(Received for publication, October 23, 1995; and in revised form, January 5, 1996)
From the
Activation of T lymphocytes requires the combined signaling of
the T cell receptor and costimulatory molecules such as CD28. The
ability of T cells to produce interleukin-2 (IL-2) is a critical
control point in T lymphocyte activation. The IL-2 enhancer contains a
functional motif named CD28 response element (CD28RE) that serves a
role as a target for mitogenic T cell activation signals. The CD28RE
sequence reveals similarity to the consensus B binding motif. Here
we demonstrate that CD28RE binds an inducible protein with a molecular
mass of approximately 35 kDa called nuclear factor of
mitogenic-activated T cells (NF-MATp35) that is clearly different from
the known NF-
B/Rel family members. Induction of NF-MATp35 was
shown to depend on de novo protein synthesis and was
restricted to T cells that received a mitogenic combination of T cell
stimuli, not necessarily including CD28 signaling. Nonmitogenic T cell
stimulation did not result in appearance of NF-MATp35. These results
indicate that mitogenic combinations of T cell activation signals are
integrated at the level of NF-MATp35 induction. Similar to its effect
on IL-2 production, cyclosporin A inhibited the induction of NF-MATp35.
Taken together, these data demonstrate that the nuclear appearance of
NF-MATp35 shows excellent correlation with IL-2 production, which is a
unique characteristic among nuclear factors implicated in the control
of IL-2 gene expression.
Proliferation and maturation of resting T lymphocytes is an
essential process in the T cell-mediated immune response. Resting T
lymphocytes can be activated via noncovalent interactions of the T cell
antigen receptor (TCR) ()complex with a peptide in
association with major histocompatibility complex molecules. Besides
TCR signaling, a second antigen-nonspecific costimulatory is required
for optimal T cell activation, which is accompanied by the production
of high levels of interleukin-2
(IL-2)(1, 2, 3) . IL-2 serves a function as
autocrine growth factor for T cells allowing them to enter the S
phase(4, 5) . Occupancy of the TCR without a
costimulatory signal leads to aborted T cell activation and the
development of functional unresponsiveness or clonal anergy of T cells
in which the T cells are incapable of producing
IL-2(1, 6) . Therefore, the ability of T cells to
synthesize IL-2 is a critical control point in determining their
participation in an immune response and consequently serves as a model
system for the analysis of molecular events in T cell activation (7) .
Considerable evidence has indicated that signals delivered through the T cell accessory molecule CD28 constitute a major costimulatory pathway (8) . This costimulatory signal is induced upon interaction of CD28 with its counterreceptors CD80 and CD86 that are expressed on the APC(9, 10) . Monoclonal antibodies directed against CD28 have been shown to serve as a valid substitute to mimic the CD80-CD86 interaction in CD28 triggering(11, 12) . Monoclonal anti-CD28 antibodies either cooperate with soluble anti-CD3, which simulates antigen-specific TCR triggering, or synergize with protein kinase C for the induction of IL-2 production and T cell proliferation. Unlike the TCR-induced signaling pathway, the CD28 signal transduction route does not involve the formation of inositol 1,4,5-trisphosphate or translocation of protein kinase C and is resistant to the immunosuppressive effects of cyclosporin A (CsA) (8, 13) . Recent evidence suggests that the CD28-induced signal is mediated via phosphatidylinositol 3-kinase(14, 15, 16) . In addition, Su and colleagues (17) reported that simultaneous activation of TCR and CD28 results in a synergistic activation of c-jun kinase. The latter observation implies that TCR and costimulatory signals become integrated at the level of c-jun kinase activation.
