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Department of Medicine, Pulmonary and Critical Care Section, Yale University School of Medicine, New Haven, CT 06520
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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or PGs, inhibits T cell activation. In effector
T cells, an increase in the level of intracellular cAMP inhibits
cytokine production in Th1 cells but stimulates cytokine production in
Th2 cells. Here we report that cAMP-induced effects in Th2 cells occur
independently of the protein kinase A pathway, which is the major
mediator of cAMP-induced signaling events in most cell types. Instead,
cAMP stimulates activation of p38 mitogen-activated protein kinase in
Th2 cells. This appears to be a Th2-selective event because cAMP barely
increased p38 phosphorylation in Th1 cells. We show that in Th2 cells,
cAMP promotes the production of both IL-5 and IL-13, which play
distinct but critical roles in asthma pathogenesis. Our data also show
that cAMP causes increased phosphorylation of the transcription factor
GATA-3, which we have shown is a critical regulator of Th2 cytokine
gene expression and, in turn, of airway inflammation in mice. Thus,
Th2-specific GATA-3 expression and p38 mitogen-activated protein kinase
activation together provide a molecular basis for the differential
effects of cAMP in the two T helper cell
subsets. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, PGE2, cholera
toxin, forskolin or by direct stimulation of cAMP pathways with
dibutyryl cAMP
(bt2cAMP)3,
inhibits IL-2 and IL-2 receptor expression thereby blocking cell cycle
progression and cell proliferation (1, 2, 3, 4, 5, 6). In effector
CD4+ T cells, elevation of intracellular cAMP is
associated with inhibition of Th1 cytokine production but augmentation
of IL-5 gene expression in Th2 cells (5, 7, 8). In
addition, intracellular cAMP-elevating agents have been shown to
inhibit IL-12 production (9).
Among the Th2 cytokines, IL-5 has been associated with eosinophilic
inflammation in many disease states, including asthma, by promoting the
proliferation, differentiation, activation, and survival of eosinophils
(10, 11, 12, 13, 14). One unique feature of IL-5 gene expression is
that cAMP-elevating agents such as IL-1, forskolin, and
PGE2 can strongly induce IL-5 gene expression
(5, 8, 15, 16), suggesting a positive regulatory role for
cAMP in IL-5 production. Interestingly, an analysis of the IL-5
promoter region reveals binding sites for several transcription factors
including GATA-3, AP-1, Oct, Ets proteins, and NFAT
(17, 18, 19) but no cAMP response element. This raises the
possibility that signal transduction pathways other than the
well described cAMP-dependent protein kinase A (PKA) pathway are
involved in cAMP-mediated IL-5 gene expression in Th2 cells.
In recent years, considerable effort has been mounted to dissect the signaling events in T cells. Full activation of T cells requires at least two signals from TCR and costimulatory receptors (20, 21). Several protein kinases such as the family of mitogen-activated protein kinases (MAPKs) are involved in the transmission of extracellular signals to intracellular targets (22, 23, 24). TCR engagement activates the extracellular signal-related kinase (ERK) pathway, and costimulation through CD28 causes c-Jun N-terminal kinase (JNK) activation, which are required for full activation of T cells (25, 26). T lymphocyte activation by cAMP was shown to be associated with down-regulation of both ERK and JNK pathways (27). Interruption of the mitogen-activated protein/extracellular signal-related kinase kinase (MEK/ERK) pathway with the pharmacological ERK inhibitor PD98059 enhanced Th2 cytokine (IL-4, IL-5, and IL-13) expression induced by PMA/CD3 or by CD3/CD28 (28). The studies using JNK1-deficient mice also showed an enhanced Th2 response (29), suggesting that JNK1 may play a negative role in Th2 cytokine production. Collectively, these studies suggest that the kinases ERK and JNK do not play significant roles in Th2 cytokine gene expression.
