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to Mediate IL-4 Gene Expression in Response to CD28 Costimulation in T Cells1
Tumor Immunology Program, German Cancer Research Center (DKFZ), Heidelberg, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, and the adaptor protein SLP76 (SH2-domain-containing
leukocyte protein of 76 kDa), induced transcription from the IL-4
promoter. Vav and PKC synergistically activated human IL-4 promoter
transcription and IL-4 mRNA production and were found to be
constitutively associated in vivo. CD3/CD28-induced IL-4 transcription
was inhibited upon coexpression of dominant negative forms of Vav, the
adaptor proteins LAT (linker for activation of T cells) and SLP76,
PKC, and components of the pathways leading to the activation of
c-Jun N-terminal kinase (mitogen-activated protein kinase kinase 7
(MKK7), mixed lineage kinase 3 (MLK3)) and NF-
B (IB kinase 
and IB kinase ß). The Vav/PKC-mediated synergistic activation
of IL-4 transcription was not inhibited by cyclosporin A. Three
independent experimental approaches revealed that Vav/PKC-derived
signals selectively target the P1 and positive regulatory element
(PRE)-I elements contained within the human IL-4 promoter. Vav/PKC
strongly activated a luciferase reporter construct controlled by
trimerized P1 or PRE-I elements and furthermore stimulated DNA binding
of nuclear proteins to the P1 and PRE-I elements. Vav/PKC-induced
transcription from the IL-4 promoter was almost completely abrogated by
mutation of either the P1 or the PRE-I element within the entire IL-4
promoter. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and TNF. The Th2 subset produces IL-4, IL-5, IL-6, and IL-10,
and is involved in humoral immunity and the allergenic response. IL-4
that is present during the priming phase plays an important role in
directing Th2 cell development and preventing the differentiation of
Th1 cells (1, 2). In contrast, IFN- and IL-12
promote the differentiation of Th1 cells (1, 3, 4). The
differential production of cytokines is a critical determinant for the
character of the immune response (5). Therefore, a
dysbalanced Th1/Th2 ratio is seen in many immunological diseases,
including infectious diseases as well as autoimmune and allergic
responses (6).
The molecular mechanisms mediating the cell type-specific expression of
IL-4 were studied intensively by analyzing multiple regulatory
sequences within the 5' and 3' flanking region of the IL-4 gene
(7, 8, 9, 10, 11). The IL-4 promoter harbors several purine-rich
motifs, the so-called P elements, that are critical for lymphocyte
activation-induced transcription (12, 13, 14, 15, 16, 17). These sites,
termed P0, P1, P2, P3, and P4, contain sequences that bind several
distinct transcription factors, including members of the NF-AT family.
Because NF-AT is present in both Th1 and Th2 cells, cell type-specific
expression of IL-4 is conferred by other transcription factors,
including c-Maf, GATA-3, and the AP-1 family member JunB, all of which
are preferentially expressed in Th2 cells (18, 19, 20). The
inducibility of IL-4 transcription is attributed to the concerted
activation of the AP-1 family members JunB, c-Jun, and JunD (10, 20); NF-AT (21); and the NF-B subunits c-Rel and
p65 (10). Such as the induced transcription of the Th1
cytokine IL-2, IL-4 expression is also augmented by triggering the
costimulatory CD28 receptor (22). Two important IL-4
enhancer elements, positive regulatory element I
(PRE-I)4 and P1,
respond to the CD28-derived signals by inducibly binding distinct AP-1
and NF-B family members (10).
CD28 provides costimulatory signals for the TCR CD3-
complex (TCR),
since the full activation of T lymphocytes necessarily requires two
distinct signals (23). The two signaling pathways derived
either from TCR or CD28 merge and synergistically stimulate the
activity of effectors such as JNK and NF-B (24, 25).
