(Received for publication, July 17, 1996, and in revised form, October 18, 1996)
From the Department of Pulmonary Diseases, G03.550, University Hospital Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands
Interleukin-5 (IL-5) is one of the major
regulators of eosinophilic granulocytes in vivo. IL-5
exerts its pleiotropic effects by binding to the IL-5 receptor, which
is composed of an IL-5-specific chain and a common
c chain
shared with the receptors for IL-3 and granulocyte-macrophage
colony-stimulating factor. Previous studies have shown that binding of
IL-5 to its receptor triggers the activation of multiple signaling
cascades, including the Ras/mitogen-activated protein kinase, the
phosphatidyl -3
-kinase, and the Janus kinase/signal transducer and
activator of transcription pathways. Here we describe that IL-5
activates the serine/threonine protein kinase Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) pathway. We show that
IL-5 activates TPA response element (TRE)-dependent transcription in transfection experiments. TRE activation by IL-5 is
mediated by a region of the
c (577-581) that is also responsible for activation of JNK/SAPK and for activation of dyad symmetry element
(DSE)-dependent transcription. Dominant-negative SAPK or
ERK kinase-1 was used to demonstrate that JNK/SAPK activation is
necessary for induction of DSE- and TRE-dependent
transcription by IL-5, whereas extracellular signal-regulated kinase 2 was not essential for TRE- and DSE-dependent transcription.
By contrast, IL-5-induced activation of the tyrosine kinase Janus
kinase 2 seems to be a prerequisite for TRE- and
DSE-dependent transcription. Taken together, we show for
the first time that IL-5 activates kinases of the JNK/SAPK family, and
that this activation is linked to IL-5-induced TRE- and
DSE-dependent transcription.
Cytokines such as interleukin (IL)1-3,
IL-5, and granulocyte-macrophage colony-stimulating factor (GM-CSF)
play an important role in hematopoiesis (1-3). IL-3 and GM-CSF are
broad specificity cytokines that have effects on multiple hematopoietic
cell lineages (3, 4). By contrast, the actions of human IL-5 are
restricted to the eosinophil and basophil lineages, because a
functional IL-5 receptor (IL-5-R) is only expressed on these cell types
(5, 6). IL-5 is essential for eosinophil differentiation (7, 8) and
plays an important role in the functioning of mature eosinophils and
basophils (9-13). The IL-5-R is a multimeric molecule composed of a
unique chain associated with a common
c chain, which is part of
the receptors for IL-3 and GM-CSF (14). Because the
c chain is
thought to play a major role in postreceptor signal transduction, it is
not surprising that IL-3, IL-5, and GM-CSF exert multiple overlapping
effects on cells that have all three receptor complexes (15).
Binding of IL-3, IL-5, or GM-CSF to their receptors leads to activation
of multiple signal transduction pathways. Within seconds to minutes,
multiple cellular proteins become phosphorylated on tyrosine residues,
an event essential for most biological functions of these cytokines
(12, 16). Because the and
c chains of these receptors do not
contain any enzymatic activity, cytoplasmic protein tyrosine kinases
are likely to mediate this process. We and others have shown that IL-5
binding leads to rapid and transient activation of the Janus kinase
JAK2 (17-19), one of the kinases involved in activation of STAT
(signal transducer and activator of transcription) proteins (reviewed
in Ref. 20). In addition, kinases such as Lyn, Syk, fyn, hck, and BTK
are also activated by cytokines of this family (21-23). Activation of
these signaling pathways is mediated by different functional domains in
the
c chain. A membrane-proximal region containing box1, a motif
found in multiple cytokine receptors, is necessary for JAK2 and STAT3 activation, induction of c-myc gene expression and
cytokine-induced proliferation (23-27). A more distal region between
amino acids 542 and 589 was shown to be involved in activation of the
RAS/MAPK pathway mediated by Shc binding to the
c chain and for
induction of transcription of the immediate-early genes
c-jun and c-fos (24, 25, 28, 29) Moreover, this
region was also implicated in activation of phosphatidylinositol
3
-kinase and p70S6 kinase by GM-CSF (25). Phosphorylation of tyrosine
577 of the
c chain, possibly by JAK2, was shown to be at least
partially responsible for activation of MAPK and c-fos,
although also other tyrosine residues are likely to play an important
role (28, 29). In addition, the proline-rich region of the
chain
was also shown to be involved in activation of different pathways, such
as the JAK2/STAT pathway and cytokine-induced proliferation (27, 30, 31).