In the past few years, several reports have established the importance
of p38 MAPK in T cell activation and development. The MKK6-p38
signaling pathway was found to be critical for negative selection of
thymocytes during T cell development (30). A specific p38
inhibitor, the pyridinyl imidazole SB203580, has been used to define
the cellular functions of p38 in many cell types including Th1 and Th2
effector cells (31, 32, 33, 34, 35, 36). Several studies have investigated
the role of p38 kinase using anti-CD3 and anti-CD28 Abs as
stimuli of T cells. In murine T cells, while one study showed that
blockade of p38 activity partially blocks TCR-stimulated IFN-
production by mouse Th1 cells without affecting Th2 cytokine (IL-4 and
IL-5) production (33), another study observed reductions
in IL-2, IL-4, and IFN- production by inhibition of p38 activity
(36). In human Th2 effector cells, SB203580 inhibited IL-4
production with lesser effects on IFN- and IL-2 production in Th1
cells (35). The substrates of p38 include the
transcription factors activating transcription factor (ATF)-2
(37, 38), CHOP (38, 39), and MEF2C
(40) and the kinases MAPKAP-2 and -3 (41, 42). However, the physiological substrate(s) of p38 MAPK in T
cells have not been identified.
In this study, we investigated the signaling events after stimulation of murine Th2 effector cells by cAMP. Our results show that cAMP-induced effects in Th2 cells are independent of the PKA pathway but instead are mediated by p38 MAPK. This appears to be a Th2-selective event because cAMP barely increased p38 kinase activity in Th1 cells. We show that cAMP not only promotes IL-5 production but also increases expression of the IL-13 gene in Th2 cells. In addition, we show that cAMP causes increased phosphorylation of the transcription factor GATA-3, which, as we have shown previously, is a critical regulator of Th2 cytokine gene expression and of airway inflammation in vivo (43, 44). We also demonstrate phosphorylation of GATA-3 by active p38 MAPK. Taken together, the differential effect of cAMP on Th1 and Th2 cytokine production can be, at least partly, explained by differential induction of p38 MAPK in Th2 cells which, in turn, triggers phosphorylation of GATA-3.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Nuclei were isolated from 5 x 107
unstimulated or stimulated D10 cells, suspended in nuclear storage
buffer (40% glycerol; 50 mM Tris-HCl, pH 8.0; 5 mM
MgCl2; 0.1 mM EDTA), quick frozen in a dry
ice-methanol bath and stored at -80°C until further use. Thawed
nuclei were incubated for 30 min at 30°C in a buffer containing 20%
glycerol; 20 mM Tris-HCl, pH 8.0; 100 mM KCl; 4.5 mM
MgCl2; 2 mM DTT; 0.4 mM each of ATP, GTP, and
CTP; and 100 µCi of [-32P]UTP (3000
Ci/mmol; Amersham, Arlington Heights, IL). Transcription was stopped by
the addition of 50 µl of termination buffer containing 50 mM
Tris-HCl, pH 8.0; 0.5 M NaCl; 5 mM EDTA; 2 U DNase I; 40 U RNasin
(Promega, Madison, WI); and incubating at 30°C for 15 min.
Transcripts were isolated by Trizol LS (Life Technologies, Grand
Island, NY) according to the instructions of the manufacturer.
Unincorporated radioactivity was removed from the final RNA pellets
using spin columns, and the incorporated radioactivity was counted by
liquid scintillation. Equal counts of radiolabeled RNA were hybridized
to cDNAs linearized with the appropriate restriction enzymes and
immobilized as slots on a nylon membrane (Schleicher & Schuell,
Keene, NH).
RNA isolation and Northern analysis
Total cellular RNA was prepared using Trizol (Life Technologies)
according to the instructions of the manufacturer. Ten micrograms of
total RNA from each sample was fractionated on a formaldehyde agarose
gel and transferred to a nylon membrane. A 
450-bp
SacI-AccI fragment of murine IL-5 cDNA was
labeled with [-32P]dCTP using a Random
Primer DNA labeling kit (Boehringer Mannheim, Indianapolis, IN).