Human T cells respond to CD28 costimulation by a dramatic
enhancement in the induction of IL-2 mRNA. Two mechanisms account for
the CD28 co-induced expression of the IL-2 gene. CD28 costimulation has
been shown to activate IL-2 gene transcription (18, 19) and to prolong IL-2 mRNA
half-life(20, 21) . Regulation of IL-2 gene
transcription is controlled by an enhancer extending from -52 to
-319 relative to the transcription initiation
site(22, 23) . Specific regulatory sequences that bind
the nuclear factors AP-1, NF-B, Oct-1, and NF-AT1 have been
implicated in regulation of IL-2 gene transcription (reviewed in (24) ). We and others have identified a sequence from
-162 to -153 (5`-AGAAATTCCA-3`) within the IL-2 enhancer
that serves a crucial role as a response element for mitogenic
combinations T cell activation signals (18, 19, 25) (Fig. 1). The sequence of
the CD28RE motif is strongly conserved between the human and murine
IL-2 gene. Comparison of the sequence with that of binding motifs of
known transcription factors reveals similarity to the consensus
NF-
B binding motif(26, 27) . Only the first and
the last (tenth) position differ from the consensus NF-
B binding
motif(19) . The significance of the sequence similarity has
been shown by cross competition studies using CD28RE and the HIV-1
B motif in combination with nuclear extracts of
mitogenic-activated cells. The HIV-1
B motif efficiently competed
for CD28RE binding activity, whereas CD28RE proved about 25-fold less
effective in competition for HIV-1
B binding activity than the
HIV-1
B motif itself. These data suggest that proteins involved in
interacting with CD28RE are also involved in binding the HIV-1
B
motif.
Figure 1:
A,
schematic representation of the IL-2 enhancer with the known protein
binding sites(24, 45, 46) . The location of
these sites are given by numbers that represent the position in base
pairs relative to the site of initiation of transcription. B,
comparison of the human and murine IL-2 CD28RE sequence. C,
sequence of CD28RE is compared with the consensus NF-B binding
sequence.
In vitro binding studies have demonstrated that
induction of CD28RE binding activity is restricted to T cells that
received a mitogenic combination of T cell activation signals, not
necessarily including CD28(25, 28) . In order to
characterize the proteins that constitute the CD28RE binding activity,
we performed UV cross-linking analysis. In this manuscript we describe
the characterization of a protein called NF-MATp35 (nuclear factor of
mitogenic activated T cells) that binds CD28RE. Although evidence is
presented that NF-MATp35 also binds to the B motif, NF-MATp35 is
clearly distinct from the known NF-
B/Rel family members. It is
demonstrated that the induction of NF-MATp35 shows excellent
correlation with IL-2 production.
DNA binding reactions were performed by incubation of 12 µg of
nuclear extract with the BrdUrd-substituted probe (1-5
10
cpm) in the presence of poly(dI-dC) as a nonspecific
competitor. Nucleoproteins were first resolved by gel retardation prior
to UV irradiation in situ (UV Stratalinker, Stratagene).
Covalently linked nucleoprotein complexes were eluted from the gel and
further analyzed by 10% SDS-polyacrylamide gel electrophoresis after
precipitation with acetone. For immunoprecipitation the eluted proteins
were mixed with 1 µl of undiluted antiserum and protein A-Sepharose
beads. The bound proteins were washed with radioimmune precipitation
buffer and analyzed by SDS-polyacrylamide gel
electrophoresis(44) .
Figure 2: Characterization of CD28RE-binding proteins. A, electrophoretic mobility shift assays with CD28RE as probe. Lane 1, free probe; lane 2, nuclear extracts from nonstimulated Jurkat cells; lane 3, nuclear extract from Jurkat cells stimulated for 8 h with anti-CD3 plus PMA; lane 4, anti-CD3 plus anti-CD28; lane 5, PMA and ionomycin. B, UV cross-linking analysis of CD28RE-binding proteins. The probe used was BrdUrd-substituted CD28RE-1. Lane 1, nuclear extract from Jurkat cells stimulated with anti-CD3 plus PMA; lane 2, anti-CD3 plus anti-CD28; lane 3, PMA and ionomycin. Molecular mass (Mw) markers are shown on the side. Similar results were obtained with BrdUrd-substituted CD28RE-2 and CD28RE-3 probes (data not shown).
To characterize the inducible nucleoprotein(s) that constitutes NF-MAT, we performed UV cross-linking experiments with a 5`-bromo-2`-deoxyuridine-substituted CD28RE probe. Nuclear extract of Jurkat cells stimulated with anti-CD3 plus anti-CD28 was incubated with this probe, and the protein-bound DNA fraction was separated by gel retardation and subjected to in situ UV irradiation. Analysis of the cross-linked protein-DNA complexes by SDS-polyacrylamide gel electrophoresis revealed the presence of two protein-DNA bands that migrated with molecular masses of 35 and 70 kDa, respectively (Fig. 2B).