Immediate-early gene expression in response to a wide array of
different extracellular stimuli is regulated by a limited amount of
cis-acting response elements in the promoters of the induced genes. Among these, the TPA-response element (TRE) binds transcription factors of the Jun/AP-1 family (reviewed in Refs. 32 and 33). This
element mediates transcriptional activation by stimuli such as phorbol
esters, UV light, tumor necrosis factor (TNF
), IL-2, and stress
induced by heat shock or protein synthesis inhibitors (33). Jun/AP-1
transcription factors are homo- or heterodimeric proteins that are
activated by serine and threonine phosphorylation. The kinases that are
responsible for these phosphorylations were described recently as a
novel family of mitogen-activated protein kinases (MAPKs), the Jun
N-terminal kinases (JNKs, also known as stress-activated protein
kinases (SAPKs); reviewed in Refs. 34-36). The JNK/SAPKs themselves
are activated by dual phosphorylation at conserved threonine and
tyrosine residues, after which they phosphorylate AP-1 members such as
c-Jun and ATF2 and stimulate their transcriptional activity (34-36).
Another cis-acting element frequently found in promoters of immediate-early genes is the dyad symmetry element (DSE) or serum response element (SRE), which was first identified in the promoter of the c-fos proto-oncogene (reviewed in Ref. 37). The DSE/SRE binds multiple transcription factors, including the serum response factor and the ternary complex factor (TCF) ELK-1 (37, 38). Mitogenic stimulation leads to a rapid phosphorylation of both serum response factor and TCF/ELK-1, leading to enhanced DNA binding by serum response factor and, more importantly, enhanced transcriptional activation by TCF/ELK-1 (37, 38). In contrast to phosphorylation of Jun proteins, phosphorylation of TCF/ELK-1 is likely to be mediated by the classical MAPKs ERK1 and ERK2 (38-40), although in was more recently shown that the SRE can also be activated in response to JNK/SAPKs (41, 42).
In this report, we have investigated the effects of IL-5 on TRE- and
DSE-mediated transcription. Here we show that IL-5 efficiently activates TRE- and DSE-containing promoters. We present evidence that
this is likely to be mediated by activation of JNK/SAPKs. Moreover, we
identify the regions in the IL-5-R and
c chains responsible for
these effects. Finally, we suggest that activation of JAK2 by IL-5
precedes and is necessary for activation of JNK/SAPKs and TRE- and
DSE-dependent transcription.
Rat-1, P19EC, and
COS-1 cells were cultured in Dulbecco's modified Eagle's medium (Life
Technologies, Inc.) supplemented with 8% heat-inactivated fetal calf
serum. Human TF-1 and mouse BaF3 cells were cultured in RPMI 1640 (Life
Technologies, Inc.) supplemented with 10% heat-inactivated fetal clone
I serum (Hyclone), 50 µm -mercapthoethanol, and human IL-5 (20 pM for TF1) or mouse IL-3 (0.1 nM for BaF3).
