Interleukin-2 Inhibits Glucocorticoid Receptor Transcriptional Activity through a Mechanism Involving STAT5 (Signal Transducer and Activator of Transcription 5) but Not AP-1

Armelle Biola, Philippe Lefebvre, Mallory Perrin-Wolff1, Marie Sturm, Jacques Bertoglio and Marc Pallardy

INSERM U461 (A.B., M.S., J.B., M.P., M.P.-W.) Faculté de Pharmacie Paris-Sud 92296 Châtenay-Malabry, France
INSERM U459 (P.L.) Faculté de Médecine Henri Warembourg 59045 Lille, France


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cytokines and glucocorticoids (GCs) signaling pathways interfere with each other in the regulation of apoptosis and gene expression in the immune system. Interleukin-2 (IL-2), through the Janus kinase/signal transducers and activators of transcription (Jak/STAT) and mitogen-activated protein kinase (MAPK) pathways, activates STAT5 and activated protein-1 (AP-1) transcription factors, respectively, which are known to repress glucocorticoid receptor (GR) activity, at least in part, through protein-protein interactions. In this work, we have analyzed the mechanisms whereby IL-2 down-regulates the GC-induced transactivation of the mouse mammary tumor virus long terminal repeat (MMTV-LTR) in murine CTLL-2 T lymphocytes. Mutagenesis studies revealed that the MMTV-LTR STAT5 binding site (-923/-914) was not required for IL-2-mediated inhibition but identified both glucocorticoid response elements (GREs) and the -104/+1 region as critical elements for this negative response. The DNA binding activities of transcription factors required for GC-mediated activation of the MMTV-LTR promoter and that bind to the -104/+1 region (nuclear factor-1, Oct-1) were not affected by IL-2 treatment. Overexpression of wild-type STAT5B enhanced the effect of IL-2 on MMTV-LTR activity, and a dominant negative form of STAT5B (Y699F) abolished the IL-2-mediated MMTV-LTR inhibition, whereas AP-1 activation had no effect in this system. Direct interaction between liganded GR and STAT5 was observed in CTLL-2 cells in a STAT5 phosphorylation-independent manner. Overexpression of nuclear coactivators CBP (CREB-binding protein) or SRC-1a (steroid receptor coactivator 1a) did not blunt IL-2 inhibitory effects. We suggest that the STAT5-repressive activity on the GC-dependent transcription may involve direct interaction of STAT5 with GR, is dependent on the promoter context and STAT5 activation level, and occurs independently of coactivators levels in T cells.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Glucocorticoids (GC) exert their biological effects through an interaction with the glucocorticoid receptor (GR), a ligand-activated transcription factor belonging to the nuclear receptor family. Two isoforms of human GR have been described: GR{alpha} and GRß. GR{alpha} is mainly located in the cytoplasm of unstimulated cells as part of a large multiprotein inactive complex with heat shock proteins and immunophilins. Hormone binding causes dissociation of this complex and translocation of the receptor into the nucleus. Then, GR stimulates hormone-dependent transcription through binding to a 15-bp glucocorticoid responsive element (GRE) present in the regulatory regions of responsive genes (1, 2). The function of GRß as a dominant negative form of GR is still a matter of debate (3, 4). The mouse mammary tumor virus (MMTV) promoter is one of the most studied GC-responsive promoters. Its transcriptional activation requires binding of GR to a cluster of four glucocorticoid response elements (GREs) located within the U3 region of the long terminal repeat (LTR) (5, 6). The promoter also contains binding sites for ubiquitous transcription factors such as nuclear factor-1 (NF-1), octamer transcription factors-1 and -2 (Oct-1 and Oct-2), and for other unknown tissue-specific regulatory factors controlling the expression of MMTV (7, 8, 9, 10). When stably transfected into mammalian cells and before stimulation, the MMTV-LTR is reproducibly packaged into a phased array of nucleosomes preventing NF-1 access to its binding site. Hormone binding initiates chromatin remodeling, and the promoter becomes accessible to NF-1. On transiently transfected MMTV-LTR plasmid DNA, NF-1 binding occurs constitutively and is not affected by GR loading (11).

To mediate their effects, steroid hormones and cytokines activate various signaling pathways through intracellular or membrane receptors, respectively. In immune cells, cross-talks between these signaling pathways affect fundamental cellular processes such as proliferation, differentiation, or apoptosis. Indeed, GCs suppress interleukin-4 (IL-4)-induced proliferation of the murine CTLL-2 cytotoxic T cell line, without affecting the IL-2-driven growth of these cells (12). In mouse T helper (Th) cell lines, the synthetic GC dexamethasone (DEX) completely inhibits IL-2-induced cell proliferation, reduces IL-4-mediated cell growth, and has no effect on IL-9-response (13). Rat CD4+ T cells transiently exposed in vitro to DEX display an altered pattern of cytokine production and develop a Th2 response (14). IL-2 has a protective role against GC-induced cell death on T cell hybridomas (15) and T lymphocytes (16, 17, 18). Moreover, IL-4 protects Th2 cells from DEX-induced apoptosis and IL-2 rescues Th1 cells from the cytolytic effect of GCs, indicating that mature T cells can be saved by their own growth factor (19). IL-9 is a potent inhibitor of GC-induced apoptosis in thymic lymphoma cell lines (20). However, mechanisms underlying interactions between GCs and cytokines signal transduction pathways in immune cells remain poorly understood.

GR can establish protein-protein interactions, independently from DNA-binding, with other transcription factors such as activated protein-1 (AP-1), NF-{kappa}B, STAT-3 (signal transducer and activator of transcription 3), and STAT-5 (21). These transcription factors are activated by cytokines, resulting in positive or negative regulation of GC-induced transcription. The outcome of cytokine stimulation on GC-mediated transcription is dependent on the promoter context and on the cell type. Indeed, in CEM, S49, and Jurkat lymphoid T cell lines, phorbol myristate acetate (PMA), through induction of AP-1, enhances DEX-induced transactivation of the MMTV-LTR, whereas it displays an inhibitory effect in NIH 3T3 fibroblasts (22). In immune cells, GCs inhibit NF-{kappa}B activation induced by tumor necrosis factor-{alpha} (TNF{alpha}), and GR was shown to physically interact with NF-{kappa}B, thereby preventing its binding to DNA (23, 24). Furthermore, GCs were shown to induce the expression of the inhibitory protein I{kappa}B{alpha}, trapping NF-{kappa}B as an inactive complex in the cytoplasm (23, 24). Overexpression of STAT-5 in PRL-activated COS-7 cells results in an increased activity of the ß-casein gene promoter upon treatment with GCs, whereas MMTV-LTR promoter activity was decreased under similar conditions (25).

The aim of this work was to evaluate the effect of IL-2 on GR transcriptional activity in lymphoid cells. We show that IL-2 strongly inhibits GC-induced transcription from the MMTV promoter, whereas IL-2 alone weakly stimulates its activity. These effects were observed in cells stably or transiently transfected with MMTV-LTR-luciferase (MMTV-LTR-luc) constructs. We also demonstrate that IL-2 does not impede binding of NF-1 or Oct-1 to their specific DNA binding sites. A STAT5 binding site was identified in the 5'-region of the promoter, which proved to be responsible for the positive regulation of the promoter by IL-2. However, deletions and mutations within the MMTV promoter showed that neither the STAT5-responsive element, nor other uncharacterized DNA sequences, play a role in IL-2 inhibition of MMTV-LTR-luc transactivation. AP-1 is not involved in the inhibitory mechanism of IL-2 as shown by PMA treatment. In CTLL-2 cells overexpressing STAT5B, IL-2 inhibition of GC-induced transcription is enhanced when compared with normal cells, whereas overexpression of a dominant negative form of STAT5B (Y699F) abolishes the IL-2- inhibitory effect. Coimmunoprecipitation experiments indicated a physical association between GR and STAT5, occurring in CTLL-2 cells treated with DEX, or DEX and IL-2. Taken together, these results strongly suggest a role for IL-2-induced STAT5 in the inhibition of GR transcriptional activity in lymphocytes. The mechanism of inhibition does not rely on a competition for limiting amounts of CBP (CREB-binding protein) or SRC-1a (steroid receptor coactivator-1a), since overexpression of the coactivators increases the IL-2 inhibitory effect.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
IL-2 Inhibition of GC-Mediated Transcription Is Promoter Context Dependent
CTLL-2, a murine T cell clone depending on IL-2 for its growth, was used to examine the effect of IL-2 on GC-induced transcriptional activation of MMTV-LTR. CTLL-2 cells were transiently transfected with the MMTV-LTR-luc plasmid, which contains the full-length MMTV LTR. Cells were treated for 12 h with 100 nM DEX, 1 ng/ml IL-2, or DEX and IL-2 (Fig. 1AGo). IL-2 alone caused a modest enhancement of the luciferase activity in the absence of DEX. In the presence of DEX, a 12-fold induction of the luciferase activity was observed. Incubation of the cells with DEX and IL-2 resulted in more than 70% inhibition of the transcriptional activity of the promoter when compared with DEX-only conditions.



