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*
The Wallenberg Laboratory, Section for Tumour Immunology, Department of Cell and Molecular Biology, University of Lund, and
Pharmacia & Upjohn, Lund, Sweden; and
Department of Biology and Molecular Biology Institute, San Diego State University, San Diego, CA 92182
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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ß reporter
construct when costimulated with B7-1 or LFA-3, but transcription of
IL-2 promoter or CD28 response element (CD28RE)-driven reporters was
superior in B7-1-costimulated cells. Combined expression of c-Jun and
p65 induced vigorous transcription of IL-2 promoter- and CD28RE-driven
reporter constructs in both LFA-3- and B7-1-costimulated Jurkat cells.
Mutating the CD28RE but not the upstream nuclear factor ß-binding
site in the IL-2 promoter reduced B7-1-driven transcription >90%. The
results implicates a major role of the CD28RE in the integration of
p65/c-Jun-mediated transcription within the IL-2 promoter. We suggest
that the transition from an autocrine LFA-3-driven immune response to a
B7--induced paracrine immune response involves the activation of c-Jun
and p65, which target the CD28RE region of the IL-2 promoter. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B, AP-1, Oct-1, and CD28RC (13, 14, 15, 16).
The NF-B/Rel family of transcription factors has been shown by
several laboratories to be important to IL-2 promoter activity. These
transcription factors share the conserved Rel homology domain, involved
in specific DNA binding and dimerization (17). Rel proteins interact
with the regulatory elements of many different inducible immune
response genes. The NF-B/Rel family of transcription factors
includes p50 (NFB1), p52 (NFB2), p65 (Rel A), Rel B, and c-Rel
(18, 19, 20). p65 and c-Rel contain different and unique C-terminal
transcription activation domains (21, 22, 23). Several studies have
demonstrated that both the p65 (22) and c-Rel (24) proteins contain a
potent trans-activating capacity. In contrast, the p50 (25, 26) subunit shows a substantial DNA-binding capacity and low
transcriptional activity, suggesting a role for p50 homodimers in
repressing p65- and c-Rel-responsive genes.
The NF-B proteins are retained in the cytoplasm in an inactive state
by complex formation with the IB-inhibitory subunits (27, 28). Cell
activation induces the phosphorylation of IB-
and its subsequent
proteolytic degradation (29). The NF-B complex is dissociated from
the negative regulator IB, is translocated to the nucleus, and binds
to the B decameric sequence motif (25, 27, 28, 29). The CD28RE within
the IL-2 promoter contains a sequence similar to but not identical with
the B site (30). Several studies have demonstrated that members of
the NF-B/Rel family proteins are involved in the nuclear protein
complex that binds to the CD28RE, suggesting that this response element
is a modified B response motif (31, 32, 33). CD28RE was originally
defined in the IL-2 promoter as a major and unique response element for
the CD28/B7-1 signal transduction pathway (31). However, recent data
suggest that induction of nuclear proteins binding to CD28RE is not
entirely exclusive for the CD28 pathway (16, 34, 35) and similar
sequences have been identified in other promoters like IL-3 and GM-CSF
(30, 36). Recently, we reported that B7-1 costimulation induced the
presence of a c-Rel-containing CD28RC complex (16) and this has been
seen by other laboratories as well (35).
The AP-1 transcription factors are composed of Jun and Fos proteins
that function as transcriptional regulators in a heterodimeric complex
(37, 38, 39). The members of this transcription factor family together with
ATF, CREB, and others belong to the class of basic-leucine zipper
(bZIP) proteins. Following cell activation, members of the Jun family
(c-Jun, Jun D, and Jun B) form complexes with various partners of the
Fos family (c-Fos, Fos B, Fra-1, and Fra-2). The formation of
heterodimers seems to increase their transcriptional activity (40). The
c-Jun but not the c-Fos protein may also form homodimers. However, the
heterodimers formed by one member of each family display higher
DNA-binding capacity and trans-activating properties than
homodimers formed by the Jun family members. The consensus AP-1-binding
site has been identified in several genes involved in T cell activation
(37, 38, 39). The AP-1 proteins have been demonstrated to complex with
NF-AT, NF-B, and Oct-1 proteins, suggesting that AP-1 may act in
concert with other transcription factors to regulate a variety of
response elements (41, 42, 43, 44, 45, 46). To elucidate the role of Rel and AP-1
proteins in costimulation of T cells, we used a model with Jurkat T
cells and CHO cells transfected with HLA-DR, B7-1, and LFA-3 as APCs.