Activation of the T cells by TCR/CD28 ligation is immediately followed
by the activation of protein tyrosine kinases (PTKs) of the Src and Syk
families. Once activated, PTKs induce the tyrosine phosphorylation of
multiple target proteins, which enables their binding to other proteins
containing Src homology 2 (SH2) domains or other
phosphotyrosine-binding domains. The induced multiprotein T cell
activation signaling complex (TASC) contains signaling molecules
including phospholipase C and Vav, but also adaptor proteins such as
LAT (linker for activation of T cells) or SLP76 (SH2-domain-containing
leukocyte protein of 76 kDa) (26). These adaptor proteins
lack any intrinsic enzymatic activity, but are required for signal
transduction (27). The TASC couples proximal PTKs to the
various downstream signaling pathways induced by T cell costimulation,
such as the activation of the Ca2+-dependent
calcineurin pathway, and the activation of cascades
triggering Ras and the GTPases Rac and Rho. Tyrosine
phosphorylation and direct binding to LAT activate the enzymatic
activity of phospholipase C, which controls the phosphatidylinositol
lipid metabolism, thereby producing inositol triphosphate and
diacylglycerols (28). Diacylglycerol mediates activation
of PKC family members (29). There is recent evidence that
the Ca2+-independent novel PKC isoform is of
special importance for T cells and contributes to JNK activity and IL-2
production, at least in leukemia cell lines, such as the widely
employed Jurkat cells (30, 31). Also, the Vav
protooncogene product can be inducibly phosphorylated by various PTKs
(32). TCR- and CD28-derived signals already merge at the
level of Vav (33), which activates
Ca2+-independent and -dependent downstream
signaling processes, such as the activation of NF-AT (32).
Tyrosine phosphorylation and phosphatidylinositol-3,4,5-triphosphate
binding of Vav activate its GDP/GTP exchange factor activity for Rac
(34, 35). The Vav-mediated activation of NF-AT and IL-2
promoter transcription (36) can be further augmented by
coexpression of Vav together with SLP76 (36, 37).
The regulatory DNA elements and transcription factors controlling
expression from the IL-4 promoter are intensively studied, but
relatively little is known on the upstream signaling events underlying
this activation. Therefore, the aim of this study was to identify and
characterize the early molecular mechanisms and signaling molecules
that deliver surface receptor-derived signals to the CD28-responsive
PRE-I and P1 elements contained within the IL-4 promoter. In this
study, we show that induced IL-4 transcription strictly relies on the
simultaneous and concerted activation of various signal transduction
pathways. These signaling cascades are not fundamentally distinct from
the pathways described for activation of IL-2 transcription in Th1
cells. Vav-induced IL-4 transcription was further augmented upon
coexpression of SLP76 and synergistically stimulated by PKC, which
was found in constitutive association with Vav. The Vav/PKC module
exerts its stimulating effects on both the P1 and PRE-I element, as
revealed by a variety of experimental approaches.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Jurkat T leukemia cell lines (Jurkat T leukemia cells expressing the large T Ag and Jurkat J16-77) were grown at 37°C and 5% CO2 in RPMI 1640 medium containing 10% (v/v) heat-inactivated FCS, 10 mM HEPES, 1% (v/v) penicillin/streptomycin, and 2 mM glutamine (all from Life Technologies, Gaithersburg, MD). Cells were grown in a humidified incubator at 37°C and 5% CO2. Jurkat cells were transfected by electroporation using a gene pulser (Bio-Rad, Richmond, CA) at 250 V/950 µF with constant amounts of DNA. Stimulation of Jurkat cells was performed in a final volume of 500 µl by adding anti-CD3 (final concentration 2 µg/ml, clone OKT3) and/or anti-CD28 (final concentration 10 µg/ml, clone 9.3).