Hybridization was performed using QuikHyb (Stratagene, La Jolla, CA)
according to the instructions of the manufacturer.
Analysis of MAPK activation
Rested Th2 cells (D10) were suspended in Bruff’s medium containing 0.5% FBS for 3 h to reduce background phosphorylation. Cells were incubated with or without H89 (Calbiochem, San Diego, CA) for 1 h before stimulating with cAMP or forskolin. Twenty minutes after stimulation, cells were washed once with cold PBS, resuspended in lysis buffer (50 mM Tris-HCl, pH 7.4; 250 mM NaCl; 2 mM EDTA; 50 mM NaF; 0.1 mM Na3VO4; 0.5% Nonidet P-40; and protease inhibitors), and incubated for 20 min on ice. The insoluble cell debris was pelleted by centrifugation at 4°C at 14,000 x g for 10 min. The protein concentration in cell lysates was determined (Bio-Rad, Hercules, CA). Equal amounts of total protein were suspended in SDS-PAGE sample buffer, fractionated on 4–20% gradient SDS polyacrylamide gels (Bio-Rad), and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA). To analyze the activation of the individual MAPKs, p38, ERK, and JNK, the blots were blocked with 5% nonfat dry milk in TBST (25 mM Tris-HCl, 0.15 M NaCl, and 0.1% Tween 20) for 1 h at room temperature and subsequently incubated with rabbit anti-phospho-p38, anti-phospho-ERK, and anti-phospho-JNK Abs (New England Biolabs, Beverly, MA) followed by anti-rabbit IgG-HRP conjugate. Blots were then visualized by chemiluminescence (Pierce, Rockford, IL). To ensure similar protein loading in each lane, the phospho blots were stripped and the membranes were reprobed with Abs against p38, ERK, and JNK.
To examine kinase activation in Th1 and Th2 cells derived from primary
CD4+ T cells, CD4+ T cells
from DO11.10 TCR transgenic mice were prepared by positive selection
using mAbs to CD4 coupled to magnetic and a magnetic-activated cell
sorting (MACS) separation column- beads (Miltenyi Biotec, Auburn, CA).
Syngeneic T cell-depleted splenocytes were used as APCs, which were
prepared by depletion of CD4 and CD8 T cells using Ab-coupled magnetic
beads, and the cells were subsequently treated with mitomycin C.
Cultures were set up in flasks containing CD4+ T
cells and T cell-depleted APCs together with
pOVA323–339 (5 µg/ml), IL-2 (50 U/ml),
anti-IL-4 and IL-12 (5 ng/ml) for Th1 polarization, and
anti-IFN- and IL-4 (200 U/ml) for Th2 polarization for 7 days.
At the end of this period, cells were recovered and stimulated with
bt2cAMP for 20 min. The phosphorylation of p38
MAPK was analyzed as described above.
Analysis of cytokine production
D10 cells (2 x 106) were cultured at 37°C for 1 h in the presence or absence of kinase inhibitor (SB203580). Cells were incubated with bt2cAMP and brefeldin A (1 µg/ml) for 4 h, and IL-5 production was evaluated by intracellular staining procedures. After washing twice in staining buffer, cells were fixed and permeabilized with Cytofix/Cytoperm solution for 20 min at 4°C. PE-anti-IL-5 was used to detect cytokine expression. Samples were then analyzed by FACS (Becton Dickinson, Mountain View, CA). The forward scatter and side scatter properties of the cells were used to exclude dead cells from the analysis.
