(Received for publication, July 6, 1995; and in revised form, August 16, 1995)
From the
The receptor for interleukin-5 (IL-5R) is composed of a unique
chain (IL-5R
) expressed on eosinophils and basophils,
associated with a
c subunit, which is shared by the receptors for
IL-3 and granulocyte macrophage-colony stimulating factor. One of the
molecular events activated via the IL-5R is the JAK/STAT signaling
pathway. Recent reports have shown that IL-5 induces tyrosine
phosphorylation of JAK2 followed by the subsequent cell type-specific
activation of either STAT1
or STAT5. To identify additional STAT
proteins activated by IL-5, we co-transfected the IL-5R with STAT cDNAs
in COS cells. We found that IL-5 induces binding of STAT3 to the
intercellular adhesion molecule-1 pIRE, and activates STAT3-dependent
transcription. Moreover, endogenous STAT3 was tyrosine phosphorylated
and activated in human IL-5-stimulated BaF3 cells ectopically
expressing the human IL-5R (BaF3/IL5R). These data imply that multiple
STAT proteins are involved in gene regulation by IL-5 in a cell
type-specific manner. We further demonstrate using C-terminal
truncations of the
and
c subunits of the IL-5R that the
membrane-proximal regions of both subunits are required for STAT
activation. Interestingly, a
c receptor mutant lacking
intracellular tyrosine residues is able to mediate STAT3 activation,
suggesting that tyrosine phosphorylation of the
c receptor is not
essential for STAT3 activation.
Hematopoiesis is tightly regulated by a complex network of
stromal interactions and by soluble polypeptide factors named
cytokines. IL-3, ()IL-5, and GM-CSF are cytokines that have
diverse effects on the proliferation, differentiation, and activation
of blood cells and their precursors (1, 2, 3) . Whereas IL-3 and GM-CSF also have
effects on other hematopoietic lineages(3, 4) , the
effect of IL-5 in humans is restricted to eosinophils and basophils.
IL-5 is essential for eosinophil differentiation (5, 6) and plays an important role in the function of
mature
eosinophils(7, 8, 9, 10, 11) .
The specific effects of IL-5 on eosinophils and basophils are due to
restricted expression of the low affinity IL-5R
receptor on these
cell types(12, 13) . The high affinity receptor for
IL-5 (IL-5R) is composed of a unique
subunit associated with a
c subunit that is shared with the receptors for IL-3 and
GM-CSF(14) . The
c subunit is essential for signal
transduction(15, 16) , but also, the
chain
transduces intracellular growth signals(17) . Therefore, the
common use of the
c subunit by IL-3, IL-5, and GM-CSF explains the
partial observed functional redundancy of these
cytokines(16, 18) . However, postreceptor signal
transduction pathways are not well defined and are likely to be
composed of both mitogenic and differentiation signals. It is known
that the
c subunit, like other cytokine receptors, does not
contain intrinsic tyrosine kinase activity(19, 20) .
However, one of the earliest events to occur after IL-3, IL-5, and
GM-CSF stimulation is induction of protein tyrosine
phosphorylation(19, 21) . This tyrosine
phosphorylation is caused by the activation of several cytoplasmic
protein tyrosine kinases such as Lyn (22, 23) and
c-Fps/Fes(24) . Recent studies have demonstrated that the
family of Janus kinases (JAKs), containing four members (JAK1, JAK2,
JAK3, and TYK2), is associated with different cytokine receptors and is
phosphorylated after ligand binding(25) . We and others have
shown that only JAK2 is activated in response to IL-3, IL-5, and GM-CSF (26, 27, 28) and specifically associates with
the membrane-proximal region of the
chain(29) . The JAK
protein-tyrosine kinases activate members of a novel family of
transcription factors named signal transducers and activators of
transcription (STATs)(25) .
STAT proteins were first
described as intermediates in the interferon-/
(IFN-
/
) signaling pathway(30, 31) . At
present, eight different STATs (STAT1
, STAT1
, STAT2, STAT3,
STAT4, STAT5A, STAT5B, STAT6) have been described and are involved in
specific cytokine
regulation.(31, 32, 33, 34, 35, 36, 37, 38, 39) .