Next we applied UV cross-linking analysis to confirm that the CD28RE-protein complex that appeared upon stimulation of Jurkat cells via different modes of mitogenic T cell activation revealed the same pattern of protein-DNA bands as we observed for the complex induced by the combination of anti-CD3 and anti-CD28 (Fig. 2B). Because the CD28RE-binding proteins selectively showed up in nuclei of mitogenic activated T cells, we refer to the proteins that correspond to the 35- and 70-kDa cross-linked adducts as NF-MATp35 and NF-MATp70, respectively. The actual molecular mass of these proteins could be slightly lower than that of the respective protein-DNA complexes due to the absence of covalently bound oligonucleotide.
Figure 3: Time course of UV irradiation for cross-linking nucleoproteins to BrdUrd-substituted CD28RE-1. Nuclear extract from Jurkat cells stimulated with anti-CD28 plus anti-CD3 was incubated with BrdUrd-substituted CD28RE-1 and subjected to gel retardation. Nucleoprotein-DNA complexes were UV irradiated for the time indicated and analyzed by 10% SDS-polyacrylamide gel electrophoresis.
Figure 4:
Immunoprecipitation of UV cross-linked
CD28RE-1-NF-MAT. A, anti-CD3 plus anti-CD28-induced NF-MAT was
UV cross-linked with BrdUrd-substituted CD28RE-1. Immunoprecipitations
of UV cross-linked NF-MATp35 and NF-MATp70 were performed with
anti-NF-B/Rel antibodies. Lane 1, without antibodies; lane 2, with anti-IL8 as negative control; lane 3,
with anti-NF
B1; lane 4, with anti-RelA; lane 5,
with anti-c-Rel. B, immunoprecipitation of BrdUrd-substituted
HIV-1
B UV cross-linked NF-
b/Rel proteins. Recombinant
NF
B1/c-Rel (lanes 1-5) and NF
B1/RelA (lanes 6-9) were immunoprecipitated by
anti-NF-
B/Rel antibodies. Lanes 1 and 6, without
antibodies; lanes 2 and 7, with anti-NF
B1; lanes 3 and 8, with anti-RelA; lanes 4 and 9, with anti-c-Rel; lane 6 with anti-IL-8 as a
control.
Figure 5:
Characterization of HIV-1 B binding
activity. A, induction of HIV-1
B binding activity.
Electrophoretic mobility shift assays were performed with end-labeled
HIV-1
B oligonucleotide and nuclear extracts from Jurkat cells
stimulated for 8 h. Lane 1, free probe; lane 2,
nuclear extract of unstimulated cells; lane 3, nuclear extract
of cells stimulated with PMA; lane 4, PMA plus anti-CD28; lane 5, anti-CD28; lane 6, anti-CD3; lane 7,
anti-CD3 plus anti-CD28; lane 8, PMA plus anti-CD3 plus anti
CD28; lane 9, anti-CD3 plus PMA. B, UV cross-linking
of HIV-1
B-binding proteins. The probes used were the
BrdUrd-substituted CD28RE-1 (lane 1) and HIV-1
B sequence (lanes 2-7). Lanes 1, 2, and 6, nuclear extract from Jurkat cells stimulated for 8 h with
anti-CD3 plus anti-CD28; lane 3, with anti-CD3; lane
4, with PMA; lane 5, with anti-CD3 plus PMA; lane
7, anti-CD3 plus anti-CD28 plus
cycloheximide.
In order to determine whether NF-MATp35 contributes to the
HIV-1 NF-B-protein complex, we performed UV cross-linking
experiments with a BrdUrd-incorporated HIV-1
B motif. With nuclear
extracts of nonmitogenic activated T cells, two major protein-DNA bands
with molecular masses of approximately 70-85 kDa and 50 kDa,
respectively, were observed (Fig. 5B). HIV-1
B-protein complexes that were formed in nuclear extracts of cells
that have received a mitogenic combination of activation signals
contained an additional protein-DNA product with a molecular mass
identical to that of the NF-MATp35-CD28RE complex. Distinct mitogenic
combinations of T cell activation conditions led to the inclusion of
this protein in the HIV-1
B-protein complex. Similar to the
NF-MATp35-CD28RE complex, the 35-kDa protein-HIV-1
B complex could
not be immunoprecipitated with anti-NF
B1, anti-RelA, and
anti-c-Rel antibodies (data not shown).