Spleens were harvested from DO11.10 TCR transgenic mice, and
CD4+ T cells were prepared by positive selection
using mAbs to CD4 coupled to magnetic beads (MACS; Miltenyi Biotec) and
a MACS separation column. Syngeneic T cell-depleted splenocytes were
used as APCs, which were prepared by depletion of CD4 and CD8 T cells
using Ab-coupled magnetic beads (MACS; Miltenyi Biotec), and the cells
were subsequently treated with mitomycin C. Cultures were set up in
flasks containing CD4+ T cells and T
cell-depleted APCs together with pOVA323–339 (5
µg/ml), IL-2 (50 U/ml), anti-IFN-, and IL-4 (200 U/ml) for 7
days. At the end of this period, cells were stimulated with
PMA+bt2cAMP or PMA+ ionomycin for 48 h in
the presence or absence of SB203580. Culture supernatants were assayed
for the presence of IL-4, IL-5, or IL-13. The lower limits of detection
for the cytokines were IL-4, 5 pg/ml; IL-5, 5 pg/ml (Endogen,
Cambridge, MA); and IL-13, 1.5 pg/ml (R&D Systems, Minneapolis,
MN).
Transfection assays
IL-5 and IL-4 reporter constructs have been described previously
(43). For the (GATA)2-Luc construct,
the GATA binding doublet present in the IL-5
promoter
(CTATCTGATTG) was dimerized and linked to the
minimal 
IL-5 promoter (5' border at -40) using BglII and
BamHI introduced into PCR primers as cloning sites. Rested
D10 cells were washed once and resuspended in serum-free RPMI 1640
medium, and transfections were conducted as described previously
(43, 44). The cells were left on ice for 15 min and then
diluted in 10 ml of Bruff’s medium at 37°C overnight. Cells were
harvested 20 h poststimulation with
PMA+bt2cAMP or PMA+ionomycin for reporter gene
assays.
Cell labeling and immunoprecipitation
D10 cells were starved in phosphorus-free RPMI 1640 medium containing 5% dialysed FCS (Life Technologies) for 1 h. 32P-labeled orthophosphate (10 mCi/ml, ICN Pharmaceuticals, Costa Mesa, CA) was added to cells (1 mCi/10 x 106 cells) in the presence or absence of bt2cAMP, and the cells were incubated for 3 h. Cells were washed and lysed in lysis buffer on ice for 20 min. Cell extracts were harvested by centrifugation at 14,000 x g for 10 min to remove cell debris. GATA-3 protein was immunoprecipitated using agarose-conjugated monoclonal anti-GATA-3 Ab (Santa Cruz Biotechnology, Santa Cruz, CA) by incubation at 4°C overnight. The immunoprecipitates were washed three times in washing buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 2 mM EDTA, 50 mM NaF, 0.1 mM Na3VO4, 0.5% Nonidet P-40, and protease inhibitor). The samples were then boiled in 2x SDS sample buffer and fractionated by SDS-PAGE. The gel was stained by Coomassie blue before autoradiography.
Production of recombinant GATA-3 and in vitro kinase assay
An EcoRI/XbaI (tagged by PCR) fragment of
murine GATA-3 cDNA was cloned into the insect cell expression vector
pIZT/V5.His (Invitrogen, Carlsbad, CA). To express recombinant GATA-3
protein, Sf9 cells were transfected with the construct pIZT/G3.V5.His
using the insectinPlus transfection kit (Invitrogen) according to the
instructions of the manufacturer. Protein expression was monitored by
GFP expression, and, at 60 h posttransfection, cells were lysed
and GATA-3 fusion protein was purified using a nickel-chelated column.
To perform in vitro kinase assay, 2 µg of GATA-3 fusion protein or 10
µg of rATF-2 (New England Biolabs) was incubated at 30°C for 30 min
with active p38 MAPK (Stratagene) in the presence of 1 µCi of
[32P]ATP (>3000 Ci/mmol; NEN, Boston, MA).