Human IL-5 (hIL-5) was a kind gift of Dr. D. Fattah (Glaxo Wellcome,
Stevenage, United Kingdom), whereas mouse IL-3 was produced in COS
cells by transfecting an expression vector containing the murine IL-3
cDNA. The 12Ca5 antibody to the HA-tag was a generous gift from
Marc van Dijk. The anti-JNK/SAPK antibody (SC-572) was purchased from
Santa Cruz Biotechnology, Inc., and the anti-Jun antibody (Ab-1) was
obtained from Oncogene Science. The MEK inhibitor PD098059 (43) was a
generous gift from David Dudley and Alan Saltiel. The JAK2 inhibitor
AG490 (44) was a generous gift from Alexander Levitzki.
pSGhIL-5R was constructed by
inserting the cDNA for the human IL-5
receptor into the
NotI/KpnI sites of pSG513, whereas pSGhIL-5R
was constructed by inserting the cDNA for the human
c subunit
into the EcoRI sites of pSG513 as described previously (27).
The expression vectors encoding
c mutants 763, 627, 542, 517, 456,
I/II, and
II and
mutants 405, 390, and 366 are described elsewhere (27). Other
c mutants were generated by progressive deletions using
c 763 and the erase-a-base kit (Promega Corp.). Stop
codons were introduced by ligating in an oligonucleotide containing
stop codons in all reading frames, and the identity of the different
mutants was verified by dideoxy-sequencing using T7 polymerase
(Pharmacia). 3xTREtkCAT, 3xDSEtkCAT (45), and the p54SAPK/JNK
expression vector (46) are described elsewhere. Dominant-negative SEK
(SEK-AL, serine 254 to alanine and threonine 258 to leucine; Refs. 46
and 47) and HA-tagged ERK2 (48) were described previously. 3xTREtataCAT
contains three copies of the collagenase TRE coupled to a synthetic
minimal promoter.
For transfection experiments, COS-1 cells were subcultured in six-well dishes (Costar), and 3 h later, the cells were transfected with 10-20 µg of supercoiled plasmid DNA as described previously (27, 49). Rat-1 and P19EC cells were transfected 16 h after splitting. Following 16-20 h exposure to the calcium-phosphate precipitate, medium was refreshed, and cells were incubated for 16 h with IL-5 (0.5 pM). Rat-1 cells were incubated in medium containing 0.5% fetal calf serum 8 h prior to the addition of IL-5. Transfected cells were subsequently harvested for CAT assays or Immuno-kinase assays. CAT assays were performed as follows. Cells were lyzed by repeated freeze-thawing in 250 mM Tris, pH 7.4, 25 mM EDTA. Twenty-five µg of cellular extract were then incubated in a total volume of 100 µl containing 250 mM Tris, pH 7.4, 2% glycerol, 0.3 mM butyryl coenzyme A, and 0.05 µCi of [14C]chloramphenicol for 2 h at 37 °C. Reaction products were then extracted using 400 µl of xylene:pristane (1:2), and the percentage of acetylated products was then determined using liquid scintillation counting.
Gel Retardation AssayNuclear extracts were prepared from
unstimulated and IL-5-stimulated cells following a procedure described
previously (27, 49). Oligonucleotides were labeled by filling in the
cohesive ends with [-32P]dCTP using Klenow fragment of
DNA polymerase I. Gel retardation assays were carried out according to
published procedures with slight modifications. Briefly, nuclear
extracts (10 µg) were incubated in a final volume of 20 µl,
containing 10 mM HEPES, pH 7.8, 50 mM KCl, 1 mM EDTA, 5 mM MgCl2, 10% (v/v)
glycerol, 5 mM dithiothreitol, 2 µg poly(dI-dC)
(Pharmacia Biotech Inc.), 20 µg of bovine serum albumin, and 1.0 ng
of 32P-labeled collagenase TRE oligonucleotide for 20 min
at room temperature. Complexes were then separated though nondenaturing
5% polyacrylamide gels and visualized by autoradiography.