Transient transfection of Jurkat cells with IL-2 promoter AP-1, NF-B
and CD28RE-driven reporter combined with Rel and AP-1 expression
plasmids was performed to study the role of these proteins in IL-2
promoter transcription. We now demonstrate that c-Jun functionally
cooperates with p65 in superinducing the transcriptional activity of
the IL-2 promoter. Indeed, c-Jun/p65-expressing Jurkat cells responded
equally well to B7-1 and LFA-3 costimulation. The CD28RE seemed to be
crucial for p65/c-Jun effects, since a mutation in this site completely
abrogated the superinduction of IL-2 promoter activity. The data
suggest that the CD28RE, flanked by an AP-1 element, integrates AP-1
and Rel nuclear factor pathways to transfer T cells from an autocrine
to a paracrine immune response.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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SEE was purchased from Toxin Technology (Madison, WI). Luciferase assay reagent, reporter lysis buffer 5X, and the pGL2 luciferase basic vector were purchase from Promega (Madison, WI). rIL-2 was obtained from Cetus (Emeryville, CA).
Transfected cell lines
CHO cells were transfected with cDNAs encoding the human HLA-DR4, B7-1, and LFA-3 cell surface molecules, as described in detail elsewhere (12, 47). Single, double, and triple transfectants expressing similar levels of the transfected molecules were established by repeated cell sortings. The surface expression of HLA-DR, LFA-3, and B7-1 was confirmed regularly by FACS analysis.
Cell culture and transfection
All experiments were done using the human T-cell Jurkat, which responds with a pattern of IL-2 production similar to those of B7-1 and LFA-3 costimulation as normal human T cells (16). The human Jurkat leukemia T cell line was maintained in culture in RPMI 1640 supplemented with 2 mM glutamine and 10% FBS (complete medium). All tests on the Jurkat T cells were performed at a concentration of 1 x 106 cells/ml in complete medium. The transfected CHO cells were irradiated (8000 rad) and added to the Jurkat cells at a concentration of 0.1 x 106 cells/ml (48).
Plasmid construction
The human IL-2 promotor-enhancer fragment (-500 to +60) was
subcloned from plasmid SV-IL-2-CAT into the luciferase vector pGL2
(Promega) (16). The AP-1-luciferase reporter plasmid driven by the rat
prolactin minimal promoter (-36 to +37) under the control of four
copies of the human AP-1 site (49) was kindly provided by M.
Rincón and R. A. Flavell (Section of Immunobiology, Howard
Hughes Medical Institute, Yale University School of Medicine, New
Haven, CT). The IL-2 promotor mutated in the CD28RE region was
generated by PCR-directed splicing overlap extension. The PCR replaced
6 bp within the CD28RE (from -159 to -164) in the IL-2 enhancer of
the pGL-2-IL-2-Luc vector with the sequence 5'-CCTCGA-3'. Plasmids
containing multimers of the recognition sites for NF-B and CD28RC
were constructed and linked to the pLuc-prolactin minimal promoter
plasmid (16). The orientation for each element was confirmed by
restriction enzyme cleavage.
