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Role of Transcriptional Repressor ICER in Cyclic AMP-mediated Attenuation of Cytokine Gene Expression in Human Thymocytes

Role of Transcriptional Repressor ICER in Cyclic AMP-mediated Attenuation of Cytokine Gene... THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 273, No. 16, Issue of April 17, pp. 9544 –9551, 1998 © 1998 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Role of Transcriptional Repressor ICER in Cyclic AMP-mediated Attenuation of Cytokine Gene Expression in Human Thymocytes* (Received for publication, November 13, 1997) Josef Bodor‡ and Joel F. Habener§ From the Laboratory of Molecular Endocrinology, Massachusetts General Hospital, Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts 02114 Proliferating human medullary thymocytes can ex- lular cAMP inhibit extracellular signal-regulated kinases 1 and 2 and c-Jun NH -terminal kinases involved in the signal trans- hibit characteristic T helper cell type 1 cytokine re- sponses exemplified by the immediate early expression duction of mitogen-activated protein kinase pathways, T cell g, tumor necrosis factor-a, of interleukin-2, interferon- extracellular signal-regulated kinases 1 and 2 are insensitive b. Here we report that cAMP-mediated and lymphotoxin- to elevated levels of intracellular cAMP (6). Moreover, the attenuation of the transcription of T helper-1-specific cAMP-mediated inhibition of c-Jun NH -terminal kinase in T cytokine genes in human medullary thymocytes corre- cells shows delayed kinetics, an observation that correlates lates with the induction of the cAMP-mediated tran- with the induction of the cAMP-inducible early repressor scriptional repressor ICER (inducible cAMP early re- ICER (7). In addition, overexpression of NFAT achieved by pressor). We show that ICER binds specifically to transfection of NFAT-encoding cDNAs to lymphoma cells ab- several NFAT/AP-1 (nuclear factor of activated T cells/ rogates the sensitivity of cAMP-mediated inhibition of IL-2 activating protein-1) composite DNA sites essential for gene expression (8, 9). Importantly, phosphorylation of amino- the activation of the interleukin (IL)-2 promoter as well terminal serines of NFAT by protein kinase A does not prevent as to a homologous DNA motif present in the proximal calcineurin-mediated translocation of NFAT to the nucleus, g promoter. In the presence of segment of the interferon- despite its ability to prevent IL-2 gene expression (10, 11). The the minimal NFAT DNA-binding domain, which is suffi- notion that a newly synthesized transcriptional repressor cient for both DNA binding and AP-1 complex formation, rather than inhibition of upstream signal transduction path- ICER and NFAT form NFAT/ICER ternary complexes on ways could be involved in the cAMP-mediated transcriptional several NFAT/AP-1 DNA composite sites previously attenuation of T helper-1 cytokine expression was further identified as essential for the expression of the immuno- strengthened by the reported alleviation of cAMP-mediated regulatory cytokines such as IL-2, IL-4, granulocyte- inhibition of IL-2 expression in the presence of inhibitors of macrophage colony-stimulating factor, and tumor ne- a. In extracts prepared from human both RNA and protein synthesis (6). crosis factor- medullary thymocytes treated with forskolin and iono- ICER is a transcriptional repressor that appears to serve as mycin, these composite sites bind endogenously ex- a generalized negative regulator of the CREB and CREM fam- pressed ICER either singly or in complexes. Moreover, ilies of transcription factors as well as other related bZIP fam- in Jurkat cells, ectopically expressed ICER represses ily members (12–15). ICER isoforms represent a unique cAMP- transcription from NFAT-mediated, phorbol ester/iono- inducible CREM subfamily of transcription factors containing phore-activated IL-2, granulocyte-macrophage colony- cAMP-response elements within an internal P2 promoter. Be- a promot- stimulating factor, and tumor necrosis factor- cause of autoregulation of the cAMP-inducible P2 promoter, the ers. We present evidence that ICER interactions with expression of ICER can be intrinsically rhythmical. The rhyth- NFAT/AP-1 composite DNA sites correlate with its ability mical expression of ICER was first described in the pineal to repress transcription. These findings provide further gland and in the hypothalamic-pituitary-gonadal axis (16, 17). insight into the mechanisms involved in cAMP-mediated However, the P2 promoter of ICER is also inducible in organs transcriptional attenuation of cytokine expression. other than the pineal and hypothalamic-pituitary gonadal axis such as in specific subsets of T lymphocytes including human medullary thymocytes (7). Importantly, in the Jurkat T cell line It is well established that cAMP signaling is inhibitory to T ectopically expressed ICER can substitute for the inhibitory cell proliferation and effector functions. In particular, cAMP effects of cAMP on the transcriptional attenuation of IL-2 pro- inhibits the expression of T helper-1 cytokine genes (1–3). moter activity (7). Earlier reported studies of fibroblasts showed that elevated The NFAT (nuclear factor of activated T cells) and AP-1 levels of intracellular cAMP inhibit upstream signal transduc- (activating protein 1) represent two major transcription factor tion pathways involved in cell growth and differentiation (4, 5). families implicated in the transcription of the IL-2 promoter in In contrast to fibroblasts, in which elevated levels of intracel- proliferating T lymphocytes (18 –20). To address the possible mechanism by which ICER down-regulates IL-2 gene expres- * This work was supported by National Institutes of Health Grant DK 25532 (to J. F. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must The abbreviations used are: ICER, inducible cAMP early repressor; therefore be hereby marked “advertisement” in accordance with 18 AP-1, activating protein 1; bZIP, basic region/leucine zipper; CRE, U.S.C. Section 1734 solely to indicate this fact. cAMP-response element; CREB, CRE-binding protein; CREM, CRE ‡ To whom correspondence should be addressed. Present address: modulator protein; GM-CSF, granulocyte-monocyte colony-stimulating Experimental Immunology Branch, Division of Basic Sciences, National factor; IFNg, interferon-g; IL, interleukin, NFAT, nuclear factor of Cancer Institute, Bldg. 10, R4B14, National Institutes of Health, 10 activated T cells; NFAT DBD; DNA binding domain of NFAT; PMA, Center Dr., Bethesda, MD 20892-1360. Tel.: 301-496-6898; Fax: 301- 12-O-tetradecanoylphorbol 13-acetate; GST, glutathione S-transferase; 496-0887; E-mail: [email protected]. TNF, tumor necrosis factor; CD28RE, CD28-responsive element; CAT, § An Investigator with the Howard Hughes Medical Institute. chloramphenicol acetyltransferase. 9544 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. ICER Represses NFAT-mediated Cytokine Gene Expression 9545 previously (30). Recombinant c-Fos (Fos-(139 –243)) and c-Jun (Jun- sion, we examined the binding of bacterially expressed ICER to (187–334)) (gift from Dr. T. Kerppola) were purified from Escherichia all five NFAT motifs of the IL-2 promoter reported to be essen- coli overexpression strains by nickel chelate affinity chromatography tial for the full induction of the IL-2 gene (21) either alone or in (31, 32). the presence of the minimal DNA-binding domain of NFAT Nuclear Extracts and Gel Mobility Shift Assay—Whole cell extracts (NFAT DBD). The highest affinity of ICER binding was found were prepared by high salt extraction using 50 mM Hepes, 250 mM on a CD28-responsive element (CD28RE; 2160 NFAT/AP-1 NaCl, 5 mM EDTA, 0.5 mM dithiothreitol, and protease inhibitors (20 mM leupeptin, 10 mg/ml aprotinin, 2 mM phenylmethylsulfonyl fluoride) composite site) and 290 site, which is the motif in the IL-2 as described previously (30). Binding reactions were performed in a promoter that has striking sequence homology with the con- 15-ml reaction volume containing 20 mM HEPES, 1 mM MgCl ,50mM served proximal region (base pairs 273 to 248) of both the KCl, 12% glycerol, 0.1 mM EDTA, 0.5 mM dithiothreitol, 0.2 mgof human and mouse promoters of the IFNg genes (21, 22). poly(dI-dC) as an unspecific competitor and recombinant proteins or Moreover, certain NFAT/AP-1 composite sites that reside whole cells extracts as indicated. P-Labeled oligonucleotides and, within the IL-4, GM-CSF, and TNF-a promoters resemble where indicated, unlabeled competitor oligonucleotides in excess were added and incubated for 10 min at room temperature. Samples were those located within the IL-2 promoter (23–27). It is believed run on a 4% polyacrylamide gel in 0.53 TBE at 200 V for 2 h following that the mechanism underlying the actions of NFAT requires a 2-h prerun at 4 °C. The dried gels were exposed for autoradiography the binding of NFAT and/or NFAT/AP-1 to the NFAT/AP-1 overnight. The oligonucleotides encompassing NFAT composite sites of composite binding motifs as ternary complexes (18). These the following human promoters were used: IL-2 (245), 59-ctagaCATTT- complexes are believed to be essential for the transcriptional TGACACCCCCATAATATTTTTCCAGAATTa-39; IL-2 (290), 59-cta- expression of immunoregulatory cytokines during T cell prolif- gaGTCTTTGAAAATATGTGTAATATGTAAAACATa-39; IL-2 (2135), 59-ctagaATCAGAAGAGGAAAAATGAAGGTAATGTTTTa-39; IL-2 eration, such as IL-2, IL-4, GM-CSF, and TNF-a (19). Here we (2160), 59-ctagaAAAGAATTCCAAAGAGTCATCAGAAa-39; IL-2 demonstrate that ICER binds to these NFAT/AP-1 composite (2280), 59-ctagaAAGAAAGGAGGAAAAACTGTTTCATACAGa-39; GM- DNA sites in vitro, either directly or indirectly via complex CSF (2330), 59-gatccCCCCATCGGAGCCCCTGAGTCAGCATGGa-39; formation with the rel homology region of NFAT (NFAT DBD). GM-CSF (2420), 59-gatccCATCTTTCTCATGGAAAGATGACATCAG- Furthermore, we detect the induction of ICER-immunoreactive GGAa-39; GM-CSF (2550), 59-gatccGAAAGGAGGAAAGCAAGAGTC- complexes in extracts prepared from human medullary thymo- ATAATAAGAa-39; IL-4 (280), 59-gatccTAACTGCAATCTGGTGTAAT- AAAATTTTCCAATGTAAACTCATa-39; TNF-a (295), 59-gatccTTCC- cytes treated with forskolin and ionomycin. Ectopically ex- TCCAGATGAGCTCATGGGTTTCTCCACGACGGAa-39. Lowercase let- pressed ICER represses transcription from the IL-2, GM-CSF, ters indicate overhangs for SpeI/XbaI recognition sites (IL-2) or BamHI/ and TNF-a promoters activated by ionomycin and phorbol es- BglII recognition sites (GM-CSF, IL-4, TNF-a). As competitors, the ter, suggesting that the induction of ICER in response to cAMP following oligonucleotides were used: nf (mouse IL-4 NFAT, positions may be responsible for the observed cAMP-mediated transcrip- 269 to 279), 59-ATAAAATTTTCCAATGTAAA-39; ap (human metallo- tional attenuation of T helper-1 cytokine responses. thionein IIA AP-1 site, positions 2114 to 288), 59-GAGCCGCAAGTG ACTCAGCGCGGGGCG-39; and cre (mouse c-fos gene oligonucleotide, MATERIALS AND METHODS surrounding CRE site in position 260) 59-gatccCAGTTCCG- Preparation of Human Medullary Thymocytes—Human thymus CCCAGTGACGTAGGAAGTCCATCa-39. Lowercase letters indicate glands were obtained from children (ages 3 months to 4 years) under- overhangs for BamHI/BglII recognition sites. going corrective cardiac surgery. Thymocytes were fractionated over GST Pull-down Assays—GST-ICER (GST-CREB) Sepharose beads discontinuous Percoll gradients (Amersham Pharmacia Biotech) (28). prepared as described above were diluted 1:10 in 50 mM Tris, pH 7.5, Cells with densities of 1.060 , o , 1.070 and p . 