Luciferase assays
Cells were harvested by centrifugation, washed twice with cold PBS buffer, and lysed in reporter lysis buffer (25 mM Tris-phosphate, 2 mM DTT, 2 mM CDTA, 10% (v/v) glycerol, and 1% (v/v) Triton X-100). The luminometer (Duo Lumat LB 9507, Berthold) was programmed to inject 50 µl of assay buffer (40 mM tricine, 2.14 mM (MgCO3)4Mg(OH)2 x 5 H2O, 5.34 mM MgSO4, 0.2 mM EDTA, 66.6 mM DTT, 540 µM CoA, 940 µM luciferin, and 1.06 mM ATP). Light emission was measured for 10 s after injection. The results were normalized to the activity of ß-galactosidase expressed by a cotransfected lacZ gene under the control of a constitutive RSV promoter.
EMSAs
A total of 1 x 107 Jurkat cells was treated as indicated, and nuclear extracts were prepared essentially as described (38). Briefly, cells were washed once with cold PBS. Cells were centrifuged and the pellet was resuspended in 400 µl cold buffer A (10 mM HEPES/KOH, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, and 0.5 mM PMSF) by gentle pipetting. After incubation for 10 min on ice, 5 µl of 10% Nonidet P-40 was added and cells were lysed by vortex mixing. The homogenate was centrifuged for 60 s in a microfuge, and the pellet containing the cell nuclei was dissolved in 80 µl buffer C (20 mM HEPES/KOH, pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.5 mM PMSF, and 1% (v/v) aprotinin). The extract was centrifuged for 5 min in a microfuge at 4°C, and 5 µg of proteins contained in the supernatant was used for band-shift assays. Binding of proteins to their cognate DNA was measured essentially as described (39). The sense sequences of the oligonucleotides were as follows: P1, 5'-ACGAAAATTTCCAATGTAAACTCATTG-3'; PRE-I, 5'-TAGCAAATTATGGTGTAATTTCCTATGCTGAA-3'.
Coprecipitation experiments and immunoblotting
Cells were washed with PBS and the pellets were resuspended on ice for 30 min in 250 µl of Nonidet P-40 lysis buffer (50 mM Tris/HCl, pH 7.5, 150 mM NaCl, 1 mM PMSF, 10 mM NaF, 0.5 mM sodium vanadate, leupeptine (10 µg/ml), 1% (v/v) Nonidet P-40, and 10% (v/v) glycerol). The extract was centrifugated with 14,000 rpm at 4°C for 10 min. Equal amounts of protein contained in the supernatant were precleared with protein A/G-Sepharose, followed by the addition of 1 µg of immunoprecipitating Abs and 25 µl of protein A/G-Sepharose. After rotation for 4 h on a spinning wheel at 4°C, the immunoprecipitates were washed five times in lysis buffer. Immunoprecipitates were boiled in 1x SDS sample buffer and separated by SDS-PAGE before immunoblotting. The proteins were detected after extensive washing with HRP-coupled secondary Abs using the ECL system (Amersham, Arlington Heights, IL), according to the instructions of the manufacturer.
Plasmids and Abs
The reporter plasmid (-269/+11) IL4-Luc PRE mut was generated
by recloning the PRE-1-mutated IL-4 promoter fragment from pCAT3
(13) into pLuc. The reporter plasmids (-269/+11) IL4-Luc,
(-269/+11) IL4-Luc P1 mut, 3 x PRE-I-Luc, and 3 x P1-Luc
were described previously (10). Expression vectors for Vav
(40), PKC, PKC K/R, and PKC A/E
(31); SLP76 and SLP76 
SH2 (41); LAT and
LAT YYFF (42); MKK7 K/L (43); MLK3 KR
(44); Myc-tagged IKK K44 M and Myc-tagged IKKß K44A
(45) are published. The Vav 319–356 expression vector
was constructed by inserting the appropriate PCR products into a
pEF-BOS-derived vector. The anti-Vav mAbs were from Upstate
Biotechnology (Lake Placid, NY), and the anti-PKC mAbs were
purchased from Transduction Laboratories (Lexington, KY).