Following incubation, the phosphorylated GATA-3 fusion protein was
immunoprecipitated with anti-GATA-3 Ab, the immunoprecipitates were
fractionated by SDS-PAGE, and the gels were analyzed by a
Phosphorimager (Cyclone; Packard, Meriden, CT).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine whether cAMP induces nascent transcription of the
IL-5 gene, nuclear run-on assays were conducted. The mitogen Con A,
which is a potent inducer of IL-5 gene expression, was used as a
positive control. As shown in Fig. 1
A, both Con A and
bt2cAMP induced new transcription of the IL-5
gene in Th2 (D10) cells. Because PKA is the major mediator of
cAMP-induced effects in many cell types, we first investigated its role
in cAMP-induced IL-5 mRNA expression. As shown in Fig. 1B,
cAMP-induced IL-5 gene expression (lane 2) was not
inhibited in the presence of the PKA inhibitor H89 (lanes
3 and 4) or HA1004 (data not shown), suggesting that
the PKA pathway is not critical for cAMP-mediated IL-5 production in
Th2 effector cells. However, the PKA signaling pathway was previously
implicated in IL-5 promoter activation in the murine thymoma line EL-4
(45). Expression of the catalytic subunit of PKA in EL-4
cells was shown to efficiently activate the IL-5 promoter in the
presence of phorbol ester (45). As shown in Fig. 1B, the involvement of the PKA pathway in EL-4 cells was
also evident in our experiments where PMA- and
bt2cAMP-stimulated IL-5 mRNA expression was
potently inhibited by H89. Thus, unlike in EL-4 cells, cAMP alone is
sufficient to efficiently activate IL-5 gene expression in Th2 cells
without dependence on the PKA signaling pathway.
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The MAPKs, p38, JNK, and ERK, have been implicated in T cell
activation. cAMP has been shown to inhibit JNK and ERK activation in T
cell lines (Jurkat and EL4 cells) (27, 46), but no study
has yet addressed the effect of cAMP on p38 MAPK activation. We
investigated the effect of cAMP on the activation of MAPKs in D10
cells. As shown in Fig. 2A, a
low level of basal phospho-p38 was detected in D10 cells, and
stimulation with bt2cAMP enhanced p38
phosphorylation. However, bt2cAMP did not induce
phosphorylation of either ERK or JNK. In fact, the phosphorylation of
JNK decreased slightly after cAMP stimulation. Our initial examination
of the kinetics of phosphorylation showed optimal p38 phosphorylation
at 20 min poststimulation; therefore, this time point was chosen in all
subsequent experiments. We did not detect ERK or JNK phosphorylation at
any of the other time points examined (from 1 to 60 min of
stimulation). These results indicated that in Th2 cells, cAMP
preferentially activates p38 kinase but not ERK or JNK. Forskolin,
which elevates intracellular cAMP by stimulating adenylate cyclase,
also enhanced p38 phosphorylation in D10 cells (Fig. 2B).
However, when cells were pretreated with H89, there was no effect on
cAMP- or forskolin-induced p38 phosphorylation. If anything, H89
enhanced p38 phosphorylation in unstimulated cells. This suggests that
the PKA pathway and the p38 MAPK signaling pathway are mutually
antagonistic in Th2 cells such that inhibition of the PKA pathway
raises the basal phosphorylation of p38. Taken together, these data
suggested that cAMP induces p38 MAPK in Th2 cells in a PKA-independent
fashion.
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C). The results were essentially the same at
other time points post stimulation. The Th2-specific activation of p38
MAPK was also evident in Th1 and Th2 cells generated by differentiation
of primary CD4+ T cells (Fig. 2D).
These results indicate that cAMP-induced p38 phosphorylation is a
Th2-specific event and strongly suggest that cAMP-induced signaling
pathways are distinct in Th1 and Th2 cells. Thus, the p38 MAPK pathway
is the dominant cAMP-induced signaling pathway stimulated in Th2 but
not in Th1 cells. Inhibition of p38 MAPK inhibits cAMP-induced IL-5 and IL-13 production in Th2 cells
Having observed the Th2-specific activation of p38 kinase by cAMP,
we next examined the role of this kinase in cAMP-induced IL-5 gene
expression. D10 cells were stimulated with PMA and cAMP for 4 h,
and IL-5 protein production in unstimulated and stimulated cells was
estimated by intracellular staining. As shown in Fig. 3A, the majority of the
unstimulated cells displayed a mean fluorescence intensity (MFI) of
<35, whereas 64% of the cells had a MFI of >100 in cAMP-stimulated
cells. However, in the presence of SB203580 IL-5 production was greatly
reduced, with 21% of the cells displaying an MFI of 54 (Fig. 3A). Instead of bt2cAMP alone, PMA+
bt2cAMP was used in this experiment as well as in
the following experiment (Fig. 4) because
the combination is a more potent stimulus for IL-5 production. However,
the overall effect of SB203580 was the same whether
bt2cAMP or PMA+ bt2cAMP was
used to induce IL-5 gene expression.