The tandem sequences used to construct the different multimers plasmids
were as follows: 1) four copies of the CD28RE (CD28RE of the human IL-2
promoter 5'-AAAGAAATTCC-3'; 2) four copies of the AP-1,
12-O-tetradecanoylphorbol-13-acetate,
5'-TCGATTGAGTCAGGGTAA3'; 3) two copies of the NF-B-binding site
of the human Ig light chain enhancer 5'-GGGACTTTCC-3'; 5) three
copies of the CD28RE/AP-1 sequence of the IL-2 promoter (-161 to
-138) was cloned in three copies upstream of the minimal promoter from
the CR version of the human T cell leukemia/lymphoma virus-1 long
terminal repeat, 3x CAGAAATTCCAAAGAG TCATCAGA (50); 6) three
copies of the CD28RE/AP-1 construct bearing the mutation for the CD28RE
(CD28REm), TTTAAAGCCATTCCAAA (50); 7) three copies of the
CD28RE/AP-1 construct bearing the mutation for the AP-1 (AP-1 REm),
CCAAAGAGAGATCAGAA (50); 8) three copies of the CD28RE/AP-1 construct
bearing a double mutation for the CD28RE (CD28REm) and AP-1 (AP-1 m),
3x CAGAAATTCCAAAGAGAGA TCAGA (50); 9) IL-2 promoter construct
bearing a mutation for the NF-B site, AAAGAGGCTTTTCACCT
(50).
DNA transfection and luciferase activity analysis
Transfection of Jurkat cells was conducted by electroporation, using an electro cell manipulator 600 (BTX, San Diego, CA) using 130 V/1700 µF capacitance. Briefly, 8 x 106 cells were transfected with 10 µg of luciferase reporter plasmid and 5 µg of each expression plasmid, and the mixture was incubated for 24 h. Cells were stimulated with the different CHO-transfected cell lines with or without 100 ng of SEE per ml for another 8 h. Cells were harvested 32 h posttransfection, washed twice in PBS, and treated with lysis buffer (luciferase assay, Promega) for 5 to 10 min on ice. Lysates were spun down for 1 min, and the total supernatants were analyzed using luciferase reagent (Promega) and measured as a duplicate in a luminometer (MicroLumat LB 96 P, Berthold) for 5 s. Background measurement was subtracted from each duplicate, and experimental values are expressed either as recorded light units of luciferase activity or as relative activity compared with extracts from unstimulated cells (16).
IL-2 bioassay
The amount of IL-2 in supernatants was determined in a microassay based on IL-2-dependent proliferation of the CTLL murine cell line as described (12).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B/Rel-driven reporter genes and high levels of IL-2
transcription, while LFA-3 costimulation results in low or moderate
activity of AP-1, NF-B, and IL-2 promoter-driven reporter genes
(16). To elucidate the role of AP-1 and Rel proteins in the
superinduction of IL-2 transcription, we transiently overexpressed
these nuclear factors in Jurkat T cells and studied their influence on
the transcriptional activity of the IL-2 promoter and multimers of the
AP-1, CD28, and NF-B response elements using reporter constructs.
The transfected Jurkat cells were costimulated with untransfected
CHO cells, CHO-DR, CHO-DR/B7-1, CHO-DR/LFA-3, and CHO-DR/B7-1/LFA-3
transfectants, in the presence and absence of the superantigen SEE. In
this system, the Jurkat T cells receive the first signal by the
interaction between TCR and the SEE/HLA-DR complex and a second signal
by the B7/CD28 or LFA-3/CD2 pathways. B7-1 but not LFA-3 costimulation superinduces IL-2 production
Large amounts of IL-2 were produced by B7-1-costimulated Jurkat T
cells in the presence of SEE, while a five- to sixfold amount was
observed after LFA-3 costimulation (Fig. 1
). Only marginal production was seen in
SEE- and CHO-DR-stimulated T cells. Production of IL-2 was further
increased when Jurkat T cells were costimulated with the triple CHO
transfectant (CHO-DR/B7-1/LFA-3), indicating a cooperative effect
between B7-1 and LFA-3 in IL-2 gene expression (Fig. 1).