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Figure 1. Effect of IL-2 on GR Transcriptional Activity in CTLL-2 Cells

A, IL-2 inhibits DEX-induced MMTV-LTR activity. CTLL-2 cells were transiently transfected with MMTV-LTR-luc plasmid and stimulated for 12 h with 1 ng/ml IL-2, with or without 100 nM DEX, or left untreated. Fold induction was calculated as the ratio of arbitrary luciferase units in cells treated with IL-2, DEX, or DEX and IL-2 compared with untreated cells. Fold induction (DEX treated cells) = (DEX activity - basal activity)/basal activity. Fold induction (DEX + IL-2 treated cells) = (DEX + IL-2 activity - IL-2 activity)/basal activity. The value 1 was consequently affected to the basal level of nontreated cells (NT). Results were obtained from two independent experiments. SEM is indicated by error bars. B, Effect of IL-2 on DEX-induced GRE5-EBV-TATA-CAT transactivation. CTLL-2 cells were transiently transfected with the GRE5-EBV-TATA-CAT plasmid driving the CAT reporter gene. Cells were stimulated for 8 h with 1 ng/ml IL-2, with or without 100 nM DEX, or left untreated. Percentage of chloramphenicol conversion represents the ratio between acetylated chloramphenicol and total chloramphenicol (acetylated and nonacetylated). A representative experiment out of three is shown here. C, Effect of IL-2 on GR DNA binding activity. The DNA-binding activity of in vitro translated recombinant rat GR was assayed by EMSA after DEX activation. CTLL-2 cells were deprived of IL-2 for 3 h and treated for 1 h with IL-2 (1 ng/ml) or left untreated (NS). Nuclear extracts were then prepared and used for competition experiments with GR for DNA binding (1 µg, 2.5 µg, 5 µg). The DNA binding specificity was determined by competition with a 50-fold excess of either a cold GRE probe, or a random probe (Rd) and by supershift experiments with 1 µg of anti-GR antibody (M20, Santa Cruz Biotechnology, Inc.) or of control antibody (C). The first lane corresponds to a nonprogrammed reticulocyte lysate. NS, Not stimulated. A representative experiment out of four is shown here.

 
We then selected a simple promoter, composed of five synthetic palindromic GREs adjacent to a minimal TATA box (GRE5-EBV-TATA-CAT), to evaluate whether the mechanism of the IL-2 inhibitory effect was mediated through GREs. CTLL-2 cells were thus transiently transfected with this reporter plasmid and incubated for 8 h in the presence or in the absence of DEX (100 nM), with or without 1 ng/ml IL-2. No inhibition of the DEX-induced promoter activity was observed in the presence of IL-2. Rather, IL-2 slightly enhanced DEX-induced GRE5-EBV-TATA-CAT transactivation (Fig. 1BGo). These results suggest that the IL-2 inhibitory effect on GC-stimulated MMTV promoter activity is probably promoter context dependent and is not uniquely mediated through GREs.

We assessed the effect of IL-2 on GR DNA-binding activity by EMSA (electrophoretic mobility shift assay) using in vitro translated recombinant GC receptor (Fig. 1CGo). At least two major complexes with different mobilities were detected, despite the unique GRE sequence of the DNA probe. This could result from a partial proteolysis of the GR in the rabbit reticulocyte lysate. These complexes bound specifically to the GRE DNA-probe, since their formation was competed by a 50-fold molar excess of unlabeled GRE DNA probe, but not by the nonspecific random DNA fragment. Furthermore, addition of the specific anti-GR antibody to the binding reaction did not block complex formation, but generated an antibody-protein-DNA ternary complex resulting in a further reduction of the mobility of all the protein-DNA complexes.

Competition experiments were performed with increasing amounts (1, 2.5, and 5 µg) of nuclear extracts of CTLL-2 cells deprived of IL-2 for 3 h and treated for 1 h with IL-2 (1 ng/ml), or left untreated. Consistent with the results presented in Fig. 1BGo, IL-2 treatment did not impede the binding of GR to DNA (Fig. 1CGo) since GR DNA-binding activity was similar in the presence of nuclear extracts from nontreated or IL-2-treated cells. Addition of nuclear extracts did not generate lower-mobility complexes, which could result from the interaction of DNA-bound GR with other cellular proteins, induced or not by IL-2. These results suggest that IL-2 stimulation of T cells does not unmask activities able to perturb GR binding to DNA.

IL-2 Inhibits DEX-Induced MMTV-LTR Activity of Stably Transfected Templates
Modifications of chromatin structure are known to affect GC-induced transcriptional activity of the MMTV promoter. IL-2 could interfere with chromatin remodeling by GR, thus affecting the activation of transcription. We therefore compared stably integrated templates and transiently transfected templates, which have been shown to display, or not, a chromatin architecture, respectively, for the effect of IL-2 on GC-induced transcriptional activity of MMTV-LTR. To this end, we stably transfected the wild-type MMTV-LTR-luc plasmid in CTLL-2 cells. Several clones were isolated and tested for their responsiveness to GC treatment. All clones tested responded similarly to IL-2 and GC stimulation, albeit with a different amplitude, which is likely to be related to the number of integrated copies. The clone showing the highest inducibility of GC-dependent transactivation (CTLL-2 pLTR 2E) was selected for further experiments. Results presented in Fig. 2Go show that treatment of these cells with DEX for 12 h results in a 20-fold increase in luciferase activity when compared with the basal transcription level. Simultaneous addition of IL-2 and DEX results in an IL-2 dose-dependent inhibition of DEX induction of the MMTV promoter activity. At a saturating dose of 1 ng/ml of IL-2, the promoter activity is almost completely abolished (91% inhibition) and reaches basal level measured in the absence of hormone stimulation. As previously observed with transiently transfected templates, IL-2 alone positively regulated MMTV-LTR activity. These results obtained with the chromatin templates are thus comparable to the transiently transfected ones, suggesting that the IL-2 effect is not affected by integration of the promoter into chromatin.



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Figure 2. IL-2 Inhibits GC-Induced MMTV-LTR-luc Transactivation in Stably Transfected CTLL-2 pLTR Cells

CTLL-2 cells stably transfected with MMTV-LTR-luc were washed three times to remove IL-2, and then stimulated for 12 h with either 0.2 ng/ml, 0.5 ng/ml, or 1 ng/ml IL-2, with or without 100 nM DEX, or left untreated. Fold induction was calculated as in Fig. 1AGo. Data from a representative experiment are expressed as a mean of triplicates. SEM is indicated by error bars.

 
MMTV-LTR Contains a STAT5 Binding Site That Is Not Involved in IL-2 Inhibition
Compared with the simple synthetic promoter of the GRE5-EBV-TATA-CAT plasmid, the composite nature of the MMTV promoter (Fig. 3Go) therefore raised the hypothesis of the existence of one (or more) regulatory element (s) necessary for the inhibitory effect of IL-2. Internal deletions of the MMTV LTR (-967 to -220, and -860 to -220) allowed us to localize a STAT5 consensus binding site (TTCGGAGAA) (Fig. 3Go).



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Figure 3. Structure of the MMTV-LTR Promoter

Numbers indicate the position relative to the transcriptional initiation site (+1). NRE, Negative regulatory element; HRE, hormone responsive element; STAT5 RE, STAT5 responsive element; AA and A, regulatory elements (44 , 46 ).

