but were shown to be distinct by gel shift experiments. Recombinant AP-1 bound strongly to an oligonucleotide encompassing the AP-1 and Ets/NFAT sites of the promoter. This binding was sequence specific and was abolished by mutation of the AP-1 site but mutation of the Ets site had no effect (Fig. 8B). The binding of recombinant Ets1 to the oligonucleotide encompassing the AP-1 and Ets sites was much weaker but was sequence specific and was abolished by mutation of the Ets site (Fig. 8B). Recombinant Ets1 was shown to bind strongly to a consensus oligonucleotide for Ets (Fig. 8B). Therefore, the failure to demonstrate strong binding with the IL-5 proximal promoter relates to this site and the conditions used and possibly a need for other proteins to be present for optimal binding. AP-1 and Ets1 were also added together but no evidence of the formation of an AP-1–Ets1 complex was obtained (data not shown). The binding of Ets1 to the proximal promoter region was followed up using ChIP assays. Specific binding of Ets1 was shown to be stimulated by PMA/cAMP stimulation (Fig. 8C). Further confirmation of Ets1 expression in the D10 cells used in these experiments was shown by western blotting (Fig. 8D). Further support for binding of Ets1 to the Ets/NFAT site in the CLE0 element has come from the previous studies of Thomas et al., (45) who demonstrated binding of Ets1 to the analogous Ets site within the CLE0 element of the granulocyte macrophage colony-stimulating factor promoter using Jurkat nuclear extracts and those of Blumenthal et al., (32) who showed binding of Ets1 to the identical Ets site in the proximal human IL-5 promoter using recombinant Ets1 produced in insect cells.


Figure 8
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  		Fig. 8. Binding of AP-1 and Ets1 proteins to the proximal region of mouse IL-5 promoter. (A) The oligonucleotides used: P1, wt IL-5 promoter; P2, IL-5 AP-1 mutant; P3, IL-5 Ets mutant; P4, Ap-1 consensus; P5, Ets consensus; P6, Ets consensus mutant; P7, non-specific sequence. (B) The radiolabeled oligonucleotide probe containing the AP-1 and Ets binding sites of the mouse IL-5 promoter (–60 to –32 bp) was incubated with 15 ng of recombinant AP-1 (c-Jun and c-Fos) or Ets1 protein. Unlabeled competitor oligonucleotides (100-fold molar excess over the probe) corresponding to the wild-type IL-5 probe (P1) or mutants containing nucleotide substitutions in the AP-1 (P2) or Ets (P3) site were added to the binding reaction mixtures as indicated. Binding reactions were fractionated on a native polyacrylamide gel and the dried gel autoradiographed at room temperature overnight. Specific protein–DNA binding is indicated with the arrows. (C) Demonstration of Ets1 binding to the proximal promoter region by ChIP using an Ets1 antibody. Primer set A flanks the Ets1 binding site in the proximal promoter region; primer set B is located 3 kb upstream. (D) Western blot of D10 cells using an Ets1 antibody.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
Antigen-activated Th2 lymphocytes expressing IL-5 appear to be the primary orchestrators of the eosinophilic inflammation seen in a number of allergic diseases such as asthma. Previous studies have suggested that the IL-5 gene and the other Th2 cytokine genes are regulated independently in Th2 lymphocytes (10, 55) despite their apparently coordinate expression in some immune responses. The work described in this paper aimed to increase our understanding of the mechanisms regulating IL-5 expression in Th2 lymphocytes and used the mouse Th2 clone D10.G4.1 as a model of normal T cells. In particular, we investigated the significance of an Ets/NFAT site which lies close to the AP-1 site in the proximal promoter region. We established a reporter gene assay in D10 cells and showed that IL-5 transcription could be induced by a combination of PMA/cAMP stimulation and strongly enhanced by specific activation of each MAP kinase pathway. Strong induction of IL-5 mRNA was also achieved by PMA/cAMP stimulation suggesting that the reporter gene expression was reflecting the expression of the endogenous IL-5 gene.