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Jurkat T cells were transiently transfected with expression
plasmids encoding c-Jun and c-Fos and reporter constructs containing
the inducible region of the IL-2 enhancer/promoter (-500 to+60) or an
AP-1 reporter gene. B7-1 costimulation was required to induce high
transcription of these constructs. The effect observed after LFA-3
costimulation was three- to fourfold lower on both IL-2 promoter and
AP-1-driven transcription. Marginal activity was induced by HL-A-DR
stimulation alone. These results correlated with the induction of IL-2
protein production presented in Figure 1
. Overexpression of c-Jun
slightly enhanced the transcriptional activity of the IL-2 promoter
(Fig. 2A) and AP-1
(Fig. 2B) reporter gene constructs. Cotransfection of
a c-Fos expression vector did not affect the IL-2 promoter (Fig. 2A) and AP-1 (Fig. 2B) reporter
constructs. A substantial increase in IL-2 promoter and AP-1
transcriptional activity was observed after combined cotransfection of
c-Jun and c-Fos. Interestingly, AP-1-driven transcription in
c-Fos/c-Jun-transfected Jurkat cells was dramatically augmented in DR
and DR/LFA-3-stimulated T cells (Fig. 2B).
Apparently, a proper signal is required to induce AP-1 reporter
activity in Jun/Fos-transfected T cells, since no activity was seen in
Jurkat cells stimulated with CHO/SEE alone.
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B and CD28RE reporter constructs in Jurkat T cells
To explore whether overexpression of the Rel family proteins p50,
c-Rel, and Rel A influences transcriptional activity of IL-2 promoter
(Fig. 3A)-, NF-B
(Fig. 3B)-, and CD28RE (Fig. 3C)-driven reporter genes, cotransfection assays with
expression vectors encoding the Rel family proteins and reporter gene
constructs were performed. The transcriptional activity of the IL-2
promoter (Fig. 3A), NF-B (Fig. 3B), and CD28RE reporter constructs was augmented by
B7-1 and B7-1/LFA-3 costimulation, while the activity induced by LFA-3
was low (Fig. 3). Marginal or no induction was observed after HLA-DR
stimulation alone. The luciferase activity expressed by the
CD28RE-driven reporter construct (Fig. 3C) was low,
compared with the luciferase activity expressed by the IL-2 promoter
(Fig. 3A) and NF-B (Fig. 3B)
reporter constructs. Overexpression of p50 and c-Rel had marginal and
low effects, respectively, on the transcriptional activity of the IL-2
promoter (Fig. 3A) and NF-B (Fig. 3B) reporter constructs. Cotransfection of p65
expression plasmids resulted in a moderate to strong increase in the
transcriptional activity of the IL-2 promoter (Fig. 3A), NF-B (Fig. 3B), and CD28RE
(Fig. 3C) reporter genes in B7-1- and
LFA-3-costimulated Jurkat T cells. A dramatic effect of the p65
proteins on the trans-activation of the NF-B reporter
construct (Fig. 3B) in DR/LFA-3-costimulated T cells
was observed, resulting in a similar activity as in B7-1-costimulated T
cells. More interestingly, a dramatic transcriptional increase was
observed after cotransfection of CD28RE reporter construct and the p65
expression plasmid (Fig. 3C). The luciferase activity
was increased about 30- to 40-fold with p65 in DR-, DR-B7-1-,
DR-LFA-3-, and DR-B7-1/LFA-3-costimulated T cells. However, the CD28RE
activity in p65 expressing Jurkat cells remained at 2.5 to threefold
lower levels when costimulated with LFA-3 compared with B7-1. c-Rel
transfection increased only two- to threefold the CD28RE-dependent
trans-activation, whereas p50 transfection failed to
influence CD28RE transcription.
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To determine whether overexpression of AP-1 and Rel proteins
cooperate to regulate the transcriptional activity of the inducible
IL-2 promoter, we performed cotransfection assays with the IL-2
promoter reporter construct and p65 (Fig. 4A), c-Rel (Fig. 4B), c-Fos and c-Jun expression plasmids (Fig. 4).