For metabolic
labeling experiments, cells were incubated for 3 h in
phosphate-free medium containing 1 mCi/ml
[32P]orthophosphate. Cells were then stimulated with
cytokines, and subsequently the cells were lyzed in RIPA buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 1% Nonidet P-40,
0.1% SDS, 0.5% sodium deoxycholate, 5 mM EDTA, 1 mM Na3VO4, 10 mg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, and 1 mM
leupeptin) for 15 min on ice. The lysate was centrifuged to remove DNA
and cellular debris. The cell lysates were incubated with the anti-Jun
polyclonal antibody for 2 h at 4 °C. Immune complexes were then
precipitated with protein A-Sepharose for 30 at 4 °C, washed five
times with lysis buffer, and boiled in 1 × Laemmli's sample
buffer. The proteins were separated on a 10% polyacrylamide gel. After
electrophoresis, the gel was fixed (50% methanol, 10% acetic acid),
dried, and exposed to X-ray films (Kodak) or analyzed using a
phosphorimager (Applied Biosystems).
For immune-kinase assays, cells were lyzed in JNK/SAPK lysis buffer (20 mM Tris, pH 7.4, 10% glycerol, 1% TX-100, 2 mM EDTA, 2 mM EGTA, 50 mM
-glycerophosphate, 1 mM Na3VO4,
10 mg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, 1 mM leupeptin, and 1 mM benzamidine), and
JNK/SAPK protein was precipitated using the 12Ca5 antibody (for
transfected cells) or the JNK/SAPK polyclonal antibody (SC-572 for
endogenous JNK/SAPK) and protein A-Sepharose beads. After extensive
washing with lysis buffer, the beads were resuspended in 20 µl of
kinase buffer (25 mM Hepes, pH 7.5, 20 mM
MgCl2, 20 mM
-glycerophosphate, and 2 mM dithiothreitol) containing 10 µg of GST-Jun and 10 µCi of [
-32P]ATP. The kinase reaction was performed
for 20 min at 30 °C, after which the reaction was terminated by
adding 1 × Laemmli's sample buffer. Proteins were then separated
on 12% polyacrylamide gels, after which the gels were fixed, dried,
and exposed to x-ray film. ERK2 immune-kinase assays were performed as
described previously (48).
IL-5,
the major cytokine involved in regulating eosinophil functions, was
demonstrated previously to be able to activate different signaling
pathways, including the RAS/MAPK pathway (50), the phosphatidylinositol
3-kinase pathway (51), and the JAK/STAT pathway (17-19, 27).
Activation of the Jun family of transcription factors by IL-5, however,
was not studied before. We, therefore, set out to determine whether
IL-5 was able to activate transcription of reporter constructs
containing a TRE, the natural Jun binding site from the collagenase
promoter (32). For this purpose, we transfected Rat-1 cells with the
IL-5 R
and
c chains and different CAT reporter constructs. IL-5
was able to increase CAT activity in cells transfected with a reporter
containing three copies of the TRE linked to the tk promoter (TREtkCAT)
but failed to activate this reporter without TRE sites (Fig.
1A). This effect was not restricted to the tk
promoter, because IL-5 also activated a CAT construct containing three
TREs coupled to a synthetic minimal tata box (TRETATACAT) but not the
enhancerless TATACAT reporter (Fig. 1A). Similar results
were obtained in P19 EC and COS-1 cells (data not shown). These results
strongly suggest that IL-5 can activate TRE-dependent
transcription in these cell types.