Mutagenesis showed that the Ets/NFAT site was critical to IL-5 expression in D10 cells and of comparable importance to the AP-1 and GATA sites. Although close to the AP-1 site, mutation of the Ets/NFAT and AP-1 sites showed that they were functionally distinct. Transactivation was used to investigate the activity of different members of the Ets/NFAT family. Ets1 showed a unique ability to strongly synergize with AP-1 suggesting that it has a role in regulating IL-5 expression and that it closely cooperates with AP-1. Real-time PCR showed that AP-1/Ets1 transactivation also induced an elevation of IL-5 mRNA levels. The expression of Ets1 in D10 cells was shown by western blotting. Although Ets1 did not bind strongly in standard gel shift assays, a specific binding of Ets1 to the proximal promoter region which was stimulated by PMA/cAMP was shown by ChIP. It has been shown previously that AP-1 and Ets proteins can physically associate in activated T cells (54), their interaction mediated through the binding of the basic domain of Jun to the Ets domain of Ets proteins. Jun, in association with Ets, is capable of interacting with Fos family members to form a trimolecular protein complex and the physical association between AP-1 and Ets1 is required for the transcriptional activity of enhancer elements containing adjacent Ets and AP-1 sites (54).

Over-expression of AP-1 alone without other stimulation was extremely effective at switching on IL-5 transcription in D10 cells. Dominant-negative AP-1 constructs showed that AP-1 was involved in normal induction of IL-5 by PMA/cAMP. Previous studies using D10 nuclear extracts have shown that transcription factor binding to the AP-1 site in the CLE0 element is strongly inducible by stimulation (27, 29) and is abolished by inhibition of protein synthesis (27). Elevation of AP-1 levels when Th2 lymphocytes are stimulated may therefore be an important part of the process of IL-5 gene induction and may explain our earlier observations that IL-5 transcription in D10 cells induced by a variety of stimulations is strongly dependent on protein synthesis (55, 56). A key role of AP-1 transcription in regulating human IL-5 induction was also postulated recently (34).

Two of the stimuli effective in inducing IL-5 expression in D10 cells (cAMP and MAP kinase activation) have been reported to have effects on AP-1, Ets1 or GATA-3 that may be relevant to their action on the IL-5 gene. cAMP stimulation of dopamine ß-hydroxylase gene expression is mediated by AP-1 (c-Fos, c-Jun and JunD) with c-Fos transcription being up-regulated by cAMP (58, 59). cAMP has also been shown to stimulate activation of p38 kinase in D10.G4.1 which can result in increased phosphorylation of GATA-3 and increased expression of IL-5 (21). In terms of MAP kinase stimulation, ERK has been shown to up-regulate c-Fos, Fra1 and c-Jun expression (60), c-Jun is known to be phosphorylated by JNK (23) and Ets1 can be phosphorylated by ERK (60). Ras has been shown to activate Ets1 and Ets2 and to induce transcription through an AP-1/Ets composite element (61, 62).

The present data support a key role for the proximal promoter region of the IL-5 gene in mediating the effects of a variety of T cell activation pathways which can induce IL-5 transcription. The absolute requirement for the GATA-3, AP-1 and Ets1 sites for IL-5 gene induction induced by transactivation with AP-1, MAP kinase activation or PMA/cAMP stimulation and the synergy demonstrated between AP-1 and Ets1 suggests transcriptional interdependence of the three transcription factors and that mouse IL-5 transcription in Th2 cells may require their close cooperation.


   Acknowledgements
 
We thank C. Vinson for A-Fos and A-Jun, D. Tremethick for recombinant AP-1, I. Kola for c-Jun, c-Fos, Ets1 and EIfI expression plasmids and recombinant Ets1, V. Rapp for MAP kinase expression constructs, J. Wallace for pEF-IRES-P, J. Engel for pGMmc5bB, D. Wilson for p MT2mGATA-4 and S. Ford for mouse IL-2.


   Abbreviations
 
cAMP   cyclic adenosine monophosphate
ChIP   chromatin immunoprecipitation
CLE0   conserved lymphokine element 0
DTT   dithiothreitol
ERK   extracellular signal-related kinase
GFP   green fluorescent protein
JNK   c-Jun N-terminal kinase
PKC   protein kinase C
PMA   phorbol myristate acetate

