©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Negative Transcriptional Regulation of the Interferon- Promoter by Glucocorticoids and Dominant Negative Mutants of c-Jun (*)

Marco Cippitelli (1), Antonio Sica (2), Vincenzo Viggiano (2), Jianping Ye (2), Paritosh Ghosh (2), Michael J. Birrer (3), Howard A. Young (2)(§)

From the (1) Biological Carcinogenesis and Development Program, Program Resources Inc./DynCorp, the (2) Laboratory of Experimental Immunology, Biological Response Modifiers Program, National Cancer Institute Frederick Cancer Research and Development Center, Frederick, Maryland 21702-1201, and (3) Biomarkers and Prevention Research Branch, Division of Cancer Prevention and Control, National Cancer Institute, Rockville, Maryland 20850

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Interferon- (IFN-) is an immunoregulatory cytokine expressed in large granular lymphocytes and T cells. However, the molecular mechanisms underlying IFN- gene transcription have not been fully defined. Here, we analyze the mechanisms responsible for the inhibition of IFN- promoter activity by the glucocorticoid hormone dexamethasone. Cotransfection assays performed in Jurkat T cells demonstrated that the activity of the initial 108 base pairs of the IFN- promoter was down-regulated in the presence of dexamethasone. Furthermore, utilizing electrophoretic mobility shift analysis, we identified activator protein 1 AP-1-cAMP response element binding protein-activating transcription factor (CREB-ATF) binding elements situated in positions of the IFN- promoter previously identified as essential for promoter activity. Moreover, dominant negative mutants of the c-Jun proto-oncogene were able to mimic the same down-regulatory effect exerted by dexamethasone, and mutations that abolished the binding of the AP-1CREB-ATF factors were able to block the glucocorticoid effect.

These results suggest a model involving the inhibition of IFN- AP-1CREB-ATF DNA binding complexes as one of the mechanisms involved in the negative regulatory action of glucocorticoids on IFN- gene expression and support the relevance of AP-1CREB-ATF binding factors during the transcriptional activation of the IFN- promoter in T cells.


INTRODUCTION

Interferon- (IFN-)() is an immunoregulatory cytokine involved in modulating nearly all phases of immune and inflammatory responses (1, 2) . Furthermore, this cytokine is relevant as a therapeutic agent for immunodeficiency states, infections, and neoplastic states and has been evoked in the pathogenesis of such disorders (1) . IFN- expression in vivo seems to be strictly regulated, as the production of messenger RNA has been detected predominantly in activated T cells and large granular lymphocytes (1, 2) . Inhibition of IFN- production has been reported to be caused by different agents, such as cyclosporin A, corticosteroids, prostaglandins, etc. (1) . Glucocorticoids in particular, already shown to play a major role in the treatment of different autoimmune, allergic, and inflammatory diseases, are able to affect the growth, the differentiation, and the function of monocytes and lymphocytes and to strongly regulate the production of different cytokines, including IFN- (3, 4, 5, 6, 7, 8) . Previous investigations have already shown that a synthetic glucocorticoid hormone dexamethasone inhibits the induction of IFN- mRNA in normal human lymphocytes (4, 5) ; in addition, nuclear transcription of the IL-2 gene in human T cells has been shown to be inhibited by the same agent (4, 5, 7, 8, 9, 10, 11) . With regard to the IL-2 gene, these results suggested that inhibition of transcription is the result of a negative interference with nuclear transcriptional factors AP-1 and NF-AT (7, 8, 9, 10, 11) , which have been demonstrated to be of crucial importance for the activity of this cytokine promoter. Moreover, a functional antagonism that involves direct protein-protein interactions between AP-1 and GR previously has been reported by several investigators in different systems (12, 13, 14, 15) .

In this report, using DNA mobility shift assays (EMSA) and transient DNA transfection assays, we have examined in Jurkat T cells the effect of the glucocorticoid hormone dexamethasone on the transcriptional activity of the human IFN- promoter. In this context, we also have analyzed the relevance of the AP-1 complex binding factors during the T cell activation by using two different inhibitors of the AP-1-mediated transactivation, dominant negative mutants of the c-Jun proto-oncogene (TAM-67 and c-Jun/1-286) (16, 17) . Our data indicate that the PMA/ionomycin-stimulated IFN- promoter activity is significantly down-regulated by dexamethasone after cotransfection with a human GR expression vector. Additionally, AP-1CREB-ATF binding sequences present in the IFN- promoter, at positions previously demonstrated to be essential for the full promoter activity, are involved in this inhibition. Moreover, dominant negative mutants of the c-Jun proto-oncogene are able to mimic the inhibitory action exerted by the GR on this promoter. These data strongly suggest a model involving inhibition of the IFN- AP-1CREB-ATF complexes, as one of the possible mechanisms of action for the GR-mediated negative regulation on the IFN- gene and support the relevance of AP-1 and CREB-ATF binding factors on the activation of the IFN- promoter in T cells.