1.070 were collected 150 mM NaCl, 0.5 mM EDTA, 10 mM Na (PO ) ,10mM NaF, 0.1% 3 4 2 and classified into large (significantly enriched for medullary thymo- Triton X-100, 2.5 mM leupeptin, 20 mM phenylmethylsulfonyl fluoride, cytes) and small (cortical) thymocytes according to an established pro- 100 mg/ml aprotinin, and recombinant protein(s) were added to a final tocol (7). Separated human thymocytes were maintained in short term volume of 250 ml and incubated at 4 °C on a nutator for 1.5 h. The beads cultures in RPMI 1640 medium, supplemented with 10% fetal calf were then washed three times with the same buffer, resuspended di- serum, 50 units/ml penicillin, and 25 mg/ml streptomycin, and treated rectly in Laemmli buffer, and loaded on 10% SDS-polyacrylamide gel. as indicated. Retained NFAT DBD protein was visualized by Western blotting de- RNase Protection Analysis—RNA extraction was performed as de- scribed below using R59 anti-NFAT DBD antiserum. scribed (Qiagen). RNA probes hCK1 and hCK3 were purchased from Transient Overexpression in Jurkat T Cells—Transfection assays 32 7 Pharmingen and labeled with [a- P]CTP using reagents from an RNA were performed by the DEAE-dextran technique. Typically, 10 Jurkat probe kit (Ambion). These probes were used for RNase protection stud- cells were transfected with 2 mg of the reporter and the same amount of ies according to the protocol provided by Ambion (RPAII ribonuclease ICER expression vector and treated 18 h posttransfection with phorbol protection assay kit). ester (10 mg/ml) and ionomycin (1 mg/ml) for 48 h. Luciferase and Western Blot Analysis—Separation of whole cell proteins (50 mg) was chloramphenicol acetyltransferase assays and quantification methods carried out by SDS-polyacrylamide gel electrophoresis (10%) for2hat are described elsewhere (Promega; Ref. 33). The percentage of conver- 40 mA in Tris/glycine buffer (25 mM Tris, 250 mM glycine, and 0.1% sion of [ C]chloramphenicol to its acetylated forms was quantified SDS) at room temperature. The proteins were electrotransferred (0.4 A) using ImageQuant (Molecular Dynamics). overnight at 4 °C in 10 mM Tris/glycine buffer with 12% methanol on Immobilon P membrane (Millipore Corp.). The membrane was blocked RESULTS in a Tris-buffered saline solution containing 0.05% Tween 20 (Sigma) cAMP-mediated Transcriptional Attenuation of T Helper-1- and 5% nonfat dry milk (Bio-Rad) for 1 h with gentle agitation at room responsive Cytokine Genes in Human Medullary Thymocytes— temperature. For immunological detection, the same solution without dry milk but containing the ICER or CREM-specific antiserum (CS4) Under the experimental conditions used, human medullary diluted 1:10,000 was agitated for 1 h, followed by three washes, with thymocytes exhibited the characteristics of naive T helper-0 subsequent incubation with horseradish peroxidase conjugated to a and T helper-1 cells with predominant IL-2 and IFNg expres- secondary antibody (Amersham Pharmacia Biotech) diluted 1:5000 for sion documented by RNase protection used for the evaluation of 1 h, followed by nine washes, and finally developed using an ECL kit mRNA levels of multiple cytokines (IL-2, IL-4, IL-5, IL-9, IL-10, (Amersham Pharmacia Biotech). IL-13, IL-14, IL-15, interferon-b, IFNg, human migration in- Expression and Purification of Recombinant Proteins—Human ICER II cDNA was subcloned into the pGEXKG vector (Amersham Pharma- hibitory factor, TNF-a, TNF-b,Ltb, and transforming growth cia Biotech) and expressed in bacteria as a glutathione S-transferase factors b1, b2, and b3) (Fig. 1). Stimulation of medullary thy- (GST) fusion protein. The pGSTagCREB construct was described pre- mocytes with a combination of phorbol ester and ionomycin viously (29). Purifications of both ICER and CREB were carried out significantly induced the synthesis of mRNAs encoding IL-2, with minor modifications according to the protocol previously estab- IFNg and to a lesser extent also TNF-a and Ltb (Fig. 1A, lane lished for CREB (29). NFATpXS-(1–187), encompassing the minimal 4; Fig. 1B, lane 4). At the same time, cotreatment with forskolin DNA-biding domain of NFATp (gift from Dr. A. Rao), was expressed in bacteria as a hexahistidine-tagged protein and purified as reported or 8-bromo-cAMP reduced the cellular mRNA levels of IL-2, 9546 ICER Represses NFAT-mediated Cytokine Gene Expression FIG.1. Cyclic AMP-mediated atten- uation of T helper-1 cytokine tran- scription in human (h) medullary thymocytes correlates with cAMP- mediated induction of the transcrip- tional repressor ICER. RNA from un- treated (U) medullary thymocytes and forskolin (F)- or 8-bromo-cAMP (Br)- treated human medullary thymocytes in the absence (lanes 2 and 3) or presence of phorbol ester and ionomycin (PMA 1 Iono; lanes 5 and 6) were scored for cyto- kine expression using RIBOQUANT probes hCK1 (A) and hCK3 (B)inthe RNase protection assay. Also shown are the corresponding RNase-protected probes following hybridization with yeast tRNA in the presence (1)(lane 7) or absence (2) (lane 8) of RNase. Templates for the anal- ysis of hL32 and hGAPDH housekeeping genes were included to allow assessments of total RNA levels (Pharmingen). Note that each probe (lane 8) migrates more slowly than its protected band; this is due to flanking sequences in the probe that are not protected by mRNA. C, Western immunoblotting using ICER-specific anti- serum generated against a peptide en- compassing the ICER-specific exon (7) shows induction of ICER in medullary thymocytes after 3 h but not after 12 h of forskolin treatment. There is no ICER protein detectable before treatment or in forskolin-treated human cortical thymo- cytes, which corresponds to a lack of de- tectable ICER mRNA reported previously in cortical thymocytes (7). IFNg, TNF-a, and Ltb (Fig. 1A, lanes 5 and 6; Fig. 1B, lanes 5 the IL-2 gene (21) (Fig. 2A). Four out of five NFAT sites in and 6). We propose that at least part of this transcriptional positions 290, 2135, 2160, and 2280 were previously charac- attenuation is based on cAMP-mediated expression of the tran- terized as NFAT/AP-1 composite sites due to their inherent scriptional repressor ICER and a subsequent blockade of ability to bind to the NFAT/AP-1 complex in a cooperative NFAT/AP-1 composite DNA sites essential for T helper-1 cyto- fashion (34). The fifth NFAT site in the most proximal position, kine expression. The inhibition by ICER may occur either di- NFAT 245, does not bind to the NFAT/AP-1 complex and was rectly through binding to the DNA element or indirectly via determined to be exclusively an NFAT binding site (34). Bac- protein-protein interactions such as to the rel homology domain terially expressed ICER, as well as ICER expressed in COS of NFAT (NFAT DBD). cells (data not shown), binds to all five NFAT sites, although to Cyclic AMP-mediated Attenuation of T Helper-1 Cytokine different extents (Fig. 2B). The strongest binding was observed Transcription in Human Medullary Thymocytes Correlates to the NFAT/AP-1 composite sites in positions 290 and 2160, with cAMP-mediated Induction of the Transcriptional Repres- an intermediate binding to the 2135 NFAT/AP-1 composite sor ICER—To test the proposed mechanism involved, we first site, and weak binding to the most proximal and distal NFAT sought evidence for the presence of ICER protein in human and NFAT/AP-1 sites in positions 245 and 2280, respectively. medullary thymocytes after forskolin treatment to explore The binding specificity of recombinant ICER was evaluated whether ICER may interact with important NFAT/AP-1 en- using a CREM-specific antiserum (CS4) that “supershifts” hancer motifs of the IL-2 and IFNg promoters. Western immu- ICER bound to specific oligonucleotides containing individually noblotting analysis using an ICER-specific antiserum (7) con- the five DNA motifs, leaving nonspecific binding unaffected firmed that ICER protein is indeed detectable in human (Fig. 2B, lanes 1–10) as well as by using control oligonucleotide medullary thymocytes but is absent in cortical thymocytes encompassing the first 21-base pair repeat (H21) of the human after3hof forskolin treatment (Fig. 1C). This finding is in T-cell lymphotrophic virus type I long terminal repeat promoter agreement with the previously observed delayed appearance of containing the CRE-like motif (35) (Fig. 2B, lanes 11 and 12). ICER mRNA in medullary thymocytes after their exposure to ICER Binding to NFAT/AP-1 Composite Sites in the Pres- forskolin and its subsequent deinduction (7). These observa- ence of NFAT DBD—To better understand the role of the in- tions suggest that the cAMP-mediated inhibition of cytokine teractions of ICER with NFAT/AP-1 composite DNA motifs, we