Previously, it was shown that activation of different signaling
pathways can be assigned to different domains of the c chain of the
IL-5 and GM-CSF receptors (24-28). We, therefore, tested a previously
described set of carboxyl-terminal deletions of the
c chain for
IL-5-dependent activation of the TRETATACAT reporter. Deletion of the most carboxyl-terminal 254 amino acids (627) did not
alter the effect of IL-5 on TRE-dependent CAT activity
(Fig. 1B). However, further deletion of 85 amino acids (542)
resulted in a complete loss of IL-5-induced TREtataCAT activity. This
region (542-627) was described previously to be involved in activation of the RAS/MAP kinase pathway by GM-CSF (28). To be able to localize
the residues of the
c chain involved in TRE activation more
precisely, we generated a new set of
c chain mutants using S1
nuclease. As shown in Fig. 1C, deletion down to amino acid 581 did not significantly decrease IL-5-induced TREtataCAT activity. Interestingly, further deletion of 4 amino acids (577) resulted in a
complete loss of IL-5-induced TRE activity (Fig. 1C), which was also observed in P19 EC cells (data not shown). The expression of
all mutants was verified by Western blotting using a rabbit anti-
c
antibody (data not shown). Therefore, the region of the
c chain
around tyrosine 577 is likely to be involved in activation of
TRE-dependent transcription upon IL-5 stimulation.
It was suggested previously that the c chain of the IL-5-R is the
major player in IL-5-induced signal transduction. We and others have,
however, shown that the cytoplasmic region of the
chain is
necessary for IL-5-induced JAK2 and STAT3 activity (27, 30, 31). To
determine whether the
chain plays a role in IL-5-induced TRE
activity, we transfected different carboxyl-terminal deletions of the
chain together with the wild-type
c chain and the TREtataCAT
reporter into Rat-1 cells. As we have described for IL-5-induced STAT3
activity (27), the region between amino acids 366 and 390 of the
chain is also necessary for IL-5-induced TREtataCAT activity (Fig.
1D). Importantly,
366 was shown previously to be able to
form a high affinity complex with the
c chain (30), showing that the
failure of this mutant to activate TRE-dependent transcription is not trivial. Taken together, activation of
TRE-dependent transcription by IL-5 is mediated by a region
containing tyrosine 577 of the
c chain and the proline-rich region
366-390 of the
chain.
The activity of transcription factor Jun/AP-1 can be
regulated at the DNA-binding level or the transcription activation
level (reviewed in Refs. 32 and 33). To discriminate between these two
possibilities, we examined Jun/AP-1 DNA binding activity in TF-1 cells
stimulated with IL-5 or IL-3. For this purpose, we isolated nuclear
extracts from these cells and assayed them for binding to the
collagenase TRE in a band shift assay. As shown in Fig. 2A,
neither IL-5 nor IL-3 significantly altered Jun/AP-1 binding activity
in TF-1 cells or BaF3 cells stably transfected with the IL-5 receptor
(data not shown). Therefore, it seemed likely that the observed
activation of TRE-dependent transcription was caused by an
IL-5-induced increase in the transcriptional activation potential of
c-Jun. As was reported previously, phosphorylation of two residues near
the amino terminus of c-Jun by kinases of the JNK/SAPK family are
thought to regulate transcriptional activation by c-Jun (33). We thus
set out to determine whether IL-5 treatment altered the phosphorylation
status of c-Jun. Orthophosphate-labeled TF-1 cells were treated with
IL-5 or the potent JNK/SAPK activators TNF and cycloheximide, after
which Jun phosphorylation was studied by immunoprecipitation. As was
expected, we observed a dose-dependent increase in Jun
phosphorylation by IL-5, albeit at lower levels than Jun
phosphorylation obtained after TNF
or cycloheximide stimulation
(Fig. 2B, CH).
To determine whether the observed increase in Jun phosphorylation upon
IL-5 stimulation was caused by an increase in JNK/SAPK kinase activity,
we performed an immune-complex kinase assay on IL-5-stimulated TF-1
cells. JNK/SAPK was precipitated from TF-1 lysates using a polyclonal
antibody against JNK2 (recognizing JNK1, JNK2, and p54), and
JNK/SAPK activity was determined using purified GST-Jun fusion protein
as a substrate. As shown in Fig. 2C, IL-5 treatment indeed
caused a 3-fold enhancement in JNK/SAPK kinase activity, which again
was lower than the activation obtained with cycloheximide
(CH). To investigate a similar enhancement in JNK/SAPK
kinase activity that was present in the cells that were used for
assaying TRE activation, we transiently transfected an epitope-tagged
version of p54SAPK/JNK together with the IL-5-R into Rat-1 and COS-1
cells. The activity of the tagged p54SAPK/JNK was then determined using
an anti-epitope antibody in an immune-complex kinase assay. Fig.