 
IL-2 activates the Janus family tyrosine kinases Jak1 and Jak3, resulting in subsequent tyrosine phosphorylation and DNA binding of STAT5 (26, 27). We first characterized the IL-2-mediated activation of STAT5 in CTLL-2 cells using transcriptional activation assays (data not shown) and EMSA. No STAT5-DNA binding activity was present in CTLL-2 cells deprived of IL-2 for 3 h (Fig. 4AGo). However, after 1 h of IL-2 stimulation (1 ng/ml), a strong STAT5-DNA binding was observed (Fig. 4AGo). Supershift experiments showed that STAT5A and STAT5B were activated in CTLL-2 cells upon IL-2 treatment. We then evaluated by EMSA whether IL-2-activated STAT5 proteins could bind to the STAT-responsive element of the MMTV promoter (-923/-914) (Fig. 4BGo). A DNA-bound complex was detected in IL-2-treated cells and could be completely supershifted with the anti-STAT5 serum (Fig. 4BGo), suggesting that STAT5 was the only component of the complex formed on the STAT/MMTV probe in CTLL-2 cells. To assess the functional activity of this STAT5 element, we mutated this sequence to disrupt STAT5-DNA binding on the MMTV-LTR distal regions of the pLTR-luc and the p-1,180/-860-luc plasmids to generate pLTRmut-luc and p-1180/-860 mut-luc plasmids. CTLL-2 cells were then transiently transfected with wild-type or mutated constructs and treated for 12 h with or without IL-2 (1 ng/ml). Results presented in Fig. 4CGo show that mutation of the STAT5 site dramatically reduces the basal activity of the promoter measured in untreated cells. In addition, the activity of the mutated constructs was not significantly affected by IL-2, strongly suggesting that this site plays a role in the positive regulatory effect of the cytokine on the MMTV promoter activity. We then investigated whether this STAT5-binding site could play a role in the IL-2-inhibitory effect on DEX-induced MMTV-LTR transactivation. To this end, CTLL-2 cells were transiently transfected with the pLTR-luc and the pLTRmut-luc plasmids and treated for 12 h with IL-2 (1 ng/ml), with or without DEX (100 nM), or left untreated. Results show that the inducibility of the mutated promoter is enhanced upon DEX treatment compared with the wild-type promoter, due to the reduced level of the basal activity. Nevertheless, the effect of IL-2 measured with pLTR-luc and pLTRmut-luc (84% and 75%, respectively) is comparable with the two constructs (Fig. 4DGo), suggesting that the STAT5-binding site does not contribute to the IL-2-inhibitory effect.



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Figure 4. Influence of the STAT5 Binding Site on IL-2 Inhibition of DEX-Induced MMTV-LTR Activity

A, Activation of STAT5A and STAT5B by IL-2 in CTLL-2 cells. Nuclear extracts were prepared from CTLL-2 cells after 3 h of IL-2 deprivation and 1 h of treatment with or without IL-2 (1 ng/ml). Competition experiments were performed in the presence of a 25-fold excess of either a cold GAS probe, or a random probe (Rd). For supershift experiments, 1.5 µl of the preimmune serum (PIS) or of the specific anti-STAT5A or anti-STAT5B sera were preincubated with nuclear extracts for 2 h at 4 C before addition of probe. B, Binding of STAT5 on the STAT response element (STAT RE) of the MMTV-LTR promoter. Nuclear extracts were prepared as described in panel A. Competition experiments were performed in the presence of a 25-fold excess of either a cold STAT/MMTV probe, or a random probe (Rd). For supershift experiments, 1 µl of the preimmune serum (PIS) or of the anti-STAT5 serum were preincubated with nuclear extracts for 2 h at 4 C before addition of probe. C, Effect of the mutation of the STAT5 RE on both the basal and IL-2-induced activity of the MMTV promoter. CTLL-2 cells were transiently transfected with the pLTR-luc, pLTRmut-luc, p-1180/-860-luc, and p-1180/-860 mut-luc plasmids and stimulated for 12 h with 1 ng/ml IL-2, or left untreated. Results are expressed in arbitrary luciferase units. Results of a representative experiment out of three. NT, Nontreated. D, Effect of the mutation of the STAT5 RE on the IL-2 inhibition of the DEX-induced MMTV promoter activity. CTLL-2 cells were transiently transfected with the pLTR-luc or the pLTRmut-luc plasmids and stimulated for 12 h with 1 ng/ml IL-2, with or without DEX, or left untreated. Fold induction was calculated as in Fig. 1AGo. Data from a representative experiment are expressed as a mean of duplicates. SEM is indicated by error bars.

 
Influence of Deletions within the MMTV-LTR on IL-2 Inhibition
We performed 5'-end deletions of the promoter up to positions -325, -223, -200, and -104 (Fig. 5AGo) to identify cis-acting sequences along the MMTV promoter involved in the IL-2-mediated inhibition. CTLL-2 cells were transiently transfected with these constructs and cultured for 12 h in the presence or in the absence of 100 nM DEX and/or IL-2 (1 ng/ml). Results show that the p104-luc plasmid, which lacks the two distal GREs, does not retain any hormone inducibility. Sequences located upstream of the -200 position can be deleted without impairing the hormone response (Fig. 5BGo). The responsiveness of p325-luc, p223-luc, and p200-luc plasmids to DEX stimulation is inhibited by 56%, 65%, and 62% upon IL-2 treatment (1 ng/ml), respectively. The p223-luc and p200-luc deletion mutants show an increased GC-induced transcriptional activity compared with the MMTV-LTR-luc plasmid, suggesting that upstream negative regulatory sequence(s) were removed (Fig. 5BGo).



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Figure 5. Influence of Deletions within the MMTV-LTR on IL-2 Inhibition of GC-Induced Transcription in Transiently Transfected CTLL-2 Cells

A, Schematic representation of the MMTV-LTR deletion mutants driving the luciferase reporter gene. LUC, luciferase. Numbers indicate the position relative to the transcriptional initiation site (+1). B, Effect of IL-2 on the DEX-induced transactivation of MMTV-LTR deletion mutants. CTLL-2 cells were transiently transfected with 10 µg of the different plasmids and were then stimulated for 12 h with 1 ng/ml IL-2, with or without 100 nM DEX, or left untreated. Fold induction was calculated as in Fig. 1AGo. Inductions were calculated from two independent experiments. SEM is indicated by error bars. NT, Not treated. C, Effect of IL-2 on the DEX-induced transactivation of p2GRE-104-luc plasmid. CTLL-2 cells were transfected as in panel B, except that the p2GRE104-luc construct was used as a reporter gene. NT, Not treated. Results from two independent experiments. SEM is indicated by error bars.

 
To investigate the role played by other functional cis-acting elements located in the region spanning from -200 to -104, we constructed the p2GRE104-luc plasmid (Fig. 5AGo) by substituting two consensus synthetic GREs for the sequence spanning from the 5' end to the -104 position within the MMTV-LTR promoter. Results from Fig. 5CGo show that the basal luciferase activity of this plasmid is very low and remains unaffected by IL-2. Very importantly, addition of IL-2 reduced by 52% the DEX-induced transcriptional activation of this promoter. These data demonstrate that the most proximal region of the promoter, including the two proximal hemi-palindromic GREs, the NF-1 and Oct-1 binding sites, as well as the TATA box, are necessary and sufficient to convey the inhibitory effect of IL-2.

IL-2 Does Not Impede NF-1 or Oct-1 Binding to Their Cognate DNA Response Elements
Given that NF-1 binding to MMTV-LTR is an absolute prerequisite for GC-induced MMTV transactivation, we evaluated whether IL-2 inhibits NF-1 binding to its specific recognition site. We conducted EMSA using a NF-1 probe whose sequence is similar to the NF-1 binding site present in the MMTV promoter. Results from Fig. 6AGo show that IL-2 does not reduce NF-1 binding activity.