EXPERIMENTAL PROCEDURES

Cell Lines and Reagents

Jurkat cells (CD4 human lymphoblastoid T cell line) were cultured in complete RPMI 1640 medium, supplemented with 10% fetal calf serum, 2 mM glutamine, and 100 units/ml penicillin-streptomycin. Antibodies against c-Jun (a rabbit affinity-purified polyclonal antibody corresponding to the highly conserved residues 247-263 within the C-terminal DNA binding domain of the c-Jun protein) and c-Fos (a mouse monoclonal antibody raised against a peptide corresponding to c-Fos amino acid residues 128-152) transcription factors were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). PMA was purchased from Sigma, and ionomycin was purchased from Calbiochem (La Jolla, CA).

Nuclear Extraction

Nuclear proteins were prepared as follows (18) . The cellular pellet was resuspended in 10-20 times its volume in buffer A (lysis buffer): 50 mM KCl, 0.5% Nonidet P-40, 25 mM Hepes buffer (pH 7.8), 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 20 µg/ml aprotinin, 100 µM dithiothreitol and subsequently incubated 4 min on ice. Cells were collected by centrifugation at 2500 rpm, and the supernatant was decanted. The nuclei were washed in buffer A without Nonidet P-40, collected at 2500 rpm, and resuspended in buffer B (extraction buffer): 500 mM KCl, 25 mM Hepes (pH 7.8), 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 20 µg/ml aprotinin, and 100 µM dithiothreitol for 5 min on ice. The samples were subsequently frozen and thawed (twice) utilizing dry ice and a 37 °C water bath, rotated 20 min at 4 °C, and centrifuged at 14,000 rpm for 20 min. The clear supernatant was collected, and the proteins were dialyzed for 2 h (4 °C) against buffer C (dialysis buffer): 50 mM KCl, 25 mM Hepes (pH 7.8), 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 20 µg/ml aprotinin, and 100 µM dithiothreitol. The amount of nuclear proteins obtained were quantified utilizing a commercial reagent (BCA, Pierce).

Electrophoretic Mobility Shift Assay

The nuclear proteins (5 µg) were incubated with radiolabeled DNA probes in a 20-µl reaction mixture containing 20 mM Tris (pH 7.5), 60 mM KCl, 2 mM EDTA, 0.5 mM dithiothreitol, 2 µg of poly(dI-dC), and 4% Ficoll. In some cases, the indicated amount of double-stranded oligomer was added as a cold competitor, and the mixture was incubated at room temperature for 10 min prior to adding the DNA probe. Nucleoprotein complexes were resolved by electrophoresis on 5% nondenaturing polyacrylamide gels in 0.5 Tris borate-EDTA buffer at 12 V/cm for 2 h at room temperature. Dried gels were exposed to Kodak XAR-5 film (Eastman Kodak Co.) at -70 °C with intensifying screens. Oligonucleotides were synthesized by the phosphoramitide method on a DNA/RNA synthesizer (Applied Biosystems, model 394). Complementary strands were denaturated at 85 °C for 5 min and annealed at room temperature.

The double-stranded probes were end-labeled using Klenow fragment (Life Technologies, Inc.) and [-P]dCTP (Amersham Corp.); approximately 1 ng of labeled DNA was used in a standard EMSA reaction. In supershift analysis, the antisera were added to the binding reaction, and the mixture was incubated for 30 min at room temperature prior to adding the labeled DNA probe.

The following double-stranded oligomers were used as labeled probes or cold competitors: IFN-/-66 to -47, 5`-gatcTTGTGAAAATACGTAATCC-3`; IFN-/-96 to -75, 5`-gatcGCCTATCTGTCACCATCTCATC-3`; IFN-/-66 to -47 (-mutant), 5`-gatcTTGTGAAAATcgcTAATCC-3`; TRE-(h-Collagenase gene, -73 to -42), 5`-agctATGAGTCAGACACCTCTGGCTTTCTGGAAGG-3`; AP-1 (hIL-2prox.), 5`-agctGAAATTCCAAAGAGTCATCAGA-3`; OCT (hIL-2prox.), 5`-agctTAATATGTAAAACATT-3`; CREB(wt), 5`-AGAGATTGCCTGACGTCAGAGAGCTAG-3`; CREB(mut.), 5`-AGAGATTGCCTGtgGTCAGAGAGCTAG-3`.

CREB (wt) and CREB (mut) consensus oligonucleotides were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Plasmid Constructions

The different deletions of the IFN- promoter pIFN-538, pIFN-339, pIFN-108 (19) were kindly provided by Dr. Christopher B. Wilson (Department of Pediatrics and Immunology, University of Washington); human GR expression vector pRShGR (20) was kindly provided by Dr. R. M. Evans (The Salk Institute, La Jolla, CA).

To prepare the NF(P)-tkCAT, three copies of the human IL-4 promoter NF(P) binding site (nucleotides -88 to -64) were subcloned into the HindIII-BamHI sites upstream to the thymidine kinase (tk) promoter in the pBLCAT2 parental vector. The plasmids 3x(-66/-47)CAT, and 3x(-96/-75)CAT, contain three copies of the regions IFN-/-66 to -47 bp and IFN-/-96 to -75 bp, subcloned into the HindIII-BamHI sites upstream to the thymidine kinase promoter in the pBLCAT2 parental vector.

