From the Ludwig Institute for Cancer Research,
Melbourne Tumour Biology Branch, P. O. Royal Melbourne Hospital,
Victoria, 3050, Australia and the ¶ University of Melbourne,
Department of Medicine, P.O. Royal Melbourne Hospital,
Victoria, 3050, Australia
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ABSTRACT |
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Cell type-specific responses to the leukemia
inhibitory factor (LIF)/interleukin 6 cytokine family are mediated by
dimerization of the LIF receptor Cytokines, such as the interleukins (IL)1 and colony
stimulating factors (CSF), regulate a
wide range of biological activities through binding to transmembrane
receptors. The type I cytokine receptor superfamily is characterized by
conserved cysteine residues and the presence of a
tryptophan-serine-X-tryptophan-serine (WSXWS) motif in the extracellular domain (1). Weak sequence similarity between
individual members of the type I receptor family extends into the
membrane-proximal Box 1 and Box 2 homology regions of the cytoplasmic
domain (2, 3). Box 1 and Box 2 homology regions associate
constitutively with Janus tyrosine kinases (JAKs) and are indispensable
for ligand-mediated JAK activation (4). Functional analysis of the
cytoplasmic domain of the IL6 signal-transducing subunit, gp130, the
ligand-binding subunit of the leukemia inhibitory factor receptor
(LIFR The pleiotropic actions of the LIF/IL6 family of cytokines include the
regulation of cell growth, apoptosis, protein secretion, and cellular
differentiation, often in a cell type-specific manner (7). In some
cases, opposing biological responses are triggered by the LIF/IL6
cytokine family such as the induction of differentiation in myeloid
leukemia M1 cells and the suppression of differentiation in embryonic
stem (ES) cells. Thus, it has been proposed that cellular responses may
be the consequence of gene expression programs elicited by an array of
different, and sometimes cell type-specific intracellular signaling
cascades, generated by the receptors for IL2, GCSF, and
granulocyte-macrophage CSF (8-10). However, in all cell types
investigated, binding of LIF to its ligand-binding chain, LIFR The LIF/IL6 cytokine family has been shown to induce tyrosine
phosphorylation and DNA binding of a subset of STAT proteins, namely
STAT1, STAT3 via a receptor binding dependent mechanism, and possibly
STAT5a via a direct interaction with JAK kinases (19-21). In gp130 and
LIFR In the present study, we attempt to identify the cytoplasmic region of
individual cytokine receptor chains required for either ligand-dependent suppression of ES cell differentiation or
promotion of cellular proliferation. To this end, we expressed chimeric receptor constructs in which the transmembrane and cytoplasmic domains
of gp130, LIFR Biological Reagents and Cell Cultures--
Recombinant
human LIF (ESGRO) was purchased from Amrad (Melbourne, Australia) and
recombinant human GCSF and murine IL6 were kindly provided by Amgen
(Thousand Oaks, CA) and R. Simpson (Ludwig Institute, Melbourne,
Australia), respectively. The monoclonal antibodies used for Western
blotting of STAT3 and anti-phosphotyrosine (4G10) were obtained from
Upstate Biotechnology Inc. (Lake Placid, NY). Immunoprecipitation
assays were carried out with polyclonal antisera directed against SHP2
(from Santa Cruz Biotechnology, Santa Cruz, CA), STAT3 and STAT5a (from
Transduction Laboratories; Lexington, KY), and Hck (gift from C. Lowell, University of California, San Francisco, CA).
Full-length chimeric receptor constructs containing the extracellular
domain of the murine GCSF-R ligated to the transmembrane and cytosolic
domain of gp130 and LIFR
All ES cell lines were derived from the HCK-hprt modified
E14TG2a cell line which contains a hypoxanthine-guanine
phosphoribosyltransferase minigene under the control of an
hck promoter fragment which is transcriptionally inactive in
differentiated ES cells (20). ES cell lines were maintained free of
feeder cells in ES cell medium (14) supplemented with 1000 units/ml LIF
which was substituted during experiments by the indicated concentration
of LIF or GCSF. The IL6-dependent murine plasmacytoma cell
line 7-TD1 was maintained in Dulbecco's modified Eagle's medium
supplemented with 10% FCS, 0.24 mM
L-asparagine, 0.55 mM L-arginine,
50 µM 2-mercaptoethanol, and 5 pM IL6 which
was replaced during experiments by the indicated concentration of
GCSF.
Cell Lines--
Stable transfected ES cell lines were obtained
by electroporating 15 µg of the linearized
6-16PGK-IRESneo-based expression constructs or 15 µg of
the linearized pEF-BOS-based constructs together with 1.5 µg of the selectable marker plasmid PGKneo (500 µfarads,
270 V, Bio-Rad Genepulser) into 1.5 × 107 ES cells.
Receptor expression level on individually expanded G418-resistant
(175 µg/ml Geneticin, Life Technologies, Inc.) colonies were carried
out by binding of radiolabeled GCSF as described (20). Cell lines with
a comparable number of receptors (800-1400 receptor/cell) were used
for experiments.
