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The Journal of Immunology, 1998, 161: 1803-1810.
Copyright © 1998 by The American Association of Immunologists

Cyclosporin A-Resistant Transactivation of the IL-2 Promoter Requires Activity of Okadaic Acid-Sensitive Serine/Threonine Phosphatases1

Gabriele Nebl2, Stefan C. Meuer and Yvonne Samstag

Institute for Immunology, Ruprecht-Karls-University, Heidelberg, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Expression of the IL-2 gene requires activation of T cells through stimulation of the TCR and costimulation through accessory receptors. We have found recently that okadaic acid-sensitive Ser/Thr phosphatases are involved in a cyclosporin A-insensitive pathway that selectively transmits costimulatory signals. In this study, we analyzed whether activities of these phosphatases are necessary for the expression of the IL-2 gene. In both activated peripheral blood T lymphocytes and activated tumorigenic T cell lines, IL-2 gene expression was blocked at the transcriptional level by okadaic acid. The transcription factors active at the IL-2 promoter were differentially influenced: upon down-modulation of okadaic acid-sensitive phosphatases, transactivation by octamer, NF-{kappa}B, and NF of activated T cells proteins was abrogated, while transactivation by AP-1 proteins was even enhanced.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Full activation of T lymphocytes requires two types of signals: those provided through the TCR/CD3 complex, and in addition, those induced by stimulation of coreceptors such as CD2, CD4, CD8, or CD28 (for review, see 1 . T cell activation in the absence of costimulation leads to anergy or apoptosis (2, 3, 4, 5). Signaling events induced by coreceptors overlap in part with the TCR signaling pathway. Recently, intracellular reactions clearly independent of the TCR have been identified (6, 7).

Expression of the IL-2 gene depends on an activation of both coreceptor- and TCR-triggered signal-transduction pathways (for review, see 8 . These influence the activities of the transcription factors that mediate IL-2 transcription, namely Oct,3 AP-1, NF-{kappa}B, and NF-AT proteins (for review, see 9 . Protein kinase C is involved in the activation of the NF-{kappa}B transcription factor proteins, which requires their release from inactive cytoplasmic complexes (10, 11, 12, 13). Members of the mitogen-activated protein kinase family regulate expression and activity of AP-1 proteins (14, 15, 16). The Ca/calmodulin-regulated Ser/Thr phosphatase 2B (PP2B)/calcineurin regulates the nuclear entry of the cytoplasmic components of the transcription factor NF-AT (17, 18, 19).

Additional phosphatases, namely okadaic acid-sensitive Ser/Thr phosphatases, were recently identified to be crucial for a cyclosporin A (CsA)-insensitive pathway, which is induced through the CD2 and CD28 coreceptors (6, 20, 21). Ser/Thr phosphatases are encoded by the gene families PPM and PPP. The PPP family comprises the subfamilies 1, 2A, 2B, and 5. The phosphatases of subfamilies 1, 2A, and 5 are sensitive to okadaic acid (22), while PP2B is inhibited by CsA.

During the CD2/CD28-induced signaling pathway, dephosphorylation of the essential phosphoprotein pp19/cofilin is catalyzed by okadaic acid-sensitive Ser/Thr phosphatases. Since dephosphorylation of pp19/cofilin correlates with IL-2 production, we addressed the question as to whether okadaic acid-sensitive Ser/Thr phosphatases influence the expression of the IL-2 gene. In this study, we show that indeed activities of okadaic acid-sensitive phosphatases are necessary to achieve transactivation of the IL-2 promoter by the NF-AT, NF-{kappa}B, and Oct transcription factor proteins.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines, tissue culture, and transfection procedure

Human primary T cells were prepared as described previously (21). EL4 cells and Jurkat cells were grown in RPMI supplemented with 5% (EL4) or 10% (Jurkat) FCS (Sigma, St. Louis, MO) and 1% penicillin/streptomycin (Life Technologies, Gaithersburg, MD). The Jurkat cell clone IL-2-luc stably transfected with the plasmid pIL-2luc2kb (23) was a gift from B. Schraven Institute for Immunology, Heidelberg, Germany). EL4 cells were transfected, as described previously (24), using 1 µg of plasmid DNA per 2 x 106 cells. The constructs used were pmoIL2–2k-luc, pmoIL2–321-luc, and the control vector pUC00luc; octp-luc, NF{kappa}B-luc, NFAT-luc, and the control vector pGL2 (25); 5x TRE-TATA CAT and the control vector TATA CAT (26); and 4x oct-luc and the control vector 4x oct mut-luc (27). Sixteen hours after transfection, cells were split in aliquots and treated with various stimulating agents. The concentrations used were 0.5 µM okadaic acid (Life Technologies); 0.1 µg/ml CsA (Sandoz, Basel, Switzerland); 10 ng/ml PMA (Sigma); 180 nM A23187 (Sigma); 10 µg/ml CD2 Abs M1, M2, and 3PT (28, 29); and 1 µg/ml CD28 Ab 9.3 (PharMingen, San Diego, CA).

