By
From the * Institute of Pathology, and the Institute of Virology and Immunobiology, University of
Würzburg, D-97080 Würzburg, Germany
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Abstract |
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The expression of the murine interleukin (IL)-2 receptor chain/CD25 is strongly induced at
the transcriptional level after T cell activation. We show here that nuclear factor of activated T
cell (NF-AT) factors are involved in the control of CD25 promoter induction in T cells. NF-ATp and NF-ATc bind to two sites around positions
585 and
650 located upstream of the
proximal CD25 promoter. Immediately 3' from these NF-AT motifs, nonconsensus sites are
located for the binding of AP-1-like factors. Mutations of sites that suppress NF-AT binding
impair the induction and strong NF-ATp-mediated transactivation of the CD25 promoter in T cells. In T lymphocytes from NF-ATp-deficient mice, the expression of CD25 is severely
impaired, leading to a delayed IL-2 receptor expression after T cell receptor (TCR)/CD3 stimulation. Our data indicate an important role for NF-AT in the faithful expression of high affinity IL-2 receptors and a close link between the TCR-mediated induction of IL-2 and IL-2 receptor
chain promoters, both of which are regulated by NF-AT factors.
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Introduction |
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The high affinity IL-2 receptor consists of three individual polypeptides, the ,
, and
chains. Although the
and
chains are shared by other lymphokine receptors,
the
chain (CD25) is restricted to the IL-2 receptor, and is
expressed by a variety of lymphoid cells (for review see reference 1). The induction of CD25 in T cells is controlled
at the transcriptional level through two DNA sequence elements, a proximal promoter/enhancer spanning the nucleotides between positions
54 and
584 in the mouse and
64 and
276 in humans, and a distal enhancer spanning ~80 nucleotides around position
1350 in the mouse and
3750 (or
4150, according to another nomenclature) in
the human CD25 gene (2). The activity of the promoter
is rapidly induced by TCR-mediated signals or IL-1, and is
controlled by an array of transcription factors, in particular
by nuclear factor (NF)-
B, Elf-1, SRF, and HMG I(Y).
The induction of the distal enhancer is controlled by IL-2,
which induces signal transducer and activator of transcription (Stat)5, a member of the family of Stat transcription
factors. Stat5 binds in concert with Elf-1, HMG I(Y), and
GATA factors to multiple sites of the distal enhancer and
contributes to its IL-2-mediated full expression in activated
peripheral T lymphocytes (4).
Nuclear factor of activated T cell (NF-AT) factors comprise a family of transcription factors that contribute to the
induced expression of numerous lymphokine and receptor
genes in T cells. Similar to NF-B factors, the nuclear
translocation and activity of NF-AT factors is stimulated by
TCR-mediated signals (for review see reference 7). The
DNA-binding domains of NF-AT and NF-
B/Rel factors
share a common architecture (8) and, therefore, recognize overlapping DNA sequence motifs. These common properties between NF-AT and NF-
B (a major regulator of the
CD25 promoter), and reports on the inhibition of CD25
expression by cyclosporin A (9) (an inhibitor of phosphatase calcineurin and, therefore, of nuclear translocation
of NF-AT; reference 7), prompted us to investigate whether
NF-AT factors participate in CD25 promoter control. We
show here that NF-ATp and NF-ATc bind to two sites located immediately upstream of the proximal CD25 promoter. Mutations within the NF-AT sites that suppress
NF-AT binding impair CD25 promoter induction. Accordingly, the induction of CD25 is markedly delayed in T
cells from NF-ATp-deficient mice. These findings implicate an important role for NF-AT factors in the inducible expression of high affinity IL-2 receptors after T cell activation.
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Materials and Methods |
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Cell Culture, Construction, and Transfection of CD25 Promoter Luciferase Plasmids.
Murine El4 T thymoma cells and human Jurkat T leukemia cells were grown in RPMI medium containing 5% FCS. 2 × 107 cells were transfected using the DEAE dextran protocol with 2.5 µg DNA of the CD25 promoter-luciferase reporter constructs alone or 0.5-2.5 µg DNA of reporter constructs (as indicated in the figure legends) along with 2 µg of a pLGP3-based vector expressing full-length murine NF-ATp (NF-AT1-C; reference 10) or an RSV-LTR vector expressing human NF-ATc. Human 293 embryonic kidney cells were cultured in DMEM and transfected using a calcium phosphate transfection protocol. The luciferase reporter gene construct contains the wild-type murine CD25 promoter spanning the nucleotides up to positionImmunofluorescence and Flow Cytometry.