To construct the plasmids(-108-36)CAT (wild-type sequence) and (-108-36)`CAT (containing the mutations for the two AP-1CREB-ATF binding sites) the appropriate DNA fragments were synthesized and subcloned into the HindIII-BamHI sites upstream to the thymidine kinase promoter in the pBLCAT2 parental vector.

The plasmid AP1-2xGRE-tk-CAT, containing two GREs in front of the thymidine kinase promoter (21) , was kindly provided by Dr. A. Gulino (University ``La Sapienza'' Rome, Italy).

The plasmid pHIV-CAT, containing a 727-bp HindIII-XhoI insert encoding the human immunodeficiency virus, type 1 long terminal repeat linked to the CAT gene (22) , was obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, National Institutes of Health. The deletion mutant of human c-Jun (TAM-67) has been described previously (16) . It was cloned as an EcoRI fragment and then blunted and ligated into the NotI site of the pCMV-gal expression vector (Clontech Laboratories, Inc., Palo Alto, CA) replacing the -galactosidase gene.

The deletion mutant of human c-Jun (c-Jun/1-286), carrying the truncation of the leucine-zipper domain, was constructed as described previously (23) and inserted into the NotI site of the pCMV-gal expression vector replacing the -galactosidase gene.

DNA Transfections

Transfections of Jurkat cells were carried out by the DEAE-dextran method (24) . For each treatment, 5 10 cells (harvested in log phase of growth) were incubated with the indicated amounts of plasmid DNA in the presence of 350 µg/ml DEAE-dextran in RPMI 1640, 50 mM Tris-Cl (pH 7.5) for 70 min at 37 °C. To decrease variations in transfection efficiency, cells were transfected in single batches, which were then separated into different drug treatment groups, and empty expression vector DNA was added as needed to maintain a constant total DNA amount in each cotransfection series. Cells were then washed with RPMI 1640, 50 mM Tris-Cl (pH 7.5) and replated in duplicate, in complete medium. After 24 h, cells were treated with different combinations of stimuli, and, after an additional 24 h, cells were harvested and washed in phosphate-buffered saline. Protein extracts were prepared for the -galactosidase assay and CAT assay, by 3 cycles of rapid freezing and thawing followed by centrifugation at 14,000 rpm (4 °C) for 15 min. Protein concentration was quantified utilizing a commercial reagent (BCA, Pierce).

-Galactosidase Assay

The -galactosidase assay was carried out according to the published procedure (25) . Enzyme activity was determined spectrophotometrically at 570 nm by the hydrolysis of chlorophenol red -D-galactopyranoside. Duplicate -galactosidase assays were normalized based on protein amount loaded at each point, and generally had variations of less than 10%. Results are expressed as percent of activity, relative to the control PMA/ionomycin-activable -galactosidase expression in each cotransfection series, without dexamethasone in the case of the GR action, or cotransfected with the empty vector in the case of the dominant negative mutants of c-Jun (TAM-67 and c-Jun/1-286).

CAT Assay

CAT assay was carried out according to the published procedure (26) by incubating different amounts of cell lysate protein for 12 h at 37 °C so that the assay was within the linear range. Acetylated and unacetylated [C]chloramphenicol were separated by thin layer chromatography and quantified by a radioactivity scanner (Ambis Inc., San Diego Ca.).


RESULTS

PMA/Ionomycin Activation of Different Deletions of the IFN- Promoter Is Down-regulated by Dexamethasone in Jurkat Cells

As for the IL-2 gene, dexamethasone has been shown to inhibit the induction of IFN- messenger RNA in T cells (4, 5) . Although a mechanism for the IL-2 gene inhibition by dexamethasone has been reported (9, 11) , it is not clear if the down-regulation of the IFN- gene expression is caused by a direct effect on the promoter activity or is mediated by other mechanisms, such as regulation of RNA stability, etc. In order to determine whether one of the possible mechanisms of dexamethasone inhibition could be the direct interference with the transcriptional activity of the human IFN- promoter, we transiently cotransfected Jurkat cells with different -galactosidase vectors in which the reporter gene transcription was directed by progressive deletions of the human IFN- promoter, together with an expression vector for the human GR.

As shown in Fig. 1, the -galactosidase activity driven by the promoter fragments -538 to +64 (pIFN-538) and the deletions -339 to +64 (pIFN-339) and -108 to +64 (pIFN-108) were all significantly inhibited by treatment with dexamethasone. These data indicate the sensitivity of the promoter to GR/dexamethasone and demonstrate that GR-sensitive element(s) were present in the promoter fragment -108 to +64 bp of the IFN- gene. The inhibition was dependent on the presence of the GR expression vector, since treatment with dexamethasone alone was not able to exert any detectable effect on the IFN- promoter activity in the Jurkat cells used in these studies (data not shown). In this context, as shown in Fig. 2A, a CAT reporter driven by two copies of a GRE, was strongly activated after dexamethasone treatment only in the presence of the cotransfected GR expression vector. These experiments indicate that Jurkat cells used in these studies were normally resistant to glucocorticoids and required cotransfection of a GR expression vector for the dexamethasone-induced inhibition of the IFN- promoter. Similar observations have been previously reported where the dexamethasone-induced inhibition of the human IL-2 promoter was dependent upon the presence of a functional cotransfected GR, in experiments utilizing murine T cell lines (BFS and EL-4) and Jurkat cells (7) .