Luciferase assay and CAT assay

Luciferase assays were performed with the Luciferase Assay System (Promega, Madison, WI). CAT assays were conducted using CAT ELISA (Boehringer Mannheim, Indianapolis, IN).

Isolation of RNA and Northern analysis

RNA was extracted from cells by a guanidinium isothiocyanate method. Before loading on a 1% agarose gel (1% in 1x MOPS; 1x MOPS equals 20 mM MOPS (3-[N-morpholino]propanesulfonic acid, 5 mM sodium acetate, 1 mM EDTA)), RNA samples were adjusted to 25% formamide, 1.1 M formaldehyde, 0.5x MOPS, 0.5% Ficoll, and 0.1% bromphenol blue. After electrophoresis, gels were blotted onto Hybond N+ filters (Amersham, Arlington Heights, IL). Prehybridization and hybridization were conducted at 65°C in solution HS (7% SDS, 1 mM EDTA, 0.5x phosphate buffer (1x phosphate buffer equals 2 M Na2HPO4·2H2O, 0.34% phosphoric acid)). For hybridization, cDNA probes (ß-actin, human IL-2, murine IL-2 (30, 31, 32)) were labeled radioactively using a random hexanucleotide priming procedure (Stratagene, La Jolla, CA). Blot washes (at 65°C) were performed with 0.04x phosphate buffer, 1 mM EDTA, 5% SDS, and 0.04x phosphate buffer, 1 mM EDTA, 1% SDS. Finally, the blots were exposed to autoradiography.

Nuclear run-on analysis

Nuclear run-on analysis was performed, as described previously (33), using cDNA probes for the ß-actin gene, the IL-2 gene, the c-jun gene, and the c-fos gene (30, 31, 34, 35) for the detection of the respective transcripts.

In vivo footprinting

In vivo footprinting was performed essentially as described (36). The oligonucleotide primers used to visualize the coding strand of the c-fos promoter were synthesized according to a previous report (37). Quantitation of band intensities was performed through scanning of the autoradiograms using a Saphir Ultra laser densitometer. From top to bottom of each lane of the autoradiogram, an intensity profile was produced using the Image Quant 3.0 software (Fuji). Subsequently, the intensity values of individual profile peaks were determined. Intensity values of affected bands were related to the intensity value of a nonaffected band in the same area of the gel to compensate for loading differences (relative intensity values). For graph representation, relative intensity values obtained from an in vitro methylated DNA band were set 1, and the relative intensity values of corresponding bands in the four in vivo DNA preparations were related to it. G-77 of the Oct site, G-84 of the OAP site, G-138 and G-140 of the NF-AT site, and G-152 and G-154 of the AP-1 site were analyzed as individual bands, while G-173 and G-174 of the ATGG site, G-205, G-206, and G-207 of the NF-{kappa}B site, and G-224 and G-225 of the TGGGC site were pooled with neighboring bands. At the ATGG, NF-{kappa}B, and TGGGC sites, in vivo methylated DNAs are hyperreactive when compared with in vitro methylated DNA (Fig. 7Go). This situation occurring at the coding strand of the IL-2 promoter has been described earlier. Its significance remains unknown (36).