Analysis of IL-4 transcription by RT-PCR
One day after transfection, cells were harvested and total RNA was extracted using the RNeasy kit (Qiagen, Chatsworth, CA), according to the instructions of the manufacturer. A total of 1 µg of RNA was reverse transcribed using oligo(dT) primers. Thirty-five PCR cycles were performed to amplify the cDNA fragments encoding IL-4 (456 bp) and ß-actin (661 bp) using specific primers. The PCR fragments were separated on a 1.5% (w/v) agarose gel in 1x TBE buffer at 50 V for 3 h. The DNA was stained with ethidium bromide and analyzed under UV light.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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synergistically stimulate IL-4 transcription
The Vav protein functions as an activator of NF-AT
(36), but also stimulates transcription factor NF-B
(46). Because Vav expression is known to trigger
transcription of the Th1-specific cytokine IL-2, we asked whether Vav
also functions for the activation of the Th2 cytokine IL-4 or whether
its activity is restricted to the control of Th1-specific genes. Jurkat
cells were transiently transfected with a luciferase reporter gene
fused to the human IL-4 promoter fragment -269 to +11 (which contains
the important regulatory elements identified to date) either alone or
together with increasing amounts of an expression vector for Vav. The
transfected cells were either left untreated or stimulated with
agonistic anti-CD3/anti-CD28 Abs. Vav expression dose
dependently increased the basal as well as CD3/CD28-elicited IL-4
transcription (Fig. 1
A). We
next tested the impact of coexpressing the adaptor protein SLP76 on
Vav-induced IL-4 transcription. Activities of the IL-4 promoter were
analyzed by transfection of Jurkat cells with the IL-4 luciferase
reporter construct alone or in different combinations with expression
vectors for Vav and SLP76. Expression of SLP76 alone was able to induce
transcription from the IL-4 promoter almost as efficient as Vav, and
coexpression of both proteins further enhanced IL-4-dependent
transcription (Fig. 1B). Similar to Vav, the PKC isoform
is also known to activate JNK in T cells in response to CD28-derived
signals (30, 31). Because PKC expression is largely
restricted to hemopoietic cells (47) and Ag stimulation
leads to its selective localization to the contact region between T
cells and APC (48), we studied the effect of transient
overexpression of Vav and PKC on the induction of IL-4
transcription. Expression vectors for Vav and a constitutively active
form of PKC (PKC A/E) were transfected either alone or in
combination along with a IL-4-luciferase reporter gene into Jurkat
cells. Expression of PKC A/E activated IL-4 transcription even more
efficiently than Vav, but coexpression of both proteins synergistically
triggered IL-4 transcription (Fig. 1B). Throughout,
comparable results were obtained when the wild-type form of PKC was
used instead of PKC A/E (data not shown). However, the
constitutively active form was used because it is more efficient in
gene activation and thus increases the sensitivity for the detection of
its regulatory functions.
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by another experimental approach, we tested the impact of
ectopically expressed Vav and PKC proteins on transcription of the
endogenous IL-4 gene. The IL-4-producing Jurkat T cell subline J16-77
was transfected with expression vectors for Vav and PKC A/E either
alone or in combination, or with the empty expression vector as a
control. Although expression of the individual proteins only moderately
increased IL-4 transcription, coexpression of Vav and PKC A/E
synergistically augmented IL-4 transcription in its natural chromatin
context, as revealed by RT-PCR (Fig. 2,
A and B). The strong functional synergism between
PKC and Vav might be explained by their physical interaction, which
was previously described in thymocytes (49). To test
whether this interaction also occurs in Jurkat cells and whether T cell
costimulation changes mutual binding of both proteins,
coimmunoprecipitation experiments were performed. Jurkat cells were
left untreated or stimulated with anti-CD3/anti-CD28 Abs. The
Vav protein was immunoprecipitated from an aliquot of the cell lysates,
and the associated proteins were detected by immunoblotting. PKC was
found to be constitutively attached to Vav (Fig. 2C),
whereas binding of the control protein SLP76 was inducible upon T cell
costimulation (data not shown). In a complementary experimental
approach, PKC was immunoprecipitated from another aliquot of the
cell lysate, followed by the detection of associated Vav protein by
Western blotting. Also, this experiment revealed the mutual and
constitutive binding of Vav and PKC.