   Notes
 
Transmitting editor: A. Kelso

Received 30 December 2004, accepted 17 November 2005.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Sanderson, C. J., Campbell, H. D. and Young, I. G. 1988. Molecular and cellular biology of eosinophil differentiation factor (interleukin-5) and its effects on human and mouse B cells. Immunol. Rev. 102:29.[CrossRef][Web of Science][Medline]
  2. Kopf, M., Brombacher, F., Hodgkin, P. D. et al. 1996. IL-5-deficient mice have a developmental defect in CD5+ B-1 cells and lack eosinophilia but have normal antibody and cytotoxic T cell responses. Immunity 4:15.[CrossRef][Web of Science][Medline]
  3. Foster, P. S., Hogan, S. P., Ramsay, A. J., Matthaei, K. I. and Young, I. G. 1996. Interleukin 5 deficiency abolishes eosinophilia, airways hyperreactivity, and lung damage in a mouse asthma model. J. Exp. Med. 183:195.[Abstract/Free Full Text]
  4. Rothenberg, M. E., Mishra, A., Brandt, E. B. and Hogan, S. P. 2001. Gastrointestinal eosinophils. Immunol. Rev. 179:139.[CrossRef][Web of Science][Medline]
  5. Perlmutter, R. M. 1993. Molecular dissection of lymphocyte signal transduction pathways. Pediatr. Res. 33:S9; discussion S13–15.[Medline]
  6. Jenkins, M. K., DeSilva, D. R., Johnson, J. G. and Norton, S. D. 1993. Costimulating factors and signals relevant for antigen presenting cell function. Adv. Exp. Med. Biol. 329:87.[Medline]
  7. Rincon, M. and Flavell, R. A. 1994. AP-1 transcriptional activity requires both T-cell receptor-mediated and co-stimulatory signals in primary T lymphocytes. EMBO J. 13:4370.[Web of Science][Medline]
  8. Lee, H. J., Koyano-Nakagawa, N., Naito, Y., Nishida, J., Arai, N., Arai, K. and Yokota, T. 1993. cAMP activates the IL-5 promoter synergistically with phorbol ester through the signaling pathway involving protein kinase A in mouse thymoma line EL-4. J. Immunol. 151:6135.[Abstract]
  9. Lee, H. J., Matsuda, I., Naito, Y., Yokota, T., Arai, N. and Arai, K. 1994. Signals and nuclear factors that regulate the expression of interleukin-4 and interleukin-5 genes in helper T cells. J. Allergy Clin. Immunol. 94:594.[CrossRef][Web of Science][Medline]
  10. Naora, H., Altin, J. G. and Young, I. G. 1994. TCR-dependent and -independent signaling mechanisms differentially regulate lymphokine gene expression in the murine T helper clone D10.G4.1. J. Immunol. 152:5691.[Abstract]
  11. Boulton, T. G., Gregory, J. S. and Cobb, M. H. 1991. Purification and properties of extracellular signal-regulated kinase 1, an insulin-stimulated microtubule-associated protein 2 kinase. Biochemistry 30:278.[CrossRef][Medline]
  12. Kyriakis, J. M., Banerjee, P., Nikolakaki, E. et al. 1994. The stress-activated protein kinase subfamily of c-Jun kinases. Nature 369:156.[CrossRef][Medline]
  13. Lee, J. C., Laydon, J. T., McDonnell, P. C. et al. 1994. A protein kinase involved in the regulation of inflammatory cytokine biosynthesis. Nature 372:739.[CrossRef][Medline]
  14. Rouse, J., Cohen, P., Trigon, S. et al. 1994. A novel kinase cascade triggered by stress and heat shock that stimulates MAPKAP kinase-2 and phosphorylation of the small heat shock proteins. Cell 78:1027.[CrossRef][Web of Science][Medline]
  15. Alberola-Ila, J., Forbush, K. A., Seger, R., Krebs, E. G. and Perlmutter, R. M. 1995. Selective requirement for MAP kinase activation in thymocyte differentiation. Nature 373:620.[CrossRef][Medline]
  16. Swan, K. A., Alberola-Ila, J., Gross, J. A., Appleby, M. W., Forbush, K. A., Thomas, J. F. and Perlmutter, R. M. 1995. Involvement of p21ras distinguishes positive and negative selection in thymocytes. EMBO J. 14:276.[Web of Science][Medline]