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  		FIGURE 7. Determination of the in vivo occupancy of the IL-2 promoter in EL4 cells. In vivo methylated genomic DNA was isolated from EL4 cells cultured in medium (lane 2, black bars), treated with okadaic acid (lane 3, bars striped longitudinal), or stimulated with PMA/A23187 for 6 h in the absence (lane 4, bars striped diagonal) or presence (lane 5, cross-striped bars) of okadaic acid. Okadaic acid was added 30 min before stimulation. The coding strand of the IL-2 promoter was footprinted from in vivo methylated DNA preparations (lanes 2–5) and in vitro methylated DNA from untreated EL4 cells (lane 1, bars shaded) using the ligation-mediated PCR protocol. The experiment is representative of three repetitions. On the left, nucleotide positions relative to the transcriptional start point are shown. In the middle, hypersensitivities (open arrows) and protections (filled arrows) that have been reported to occur at the IL-2 promoter (36) as well as the corresponding binding transcription factors (NF-AT, TGGGC, NF-{kappa}B, ATGG, AP-1, NF-AT, OAP, Oct) are marked. Densitometric analysis of the autoradiogram is presented on the right-hand side. Relative intensity values of the bands were determined, as described in Materials and Methods. For graph representation, relative intensity values derived from in vivo methylated DNAs were normalized to that from in vitro methylated DNA, which was set 1. Densitometric analysis of the protected G-285 and the hypersensitive G-287 at the distal NF-AT site was impaired by the weak resolution on top of the gel.

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Okadaic acid prevents IL-2 expression in human peripheral blood T lymphocytes

Activation of human peripheral blood T lymphocytes (PBL-T) was performed using a combination of mitogenic Abs directed at the CD2 coreceptor or, alternatively, by phorbolester and Ca ionophore (PMA + A23187). CD2 triggering provides an activation signal to T cells (28, 38). Phorbolesters induce the activation of protein kinase C, whereas Ca ionophores lead to an elevation of intracellular calcium. This treatment mimics intracellular signals induced by antigenic stimulation of T cells via TCR/CD3 and coreceptors (39).

As expected, Northern blot analysis showed that both stimulation protocols induced the accumulation of IL-2 mRNA (Fig. 1Go, lanes 3 and 5), which was prevented completely when okadaic acid was added 30 min before stimulation at concentrations known to inhibit the okadaic acid-sensitive Ser/Thr phosphatases of subfamilies 1, 2A, and 5 (22, 27, 40, 41) (Fig. 1Go, lanes 4 and 6). This treatment did not generally affect the functional activity of the cells, since a nuclear run-on analysis (see below, Fig. 2Go) revealed that other transcriptional processes were ongoing throughout cell treatment.



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  		FIGURE 1. Okadaic acid prevents IL-2 mRNA expression in PBL-T. Northern blot analysis was performed with RNA isolated from PBL-T. Cells were cultured in medium (lane 1) or treated with okadaic acid (lane 2), mitogenic CD2 Abs (lane 3), okadaic acid and CD2 Abs (lane 4), PMA plus A23187 (lane 5), or okadaic acid and PMA plus A23187 (lane 6). Okadaic acid was added 30 min before the stimulating agents; cells were then triggered for 6 h. Filters were hybridized with radioactively labeled cDNA probes for the human IL-2 gene and the ß-actin gene.

 


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  		FIGURE 2. Okadaic acid blocks IL-2 transcription in PBL-T. Transcription of the IL-2, c-fos, c-jun, and ß-actin genes in PBL-T was determined by nuclear run-on analysis. Cells were incubated with medium (lanes 1 and 5) or treated with okadaic acid (lanes 2 and 6). For activation, cells were stimulated for 6 h via CD2 triggering (lanes 3 and 4) or PMA/A23187 treatment (lanes 7 and 8), each in the absence (lanes 3 and 7) or presence (lanes 4 and 8) of okadaic acid. Okadaic acid was added 30 min before stimulation. IL-2, c-fos, c-jun, and ß-actin transcripts were detected with isolated cDNA probes from the respective genes.

 
These results show that inhibition of okadaic acid-sensitive phosphatases abrogates the expression of the IL-2 gene in activated PBL-T cells.

Okadaic acid blocks IL-2 expression at the transcriptional level

We then analyzed the mechanisms underlying the inhibition of IL-2 expression by okadaic acid. Specifically, we addressed the question as to whether okadaic acid treatment results in mRNA destabilization and/or reduced mRNA synthesis. To discriminate between these possibilities, the transcription rate of the IL-2 gene was determined in nuclear run-on experiments (Fig. 2Go).

To this end, PBL-T cells were activated through CD2 or by treatment with PMA + A23187, in the presence or absence of okadaic acid. Both CD2 triggering and PMA + A23187 treatment induced transcription of the IL-2 gene (Fig. 2Go, lanes 3 and 7, IL-2), which was inhibited by okadaic acid (Fig. 2Go, lanes 4 and 8, IL-2). This effect was not due to a general down-regulation of transcriptional processes by okadaic acid, since the ß-actin gene was transcribed at similar levels in both groups of differently treated cells (Fig. 2Go, ß-actin). Moreover, transcription of the AP-1 transcription factors c-Fos and c-Jun, detectable in resting and activated PBL-T cells (Fig. 2Go, lanes 5 to 8), was even strongly induced by okadaic acid (Fig. 2Go, lanes 6 and 8).