expression may occur in a stage-specific manner in cells of T examined the effectiveness of ICER binding to the DNA motifs cell lineage. in the presence of NFAT DBD. This domain of NFAT, which ICER Binds to NFAT/AP-1 Composite Sites of IL-2 Promot- has the highest degree of conservation among NFAT family er—To further address the possible mechanism by which ICER members (36), is both necessary and sufficient for DNA binding down-regulates IL-2 gene expression, we examined the binding as well as to associate AP-1 with NFAT (30). All NFAT motifs of bacterially expressed ICER to all five NFAT motifs of the on the IL-2 promoter examined, with the exception of the IL-2 promoter reported to be essential for the full induction of NFAT/AP-1 composite site in position 290, bound NFAT effi- ICER Represses NFAT-mediated Cytokine Gene Expression 9547 FIG.2. ICER binding and formation of an NFAT/ICER complex with NFAT/AP-1 composite DNA-binding sites of the IL-2 promoter. A, list of NFAT/AP-1 composite sites of IL-2 promoter delineated previously to be essential for IL-2 expression (34) used in electromobility shift assay analysis. NFAT and AP-1 (top) denote domains of homology between NFAT/AP-1 composite sites and consensus sequences for NFAT and AP-1 in human IL-2 promoter, respectively. Numbers on the left correspond to the relative distance of the depicted DNA-binding motifs from the TATA box of the IL-2 promoter. B, bacterially expressed ICER binds in boiled total bacterial lysate specifically to 290 (lanes 3 and 4) and 2160 CD28RE motifs (lanes 7 and 8) and to a limited extent also to the rest of the motifs. The binding of ICER to these motifs is specific, since it is recognized by CS4 CREM-specific antiserum (CS4), causing a specific supershift (sICER), while the nonspecific complex (NS) remains unperturbed both in the presence of CS4 (1) and normal rabbit antisera (2). The control CRE consists of an oligonucleotide encompassing the 21-base pair repeat of human T-cell lymphotrophic virus type I long terminal repeat (H21 CRE) (35). C, in vitro binding of purified recombinant ICER and NFAT DBD (NFAT) proteins yields NFAT/ICER ternary complex (NF/IC) on CD28RE motif 2160 (lane 12) and to a lower extent also on the NFAT 245 motif (lane 3). D, ICER and truncated Fos and Jun proteins (AP) form similar complexes (NF/IC versus NF/AP) in the presence of NFAT DBD (NFAT)onthe 2160 motif of the IL-2 promoter (lanes 3 and 9), which are recognized by the CS4 anti-CREM (C)(lane 4) and DX anti-Fos (D)(lane 10) or K25 anti-Jun (K)(lane 11) and to a limited extent also by R59 NFAT (R) antisera (lane 12), respectively. Both unlabeled oligonucleotides NFAT (nf)(lanes 6 and 13) and AP-1 (ap)(lanes 7 and 14) efficiently compete for complex. ciently (Fig. 2C). The most pronounced ability of NFAT to associate with ICER was observed on the sites with the highest DNA binding efficiencies to both proteins, particularly at site position 2160 (CD28RE), and to a lesser extent to the NFAT site in position 245. Interestingly, this NFAT 245 site tends to show a stronger binding of ICER to NFAT in the NFAT/ICER complex compared with ICER by itself (Fig. 2C, lanes 1–3). Because the NFAT 245 of the IL-2 promoter is the only one of the five sites examined without an adjacent AP-1 site, the finding that this site can form an NFAT/ICER complex sug- gests that ICER itself may tether to NFAT by a protein-protein interaction in addition to a protein-DNA action. ICER Can Interact Directly with NFAT DBD—To examine whether or not ICER and NFAT may directly interact in the absence of DNA, GST pull-down assays were performed using a GST-ICER fusion protein linked to a Sepharose matrix in the FIG.3. ICER protein can interact directly with NFAT DBD. presence of a truncated NFAT consisting of the NFAT DBD Decreasing amounts of NFAT DBD protein retained on Sepharose ma- (Fig. 3, lane 1). GST-ICER formed a complex with NFAT DBD trix with equal amounts of GST-linked ICER (GST-ICER, lanes 1, 3, as demonstrated by retention of NFAT DBD. Interestingly, and 6) beside a negative control represented by equivalent amounts of GST-CREB failed to associate with NFAT DBD under the same GST matrix alone (GST, lanes 2, 4, and 7) interact specifically in a GST conditions (Fig. 3, lane 10). To examine the specificity of both pull-down assay. Lane 5 represents the input of NFAT DBD, which is components in the NFAT/ICER ternary complex found on the equivalent to protein added to GST-ICER beads retained in GST pull- down in lane 3. In contrast, GST-CREB (lane 10) does not interact with CD28RE (2160 motif) of the IL-2 promoter, we used both comparable amounts of NFAT DBD protein (input, lane 8). Lane 9 CREM-specific antiserum (CS4) and antiserum raised against represents GST-CREB beads alone. NFAT DBD retained on GST-ICER the minimal DNA-binding domain of NFAT (R59). Both anti- Sepharose beads was separated by SDS-polyacrylamide gel electro- sera either prevented or reduced the amount of the NFAT/ phoresis and visualized by Western blotting using the NFAT-specific ICER complex (Fig. 2D, lanes 4 and 5, respectively). Competi- antibody R59. 9548 ICER Represses NFAT-mediated Cytokine Gene Expression Studies performed on the conserved proximal motifs of both human and mouse IFNg promoters demonstrated high affinity ICER binding and a lack of NFAT binding or NFAT/ICER complex formation in the presence of NFAT/DBD (Fig. 4), a situation similar to that observed on the homologous 290 motif of the IL-2 promoter (Fig. 2). NFAT/AP-1 Composite Sites in the Context of the GM-CSF, IL-4, and TNF-a Promoters Bind ICER either Alone or in Com- plexes—NFAT/AP-1 binding sites have been shown previously to be essential for the efficient activation of the GM-CSF, IL-4, and TNF-a promoters. Therefore, we examined the binding to these sites of ICER and NFAT, both as purified recombinant proteins and in extracts prepared from human medullary thy- mocytes treated with forskolin and ionomycin (Fig. 5). These studies demonstrated that ICER can bind either by itself or in complexes with NFAT DBD to these composite sites in the promoters of the GM-CSF, IL-4, and TNF-a promoters, similar to the experiments using the binding site motifs of the IL-2 and IFNg promoters. The GM-420 DNA motif strongly bound the purified NFAT/ ICER complex (Fig. 5B, lane 6), whereas the GM-330 and FIG.4. ICER binds to a conserved proximal element of the GM-550 motifs bound the complex much more weakly (Fig. 5B, IFNg promoter homologous to the 290 motif of IL-2 promoter. A, lanes 3 and 9). It is noteworthy that the GM-420 motif has been ICER binds to both human and mouse motifs of the IFNg promoter shown to constitute the essential enhancer core of the GM-CSF (lanes 2 and 9), despite the fact that these motifs fail to bind NFAT DBD promoter (23). Likewise, NFAT/ICER readily formed a complex (NFAT)(lanes 3 and 10) in a fashion analogous to the 290 motif of the IL-2 promoter. No complex formation between ICER and NFAT was with the 280 element of the IL-4 and 295 element of the detected in electromobility shift assay (lanes 4 and 11). ICER bound to TNF-a promoter (Fig. 5B, lanes 12 and 15, respectively). Inter- the proximal element of the IFNg promoter is specifically “supershifted” estingly, the k3 motif of the TNF-a promoter, which contains an (sICER) by a CS4 CREM-specific antibody (C)(lanes 5 and 12). The “inverted CRE” motif adjacent to the NFAT composite site (27), mobility of the ICER complex is altered using competition with unla- beled CRE oligonucleotide (cre)(lanes 6 and 13) but unchanged in the created a complex with an electrophoretic mobility different presence of unlabeled NFAT oligonucleotide (nf)(lanes 7 and 14). B, from those observed on the motifs of the IL-2, IL-4, and GM- sequences of human and mouse proximal elements of the IFNg pro- CSF promoters (Fig. 2C and Fig. 5B). Furthermore, NFAT moter show significant homology to the 290 motif of the human IL-2 DBD alone formed a much slower mobility complex (lane 14), promoter. suggesting that NFAT may bind to the TNF-a motif as oligomers. The treatment of isolated human medullary thymocytes with tion with unlabeled oligonucleotides containing NFAT (nf forskolin and ionomycin readily induced the expression of oligonucleotide spanning mouse IL-4 NFAT, positions 269 to ICER (Fig. 5, D and E) not seen in uninduced thymocytes (Fig. 279) or the AP-1 motif (ap oligonucleotide spanning the human 5C). The binding of ICER to the oligonucleotides containing the metallothionein IIA AP-1 site, positions 2114 to 288) elimi- NFAT/AP-1 composite sites is inhibited by competition of the nated the NFAT/ICER complex (Fig. 2D, lanes 6 and 7, respec- binding with a CRE-containing oligonucleotide (Fig. 5D, lanes tively), suggesting that both ICER and NFAT are essential 4, 7, and 10) or interference of the binding with antiserum to components of the observed complex. ICER (C-Ab) (Fig. 5E, lanes 4, 7, and 10). NFAT/ICER Ternary Complex Shows Distinct Features in Ectopically Expressed ICER Represses NFAT-mediated Acti- Comparison with NFAT/AP-1 Complex on Various NFAT Mo- vation of IL-2, GM-CSF, and TNF-a Promoters—To determine tifs of IL-2 Promoter—Analogous experiments performed with whether ICER expression could supplant the effect of forskolin the NFAT DBD and truncated forms of Fos and Jun confirmed in transcriptional attenuation of various cytokine promoters in vitro that both NFAT/AP-1 and NFAT/ICER complexes could observed in medullary thymocytes, ICER (isoform II) was ex- exist in the context of the CD28RE (2160 NFAT/AP-1 compos- pressed in Jurkat T cells in transient transfection assays. Ex- ite site; Fig. 2D, lanes 8 –14). Interestingly, in the presence of pression of ICER down-regulated the human IL-2, GM-CSF, NFAT DBD, the majority of ICER protein was retained in the and TNF-a promoters activated by the combined treatment of complex with NFAT DBD, although NFAT/ICER and NFAT/ the cells with PMA and ionomycin, whereas ectopic expression AP-1 complexes appear to show unequal binding affinities to of neither isoform of ICER did not prove to have any significant different NFAT/AP-1 motifs. For