For Ab stainings, 2-8 × 105 cells were incubated on ice with mAbs at saturating concentrations. Fluorescein- and PE-labeled mAbs (Pharmingen, San Diego, CA) were used for two- and three-color immunofluorescence. For three-color flow cytometry, cells were stained first with biotinylated mAbs (PharMingen) for 15 min and were subsequently incubated with streptavidin-Red670 (GIBCO BRL, Eggenstein, Germany) and FITC- and PE-labeled mAbs for 15 min. Results obtained after analysis on a FACScan® flow cytometer (Becton Dickinson, Mountain View, CA) using Lysys II software (Becton Dickinson) are shown as log dot-plots or histograms.DNase I Footprint Protection Assays and EMSAs.
In DNase I footprint protection assays, end-labeled DNA probes were prepared using [ ![]() |
Results and Discussion |
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To determine whether the CD25 promoter is a target
for NF-AT, we cotransfected a CD25 promoter-driven luciferase reporter gene with NF-ATp- and NF-ATc-specific expression vectors into El4 T and 293 cells. Treatment
of 293 cells with TPA plus ionomycin (T+I) led to a <2-fold, and treatment of El4 cells with T+Con A led to an
8-9-fold, induction of activity of CD25 promoter spanning the nucleotides up to position 2556 (4), and to a 12-fold induction of a shorter CD25 promoter reaching up to
800. Cotransfection of an NF-ATp expression vector
into El4 cells resulted in a strong, 40-fold induction of activity of the longer CD25 promoter and in an up to 60-fold
induction of the shorter CD25 promoter fragment after
T+Con A treatment of cells (Fig. 1). Cotransfection with the NF-ATc vector gave rise to only a slight increase in
promoter activity. In 293 cells, the overexpression of both
NF-AT factors resulted in a six- to ninefold increase in
CD25 promoter activity (Fig. 1).
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To demonstrate the binding of NF-ATp to the CD25
promoter, GST-NF-ATp encoding its DNA-binding domain was incubated with DNA fragments containing the
first 800 bp of the promoter region in DNase I footprint
protection assays. Two prominent footprints were detected, spanning the nucleotides from 577 to
587 and
639 to
658, respectively (Fig. 2 A). These comprise the
NF-AT "core" binding sequence TGGAA (7) in opposite
orientations (Fig. 2 B). When probes of these oligonucleotides were incubated with GST-NF-ATp in EMSAs,
both probes were bound by NF-ATp, whereas a third
probe spanning the unprotected nucleotides from
619 to
636 was unable to bind (Fig. 2 C, lanes 13-15). Using
nuclear proteins from murine splenocytes, the generation
of typical inducible NF-AT complexes was detected with
the
639/
658 probe (Fig. 2 C, lanes 1-6) and, although
far more weakly, with the
577/
587 probe (Fig. 2 C,
lanes 7-12). The generation of these complexes was enhanced
after induction of cells with T+I (lanes 1, 2, 7, and 8) and
efficiently competed with a 100-fold molar excess of the distal IL-2 NF-AT site (Fig. 2 C, lanes 6 and 12). Moreover, the complexes were supershifted in EMSAs using NF-ATp- and NF-ATc-specific Abs (lanes 3, 4, 9, and 10).
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The 639/
658 site corresponds to a high-affinity NF-AT binding site. This could be seen best in EMSA competition assays comparing the binding of NF-AT factors in
nuclear protein preparations from T+I-induced Jurkat cells
to this site and the distal NF-AT site of the murine IL-2
promoter (Fig. 2 D). Although 5-10 ng of the
639/
658
site was sufficient to suppress almost all NF-AT binding, 10-50 ng of the distal IL-2 NF-AT site was necessary to see
the same effect (Fig. 2 D, lanes 6-11). 50 ng of an AP-1
site was also able to suppress NF-AT complex formation,
whereas the same amount of the upstream promoter site,
i.e., an efficient octamer but poor AP-1 site from the IL-2
promoter (11), was without effect on factor binding (Fig. 2
D, lanes 12-15). In contrast to the
639/
658 site, the
577/
587 NF-ATp binding site is a low-affinity NF-AT
site (Fig. 2 C, lanes 7-12; note that lanes 7-12 were exposed five times longer than lanes 1-6).