  17. Su, B., Jacinto, E., Hibi, M., Kallunki, T., Karin, M. and Ben-Neriah, Y. 1994. JNK is involved in signal integration during costimulation of T lymphocytes. Cell 77:727.[CrossRef][Web of Science][Medline]
  18. Dickens, M., Rogers, J. S., Cavanagh, J. et al. 1997. A cytoplasmic inhibitor of the JNK signal transduction pathway. Science 277:693.[Abstract/Free Full Text]
  19. Hoffmeyer, A., Flory, A., Weber, C. K., Serfling, E. and Rapp, U. R. 1998. The GABP-responsive element of the interleukin-2 enhancer is regulated by JNK/SAPK-activating pathways in T lymphocytes. J. Biol. Chem. 273:10112.[Abstract/Free Full Text]
  20. Mori, A., Kaminuma, O., Miyazawa, K., Ogawa, K., Okudaira, H. and Akiyama, K. 1999. p38 mitogen-activated protein kinase regulates human T cell IL-5 synthesis. J. Immunol. 163:4763.[Abstract/Free Full Text]
  21. Chen, C. H., Zhang, D. H., LaPorte, J. M. and Ray, A. 2000. Cyclic AMP activates p38 mitogen-activated protein kinase in Th2 cells: phosphorylation of GATA-3 and stimulation of Th2 cytokine gene expression. J. Immunol. 165:5597.[Abstract/Free Full Text]
  22. Egerton, M., Fitzpatrick, D. R. and Kelso, A. 1998. Activation of the extracellular signal-regulated kinase pathway is differentially required for TCR-stimulated production of six cytokines in primary T lymphocytes. Int. Immunol. 10:223.[Abstract/Free Full Text]
  23. Karin, M. 1995. The regulation of AP-1 activity by mitogen-activated protein kinases. J. Biol. Chem. 270:16483.[Free Full Text]
  24. Miyatake, S., Shlomai, J., Arai, K. and Arai, N. 1991. Characterization of the mouse granulocyte-macrophage colony-stimulating factor (GM-CSF) gene promoter: nuclear factors that interact with an element shared by three lymphokine genes—those for GM-CSF, interleukin-4 (IL-4), and IL-5. Mol. Cell. Biol. 11:5894.[Abstract/Free Full Text]
  25. Lee, H. J., Masuda, E. S., Arai, N., Arai, K. and Yokota, T. 1995. Definition of cis-regulatory elements of the mouse interleukin-5 gene promoter. Involvement of nuclear factor of activated T cell-related factors in interleukin-5 expression. J. Biol. Chem. 270:17541.[Abstract/Free Full Text]
  26. Bourke, P. F., van Leeuwen, B. H., Campbell, H. D. and Young, I. G. 1995. Localization of the inducible enhancer in the mouse interleukin-5 gene that is responsive to T-cell receptor stimulation. Blood 85:2069.[Abstract/Free Full Text]
  27. Naora, H., van Leeuwen, B. H., Bourke, P. F. and Young, I. G. 1994. Functional role and signal-induced modulation of proteins recognizing the conserved TCATTT-containing promoter elements in the murine IL-5 and GM-CSF genes in T lymphocytes. J. Immunol. 153:3466.[Abstract]
  28. Stranick, K. S., Payvandi, F., Zambas, D. N., Umland, S. P., Egan, R. W. and Billah, M. M. 1995 Transcription of the murine interleukin 5 gene is regulated by multiple promoter elements. J. Biol. Chem. 270:20575.[Abstract/Free Full Text]
  29. Zhang, D. H., Cohn, L., Ray, P., Bottomly, K. and Ra, A. 1997. Transcription factor GATA-3 is differentially expressed in murine Th1 and Th2 cells and controls Th2-specific expression of the interleukin-5 gene. J. Biol. Chem. 272:21597.[Abstract/Free Full Text]
  30. Siegel, M. D., Zhang, D. H., Ray, P. and Ray, A. 1995. Activation of the interleukin-5 promoter by cAMP in murine EL-4 cells requires the GATA-3 and CLE0 elements. J. Biol. Chem. 270:24548.[Abstract/Free Full Text]
  31. Karlen, S., D'Ercole, M. and Sanderson, C. J. 1996. Two pathways can activate the interleukin-5 gene and induce binding to the conserved lymphokine element 0. Blood 88:211.[Abstract/Free Full Text]