To further elucidate the mechanisms underlying okadaic acid-mediated inhibition of IL-2 transcription, we performed IL-2 promoter studies using transient transfections of reporter gene constructs or the in vivo footprinting technique. Since PBL-T cells could not be used due to their limited experimental manipulability, the tumorigenic murine T lymphoma line EL4 and the human T cell line Jurkat were employed.

As shown before in PBL-T cells, the high level of IL-2 mRNA expression detectable in PMA + A23187-activated cells (Fig. 3Go, lanes 3 and 7) was not induced in Jurkat and EL4 cells when the cells were activated in the presence of okadaic acid (Fig. 3Go, lanes 4 and 8). Thus, the effects of okadaic acid on IL-2 expression are independent of tumorigenic transformation and the differentiation state of the T cell lines used in this study.



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  		FIGURE 3. Okadaic acid inhibits IL-2 mRNA expression in the tumorigenic cell lines Jurkat and EL4. Northern blot analysis was performed with RNA isolated from differently treated Jurkat (lanes 1–4) or EL4 (lanes 5–8) cells. Cells were cultured in medium (lanes 1 and 5) or treated with okadaic acid (lanes 2 and 6). For stimulation, cells were incubated for 6 h with PMA/A23187 in the absence (lanes 3 and 7) or presence (lanes 4 and 8) of okadaic acid. Okadaic acid was added 30 min before stimulation. For detection of transcripts, cDNA probes of the human IL-2 gene (Jurkat cells), the murine IL-2 gene (EL4 cells), and the ß-actin gene were used.

 
In line with a previous report (42), okadaic acid treatment of nonstimulated EL4 cells alone induced a low level of IL-2 expression (Fig. 3Go, lane 6). This effect seems to be peculiar to EL4 cells since it was observed neither in PBL-T cells (compare Fig. 1Go, lane 2) nor in Jurkat cells (Fig. 3Go, lane 2). Intriguingly, although okadaic acid treatment alone induced IL-2 expression in EL4 cells, this compound completely prevented IL-2 expression in EL4 cells following stimulation with PMA + A23187 (Fig. 3Go, lane 8).

Similar results were obtained upon transient transfection of EL4 cells with IL-2 promoter reporter constructs, in which the luciferase gene is driven by the murine IL-2 promoter. Two IL-2 promoter luciferase constructs containing either the 321-bp IL-2 enhancer (pmoIL2–321-luc) or 2-kb upstream sequence of the IL-2 gene (pmoIL2–2k-luc) and a corresponding control vector were used (25). The transfected cells were cultured with medium (-), okadaic acid (OA), PMA + A23187 (P/A), or a combination of the three agents (P/A + OA), respectively. In EL4 cells transfected with the control vector, only very low levels of luciferase activity were detected, which were not altered significantly by cell treatment (Fig. 4Go, vector). Irrespective of which of the two IL-2 promoter luciferase constructs was transfected (Fig. 4Go, -2k, -321), luciferase activity was slightly induced by okadaic acid (Fig. 4Go, OA) and, to a much higher level, by PMA + A23187 (Fig. 4Go, P/A). Addition of okadaic acid completely inhibited the PMA + A23187-inducible activity of both IL-2 promoter constructs (Fig. 4Go, P/A + OA). Taken together, these data show that IL-2 transcription in transformed murine and human T cell lines as well as in primary human blood T cells is influenced strongly by okadaic acid-sensitive Ser/Thr phosphatases.



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  		FIGURE 4. Okadaic acid prevents IL-2 transcription in EL4 cells. EL4 cells were transiently transfected with a control vector (00-luc) or two reporter plasmids expressing the luciferase gene under the control of fragments of the murine IL-2 promoter. -321-luc contains the 321 bp of the IL-2 enhancer, and -2k-luc contains the 2 kb upstream sequence of the IL-2 gene. Thirty-six hours after transfection, cells were split into four aliquots. Cells were cultured in medium (-), treated with okadaic acid (OA), or stimulated for 6 h with PMA/A23187 in the absence (P/A) or presence (P/A + OA) of okadaic acid. Okadaic acid was added 30 min before stimulation. Luciferase activities of lysates from the different cell populations were measured and normalized to the amount of protein in each probe. The figure shows the normalized luciferase activities expressed as multiples of the value measured in untreated cells, which was set 1. The data reflect results of five independent experiments. Error bars indicate SE of the mean.