example, the 2280 motif of effect on VP16-mediated transactivation of (33 GAL4)-CR- the IL-2 promoter, known from NFAT studies as the principal CAT under the same conditions (Fig. 6). Thus, ICER can be site for binding of the NFAT/AP-1 complex, binds NFAT/AP-1 induced by, and substituted for, forskolin in the transcriptional complexes effectively (32, 37), showing little or no detectable down-regulation of the calcineurin-dependent, NFAT/AP-1-me- formation of an NFAT/ICER complex (Fig. 2C, lanes 13–15). In diated transactivation of IL-2, GM-CSF, and TNF-a promoters contrast, the CD28RE (2160 NFAT/AP-1 composite site) of when induced by PMA and ionomycin. human IL-2 promoter creates a ternary NFAT/ICER complex DISCUSSION with an equal or slightly higher efficiency than the NFAT/AP-1 complex (Fig. 2D). The mechanism of cAMP-mediated inhibition of cytokine ICER Binds Directly to the Conserved Proximal Motif of expression in proliferating T lymphocytes has been attributed IFNg Promoter—Unlike the CD28RE (2160 motif), which to cAMP-mediated inactivation of upstream signal transduc- shows equally high affinity for both ICER and NFAT, the tion pathways directing the proliferation of T lymphocytes. NFAT/AP-1 motif of the IL-2 promoter in position 290, which Although this hypothesis was supported by studies of fibro- has striking homology to the conserved proximal element of the blasts (5), it was not confirmed by studies of T cells (6). Sur- INFg promoter, does not interact with NFAT DBD (Fig. 4). prisingly, several protein kinases required for T cell prolifera- ICER Represses NFAT-mediated Cytokine Gene Expression 9549 FIG.5. ICER forms complexes on several NFAT/AP-1 composite sites in GM-CSF, IL-4, and TNF-a promoters. A, a list of NFAT/AP-1 composite sites previously identified as essential for the expression of GM-CSF (25), IL-4 (23), and TNF-a (26, 27) promoters used in electromobility shift assay analysis. B, purified ICER and truncated NFAT DBD proteins form in vitro NFAT/ICER complexes on the enhancer core of GM-CSF promoter GM-420 (lane 6), the proximal NFAT/AP-1 motif of the IL-4 promoter (IL4(280))(lane 12), and the k3 motif of TNF-a (kTNF-a)(lane 15) with affinities comparable with the CD28RE motif (2160) of the IL-2 promoter (Fig. 2). C–E, NFAT/AP-1 composite sites in GM-CSF, IL-4, and TNF-a promoters bind to ICER and form ICER containing complexes in whole cell extracts prepared from freshly prepared human medullary thymocytes. Bacterially expressed, purified ICER II and ectopically expressed full-length NFAT1 protein in COS cells served as controls (lanes 1 and 2, respectively) for evaluation of extracts (lanes 3–11) prepared from untreated human medullary thymocytes (C) or human medullary thymocytes treated with forskolin (3 h) and ionomycin (15 min) (D and E). ICER and ICER-containing complexes were competed by unlabeled oligonucleotides containing CRE motifs (cre)(lanes 4, 7, and 10) or NFAT motifs (nf)(lanes 5, 8, and 11)(panels C and D) and were immunoreactive with CS4 CREM-specific antisera (C)(panel E, lanes 4, 7, and 10), whereas the R59 NFAT-specific antisera (R)(lanes 5, 8, and 11) affected the mobility of numerous complexes but showed little effect on the binding of ICER (panel E). tion were found to be insensitive or to exhibit a delayed indirect role of ICER has been demonstrated in transgenic mice response to high levels of intracellular cAMP (6). Here we overexpressing the dominant negative CREB mutant (a func- provide evidence that in human medullary thymocytes expres- tional homologue of ICER), which impairs the expression of sion of the transcriptional repressor ICER correlates with a IL-2 in thymocytes (44). Since the induction of IL-2 and IFNg delayed cAMP-mediated transcriptional attenuation of T help- expression is dependent on the activity conferred by each of the er-1 cytokine responses. individual DNA motifs (22, 34), a demonstration of a direct Footprinting and electrophoretic mobility shift analysis of binding of ICER and/or the formation of an inhibitory NFAT/ the IL-2 promoter revealed (34) that the originally defined ICER complex on any of these NFAT/AP-1 composite sites AP-1 site at position 2150 of the IL-2 promoter (37, 38), a major could provide an explanation for the mechanism involved in the CD28RE that contains an upstream NFAT binding site (39, transcriptional attenuation of IL-2 and IFNg expression medi- 40), represents a novel NFAT/AP-1 composite site at position ated by cAMP. These findings correlate with observations in 2160 (34). A reexamination of the original observations in which the conserved proximal motif of the IFNg promoter was which NF-kB was identified as a major component of the com- reported to be inhibited by forskolin in proliferating thymo- plex (41, 42) determined that NFAT is a prevalent component cytes of mice made transgenic with an IFNg promoter-lucif- of the complex that binds the CD28RE in vivo (34, 43). Our erase reporter gene (45). These findings further suggest that findings indicate that the CD28RE (2160 composite site of IL-2 both NFAT/AP-1 motifs, either those that directly bind ICER or promoter) effectively binds ICER either alone or in an NFAT/ those that form NFAT/ICER complexes, could convey ICER- ICER complex. These findings may be important for obtaining mediated transcriptional attenuation. a better understanding of a direct cAMP-mediated transcrip- It appears that numerous NFAT/AP-1 composite sites previ- tional attenuation of IL-2 expression. In addition, a potential ously identified in the context of GM-CSF, IL-4, and TNF-a 9550 ICER Represses NFAT-mediated Cytokine Gene Expression that these sites, previously shown to be essential for efficient expression (23, 26, 27), bind ICER either alone or in complexes similarly to the motifs of the IL-2 and IFNg promoters. It remains to be determined whether the induction of ICER can selectively modulate T helper-1 versus T helper-2 cytokine ex- pression in peripheral blood T lymphocytes. We have reported previously that human medullary but not cortical thymocytes synthesize ICER mRNA after3hof fors- kolin treatment (7). Western immunoblot analysis using an ICER-specific antiserum confirmed that in these conditions the ICER mRNA is translated efficiently into ICER protein. More- over, endogenously expressed ICER protein was detected in extracts prepared from human medullary thymocytes treated with forskolin and ionomycin using oligonucleotide probes con- taining NFAT/AP-1 DNA motifs that are able to form NFAT/ ICER complexes in vitro. In contrast to bacterially expressed ICER, endogenously expressed ICER in medullary thymocytes shows an altered mobility in gel shift assays, suggesting that posttranslational modification(s) may be involved in the regu- lation of the binding properties of ICER and/or degradative pathways involved in its proteolysis in vivo. The ICER-contain- ing complexes that are immunoreactive to ICER-supershifting antisera are efficiently competed by oligonucleotides contain- ing CRE or NFAT motifs. NFAT antisera that are unable to recognize directly bound ICER still affect the mobility of ICER- containing complexes, suggesting the possibility of the forma- tion of NFAT/ICER complexes in vivo. The ambiguity of the DNA-protein complexes in extracts of thymocytes observed on gel shift assays may be due to posttranslational modifications of the proteins involved (data not shown) and/or their potential consequences for DNA binding. At this point, the possibility cannot be excluded that proteins other than ICER and NFAT that contain homologous bZIP or rel homology regions may also participate in the formation of ICER-containing complexes. Finally, ectopic expression of ICER in Jurkat cells demon- strates that ICER, in agreement with its binding capabilities, can also effectively inhibit NFAT-mediated, phorbol ester/ ionophore-induced expression of IL-2, GM-CSF, and TNF-a FIG.6. ICER isoform II represses transcription from NFAT/ promoters. AP-1-activated cytokine promoters stimulated by PMA and In conclusion, inducible ICER expression in developing hu- ionomycin (P 1 I) treatment. A, promoter-reporter, IL-2-CAT (hu- man interleukin-2 (32)); CD28RE (2160 AP-Luc; gift from A. Rao); man medullary thymocytes as well as in certain subset(s) of GM-CSF-CAT (25) (human granulocyte-macrophage colony stimulating human peripheral blood lymphocytes (7) and monocytes (work factor), TNF-a-Luc (contains the sequence from 2614 to 120 of the in progress) could significantly influence their respective effec- human tumor necrosis factor-a in pGL2; gift from S. L. McKnight). A tor functions(s). The proposed inhibitory effects on effector control (33 GAL4)-CR-CAT (with three GAL-4 binding sites substitut- ing 21-base pair repeats in the human T-cell lymphotrophic virus type function of the immune system mediated by ICER may be I long terminal repeat (35)) transactivated by GAL4VP16 (46) is not related to its ability to bind (mask) a wide range of CRE and affected in Jurkat cells by isoforms of ICER (ICER II, ICER IIg, ICER AP-1 motifs and/or its ability to inactivate certain transcription I). B, amounts of respective cytokine reporters and ICER expression complexes via protein-protein interactions. constructs in transient transfections were kept constant (2 mg). Error bars represent S.D. values calculated from three or more experiments. Acknowledgments—We thank Dr. A. Rao for the generous gift of truncated NFAT DBD proteins and NFAT antibodies; Dr. T. K. promoters as essential determinants of their expression (23, 25, Kerppola for the gift of truncated Fos and Jun proteins; Drs. J. L. 27) can associate with ICER. This property does not seem to be Strominger, David Weng, Peter A. Cohen, K. Shaw, P. McCaffrey, Cathy Bare, and Ronald E. Gress for stimulating discussions; E. a universal feature shared by all NFAT/AP-1 composite sites Burgeon for expert advice on GST pull-down assays; and T. 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Role of Transcriptional Repressor ICER in Cyclic AMP-mediated Attenuation of Cytokine Gene Expression in Human Thymocytes

Bodor, Josef; Habener, Joel F.