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B,
PMA+bt2cAMP increased IL-5 and IL-13, but not
IL-4, production in effector Th2 cells (DO11.10), and the increase in
cAMP-induced IL-5 and IL-13 production was suppressed by SB203580 in a
dose-dependent fashion. The inhibitor SB203580 did not affect basal
expression of any of the cytokines in the cells. To show the specific
effect of cAMP on IL-5 and IL-13 expression, the effector Th2 cells
(DO11.10) were stimulated with PMA and ionomycin, an efficient inducer
of IL-4 gene expression. As shown in Fig. 4B, IL-4
production was inhibited in the presence of SB203580, suggesting the
involvement of p38 MAPK in calcium-induced IL-4 production. These data
confirm that cAMP is a poor inducer of IL-4 but a strong inducer of
IL-5 and suggest that full induction of IL-4 gene expression requires
more than p38 MAPK activation. These results also show for the first
time that cAMP can induce IL-13 production and provide evidence that
p38 kinase is involved in cAMP-induced IL-5 and IL-13 production in
effector Th2 cells. Inhibition of GATA-3-dependent promoter activation in Th2 cells
To further examine the role of p38 in IL-4 and IL-5 gene
expression, we tested the ability of SB203580 to inhibit IL-4 and IL-5
promoter activation in Th2 cells. As shown in Fig. 4A, the
p38 MAPK inhibitor SB203580, but not the ERK inhibitor PD98059,
resulted in a dose-dependent decrease in IL-5 promoter activity with a
50% decrease at the highest dose of the inhibitor (10 µM) (Fig. 4A). However, there was no inhibition of
PMA+ionomycin-induced IL-4 promoter activity in the presence of either
SB203580 or PD98059 (Fig. 4A). These results indicate that
cAMP-induced p38 kinase is involved in IL-5 promoter activation
probably through phosphorylation of specific transcription factor(s)
activated by this kinase. It is to be noted that multiple isoforms of
p38 kinase, , 
, , and 
have been described and the p38
kinase inhibitor SB203580 can only inhibit the and forms of
p38. Recently, CD4+ T cells were shown to
preferentially express p38 and p38 (47). Thus, the
inability of SB203580 to completely inhibit IL-5 promoter activity may
be due to residual cAMP-stimulated phospho-p38 activity in D10
cells.
The selective inhibition of IL-5 promoter activity by SB203580 together
with our previous studies demonstrating a direct activation of the IL-5
but not the IL-4 promoter by GATA-3 (48) raised the
possibility that GATA-3 was the downstream substrate of the p38
signaling pathway activated by cAMP. To test this hypothesis, a
(GATA)2-luciferase gene construct was transfected
into D10 cells. Interestingly, (GATA)2-directed
luciferase activity could be induced by stimulation with
PMA+bt2cAMP (or bt2cAMP
alone) but not with PMA+ionomycin (Fig. 4B), suggesting that
the activation was mediated by a specific selective pathway induced by
cAMP. Similar to its effects on the IL-5 promoter, SB203580 caused a
dose-dependent decrease in (GATA)2-luciferase
activity (Fig. 4B). As discussed earlier, the inability of
the p38 inhibitor to fully repress reporter gene activation is probably
because of residual activities of other p38 isoforms such as p38 in
the cells that may also be activated by cAMP. In contrast, the ERK
inhibitor PD98059 did not affect
(GATA)2-luciferase activity.