The proliferative capacities of the chimeric gp130 and LIFR Cell Culture Assays--
The extent to which ES cell
differentiation was prevented through GCSF-dependent
formation of mutant receptor homodimers was determined as described by
morphological appearance (14, 51) as well as by selection against
differentiated cells in the presence of HAT-containing (200 µM hypoxanthine, 0.8 µM aminopterin, 32 µM thymidine) culture medium and measuring the conversion
of MTT (3-(4,5-dimethlydiazol-2-yl)-2,5-diphenyltetrazolium bromide; Sigma, 0.5 mg/ml final concentration) to a product with maximal optical
absorbance at 540 nm (20). AS oligonucleotide experiments were carried
out by incubating 24-h cultures (inoculated at 5000 cells/cm2) for 10 min with streptolysin-O-containing (5 units/ml, Sigma) permeabilization buffer (150 mM KCl, 37.5 mM NaCl, 6.25 mM MgCl2, 0.8 mM EGTA, 1 mM CaCl2, 1.24 mM ATP, 12.5 mM PIPES, pH 7.5) supplemented with 3 µM AS oligonucleotide. Cultures were then rinsed
carefully several times with ES cell medium and incubated for 2 days in the presence of 3 µM AS oligonucleotide in ES cell medium
supplemented with the indicated concentration of LIF. At this time, the
medium was replaced with fresh medium containing 3 µM AS
oligonucleotide and the cultures were incubated for an additional 3 days.
The GCSF-dependent survival/proliferation of
G418-resistant, chimeric receptor expressing 7-TD1 cells was assessed
by determining cell numbers following an 8-day exposure of cultures
(inoculated in 6-well multiculture dishes at a concentration 2 × 104 cells/well) to either GCSF (50 ng/ml) or 5 pM IL6 as a control. The mitogenic activity of
GCSF-R-expressing Ba/F03 cells was determined by thymidine
incorporation. For this purpose, the cells were inoculated in 96-well
multiculture dishes at a density of 104 cells/well,
stimulated for 48 h with GCSF (10 ng/ml) in RPMI 1640 medium
supplemented with 10% FCS. The cultures were pulsed for 4 h with
0.5 µCi/well [methyl-3H]thymidine (NEN Life
Science Products Inc.) prior to harvesting and incorporated
radioactivity was measured using a Betaplate liquid scintillation
counter (Pharmacia Biotech Inc.).
Immunoblotting, Electrophoretic Mobility Shift Assay, and in
Vitro Kinase Assays--
For all assays, confluent cultures of
undifferentiated ES cells were starved of LIF in ES cell medium
containing reduced FCS (1%) levels. Immunoprecipitation experiments
were carried out with anti-STAT3 or SHP2 antisera on 500 µg of cell
lysates in lysis buffer (1% Triton X-100, 150 mM NaCl, 50 mM Tris, pH 7.5, 1 mM EDTA, 1 mM
phenylmethylsulfonyl fluoride, 1% Trasylol, 0.1 mM
Na3VO4) prepared from cultures stimulated for
10 min with LIF (500 units/ml), GCSF (10 ng/ml), or saline as described
previously (51). The resulting immunoprecipitates were separated by
reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(PAGE) and transferred to nitrocellulose (Schleicher & Schull). The
membranes were incubated with anti-phosphotyrosine antibodies (1:1500
dilution) and the proteins visualized with peroxidase-conjugated rabbit anti-mouse immunoglobulin (1:20000; Bio-Rad) using the ECL system (Amersham Corp.). Before reprobing the membranes with anti-STAT3 (1:500
dilution) or anti-SHP2 (1:2500) antibodies, they were stripped of
antibodies in 62.5 mM Tris-HCl, pH 7.5, 2% SDS, 100 mM 2-mercaptoethanol. Sixty µg of total cell lysates were
also used for MAPK mobility shift assays as described previously
(14).
Electrophoretic mobility shift assays using the high affinity
c-sis, factor inducible-binding site m67 were carried out as described previously (20, 30) with 16 µg of extracts prepared from ES
cultures stimulated for 15 min with LIF (500 units/ml), GCSF (10 ng/ml), or saline and, where indicated, with STAT1 or STAT3 antisera.
The DNA-binding complexes were then subjected to electrophoresis and
visualized using a PhosphorImager (Molecular Dynamics).