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, and NF-B
To gain insight into the relative contribution of the various
pathways leading to the induced transcription of IL-4, we tested the
effects of expressing various dominant negative variants of signaling
molecules with pathway specificity on CD3/CD28-induced IL-4
transcription. Jurkat cells were transfected with the IL-4 luciferase
reporter gene construct together with expression vectors for dominant
negative forms of Vav, LAT, SLP76, PKC, MLK3, and MKK7. Each of
these dominant negative signaling molecules significantly impaired
CD3/CD28-induced IL-4 transcription (Fig. 3A). This reveals that adaptor
proteins (LAT, SLP76), the JNK signaling pathway (MLK3 and MKK7), as
well as PKC and Vav are not only involved, but required and
necessary for T cell costimulation-triggered activation of IL-4
transcription. NF-B participates in IL-4 expression, but neither the
relative contribution of this transcription factor nor the involved
mechanism of NF-B activation is known. To test whether
IL-4-activating NF-B subunits are generated via activation of the
IB kinases (IKKs) or by one of the recently described
IKK-independent mechanisms (50, 51, 52), we tested the impact
of expressing increasing amounts of dominant negative forms of IKK
and IKKß on Vav/PKC A/E-induced IL-4 transcription in Jurkat
cells. The dominant negative form of IKKß inhibited the
Vav/PKC-submitted IL-4 luciferase activation more completely than
IKK KM, suggesting that IKKß is more important for this activation
process. A comparable result was obtained, when IL-4 transcription was
induced by CD3/CD28 stimulation (data not shown). Furthermore, this
experiment shows that both the Vav- and PKC-derived signals
independently lead to the activation of IKKß.
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-submitted signals rely on
Ca2+-dependent signaling events, which are known
to contribute to CD3/CD28-induced IL-4 transcription (53).
Jurkat cells were transfected with a IL-4-dependent luciferase reporter
construct and various combinations of expression vectors for Vav and/or
constitutively active PKC. The transfected cells were further grown
in the absence or presence of CsA, which blocks the
Ca2+-dependent activation of the phosphatase
calcineurin and thereby inhibits Ca2+-dependent
signaling events (54). Basal and PKC-elicited IL-4
transcription were unchanged, but Vav-mediated IL-4 transcription was
strongly impaired in the presence of CsA (Fig. 3
C).
Interestingly, the Vav/PKC-mediated synergistic activation of IL-4
transcription was not influenced by CsA. This reveals that Vav-derived
signals mediating the synergism with PKC do not rely on
Ca2+-dependent processes.
Vav and PKC activate the CD28-responsive PRE-I and P1 elements
contained within the IL-4 promoter
Because both Vav and PKC respond to signals from the
CD28 receptor (30, 55), we tested whether they could act
on the CD28-responsive PRE-I and P1 elements. Luciferase reporter genes
controlled by three copies of the PRE-I (3x PRE-I-luciferase) or P1
(3x P1-luciferase) element, respectively, were cotransfected with
expression vectors for Vav and/or PKC A/E into Jurkat cells (Fig. 4A). The PRE-I- and the
P1-controlled reporter genes were efficiently induced upon individual
expression of Vav or PKC. Simultaneous expression of both activators
resulted in the synergistic activation of both reporter genes, showing
that the behavior of the IL-4 promoter is well reflected by the
individual PRE-I and P1 elements (Fig. 4A). Subsequently, we
investigated the relative importance of the P1 and PRE-I elements for
Vav/PKC-induced transcription of the IL-4 gene. Jurkat cells were
transfected with expression vectors for Vav and PKC A/E, together
with the wild-type IL-4 luciferase construct ((-269/+11) IL4-Luc) or
IL-4 reporter constructs mutated either in the PRE-I sequence
((-269/+11) IL4-Luc PRE mut) or the P1 element ((-269/+11) IL4-Luc P1
mut). Vav/PKC-elicited transcription was severely
impaired upon mutation of the P1 or the PRE-I element,
respectively (Fig. 4B). This shows that induced IL-4
transcription necessarily relies on the integrity of each of both
sites. Furthermore, this experiment reveals that the
Vav/PKC-triggered signals act mainly at the P1 and PRE-I elements
and not at other elements within the minimal essential IL-4 promoter.