Figure 1: Effect of dexamethasone (Dex) on different IFN- promoter deletions. 5 10 Jurkat T cells were cotransfected with 10 µg of the indicated reporter gene vector plus 2 µg of GR expression vector as described under ``Experimental Procedures.'' 24 h after transfection, cells were stimulated with 10 ng/ml PMA and 1 µg/ml ionomycin in the presence or in the absence of 1 µM dexamethasone. After a further 24 h, cells were harvested, and protein extracts were prepared for the -galactosidase assay. The percentage of activation, relative to the controls in the absence of dexamethasone (considered as 100%), represents the mean value (X ± S.E.) from at least four individual experiments. -galactosidase activities with PMA/ionomycin treatment for each construct were, respectively, (units/µg of protein) 0.1 10 ± 0.016 (pIFN-538), 0.068 10 ± 0.009 (pIFN-340), 0.088 10 ± 0.013 (pIFN-108).




Figure 2: Effect of dexamethasone (Dex) on the expression of AP1-2xGRE-tk-CAT (A) and pHIV-CAT (B). 5 10 Jurkat T cells were cotransfected with 10 µg of the indicated reporter gene vector plus 2 µg of GR expression vector as described under ``Experimental Procedures.'' 24 h after transfection, cells were stimulated with 1 µM dexamethasone (A), or 10 ng/ml PMA and 1 µg/ml ionomycin in the presence or in the absence of 1 µM dexamethasone (B). After a further 24 h, cells were harvested and protein extracts were prepared for the CAT assay.



As a further control for the specificity of GR/dexamethasone action on the PMA/ionomycin-mediated gene activation, a CAT reporter driven by the human immunodeficiency virus, type 1 long terminal repeat was used; as shown in Fig. 2B, the PMA/ionomycin inducibility of this reporter was not modified by the presence of the GR expression vector and dexamethasone treatment. These data are in agreement with previous observations by Vacca et al.(7) where the PMA/ionomycin-mediated activation of different promoters (SV40 early promoter, Rous sarcoma virus and human T cell lymphotrophic virus long terminal repeats) was unaffected by GR/dexamethasone.

AP-1CREB-ATF Binding Elements Are Present in the IFN- Promoter

Several recent reports have clearly demonstrated that inhibition of the IL-2 promoter by GR in T cells involves a functional impairment of the NF-AT and the proximal AP-1 binding sites (9, 10, 11) . Moreover, AP-1 recently has been found to be a component of NF-AT (27) , thus indicating the AP-1 complex as the primary target for GR-mediated IL-2 promoter inhibition.

In order to determine whether AP-1 related binding elements were present in the IFN- promoter, a sequence homology search was done by comparison of the -538/+64 IFN- fragment sequence, with the canonical consensus sequences for AP-1 and CREB-ATF DNA binding factors (28). Based on this analysis () and on the observation that a GR-mediated negative response was identified at the level of the -108 to +64 bp promoter fragment, two different noncanonical sequences were identified to form specific AP-1CREB-ATF-related DNA-bound protein complexes in band-shift assays using nuclear extracts prepared from Jurkat cells (Figs. 3-5) and fresh T cells (data not shown).

Interestingly, these two identified binding sites showing homology with the AP-1CREB-ATF DNA consensus sequences, were situated at positions (-96 to -75) and (-66 to -47) (), previously demonstrated by Penix and co-workers (19) to be critical for the full transcriptional activation of this promoter and named ``distal'' and ``proximal'' essential elements.

Fig. 3 shows the DNA binding pattern obtained using nuclear extracts from unstimulated and PMA/ionomycin-treated Jurkat cells in the presence of a labeled probe spanning nucleotides from -96 to -75 (, the distal element of the IFN- promoter). Three specific and constitutively expressed DNA-protein(s) complexes were detected and designated here as complex a, b, and c (lane1). A slower migrating complex, designated here as AP-1, was induced after the PMA/ionomycin stimulation (lane6) and specifically competed either by a molar excess of a cold competitor specific for CREB-ATF or by the proximal AP-1 site of the human IL-2 promoter (lanes8 and 10) but not by a mutated version of the CREB-ATF oligonucleotide (lane9) or a nonrelated cold competitor specific for the SP-1 binding factor (data not shown). Interestingly, the complex a was partially competed by the cold oligonucleotide specific for CREB-ATF (lanes3 and 8), while both the mutant CREB-ATF oligonucleotide and the IL-2/AP-1 cold oligonucleotide failed to compete in the same manner (lanes4 and 10). Complexes b and c were observed to be specifically competed by a typical DNA binding sequence for the GATA binding factors (29, 30, 31) , present in the human T cell receptor -chain promoter (31) (data not shown).