This family of STAT proteins comprises a new class of transcription
factors that contain Src homology 2 (SH2), SH3-like domains, and a
carboxyl-terminal tyrosine phosphorylation site(25) . STAT
proteins normally exist as inactive monomers in the cytoplasm, but
after phosphorylation on tyrosine, they form homo- or heterodimers,
which translocate to the nucleus and bind to specific DNA
motifs(40, 41, 42, 43, 44) .
We recently found that STAT3 is also phosphorylated on serine
residues(45) . This serine phosphorylation seems to be
necessary for the regulation of the transactivation potential of STAT3.
We have previously shown that IL-5 activates STAT1 together
with an unidentified DNA-binding protein in human
eosinophils(27) . Therefore, we were interested whether other
known STAT proteins can be activated by IL-5. For this purpose, we
reconstituted both subunits of the IL-5 receptor together with
different STAT cDNAs in COS cells. We demonstrated that besides
STAT1
, STAT3 was also activated by IL-5 in these transfected COS
cells. In addition, endogenous STAT3 was tyrosine phosphorylated and
activated by hIL-5 in BaF3 cells stably expressing the hIL-5R
(BaF3/IL5R). Finally we demonstrate, using carboxyl-terminal
truncations of both the
and
subunits, that the
membrane-proximal region of both subunits are necessary for STAT3
activation.
Figure 1:
Transactivation by STAT3 is induced in
response to hIL-5. Monkey COS cells were transiently transfected by
calcium phosphate precipitation with 2 µg of 2xIREtkluc reporter
construct, 0.5 µg of hIL-5R cDNA, 0.5 µg of hIL-5R
cDNA, 4 µg of pSG5 (lane 1), 4 µg of hSTAT1
cDNA (lane 2), 4 µg of hSTAT3 cDNA (lane 3), 4 µg
of mSTAT4 cDNA (lane 4), 4 µg of shSTAT5 cDNA (lane
5), 4 µg of hSTAT6 cDNA (lane 6) and 2 µg of a
-galactosidase expression vector (pSV-lacZ) as a control for
transfection efficiency. 1 day after transfection, cells were incubated
for 16 h with IL-5. The cells were then harvested, and
-galactosidase and luciferase activities were determined.
Luciferase activities were normalized to
-galactosidase
activities. -Fold induction of luciferase activity in IL-5 treated (gray bars) compared with untreated control cells (black
bars) is shown. Values represent the averages of four different
experiments ± S.E. IL-5 clearly enhances IL-6/IFN-
response
element activation by STAT1
and STAT3.
Furthermore, we observed no effect
of IL-5 on luciferase activity after cotransfection of the cDNAs
encoding for STAT4, STAT5, and STAT6 (Fig. 1). These results
show that IL-5 can activate the transactivation potential of both
STAT1 and STAT3 in COS cells.
Since DNA-binding is a
prerequisite for transactivation, we tested nuclear extracts from
untreated and IL-5-stimulated COS cells transfected with STAT3. Indeed,
when COS cells were stimulated with IL-5 for 30 min, a single DNA
binding complex is induced using the ICAM-1 pIRE as a probe (Fig. 2). This is a specific binding complex because it could be
competed with an excess of unlabeled pIRE, whereas a nonspecific
competitor (SP1) was unable to compete for binding. To confirm that
this DNA-binding protein was STAT3, we preincubated nuclear extracts
with antibodies against STAT3. As shown in Fig. 2, this antibody
against STAT3 binds to the DNA-protein complex and is therefore not
able to migrate into the gel. In contrast, a control preincubation with
STAT1 antibodies had no effect on this DNA-protein complex. These
results indicate that IL-5 activates STAT3 binding to the ICAM-1 pIRE.
Figure 2:
hIL-5 induces a DNA binding complex
containing STAT3. The hIL-5 receptor and STAT3 were expressed in COS
cells. These cells were either untreated or treated for 30 min with
IL-5, after which nuclear extracts were prepared. Nuclear extract were
assayed for binding to the P-labeled ICAM-1 pIRE in
bandshift experiments. For competition experiments, extracts were
preincubated for 5 min with a 50-fold molar excess of unlabeled
oligonucleotide as indicated. For identification of the STAT proteins,
the extracts were incubated with either anti-STAT1
, anti-STAT3
antibodies, or nonimmune serum for 30 min before the addition of the
P-labeled ICAM-1 pIRE. IL-5 clearly induces binding of
STAT3 to the ICAM-1 pIRE.