2D shows that in both cell types, IL-5 significantly enhanced the activity of the transfected p54SAPK/JNK. Taken together, it seems likely that the observed activation of
TRE-dependent transcription by IL-5 was caused by
phosphorylation of c-Jun by JNK/SAPK.
To provide further evidence for this model, we tested the activity of
the different c chain mutants in the transient p54SAPK/JNK activation assay. Fig. 3A shows that the
region between 542 and 627 is necessary for IL-5-induced enhancement of
p54SAPK/JNK kinase activity. Moreover, as we described above for the
activation of TRE-dependent transcription, mutant 581, but
not 577, fully supported IL-5-induced p54SAPK/JNK activation (Fig.
3B). This again supports the hypothesis that IL-5-induced
TRE activation is mediated by JNK/SAPK.
Activation of DSE-dependent Transcription by IL-5
It was shown recently that besides c-Jun, JNK/SAPK also
phosphorylates and activates p62TCF/ELK-1, one of the transcription factors regulating the activity of the dyad symmetry element (DSE) in
the c-fos promoter (41, 42). Therefore, we set out to
determine whether IL-5 will also activate DSE-dependent
transcription in our transient system. A reporter construct containing
three copies of the DSE coupled to tkCAT was transfected in Rat-1 cells
together with the IL-5-R and
c chains. Interestingly, IL-5
causes a 4-fold increase in DSE-dependent transcription in
these cells (Fig. 4). Moreover, as we demonstrated for
TRE and p54SAPK/JNK activation,
c 581 was able to activate DSEtkCAT
activity, whereas
c 577 had lost this ability (Fig. 4).
ERK2 Is Not Involved in Activation of TRE- or DSE-dependent Transcription by IL-5
Activation of
DSE-dependent transcription can also be accomplished by the
RAS-ERK2 kinase pathway (40, 51). Moreover, it was shown recently that
tyrosine 577 of the c chain is involved in activation of this
pathway by GM-CSF (28). We, therefore, determined whether activation of
MAP kinase was involved in the observed regulation of TRE- and
DSE-dependent transcription by IL-5. For this purpose, we
used PD098059, a potent and selective inhibitor of the MAP kinase
kinase MEK (43). PD098059 was indeed able to block ERK2/MAP kinase
hyperphosphorylation induced by IL-5 in Rat-1 cells (Fig.
5A). Moreover, PD098059 completely blocks IL-5-induced ERK2/MAP kinase activity in Rat-1 cells (Fig.
5B). However, Fig. 6A shows that preincubation
with PD098059 did not alter TRE-dependent CAT activity in
IL-5-treated cells. Moreover, PD098059 pretreatment did also not
influence IL-5-induced DSE activation (Fig.
6B) or IL-5-induced p54SAPK/JNK kinase
activity (Fig. 6C). These results and those described
previously strongly suggest that activation of TRE- and
DSE-dependent transcription is mediated by JNK/SAPK but not
by the ERK2/MAP kinase pathway.
To further investigate the role of JNK/SAPK in IL-5 induced TRE and DSE
activation, we used a construct expressing dominant-negative SEK
(SEK-AL, made by mutating serine 254 to alanine and threonine 258 to
leucine; Refs. 46 and 47), an efficient repressor of JNK/SAPK activity.
Indeed, when dominant-negative SEK was transfected in Rat-1 cells,
IL-5-induced JNK/SAPK activity was strongly repressed (Fig.
7, right panel). Interestingly,
dominant-negative SEK also reduces IL-5-induced TRE- and
DSE-dependent transcription, suggesting that TRE and DSE
activation by IL-5 are at least partially mediated by JNK/SAPK.