Figure 3: Electrophoretic mobility shift assay of the distal IFN- promoter region -96 to -75 bp. EMSA was performed using the indicated P-labeled oligonucleotide as a probe in the presence of nuclear extracts from unstimulated or PMA/ionomycin-treated Jurkat cells. Lanes1-5, untreated cells; lanes6-10, 4 h of PMA/ionomycin treatment.



A more complex binding pattern was observed with a oligonucleotide encompassing the proximal DNA binding element of the IFN- promoter (nucleotides -66 to -47) (Fig. 4) (). Several specific DNA binding complexes were present using nuclear extracts from unstimulated Jurkat cells in EMSA (lane1), and the stimulation with PMA/ionomycin was able to induce the binding of other complex(es) over the basal DNA binding activity (lane2). Interestingly, a cold oligonucleotide specific for CREB-ATF binding factors was able to totally compete all of these complexes (lane6), with the exception of one designated here as complex 1. This complex was recognized to contain the Oct-1 binding protein by specific competition with a cold oligonucleotide encompassing the proximal octamer binding site of the human IL-2 promoter (lane8) and by supershift assay using an antibody able to specifically recognize only the Oct-1 DNA binding factor (data not shown). When a cold competitor specific for the proximal AP-1 binding site of the human IL-2 promoter was used in this EMSA, only the PMA/ionomycin-induced complex(es) were specifically competed (lane5). These data suggest that the overlapping DNA binding activity observed with stimulated Jurkat cell nuclear extracts was caused by induction of AP-1 complex-related binding proteins (designated here as complex 2), already known to be able to bind with different affinities to the CREB-ATF DNA binding sequences (32, 33) .


Figure 4: Electrophoretic mobility shift assay of the proximal IFN- promoter region -66 to -47 bp. EMSA was performed using the indicated P-labeled oligonucleotide as a probe in the presence of nuclear extracts from unstimulated or PMA/ionomycin-treated Jurkat cells. Lane1, untreated cells; lanes2-9, 4 h of PMA/ionomycin treatment.



The constitutively present complexes not competed by the AP-1 oligonucleotide were probably other CREB-ATF-related DNA-binding proteins that have a more specific affinity for this particular noncanonical CREB-ATF consensus sequence than the AP-1 binding sequence.

Interestingly, a cold oligonucleotide encompassing the distal element of the IFN- promoter (nucleotides -96 to -75) was able to compete for the overlapping PMA/ionomycin-induced AP-1 related binding but not the constitutively present CREB-ATF-related DNA binding activity (lane9).

The IFN- Promoter AP-1CREB-ATF-like Motifs Bind Complexes Containing Jun and Fos Proteins in Jurkat Cells

GR has been shown to inhibit the transcriptional activity of AP-1 cis-acting elements by interfering with Jun and Fos proteins, two normal components of the AP-1 complex. Since both the sequence homology search and the EMSAs strongly indicated that AP-1-related proteins were specific components of the induced complexes described above, we wanted to determine whether Jun/Fos proteins were actually present in these PMA/ionomycin-induced bands.

In Fig. 5, a supershift analysis in the presence of anti-Jun or anti-Fos antibodies is shown; both of the antibodies specific for Jun and Fos family members were able to compete for the binding when used in the presence of nuclear extracts from PMA/ionomycin-stimulated Jurkat cells, while a nonrelated antibody was not able to modify the binding capability or the migration of the complexes in EMSA. The figure also shows a comparison between canonical AP-1 and CREB-ATF binding sequences with the two ``essential'' IFN- promoter binding elements (28) . The position indicated here as AP-1 shows the induced complexes specifically competed by antibodies for Jun and Fos, overlapping with other CREB-ATF family members in the case of the proximal element of the IFN- promoter (nucleotides -66 to -47). A more detailed mutational analysis and characterization of the proteins that form the CREB-ATF-related complexes in this proximal regulatory element are the subject of a separate report.() Cotransfection of Dominant Negative Mutants of c-Jun (TAM-67 and c-Jun/1-286) Is Able to Down-regulate the Activity of the IFN- Promoter-GR has been demonstrated to down-regulate a number of different gene promoters by interaction with transcriptional factors, including AP-1 family members, Rel-A, CREB family members, Oct-2A, and GATA-1 (12, 13, 14, 15, 34, 35, 36, 37) . In addition, other mechanisms of GR-mediated transcriptional repression include specific binding to different GR noncanonical sequences, as negative GR response elements (38) or interaction with GR binding sites overlapping important regulatory elements such as the TATA box region (39, 40, 41) .