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Figure 6. Effect of IL-2 and DEX on NF-1 or Oct-1 DNA Binding Activity

Nuclear extracts were prepared from CTLL-2 cells after 3 h of IL-2 deprivation and 1 h of treatment with DEX (100 nM) and/or IL-2 (1 ng/ml). A, NF-1 DNA binding activity. Competition experiments were performed in the presence of a 25-fold excess of either a cold NF-1 probe, or a random probe (Rd). B, Oct-1 DNA binding activity. Competition experiments were performed in the presence of a 25-fold excess of either a cold Oct-1 probe, or a random probe (Rd). For supershift experiments, 3 µg of the control IgG or of the specific anti-Oct-1 IgG were preincubated with nuclear extracts for 2 h at 4 C before addition of probe.

 
Oct-1 is a POU family transcription factor constitutively expressed in many cell types, which specifically interacts with the octamer motif ATGCAAAT. The MMTV promoter contains two functional octamer-related elements involved in basal and steroid-induced transcription. Mutation of both octamer motifs was shown to strongly reduce GC-induced MMTV promoter activity (28). To determine the effect of IL-2 on Oct-DNA binding activity, we performed EMSA experiments with an oligonucleotide derived from MMTV sequence between -61 and -32 positions. Results show that IL-2 does not significantly affect the binding of Oct proteins to their specific DNA sequence (Fig. 6BGo). To characterize the nature of the Oct family member bound to the probe in CTLL-2 cells, we performed supershift experiments with a specific anti-Oct-1 polyclonal antibody directed against the carboxy terminus of the protein. The anti-Oct-1 IgG, but not the control IgG, completely displaced the specific complex for all tested treatments, indicating that Oct-1 is an integral part of the complex formed on the Oct probe in CTLL-2 cells.

AP-1 Is Not Involved in the IL-2 Inhibition of GC-Induced MMTV-LTR Transactivation
These results prompted us to determine which IL-2-dependent signal transduction pathway was involved in the inhibition of GC-induced transcription. The AP-1 transcription factor, which plays a crucial role in cell cycle control and survival of lymphoid cells, has been shown to physically interact with the GR, resulting in a mutual transcriptional repression (29, 30, 31). IL-2 and PMA have been shown to increase AP-1 DNA binding and transcriptional activities in CTLL-2 cells (Ref. 32 and Fig. 7AGo). Results showed that PMA did not repress the GR transcriptional activity assessed with the MMTV-LTR-luc plasmid (Fig. 7BGo). These results were obtained in stably transfected CTLL-2 pLTR cells (Fig. 7BGo), as well as in transiently transfected CTLL-2 cells (data not shown).



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Figure 7. Effect of IL-2 and PMA on GC-Induced MMTV-LTR-luc Transactivation in Stably Transfected CTLL-2 pLTR Cells

A, IL-2 and PMA activate AP-1. CTLL-2 cells were transiently transfected with the PMA-inducible 5XTRE-tk-CAT plasmid driving the CAT reporter gene. Cells were stimulated for 8 h with 1 ng/ml IL-2, or 50 ng/ml PMA, or left untreated. Percentage of chloramphenicol conversion represents the ratio between acetylated chloramphenicol and total chloramphenicol (acetylated and nonacetylated). Induction was calculated as the ratio of percentage of chloramphenicol conversion in cells treated with IL-2 compared with untreated cells (x5) or in cells treated with PMA (x4) compared with untreated cells. A representative experiment out of three is shown here. B, Effect of IL-2 and PMA on DEX-induced MMTV-LTR transactivation. CTLL-2 cells stably transfected with MMTV-LTR-luc were washed three times to remove IL-2, and then stimulated for 12 h with 1 ng/ml IL-2, or 50 ng/ml PMA with or without 100 nM DEX, or left untreated. Fold induction was calculated as in Fig. 1AGo, except for DEX + PMA induction = (DEX + PMA activity - PMA activity)/basal activity.

 
Role of STAT5B in IL-2 Inhibition of GC-Induced MMTV-LTR Transactivation
We demonstrated above that STAT5 DNA binding on the distal region of the promoter could not account for the inhibitory effect of IL-2. Since the proximal part of the promoter contains no STAT5 binding site, we assessed whether STAT5 could alter the promoter activity by engaging protein-protein interactions with other transcription factors. For this purpose, we used two complementary approaches. CTLL-2 cells were stably transfected with either the wild-type form of STAT5B (clone 15A) or a mutated dominant negative STAT5B protein under the control of a tetracycline-regulated (Tet-Off) gene expression system (33). The dominant negative form of STAT5B cannot be activated by IL-2 since it possesses one mutation on tyrosine 699 (Y699F). Both STAT5B proteins were expressed as fusion proteins (Myc-STAT5B and Myc-STAT5B Y699F) with a Myc tag to screen their expression independently of the endogenous form of STAT5B, using the 9E10 anti-Myc antibody.

Overexpression of wild-type Myc-STAT5B protein was achieved after 48 h of culture in the absence of tetracycline. In the presence of tetracycline (1 µg/ml), the expression of this protein was fully repressed (Fig. 8AGo, right insert). Cells were then transiently transfected with the p2GRE104-luc construct and stimulated for 12 h in the presence or in the absence of 100 nM DEX and/or IL-2 (1 ng/ml and 10 ng/ml). In STAT5B overexpressing cells, IL-2 (1 ng/ml) inhibition of GC-induced p2GRE104-luc transactivation was enhanced by almost 20% (54% inhibition in overexpressing STAT5B cells compared with 36% inhibition in wild-type cells). This difference was not detectable when a 10-fold higher concentration of IL-2 (10 ng/ml) was used, leading to a maximal inhibition of DEX-induced p2GRE104-luc transactivation (Fig. 8AGo).



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Figure 8. Role of STAT5B in IL-2 Inhibition of GC-Induced p2GRE104-luc Transactivation

A, Effect of wild-type STAT5B overexpression. CTLL-2 Myc-STAT5B 15 A cells were cultured for 48 h in complete medium containing 1 ng/ml IL-2, in the presence or in the absence of tetracycline (1 µg/ml). Cells were then transiently transfected with 20 µg of the p2GRE104-luc construct and stimulated for 12 h with either 1 ng/ml or 10 ng/ml IL-2, with or without 100 nM DEX, or left untreated. Results are expressed in percentage of IL-2 inhibition of GC-induced p2GRE-104-luc transactivation. At the end of the incubation period, Myc-STAT5B overexpression was characterized using the monoclonal 9E10 anti-myc antibody (right inset). Results of a representative experiment out of three. Tet, Tetracycline. B and C, Expression of the STAT5B Y699F protein in clones 9, 4, and 5. CTLL-2 Myc-STAT5B Y699F cells (clones 4, 5, and 9) were cultured for 48 h in complete medium containing 1 ng/ml IL-2, in the absence of tetracycline. For all points, cells were deprived of IL-2 for 2 h and stimulated for 12 h with 500 pg/ml IL-2. Whole cell lysates were prepared and probed with the monoclonal 9E10 anti-myc antibody to control Myc-STAT5B Y699F expression compared with untransfected CTLL-2 cells (C in panel B). The same extracts were assayed for STAT5B activation using the DNA affinity precipitation method. Bound proteins were probed with a specific anti-STAT5B antibody (panel C). Tet, Tetracycline; C, CTLL-2 cells; WCE, whole-cell extracts. D, Effect of Myc-STAT5B Y699F mutant overexpression. CTLL-2 Myc-STAT5B Y699F cells (clones 4, 5, and 9) were cultured for 48 h in complete medium containing 1 ng/ml IL-2, in the absence of tetracycline. Cells were then deprived of IL-2 for 2 h, transiently transfected with 20 µg of the p2GRE104-luc construct, and stimulated for 12 h with 500 pg/ml IL-2, with or without 100 nM DEX, or left untreated. Results are expressed in percentage of IL-2 inhibition of GC-induced p2GRE-104-luc transactivation. Results from a representative experiment out of two. E, Physical interaction between STAT5 and the GR. CTLL-2 cells were deprived of IL-2 for 3 h and treated for 1 h with IL-2 (1 ng/ml) with or without 100 nM DEX, or left untreated. Lysates were immunoprecipitated with an anti-GR antibody (BuGR2, Affinity BioReagents, Inc.). Immunocomplexes were separated by SDS-PAGE, transferred to PVDF membrane, and detected with an anti-STAT5 antibody (upper panel) or an anti-GR antibody (lower panel). IP, Immunoprecipitation; IB, immunoblot. F, Physical interaction between STAT5B Y699F and the GR. CTLL-2 Myc-STAT5B Y699F cells (clone 5) were cultured for 48 h in complete medium containing 1 ng/ml IL-2, in the absence of tetracycline. Cells were then deprived of IL-2 for 3 h, treated for 1 h with DEX (100 nM) and IL-2 (1 ng/ml). Lysates were immunoprecipitated with an anti-GR antibody (BuGR2, Affinity BioReagents, Inc.). Immunocomplexes were separated by SDS-PAGE, transferred to PVDF membrane, and detected with an anti-myc antibody (9E10). WCE, Whole cell extract; IP, Immunoprecipitation; IB, immunoblot.

