(Received for publication, August 18, 1995; and in revised form, November 8, 1995)
From the
Granulocyte-macrophage colony-stimulating factor (GM-CSF),
supports proliferation, differentiation, and functional activation of
hemopoietic cells by its interaction with a heterodimeric receptor.
Although GM-CSF receptor is devoid of tyrosine kinase enzymatic
activity, GM-CSF-induced peripheral blood polymorphonuclear leukocytes
(PMN) functional activation is mediated by the phosphorylation of a
large number of intracellular signaling molecules. We have previously
shown that JAK2 becomes tyrosine-phosphorylated in response to GM-CSF
in PMN. In the present study we demonstrate that also the signal
transducers and activators of transcription (STAT) family members STAT1
p91 and STAT3 p92 and the product of the c-fps/fes protooncogene become tyrosine-phosphorylated upon GM-CSF
stimulation and physically associated with both GM-CSF receptor
common subunit and JAK2. Moreover GM-CSF was able to induce JAK2 and
p93
catalytic activity. We also demonstrate that
the association of the GM-CSF receptor
common subunit with JAK2
is ligand-dependent.
Finally we demonstrate that GM-CSF induces a DNA-binding complex that contains both p91 and p92. These results identify a new signal transduction pathway activated by GM-CSF and provide a mechanism for rapid activation of gene expression in GM-CSF-stimulated PMN.
Granulocyte-macrophage colony-stimulating factor (GM-CSF) ()regulates proliferation and differentiation of hemopoietic
progenitor cells and functionally activates polymorphonuclear
leukocytes (PMN)(1) . In particular, GM-CSF exerts several
direct actions on neutrophils, including stimulation of changes in
surface expression of both chemotactic receptors and adherence
proteins(2, 3, 4, 5) , as well as
hydrogen peroxide production by neutrophils adhered to extracellular
matrix components(6, 7) . Moreover GM-CSF has indirect
effects on neutrophils, such as ``priming'' these cells for
enhanced responses to a number of physiologically relevant stimuli such
as ingestion of Staphylococcus aureus(8) ,
serum-opsonized particles(3) , antibody-dependent
cytotoxicity(3) , fMet-Leu-Phe (FMLP)-stimulated intracellular
calcium mobilization(9) , and oxyradical (2, 10) and platelet-activating factor (11) production as well as leukotriene
synthesis(12, 13, 14) . More recently it has
been reported that GM-CSF inhibits programmed cell death both in human
eosinophils and neutrophils (15) and that this effect is
mediated by tyrosine phosphorylation of intracellular
substrates(15) .
All GM-CSF effects are mediated by a
heterodimeric receptor comprised of a ligand binding subunit, denoted
(16) , and of a transducing subunit designated as
(17) , which is also shared with interleukin-3 (IL-3) (17) and IL-5 receptor(18) . Although GM-CSF receptor
does not possess an intrinsic tyrosine kinase domain, several lines of
evidence indicate that signaling processes initiated by ligand binding
to the receptor induce activation of cellular tyrosine
kinases(19) . Studies on the biochemical interaction involved
in signaling from the GM-CSF receptor have demonstrated that a number
of transducing molecules such as
Shc(20, 21, 22) , Grb2(20) ,
Sos1(20) , Ras(23) , Raf-1 (24) , and
mitogen-activated protein kinase (25) become activated upon
GM-CSF stimulation. It has also been reported that a nonreceptor
tyrosine kinase, the c-fps/fes protooncogene product, is
phosphorylated in response to GM-CSF(26) . More recently the
receptor-associated protein JAK2 (27, 28) has been
reported to be rapidly phosphorylated upon GM-CSF receptor
activation(29, 30) . Recent data suggest that at least
two components of latent cytoplasmic proteins termed signal transducers
and activators of transcription (STATs)(31) , which become
activated upon ligand binding, are substrates of JAK family
members(32, 33, 34) . In order to
characterize the tyrosine-phosphorylated proteins involved in
GM-CSF-mediated PMN activation, we examined the role of two STAT
proteins, STAT1
(p91) and STAT3 (p92) and of
c-fps/fes protooncogene product
(p93
) in this process. We demonstrate that, upon
GM-CSF stimulation, both STAT proteins and p93
become tyrosine-phosphorylated and physically associate with
GM-CSF receptor
common subunit as well as with JAK2. GM-CSF
stimulation was also able to induce p93
and JAK2
catalytic activity. Moreover we demonstrate that, as previously
reported for erythropoietin receptor (35) , JAK2 association
with the
common subunit is ligand-dependent. Finally we
demonstrate that the DNA-binding proteins p91 and p92 are early targets
of the GM-CSF-induced DNA-binding complex. These results identify a
signal transduction pathway that is activated in response to GM-CSF in
human PMN and provide evidence for the role of STAT proteins in
GM-CSF-mediated rapid modulation of gene expression in functionally
activated nonproliferating cells.