The effects of Vav/PKC on the PRE-I and P1 elements were further
characterized by EMSAs. Jurkat cells were transfected with different
combinations of expression vectors for Vav, PKC A/E, or the empty
expression plasmid as a control. The next day, cells were either left
untreated or stimulated with anti-CD3/anti-CD28 Abs, and
DNA-binding activity in nuclear extracts was determined by EMSAs using
either the P1 element (position -79 to -54) or the PRE-I sequence
(position -250 to -221) as a probe. EMSAs with the labeled P1 element
showed that expression of either Vav or PKC A/E alone resulted in a
weak induction of DNA binding of proteins contained in the inducible
DNA/protein complex I. Formation of this complex could be further
triggered upon CD3/CD28 stimulation. Coexpression of Vav and PKC A/E
strongly induced the DNA-binding activities of complex I, showing that
the synergistic behavior is also seen at the level of DNA binding (Fig. 5A). Vav/PKC-induced
binding of complex I was further enhanced upon CD28 stimulation (data
not shown) and CD3/CD28 ligation. In contrast to complex I, DNA/protein
complex II was already present in extracts from the untransfected,
nonstimulated cells, and showed only a weak inducibility upon
Vav/PKC A/E coexpression and T cell activation. The relatively
moderate effects of Vav/PKC on induced binding of proteins to the P1
element can be attributed to the limited transfection efficiency of
Jurkat cells. In parallel, we tested proteins contained in the nuclear
extracts for binding to the labeled PRE-I element. From four different
DNA/protein complexes, only complex I was inducible (Fig. 5B). Induction of complex I was apparent by expression of
Vav or PKC A/E and further enhanced by CD3/CD28 ligation.
Coexpression of both proteins synergistically augmented complex I
formation. T cell costimulation even further enhanced binding of
proteins contained in complex I. These results show that the Vav/PKC
module exert their stimulatory effects on IL-4 transcription by
triggering pathways leading to the enhanced recruitment of
transcription factors to the P1 and PRE-I elements.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and SLP76 as activators of
IL-4 mRNA production. Our findings indicate that the basic mechanisms
leading to the transcriptional activation of the Th2 cytokine IL-4 are
not fundamentally distinct from those leading to the transcription of
the Th1 cytokine IL-2. A further parallel between IL-2 and IL-4
activation is the complete dependence of IL-4 transcription on the
coordinated activation of several transcription factors. Therefore,
IL-4 expression does not occur upon inhibition of each of the various
signaling cascades. Inhibition of the JNK activation pathway by
expression of dominant negative MKK7 and MLK3, which prevents the
induced phophorylation of c-Jun and JunB (20) and thereby
the activation of AP-1 (56), efficiently blocked the
CD3/CD28-triggered transcription from the IL-4 promoter. Similarly,
interference with the activation NF-B pathway by a dominant negative
form of IKKß completely precluded IL-4 transcription induced by
Vav/PKC or CD3/CD28 ligation. This observation shows that the
Vav/PKC-derived signal activates NF-B via the IB kinase
complex. Furthermore, this result is in good accordance with the
predominant role of IKKß for the stimulus-induced phosphorylation of
IB, as revealed by gene disruption experiments (57, 58). The importance of NF-B for IL-4 transcription is also
evident from transgenic mice constitutively expressing a transdominant
negative form of IB- in T lineage cells, which fail to produce
IL-4 induced by stimulation of T cells with phorbol ester and ionomycin
(59). Besides AP-1 and NF-B, NF-AT also contributes to
IL-4 transcription, as evident from the impaired IL-4 production in
mice lacking the NF-ATc gene (60, 61). The impairment of
CD3/CD28-induced IL-4 transcription by dominant negative forms of Vav
and PKC reveals their importance, but does not allow conclusions on
their relative contribution in signal transduction, because it is
reasonable to assume that further molecules participate in this
activation process (62).