For Hck in vitro kinase experiments, 500 µg of cell lysate
were immunoprecipitated with an Hck-specific antiserum 1077 (1:500 dilution, gift of C. Lowell, University of California, San Francisco, CA) and the tyrosine autophosphorylation reaction was carried out in
the presence of 10 µCi of [ The Membrane-distal Cytoplasmic Receptor Domains Are Required for
the Regulation of ES Cell Differentiation--
In order to test for
the independent signaling capacity of the two components of the native
heterodimeric LIF receptor complex, we exploited the fact that
undifferentiated ES cells do not express GCSF-R. Cell lines expressing
chimeric receptor constructs, in which the intracellular domain of
either gp130 or LIFR
Since the YXXQ motifs in the COOH-terminal region of gp130
are required for the induction of differentiation in myeloid M1 cells
in response to IL6 (18, 26), we next tested the biological effect
of carboxyl-terminal truncations of the cytoplasmic tail of gp130 and
LIFR
We next investigated the potential of wild-type and truncated chimeric
receptor homodimers to mediate a proliferative response in the
IL6-dependent cell line 7-TD1. Unlike the traditionally employed, factor-dependent pro-B cell line Ba/F03, the
plasmacytome cell line 7-TD1 shows marked induction of proliferation
upon activation of gp130 mediated signaling. As revealed in Fig.
2, both full-length, as well as the most
truncated receptors, mediated a GCSF-dependent increase in
cell number which was independent of STAT3 phosphorylation as assessed
by anti-phosphotyrosine blotting of cell lysates. These data,
therefore, suggest that the carboxyl-terminal membrane-distal regions
of gp130 and LIFR Activation of Intermediate Signaling Molecules--
The
heterodimeric LIFR
Since activation of the most extensively truncated receptors
(G/gp130 STAT3 Is Required for the Suppression of ES Cell
Differentiation--
The heterodimeric LIFR
Receptor-dependent engagement of particular STAT family
members is one of the mechanisms by which different biological outcomes may be achieved through JAK-STAT signal transduction pathways (4).
Consistent with this finding, stimulation of the interferon- Tyrosine Substitution in GCSF-R Impairs the Regulation of ES Cell
Differentiation--
Our limited survey of cytokine receptors suggests
that activation of the Box 3-containing cytoplasmic GCSF-R domain can
suppress ES cell differentiation (Fig. 1). Since the tyrosine residues within the cytoplasmic tail of the GCSF-R contribute to specific signaling pathways (31), we focused on the two tyrosine residues which
showed the most prominent impairment of GCSF-mediated differentiation in the myeloid M1 cell line (29, 30). Stimulation of a GCSF-R mutant
with a phenylalanine substitution of the membrane most proximal
tyrosine residue (Y74F) suppressed ES cell differentiation to a similar
extent as stimulation of the wild-type GCSF-R (Fig. 7). By contrast, substitution of the
tyrosine residue within Box 3 (Y144F) decreased the capacity to inhibit
differentiation in a GCSF-dependent manner by approximately
80%. The capacity to suppress differentiation was entirely lost in ES
cells expressing GCSF-R containing the double substitution
(Y74F,Y144F). In all cases, stimulation through the endogenous
LIFR
We also tested whether the tyrosine residues in GCSF-R required for
signaling in ES cells contribute to propagation of mitogenic signals.
For this purpose, we introduced the various GCSF-R expression constructs into the factor-dependent Ba/F03 cell line and
screened for surface expression of GCSF-R protein by flow cytometry
(data not shown) and representative cell lines were chosen which showed comparable levels of receptor expression. With all GCSF-R constructs tested, we were able to derive GCSF-dependent Ba/F03 cell
lines. Moreover, GCSF-dependent [3H]thymidine
incorporation was recorded in Ba/F03 cells expressing wild-type, Y74F,
and Y144F GCSF-R, respectively, while some of the cell lines containing
the Y74F,Y144F double substitution responded slightly less than
wild-type receptor expressing cells (Fig.
8). These results suggest that
Tyr144 within the GCSF-R Box 3 region is absolutely
required for the regulation of ES cell differentiation, but is
dispensable for the transmission of a mitogenic signal.
Analysis of STAT3 phosphorylation indicated reduced tyrosine
phosphorylation following activation of the GCSF-R mutants Y144F when
compared with the pattern observed with the wild-type and the Y74F
receptor, respectively (Fig. 9).
Furthermore, we could not detect significant STAT3 phosphorylation
following stimulation of ES cell lines expressing GCSF-R (Y74F,Y114F).
Similar results were obtained in electrophoretic mobility shift assays
using the SIE probe (data not shown). Thus, the decreased STAT3
phosphorylation and DNA-binding observed with the GCSF-R(Y74F,Y144F)
correlates with an inability to prevent differentiation of ES
cells.