HEPG2 cells were maintained in RPMI 1640 medium supplemented with 10% bovine calf serum and serum-starved overnight before being treated with rhIL-6 (30 ng/ml).
GM-CSF plays an important role in host defense by enhancing
the functional activities of mature leukocytes and, in particular,
neutrophils(2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14) .
The binding of GM-CSF to its heterodimeric receptor, which is devoid of
intrinsic kinase activity, leads to tyrosine phosphorylation of
cellular substrates(19) . It has been reported that, in growth
factor-dependent cell lines, JAK2 is constitutively associated with the
subunit(29, 30) . Moreover JAK2 has been shown
to be phosphorylated upon growth factor stimulation not only in
proliferating cells but also in PMN and eosinophils functionally
activated by GM-CSF (30) and IL-5(38) , respectively.
Moreover, a ligand-dependent association of JAK2 with erythropoietin
receptor has also been reported(35) . To further elucidate the
interaction between the
subunit and JAK2 we performed
co-immunoprecipitation experiments in unstimulated and
GM-CSF-stimulated PMN. The results, reported in Fig. 1,
demonstrate that in PMN p130 JAK2 physically associates with the
subunit only upon GM-CSF stimulation, suggesting that, under
physiological conditions, the association between JAK2 and the receptor
may not be constitutive. Kinetic analysis of JAK2 activation, upon
GM-CSF stimulation, reported in Fig. 2, demonstrates a transient
JAK2 tyrosine phosphorylation peaking at 5 min and disappearing after
10 min.
Figure 1:
JAK2 association with the
GM-CSF receptor common subunit in PMN. Cell lysates from
unstimulated(-) or GM-CSF-stimulated (10 ng/ml for 5 min)
(+) PMN were immunoprecipitated with anti-
antiserum. The
cells were also immunoprecipitated with anti-JAK2 antiserum to indicate
the p130 JAK2 protein. The filter was probed with the anti-JAK2
antiserum (upper panel) and reprobed with the anti-
antiserum (lower panel). The p130 JAK2 is indicated. IP, immunoprecipitated; IB,
immunoblotted.
Figure 2: Kinetic analysis of GM-CSF-induced JAK2 activation in human PMN. PMN were incubated in 1640 RPMI medium in the absence or in the presence of GM-CSF (10 ng/ml) for the indicated time, lysed, and immunoprecipitated with the anti-JAK2 antiserum. The filter was probed with 4G10 anti-phosphotyrosine monoclonal antibody (upper panel) and reprobed with the antiserum against JAK2 (lower panel). The positions of the p130-JAK2 and the p90 phosphotyrosine proteins are indicated. IP, immunoprecipitated; IB, immunoblotted; P-tyr, phosphotyrosine.
A likely set of substrates for the JAKs is the family of
latent cytoplasmic transcription factors termed STATs(31) .
Ligand binding to several cytokine receptors induces tyrosine
phosphorylation of STAT family members that, subsequently, translocate
to the nucleus, bind to related DNA sequences, and promote
transcription(39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52) .
The anti-phosphotyrosine blot of anti-JAK2 immunoprecipitates from
GM-CSF-treated PMN revealed, together with the phosphorylated p130
JAK2, a marked band of phosphotyrosine-containing protein(s) of
approximately 90 kDa (Fig. 2). It is known that among the STAT
proteins STAT1 and STAT3 exhibit a molecular mass of 91 and 92 kDa,
respectively. Therefore we sought to determine whether the JAK2
co-precipitating p90 phosphoprotein(s) included one or both STATs. It
has also been demonstrated that a p93 myeloid-specific protein, the
product of the protooncogene c-fps/fes, becomes phosphorylated
and associated to the GM-CSF receptor common subunit upon GM-CSF
stimulation in a growth factor-dependent cell line(26) .