Following their activation by TCR/CD28 stimulation, Vav, SLP76, and
PKC trigger IL-4 production by overlapping and separate pathways.
The involvement of the LAT protein in CD3/CD28-induced IL-4
transcription remains uncertain, because expression of wild-type LAT
failed to induce IL-4 expression (data not shown). This finding might
be explained by the relative abundance of this protein, since on the
other hand CD3/CD28-elicited IL-4 transcription was inhibited upon
expression of dominant negative LAT (compare Fig. 3A). The
IL-4 promoter activating SLP76 protein is comprised of three motifs
allowing for protein/protein interactions: an N-terminal acidic SAM
domain, a middle proline-rich motif that binds to the SH3 domain of
Grb2 family proteins, and a C-terminal SH2 domain (63).
The importance of SLP76 for T cell activation is seen in a variety of
experimental models: TCR-mediated signals are abrogated in a mutant
Jurkat cell line that has lost expression of SLP76 (64).
SLP76-/- mice show a block in pre-TCR
signaling, as these mice exhibit arrest of thymocyte development
(65, 66). Because all three domains of SLP76 are important
for SLP76-induced activation of NF-AT and IL-2 transcription in Jurkat
(41), the function of SLP76 for signaling might be
attributed to its structural role as an important architectural
component of the TASC. There is recent evidence that SLP76 and Vav
induce IL-2 transcription by overlapping and distinct signaling
pathways. Cotransfection experiments using a mutated form of SLP76
unable to interact with Vav show that both proteins are still able to
synergistically trigger IL-2 expression (37). The Vav
protein is known to exert its activating function by several
mechanisms, including the induction of
Ca2+-dependent NF-AT activation (67)
and the Ca2+-independent direct stimulation of
Rac (34, 35). This study reveals that the Vav-derived
signals mediating the synergism with PKC are
Ca2+ independent. The Vav-submitted and
Rac-transmitted signals then activate the MAPKs JNK and p38 as well as
transcription factor NF-B (33, 46). The concept that
Vav delivers its signals to more than one pathway is corroborated by a
recent study describing that the restoration of intracellular
Ca2+ fluxes by ionomycin in
Vav-/- T cells rescues only the activation of
NF-AT, but not of NF-B (46). The role of Vav for IL-4
production is also seen in Vav-/- mice, in
which defective IgE Ig class switching can be attributed to compromised
T cell help due to impaired IL-4 transcription (68).
In this study, we describe the physical and functional interaction
between Vav and PKC. This finding is in good agreement with a
previous report showing that PMA, a pleiotropic PKC activator, induces
the release of IL-4 from human PBL in the presence of ionomycin
(69). However, this study also revealed that PMA/ionomycin
fails to trigger IL-4 production in human basophils, raising the
possibility that PKC may not be operational in all IL-4-producing
cell types. It is not likely that other PKC isoforms are involved in
IL-4 expression, because there is ample evidence that PKC, 
, and
/
and further isotypes are unimportant in the process of T cell
activation (30, 31, 48). It is currently not clear whether
the mutual binding of Vav and PKC is required for their synergistic
behavior. The activation-induced translocation of PKC to the cell
membrane is prevented by the 14-3-3-
protein (70), and
it remains to be seen whether Vav interferes with this regulatory
protein/protein interaction. PKC-induced activation of JNK is
independent of MEKK1 and SEK (31), suggesting that it
targets this effector kinase by an alternative pathway. Besides its
functional synergism with Vav described in this work, PKC also
cooperates with calcineurin and together their signals converge on Rac,
leading to the activation of JNK (30). Therefore, it seems
that PKC, such as Vav, is placed upstream from Rac within the
signaling cascade. However, in the absence of well-defined substrates
for PKC, the exact mechanisms by which this kinase triggers T cell
signaling processes remain incompletely defined. Future studies must
show whether Vav and PKC act in parallel, consecutive, or
overlapping pathways.