In this study we demonstrate that the membrane-proximal region of
the cytoplasmic region of gp130 and LIFR Many cytokine receptors, including those for the ILF/IL6 family,
initiate divergent intracellular signaling pathways through domains
that are specifically required for regulation of proliferation, differentiation, or apoptosis (33-35). Signaling by most type I cytokine receptors depends minimally on the Box 1 motif which is
required for the association with JAKs. Transduction of mitogenic signals from the GCSF-R (36) and gp130 (27) depends on the membrane-proximal region extending over Box 1 and Box 2 domains. In
particular, phosphorylation of SHP2 via the YXXV motif,
which is located distal to the Box 2 and proximal to the Box 3 motif in
gp130, is absolutely required for mitogenesis in Ba/F03 cells (27).
Here, we show that the truncated G/gp130 Specificity of the JAK-STAT pathway is controlled at the level of JAK
kinase engagement (14, 41), SH2 domain dependent and independent
interaction of STAT proteins with specific cytokine receptors, STAT
dimerization, and the resulting specificity in DNA binding (42-44).
Receptor phosphotyrosine-independent STAT5 activation and cell
type-specific differences in STAT expression patterns may therefore
account for the fact that the truncated G/gp130 Based on the severity of the phenotype observed in mice with deletions
for either STAT3 or gp130, STAT3 appears to be a crucial signaling
intermediate for a number of growth factor and cytokine receptors in
addition to those for the LIF/IL6 cytokine family. STAT3 plays an
important role in self-renewal of lymphocytes (46) and is critically
involved in transducing some of the biological responses of the LIF/IL6
cytokine family, including IL6-induced terminal differentiation, growth
arrest, and macrophage differentiation of myeloid leukemia M1 cells
(18, 26) and prevention of apoptosis in factor-dependent
Ba/F03 cells (27). Similarly, phenylalanine substitutions or deletions
of all YXXQ motifs in gp130 or LIFR The presence of the common Box 3 motif in the cytoplasmic domain
of GCSF-R, LIFR While distinct, phosphotyrosine-mediated intracellular signaling
cascades appear to be the predominant mechanism by which mitogenic
signals are dissociated from those not affecting cell growth, we
present evidence that seemingly identical signaling mechanisms may
regulate signals that suppress as well as promote cellular
differentiation. Further studies are required in order to
establish whether differences in the strength of activation of a
particular signaling pathway (as observed here for STAT3) may
help to distinguish between the diverse biological outcomes mediated by
these signaling intermediates.
-chain (LIFR
) with the signal
transducer gp130 or of two gp130 molecules followed by activation of
the JAK/STAT and Ras/mitogen-activated protein kinase cascades. In order to dissect the contribution of gp130 and LIFR
individually, chimeric molecules consisting of the extracellular domain of the granulocyte colony stimulating factor receptor (GCSF-R) and various mutant forms of the cytoplasmic domains of gp130 or LIFR
were expressed in embryonic stem (ES) cells to test for suppression of
differentiation, or in a factor-dependent plasma cytoma
cell line to assess for induction of proliferation. Carboxyl-terminal domains downstream of the phosphatase (SHP2)-binding sites were dispensable for mitogen-activated protein kinase activation and the
transduction of proliferative signals. Moreover, carboxyl-terminal truncation mutants which lacked intact Box 3 homology domains showed
decreased STAT3 activation, failed to induce Hck kinase activity and
suppress ES cell differentiation. Moreover, STAT3 antisense
oligonucleotides impaired LIF-dependent inhibition of differentiation. Substitution of the tyrosine residue within the Box 3 region of the GSCF-R abolished receptor-mediated suppression of
differentiation without affecting the transduction of proliferative signals. Thus, distinct cytoplasmic domains within the LIFR
, gp130,
and GCSF-R transduce proliferative and differentiation suppressing signals.
INTRODUCTION
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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), the granulocyte CSF receptor (GCSF-R), and the receptor for
thrombopoietin (c-Mpl) has revealed an additional region of homology
(Box 3) which is specific to gp130, LIFR
, and GCSF-R but not found
in c-Mpl (5, 6). Mutational analysis revealed that the Box 3 homology
region is required for the induction of some specific biological
responses, such as activation of acute-phase protein expression in
transfected liver cells, thereby suggesting that distinct regions of
the cytoplasmic domain may be required for the induction of specific
biological responses (5, 6).