Journal of Biological Chemistry – Apr 1, 1998
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10.1074/jbc.273.16.9544
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Abstract

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 273, No. 16, Issue of April 17, pp. 9544 –9551, 1998 © 1998 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Role of Transcriptional Repressor ICER in Cyclic AMP-mediated Attenuation of Cytokine Gene Expression in Human Thymocytes* (Received for publication, November 13, 1997) Josef Bodor‡ and Joel F. Habener§ From the Laboratory of Molecular Endocrinology, Massachusetts General Hospital, Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts 02114 Proliferating human medullary thymocytes can ex- lular cAMP inhibit extracellular signal-regulated kinases 1 and 2 and c-Jun NH -terminal kinases involved in the signal trans- hibit characteristic T helper cell type 1 cytokine re- sponses exemplified by the immediate early expression duction of mitogen-activated protein kinase pathways, T cell g, tumor necrosis factor-a, of interleukin-2, interferon- extracellular signal-regulated kinases 1 and 2 are insensitive b. Here we report that cAMP-mediated and lymphotoxin- to elevated levels of intracellular cAMP (6). Moreover, the attenuation of the transcription of T helper-1-specific cAMP-mediated inhibition of c-Jun NH -terminal kinase in T cytokine genes in human medullary thymocytes corre- cells shows delayed kinetics, an observation that correlates lates with the induction of the cAMP-mediated tran- with the induction of the cAMP-inducible early repressor scriptional repressor ICER (inducible cAMP early re- ICER (7). In addition, overexpression of NFAT achieved by pressor). We show that ICER binds specifically to transfection of NFAT-encoding cDNAs to lymphoma cells ab- several NFAT/AP-1 (nuclear factor of activated T cells/ rogates the sensitivity of cAMP-mediated inhibition of IL-2 activating protein-1) composite DNA sites essential for gene expression (8, 9). Importantly, phosphorylation of amino- the activation of the interleukin (IL)-2 promoter as well terminal serines of NFAT by protein kinase A does not prevent as to a homologous DNA motif present in the proximal calcineurin-mediated translocation of NFAT to the nucleus, g promoter. In the presence of segment of the interferon- despite its ability to prevent IL-2 gene expression (10, 11). The the minimal NFAT DNA-binding domain, which is suffi- notion that a newly synthesized transcriptional repressor cient for both DNA binding and AP-1 complex formation, rather than inhibition of upstream signal transduction path- ICER and NFAT form NFAT/ICER ternary complexes on ways could be involved in the cAMP-mediated transcriptional several NFAT/AP-1 DNA composite sites previously attenuation of T helper-1 cytokine expression was further identified as essential for the expression of the immuno- strengthened by the reported alleviation of cAMP-mediated regulatory cytokines such as IL-2, IL-4, granulocyte- inhibition of IL-2 expression in the presence of inhibitors of macrophage colony-stimulating factor, and tumor ne- a. In extracts prepared from human both RNA and protein synthesis (6). crosis factor- medullary thymocytes treated with forskolin and iono- ICER is a transcriptional repressor that appears to serve as mycin, these composite sites bind endogenously ex- a generalized negative regulator of the CREB and CREM fam- pressed ICER either singly or in complexes. Moreover, ilies of transcription factors as well as other related bZIP fam- in Jurkat cells, ectopically expressed ICER represses ily members (12–15). ICER isoforms represent a unique cAMP- transcription from NFAT-mediated, phorbol ester/iono- inducible CREM subfamily of transcription factors containing phore-activated IL-2, granulocyte-macrophage colony- cAMP-response elements within an internal P2 promoter. Be- a promot- stimulating factor, and tumor necrosis factor- cause of autoregulation of the cAMP-inducible P2 promoter, the ers. We present evidence that ICER interactions with expression of ICER can be intrinsically rhythmical. The rhyth- NFAT/AP-1 composite DNA sites correlate with its ability mical expression of ICER was first described in the pineal to repress transcription. These findings provide further gland and in the hypothalamic-pituitary-gonadal axis (16, 17). insight into the mechanisms involved in cAMP-mediated However, the P2 promoter of ICER is also inducible in organs transcriptional attenuation of cytokine expression. other than the pineal and hypothalamic-pituitary gonadal axis such as in specific subsets of T lymphocytes including human medullary thymocytes (7). Importantly, in the Jurkat T cell line It is well established that cAMP signaling is inhibitory to T ectopically expressed ICER can substitute for the inhibitory cell proliferation and effector functions. In particular, cAMP effects of cAMP on the transcriptional attenuation of IL-2 pro- inhibits the expression of T helper-1 cytokine genes (1–3). moter activity (7). Earlier reported studies of fibroblasts showed that elevated The NFAT (nuclear factor of activated T cells) and AP-1 levels of intracellular cAMP inhibit upstream signal transduc- (activating protein 1) represent two major transcription factor tion pathways involved in cell growth and differentiation (4, 5). families implicated in the transcription of the IL-2 promoter in In contrast to fibroblasts, in which elevated levels of intracel- proliferating T lymphocytes (18 –20). To address the possible mechanism by which ICER down-regulates IL-2 gene expres- * This work was supported by National Institutes of Health Grant DK 25532 (to J. F. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must The abbreviations used are: ICER, inducible cAMP early repressor; therefore be hereby marked “advertisement” in accordance with 18 AP-1, activating protein 1; bZIP, basic region/leucine zipper; CRE, U.S.C. Section 1734 solely to indicate this fact. cAMP-response element; CREB, CRE-binding protein; CREM, CRE ‡ To whom correspondence should be addressed. Present address: modulator protein; GM-CSF, granulocyte-monocyte colony-stimulating Experimental Immunology Branch, Division of Basic Sciences, National factor; IFNg, interferon-g; IL, interleukin, NFAT, nuclear factor of Cancer Institute, Bldg. 10, R4B14, National Institutes of Health, 10 activated T cells; NFAT DBD; DNA binding domain of NFAT; PMA, Center Dr., Bethesda, MD 20892-1360. Tel.: 301-496-6898; Fax: 301- 12-O-tetradecanoylphorbol 13-acetate; GST, glutathione S-transferase; 496-0887; E-mail: [email protected]. TNF, tumor necrosis factor; CD28RE, CD28-responsive element; CAT, § An Investigator with the Howard Hughes Medical Institute. chloramphenicol acetyltransferase. 9544 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. ICER Represses NFAT-mediated Cytokine Gene Expression 9545 previously (30). Recombinant c-Fos (Fos-(139 –243)) and c-Jun (Jun- sion, we examined the binding of bacterially expressed ICER to (187–334)) (gift from Dr. T. Kerppola) were purified from Escherichia all five NFAT motifs of the IL-2 promoter reported to be essen- coli overexpression strains by nickel chelate affinity chromatography tial for the full induction of the IL-2 gene (21) either alone or in (31, 32). the presence of the minimal DNA-binding domain of NFAT Nuclear Extracts and Gel Mobility Shift Assay—Whole cell extracts (NFAT DBD). The highest affinity of ICER binding was found were prepared by high salt extraction using 50 mM Hepes, 250 mM on a CD28-responsive element (CD28RE; 2160 NFAT/AP-1 NaCl, 5 mM EDTA, 0.5 mM dithiothreitol, and protease inhibitors (20 mM leupeptin, 10 mg/ml aprotinin, 2 mM phenylmethylsulfonyl fluoride) composite site) and 290 site, which is the motif in the IL-2 as described previously (30). Binding reactions were performed in a promoter that has striking sequence homology with the con- 15-ml reaction volume containing 20 mM HEPES, 1 mM MgCl ,50mM served proximal region (base pairs 273 to 248) of both the KCl, 12% glycerol, 0.1 mM EDTA, 0.5 mM dithiothreitol, 0.2 mgof human and mouse promoters of the IFNg genes (21, 22). poly(dI-dC) as an unspecific competitor and recombinant proteins or Moreover, certain NFAT/AP-1 composite sites that reside whole cells extracts as indicated. P-Labeled oligonucleotides and, within the IL-4, GM-CSF, and TNF-a promoters resemble where indicated, unlabeled competitor oligonucleotides in excess were added and incubated for 10 min at room temperature. Samples were those located within the IL-2 promoter (23–27). It is believed run on a 4% polyacrylamide gel in 0.53 TBE at 200 V for 2 h following that the mechanism underlying the actions of NFAT requires a 2-h prerun at 4 °C. The dried gels were exposed for autoradiography the binding of NFAT and/or NFAT/AP-1 to the NFAT/AP-1 overnight. The oligonucleotides encompassing NFAT composite sites of composite binding motifs as ternary complexes (18). These the following human promoters were used: IL-2 (245), 59-ctagaCATTT- complexes are believed to be essential for the transcriptional TGACACCCCCATAATATTTTTCCAGAATTa-39; IL-2 (290), 59-cta- expression of immunoregulatory cytokines during T cell prolif- gaGTCTTTGAAAATATGTGTAATATGTAAAACATa-39; IL-2 (2135), 59-ctagaATCAGAAGAGGAAAAATGAAGGTAATGTTTTa-39; IL-2 eration, such as IL-2, IL-4, GM-CSF, and TNF-a (19). Here we (2160), 59-ctagaAAAGAATTCCAAAGAGTCATCAGAAa-39; IL-2 demonstrate that ICER binds to these NFAT/AP-1 composite (2280), 59-ctagaAAGAAAGGAGGAAAAACTGTTTCATACAGa-39; GM- DNA sites in vitro, either directly or indirectly via complex CSF (2330), 59-gatccCCCCATCGGAGCCCCTGAGTCAGCATGGa-39; formation with the rel homology region of NFAT (NFAT DBD). GM-CSF (2420), 59-gatccCATCTTTCTCATGGAAAGATGACATCAG- Furthermore, we detect the induction of ICER-immunoreactive GGAa-39; GM-CSF (2550), 59-gatccGAAAGGAGGAAAGCAAGAGTC- complexes in extracts prepared from human medullary thymo- ATAATAAGAa-39; IL-4 (280), 59-gatccTAACTGCAATCTGGTGTAAT- AAAATTTTCCAATGTAAACTCATa-39; TNF-a (295), 59-gatccTTCC- cytes treated with forskolin and ionomycin. Ectopically ex- TCCAGATGAGCTCATGGGTTTCTCCACGACGGAa-39. Lowercase let- pressed ICER represses transcription from the IL-2, GM-CSF, ters indicate overhangs for SpeI/XbaI recognition sites (IL-2) or BamHI/ and TNF-a promoters activated by ionomycin and phorbol es- BglII recognition sites (GM-CSF, IL-4, TNF-a). As competitors, the ter, suggesting that the induction of ICER in response to cAMP following oligonucleotides were used: nf (mouse IL-4 NFAT, positions may be responsible for the observed cAMP-mediated transcrip- 269 to 279), 59-ATAAAATTTTCCAATGTAAA-39; ap (human metallo- tional attenuation of T helper-1 cytokine responses. thionein IIA AP-1 site, positions 2114 to 288), 59-GAGCCGCAAGTG ACTCAGCGCGGGGCG-39; and cre (mouse c-fos gene oligonucleotide, MATERIALS AND METHODS surrounding CRE site in position 260) 59-gatccCAGTTCCG- Preparation of Human Medullary Thymocytes—Human thymus CCCAGTGACGTAGGAAGTCCATCa-39. Lowercase letters indicate glands were obtained from children (ages 3 months to 4 years) under- overhangs for BamHI/BglII recognition sites. going corrective cardiac surgery. Thymocytes were fractionated over GST Pull-down Assays—GST-ICER (GST-CREB) Sepharose beads discontinuous Percoll gradients (Amersham Pharmacia Biotech) (28). prepared as described above were diluted 1:10 in 50 mM Tris, pH 7.5, Cells with densities of 1.060 , o , 1.070 and p . 