The coexpression of p65 with c-Jun dramatically enhanced the IL-2
promoter activity (Fig. 4), whereas c-Jun/c-Rel moderately influenced
the activity. In contrast, expression of c-Fos suppressed the potent
trans-activation by p65 (Fig. 4A) but did
not affect the increase induced by c-Rel and c-Rel/c-Jun coexpression
(Fig. 4B). Interestingly, LFA-3 costimulation of
p65/c-Jun cotransfected cells induced >20-fold increase in the
transcriptional activity of the IL-2 promoter, resulting in levels
similar to that observed in B7-1-costimulated cells (Fig. 4A).
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Previous studies have suggested that the IL-2 promoter
proximal AP-1-binding site influences the activity of the CD28RE (35, 50, 51). To determine whether the cis-acting AP-1 site of
the IL-2 promoter could influence the moderate luciferase activity
expressed by reporter construct containing the multimer of the CD28RE,
we used luciferase reporter constructs containing a multimer of a
linked CD28RE-AP-1(wild-type) construct and a panel of substitution
mutants, including CD28REmt-AP-1 (CD28RE mutant), CD28RE-AP-1 mt (AP-1
mutant), and CD28REmt-AP-1 mt (CD28RE-AP-1 double mutant). The pGL-2
vector was used as a negative control. Jurkat T cells transfected with
the CD28RE/AP-1 (wild-type) responded strongly to costimulation induced
by B7-1 and B7-1/LFA-3 (Fig. 5).
Interestingly, mutations introduced into the CD28RE only marginally
affected the activity induced by DR/LFA-3 but strongly reduced the
response to B7-1 costimulation. The AP-1 mutation reduced the response
dramatically after B7-1 costimulation and moderately after LFA-3
costimulation (Fig. 5). The luciferase activity expressed by either the
CD28REmt or the AP-1 mt is grossly in the same range as the activity
expressed by the reporter construct multimers of AP-1 or CD28RE sites
alone in this system. The linkage of the CD28RE/AP-1 site creates a
strong transcriptional reporter gene activity in B7-1-costimulated
Jurkat T cells and supports the hypothesis of cooperative effects
between these cis-acting sites.
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To determine whether the combined action of p65 and c-Jun could
cooperate to up-regulate the transcriptional activity of the
CD28RE/AP-1 reporter construct, we transiently expressed in Jurkat T
cells the CD28RE/AP-1 reporter construct with p65, c-Fos, and c-Jun
expression plasmids. The luciferase activity in Jurkat T cells
costimulated with DR, DR/B7-1, DR/LFA-3, and DR/B7-1/LFA-3 was
moderately increased by p65 and strongly increased by the combined
action of p65/c-Jun (Fig. 6)
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To study whether the p65- and p65/c-Jun mediated superinduction of
the IL-2 gene promoter involved the CD28RE, we performed cotransfection
assays with wild-type IL-2 promoter (Fig. 7A) and CD28RE-mutated
IL-2 promoter (Fig. 7B) luciferase reporter constructs together with
expression vectors for p65, c-Fos, c-Jun, and the combination of
c-Fos/p65 and c-Jun/p65. The luciferase activity was strongly reduced
in the IL-2 mutant (Fig. 7B), and the coexpression of
p65/c-Jun only marginally influenced this response and failed to
restore the transcriptional activity. These data demonstrated the
requirement for a functional CD28RE to superinduce IL-2 transcription
in p65/c-Jun-stimulated T cells. This requirement was further
demonstrated with an IL-2 promoter wild-type (Fig. 8A) and an IL-2
promoter bearing a mutation at the NF-B binding site (Fig. 8B). The results demonstrated that the
transcriptional activity in the mutated IL-2 promoter was diminished by
30%, and it could not only be restored but also be superinduced by the
combination of c-Jun/p65.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this work, we have demonstrated that the overexpression of c-Jun and