 
Okadaic acid inhibits a CsA-insensitive pathway of IL-2 expression

Like okadaic acid, the PP2B/calcineurin inhibitor CsA prevents IL-2 expression via a transcriptional block (43). To exclude the possibility that the blocking activity of okadaic acid on IL-2 transcription was mediated via inhibition of PP2B/calcineurin, we analyzed whether the PP2B/calcineurin-independent pathway of IL-2 expression induced by a combination of PMA and the activating CD28 Ab 9.3 (44) could also be inhibited by okadaic acid.

To investigate this point, a Jurkat cell clone stably transfected with the IL-2 enhancer luciferase reporter construct pIL-2luc2kb (23) was stimulated by a combination of PMA plus CD28 Ab. This resulted in an induction of luciferase activity, while treatment with each agent alone led to no significant change in the basal level of luciferase activity (Fig. 5Go, compare lanes 1, 4, 5, and 6). Treatment with okadaic acid, but not with CsA, completely abrogated the PMA/anti-CD28-inducible promoter activity (Fig. 5Go, lanes 7 and 8). In contrast, luciferase activity, induced by a combination of phorbolester and Ca ionophore (Fig. 5Go, lane 9), was prevented completely by both okadaic acid and CsA (Fig. 5Go, lanes 10 and 11). These data show that okadaic acid inhibits a PP2B/calcineurin-independent pathway involved in IL-2 expression.



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  		FIGURE 5. Okadaic acid inhibits a CsA-insensitive pathway of IL-2 expression. Cells from the Jurkat clone Jurkat-IL-2-luc stably transfected with a IL-2 promoter reporter construct were cultured in medium (lane 1, -), or treated with okadaic acid (lane 2, OA), CsA (lane 3, CsA), PMA (lane 4, P), the CD28 Ab 9.3 (lane 5, 28), or with combinations of PMA plus CD28 Ab (lanes 6–8, P/28) or PMA plus A23187 (lanes 9–11, P/A). Stimulations via P/28 or P/A treatment were performed in the absence of OA and CsA (lanes 6 and 9) or in the presence of OA (lane 7, P/28 + OA, and lane 10, P/A + OA) or CsA (lane 8, P/28 + CsA, and lane 11, P/A + CsA). Okadaic acid and CsA were added 30 min before stimulation; cells were then triggered for 6 h. Luciferase activities of lysates from the different cell populations were measured and normalized to the amount of protein in each probe. The figure shows the normalized luciferase activities expressed as multiples of the value measured in untreated cells, which was set 1. The data reflect results of four independent experiments. Error bars indicate SE of the mean.

 
Oct-, NF-{kappa}B-, NF-AT-, but not AP-1-mediated transcription depends on okadaic acid-sensitive Ser/Thr phosphatases

To determine whether okadaic acid differentially influences the transactivation by individual transcription factors regulating IL-2 promoter activity, luciferase reporter constructs driven by multimeric NF-AT-, NF-{kappa}B-, or Oct-binding elements derived from the IL-2 promoter (25) or the respective control vector were transfected into EL4 cells. After transfection, cells were treated with okadaic acid (OA), PMA + A23187 (P/A), or okadaic acid and PMA + A23187 (P/A + OA), respectively. In the case of okadaic acid treatment of EL4 cells, predominantly the Oct reporter was stimulated, whereas the extent of NF-{kappa}B- and NF-AT-reporter induction was very low (Fig. 6GoA, OA). PMA + A23187 treatment activated the Oct-, NF-{kappa}B-, and NF-AT-reporter constructs (Fig. 6GoA, P/A), while application of okadaic acid inhibited the PMA + A23187-inducible promoter activities of all three constructs (Fig. 6GoA, P/A + OA).