We further characterized the DNA-binding specificity of the
IL-5-induced STAT3 complex. These experiments were performed with
nuclear extracts from COS cells transfected with the IL-5R and STAT3
and treated with IL-5 for 30 min. The ICAM-1 pIRE was used as a
radioactive probe, and as competitors we used the IFN- activating
site elements from the c-fos (SIEm67) (40) and the Fc
RI
promoter (GRR)(41) . Fig. 3A shows that
competition with a 50-fold molar excess of either the pIRE or SIEm67
oligonucleotides completely inhibited binding of STAT3 to the ICAM-1
pIRE, whereas a mutant ICAM-1 pIRE was unable to compete for binding.
Competition with the GRR sequence resulted in a decrease of STAT3
binding to the pIRE, suggesting that the pIRE is a better binding site
for STAT3 than the GRR. We also tested the transcriptional activity of
STAT3 mediated on the GRR sequence. We therefore inserted four copies
of the GRR sequence in front of a tkCAT reporter plasmid. This
4xGRRtkCAT reporter construct was transfected in COS cells together
with cDNAs encoding the IL-5R
and
c subunits and STAT3. As
shown in Fig. 3B, overexpression of the IL-5R and STAT3
in the presence of IL-5 resulted in a 40-fold induction of luciferase
activity mediated via the GRR sequence. The increase in luciferase
activity is comparable with the IL-5-induced transactivation of a
4xpIREtkCAT reporter construct. Taken together, these results show that
IL-5 is capable of activating STAT3 in COS cells.
Figure 3:
hIL-5 induced STAT3 activity is not
restricted to the ICAM-1 pIRE. A, COS cells expressing the
hIL-5 receptor and STAT3 were stimulated with IL-5 for 30 min. Nuclear
extracts were prepared and incubated with a P-labeled
ICAM-1 pIRE. For competition experiments, extracts were pre-incubated
for 5 min with a 50-fold molar excess of unlabeled oligonucleotide as
indicated. IL-5-induced STAT3 can bind to other STAT binding sites. B, COS cells were transient transfected with the IL-5R
,
IL-5R
, and STAT3 cDNAs together with either a 4xIREtkCAT or a
4xGRRtkCAT reporter construct. 1 day after transfection, cells were
stimulated with IL-5 and harvested after 16 h. Chloramphenicol
acetyltransferase assays were performed with
-galactosidase-normalized samples, and -fold induction was
calculated relative to untreated cells. Values represent the averages
of three different experiments ± S.E. IL-5 induces activation of
both IRE- and GRR-containing promoters by
STAT3.
Figure 4:
Tyrosine phoshorylation and activation of
STAT3 by hIL-5 in BaF3/IL5R cells. BaF3 cells stably expressing the
hIL-5 receptor (BaF3/IL5R) were untreated or treated with hIL-5 for 15
min. A, endogenous STAT3 was immunoprecipitated from these
BaF3/IL5R cells with a monoclonal antibody specific against STAT3.
Immunoprecipitates were analyzed by Western blotting with the
phosphotyrosine-specific antibody 4G10 (left panel). The
membrane was stripped and reprobed with the STAT3 specific antibody (right panel). hIL-5 clearly induces phosporylation of STAT3
on tyrosine residues. B, nuclear extracts from these BaF3/IL5R
cells were analyzed in a bandshift assay using the ICAM-1 pIRE as a
probe. The extracts from hIL-5-stimulated cells were incubated with
either anti-STAT1, anti-STAT3 antibodies, or nonimmune serum. The
hIL-5-induced pIRE binding complex contains both STAT1
and
STAT3.
Subsequent to
tyrosine phosphorylation, STATs dimerize and translocate to the nucleus
were they bind to specific DNA sequences. Therefore, nuclear extracts
from untreated and hIL-5-stimulated BaF3/IL5R cells were incubated with
a P-labeled pIRE oligonucleotide and analyzed in a
bandshift assay. Fig. 4B shows that stimulation with
hIL-5 for 15 min results in the formation of four binding complexes.
The same complexes were observed in mouse IL-3-treated parental BaF3
cells.