,
results in the formation of a heterodimeric receptor complex with gp130
which in turn triggers the activation of gp130-associated JAKs and
other cytoplasmic tyrosine kinases (11-14). In ES cells, ostensibly
identical signaling events are initiated following IL6-induced
homodimerization of gp130 molecules leading to activation of JAK family
members Jak1, Jak2, and Tyk2, as well as of the Src-related tyrosine
kinase Hck (14, 15). The kinases are thought to undergo
autophosphorylation and at least Jak1 has been shown to subsequently
phosphorylate tyrosine residues on gp130 and LIFR
(12, 13). In turn,
the phosphotyrosine residues on the cytokine receptors recruit various
Src homology (SH2) domain-containing intermediate signal transducing
molecules which often result in their subsequent tyrosine
phosphorylation and activation. Two such molecules, the tyrosine
phosphatase SHP2 and the latent signal transducer and activator of
transcription 3 (STAT3), are phosphotyrosine binding partners for gp130
and LIFR
(16, 17), which contain 6 and 5 tyrosine residues in their
respective cytoplasmic domains (15, 18). In the case of gp130 and
LIFR
, activation of STAT3 depends on phosphorylation of the most
carboxyl-terminal tyrosine residues while a different, more
membrane-proximal located phosphotyrosine residue is required for
activation of SHP2 (15, 18).
, tyrosine phosphorylation and activation of STAT3 appears
dependent on the presence of phosphotyrosine residues with a glutamine
at position +3 relative to the tyrosine (YXXQ) thereby
contrasting the sequence requirement for the activation of SHP2 which
requires a valine at position +3 (YXXV) (15). The relative
specificity of STAT activation by individual cytokine receptors
prompted the notion that individual STAT family members determine the
specificity of signal transduction from a particular cytokine receptor.
Support for this view has recently been provided by genetic deletion
studies in mice which point to a critical role for STAT1 in the innate
immunity elicited by interferon-
and -
(22, 23). By contrast,
lack of STAT3 expression results in early embryonic lethality (24),
while STAT5a-deficient mice lack terminally differentiated mammary
glands (25). In vitro, activation of STAT3 appears essential
for IL6-mediated differentiation of mouse leukemia M1 cells (18, 26)
and the prevention of apoptosis in factor-dependent Ba/F03
cells (27).
, or GCSF-R were fused to the extracellular domain of
the GCSF-R. This approach permits GCSF-dependent
homodimerization of chimeric receptor subunits independent of the
corresponding endogenous receptor chains (5, 6, 19, 20, 27). Our results demonstrate that carboxyl-terminal truncations or mutation of
specific tyrosine residues within Box 3-containing receptor chains have
no effect on the transduction of proliferative signals, while reducing
the capacity to suppress ES cell differentiation. This response
correlates with a decrease in Hck kinase activity and STAT3
phosphorylation. A critical role for STAT3 is supported by our finding
that the presence of STAT3 antisense (AS)-oligonucleotides impairs the
differentiation-inhibiting activity of full-length gp130 and LIFR
chains on ES cells in vitro.
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ABSTRACT
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DISCUSSION
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have been described previously (20). The
full-length G/mpl and the truncated chimeric receptor were obtained by
fusing the extracellular domain of the GCSF-R via an introduced
BamHI site (20) with fragments encoding the transmembrane
and cytoplasmic domains of murine c-Mpl (28), gp130, and the LIFR
,
respectively. These fragments were generated by polymerase chain
reaction and contained at their 5'-end a silent BamHI
restriction site over the second last amino acid of the extracellular domain and at their 3'-end translational stop codons at
amino acid positions 220, 156, and 130 in gp130 (counting from the most
membrane-proximal amino acid of the cytoplasmic domains) and position
136 in LIFR
yielding construct G/gp130
220,
G/gp130
156, and G/gp130
130F, and
G/LIFR
136 (Table I). In the
G/gp130
130F construct, the tyrosine (Y) residue in the
putative STAT3 binding sequence Y126XXQ in Box 3 was mutated to phenylalanine (F). In order to facilitate subcloning of
isolated fragments into the mammalian expression vectors, the
3'-polymerase chain reaction oligonucleotides also contained a
XbaI restriction site. All newly generated chimeric receptor
cDNA constructs were sequenced throughout their coding regions on
an Applied Biosystem 373A DNA sequencing system and subcloned into the
XbaI site of the mammalian expression vector 6-16PGK-IRESneo designed to give rise to dicystronic
mRNAs (20). For analysis of the full-length GCSF-R, Tyr
Phe
substitutions were introduced by site-directed mutagenesis at
Tyr74, Tyr114, and at both tyrosine residues
(Tyr74, Tyr144) as described previously (29).