JAK2 Is Essential for IL-5-induced TRE- and DSE-dependent Transcription
We and others have shown
previously that IL-5 causes a strong and rapid activation of the JAK2
tyrosine kinase in different IL-5-responsive cell types (17-19).
Because IL-5 is known to induce tyrosine phosphorylation of the c
chain, a process that might well be mediated by JAK2 (52), we wanted to
determine whether JAK2 activation by IL-5 is involved in the activation
of TRE- and DSE-dependent transcription. For this purpose,
we used two different approaches. (i) We transfected two different
mutations of the
c chain,
I/II and
II, into Rat-1 cells and
tested their activity on TRE- and DSE-containing reporters. Although
both mutants are wild-type around tyrosine 577, mutant
I/II contains
an internal deletion that removes both box1 and box2 from the
full-length
c receptor, whereas
II is only lacking box2 in the
full-length
c receptor. Deletion of both box1 and box2 completely
blocked IL-5-induced TRE and DSE activation, whereas deletion of only box2 did not have any effect (Fig. 8). Interestingly,
box1, but not box2, was shown previously to be involved in JAK2 binding and activation (19, 52). (ii) We used the tyrphostin AG490, which was
shown previously to be an inhibitor of JAK2 (44). Fig. 8
shows that this inhibitor indeed causes a dose-dependent decrease in IL-5-induced TRE and DSE activity, although we failed to
completely block these processes. Control experiments showed that AG490
caused a significant but not complete block in IL-5-induced JAK2
activation (data not shown). We, therefore, conclude that JAK2 is
likely to be involved in IL-5-induced activation of TRE- and
DSE-dependent transcription (Fig. 9).
IL-5 binding to its receptor results in the activation of multiple
signaling pathways, including the RAS/MAPK pathway (50), the
phosphatidylinositol 3-kinase pathway (51) and the JAK/STAT pathway
(17-19, 27). Here we show that IL-5 activates TRE- and DSE-dependent transcription via activation of JNK/SAPK.
Moreover, we present evidence that activation of JAK2 is necessary for
this process.
Immediate-early gene induction in response to growth factors and phorbol esters can be mediated by a DSE/SRE cis-acting elements present in the promoters of induced genes (reviewed in Ref. 37). The DSE/SRE binds serum response factor and proteins from the TCF family including Elk-1, SAP-1, and NET-1/ERP/SAP-2 (reviewed in Ref. 38). Upon growth factor signaling, Elk-1 is phosphorylated by the ERK group of MAP kinases, resulting in increased ternary complex formation as well as activation of the transcriptional activation domain of Elk-1 (38-40). Because IL-5 efficiently activates ERK2 (50)(Fig. 5), it is surprising that blocking this process with PD098059 does not influence IL-5-induced DSE activation (Fig. 6). At present, we can only speculate on this apparent contradiction. It is possible that phosphorylation and activation of Elk-1 by ERK2 is a cell type-specific process that involves components that might not be available in our IL-5-induced cellular system. However, because there are no examples of cell type-specific Elk-1 phosphorylation, it seems more likely that both ERK and JNK/SAPK are capable of activating Elk-1 in IL-5-stimulated cells. Activation of Elk-1 by JNK/SAPK is not unprecedented, because it was recently shown that JNK/SAPK activators, such as UV or IL-1, efficiently activate Elk-1 though phosphorylation on sites identical to those phosphorylated by ERK2 (41, 42). Blocking either the ERK or JNK/SAPK pathway, therefore, does not significantly alter IL-5-induced DSE activity because the other pathway is redundant. This redundancy was also suggested by Cano et al. (53), who showed that activation of a single MAPK subtype (ERK or JNK/SAPK) is sufficient to elicit a complete nuclear response. Further demonstration of this functional redundancy awaits the availability of an efficient JNK/SAPK inhibitor.