To determine the identity of the DNA binding
proteins induced by hIL-5, we preincubated nuclear extracts with
antibodies against STAT1
and STAT3. We found that anti-STAT1
antibodies diminished complex C2 and C3, whereas anti-STAT3 antibodies
blocked the formation of complex C1 and C2 (Fig. 4B).
The upper complex C4 was not affected by either STAT1
or STAT3
antibodies. We conclude from these data that in BaF3/IL5R cells, hIL-5
activates both STAT1
and STAT3, which bind both as homo- and
heterodimers to the ICAM-1 pIRE.
Figure 5:
Analysis of c-terminally truncated hIL-5
receptor mutants for STAT3 activation. A, schematic
representation of the wild type and carboxyl-terminal deletions mutants
of the hIL-5 receptor and
c subunits. The box1/2 homology
regions are shown for the
c chain. A proline-rich region of
homology between the
subunits of receptors for GM-CSF, IL-3, and
IL-5 is also shown. B, COS cells were transiently transfected
with the wild type hIL-5R
together with the wild type human
c
chain or one of the
c chain deletion mutants. Furthermore, the
expression vectors for STAT3 and a
-galactosidase and the
2xpIREtkluc reporter plasmid were co-transfected. 1 day after
transfection, cells were incubated for 16 h with IL-5. The cells were
then harvested, and
-galactosidase and luciferase activities were
determined. Luciferase activities were normalized to galactosidase
activities. -Fold induction of luciferase activity in IL-5-treated (gray bars) compared with untreated control (black
bars) cells is shown. Values represent the averages of five
different experiments ± S.E. The region between 456 and 516 is
necessary for STAT3 activation by IL-5. C, transfections were
performed with the wild type
c chain together with the the wild
type
subunit or one of the
chain deletion mutants as
described above. The region between 366 and 390 is necessary for STAT3
activation by IL-5.
We next examined whether the cytoplasmic
domain of the hIL-5R is involved in STAT3 activation. A schematic
representation of the constructed carboxyl-terminal
subunit
mutants
405,
390, and
366 is shown in Fig. 5A. Transfection of COS cells with these truncated
receptor mutants together with the wild type
c subunit shows
that only mutant
366 was unable to mediate STAT3 activation (Fig. 5C). This region between amino acids 366 and 390
contains a proline-rich region, which has some similarity with the box1
region in other receptors. Furthermore, it has been shown that this
region is necessary for JAK2 activation, although JAK2 is not
associated with the
subunit(17, 29) . Taken
together, we found that the cytoplasmic proline-rich region of
IL-5R
and the box1 region of the
c subunit are essential for
STAT3 activation.
Our results demonstrate that besides STAT1 and
STAT5(27, 36, 37) , STAT3 also plays a role
in IL-5 signal transduction. This extends the list of cytokines that
are capable to activate the STAT3 protein (32, 56, 57) . STAT3 was originally
identified as the acute phase response factor, which is activated in
response to IL-6 and plays a role in the activation of the acute phase
response genes(58) . Later on it became clear that this protein
shows homology (40-50%) to STAT1
/
(32) and was
therefore renamed STAT3.
The studies presented here demonstrate that
STAT3 is activated in response to IL-5 both at the DNA-binding and
transcription-activation level. We performed these experiments in COS
cells by reconstitution of the IL-5R together with several members of
the STAT family. We found that IL-5 induces specifically the
transactivation potential of STAT1 and STAT3 mediated via the
pIRE. Although we were not able to detect an effect of IL-5 to activate
the transcriptional potential of STAT5, we found an IL-5-induced
increase in DNA-binding of STAT5 to the GRR.