Therefore, we tried to assess whether p93
was also
included in JAK2 co-precipitating p90 phosphoprotein(s). To test these
possibilities we first evaluated tyrosine phosphorylation of p91, p92,
and p93
upon GM-CSF stimulation. As shown in Fig. 3, both STAT proteins, p91 and p92, and p93
become phosphorylated after 5 min of GM-CSF treatment. In
addition, in the anti-p91 and -p92 immunoprecipitates together with the
marked band of approximately 90 kDa a faint band of approximately 130
kDa was detected. Taken together these results strongly suggest that at
least the two STAT proteins physically associated with JAK2. To confirm
this hypothesis, anti-JAK2 co-immunoprecipitates from unstimulated and
GM-CSF-stimulated PMN were divided into four aliquots, resolved by
SDS-polyacrylamide gel electrophoresis, and independently blotted with
the antibodies of interest. In the anti-phosphotyrosine immunoblot
reported in Fig. 4A a large band of approximately 90 kDa can
be detected only in GM-CSF-stimulated PMN. The anti-p91, anti-p92, and
anti-p93
immunoblots of anti-JAK2 immunoprecipitates from
untreated and GM-CSF-treated PMN are shown in Fig. 4, B, C, and D, respectively, demonstrating
that the two STAT proteins together with the p93
are
physically associated with JAK2 only upon GM-CSF stimulation. The
specificity of these results was further confirmed by the inability of
anti-JAK1 antiserum to co-immunoprecipitate these proteins (data not
shown). Moreover the correlation between the biochemical events induced
by GM-CSF stimulation and its biological effects on PMN was supported
by the observation that, upon IL-3 stimulation, neither functional
activation (data not shown) or protein tyrosine phosphorylation were
detected in anti-JAK2 immunoprecipitates (Fig. 5). The
observation that JAK2 physically associates with the
common
subunit as well as with p91 and p92 STATs and p93
implies
that the latter three proteins are also directly or indirectly, via
JAK2, associated with the
common. To evaluate this hypothesis
anti-beta co-immunoprecipitation experiments were performed. The
anti-phosphotyrosine immunoblot of anti-
common immunoprecipitates
from unstimulated and GM-CSF-stimulated PMN, shown in Fig. 6A, demonstrates the presence of approximately 90-kDa
tyrosine-phosphorylated protein(s) only in GM-CSF-stimulated cells.
Moreover when aliquots of the same samples were resolved by
SDS-polyacrylamide gel electrophoresis and independently blotted with
the anti-p91 (Fig. 6B), anti-p92 (Fig. 6C),
and anti-p93
(Fig. 6D) antibodies, the
two STAT proteins together with the p93
were found to be
physically associated, upon ligand binding, with the
common
subunit.
Figure 3:
GM-CSF-induced tyrosine phosphorylation of
STATp91, STATp92, and p93 in PMN.
Untreated(-) and GM-CSF-treated (for 5 min with 10 ng/ml of
GM-CSF) (+) PMN were immunoprecipitated with anti-p91 (A)
anti-p93
(B), and anti-p92 (C)
antibodies. The filters were probed with 4G10 anti-phosphotyrosine
monoclonal antibody (upper panels) and reprobed with anti-p91 (A), anti-p93
(B), and
anti-p91 (C) antibodies (lower panels). The position
of p91, p93
, and p92 are indicated. IP,
immunoprecipitated; IB, immunoblotted; P-tyr,
phosphotyrosine.
Figure 4:
p91, p92, and p93 are physically associated with JAK2. Unstimulated(-)
and GM-CSF-stimulated (+) PMN were lysed and immunoprecipitated
with anti-JAK2 antiserum. The immunoprecipitates were divided into four
aliquots that were resolved in SDS-polyacrylamide gel and blotted
independently. A, the filter was probed with 4G10
anti-phosphotyrosine monoclonal antibody (upper panel) and
reprobed with anti-JAK2 antiserum (lower panel). B,
the filter was probed with anti-p91 antiserum. The cells were also
immunoprecipitated with anti-p91 antiserum to indicate the migration of
the p91 protein. C, the filter was probed with anti-p92
antibody. The cells were also immunoprecipitated with anti-p92
antiserum to indicate the migration of the p92 protein. D, the
filter was probed with anti-p93
antiserum. The
cells were also immunoprecipitated with anti-p93
antiserum to indicate the migration of the p93
protein. The position of p91, p92, and p93
are indicated. IP, immunoprecipitated; IB,
immunoblotted; P-tyr,
phosphotyrosine.
Figure 5: GM-CSF-induced JAK2 tyrosine phosphorylation in human PMN. Cell lysates from unstimulated(-) and IL-3- and GM-CSF-stimulated (10 ng/ml each for 5 min) (+) PMN were immunoprecipitated with anti-JAK2 antiserum. The filter was probed with 4G10 anti-phosphotyrosine monoclonal antibody (upper panel) and reprobed with the antiserum against JAK2 (lower panel). The positions of the p130-JAK2 and the p90 phosphotyrosine proteins are indicated. IP, immunoprecipitated; IB, immunoblotted; P-tyr, phosphotyrosine.