Figure 5: Supershift analysis of the DNA-protein complexes binding to the IFN- promoter regions -96 to -47 and -66 to -47 bp. EMSA was performed using the indicated P-labeled oligonucleotides as probes in the presence of nuclear extracts from PMA/ionomycin-treated Jurkat cells. 1 µg of purified anti-c-Jun, anti-c-Fos, or nonspecific antibody was added to the reaction where indicated, as described under ``Experimental Procedures.'' Lanes14-15 and 17-18 contain the two different probes indicated above, plus the antibody for c-Jun (14, 17) or c-Fos (15, 18) without nuclear extracts as controls.



As a further test of the hypothesis that the identified AP-1CREB-ATF complexes play an important role during the activation of the IFN- promoter and are potential targets for GR negative regulation of the IFN- transcription, we utilized two different dominant negative mutants of the c-Jun proto-oncogene (TAM-67 and c-Jun/1-286), potent inhibitors of AP-1 mediated transactivation, in cotransfection experiments (16, 17, 23) . In particular, TAM-67 already has been shown to selectively inhibit the IL-2 promoter by directly interfering with the NF-AT complex activity in Jurkat T cells, but not with other DNA binding regions including the NF-IL2A or the NF-B sites (42) . Cotransfection assays, in the presence of the different IFN- promoter -galactosidase reporters described above showed that both TAM-67 and c-Jun/1-286 were able to mimic the same kind of suppression observed in Jurkat cells by cotransfection of the GR expression vector followed by dexamethasone stimulation (Fig. 6), with all the progressive deletions significantly inhibited. These data indicate that selective impairment of the c-Jun-mediated transactivation is able to inhibit the activation triggered by PMA/ionomycin stimulation of the IFN- promoter.


Figure 6: 5 10 Jurkat T cells were cotransfected with 10 µg of the indicated reporter gene vector plus 5 µg of Tam-67 expression vector or c-Jun/1-286 expression vector as described under ``Experimental Procedures.`` In the case of the control samples, the same amount of empty expression vector DNA was added. Cells were treated 24 h later with 10 ng/ml PMA and 1 µg/ml ionomycin, and protein extracts were prepared for the -galactosidase assay as described before. The percentage of activation, relative to the controls transfected with empty vector (considered as 100%), represents the mean value (X ± S.E.) from at least four individual experiments. -galactosidase activities with PMA/ionomycin treatment for each construct were, respectively (units/µg of protein), 0.085 10 ± 0.009 (pIFN-538), 0.064 10 ± 0.007 (pIFN-340), 0.084 10 ± 0.007 (pIFN-108).



In order to test the specificity of these two dominant negative mutants, we cotransfected (using a CAT-reporter/dominant negative vector ratio of 1:1) TAM-67 or c-Jun/1-286 together with a CAT vector in which the reporter gene transcription was directed by three copies of the human NF(P) regulatory element, present in the promoter of the IL-4 gene (43) . The PMA/ionomycin induction of this reporter was not significantly inhibited by TAM-67 and c-Jun/1-286 expression (data not shown), indicating the selective specificity for the AP-1 inhibition of these vectors. The Proximal and Distal AP-1CREB-ATF Binding Sites in the -108-bp Fragment of the IFN- Promoter Are Responsive to the Inhibitory Action of GR/Dexamethasone-In order to better characterize the mechanism by which GR/dexamethasone are able to negatively interfere with the IFN- promoter-activation process, we studied in cotransfection assay the behavior of multimers of the two identified AP-1CREB-ATF binding sites in the presence of the GR expression vector and different stimulations. As shown in Fig. 7A, both the CAT reporters studied, 3x(-66/-47)CAT and 3x(-96/-75)CAT, were responsive to the PMA/ionomycin treatment in Jurkat cells, and dexamethasone was able to significantly inhibit this activity, while the normal basal induction of the parental pBL2CAT vector was unchanged.


Figure 7: A, effect of dexamethasone treatment on the thymidine kinase promoter-CAT activity driven by multimers of the IFN- proximal and distal elements. 5 10 Jurkat T cells were cotransfected with 10 µg of the indicated reporter gene vector plus 2 µg of GR expression vector as described under ``Experimental Procedures.'' 24 h after transfection, cells were stimulated with 10 ng/ml PMA and 1 µg/ml ionomycin in the presence or in the absence of 1 µM dexamethasone. After a further 24 h, cells were harvested, and protein extracts were prepared for the CAT assay. Results are expressed as the average (X ± S.E. of at least three individual experiments) -fold induction of the CAT activity measured in PMA/ionomycin-treated samples versus untreated, in the absence (Control) or in the presence (+Dex) of 1 µM dexamethasone. B, sequence of the oligonucleotides used as multimers in the vectors 3x(-66/-47)CAT and 3x(-96/-75)CAT.