The GCSF-R constructs were cloned into the XbaI site of the
mammalian expression vector pEF-BOS (29) and co-transfected
with the resistance marker plasmids PGKneo (14). High
performance liquid chromatography-purified phosphothioate AS
oligonucleotides directed against the amino-terminal region of mouse
STAT3 (5'-GCCAGGAACTGCCGCAGC-3') or consisting of a randomized sequence
of a similar nucleotide composition (5'-AGTCCAGGCCCAGGTCCG-3') were
purchased from Bresatec (Australia).
receptor
constructs were assessed in stable transfected 7-TD1 cells which were
obtained following electroporation (960 µfarads, 270 V) of 5 × 105 cells with 15 µg of linearized plasmid. Wild-type and
mutant GCSF-R expressing IL3-dependent Ba/F03 cell lines
were obtained essentially as described (20) by electroporating (960 µfarads, 270 V) 15 µg of linearized receptor plasmid together with
1.5 µg of the selectable marker plasmid PGKneo. Receptor
expression on clonally derived neomycin-resistant (20 µg/ml) Ba/F03
cell lines was assessed by flow cytometry (FACScan, Becton Dickinson) as described (29).
-32P]ATP (3000 Ci/mmol,
Bresatec, Australia) as described previously (14, 51).
RESULTS
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ABSTRACT
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RESULTS
DISCUSSION
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was fused to the extracellular domain of the
GCSF-R, were established (Table I). This
strategy permits GCSF-dependent dimerization and activation of chimeric receptor chains without the interference of the endogenous LIFR
and gp130 receptor chains. Clonally derived ES cell lines, expressing comparable numbers of chimeric and endogenous receptor chains (800-1400 per cell) as assessed by binding of radiolabeled GCSF
and LIF (Ref. 20, and data not shown) were chosen for subsequent analysis. The ability of the introduced receptor chains to suppress ES
cell differentiation was determined in two independent assay systems.
The first takes advantage of the striking morphological differences
between the densely packed colonies of undifferentiated ES cells and
the flattened morphology of differentiated cells (14, 51).
Homodimerization of the full-length cytoplasmic domain of gp130
(G/gp130) or LIFR
(G/LIFR
), respectively, prevented ES cell
differentiation in a GCSF-dependent manner (Fig.
1a). Moreover, GCSF also
prevented differentiation of ES cells transfected with the full-length
GCSF-R (Fig. 1c), but had no effect on mock-transfected wild-type cells (20). Signaling through the endogenous LIFR
/gp130 receptor heterodimer, formed in response to stimulation with LIF, prevented differentiation of approximately 95% of colonies in all ES
cell lines investigated, indicating that the presence of chimeric
receptors did not interfere with activation of the endogenous receptor
chains. Since the scoring of cell morphology is potentially subject to
investigator bias, we also carried out a colony assay based on
selection against differentiated cells. This sensitive "killing
assay" (Fig. 1, b and d) facilitated direct
measurement of the proportion of undifferentiated ES cells after
pulsing the cultures with the MTT dye. For this purpose, we exploited
the HCK-hprt ES cell line which harbors a hprt
minigene under control of the murine hck promoter that is
transcriptionally inactivated in differentiating ES cells (20, 51).
Thus, differentiating cells are killed when incubated continuously in
HAT-containing medium, while undifferentiated, HAT-resistant cells
proliferate and metabolize the MTT dye to a product which can be
measured photometrically.
Schematic diagrams of receptor constructs used in this study
are white,
while domains representing c-Mpl are black. The cytoplasmic homology
motifs Box 1, Box 2, and Box 3 are shown in black (white in the case of
c-Mpl) and all intracellular tyrosine (Y) residues are shown; those
conforming to the YXXQ and YXXV motif are
highlighted. The numbers refer to the amino acid position counting from
the most membrane proximal intracellular residue. GCSF-R protein
domains are depicted as light grey, gp130 domains as dark grey, LIFR
domains as white, while domains representing c-Mpl are
black.
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Fig. 1.
Differentiation inhibiting activity of
chimeric receptors in ES cells. a and c,
assessment by cell morphology. Transfected ES cells were cultured for 5 days with the indicated concentration of GCSF. The proportion of
undifferentiated ES cell colonies was calculated by scoring the
morphology of 300 randomly chosen colonies in triplicate culture dishes
and expressed as the proportion of undifferentiated cells observed in
the presence of 250 units/ml LIF. Results are shown for one
representative cell line for each of the indicated receptor constructs.
b and d, assessment by chemical selection.
Transfected cells were plated in 24-well plates and grown for 6 days in
ES cell medium supplemented with HAT (see "Experimental
Procedures") and the indicated concentration of GCSF. Quadruplicate
cultures were incubated for the last 3 h with MTT and the
reduction of the MTT dye was quantitated by optical absorbance and
expressed as a percentage of the maximal absorbance measured in
undifferentiated cultures maintained in 250 units/ml LIF. The results
for one representative cell line transfected with the indicated
receptor construct are shown. Mean ± S.D.