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  		FIGURE 6. Okadaic acid-sensitive Ser/Thr phosphatases differentially influence individual transcription factors. A, EL4 cells were transfected with reporter constructs containing multimerized Oct-, NF-{kappa}B-, or NF-AT-binding elements derived from the murine IL-2 promoter or a control vector. After transfection, treatment of the cells and determination of luciferase activities were performed, as described in Figure 4Go. B, For analysis of Oct-specific transactivation, the Oct-reporter 4x oct-luc containing a multimerized Oct consensus site (Oct (cons.)) was transfected into EL4 cells. Cells were treated as described above. C, EL4 cells were transfected with the AP-1 reporter 5x TRE-TATA-CAT containing the AP-1 site from the human collagenase gene (AP-1 (coll.)) or a control vector. After transfection, cells were treated as described before (see Fig. 4Go). CAT activities in lysates of the different cell populations were determined and normalized to the amount of protein in each probe. In lysates from cells transfected with the control vector and untreated cells transfected with the AP-1 reporter, no CAT activities were detected. The normalized CAT activities in the different cell populations are therefore shown as multiples of the value measured in P/A-activated cells, which was set 1. The data reflect results of three independent experiments. Error bars indicate SE of the mean.

 
The Oct and NF-AT reporters used in Figure 6GoA contain the composite binding elements of the IL-2 promoter that allow recruitment of AP-1 proteins together with Oct and NF-AT proteins (45, 46). In addition Oct- and AP-1-dependent transcription was analyzed using Oct- and AP-1-specific reporter constructs (Fig 6Go, B and C). The Oct reporter used contains a multimerized octamer motif (27). It had a high constitutive activity in nonactivated cells (Fig. 6GoB, -) that was not further inducible by PMA + A23187 (Fig. 6GoB, P/A). Okadaic acid treatment strongly reduced this activity both in nonactivated and activated cells (Fig. 6GoB, P/A + OA).

The AP-1 reporter contains a multimerized AP-1 binding site derived from the human collagenase gene (26). The AP-1 reporter was induced by okadaic acid (Fig. 6GoC, OA) or PMA + A23187 treatment (Fig. 6GoC, P/A), respectively. In contrast to the results obtained with the Oct-, NF-{kappa}B-, and NF-AT-reporter constructs, simultaneous addition of okadaic acid and PMA + A23187 further enhanced the activity of the AP-1 reporter (Fig. 6GoC, P/A + OA). These observations imply that the inhibition of the Oct and NF-AT reporters derived from the IL-2 promoter sequence (Fig. 6GoA) is mediated by repression of Oct and NF-AT activity, and not by inhibition of AP-1.

Determination of the in vivo occupancy of the IL-2 promoter in EL4 cells

To analyze the events at the endogenous IL-2 promoter that are influenced by okadaic acid treatment, we used the in vivo footprinting technique by which protein-DNA interactions in intact cells can be visualized. To this end, EL4 cells were treated in culture with the membrane-permeable alkylating agent DMS. After DMS treatment, piperidine was used to produce DNA cleavage at alkylated G residues. Resulting fragments spanning the IL-2 promoter region were visualized by the ligation-mediated PCR technique (47), giving a G-specific sequence ladder of the promoter. DMS footprints (i.e., protected or hypersensitive G residues) indicative of protein binding to the DNA were revealed by comparing the patterns of G-specific sequence ladders obtained from differently stimulated living EL4 cell populations with those obtained from purified and in vitro methylated genomic DNA of EL4 cells. PMA + A23187 treatment of EL4 cells resulted in a change of the pattern of the G-specific sequence ladder as compared with the one detected in unstimulated cells (Fig. 7Go, compare lanes 2 and 4). Footprints (i.e., reproducible changes of band intensities of at least 25%), as described before (36), were visible within the coding strand at the Oct site (protection at G-77), the OAP site (protection at G-84), the proximal NF-AT site (protection at G-138 and hypersensitivity at G-140), the AP-1 site (hypersensitivities at G-152 and G-154), the ATGG site (protections at G-173 and G-174), the NF-{kappa}B site (protections at G-205, G-206, and G-207), the TGGGC site (protections at G-224 and G-225), and the distal NF-AT site (protection at G-285 and hypersensitivity at G-287) (Fig. 7Go, lane 4). Okadaic acid treatment of EL4 cells alone reproducibly induced a protection at G-77 within the Oct site, at G-84 within the OAP site, and two hypersensitivities at G-152 and G-154 indicative of protein binding to the AP-1 site of the IL-2 promoter (Fig. 7Go, lane 3). This observation correlated with the induction of low levels of IL-2 expression under these conditions (compare Fig. 3Go and 42 as well as with the induction of AP-1 expression (compare Fig. 2Go) and activity (compare Fig. 6GoC) in okadaic acid-treated cells. Surprisingly, binding of Oct and AP-1 proteins seemed to be sufficient to induce a low level of IL-2 transcription.