 
Three stable cell lines (clones 4, 5, and 9) expressing different levels of the dominant negative Myc-STAT5B Y699F protein were established (Fig. 8BGo), since no clone displaying high regulatable levels of Myc-STAT5B Y699F could be obtained. Analysis of STAT5 activation in these clones, by DNA affinity precipitation with a 5'-biotinylated IFN{gamma}-activated sequence (GAS) oligonucleotide, revealed that a dominant negative effect of STAT5B Y699F was only achieved with clone 5, which showed the highest level of expression of this protein (Fig. 8CGo).

CTLL-2 Myc-STAT5B Y699F cells (clones 4, 5, and 9) were cultured for 48 h in the absence of tetracycline. Cells were then transiently transfected with the p2GRE104-luc construct and stimulated for 12 h in the presence or in the absence of 100 nM DEX and/or IL-2 (500 pg/ml). In clones 4 and 9, DEX-induced p2GRE104-luc transactivation was still inhibited (-55% and -51%, respectively) despite expression of the dominant negative form of STAT5B (Fig. 8DGo). These results were correlated with STAT5 activation levels (Fig. 8CGo). IL-2 inhibition of DEX-induced p2GRE104-luc transactivation, however, was almost completely abolished (-6%) in clone 5, which expresses the highest level of the Myc-STAT5B Y699F protein (Fig. 8DGo). These results showed that activation of STAT5 is a key event in the inhibition of GC-induced MMTV transactivation by IL-2.

We postulated that STAT5 might physically interact with the GR, explaining the transcriptional interference existing between these two factors. Coimmunoprecipitation experiments were performed with an anti-GR antibody using whole-cell extracts from CTLL-2 cells deprived of IL-2 for 3 h and treated for 1 h with IL-2 (1 ng/ml), DEX (100 nM), DEX and IL-2 (1 ng/ml), or left untreated. Blotting of the GR immunocomplex with an anti-STAT5 antibody allowed us to detect coprecipitated STAT5 in DEX- or DEX and IL-2-treated cells (Fig. 8EGo). These results suggested that GR activation, but not STAT5 activation, is a prerequisite for GR-STAT5 complex formation. To confirm this observation, we assessed whether STAT5B Y699F could interact with the GR. For this purpose, CTLL-2 STAT5B Y699F cells (clone 5) were cultured for 48 h in the absence of tetracycline, deprived of IL-2 for 3 h, and then treated for 1 h with DEX (100 nM) and IL-2 (1 ng/ml). Cell lysates were then immunoprecipitated with an anti-GR antibody, and GR immunocomplexes were separated by SDS-PAGE and probed with an anti-myc antibody. Results presented in Fig. 8FGo show that STAT5B Y699F physically interacts with GR, confirming that STAT5B tyrosine phosphorylation is not necessary for GR-STAT5 association.

Role of Coactivators in the IL-2 Inhibition of GC-Induced MMTV-LTR Transactivation
Since CBP was described to interact with both the GR and STAT5, we tested the hypothesis of a competition between GR and STAT5 for limiting amounts of the coactivator. CTLL-2 cells were transiently transfected with p2GRE104-luc and pCMV-2N3T-CBP or pCMV-2N3T plasmids, cultured for 48 h in complete medium containing 1 ng/ml IL-2, and then treated for 12 h with DEX (100 nM), IL-2 (1 ng/ml), DEX and IL-2, or left untreated (Fig. 9AGo). The transfection of increasing quantities of CBP (1, 5, and 10 µg plasmid) resulted in an enhancement of the IL-2 inhibitory effect on GC-induced p2GRE104-luc transactivation (62%, 69%, and 72%, respectively, compared with 42% with the control vector), ruling out the possibility of a squelching of CBP by STAT5 as a mechanism of IL-2 inhibition (Fig. 9AGo). Moreover, we could determine that transfection of 1 µg pCMV-2N3T-CBP leads to an enhancement of DEX-induced p2GRE104-luc transactivation, showing that CBP participates in GC-induced transactivation. However, transfection of 10 µg of plasmid has a negative effect on the promoter activity. Taken together, these results suggest that CBP is a modulator of MMTV promoter activity. We then evaluated the role of another GR coactivator, SRC-1a, in the IL-2 inhibitory effect (Fig. 9BGo). Overexpression of SRC-1a causes a slight enhancement of the IL-2 inhibition of DEX-induced p2GRE104-luc transactivation (47% vs. 35% for 5 µg of plasmid), indicating that the mechanism of inhibition does not rely on a competition for limiting amounts of SRC-1a (Fig. 9BGo). Again, transfection of high amounts of SRC-1a leads to a down-regulation of the MMTV promoter activity.



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Figure 9. Role of Coactivators in IL-2 Inhibition of GC-Induced p2GRE104-luc Transactivation

A, Effect of CBP overexpression on the IL-2 inhibitory effect. CTLL-2 cells were transiently transfected with the p2GRE104-luc plasmid, and pCMV-2N3T (control vector, 10 µg) or pCMV-2N3T-CBP (1, 5, or 10 µg). Cells were cultured for 48 h in complete medium containing 1 ng/ml IL-2 and then stimulated for 12 h with 1 ng/ml IL-2, with or without DEX, or left untreated. Fold induction was calculated as in Fig. 1AGo. Representative experiment out of three. NT, Not treated. B, Effect of SRC-1a overexpression on the IL-2 inhibitory effect. CTLL-2 cells were transiently transfected with the p2GRE104-luc plasmid and pCR3.1 (control vector, 5 µg) or pCR3.1-SRC-1a (0.5, 1, or 5 µg). Cells were cultured for 48 h in complete medium containing 1 ng/ml IL-2 and then stimulated for 12 h with 1 ng/ml IL-2, with or without DEX, or left untreated. Fold induction was calculated as in Fig. 1AGo. Results of a representative experiment out of three. NT, Not treated.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cross-talks between signal transduction pathways controlled by GCs and cytokines are believed to play an important role in different pathologies such as asthma or lymphoma (34). In addition, how cytokines modulate GC-regulated genes is still a matter of debate in the field of apoptosis (35). We and others have previously found that IL-2 was able to inhibit GC-induced apoptosis in murine IL-2-dependent CTLL-2 cells (17, 18, 32). In these lymphoid T cells, apoptosis provoked by GC is dependent upon the transcriptional activity of GR (A. Biola, unpublished data). IL-2 activates three major transduction pathways: the phosphatidylinositol-3 kinase (PI3K) pathway, the Jak-STAT pathway leading to STAT-3 and STAT-5 activation, and the Ras-MAPK (mitogen-activated protein kinase) pathway resulting in AP-1 activation (Refs. 36, 37 for review). In the present paper, we investigated the mechanism underlying IL-2 inhibition of GC-induced transactivation of MMTV-LTR. Since we were interested in evaluating this effect in physiological conditions, we used murine lymphoid cells expressing endogenous levels of the GR and of IL-2 receptor components and transduction partners.

DEX-induced MMTV-LTR transcriptional activity was significantly reduced upon IL-2 addition at saturating concentrations. However, GC-dependent transactivation measured with the GRE5-EBV-TATA-CAT plasmid (38) was not impaired by IL-2, suggesting that this effect depends on the promoter context, and that IL-2 did not prevent binding of DEX to the GR or translocation of the GR to the nucleus. Moreover, GR DNA binding activity was not affected in the presence of nuclear extracts of IL-2-treated CTLL-2 cells, showing that IL-2 did not induce the appearance of inhibitory factors.