Similar to Vav, PKC also further augments signals from the CD28
receptor (30, 31). Accordingly, Vav and PKC trigger
IL-4 transcription by targeting the CD28-responsive P1 and PRE-I
elements, although it cannot be ruled that further elements outside
from the minimal essential promoter portion (-269 to +11) are also
affected. In this study, we show that the Vav/PKC module triggers
binding of transcription factors to the P1 and PRE-I elements. This may
be explained by JNK-induced phosphorylation of JunB, which was shown to
allow cooperative DNA binding with c-Maf to the P1 site
(20). Such a mechanism may also apply for the PRE-I site,
which is also contacted by AP-1 family members (10).
Accordingly, the simultaneous expression of Vav and PKC
synergistically stimulated the activity of JNK1 (S. P. Hehner and
M. L. Schmitz, unpublished data). Further candidates for
Vav/PKC-induced DNA-binding proteins are NF-B family members,
which contact the P1 site, and NF-AT proteins, which bind both
elements. Studies are underway to further our understanding on the
molecular mechanisms underlying the synergism between Vav and
PKC.
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Acknowledgments |
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Footnotes |
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2 S.P.H. and M.L.-W. contributed equally to this paper.
3 Address correspondence and reprint requests to Dr. M. Lienhard Schmitz, Department of Immunochemistry (G0200), German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany. E-mail address:
4 Abbreviations used in this paper: PRE-I, positive regulatory element I; CsA, cyclosporin A; IKK, IB kinase; JNK, c-Jun N-terminal kinase; LAT, linker for activation of T cells; MKK, mitogen-activated protein kinase kinase; MLK3, mixed lineage kinase 3; PKC, protein kinase C; PTK, protein tyrosine kinase; SH2, Src homology 2; SLP76, SH2-domain-containing leukocyte protein of 76 kDa; TASC, T cell activation signaling complex.
Received for publication September 16, 1999. Accepted for publication January 24, 2000.
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ß T-cell-receptor transgenic system. Proc. Natl. Acad. Sci. USA 89:6065. production and diminishes interleukin 4 inhibition of such priming. Proc. Natl. Acad. Sci. USA 90:10188.B transcription factor activation in T cells. Ann. NY Acad. Sci. 766:245.[Medline]
to activate JNK and IL-2 promoter in T lymphocytes. EMBO J. 17:3101.[Medline]
, a selective upstream regulator of JNK/SAPK and IL-2 promoter activation in Jurkat T cells. Eur. J. Immunol. 29:132.[Medline]
B activation is antagonized by E1B 19K. Mol. Cell. Biol. 16:4052.[Abstract]
and cellular stresses. EMBO J. 16:7045.[Medline]
B kinase-ß: NF-B activation and complex formation with IB kinase- and NIK. Science 278:866.B pathways. Proc. Natl. Acad. Sci. USA 96:3035., a novel member of the protein kinase C (PKC) gene family expressed predominantly in hematopoietic cells. J. Biol. Chem. 268:4997. during T-cell activation. Nature 385:83.[Medline]
B activation. Proc. Natl. Acad. Sci. USA 96:429.B by UV. EMBO J. 17:5170.[Medline]
B through two distinct mechanisms. Proc. Natl. Acad. Sci. USA 95:13012.B activation in IKK-ß-deficient mice. Immunity 10:421.[Medline]
B kinase 2 gene. Science 284:321.B in T-lineage cells leads to a dramatic decrease in cell proliferation and cytokine production and to increased cell apoptosis in response to mitogenic stimuli, but not to abnormal thymopoiesis. J. Immunol. 162:6442.1 in an SLP-76-deficient T cell. Science 281:413. (PKC ) and 14-3-3 in T cells: 14-3-3 overexpression results in inhibition of PKC translocation and function. Mol. Cell. Biol. 16:5782.[Abstract]
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