p65 nuclear factors in Jurkat T cells superinduces IL-2 production in
LFA-3-costimulated T cells. Overexpression of c-Jun or p65 alone only
moderately enhanced IL-2 promoter activity in LFA-3-costimulated cells,
whereas coexpression of these proteins strongly augmented IL-2 activity
in LFA-3-costimulated cells and induced transcriptional levels
severalfold higher than the levels seen in B7-1-costimulated
untransfected T cells. Indeed, c-Jun/p65-expressing T cells transcribed
the IL-2 promoter during LFA-3 costimulation almost as efficiently as
during B7-1 costimulation. The ability of Fos/Jun and Rel proteins to
cross-couple and potentiate transcription has been reported also in
other systems (46, 53). However, the combined action of p65/c-Jun was
unable to substantially improve the low luciferase activity observed in
an IL-2 promoter reporter construct containing a mutation in the CD28RE
site but strongly induced the transcriptional activity of a
NF-B-mutated IL-2 promoter, suggesting a requirement for an intact
CD28RE for the effect of c-Jun/p65 complex in the up-regulation of IL-2
promoter activity. Our present data support a role for the CD28RE in
integrating AP-1- and Rel-mediated transcriptional events which seems
to be crucial in the transition from low (autocrine) to strong IL-2
(paracrine) transcription. Recent studies have suggested that the
CD28RE may work in conjunction with the IL-2 promoter-proximal AP-1
site (35, 50, 51). Our analysis using CD28RE/AP-1 mutant constructs
demonstrates that both sites are required for optimal transcription and
that the AP-1-proximal sequence cooperates with the NF-B like CD28RE
to increase the IL-2 transcriptional activity in B7-1-costimulated
cells. How do the p65 and c-Jun proteins cooperate? One possibility is
that c-Jun itself or in conjunction with a cofactor protein binds to
the AP-1 site flanking the CD28RE and stabilizes the binding of p65. A
role for AP-1 proteins in stabilizing Rel-like proteins in the IL-2
promoter is supported by the fact that AP-1 participation in the NF-AT
complex confers increased DNA binding stability of NF-ATp (46).
Moreover, a physical interaction between members of the bZip protein
family of transcription factors which includes AP-1, CREB, ATF, and
SRF, and Rel family proteins has been reported in several other
promoters (53). Gel shift experiments have indicated that CD28RE has a
low affinity for p65 (16) but displays a more prominent binding to
c-Rel (16). Our data further suggest that the different members of the
Fos/Jun and Rel families may affect the transcriptional activity of the
IL-2 promoter in a distinct manner (16). Whereas coexpression of c-Rel
and c-jun only moderately enhanced the luciferase activity in LFA-3
costimulated cells, coexpression of p65 and c-jun superinduced the
transcriptional activity of IL-2 promoter. The p50 subunit was unable
to increase the transcriptional activity of the IL-2 promoter or
NF-B or CD28REs, suggesting that the p50 subunit contains a weak
trans-activation domain or that it may bind to suppressive
factors. In contrast to p50, c-Rel and p65 contain strong
trans-activating domains (21, 22). The p65
trans-activating subunit of NF-B has been extensively
characterized (17, 54), but little is known about its interaction with
other nuclear factors. Recently, it has been demonstrated that the
binding of the high mobility group I (Y) proteins to the CD28RE within
the IL-2 strongly potentiated the binding activity of c-Rel (55), while
the binding of Rel-A was not increased. This suggests that the
involvement of cofactors can modulate the activity of various Rel
proteins in a distinct manner. It remains to be delineated whether high
mobility group-like proteins that interact preferentially with p65 are
present in c-Jun/p65-transfected Jurkat cells.