In the MMTV-LTR promoter, binding of the activated GR to GREs initiates a remodeling of the chromatin that displaces nucleosome B and makes the promoter accessible to NF-1, a step necessary for GC-dependent transcription to occur. This mechanism is not observed on chromatin-free templates (39, 11). Binding of the octamer motifs in vivo was also observed to be strictly hormone dependent, and Oct/GR interactions result in a transcriptional cooperativity between these two factors (40). We observed that IL-2 inhibition was still effective when MMTV-LTR was stably integrated into chromatin, and that IL-2 did not alter NF-1 or Oct-1 binding capacity to their cognate DNA sequences, indicating that inhibition does not occur through modulation of either NF-1 or Oct-1 DNA binding activities.

Another mechanism that could account for IL-2 inhibition is the activation of a trans-regulatory factor acting by binding to the MMTV-LTR. Several sequences located within the U3 region of the MMTV-LTR have been shown to regulate its activity (41). The MMTV promoter contains four HREs (hormone responsive elements): two distal palindromic sites (located between -184 and -114 positions) and two proximal hemipalindromes (located between positions -98 and -78) (see Fig. 3Go). Deletion of the two distal GREs/HREs is sufficient to abolish GC-induced transactivation (see Fig. 5BGo) (42). A region around –1,090 to -900 within the MMTV-LTR has been delimited as an enhancer that seems to be mainly involved in mammary specificity (41, 43). Deletion of a regulatory element located between -294 to -200 termed AA element has also been shown to decrease GC-induced transactivation (42). Moreover, within this AA element a 20-bp region located between -223 and -201 seems to play a regulatory role (44). At least three negative regulatory elements have been described between -861 and -364 (41). An AP-1 site has also been described between position -766 and -737 (45). We identified in this work a consensus STAT5-binding site between positions -923 and -914 of the MMTV-LTR and showed the involvement of this sequence in the IL-2 positive regulation of the promoter activity, but not in the IL-2 inhibitory effect on DEX-induced MMTV transactivation. This result rules out a role for a STAT5 DNA-binding element in the negative regulation of MMTV-LTR by IL-2. Deletion of the entire 5'-end of the LTR up to the indicated position allowed us to rule out the involvement of regions upstream of position -325 (enhancer region, negative regulatory elements, AP-1 site), between -325 and -223 and of the 20-bp sequence within the AA element, respectively, in the IL-2-inhibitory effect. However, this approach did not evaluate whether sequences located between GREs could play a role. Indeed, a sequence located between the two distal GREs (-163 to -147) and termed A element has been described to regulate negatively MMTV-LTR activity upon binding of a trans-negative modulator named C1 in 6.10.2 rat hepatoma cells (46). However, data generated with the p2GRE-104 plasmid did not confirm this hypothesis in CTLL-2 lymphocytes. Taken together, our results obtained with these deletion mutants suggested that the -104/+1 region is critical for the IL-2 negative response and that a trans-acting IL-2-activated factor is not involved in the regulation of MMTV-LTR activity by IL-2.

We then decided to identify which component of the IL-2 signal transduction pathway was involved in the IL-2-inhibitory effect. This approach could also clarify whether an IL-2-induced factor could interact with GR-transcriptional activity without binding on the DNA.

IL-2 leads to AP-1 activation in CTLL-2 cells (Ref. 32 and Fig. 7AGo). AP-1 and GR have been shown to mutually interfere with their transactivating functions (29, 30, 31). Elevated c-Jun or c-Fos levels can inhibit GR-dependent transcription from the MMTV-LTR promoter or from promoters carrying only GREs (29, 30, 31). However, when CTLL-2 cells were treated with PMA and DEX, no inhibition of MMTV-LTR transactivation was found, although AP-1 activity was clearly induced as assayed by gene reporter assays. We note that the composition of the AP-1 complex could differ between IL-2 or PMA treatment (32), explaining this result. Indeed, cell-specific factors and AP-1 composition may affect the outcome of the effect of AP-1 on GR-transcriptional activity (22, 47, 48).

The Jak-STAT pathway has been recently described to interact with GC-dependent signaling. In COS-7 cells stimulated with PRL, STAT5 appears to synergize with GC on the ß-casein promoter (25, 49, 50) but to antagonize GC-induced MMTV-LTR promoter activity (25, 51). Moreover, in the rat hepatoma cell line H4IIE and in COS-7 cells stimulated with IL-6, STAT3 was shown to synergize with GC on the rat {gamma}-fibrinogen promoter and on the MMTV-LTR promoter (52). Noticeably, many of these studies have been performed with overexpressed factors (STAT or GR) in cells that do not normally express either STAT or GR proteins. Our experiments showed that, in CTLL-2 cells expressing endogenous levels of STAT5 and GR, STAT5B is necessary for IL-2 inhibition of MMTV promoter activity. Moreover, tyrosine phosphorylation of STAT5B seems to be an important step in this mechanism. A physical association between GR and STAT5 was present only if the GR was activated but independently of STAT5 activation. Formation of complexes between STAT5 and GR has previously been described in both COS-7 cells and in HC11 mammary cells, and this association is dependent upon ligand-induced activation of STAT5 in COS cells but not in HC11 cells (25, 53).

In light of these observations, STAT5 appears to play a major role in the IL-2 inhibitory effect, without binding on a specific DNA sequence located on the MMTV promoter. Our results with the deletion mutants of the promoter argue that integrity of sequences from -104 to +1 is required for IL-2 inhibition. However, the MMTV-LTR proximal promoter does not contain STAT5-specific DNA-binding sequences, suggesting that STAT5-mediated inhibition could occur through interference with specific factors loading on the MMTV promoter rather than through binding to a specific DNA sequence, probably by protein-protein interaction. Indeed, we detected a physical association between GR and STAT5 in CTLL-2 cells. GR/STAT5 complex formation could interfere with contacts between the GR and coactivators of the basal transcription machinery. It has been shown in COS cells that overexpression of CBP/p300 did not alter STAT5 inhibition of MMTV-LTR activity, despite interaction of p300 with STAT5 and p300-dependent enhancement of MMTV-LTR activity (51). In CTLL-2 cells, we have also found that overexpression of CBP or SRC-1a does not alter IL-2 inhibitory effect. Although these results rule out a mechanism of squelching, where GR and STAT5 could compete for a limiting amount of this coactivator, they do not exclude the possibility that another mechanism involving CBP/p300 could take place. The tyrosine phosphorylation of STAT5 appears as an essential step for IL-2 inhibition, either by altering the conformation of the GR-STAT5 complex, or by allowing the recruitment of other partners. GR and STAT5 could both interact with a cofactor like CBP/p300, and/or SRC-1a, and form a complex devoid of transcriptional activity, as recently hypothesized for the mutual antagonism between GR and NF-{kappa}B (54).

All together, these results indicate that in lymphocytes expressing endogenous levels of STAT5 and GR, STAT5 plays a critical role in IL-2 regulation of GC-dependent transactivation. This could be of importance in elucidating how cytokines modulate expression of GC-regulated genes in pathological situations, i.e. asthma or lymphoma, or in physiological situations, i.e. apoptosis (55).


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Chemicals and Reagents
Recombinant human IL-2 (Proleukin) was a gift from Chiron Corp. ( Amsterdam, The Netherlands). DEX, tetracycline, and PMA were purchased from Sigma (L’Isle D’Abeau, France), and G418 and Hygromycin B were from Life Technologies, Inc. (Cergy-Pontoise, France). The luciferase assay system was purchased from Promega Corp. (Madison, WI).