Phosphorylation of GATA-3 by activated p38
Inhibition of GATA-3-dependent promoter activity by the p38 kinase
inhibitor indicated that p38 kinase may contribute to GATA-3 activation
either directly or indirectly. Because there was no report on
posttranslational modifications of GATA-3, we first investigated
whether GATA-3 could be phosphorylated after cAMP stimulation of Th2
cells. D10 cells were metabolically labeled with inorganic
32P in the presence or absence of cAMP, and
GATA-3 protein was recovered by immunoprecipitation. As shown in Fig. 5A, a phosphorylated protein
of expected molecular size (55 kDa) was obtained using anti-GATA-3
but not control Ab. Although a basal level of GATA-3 phosphorylation
was observed in Th2 cells, cAMP augmented the level of phosphorylation.
These results suggested that GATA-3 exists as a phosphoprotein in Th2
cells, and cAMP signaling is able to increase GATA-3
phosphorylation.
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B, phosphorylation of GATA-3 and ATF-2 occurred in a p38
kinase-dependent fashion; no phosphorylation of either protein was
detected in the presence of only [-32P]ATP.
Also, no phosphorylated band was detected in the absence of any added
substrate. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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highlight the following points: 1)
cAMP activates p38 MAPK in a dominant fashion in Th2 but not Th1 cells;
and 2) GATA-3 is a substrate of p38 kinase, which explains, at least
partly, the distinct effect of cAMP in Th2 cells.
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A novel finding in our studies is the differential activation of p38
MAPK in Th2 and Th1 cells in response to cAMP (Fig. 6). p38 MAPK was
first identified as a major tyrosine-phosphorylated protein of 38 kDa
when murine macrophage cell lines were induced by LPS
(52). Thus far, four mammalian isoforms have been
described: p38, p38, p38, and p38 (26, 31, 41, 52, 53, 54, 55, 56, 57, 58, 59, 60). Although p38 MAPK was first implicated in the
production of proinflammation cytokines (e.g., TNF- and IL-1) by
monocytes/macrophages (31), the activation of p38 kinase
has also been described in activated T cells in both human and murine
systems. An interesting finding in these studies is that p38 is an
important integrator of TCR/CD28 stimulation of cell proliferation and
cytokine production in primary naive mouse CD4+ T
cells (36). Thus, stimulation of naive primary
CD4+ T cells through the TCR in the presence of
costimulation was shown to activate this signaling pathway, and its
blockade by SB203580 reduced the production of IL-2, IL-4, and IFN-
(36). In effector cells, although activation of p38 kinase
was shown to be a critical requirement for Th1 (IFN-) but not Th2
cytokine production in response to TCR/CD28 stimulation in murine cells
(33), studies of human Th2 effector cells show a
requirement for this kinase in IL-4 production in response to TCR/CD28
costimulation (35). This difference has been attributed to
the dependence of IL-4 on CD28 costimulation in human but not murine
effector Th2 cells (36). Our data demonstrate the
importance of p38 kinase in cAMP-induced effects in Th2 cells.
The p38 inhibitor SB203580 blocks the activity of p38 by binding to the
ATP-binding site on p38 MAPK (31). However, this inhibitor
is specific for p38 and p38 and does not inhibit p38 and
p38 (55, 56, 60, 61, 62). A recent study showed that both
p38 and p38 isoforms are highly expressed in T cells
(47). Thus, the inability of SB203580 to completely
inhibit reporter gene activity (Fig. 4) may be due to the fact that D10
cells express both isoforms, and both may contribute toward activation
of GATA-3. However, the complete inhibition of IL-5 and IL-13
production by SB203580 in in vitro differentiated Th2 cells suggests
that the major form of p38 in Th2 cells is p38.