In agreement with our results, it was shown previously that box 1 and
tyrosine 577 of the c chain are involved in the activation of the
c-fos promoter by GM-CSF (28, 52). However, mutation of
tyrosine 577 (Y577F) in the context of the full-length
c chain resulted in only partial inhibition of c-fos induction by
GM-CSF. This phenomenon was explained by the fact that other
phosphorylated tyrosines might play a role in c-fos
induction by GM-CSF (28). It was also suggested that the RAS/ERK
cascade was essential for GM-CSF-induced c-fos expression,
because dominant-negative RAS expression efficiently blocks
c-fos induction by GM-CSF (28, 54). Our results with the MEK
inhibitor PD098059 (Figs. 5 and 6) suggest that the ERK2/MAP kinase
cascade is not involved in DSE (and c-fos) induction by
IL-5. Although these results seem to be in contrast, they might be
explained by the fact that activated RAS also stimulates the JNK/SAPK
pathway (55, 56), although TNF
induction of JNK/SAPK occurs in a
RAS-independent manner (56). Because PD098059 efficiently blocks
ERK2 activation, but not RAS activation, the result with the
dominant-negative RAS might be explained by inhibition of JNK/SAPK
and, therefore, inhibition of GM-CSF-dependent
c-fos expression.
Inhibition of IL-5-induced JAK2 activation by using c chain mutants
or the tyrphostin AG490 resulted in a decrease in IL-5-induced TRE and
DSE activation (Fig. 8). Therefore, JAK2 activation seems to play a
role in IL-5 signaling apart from STAT activation. In agreement with
our results, Watanabe et al. (52) showed recently that
blocking JAK2 with dominant-negative JAK2 forms inhibits c-fos promoter activation by GM-CSF. It is worthwhile to
mention that JAK2 was also shown to be necessary for ERK2 activation by growth hormone (57). Because the
c chain is phosphorylated on
multiple tyrosine residues after IL-5 stimulation, including tyrosine
577 (28), it seems likely that JAK2 is the kinase responsible for this
phenomenon. Indeed, dominant-negative JAK2 expression resulted in
abrogation of
c phosphorylation after GM-CSF addition (57). The
phosphorylated tyrosine 577 might well be the binding site for adapter
proteins such as Shc, Grb2, P80, and other proteins responsible for
transmitting the IL-5/GM-CSF-induced signal from the
c chain through
the cytoplasm (28, 29, 58). Verification of this model awaits the
precise identification of
c residues that are phosphorylated by JAK2
and the characterization of proteins binding to these residues.
Although the cytoplasmic domain of the c chain of the
IL-5/IL-3/GM-CSF receptors is essential for all previously described intracellular signaling events, we have shown that amino acids 366-390
of the
chain are also necessary for IL-5-induced TRE and DSE
activation (Fig. 1D). This is not trivial, because
366 is
able to form a high affinity binding receptor with the
c chain (30).
We have shown previously that this region is essential for IL-5-induced
STAT3 activation (27). This region contains conserved proline residues
that are also found in the IL-3-R
, GM-CSF-R
, prolactin receptor
and growth hormone receptor. Site-directed mutagenesis showed that
these prolines are essential for IL-5-induced proliferation and
c-jun and c-fos induction as well as JAK2
activation (30, 31). Recent evidence suggests that the cytoplasmic
domain of the
chain is necessary for the activation of a preformed
c dimeric complex (59). However, the molecular mechanism responsible for this process remains to be determined.
Taken together, we describe for the first time that besides ERK2, IL-5 also activates MAP kinases of the JNK/SAPK family. The underlying mechanism as well as the functional consequence of this process for IL-5-induced responses in eosinophils are objectives of future study.
We thank David T. Dudley, Alan R. Saltiel, Alexander Levitzki, Jim Woodgett, Jan Tavernier, Dilnya Fattah, Marc van Dijk, and Roberto Solari for the kind gifts of materials and plasmids.