. Ligand-dependent suppression of
differentiation was fully maintained by the
G/gp130
220 receptor retaining two out of four
YXXQ motifs, but was significantly reduced with the
G/gp130
156 chimera in which only the single
YXXQ motif within Box 3 is retained (Figs. 1, a
and c). Extending the truncation further to the
membrane-distal border of Box 3 in conjunction with a phenylalanine
substitution of the fourth YXXQ motif
(F126XXQ) within Box 3 abolished the capacity of
the G/gp130
130F receptor to suppress differentiation.
Similarly, a truncated LIFR
with a deletion of the Box 3 region and lacking two out of three YXXQ motifs
(G/LIFR
136), was no longer capable of preventing ES
cell differentiation (Fig. 1, b and d).
Furthermore, GCSF-dependent activation of a chimeric
receptor containing the full-length cytoplasmic domain of the
thrombopoietin receptor c-Mpl, which lacks the Box 3 homology region but contains one YXXQ motif, was also unable to
maintain the undifferentiated ES cell phenotype.
which contain the Box 3 homology region as well as
several YXXQ motifs play a critical role in the signal transduction pathways required to suppress differentiation of ES cells
in vitro. By contrast, the carboxyl-terminal regions of
gp130 and the LIFR
are dispensable for the transduction of proliferative signals in 7-TD1 cells which appears to be independent of
STAT3 phosphorylation.
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Fig. 2.
Proliferation promoting activity of chimeric
receptors in 7-TD1 cells. Clonal 7-TD1 cells lines, expressing the
indicated chimeric receptors, were grown for 8 days in the presence of
50 ng/ml GCSF. The number of viable cells was then determined by trypan
blue exclusion in triplicate cultures. The results for one
representative cell line transfected with the indicated receptor
construct are shown. Mean ± S.D.
·gp130 receptor complex is physically and
functionally associated with the Src-related tyrosine kinase Hck and
the JAK/STAT signaling cascade in ES cells (14, 51). We therefore
investigated the capacity of the chimeric receptors to activate
p56/59Hck and to phosphorylate STAT3, the predominant STAT
protein activated in response to the LIF/IL6 family of cytokines. GCSF
stimulation of cells expressing the full-length G/gp130, G/LIFR
, or
the truncated G/gp130
220 receptors led to
increased in vitro autophosphorylation of the two Hck
isoforms (p56/59Hck) in cell lysates immunoprecipitated
with an anti-Hck antiserum comparable to the increase observed after
LIF-dependent stimulation of the endogenous gp130·LIFR
complex (Fig. 3). By contrast, the truncated G/gp130
156, G/gp130
130F, nor
G/LIFR
136 receptors were capable of mediating
significant activation of Hck. Furthermore, Hck kinase activity
remained unaffected after stimulation of the G/mpl receptor, indicating
that the most carboxyl-terminal sequences of the cytoplasmic tails of
gp130 and the LIFR
are required for ligand-dependent Hck
activation. Immunoprecipitation of STAT3 proteins followed by Western
blotting with anti-phosphotyrosine antibody showed a prominent increase
in tyrosine phosphorylation of STAT3 in response to GCSF in ES cells
expressing the full-length G/gp130, G/LIFR
, or the truncated
G/gp130
220 receptor (Fig. 3). Weak
ligand-dependent phosphorylation of STAT3 was registered
following stimulation of chimeric receptor with a single
YXXQ motif (G/gp130
156,
G/LIFR
136, G/mpl), while no response was observed in
G/gp130
130F expressing cells. Reprobing of the blots
with anti-STAT3 antibodies demonstrated comparable amounts of STAT3
protein loaded.
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Fig. 3.
Ligand-dependent activation of
intermediate signaling molecules. Confluent cultures of ES cells,
expressing the indicated chimeric receptors, were starved of LIF for
12 h in medium containing 1% FCS. Cultures were subsequently
stimulated for 10 min with 20 ng/ml GCSF (G), 500 units/ml
LIF (L), or saline (c) and cell lysates were
prepared. Autophosphorylation activity of p56/59Hck
(left panel) was determined by immunoprecipitating Hck from
500 µg of ES cell lysates followed by an in vitro
autophosphorylation assay in the presence [ -32P]ATP.
The reaction products were separated by SDS-PAGE and visualized by
autoradiography. The two bands represent the two protein isoforms
p56Hck and p59Hck. Tyrosine phosphorylation of
STAT3 was assessed (right panel) by immunoprecipitating 500 µg of ES cell lysates with anti-STAT3 antibodies, separated by
SDS-PAGE, transferred to nitrocellulose and probed with
anti-phosphotyrosine (Tyr(P)) antibodies. The membranes were stripped
and reprobed to assess for equal amounts of STAT3 transferred.