It is interesting that no increase in IL-2 promoter or CD28RE/AP-1 activity can be detected in c-Rel-transfected Jurkat T cells because of a large amount of evidence that this protein is important to the expression of this gene. Several laboratories have now reported c-rel-specific binding to the CD28RE (16, 35, 51, 55) and c-Rel, but not p65, can be found in a CD28RE/AP-1 specific complex also containing c-Fos/c-Jun (35). In conjunction with the decrease in IL-2 production observed in c-Rel-deficient mice, these data strongly suggest that c-Rel is important to IL-2 promoter activity in vivo. However, recent studies have also suggested that c-Rel levels in the nucleus are high under conditions where IL-2 promoter activity is low (35), strongly suggesting that c-Rel is not a limiting factor in IL-2 production but that some other mechanisms limit the activity of this promoter in suboptimal stimulation conditions. This could explain the inability of c-Rel to efficiently trans-activate the IL-2 promoter in transfected Jurkat T cells despite playing a role in the expression of this gene in vivo.
The relevance of the interaction between members of the B/Rel family
and AP-1 transcription factors families for regulation of gene
expression has been demonstrated in a previous study showing that c-Fos
and c-Jun act synergistically with NF-B to trans-activate
a B enhancer element or an AP-1 site (53). In addition we found that
the synergistic effect on the NF-B-like CD28RE was restricted to
c-Jun and p65. In contrast, overexpression of c-Fos suppressed the
potent trans-activation of the IL-2 promoter by p65.
Positive and negative effects of c-fos have been reported previously
(56, 57, 58, 59). Accordingly, it is tempting to propose that the c-fos
suppresses the strong transcriptional activity of p65. Whether this
involves a direct protein-protein interaction between p65 and c-Fos
that decreases the transcriptional activity of p65, sequestering of
c-Jun from p65 by formation of c-Jun/c-Fos heterodimers or other
indirect effects remains to be determined. The activity of a
c-Rel/c-Jun combination was not reduced by c-Fos expression, suggesting
that the negative effect of c-Fos is selective for the p65 Rel family
protein. Thus, both c-Jun and c-Fos are able to affect p65 activity
causing a dynamic modulation of p65-mediated transcription of the
CD28RE. T cells transfected with c-Jun/c-Fos expressed high levels of
an AP-1 reporter gene activity when stimulated with DR/superantigen but
failed to respond to CHO + SEE. This indicates that signal 1 is
required and sufficient for posttranscriptional activation of
c-Jun/c-Fos proteins when these are expressed in high levels. Thus, the
failure of signal 1 to drive the AP-1 reporter in untransfected cells
may relate to the low expression of these gene products.
The fact that B7-1 costimulation superinduced AP-1 activity in
c-Jun/c-Fos-overexpressing cells may reflect that CD28/B7-1 interaction
augment the JNK protein required for translational activation of
c-Jun (60), which may be a rate-limiting step in
c-Jun/c-Fos-overexpressing cells. Indeed, our preliminary data
demonstrate profound induction of JNK activity in B7-1- but not
LFA-3-costimulated Jurkat T cells (data not shown). p65-transfected
Jurkat T cells expressing a NF-B reporter responded only moderately
to signal 1 induction. B7-1 or LFA-3 was required for strong
transcription in these cells. Whether this reflects insufficient
degradation of IB protein is presently under evaluation. LFA-3 and
B7-1 costimulation of p65-expressing Jurkat cells induced similar
levels of NF-B, while the response of a CD28RE reporter was
severalfold higher in B7-1 cells. Possibly, this may reflect a more
efficient activation of endogenous AP-1 protein in B7-1-costimulated T
cells.
In conclusion, our data suggest that the molecular base for transition from a LFA-3-dependent autocrine response to a B7-1 paracrine response involves induction of p65/c-Jun and targeting of the CD28RE within the IL-2 promoter.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Mikael Dohlsten, Astra Draco AB, Preclinical R&D, Box 34, S-221 00 Lund, Sweden. E-mail address:
3 Abbreviations used in this paper: NF-AT, nuclear factor of activated T lymphocytes; CHO, Chinese hamster ovary; SEE, staphylococcal enterotoxin E; AP-1, activation protein-1; CD28RE, CD28 response element; JNK, Jun N-terminal kinase; RLU, arbitrary light units.
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