The IL-2 promoter of cells treated with PMA + A23187 in the presence of okadaic acid showed no footprints at all (Fig. 7Go, lane 5). Thus, in PMA + A23187 plus okadaic acid-treated EL4 cells also AP-1 proteins, although activated (compare Fig. 6GoC, P/A + OA), do not bind to the IL-2 promoter. The absence of footprints at the IL-2 promoter was gene specific since the cellular c-fos promoter was still occupied under these conditions. Thus, in vivo footprinting of the same DNA preparations, as used for the IL-2 promoter analysis, revealed the characteristic footprint pattern at the SRE binding region of the c-fos promoter (data not shown) (37).

These data demonstrate that okadaic acid completely prevents transcription factor binding to the IL-2 promoter in PMA + A23187-stimulated EL4 cells.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
IL-2 is produced by activated T cells. Its expression is regulated at the transcriptional level. In addition, posttranscriptional effects on IL-2 mRNA stability have been described (48). In this study, it is demonstrated for the first time that in activated T cells, the inhibitor of Ser/Thr phosphatases of the subfamilies 1, 2A, and 5, okadaic acid, prevents IL-2 expression through a transcriptional block. Okadaic acid treatment caused no general inhibition of transcription since the ß-actin, c-fos, and c-jun genes (Fig. 2Go) and AP-1-dependent reporter genes (Fig. 6GoC) were still transcribed. Therefore, neither RNA polymerase II nor the basic transcriptional machinery is affected directly by inhibition of these Ser/Thr phosphatases.

Interestingly, okadaic acid treatment differentially affects the potencies of individual transcription factors regulating IL-2 transcription. Thus, while okadaic acid inhibits NF-{kappa}B-, Oct-, and NF-AT-mediated transactivation processes during T cell activation, AP-1 activity is even enhanced.

Okadaic acid treatment enhances AP-1 activity through an induction of the expression of AP-1 proteins (Fig. 2Go; see also 49 . Unexpectedly, this AP-1 expression was still detectable at late times after activation. In both activated and nonactivated PBL-T cells, okadaic acid treatment resulted in a high transcription rate of AP-1 genes 6 h after stimulation (see Fig. 2Go). Okadaic acid treatment rapidly induces expression of the AP-1 genes c-fos, c-jun, junB, and junD in EL4 cells. This expression was still detectable 8 h after stimulation (unpublished data). This kinetics of the AP-1 expression differs from that under other experimental conditions of T cell activation, in which AP-1 expression occurs as an early event followed by a rapid shut-off (see Ref. 9 for summary). At present, the regulation of this shut-off is not well understood. One explanation for the AP-1 expression pattern in T cells described in this study could be that okadaic acid-sensitive phosphatases are also involved in the down-regulation of AP-1 expression.

In activated T cells, IL-2 transcription was inhibited completely by okadaic acid. AP-1 activity, which was enhanced under these conditions, was clearly not sufficient to overcome the inhibitory effect of okadaic acid on the IL-2 promoter. This could be based on the finding that IL-2 transcription seems to depend on the binding of a complete set of transcription factors to the promoter (36), most likely because the IL-2 promoter contains nonconsensus binding sites that favor cooperative transcription factor binding (24). Then, AP-1 proteins, although induced, could not transactivate the IL-2 promoter because their binding affinities toward the nonconsensus AP-1-binding elements would not be strong enough under conditions in which Oct, NF-{kappa}B, and NF-AT activities are missing. Indeed, in vivo footprinting analysis (Fig. 7Go) showed that the IL-2 promoter in okadaic acid-treated EL4 cells stimulated with PMA + A23187 is not occupied, indicating that transcription was blocked due to complete inhibition of activator binding. Moreover, no evidence for binding of repressor proteins to the IL-2 promoter was found.

In nonactivated EL4 cells, however, okadaic acid treatment led to low expression of IL-2. Note that this was the case only in EL4 cells, not in the other transformed and nontransformed T cells analyzed in this study. Okadaic acid-induced IL-2 expression in EL4 cells correlated with footprints only at the Oct site (G-77), at the noncomposite AP-1 site at nucleotides G-152 and G-154, and at the OAP site (G-84) that is bound by AP-1 proteins (46) (compare Fig. 7Go). Thus, in nonactivated EL4 cells, Oct and AP-1 proteins can induce transcription at the IL-2 promoter in the absence of other transcription factors.