1.070 were collected 150 mM NaCl, 0.5 mM EDTA, 10 mM Na (PO ) ,10mM NaF, 0.1% 3 4 2 and classified into large (significantly enriched for medullary thymo- Triton X-100, 2.5 mM leupeptin, 20 mM phenylmethylsulfonyl fluoride, cytes) and small (cortical) thymocytes according to an established pro- 100 mg/ml aprotinin, and recombinant protein(s) were added to a final tocol (7). Separated human thymocytes were maintained in short term volume of 250 ml and incubated at 4 °C on a nutator for 1.5 h. The beads cultures in RPMI 1640 medium, supplemented with 10% fetal calf were then washed three times with the same buffer, resuspended di- serum, 50 units/ml penicillin, and 25 mg/ml streptomycin, and treated rectly in Laemmli buffer, and loaded on 10% SDS-polyacrylamide gel. as indicated. Retained NFAT DBD protein was visualized by Western blotting de- RNase Protection Analysis—RNA extraction was performed as de- scribed below using R59 anti-NFAT DBD antiserum. scribed (Qiagen). RNA probes hCK1 and hCK3 were purchased from Transient Overexpression in Jurkat T Cells—Transfection assays 32 7 Pharmingen and labeled with [a- P]CTP using reagents from an RNA were performed by the DEAE-dextran technique. Typically, 10 Jurkat probe kit (Ambion). These probes were used for RNase protection stud- cells were transfected with 2 mg of the reporter and the same amount of ies according to the protocol provided by Ambion (RPAII ribonuclease ICER expression vector and treated 18 h posttransfection with phorbol protection assay kit). ester (10 mg/ml) and ionomycin (1 mg/ml) for 48 h. Luciferase and Western Blot Analysis—Separation of whole cell proteins (50 mg) was chloramphenicol acetyltransferase assays and quantification methods carried out by SDS-polyacrylamide gel electrophoresis (10%) for2hat are described elsewhere (Promega; Ref. 33). The percentage of conver- 40 mA in Tris/glycine buffer (25 mM Tris, 250 mM glycine, and 0.1% sion of [ C]chloramphenicol to its acetylated forms was quantified SDS) at room temperature. The proteins were electrotransferred (0.4 A) using ImageQuant (Molecular Dynamics). overnight at 4 °C in 10 mM Tris/glycine buffer with 12% methanol on Immobilon P membrane (Millipore Corp.). The membrane was blocked RESULTS in a Tris-buffered saline solution containing 0.05% Tween 20 (Sigma) cAMP-mediated Transcriptional Attenuation of T Helper-1- and 5% nonfat dry milk (Bio-Rad) for 1 h with gentle agitation at room responsive Cytokine Genes in Human Medullary Thymocytes— temperature. For immunological detection, the same solution without dry milk but containing the ICER or CREM-specific antiserum (CS4) Under the experimental conditions used, human medullary diluted 1:10,000 was agitated for 1 h, followed by three washes, with thymocytes exhibited the characteristics of naive T helper-0 subsequent incubation with horseradish peroxidase conjugated to a and T helper-1 cells with predominant IL-2 and IFNg expres- secondary antibody (Amersham Pharmacia Biotech) diluted 1:5000 for sion documented by RNase protection used for the evaluation of 1 h, followed by nine washes, and finally developed using an ECL kit mRNA levels of multiple cytokines (IL-2, IL-4, IL-5, IL-9, IL-10, (Amersham Pharmacia Biotech). IL-13, IL-14, IL-15, interferon-b, IFNg, human migration in- Expression and Purification of Recombinant Proteins—Human ICER II cDNA was subcloned into the pGEXKG vector (Amersham Pharma- hibitory factor, TNF-a, TNF-b,Ltb, and transforming growth cia Biotech) and expressed in bacteria as a glutathione S-transferase factors b1, b2, and b3) (Fig. 1). Stimulation of medullary thy- (GST) fusion protein. The pGSTagCREB construct was described pre- mocytes with a combination of phorbol ester and ionomycin viously (29). Purifications of both ICER and CREB were carried out significantly induced the synthesis of mRNAs encoding IL-2, with minor modifications according to the protocol previously estab- IFNg and to a lesser extent also TNF-a and Ltb (Fig. 1A, lane lished for CREB (29). NFATpXS-(1–187), encompassing the minimal 4; Fig. 1B, lane 4). At the same time, cotreatment with forskolin DNA-biding domain of NFATp (gift from Dr. A. Rao), was expressed in bacteria as a hexahistidine-tagged protein and purified as reported or 8-bromo-cAMP reduced the cellular mRNA levels of IL-2, 9546 ICER Represses NFAT-mediated Cytokine Gene Expression FIG.1. Cyclic AMP-mediated atten- uation of T helper-1 cytokine tran- scription in human (h) medullary thymocytes correlates with cAMP- mediated induction of the transcrip- tional repressor ICER. RNA from un- treated (U) medullary thymocytes and forskolin (F)- or 8-bromo-cAMP (Br)- treated human medullary thymocytes in the absence (lanes 2 and 3) or presence of phorbol ester and ionomycin (PMA 1 Iono; lanes 5 and 6) were scored for cyto- kine expression using RIBOQUANT probes hCK1 (A) and hCK3 (B)inthe RNase protection assay. Also shown are the corresponding RNase-protected probes following hybridization with yeast tRNA in the presence (1)(lane 7) or absence (2) (lane 8) of RNase. Templates for the anal- ysis of hL32 and hGAPDH housekeeping genes were included to allow assessments of total RNA levels (Pharmingen). Note that each probe (lane 8) migrates more slowly than its protected band; this is due to flanking sequences in the probe that are not protected by mRNA. C, Western immunoblotting using ICER-specific anti- serum generated against a peptide en- compassing the ICER-specific exon (7) shows induction of ICER in medullary thymocytes after 3 h but not after 12 h of forskolin treatment. There is no ICER protein detectable before treatment or in forskolin-treated human cortical thymo- cytes, which corresponds to a lack of de- tectable ICER mRNA reported previously in cortical thymocytes (7). IFNg, TNF-a, and Ltb (Fig. 1A, lanes 5 and 6; Fig. 1B, lanes 5 the IL-2 gene (21) (Fig. 2A). Four out of five NFAT sites in and 6). We propose that at least part of this transcriptional positions 290, 2135, 2160, and 2280 were previously charac- attenuation is based on cAMP-mediated expression of the tran- terized as NFAT/AP-1 composite sites due to their inherent scriptional repressor ICER and a subsequent blockade of ability to bind to the NFAT/AP-1 complex in a cooperative NFAT/AP-1 composite DNA sites essential for T helper-1 cyto- fashion (34). The fifth NFAT site in the most proximal position, kine expression. The inhibition by ICER may occur either di- NFAT 245, does not bind to the NFAT/AP-1 complex and was rectly through binding to the DNA element or indirectly via determined to be exclusively an NFAT binding site (34). Bac- protein-protein interactions such as to the rel homology domain terially expressed ICER, as well as ICER expressed in COS of NFAT (NFAT DBD). cells (data not shown), binds to all five NFAT sites, although to Cyclic AMP-mediated Attenuation of T Helper-1 Cytokine different extents (Fig. 2B). The strongest binding was observed Transcription in Human Medullary Thymocytes Correlates to the NFAT/AP-1 composite sites in positions 290 and 2160, with cAMP-mediated Induction of the Transcriptional Repres- an intermediate binding to the 2135 NFAT/AP-1 composite sor ICER—To test the proposed mechanism involved, we first site, and weak binding to the most proximal and distal NFAT sought evidence for the presence of ICER protein in human and NFAT/AP-1 sites in positions 245 and 2280, respectively. medullary thymocytes after forskolin treatment to explore The binding specificity of recombinant ICER was evaluated whether ICER may interact with important NFAT/AP-1 en- using a CREM-specific antiserum (CS4) that “supershifts” hancer motifs of the IL-2 and IFNg promoters. Western immu- ICER bound to specific oligonucleotides containing individually noblotting analysis using an ICER-specific antiserum (7) con- the five DNA motifs, leaving nonspecific binding unaffected firmed that ICER protein is indeed detectable in human (Fig. 2B, lanes 1–10) as well as by using control oligonucleotide medullary thymocytes but is absent in cortical thymocytes encompassing the first 21-base pair repeat (H21) of the human after3hof forskolin treatment (Fig. 1C). This finding is in T-cell lymphotrophic virus type I long terminal repeat promoter agreement with the previously observed delayed appearance of containing the CRE-like motif (35) (Fig. 2B, lanes 11 and 12). ICER mRNA in medullary thymocytes after their exposure to ICER Binding to NFAT/AP-1 Composite Sites in the Pres- forskolin and its subsequent deinduction (7). These observa- ence of NFAT DBD—To better understand the role of the in- tions suggest that the cAMP-mediated inhibition of cytokine teractions of ICER with NFAT/AP-1 composite DNA motifs, we