The latter
finding is in line with recent studies, which demonstrated an increase
in DNA-binding but not in transactivation mediated by mSTAT5 in
response to growth hormone and erythropoietin in COS
cells(37) . This means that probably cell type-specific factors
play an additional role in the transactivation of the STAT proteins. We
reported recently that serine phosphorylation is necessary to mediate
STAT3 transactivation(45) . Because IL-5 plays a restricted
role in hematopoiesis, it can be envisaged that cell type-specific
serine/threonine kinases are necessary to activate the transactivation
potential of some STAT proteins. A recent report showed induced
tyrosine phosphorylation of STAT6 by IL-3 in murine IL-3-dependent
myeloid DA-3 cells(39) . Although IL-3 and IL-5 use the common
c subunit, we found no activation of STAT6 by IL-5 after
overexpression of STAT6 in COS cells. This observation also supports
the hypothesis that cell type-specific factors are required for STAT
activation. The involvement of STAT3 in IL-5 signaling is further
supported by the observation that STAT3 was tyrosine phosphorylated in
BaF3/IL5R cells after treatment with hIL-5. The tyrosine
phosphorylation of STAT3 is necessary for binding of the STAT3 protein
to the ICAM-1 pIRE as can be seen in Fig. 4B. We found
that hIL-5 induced four DNA-binding complexes in these BaF3/IL5R cells.
Using different STAT antibodies, we identified complex C1 as a STAT3
homodimer, complex C2 as a STAT1
/STAT3 heterodimer, and C3 as a
STAT1
homodimer, whereas C4 was not supershifted by both
antibodies. Recently a similar DNA binding pattern was found when human
breast carcinoma T47D cells were treated with IL-6. However, they
identified the C4 complex as a multimeric complex composed of STAT3, a
91-kDa (not STAT1
), and a 46-kDa protein(59) . In
conclusion, IL-5 activates both STAT1
and STAT3 in COS and BaF3
cells. However, the previously described unidentified DNA-binding
protein induced by IL-5 in mature human eosinophils was not recognized
by a STAT3 antibody.
The activation of different STAT
proteins by a single cytokine such as IL-5 provides the cell with a
mechanism to activate different subsets of target genes depending on
the cellular context. For example, STAT3 might play a role in the
precursor cells of the eosinophil. This hypothesis is presently tested
by differentiating hematopoietic stem cells from cord blood with IL-5
to mature eosinophils.
Signal transduction by the IL-5 receptor is
rather poorly characterized. The cytoplasmic domain of the c
subunit is well characterized in the context of the GM-CSF receptor and
contains two distinct regions that are responsible for different
signals(19) . A membrane proximal region of the approximately
60 amino acids is essential for induction of the c-myc and pim-1 genes as well as for inducing mitogenesis. A distal
region of 140 amino acid residues is required for activation of Ras,
Raf-1, mitogen-activated protein kinase and p70 S6 kinase. Recently, it
has been shown that a cytoplasmic portion containing box1 of the
c
subunit is required for JAK2 activation. Moreover, JAK2 is
constitutively associated with the membrane proximal region of the
c chain(29) . Deletion analysis of the receptors for
growth hormone, erythropoietin, and the interleukin-6 receptor signal
transducing protein gp130, showed an absolute requirement of a six
amino acid PXXPXP sequence in box1 for JAK kinase
association(60) . Furthermore, a proline-rich region in the
IL-5
receptor that resembles box1 in the
c subunit is also
important for JAK2 activation(17) , although JAK2 is not
associated with this region (29) . However, not much is known
about the involvement of the IL-5R in the activation of STAT proteins.
Our results using a series of c-terminally truncated deletion mutants
of the
and
chain demonstrate that the membrane-proximal
region of both subunits are required for STAT activation. Recently, the
involvement of a specific tyrosine-based motif in the gp130 receptor
has been described in the phosphorylation of STAT3(55) .
However, receptor deletion mutant
516 contains no tyrosine
phosphorylation sites but was still able to activate STAT3. This means
that tyrosine-phosphorylation of the
c subunit is not important
for STAT3 activation. The same observation was previously shown for
STAT1
activation accomplished via the growth hormone
receptor(61) . An alternative mechanism could be that the SH2
domain of the STAT3 protein binds to the tyrosine-phosphorylated JAK
kinase, or that the SH3 domain of STAT3 binds to the proline-rich box1
region of the
c subunit. Such a mechanism can be explained by the
requirement of the membrane-proximal region of the
c subunit for
both JAK and STAT activation. This suggests that the precise mechanism
by which STAT proteins are activated probably depends on the
association of the different JAK kinases to cytokine receptors and on
specific binding sites for STAT proteins on the cytokine receptor.
Therefore, further studies are required to determine the precise
molecular mechanism by which STAT proteins are activated via the
c
subunit of the IL-5 receptor.