In previous studies, we and others demonstrated Th2-specific expression of the transcription factor GATA-3 (43, 63, 64) and showed a critical role for this transcription factor in IL-5 gene expression (19, 43, 64). Furthermore, we and others have shown that GATA-3 directly activates the IL-5 but not the IL-4 promoter (48, 65). It is important to note that GATA-3 is also important for IL-4 and IL-13 gene expression as we have recently demonstrated using transgenic mice expressing a dominant-negative mutant of GATA-3 (44). However, the mechanisms involved in GATA-3-induced IL-4 and IL-13 gene expression are not well understood. The transcription factor c-Maf has been shown to be critical for IL-4 gene expression and directly activates the IL-4 promoter (66, 67). However, c-Maf does not appear to directly regulate IL-5 or IL-13 gene expression (67). Thus, the differential responsiveness of the IL-4 and the IL-5/IL-13 genes to cAMP in effector Th2 cells may be due to the inability of cAMP to activate c-Maf. Also, although cAMP-induced p38 MAPK activation alone is not sufficient to cause IL-4 gene expression, calcium-induced IL-4 production involves p38 MAPK.
Six members of GATA family have been cloned, and GATA-1 and GATA-2 have been shown to exist as phosphoproteins (68, 69). By phosphorylation mapping, GATA-1 was shown to be phosphorylated on six serine residues in uninduced cells and, after stimulation, a seventh serine residue became phosphorylated (68). Similarly, the phosphorylation of GATA-2 was increased after IL-3 stimulation (69). Here we show that GATA-3 also exists as a phosphoprotein in unstimulated cells. Most importantly, we demonstrate that cAMP can induce GATA-3 phosphorylation via p38 MAPK and stimulate GATA-3-dependent promoter activity. It is unlikely that phosphorylation will affect the DNA-binding ability of GATA-3 because the GATA-3 protein recovered from unstimulated Th2 cells or in vitro translated GATA-3 are able to bind the IL-5 GATA doublet with high affinity. The p38 kinase-induced phosphorylation of GATA-3 may be important for its ability to interact with other regulatory factors to promote IL-5 gene expression or to make the IL-4/IL-13 locus more accessible to other factors. The optimum consensus sequence for MAPK has been identified as PPTP, and one such motif exists in the GATA-3 coding region. The retention of wild-type activity by the GATA-3 mutant T155A suggests that this site may not be the correct site for the response to cAMP (J. M. LaPorte and C.-H. Chen, unpublished observations). There are at least 12 potential phosphorylation sites ((S/T)P) for MAPK on GATA-3, and which of these sequences is important for phosphorylation by p38 kinase is currently under investigation. It is also possible that p38 MAPK phosphorylates an unconventional site in GATA-3. The signaling intermediate that couples intracellular cAMP to the p38 MAPK pathway in Th2 cells remains to be identified.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Anuradha Ray, Department of Medicine, Pulmonary and Critical Care Section, Yale University School of Medicine, 333 Cedar Street, LCI 105, New Haven, CT 06520.
3 Abbreviations used in this paper: bt2cAMP, dibutyryl cAMP; MAPK, mitogen-activated protein kinase; PKA, protein kinase A; ERK, extracellular signal-related kinase; JNK, c-Jun N-terminal kinase; ATF, activating transcription factor; MACS, magnetic-activated cell sorting; MFI, mean fluorescence intensity.
Received for publication March 10, 2000. Accepted for publication August 25, 2000.
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gene expression at multiple levels. J. Immunol. 148:2845.[Abstract]
, but not on eosinophils. J. Immunol. 162:6178. expression by Th1 effector T cells mediated by the p38 MAP kinase signaling pathway. EMBO J. 17:2817.[Medline]
mitogen-activated protein kinase is activated by CD28-mediated signaling and is required for IL-4 production by human CD4+CD45RO+ T cells and Th2 effector cells. J. Immunol. 162:7110., , , and in inflammatory cell lineages. J. Immunol. 162:4246.). J. Biol. Chem. 271:17920.This article has been cited by other articles:
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