Figure 6:
p91, p92, and p93 physically associate with the GM-CSF receptor
common
subunit. Unstimulated(-) and GM-CSF-stimulated (+) PMN were
lysed and immunoprecipitated with anti-GM-CSF receptor
common
subunit antiserum. The immunoprecipitates were divided into four
aliquots that were resolved in SDS-polyacrylamide gel and blotted
independently. A, the filter was probed with 4G10
anti-phosphotyrosine monoclonal antibody (upper panel) and
reprobed with anti-GM-CSF receptor
common subunit antiserum (lower panel). B, the filter was probed with anti-p91
antiserum. The cells were also immunoprecipitated with anti-p91
antiserum to indicate the migration of the p91 protein. C, the
filter was probed with anti-p92 antibody. The cells were also
immunoprecipitated with anti-p92 antiserum to indicate the migration of
the p92 protein. D, the filter was probed with
anti-p93
antiserum. The cells were also
immunoprecipitated with anti-p93
antiserum to
indicate the migration of the p93
protein. The
positions of p91, p92, and p93
are indicated. IP, immunoprecipitated; IB, immunoblotted; P-tyr, phosphotyrosine.
Tyrosine phosphorylation of various tyrosine kinases is
commonly associated with the activation of their catalytic
activity(53) . An in vitro kinase assay was performed
to examine whether phosphorylation of JAK2 and p93 correlates with their intrinsic kinase activity. As shown in Fig. 7, anti-JAK2 (panel A) and anti-p93
(panel B) immunoprecipitates from GM-CSF-stimulated, but
not from IL-3-stimulated, PMN have a detectable in vitro kinase activity.
Figure 7:
In vitro JAK2 and p93 kinase activity following IL-3 and GM-CSF stimulation in
human PMN. Anti-JAK2 (panel A) and anti-p93
(panel B) immunoprecipitates from
unstimulated(-) and IL-3- and GM-CSF-stimulated (10 ng/ml each
for 5 min) (+) PMN were washed and divided in two aliquots. The
first one was resuspended in the kinase assay buffer containing
[
-
P]ATP for 30 min at room temperature,
washed and eluted with sample buffer for SDS-polyacrylamide gel
electrophoresis, separated on 8% gel, and detected by autoradiography (upper panels). The second one was separated on 8% gel and
probed with anti-JAK2 and anti-p93
antisera,
respectively (lower panels). The p130-JAK2 and the
p93
proteins are indicated. IP,
immunoprecipitated; IB,
immunoblotted.
PMN are terminally differentiated cells and do
not undergo proliferation; however, tyrosine phosphorylation of
intracellular substrates has been implicated in a number of functional
activities such as superoxide anion
production(54, 55, 56, 57, 58) ;
regulation of integrin surface expression, leading to adherence of PMN
to endothelial cells (59) ; regulation of microvascular
permeability, leading to migration of PMN into inflammatory
tissue(60, 61) ; and modulation of apoptotic
process(15) . The role of protein tyrosine phosphorylation in
physiological agonist-mediated or GM-CSF-mediated PMN activation is
further supported by the observation that PMN biological responses are
prevented by the addition of tyrosine kinase
inhibitors(15, 55, 56, 59, 62) .
It has been shown that, in PMN, GM-CSF causes a rapid tyrosine
phosphorylation of intracellular molecules including both 90- and
130-kDa proteins(15, 55, 62, 63) .
In agreement with these findings, the present study demonstrates that a
set of 90-kDa proteins namely STAT1, STAT3, and p93 and a
130-kDa protein, identified as JAK2, become phosphorylated upon GM-CSF
stimulation. Therefore, it is reasonable to assume that at least some
PMN functional activities may be regulated by the JAK/STAT signaling
pathway.