Figure 6: Influence of cycloheximide and CsA on the appearance of NF-MAT. Gel retardation analysis was performed with CD28RE as a probe, and nuclear extract from Jurkat cells was stimulated for 8 h in the absence (lane 1) or the presence of either cycloheximide (lanes 2-4) or CsA (lanes 5-7). Lane 1, cells stimulated with anti-CD3 plus PMA; lanes 2 and 5, anti-CD3 plus anti-CD28; lanes 3 and 6, anti-CD3 plus PMA; lanes 4 and 7, anti-CD28 plus PMA.
When the HIV-1 B motif was used as a probe,
cycloheximide selectively inhibited the appearance of the fast
migrating moiety (C2) of the mitogenic induced complex (Fig. 7A). The addition of anti-serum directed against
NF
B1 reveals that the complex that appears with extracts of nuclei
from mitogenic stimulated (anti-CD3 plus anti-CD28) cells in the
presence of cycloheximide resembles the complex induced upon
nonmitogenic stimulation with anti-CD3 alone (Fig. 7B).
Figure 7:
Influence of cycloheximide and CsA on the
appearance of mitogenic induced HIV-1 B binding activity. A, gel retardation analysis was performed with HIV-1
B as
a probe, and nuclear extract from Jurkat cells was stimulated for 8 h
in the absence (lane 1) or the presence of either
cycloheximide (lanes 2-4) or CsA (lanes
5-7). Lane 1, cells stimulated with anti-CD3 plus
PMA; lanes 2 and 5, anti-CD3 plus anti-CD28; lanes 3 and 6, anti-CD3 plus PMA; lanes 4 and 7, anti-CD28 plus PMA. B, gel retardation
assay was performed with HIV-1
B as probe in the absence (lanes 1-4) or the presence (lanes 5-8)
of antibodies directed against NF
B1 with nuclear extract from
stimulated Jurkat cells. Lanes 1 and 5, cells
stimulated for 8 h with anti-CD3; lanes 2 and 6,
anti-CD3 plus anti-CD28; lanes 3 and 7, anti-CD3 in
the presence of cycloheximide; lanes 4 and 8,
anti-CD3 plus anti-CD28 in the presence of
cycloheximide.
UV cross-linking analysis with a BrdUrd-substituted HIV-1 B
motif shows that the complex that appears in the presence of
cycloheximide is devoid of the p35 protein (Fig. 6B).
This result is in accordance with the NF-MAT data and substantiates the
conclusion that the protein that constitutes the 35-kDa protein-HIV-1
B complex is identical to NF-MATp35.
As
anticipated we found a similar effect with CsA on the appearance of the
fast migrating moiety (C2) of the HIV-1 B-protein complex (Fig. 7B). This result further substantiates the
conclusion that this moiety represents the NF-MATp35 containing part of
the complex.
In this study we describe an approximately 35-kDa inducible protein, named NF-MATp35, which interacts with the CD28RE within the IL-2 enhancer. Distinct mitogenic combinations of T cell activation signals contribute to the nuclear appearance of NF-MATp35, indicating that there is a redundancy in the activation requirements of NF-MATp35.
We previously reported that the CD28RE sequence displays similarity
with the B enhancer consensus motif. The Rel-related proteins
c-Rel, NF
B1, and RelA constitute a major class of
B
enhancer-binding proteins. In line with this Ghosh et al.(28) were able to detect the presence of NF
B1, RelA,
and c-Rel among the proteins that bind CD28RE. However, the affinity
for NF-
B/Rel proteins is low as judged from competition
experiments and UV cross-linking studies performed by us and
others(19, 30, 31) .
Our data clearly
demonstrate that NF-MATp35 is distinct from NFB1, RelA, and c-Rel.