expression may occur in a stage-specific manner in cells of T examined the effectiveness of ICER binding to the DNA motifs cell lineage. in the presence of NFAT DBD. This domain of NFAT, which ICER Binds to NFAT/AP-1 Composite Sites of IL-2 Promot- has the highest degree of conservation among NFAT family er—To further address the possible mechanism by which ICER members (36), is both necessary and sufficient for DNA binding down-regulates IL-2 gene expression, we examined the binding as well as to associate AP-1 with NFAT (30). All NFAT motifs of bacterially expressed ICER to all five NFAT motifs of the on the IL-2 promoter examined, with the exception of the IL-2 promoter reported to be essential for the full induction of NFAT/AP-1 composite site in position 290, bound NFAT effi- ICER Represses NFAT-mediated Cytokine Gene Expression 9547 FIG.2. ICER binding and formation of an NFAT/ICER complex with NFAT/AP-1 composite DNA-binding sites of the IL-2 promoter. A, list of NFAT/AP-1 composite sites of IL-2 promoter delineated previously to be essential for IL-2 expression (34) used in electromobility shift assay analysis. NFAT and AP-1 (top) denote domains of homology between NFAT/AP-1 composite sites and consensus sequences for NFAT and AP-1 in human IL-2 promoter, respectively. Numbers on the left correspond to the relative distance of the depicted DNA-binding motifs from the TATA box of the IL-2 promoter. B, bacterially expressed ICER binds in boiled total bacterial lysate specifically to 290 (lanes 3 and 4) and 2160 CD28RE motifs (lanes 7 and 8) and to a limited extent also to the rest of the motifs. The binding of ICER to these motifs is specific, since it is recognized by CS4 CREM-specific antiserum (CS4), causing a specific supershift (sICER), while the nonspecific complex (NS) remains unperturbed both in the presence of CS4 (1) and normal rabbit antisera (2). The control CRE consists of an oligonucleotide encompassing the 21-base pair repeat of human T-cell lymphotrophic virus type I long terminal repeat (H21 CRE) (35). C, in vitro binding of purified recombinant ICER and NFAT DBD (NFAT) proteins yields NFAT/ICER ternary complex (NF/IC) on CD28RE motif 2160 (lane 12) and to a lower extent also on the NFAT 245 motif (lane 3). D, ICER and truncated Fos and Jun proteins (AP) form similar complexes (NF/IC versus NF/AP) in the presence of NFAT DBD (NFAT)onthe 2160 motif of the IL-2 promoter (lanes 3 and 9), which are recognized by the CS4 anti-CREM (C)(lane 4) and DX anti-Fos (D)(lane 10) or K25 anti-Jun (K)(lane 11) and to a limited extent also by R59 NFAT (R) antisera (lane 12), respectively. Both unlabeled oligonucleotides NFAT (nf)(lanes 6 and 13) and AP-1 (ap)(lanes 7 and 14) efficiently compete for complex. ciently (Fig. 2C). The most pronounced ability of NFAT to associate with ICER was observed on the sites with the highest DNA binding efficiencies to both proteins, particularly at site position 2160 (CD28RE), and to a lesser extent to the NFAT site in position 245. Interestingly, this NFAT 245 site tends to show a stronger binding of ICER to NFAT in the NFAT/ICER complex compared with ICER by itself (Fig. 2C, lanes 1–3). Because the NFAT 245 of the IL-2 promoter is the only one of the five sites examined without an adjacent AP-1 site, the finding that this site can form an NFAT/ICER complex sug- gests that ICER itself may tether to NFAT by a protein-protein interaction in addition to a protein-DNA action. ICER Can Interact Directly with NFAT DBD—To examine whether or not ICER and NFAT may directly interact in the absence of DNA, GST pull-down assays were performed using a GST-ICER fusion protein linked to a Sepharose matrix in the FIG.3. ICER protein can interact directly with NFAT DBD. presence of a truncated NFAT consisting of the NFAT DBD Decreasing amounts of NFAT DBD protein retained on Sepharose ma- (Fig. 3, lane 1). GST-ICER formed a complex with NFAT DBD trix with equal amounts of GST-linked ICER (GST-ICER, lanes 1, 3, as demonstrated by retention of NFAT DBD. Interestingly, and 6) beside a negative control represented by equivalent amounts of GST-CREB failed to associate with NFAT DBD under the same GST matrix alone (GST, lanes 2, 4, and 7) interact specifically in a GST conditions (Fig. 3, lane 10). To examine the specificity of both pull-down assay. Lane 5 represents the input of NFAT DBD, which is components in the NFAT/ICER ternary complex found on the equivalent to protein added to GST-ICER beads retained in GST pull- down in lane 3. In contrast, GST-CREB (lane 10) does not interact with CD28RE (2160 motif) of the IL-2 promoter, we used both comparable amounts of NFAT DBD protein (input, lane 8). Lane 9 CREM-specific antiserum (CS4) and antiserum raised against represents GST-CREB beads alone. NFAT DBD retained on GST-ICER the minimal DNA-binding domain of NFAT (R59). Both anti- Sepharose beads was separated by SDS-polyacrylamide gel electro- sera either prevented or reduced the amount of the NFAT/ phoresis and visualized by Western blotting using the NFAT-specific ICER complex (Fig. 2D, lanes 4 and 5, respectively). Competi- antibody R59. 9548 ICER Represses NFAT-mediated Cytokine Gene Expression Studies performed on the conserved proximal motifs of both human and mouse IFNg promoters demonstrated high affinity ICER binding and a lack of NFAT binding or NFAT/ICER complex formation in the presence of NFAT/DBD (Fig. 4), a situation similar to that observed on the homologous 290 motif of the IL-2 promoter (Fig. 2). NFAT/AP-1 Composite Sites in the Context of the GM-CSF, IL-4, and TNF-a Promoters Bind ICER either Alone or in Com- plexes—NFAT/AP-1 binding sites have been shown previously to be essential for the efficient activation of the GM-CSF, IL-4, and TNF-a promoters. Therefore, we examined the binding to these sites of ICER and NFAT, both as purified recombinant proteins and in extracts prepared from human medullary thy- mocytes treated with forskolin and ionomycin (Fig. 5). These studies demonstrated that ICER can bind either by itself or in complexes with NFAT DBD to these composite sites in the promoters of the GM-CSF, IL-4, and TNF-a promoters, similar to the experiments using the binding site motifs of the IL-2 and IFNg promoters. The GM-420 DNA motif strongly bound the purified NFAT/ ICER complex (Fig. 5B, lane 6), whereas the GM-330 and FIG.4. ICER binds to a conserved proximal element of the GM-550 motifs bound the complex much more weakly (Fig. 5B, IFNg promoter homologous to the 290 motif of IL-2 promoter. A, lanes 3 and 9). It is noteworthy that the GM-420 motif has been ICER binds to both human and mouse motifs of the IFNg promoter shown to constitute the essential enhancer core of the GM-CSF (lanes 2 and 9), despite the fact that these motifs fail to bind NFAT DBD promoter (23). Likewise, NFAT/ICER readily formed a complex (NFAT)(lanes 3 and 10) in a fashion analogous to the 290 motif of the IL-2 promoter. No complex formation between ICER and NFAT was with the 280 element of the IL-4 and 295 element of the detected in electromobility shift assay (lanes 4 and 11). ICER bound to TNF-a promoter (Fig. 5B, lanes 12 and 15, respectively). Inter- the proximal element of the IFNg promoter is specifically “supershifted” estingly, the k3 motif of the TNF-a promoter, which contains an (sICER) by a CS4 CREM-specific antibody (C)(lanes 5 and 12). The “inverted CRE” motif adjacent to the NFAT composite site (27), mobility of the ICER complex is altered using competition with unla- beled CRE oligonucleotide (cre)(lanes 6 and 13) but unchanged in the created a complex with an electrophoretic mobility different presence of unlabeled NFAT oligonucleotide (nf)(lanes 7 and 14). B, from those observed on the motifs of the IL-2, IL-4, and GM- sequences of human and mouse proximal elements of the IFNg pro- CSF promoters (Fig. 2C and Fig. 5B). Furthermore, NFAT moter show significant homology to the 290 motif of the human IL-2 DBD alone formed a much slower mobility complex (lane 14), promoter. suggesting that NFAT may bind to the TNF-a motif as oligomers. The treatment of isolated human medullary thymocytes with tion with unlabeled oligonucleotides containing NFAT (nf forskolin and ionomycin readily induced the expression of oligonucleotide spanning mouse IL-4 NFAT, positions 269 to ICER (Fig. 5, D and E) not seen in uninduced thymocytes (Fig. 279) or the AP-1 motif (ap oligonucleotide spanning the human 5C). The binding of ICER to the oligonucleotides containing the metallothionein IIA AP-1 site, positions 2114 to 288) elimi- NFAT/AP-1 composite sites is inhibited by competition of the nated the NFAT/ICER complex (Fig. 2D, lanes 6 and 7, respec- binding with a CRE-containing oligonucleotide (Fig. 5D, lanes tively), suggesting that both ICER and NFAT are essential 4, 7, and 10) or interference of the binding with antiserum to components of the observed complex. ICER (C-Ab) (Fig. 5E, lanes 4, 7, and 10). NFAT/ICER Ternary Complex Shows Distinct Features in Ectopically Expressed ICER Represses NFAT-mediated Acti- Comparison with NFAT/AP-1 Complex on Various NFAT Mo- vation of IL-2, GM-CSF, and TNF-a Promoters—To determine tifs of IL-2 Promoter—Analogous experiments performed with whether ICER expression could supplant the effect of forskolin the NFAT DBD and truncated forms of Fos and Jun confirmed in transcriptional attenuation of various cytokine promoters in vitro that both NFAT/AP-1 and NFAT/ICER complexes could observed in medullary thymocytes, ICER (isoform II) was ex- exist in the context of the CD28RE (2160 NFAT/AP-1 compos- pressed in Jurkat T cells in transient transfection assays. Ex- ite site; Fig. 2D, lanes 8 –14). Interestingly, in the presence of pression of ICER down-regulated the human IL-2, GM-CSF, NFAT DBD, the majority of ICER protein was retained in the and TNF-a promoters activated by the combined treatment of complex with NFAT DBD, although NFAT/ICER and NFAT/ the cells with PMA and ionomycin, whereas ectopic expression AP-1 complexes appear to show unequal binding affinities to of neither isoform of ICER did not prove to have any