Plasmids
pLTR-luc plasmid contains the entire MMTV-LTR GC-responsive promoter from the C3H strain coupled to the luciferase reporter gene (43). p325-luc, p223-luc, p200-luc, and p104-luc plasmids display sequential 5'-end deletions up to positions -325, -223, -200, and -104, respectively (Fig. 5AGo). p-1,180/-860-luc plasmid displays an internal deletion between positions -860 and -220 (43). p200-luc was obtained by deletion of the HindIII/AflII fragment from the pLTR-luc plasmid, filling with Klenow, and ligation. p2GRE-104-luc plasmid was constructed by inserting two synthetic consensus GREs immediately upstream of the -104 promoter. Briefly, the HindIII/SacI fragment of the pLTR-luc plasmid was ligated to the HindIII/SacI double-stranded 5'-phosphorylated oligonucleotide (5'-AGCTTTGTACAGGATGTTCTAGATCTTGTACAGGATGTTCTGAGCT-3'). These constructs were then verified by sequencing. The GRE5-EBV-TATA-CAT plasmid was a kind gift of S. Mader. The 5XTRE-tk-CAT contains five TRE sequences upstream from the CAT (chloramphenicol acetyltransferase) gene and was a kind gift of B. Binetruy.

The NdeI/HindIII fragment of STAT 5B was amplified by PCR by using RSV-STAT5B (a kind gift of F. Gouilleux) as template and oligonucleotides 5BSTART (5'-GGGAATTCCATATGGCTATGTGGATACAGGCTCAG-3') and 5BSTOP (5'-CCCAAGCTTGAATTCTCATGACTGTGCGTGAGGGAT-3') as primers. The NdeI/HindIII fragment of STAT5B was inserted at the NdeI/HindIII sites of pGEM-Myc. The EcoRI/EcoRI fragment of Myc-STAT5B was subsequently inserted into the PUHD 10–3 plasmid (33) at the EcoRI site in the sense orientation, generating the construct pTRE-Myc-STAT5B.

A point mutation in the STAT5B sequence from tyrosine to phenylalanine (Y699F) was introduced using the Quick-change site-directed mutagenesis kit (Stratagene, Amsterdam, The Netherlands). The pTRE-Myc-STAT5B construct was used as a template and the complementary oligonucleotides STAT5B Y699F sense (5'-TGACGGATTCGTGAAGCCACAGAT-3') and STAT5B Y699F antisense (5'-ATCTGTGGCTTCACGAATCCGTCA-3') as primers. The PCR was run under conditions recommended by the manufacturer for 12 cycles (30 sec at 95 C, 1 min at 55 C, and 13 min at 68 C). The mutation in the STAT/MMTV sequence (TTCGGAGAA 224 GGCGGAGAA) was introduced using the same kit, with the pLTR-luc and p-1,180/-860-luc plasmids as templates and complementary oligonucleotides STAT/MMTV sense (5'-ACAATCTAAACAAGGCGGAGAACTCGACCTTCCTCCTG-3') and STAT/MMTV antisense (5'-CAGGAGGAAGGTCGAGTTCTCCGCCTTGTTTAGATTGT-3') as primers. The PCR was run for 16 cycles (30 sec at 95 C, 1 min at 55 C, and 15 min at 68 C).

The pCMV-2N3T and pCMV-2N3T-CBP plasmids were a kind gift of A. Harel-Bellan, the pCR3.1 and pCR3.1-SRC-1a plasmids were a kind gift of S. Tsai.

Cell Culture, Transfections, and Tet-Off Gene Expression System
The murine IL-2-dependent cytotoxic T cell line CTLL-2 was cultured in complete medium: RPMI 1640 medium (Life Technologies, Inc.) containing 2 mM L-glutamine, 0.1 mg/ml streptomycin, 100 U/ml penicillin (Life Technologies, Inc.), 50 µM 2-mercaptoethanol (Sigma), 1% sodium pyruvate (Life Technologies, Inc.), 10% FCS (Life Technologies, Inc.), and 1 ng/ml of human recombinant IL-2.

Transfections were performed using the electroporation method. Exponentially growing CTLL-2 cells (107) were washed in RPMI 1640 buffer and resuspended in 150 µl of RPMI 1640 containing 10 µg of plasmid. After 10 min incubation on ice, cells were electroporated using a Bio-Rad Laboratories, Inc. gene pulser (Ivry-sur-Seine, France) set at 250 V and 960 µF. Cells were then maintained on ice for 10 min and resuspended in complete medium.

For transient transfection assays with MMTV constructs, cells were cultured with 1 ng/ml IL-2 for 48 h before transfection. After electroporation, cells were stimulated with DEX (100 nM), IL-2 (1 ng/ml), IL-2 plus DEX, or left untreated. After 12 h of incubation, proteins were extracted and assayed for luciferase activity.

Selection of stably transfected cells was initiated 48 h after electroporation using 800 µg/ml G418 (Life Technologies, Inc.) for the cotransfections with the pMC1 plasmid (conferring resistance to neomycin) or 800 µg/ml hygromycin B (Life Technologies, Inc.) for the cotransfections with the pTK-hygromycin plasmid encoding an hygromycin resistance gene (CLONTECH Laboratories, Inc. Palo Alto, CA). Stably transfected CTLL-2 pLTR cells were selected by a 2-week treatment with G418 and cloned by limiting dilution. Clones were then screened for their GC-stimulated luciferase activity.

Development of a stable tTA (tetracycline transactivator) cell line was initiated by cotransfection of CTLL-2 cells with the plasmid pUHD 15–1 (33) and with pMC1, encoding a neomycin resistance gene. Stably transfected cells were selected in the presence of 800 µg/ml G418 for 2 weeks, after which CTLL-2 Tet-Off cells stably expressing the tTA were transfected with the plasmids pTRE-Myc-STAT5B or pTRE-Myc-STAT5B Y699F, and with pTK-hygromycin (CLONTECH Laboratories, Inc.). Cells were cultured in the presence of 1 µg/ml tetracycline and 800 µg/ml hygromycin. Expression of the fusion proteins Myc-STAT5B or Myc-STAT5B Y699F was turned on by removal of tetracycline for 48 h, and analyzed by Western blotting using the 9E10 anti-Myc antibody.

Reporter Gene Activity Assays
Luciferase Assay.
Luciferase levels were measured according to the manufacturer’s protocol (Promega Corp.). Briefly, extracts were prepared by three cycles of freezing and thawing of cells resuspended in a lysis buffer containing 25 mM Tris-phosphate, pH 7.8, 2 mM CDTA, 2 mM dithiothreitol (DTT), and 10% glycerol. Protein extracts in equivalent protein concentration samples were mixed with 100 µl of luciferase assay reagent (Promega Corp.). Luciferase activity was determined at 25 C after 1 min with a luminometer (LKB Wallac, Inc. Turku, Finland). Results are expressed in relative luciferase units (RLU) relative to the basal level to which the value 1 was arbitrarily affected. Fold induction was calculated as the ratio of arbitrary luciferase units in cells treated with IL-2, DEX, or DEX and IL-2 compared with untreated cells. Fold induction (DEX-treated cells) = (DEX activity - basal activity)/basal activity.

Fold induction (DEX + IL-2-treated cells) = (DEX + IL-2 activity - IL-2 activity)/basal activity.

CAT Assay.
Extracts were prepared by three cycles of freezing and thawing of cells resuspended in hypotonic buffer (0.25 M Tris HCl, pH 8). Protein extracts (40 µg) were incubated with (14C)-chloramphenicol (60 mCi/mmol, Amersham Pharmacia Biotech, Orsay, France) in the presence of 2 mM acetyl coenzyme A (Sigma) for 1 h at 37 C. Acetylated chloramphenicol was extracted in ethyl acetate and separated from unmodified chloramphenicol by TLC. Conversion of chloramphenicol was quantified using a Storm 840 phosphorimager and the Imagequant software (Molecular Dynamics, Inc., Sunnyvale, CA). Percentage of chloramphenicol conversion represents the ratio between acetylated chloramphenicol and total chloramphenicol (acetylated and nonacetylated).