  32. Blumenthal, S. G., Aichele, G., Wirth, T., Czernilofsky, A. P., Nordheim, A. and Dittmer, J. 1999. Regulation of the human interleukin-5 promoter by Ets transcription factors. Ets1 and Ets2, but not Elf-1, co-operate with GATA3 and HTLV-I Tax1. J. Biol. Chem. 274:12910.[Abstract/Free Full Text]
  33. Salerno, M. S., Schwenger, G. T., Sanderson, C. J. and Mordvinov, V. A. 2001. Binding of octamer factors to the murine IL-5 CLE0 in primary T-cells and a T-cell line. Cytokine 15:4.[CrossRef][Web of Science][Medline]
  34. Schwenger, G. T., Kok, C. C., Arthaningtyas, E., Thomas, M. A., Sanderson, C. J. and Mordvinov, V. A. 2002. Specific activation of human interleukin-5 depends on de novo synthesis of an AP-1 complex. J. Biol. Chem. 277:47022.[Abstract/Free Full Text]
  35. Lee, H. J., O'Garra, A., Arai, K. and Arai, N. 1998. Characterization of cis-regulatory elements and nuclear factors conferring Th2-specific expression of the IL-5 gene: a role for a GATA-binding protein. J. Immunol. 160:2343.[Abstract/Free Full Text]
  36. Stranick, K. S., Uss, A. S., Zambas, D. N., Egan, R. W., Billah, M. M. and Umland, S. P. 1998. Characterization of the mouse interleukin-5 promoter in a mouse TH2 T cell clone. Biochem. Biophys. Res. Commun. 252:56.[CrossRef][Web of Science][Medline]
  37. Zhang, D. H., Yang, L. and Ray, A. 1998. Differential responsiveness of the IL-5 and IL-4 genes to transcription factor GATA-3. J. Immunol. 161:3817.[Abstract/Free Full Text]
  38. Zheng, W. and Flavell, R. A. 1997. The transcription factor GATA-3 is necessary and sufficient for Th2 cytokine gene expression in CD4 T cells. Cell 89:587.[CrossRef][Web of Science][Medline]
  39. Yamagata, T., Nishida, J., Sakai, R. et al. 1995. Of the GATA-binding proteins, only GATA-4 selectively regulates the human interleukin-5 gene promoter in interleukin-5-producing cells which express multiple GATA-binding proteins. Mol. Cell. Biol. 15:3830.[Abstract/Free Full Text]
  40. Yamagata, T., Mitani, K., Ueno, H., Kanda, Y., Yazaki, Y. and Hirai, H. 1997. Triple synergism of human T-lymphotropic virus type 1-encoded tax, GATA-binding protein, and AP-1 is required for constitutive expression of the interleukin-5 gene in adult T-cell leukemia cells. Mol. Cell. Biol. 17:4272.[Abstract/Free Full Text]
  41. Bert, A. G., Burrows, J., Osborne, C. S. and Cockerill, P. N. 2000. Generation of an improved luciferase reporter gene plasmid that employs a novel mechanism for high-copy replication. Plasmid 44:173.[CrossRef][Web of Science][Medline]
  42. Ko, L. J. and Engel, J. D. 1993. DNA-binding specificities of the GATA transcription factor family. Mol. Cell. Biol. 13:4011.[Abstract/Free Full Text]
  43. Hobbs, S., Jitrapakdee, S. and Wallace, J. C. 1998. Development of a bicistronic vector driven by the human polypeptide chain elongation factor 1alpha promoter for creation of stable mammalian cell lines that express very high levels of recombinant proteins. Biochem. Biophys. Res. Commun. 252:368.[CrossRef][Web of Science][Medline]
  44. Bielinska, M. and Wilson, D. B. 1995. Regulation of J6 gene expression by transcription factor GATA-4. Biochem. J. 307:183.[Medline]
  45. Thomas, R. S., Tymms, M. J., McKinlay, L. H., Shannon, M. F., Seth, A. and Kola, I. 1997. ETS1, NFkappaB and AP1 synergistically transactivate the human GM-CSF promoter. Oncogene 14:2845.[CrossRef][Web of Science][Medline]
  46. Hoey, T., Sun, Y. L., Williamson, K. and Xu, X. 1995. Isolation of two new members of the NF-AT gene family and functional characterization of the NF-AT proteins. Immunity 2:461.[CrossRef][Web of Science][Medline]
  47. Olive, M., Krylov, D., Echlin, D. R., Gardner, K., Taparowsky, E. and Vinson, C. 1997. A dominant negative to activation protein-1 (AP1) that abolishes DNA binding and inhibits oncogenesis. J. Biol. Chem. 272:18586.[Abstract/Free Full Text]