The essential role of these two AP-1CREB-ATF binding sites during the normal activation process, and the correlation with the negative action exerted by GR/dexamethasone on the IFN- promoter, was further investigated by using mutations able to selectively abolish the binding of these complexes on the promoter. The mutation able to eliminate the binding activity of the distal AP-1CREB-ATF element, present in the promoter segment from nucleotides -96 to -75 (named here as `) has been already described by Penix et al.(19) (Fig. 8B). The mutation able to block the binding of AP-1CREB-ATF binding proteins, at the level of the proximal site present in the promoter segment from nucleotides -66 to -47, is shown in Fig. 8B and 9. Only a single DNA-binding complex, designated as 1 and corresponding to the transcriptional factor Oct-1 described above, was able to bind this mutated sequence (named here as ) in EMSA, both in unstimulated and PMA/ionomycin-stimulated Jurkat cells nuclear extracts (Fig. 9).


Figure 8: A, mutations of the IFN- proximal and distal elements are able to abolish the transactivating capability of the promoter fragment (-108 to -36) and dexamethasone (Dex) induced negative regulation. Results are expressed as the average (X ± S.E. of at least three individual experiments) -fold induction of the CAT activity measured in PMA/ionomycin-treated samples versus untreated, in the absence (Control) or in the presence (+Dex) of 1 µM dexamethasone. B, mutations introduced in the (-108-36)`CAT vector.




Figure 9: EMSA was performed using the wild-type IFN- proximal region (-66 to -47 bp) or the -mutant oligonucleotides as labeled probes, in the presence of nuclear extracts from unstimulated or PMA/ionomycin-treated Jurkat cells. Lanes1 and 3, untreated cells; lanes2 and 4, 4 h of PMA/ionomycin treatment.



The simultaneous site mutation, or deletion, of these two binding elements is able to dramatically reduce the -galactosidase activity of the IFN- promoter reporters described above to levels not suitable for studying negative regulations in transfection assays (data not shown and Ref. 19). Thus the wild-type IFN- promoter region encompassing the two essential regulatory elements (nucleotides -108 to -36), and the double mutant for these elements, were subcloned into the HindIII-BamHI sites upstream to the thymidine kinase promoter in the pBLCAT2 parental vector.

Fig. 8A, shows that the transcriptional activity of the (-108-36)CAT reporter was enhanced by PMA/ionomycin, and the treatment with dexamethasone was able to significantly inhibit this activation. On the contrary, the mutations of the two AP-1CREB-ATF binding elements in the(-108-36)`CAT reporter, strongly reduced the activation level after PMA/ionomycin, and the treatment with dexamethasone was not able to modulate the residual CAT activity driven by the mutated promoter fragment and the thymidine kinase promoter of the parental vector.

All of these data taken together suggest that the two AP-1CREB-ATF binding sites identified in the first 108 bp of the IFN- promoter, function normally as enhancers during the activation process through a mechanism mediated by the cooperation of AP-1CREB-ATF proteins and represent sensitive elements for the GR/dexamethasone-induced down-regulation of the promoter.


DISCUSSION

Physiological activation of T cells requires the interaction of antigen with the T cell receptor and the cooperation of a second signal provided by an antigen presenting cell, a process that can also be mimicked by treating the cells with PMA and calcium ionophore. The typical hallmark of this phenomena involves several distinct steps that, in the case of CD4 Th1 cell lines, include induction of immediate early genes including c-jun, c-fos, CD25 and induction of cytokine genes such as interleukin-2 and IFN- (44, 45). With regard to the IFN- gene, the molecular mechanisms involved in its transcriptional activation have been only partially characterized. Along with other investigators, our laboratory has reported the presence of positive and negative cis-acting promoter elements in a region 500 bp 5` to the transcription start site (1, 46, 47, 48, 49, 50) . Moreover, recent results obtained using reporter vectors driven by the first 538 or 108 bp of the IFN- promoter appeared to faithfully mirror the endogenous gene's requirements for specific induction by exogenous stimuli and suppression by cyclosporin A (19) . Two essential highly conserved elements within the first 108 bp of the transcription initiation site in the IFN- genomic DNA have been demonstrated to sufficiently confer activation-specific promoter function in T cells (19) , and recently our laboratory also has shown that the proximal region spanning nucleotides -71 to -49 bp of the IFN- promoter appears to be differently methylated in murine CD4 TH1 and TH2 T cell clones, and this difference correlates with the transcriptional activity of the gene (51) .

In this report, we focused our interest on the inhibition of IFN- promoter activity by the glucocorticoid hormone dexamethasone. To address the molecular mechanism involved in this transcriptional repression, we have investigated the relevance of AP-1CREB-ATF DNA binding complexes in this inhibition and in the overall regulation of IFN- gene transcription.

The AP-1 complex, composed of Jun and Fos family proteins (52) , is intimately involved in T cell activation. The activity of this complex is regulated both at the level of jun/fos gene transcription and by post-translational modifications of these proteins (52-56). For example, activation of the IL-2 promoter in T cells has been shown to be regulated by the cooperative interaction of several transcriptional factors, such as AP-1, NF-B, NF-AT, NF-IL-2A (44) . In particular, the AP-1 complex, although directly involved at a functionally relevant AP-1 site in the IL-2 promoter (57) , also participates in the formation of the NF-AT and NF-IL-2A regulatory elements (28, 58, 59, 60) . Moreover the AP-1 binding complex also may be a target for T cell clonal anergy (61) , and, in different experimental systems, its activity can be strongly inhibited by direct interference with hormone receptors such as the GR or retinoid receptors (7, 8, 9, 10, 11, 12, 13, 14, 15, 60, 62) .