significant different NFAT/AP-1 motifs. For example, the 2280 motif of effect on VP16-mediated transactivation of (33 GAL4)-CR- the IL-2 promoter, known from NFAT studies as the principal CAT under the same conditions (Fig. 6). Thus, ICER can be site for binding of the NFAT/AP-1 complex, binds NFAT/AP-1 induced by, and substituted for, forskolin in the transcriptional complexes effectively (32, 37), showing little or no detectable down-regulation of the calcineurin-dependent, NFAT/AP-1-me- formation of an NFAT/ICER complex (Fig. 2C, lanes 13–15). In diated transactivation of IL-2, GM-CSF, and TNF-a promoters contrast, the CD28RE (2160 NFAT/AP-1 composite site) of when induced by PMA and ionomycin. human IL-2 promoter creates a ternary NFAT/ICER complex DISCUSSION with an equal or slightly higher efficiency than the NFAT/AP-1 complex (Fig. 2D). The mechanism of cAMP-mediated inhibition of cytokine ICER Binds Directly to the Conserved Proximal Motif of expression in proliferating T lymphocytes has been attributed IFNg Promoter—Unlike the CD28RE (2160 motif), which to cAMP-mediated inactivation of upstream signal transduc- shows equally high affinity for both ICER and NFAT, the tion pathways directing the proliferation of T lymphocytes. NFAT/AP-1 motif of the IL-2 promoter in position 290, which Although this hypothesis was supported by studies of fibro- has striking homology to the conserved proximal element of the blasts (5), it was not confirmed by studies of T cells (6). Sur- INFg promoter, does not interact with NFAT DBD (Fig. 4). prisingly, several protein kinases required for T cell prolifera- ICER Represses NFAT-mediated Cytokine Gene Expression 9549 FIG.5. ICER forms complexes on several NFAT/AP-1 composite sites in GM-CSF, IL-4, and TNF-a promoters. A, a list of NFAT/AP-1 composite sites previously identified as essential for the expression of GM-CSF (25), IL-4 (23), and TNF-a (26, 27) promoters used in electromobility shift assay analysis. B, purified ICER and truncated NFAT DBD proteins form in vitro NFAT/ICER complexes on the enhancer core of GM-CSF promoter GM-420 (lane 6), the proximal NFAT/AP-1 motif of the IL-4 promoter (IL4(280))(lane 12), and the k3 motif of TNF-a (kTNF-a)(lane 15) with affinities comparable with the CD28RE motif (2160) of the IL-2 promoter (Fig. 2). C–E, NFAT/AP-1 composite sites in GM-CSF, IL-4, and TNF-a promoters bind to ICER and form ICER containing complexes in whole cell extracts prepared from freshly prepared human medullary thymocytes. Bacterially expressed, purified ICER II and ectopically expressed full-length NFAT1 protein in COS cells served as controls (lanes 1 and 2, respectively) for evaluation of extracts (lanes 3–11) prepared from untreated human medullary thymocytes (C) or human medullary thymocytes treated with forskolin (3 h) and ionomycin (15 min) (D and E). ICER and ICER-containing complexes were competed by unlabeled oligonucleotides containing CRE motifs (cre)(lanes 4, 7, and 10) or NFAT motifs (nf)(lanes 5, 8, and 11)(panels C and D) and were immunoreactive with CS4 CREM-specific antisera (C)(panel E, lanes 4, 7, and 10), whereas the R59 NFAT-specific antisera (R)(lanes 5, 8, and 11) affected the mobility of numerous complexes but showed little effect on the binding of ICER (panel E). tion were found to be insensitive or to exhibit a delayed indirect role of ICER has been demonstrated in transgenic mice response to high levels of intracellular cAMP (6). Here we overexpressing the dominant negative CREB mutant (a func- provide evidence that in human medullary thymocytes expres- tional homologue of ICER), which impairs the expression of sion of the transcriptional repressor ICER correlates with a IL-2 in thymocytes (44). Since the induction of IL-2 and IFNg delayed cAMP-mediated transcriptional attenuation of T help- expression is dependent on the activity conferred by each of the er-1 cytokine responses. individual DNA motifs (22, 34), a demonstration of a direct Footprinting and electrophoretic mobility shift analysis of binding of ICER and/or the formation of an inhibitory NFAT/ the IL-2 promoter revealed (34) that the originally defined ICER complex on any of these NFAT/AP-1 composite sites AP-1 site at position 2150 of the IL-2 promoter (37, 38), a major could provide an explanation for the mechanism involved in the CD28RE that contains an upstream NFAT binding site (39, transcriptional attenuation of IL-2 and IFNg expression medi- 40), represents a novel NFAT/AP-1 composite site at position ated by cAMP. These findings correlate with observations in 2160 (34). A reexamination of the original observations in which the conserved proximal motif of the IFNg promoter was which NF-kB was identified as a major component of the com- reported to be inhibited by forskolin in proliferating thymo- plex (41, 42) determined that NFAT is a prevalent component cytes of mice made transgenic with an IFNg promoter-lucif- of the complex that binds the CD28RE in vivo (34, 43). Our erase reporter gene (45). These findings further suggest that findings indicate that the CD28RE (2160 composite site of IL-2 both NFAT/AP-1 motifs, either those that directly bind ICER or promoter) effectively binds ICER either alone or in an NFAT/ those that form NFAT/ICER complexes, could convey ICER- ICER complex. These findings may be important for obtaining mediated transcriptional attenuation. a better understanding of a direct cAMP-mediated transcrip- It appears that numerous NFAT/AP-1 composite sites previ- tional attenuation of IL-2 expression. In addition, a potential ously identified in the context of GM-CSF, IL-4, and TNF-a 9550 ICER Represses NFAT-mediated Cytokine Gene Expression that these sites, previously shown to be essential for efficient expression (23, 26, 27), bind ICER either alone or in complexes similarly to the motifs of the IL-2 and IFNg promoters. It remains to be determined whether the induction of ICER can selectively modulate T helper-1 versus T helper-2 cytokine ex- pression in peripheral blood T lymphocytes. We have reported previously that human medullary but not cortical thymocytes synthesize ICER mRNA after3hof fors- kolin treatment (7). Western immunoblot analysis using an ICER-specific antiserum confirmed that in these conditions the ICER mRNA is translated efficiently into ICER protein. More- over, endogenously expressed ICER protein was detected in extracts prepared from human medullary thymocytes treated with forskolin and ionomycin using oligonucleotide probes con- taining NFAT/AP-1 DNA motifs that are able to form NFAT/ ICER complexes in vitro. In contrast to bacterially expressed ICER, endogenously expressed ICER in medullary thymocytes shows an altered mobility in gel shift assays, suggesting that posttranslational modification(s) may be involved in the regu- lation of the binding properties of ICER and/or degradative pathways involved in its proteolysis in vivo. The ICER-contain- ing complexes that are immunoreactive to ICER-supershifting antisera are efficiently competed by oligonucleotides contain- ing CRE or NFAT motifs. NFAT antisera that are unable to recognize directly bound ICER still affect the mobility of ICER- containing complexes, suggesting the possibility of the forma- tion of NFAT/ICER complexes in vivo. The ambiguity of the DNA-protein complexes in extracts of thymocytes observed on gel shift assays may be due to posttranslational modifications of the proteins involved (data not shown) and/or their potential consequences for DNA binding. At this point, the possibility cannot be excluded that proteins other than ICER and NFAT that contain homologous bZIP or rel homology regions may also participate in the formation of ICER-containing complexes. Finally, ectopic expression of ICER in Jurkat cells demon- strates that ICER, in agreement with its binding capabilities, can also effectively inhibit NFAT-mediated, phorbol ester/ ionophore-induced expression of IL-2, GM-CSF, and TNF-a FIG.6. ICER isoform II represses transcription from NFAT/ promoters. AP-1-activated cytokine promoters stimulated by PMA and In conclusion, inducible ICER expression in developing hu- ionomycin (P 1 I) treatment. A, promoter-reporter, IL-2-CAT (hu- man interleukin-2 (32)); CD28RE (2160 AP-Luc; gift from A. Rao); man medullary thymocytes as well as in certain subset(s) of GM-CSF-CAT (25) (human granulocyte-macrophage colony stimulating human peripheral blood lymphocytes (7) and monocytes (work factor), TNF-a-Luc (contains the sequence from 2614 to 120 of the in progress) could significantly influence their respective effec- human tumor necrosis factor-a in pGL2; gift from S. L. McKnight). A tor functions(s). The proposed inhibitory effects on effector control (33 GAL4)-CR-CAT (with three GAL-4 binding sites substitut- ing 21-base pair repeats in the human T-cell lymphotrophic virus type function of the immune system mediated by ICER may be I long terminal repeat (35)) transactivated by GAL4VP16 (46) is not related to its ability to bind (mask) a wide range of CRE and affected in Jurkat cells by isoforms of ICER (ICER II, ICER IIg, ICER AP-1 motifs and/or its ability to inactivate certain transcription I). B, amounts of respective cytokine reporters and ICER expression complexes via protein-protein interactions. constructs in transient transfections were kept constant (2 mg). Error bars represent S.D. values calculated from three or more experiments. Acknowledgments—We thank Dr. A. Rao for the generous gift of truncated NFAT DBD proteins and NFAT antibodies; Dr. T. K. promoters as essential determinants of their expression (23, 25, Kerppola for the gift of truncated Fos and Jun proteins; Drs. J. L. 27) can associate with ICER. This property does not seem to be Strominger, David Weng, Peter A. Cohen, K. Shaw, P. McCaffrey, Cathy Bare, and Ronald E. Gress for stimulating discussions; E. a universal feature shared by all NFAT/AP-1 composite sites Burgeon for expert advice on GST pull-down assays; and T. 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Published: Apr 1, 1998

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