  48. Ahn, S., Olive, M., Aggarwal, S., Krylov, D., Ginty, D. D. and Vinson, C. 1998. A dominant-negative inhibitor of CREB reveals that it is a general mediator of stimulus-dependent transcription of c-fos. Mol. Cell. Biol. 18:967.[Abstract/Free Full Text]
  49. Schreiber, E., Matthias, P., Muller, M. M. and Schaffiner, W. 1989. Rapid detection of octamer binding proteins with ‘mini-extracts’ prepared from a small number of cells. Nucleic Acids Res. 17:6419.[Free Full Text]
  50. Ng, K. W., Ridgway, P., Cohen, D. R. and Tremethick, D. J. 1997. The binding of a Fos/Jun heterodimer can completely disrupt the structure of a nucleosome. EMBO. J. 16:2072.[CrossRef][Web of Science][Medline]
  51. Himes, S. R., Coles, L. S., Katsikeros, R., Lang, R. K. and Shannon, M. F. 1993. HTLV-1 tax activation of the GM-CSF and G-CSF promoters requires the interaction of NF-kB with other transcription factor families. Oncogene 8:3189.[Web of Science][Medline]
  52. Young, I. G., Tan, C. L., Fisher, J. A., van Leeuwen, B. H. and Matthaei, K. I. 1999. Regulation of interleukin-5 expression in T lymphocytes: repression by dexamethasone. In Sanderson C. J., ed., Interleukin-5: From Molecule to Drug Target for Asthma, p. 289. Marcel Dekker, Inc, New York.
  53. Hoffmeyer, A., Grosse-Wilde, A., Flory, E., Neufeld, B., Kunz, M., Rapp, U. R. and Ludwig, S. 1999. Different mitogen-activated protein kinase signaling pathways cooperate to regulate tumor necrosis factor alpha gene expression in T lymphocytes. J. Biol. Chem. 274:4319.[Abstract/Free Full Text]
  54. Bassuk, A. G. and Leiden, J. M. 1995. A direct physical association between ETS and AP-1 transcription factors in normal human T cells. Immunity 3:223.[CrossRef][Web of Science][Medline]
  55. Naora, H. and Young, I. G. 1995. Comparison of the mechanisms regulating IL-5, IL-4, and three other lymphokine genes in the Th2 clone D10.G4.1. Exp. Hematol. 23:597.[Web of Science][Medline]
  56. Tan, C. L. 1998. Transcriptional Regulation of the Mouse Interleukin-5 Gene in T-Lymphocytes. PhD Thesis. Australian National University, Australia.
  57. Oosterwegel, M., Timmerman, J., Leiden, J. and Clevers, H. 1992. Expression of GATA-3 during lymphocyte differentiation and mouse embryogenesis. Dev. Immunol. 3:1.[Medline]
  58. Swanson, D. J., Zellmer, E. and Lewis, E. J. 1998. AP1 proteins mediate the cAMP response of the dopamine beta-hydroxylase gene. J. Biol. Chem. 273:24065.[Abstract/Free Full Text]
  59. Griffiths, M. R., Black, E. J., Culbert, A. A., Dickens, M., Shaw, P. E., Gillespie, D. A. and Tavare, J. M. 1998. Insulin-stimulated expression of c-fos, fra1 and c-jun accompanies the activation of the activator protein-1 (AP-1) transcriptional complex. Biochem. J. 335:19.[Medline]
  60. Rabault, B., Roussel, M. F., Quang, C. T. and Ghysdael, J. 1996. Phosphorylation of Ets1 regulates the complementation of a CSF-1 receptor impaired in mitogenesis. Oncogene 13:877.[Web of Science][Medline]
  61. Wasylyk, C., Bradford, A. P., Gutierrez-Hartmann, A. and Wasylyk, B. 1997. Conserved mechanisms of Ras regulation of evolutionary related transcription factors, Ets1 and Pointed P2. Oncogene 14:899.[CrossRef][Web of Science][Medline]
  62. Patton, S. E., Martin, M. L., Nelsen, L. L., Fang, X., Mills, G. B., Bast, R. C. Jr and Ostrowski, M. C. 1998. Activation of the ras-mitogen-activated protein kinase pathway and phosphorylation of ets-2 at position threonine 72 in human ovarian cancer cell lines. Cancer Res. 58:2253.[Abstract/Free Full Text]

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