Preparation of Nuclear Extracts
Nuclear extracts were prepared by a modification of the method described by Dignam et al. (56). Cells were deprived of IL-2 for 3 h and stimulated at 37 C for 1 h with DEX, IL-2, or DEX plus IL-2. Cells were then pelleted, washed with ice-cold 1x PBS, and maintained for 10 min on ice in a hypotonic buffer containing 10 mM HEPES, pH 7.8, 15 mM KCl, 2 mM MgCl2, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 mM DTT. Cytoplasmic membranes were lysed by 50 strokes using a Kontes all-glass Dounce homogenizer (B type pestle). The lysate was centrifuged at 1,000 x g for 5 min at 4 C, and the nuclear pellet was resuspended in a high-salt buffer (20 mM HEPES, pH 7.8, 1.5 mM MgCl2, 0.2 mM EDTA, 25% (vol/vol) glycerol, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 mM DTT, 400 mM NaCl). Nuclear extracts were centrifuged at 15,000 x g for 20 min at 4 C.

EMSA
Oligonucleotides were purchased from Oligo Express (Paris, France). Complementary sequences were annealed at 80 C for 10 min and 65 C for 10 min and then were end-labeled using (32P)-ATP with T4 polynucleotide kinase (Life Technologies, Inc.) and used for EMSA after ethanol precipitation.

The 5'-TCTTTTGGAATTTATCCAAATCTTAT-3' probe was used for NF-1 binding. The 5'-ATCTTATGTAAATGCTTATGTAAACCAAGA- 3' probe was used for Oct binding. These probes correspond, respectively, to the NF-1 and Oct binding sites found in the MMTV promoter between positions -80 and -55 for NF-1, -61 and -32 for Oct-1. The GAS probe from the GAS site of the Fc{gamma}R promoter (5'-GTATTTCCCAGAAAAGGAAC-3') was used for STAT5 binding, and the STAT/MMTV probe (5'-ATCTAAACAATTCGGAGAACTCGACCTTC-3') corresponds to the STAT response element located between positions -923 and -914 of the MMTV promoter. Specificity was determined by using a 25-fold molar excess of cold probe or random probe (5'-CCTCCATGACTCCAGAACTAACCTCCATGAC-3').

End-labeled oligonucleotides were incubated at 25 C for 30 min with 15 µg of nuclear proteins in the presence of 1 µg of sonicated salmon sperm DNA in 20 µl of binding buffer (12% glycerol, 12 mM HEPES, pH 7.8, 60 mM KCl, 1 mM EDTA, and 1 mM DTT). Protein-DNA complexes were separated from free probe on a 5% polyacrylamide gel in 0.5x TBE running buffer at 200 V. For supershift experiments, 3 µg of the control IgG or of the specific anti-Oct-1 IgG (C-21, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), or 1.5 µl of the anti-STAT5A and anti-STAT5B sera were preincubated with nuclear extracts for 2 h at 4 C before addition of the probe.

Production and DNA Binding Activity of Recombinant GR
The recombinant rat GR was produced using the TNT Quick coupled transcription/translation system as recommended by the manufacturer (Promega Corp.). Briefly, 1 µg of pET30rGR (containing the rat GR gene under the control of the T7 promoter) was added to an aliquot of TNT Quick master mix and incubated in a 50 µl reaction volume for 90 min at 30 C. The synthesized GR was then activated for 1 h at 4 C and 1 additional hour at 30 C in a buffer containing 10 mM HEPES, pH 7.4, 20 mM ß-mercaptoethanol, 5% glycerol, 50 mM NaCl, and 1 µM DEX. The 5'-ATCTCTGCAGAACAGGATGTTCTAGCTACTT-3' probe was used for GR DNA binding. Specificity was determined by using a 50-fold molar excess of cold probe or random probe. End-labeled GRE oligonucleotides were incubated at 25 C for 1 h with 2 µl of activated lysate in the presence of 1 µg of sonicated salmon sperm DNA in 20 µl of binding buffer (12% glycerol, 12 mM HEPES, pH 7.8, 60 mM KCl, 1 mM EDTA, and 1 mM DTT). For competition experiments, 1 µg, 2.5 µg, or 5 µg of nuclear extracts from CTLL-2 cells deprived of IL-2 for 3 h and treated or not with IL-2 for 1 h were incubated with the activated lysate. For supershift experiments, 1 µg of the control IgG or of the specific anti-GR (M-20, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were preincubated with GR for 30 min at 4 C before addition of the probe.

DNA Affinity Precipitation of STAT Proteins
Cells were deprived of IL-2 for 2 h and treated with IL-2 (500 pg/ml) for 12 h at 37 C. Cells were then collected by centrifugation, washed in 1x PBS, and resuspended in NP40 buffer (50 mM Tris HCl, pH 8, 0.5% NP40, 150 mM NaCl, 0.1 mM EDTA, 10 mM NaF, 1 mM PMSF, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 mM DTT). The double-stranded 5'-biotinylated oligonucleotide GAS was coupled to streptavidin-agarose beads (Sigma) for 1 h at 4 C. Whole-cell extracts were then incubated with the precoated beads for 1 h at 4 C. The beads were then washed three times with the NP40 lysis buffer and boiled in reducing sample buffer to elute the complexes. Bound proteins were then separated on 8% polyacrylamide gel and electroblotted onto Amersham Pharmacia Biotech PVDF (polyvinylidene difluoride) membranes. Western blot analysis was performed with the specific anti-STAT5B antibody (ref 06–969, Upstate Biotechnology, Inc. Lake Placid, NY).

Coimmunoprecipitation Assays
CTLL-2 cells were deprived of IL-2 for 3 h and then stimulated for 1 h with IL-2 (1 ng/ml) and/or DEX (100 nM), or left untreated. Cell lysates were first incubated for 30 min at 4 C with protein A sepharose beads (Sigma) and preimmune serum and then centrifuged at 4,000 rpm for 1 min. The supernatant (precleared lysate) was incubated overnight at 4 C with the anti-GR antibody (BuGR2, Affinity BioReagents, Inc., Golden, CO) precoupled to Protein A sepharose beads. Immune complexes were washed three times with lysis buffer and analyzed by SDS-PAGE using anti-STAT5 antiserum.

Western Blot
Cells were collected by centrifugation, washed in 1xPBS, and resuspended in NP40 buffer (50 mM Tris HCl, pH 8, 0.5% NP40, 150 mM NaCl, 0.1 mM EDTA, 10 mM NaF, 1 mM PMSF, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 mM DTT). Cell lysates were resolved by SDS-PAGE on 8% polyacrylamide gels and electroblotted onto Amersham Pharmacia Biotech PVDF membranes. After saturation of nonspecific binding sites with dry low-fat milk in TBS-Tween 20 (0.2%) for 2 h, membranes were probed with the monoclonal 9E10 anti-Myc monoclonal antibody and developed with ECL (Amersham Pharmacia Biotech).


    ACKNOWLEDGMENTS
 
The authors specially acknowledge Karine Andréau and Marie-Liesse Asselin for helpful discussion and participation in some of the experiments. The authors thank Michel Renoir, Véronique Marsaud, and José Luis-Zugaza for critical reading of this manuscript. The authors thank Fabrice Gouilleux for the kind gift of RSV-STAT5B plasmid and anti-STAT5A and STAT5B sera, Sylvie Mader for the pGRE5-EBV-TATA-CAT plasmid, Bernard Binetruy for the 5XTRE-tk-CAT plasmid, Annick Harel-Bellan for the pCMV-2N3T and pCMV-2N3T-CBP plasmids, Sophia Tsai for the pCR3.1 and pCR3.1-SRC-1a plasmids, and Josiane Pierre for making available the tet-system components (pUHD 15–1, pUHD 10–3, and pUHC 13–3 plasmids) of Hermann Bujard (Zentrum für Molekulare Biologie der Universität Heidelberg). We thank Sophie Amsellem for raising the anti-STAT5 antibody and gratefully acknowledge the technical assistance of Sophie Gruel and Chantal Broch.


    FOOTNOTES
 
Address requests for reprints to: Marc Pallardy, INSERM U461, 5 rue Jean-Baptiste Clément, 92296 Châtenay-Malabry cedex, France. E-mail: marc.pallardy{at}cep.u-psud.fr

This research was supported by INSERM and by a fellowship from the Association pour la Recherche sur le Cancer to Armelle Biola.

1 Present address: INSERM, 101 rue de Tolbiac, 75654 Paris, France. Back

Received for publication June 9, 2000. Revision received March 7, 2001. Accepted for publication March 12, 2001.


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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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