First, the activation requirements of NF-MATp35 are more stringent than
those for the induction of the NF-
B/Rel family members. Second,
the molecular mass of NF-MATp35 does not match those of the individual
NF-
B/Rel proteins. Third, antibodies directed against NF
B1,
RelA, or c-Rel have not been successful in immunoprecipitating
NF-MATp35 covalently linked to CD28RE. Finally, the appearance of
NF-MATp35 is dependent on protein synthesis. It is interesting that
Fraser and Weiss (30) reported that the predominant protein
species present in the CD28RC constitute three proteins of 35, 36, and
44 kDa. The most abundant were the 35/36-kDa proteins. It could be that
NF-MATp35 is related to one of these proteins; however, in contrast to
NF-MATp35, the CD28RC proteins require the contribution of the CD28
signaling for their nuclear appearance. In addition, none of these
proteins appears upon stimulation with anti-CD28 plus PMA but requires
the addition of a third stimulus, i.e. ionomycin. Furthermore,
Li and Siekevitz (31) described a 45-kDa Tax inducible protein
(TxREF) distinct from NF-
B/Rel that specifically interacts with
CD28RE. This protein could be related to the 44-kDa protein described
by Fraser and Weiss(30) .
The low affinity of CD28RE for
binding NF-B/Rel proteins could be explained by differences in the
5` and 3` extremities between CD28RE and NF-
B/Rel binding motifs.
Analysis of the various NF-
B/Rel binding motifs has showed that
the 5` and 3` ends of the recognition motif are the best conserved
parts(32) . Based on crystallographic analysis of NF-
B-DNA
complexes, it is believed that these parts of the
B binding motif
in particular are involved in NF-
B/Rel
binding(33, 34) . The fact that NF-MATp35 binds to the
B elements as well as to CD28RE, whereas NF-
B/Rel proteins
have a preference for the consensus-matching sequences, indicates that
less variation is tolerated for binding of NF-
B/Rel proteins than
for NF-MATp35. This might implicate that every
B element is in
potency able to bind NF-MATp35.
A functional role of CD28RE as a mitogen response element in the activation of the IL-2 enhancer stems from studies wherein a mutant enhancer with a nonfunctional CD28RE site was used in transient expression studies(18, 19) . In accordance with the requirements for NF-MATp35 induction, mutations within the CD28RE motif affected the IL-2 enhancer in its capacity to respond to mitogenic T cell activation signals. Because CD28RE serves a role as a response element for distinct mitogenic combinations of T cell activation signals not necessarily including CD28 signaling, we propose to name the element MARS, which stands for mitogenic activation responsive sequence (35) .
IL-2 production is either
completely resistant (anti-CD28 plus PMA) or sensitive (anti-CD3 plus
either anti-CD28 or PMA) to the immunosuppressive action of
CsA(13, 36, 37) . Inhibition of IL-2 gene
expression by CsA is explained by the negative effects of CsA on the
nuclear expression of the cytoplasmic component of NF-AT and the
activation of Oct-1/AP1, both known to be TCR-responsive nuclear
proteins(37, 38, 39, 40) . Here we
demonstrated that nuclear appearance of NF-MATp35 is also sensitive to
CsA. The sensitivity of NF-MATp35 for CsA is, similar to IL-2 gene
expression, dependent on the mode of mitogenic stimulation. When
Ca influx is not involved in the signaling regime,
NF-MAT still appears. Taken together the above mentioned results show
that the induction of NF-MATp35 reveals excellent correlation with
activation of IL-2 gene expression (Table 1). This is a unique
characteristic among nuclear proteins implicated in IL-2 gene
regulation and suggests a crucial role for NF-MATp35 in IL-2 gene
regulation.
Similar to the induction of NF-MATp35, the activation of
c-jun kinase revealed excellent correlation with IL-2
production(17) . However, based on supershift analysis and
immunoprecipitation experiments with anti-c-jun antibodies, we
could exclude the possibility that NF-MATp35 is identical to c-jun.()c-jun kinase is the only protein kinase
among the kinases involved in mitogenic signaling described so far that
requires mitogenic combinations of T cell stimuli for its activation.
This indicates that signal integration occurs at an even more proximal
step in the events that lead to IL-2 gene transcription than NF-MATp35
induction. Therefore, it is of interest to investigate whether
NF-MATp35 is somehow involved in c-jun kinase signaling.