One possibility to explain the opposite effects of okadaic acid on IL-2 transcription in activated and nonactivated EL4 cells could be that the accessibility of the IL-2 promoter is different. Since the binding of AP-1 proteins to the IL-2 promoter (Fig. 7Go) is distinct in activated and nonactivated EL4 cells while transactivating capacities of AP1 proteins are detectable in both states (Fig. 6GoC), perhaps this means that, comparable with the situation that is found at a growing number of cellular promoters (50, 51), the activity of the IL-2 promoter is regulated by its nucleosomal architecture as well. In this case, T cell activation would induce the establishment of an open form of IL-2 promoter packaging. The inhibition of okadaic acid-sensitive phosphatases during activation may either alter or disturb this process. At present, it is not clear whether, and if so how, okadaic acid treatment induces (in activated EL4 cells) or fixes (in other T cells) a promoter structure similar to the one in resting T cells that does not permit access of single transcription factors (36). Alternatively, simultaneous cell activation and okadaic acid treatment may establish a new inhibitory form of IL-2 promoter packaging. Taken together, the characteristic features of IL-2 transcription in EL4 cells indicate that besides transcription factor activity, changes of the chromatin structure are probably involved in the regulation of IL-2 expression in T cells.

The signaling pathway investigated in this study does not involve PP2B/calcineurin. Thus, inhibition of okadaic acid-sensitive Ser/Thr phosphatases abrogated both the PP2B/calcineurin-dependent and independent pathways of IL-2 expression (Fig. 5Go). The similarities of the effects of okadaic acid and the PP2B/calcineurin inhibitor CsA are, however, remarkable: loss of activator binding to the IL-2 promoter was also observed when cells were stimulated in the presence of CsA (36). CsA is known to block NF-AT activation and, in addition, to exert inhibitory effects on Oct and NF-{kappa}B proteins (17, 46, 52). As shown in this study, okadaic acid also inhibits NF-AT, Oct, and NF-{kappa}B activities (Fig. 6GoA). One conclusion from these observations is to predict a convergence of the pathways depending on okadaic acid-sensitive Ser/Thr phosphatases and on PP2B/calcineurin. Perhaps this occurs downstream of PP2B/calcineurin, since there is evidence that PP2B/calcineurin may activate the type 1 Ser/Thr phosphatase PP1 by inactivation of the PP1 inhibitor 1 (53).

The dependency of IL-2 transcription on okadaic acid-sensitive phosphatases is conserved during tumorigenic transformation and evolution, since the inhibitory effect of okadaic acid was observed in nontransformed primary human T cells as well as in malignant T cell lines from mice and humans. This stresses the importance of these phosphatases for the regulation of IL-2 expression. Future work should focus on the identification of the individual phosphatases and proteins targeted by them. In this regard, the essential actin-binding protein pp19/cofilin has been identified as one of the substrates of okadaic acid-sensitive phosphatases. Dephosphorylation and nuclear translocation of pp19/cofilin as a consequence of costimulatory signals correlate with the induction of IL-2 production (20). Whether effects of cofilin on the nucleoskeleton are involved in changes of the chromatin structure influencing transcriptional activation remains to be determined.


   Acknowledgments
 
We thank H. Kirchgessner and B. Schraven for the Jurkat clone IL-2-luc, T. Tsuruta and T. Yokota for the IL-2 promoter reporter constructs, P. Quehl for preparation of the CD2 antibodies, and H. König for critical reading of the manuscript.


   Footnotes
 
1 This work was supported by Deutsche Forschungsgemeinschaft (Grants DFG-Sa393/2-1 (to Y.S.) and SFB405/A4). Back

2 Address correspondence and reprints to Dr. Gabriele Nebl, Institute for Immunology, Ruprecht-Karls-University, Im Neuenheimer Feld 305, 69120 Heidelberg, Germany. E-mail address: Back

3 Abbreviations used in this paper: Oct, octamer; AP-1, activator protein-1; CAT, chloramphenicol acetyl transferase; CsA, cyclosporin A; NF-AT, nuclear factor of activated T cells; NF-{kappa}B, nuclear factor-{kappa}B; OAP, octamer-1-associated protein; PBL-T, peripheral blood T lymphocytes. Back

Received for publication December 19, 1997. Accepted for publication April 17, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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