It has been reported that treatment of cells with different
cytokines results in rapid STAT protein phosphorylation. Activated
STATs (one or more) form dimers that migrate in the nucleus and form
stable complexes with specific DNA sequences (response elements) and
stimulate transcription(31) . Three discrete complexes between
activated STAT proteins and DNA response elements have been
demonstrated upon EGF treatment(50) . These complexes seem to
be formed by STAT1 or STAT3 homodimers or by heterodimers between the
two STATs(50) . In contrast, in interferon--(44) ,
IL-6-(50) , and GM-CSF-treated cells(51) , only one
complex can be detected containing either the STAT1 (45) or the
STAT3 homodimers(51) . The rapid tyrosine phosphorylation of
p91 and p92 observed in GM-CSF-stimulated PMN led us to evaluate, by
gel retardation assay, the formation of DNA-protein complexes in
nuclear extract of untreated and treated cells. As shown in Fig. 8A, both in IL-6-treated HEPG2 cells and in
GM-CSF-treated PMN a DNA-protein complex appears. Moreover when the
same nuclear extracts were incubated with an excess of unlabeled
oligonucleotide, both the IL-6- and GM-CSF-induced complexes are
competed (Fig. 8A) demonstrating its sequence specificity.
Moreover it is also clear that the DNA-binding complex observed in
IL-6-stimulated HEPG2 cells shows a slower migration than that observed
in GM-CSF-stimulated PMN. It has been reported that in HEPG2 cells IL-6
induces only the formation of a major complex, defined also as SIF-A (40, 45) , corresponding to the complex containing p92
homodimers(50) . In contrast, EGF-activated proteins have been
shown to form three complexes, designated as SIF-A, SIF-B, and
SIF-C(50) , with the serum-inducible element of c-fos (in its mutated, hyperactive form)(40, 45) .
Therefore, it is possible that the faster migrating complex observed in
GM-CSF-stimulated PMN contains either STAT1
STAT3 heterodimers
and/or STAT1 homodimers. We thus tested the GM-CSF-induced complex for
reactivity with anti-STAT1 p91 and anti-STAT3 p92 antibodies. As shown
in Fig. 8B, when anti-p91 and anti-p92 antisera were
added to GM-CSF-treated nuclear extract, a new band, which was not
present in the binding reaction with preimmune serum, appeared in the
upper part of the gel, thus demonstrating the formation of a
supershifted species. A supershifted species appears also when nuclear
extract from IL-6-stimulated HEPG2 cells was preincubated with anti-p92
antiserum (Fig. 8B) The presence of the supershifted
complex observed both in anti-p91 and anti-p92-pretreated nuclear
extract suggests that, in PMN, GM-CSF can rapidly modulate gene
expression by the induction of a DNA-binding complex containing p91 and
p92 heterodimer.
Figure 8: A, induction of DNA-binding activity by GM-CSF and IL-6 in PMN and in HEPG2 cells. Nuclear extracts of untreated(-) or 15-min IL-6-treated HEPG2 cells and GM-CSF-treated PMN (+) were either treated (+) or not(-) treated with a 50-fold excess of unlabeled oligonucleotide (competitor) for 30 min before the addition of radiolabeled oligonucleotide. The complexes were then resolved by nondenaturing polyacrylamide gel electrophoresis. The DNA-binding complexes are indicated. B, the GM-CSF-induced DNA-binding complex is antigenically related to p91 and p92. Nuclear extracts from 15-min GM-CSF-treated PMN or IL-6-treated HEPG2 cells were preincubated for 1 h at 4 °C with preimmune serum (PI), anti-p91 antiserum, or anti-p92 antibodies before incubation with radiolabeled oligonucleotide and separated on a nondenaturing polyacrylamide gel electrophoresis. The IL-6- and GM-CSF-induced DNA-protein complexes and the supershifted specie are indicated.
It has been shown that tyrosine-phosphorylated proteins are involved in GM-CSF-mediated PMN functional activation and c-fos gene transcription(55) . Moreover the role of tyrosine kinases in controlling GM-CSF-induced c-fos gene expression in PMN, has been demonstrated by the use of a tyrosine kinase inhibitor(55) . Our finding that at least two of the STAT proteins that become phosphorylated upon GM-CSF stimulation are involved in the formation of a complex with the serum-inducible elements of c-fos, supports the hypothesis that GM-CSF can regulate the transcription of this gene via STAT1 and STAT3 activation.
In conclusion, our study demonstrates that in PMN both STAT1 p91 and
STAT3 p92 and the myeloid-specific p93 become
phosphorylated upon GM-CSF stimulation and are co-immunoprecipitated by
anti-JAK2 and anti-
common subunit antibodies, and that GM-CSF
induces the formation of a DNA-protein complex containing both p91 and
p92.
The redundancy of growth factors inducing the same DNA-responsive element to stimulate both cell proliferation and functional activation raises the question of how their specificity can be determined. The answer could be obtained by the identification of more genes whose transcription can be activated by the binding of known or unknown proteins.