Recent data have also shown that the Jun/Fos transcription factors are also able to cross-heterodimerize with CREB-ATF DNA binding factors through the ``leucine zipper'' motif (32, 33, 63) . These resulting heterodimers have been shown to display distinct DNA binding specificities from the parental homo/heterodimers (32, 33) .

We have demonstrated here that the IFN- promoter contains different AP-1CREB-ATF binding elements, in positions previously shown to be critical for its full transcriptional activity. Noteworthy, at the position corresponding to the proximal regulatory element (nucleotides from -66 to -47), we detected in EMSA the simultaneous presence of a basal CREB-ATF-related DNA binding activity and an inducible AP-1-related binding activity ( Fig. 4and Fig. 5 ), thus suggesting a further level of possible interaction/regulation between these two families of DNA-binding proteins in this context.

The similar down-regulation observed in cotransfection assays, utilizing a GR expression vector and dexamethasone treatment, or two different dominant negative mutants of c-Jun (TAM-67 and c-Jun/1-286), correlates well with the presence of AP-1CREB-ATF binding sequences in the promoter and highlights the relevance of these binding complexes for the transcriptional regulation of this cytokine.

As a negative regulation involving direct protein-protein interference between AP-1 and GR has been reported by several investigators in different systems (12, 13, 14, 15, 64) , we propose here a similar model involving a functional impairment mediated by GR on the ``essential'' AP-1CREB-ATF binding complexes of the IFN- promoter. In support of this model, specific mutation of these binding elements correlated with a significant inhibition of the promoter activation and with the loss of the glucocorticoid-mediated down-regulation. A similar model has also been proposed for the IL-2 gene promoter, where the GR interferes with the activity of NF-AT and proximal AP-1 binding sites, two regulatory elements shown to be necessary for the transcriptional activation of this gene (9) .

The inhibitory mechanism of the c-Jun dominant negative mutant TAM-67 has been only partially characterized. This inhibitor is in fact able to form stable complexes with Jun and Fos proteins, and these complexes bind DNA with the same affinity as the normal Jun and Fos heterodimers (17). In addition, recent data obtained by analysis of different TAM-67 chimeric proteins suggest a model involving a ``quenching mechanism'' rather than a ``blocking mechanism,'' through dimerization of TAM-67 with wild-type Jun and Fos proteins, this interfering with the normal AP-1 transcriptional activation (17) .

A different mechanism could be evoked for the negative mutant c-Jun/1-286, that bears a selective deletion of the leucine zipper domain; in this case, a direct interference involving a ``squelching mechanism'' via the transactivation domain on the IFN- AP-1CREB-ATF complexes might be involved (65) , since this deletion mutant is not able to bind DNA or to form dimers with wild-type Jun and Fos proteins via the leucine zipper domain. The fact that both of the mutants of c-Jun were effective in suppressing the IFN- promoter transcriptional activity underlines the relevance of the dimerization of c-Jun with Fos and/or CREB-ATF family proteins (i.e. a quenching mechanism proposed for TAM-67), and suggests the requirement of ``accessory factors'' that do not bind directly to the DNA during the activation process (i.e. a squelching mechanism proposed for c-Jun/1-286) (16, 17, 23, 65) .

In conclusion, these results further emphasize the transcriptional control complexity of the IFN- gene, and demonstrate the sensitivity of the IFN- promoter to the suppressive action caused by glucocorticoid hormone treatment during T cell activation.

  
Table: Sequence homology between IFN- AP-1CREB-ATF binding sites and canonical consensus sequences for AP-1 and CREB-ATF



FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: NCI-FCRDC, Bldg. 560, Rm. 31-93, Frederick, MD 21702-1201. Tel.: 301-846-5700; Fax: 301-846-1673.

The abbreviations used are: IFN, interferon; IL, interleukin; GR, glucocorticoid receptor; PMA, phorbol 12-myristate 13-acetate; OCT, octamer; TRE, TPA-responsive element; EMSA, electrophoretic mobility shift assay; GRE, GR-responsive element; CAT, chloramphenicol acetyltransferase; tk, thymidine kinase; bp, base pair(s); CREB, cAMP response element binding protein; ATF, activating transcription factor; AP-1, activator protein 1; NF-AT, nuclear factor of activated T cells.

L. Penix, M. Sweetser, M. Weaver, J. Hoeffler, T. Kerpolla, and C. B. Wilson, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Dr. Christopher B. Wilson for providing plasmids pIFN-538,339,108, Dr. Ronald M. Evans for providing the human GR expression vector pRShGR, Dr. Alberto Gulino for providing the AP1-2xGRE-tk-CAT vector, Dr. John R. Ortaldo and Dr. Kathrin Muegge for critical comments regarding this manuscript, and Joyce Vincent for editorial assistance.


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