To demonstrate a functional role for the two NF-ATp sites, we introduced mutations into the NF-AT motifs of each site, or into both sites, in the context of the 800-bp wild-type CD25 promoter fragment. These mutations led to a loss of NF-AT binding in EMSAs using nuclear proteins from induced Jurkat cells (see Fig. 2 D, lanes 18-20) or GST-NF-ATp (data not shown). When the mutated CD25 promoter/luciferase constructs were transfected into El4 T cells alone or with an NF-ATp expression vector their induction was severely impaired compared with the wild-type promoter. The T+Con A-mediated 16-fold induction of the 800-bp CD25 promoter fragment was almost abolished (Fig. 3 A) and its >25-fold transactivation by NF-ATp was reduced to a 2-3-fold increase for the mutated promoter (Fig. 3 B).
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The importance of NF-ATp sites for the CD25 expression is underlined by defects in the CD25 surface expression on LN T cells from NF-ATp/
mice established in
our laboratory (13). When LN T cells from wild-type mice
were stimulated with plate-bound
-CD3 Abs for 2-24 h
in vitro, a marked increase of CD25 surface expression was
detected after 6-12 h, which became even more pronounced after 24 h. Due to the strong stimulation of the
CD25 promoter by secreted IL-2 (2), >50% of T cells express large amounts of CD25 48 h after stimulation (Fig. 4).
On NF-ATp
/
LN T cells, CD25 expression was found to
be distinctly delayed, becoming clearly detectable only 24 h
after induction in spite of high, unimpaired IL-2 production
of NF-ATp
/
T cells (13). In addition, fewer cells expressed high levels of CD25 after induction for 48 h (Fig. 4).
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The two NF-ATp binding sites are located near the CD25 promoter and therefore appear to be involved in the rapid induction of the murine CD25 gene in resting T cells. The core sequences of these sites, TGGAA, differ slightly from the AGGAAAA core motifs of the IL-2 and IL-4 promoters and are the strongest NF-ATp binding sites in the human GM-CSF enhancer (14). As indicated in Fig. 2 B, 7-9 bp 3' to the NF-AT motifs are situated TPA- responsive element-like sequences that might allow the concerted binding of AP-1 and NF-AT. In EMSAs we detected a specific binding of GST-c-Jun to both sites (data not shown), but it remains to be shown which proteins of the AP-1 family bind and regulate the CD25 promoter in vivo.
Finally, it should be pointed out that several properties of
NF-ATp/
mice, such as the impaired clonal deletion of
T cells and expansion of lymphoid organs (13, 15, 16), are
shared by the mice deficient for IL-2 and IL-2 receptors
(17). We assume that the impaired CD25 expression
might contribute to the development of this phenotype in
NF-ATp
/
mice, which is reminiscent of other mice with
defects in the IL-2 signaling system.
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Footnotes |
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Address correspondence to Edgar Serfling, Department of Molecular Pathology, Institute of Pathology, University of Würzburg, Josef-Schneider-Str. 2, D-97080 Würzburg, Germany. Phone: 49-931-201-34-29/95; Fax: 49-931-201-34-40; E-mail: path015{at}mail.uni-wuerzburg.de
Received for publication 27 January 1998 and in revised form 29 July 1998.
The first two authors contributed equally to this paper.We are indebted to Daniela Räth and Heidi Runknagel for excellent technical assistance. We wish to thank Dr. M. Nabholz (Swiss Institute for Experimental Cancer Research, Epalinges, Switzerland) and Dr. A. Rao (Center for Blood Research and Department of Pathology, Harvard Medical School, Boston, MA) for DNA constructs.
This work was supported by grants from the Wilhelm-Sander-Stiftung (to E. Serfling) and the Deutsche Forschungsgemeinschaft, SFBs 165 and 465 (to A. Schimpl and E. Serfling).
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