130F and G/LIFR
136) still
induced proliferation of 7-TD1 cells (Fig. 2), we investigated ligand-dependent activation of the mitogen-activated
protein kinase (MAPK). GCSF treatment of 7-TD1 cells led to an increase
in MAPK activity as assessed by the
phosphorylation-dependent mobility shift of
p42/44MAPK of the full-length and the truncated receptors
(Fig. 4a, top). A similar
change in MAPK mobility was observed in serum-starved ES cells
expressing chimeric receptors following LIF or GCSF stimulation (Fig.
4a, bottom), confirming that gp130 and
LIFR
-dependent activation of MAPK occurs independently
of YXXQ motifs (31). Furthermore, ligand-dependent activation of MAPK in ES cells correlated
with an increase in tyrosine phosphorylation of the phosphatase SHP2 (Fig. 4b) which has been suggested as a molecular mechanism
by which gp130 connects to the Ras/MAPK pathway (27). Thus,
ligand-dependent activation of SHP2 and MAPK correlates
with the receptors' capacity to stimulate proliferation, however,
activation of this pathway in isolation does not suppress ES cell
differentiation.
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Fig. 4.
Ligand-dependent activation of
the MAPK pathway. Confluent cultures of ES cells or 7-TD1 cells
expressing the indicated chimeric receptors were starved for 12 h
of LIF (ES cells) or IL6 (7-TD1 cells) in medium containing 1% FCS.
Cultures were then stimulated for 10 min with 20 ng/ml GCSF
(G), 500 units/ml LIF (L), or saline
(c) and cell lysates were prepared. a, MAPK
mobility shift in 7-TD1 cells (upper panel) and ES cells
(lower panel). Sixty µg of cell lysate was separated by
SDS-PAGE and transferred to nitrocellulose and blotted with Erk-1
antibodies. The phosphorylated, retarded forms of
p42/44MAPK are indicated by arrowheads.
b, tyrosine phosphorylation of SHP2. Five hundred µg of ES
cell lysates were immunoprecipitated with anti-SHP2 antibodies,
separated by SDS-PAGE, transferred to nitrocellulose, and blotted with
anti-phosphotyrosine antibodies (Tyr(P)). Blots were stripped and
reprobed to assess for equal amounts of SHP2.
·gp130 receptor
complex in ES cells is known to induce binding of STAT1 and STAT3
containing protein complexes to the high affinity
sis-inducible SIE element (17, 20). Based on the retardation
of these complexes by STAT antibodies, the most prominent SIF-A complex
consists of STAT3 homodimers whereas the less abundant SIF-B and SIF-C
complexes represent STAT3/STAT1 heterodimers and STAT1 homodimers,
respectively (19, 20). Consistent with decreased or absent STAT3
phosphorylation following GCSF stimulation of the
G/LIFR
136, G/mpl, or G/gp130
130F
receptors, we observed reduced STAT-DNA binding in cell lines expressing G/LIFR
136 or G/mpl, when compared with the
pattern obtained after activation of the full-length G/gp130 and
G/LIFR
, respectively (Fig. 5). Activation of the G/gp130
130F receptor failed to induce
STAT binding activity all together.
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Fig. 5.
Ligand-dependent induction of
STAT-DNA binding in ES cells. Confluent cultures of ES cells,
expressing the indicated chimeric receptors, were starved of LIF for
12 h in medium containing 1% FCS. Cultures were then stimulated
for 15 min with either saline (c) or 20 ng/ml GCSF
(G), respectively, and lysates were incubated with a
radiolabeled m67 (SIE) element to test for STAT-specific DNA
binding activity in electrophoretic mobility shift assays. SIF-A,
SIF-B, and SIF-C refer to the STAT3·STAT3,
STAT3·STAT1 and STAT1·STAT1 complexes as described by Lai et
al. (18).
receptor, which activates STAT1 and STAT2, fails to suppress ES cell
differentiation (14). We therefore focused on possible functions of
STAT3 by attempting to reduce the intracellular protein levels of STAT3
by exploiting phosphothioate AS oligonucleotides directed against the
amino-terminal sequences of murine STAT3. A 5-day treatment of
undifferentiated ES cells with 3 µM STAT3-AS oligonucleotides specifically decreased the STAT3 protein level by more
than 90% when compared with cells exposed to a control AS
oligonucleotide consisting of a randomized sequence with a similar
nucleotide composition (Fig.
6a). Furthermore, the effect of the STAT3 AS oligonucleotide was specific, since in the same cells
the protein level of STAT5a was not affected. The addition of STAT3 AS
oligonucleotides significantly decreased the proportion of
undifferentiated wild-type ES cells in LIF-treated cultures, while the
randomized control AS oligonucleotide was without effect (Fig.
6b). Addition of STAT3 AS oligonucleotides also decreased the proportion of undifferentiated colonies in GCSF-treated cultures of
cells expressing either G/gp130 or G/LIFR
(Fig. 6b and
data not shown). We also tested the effect of the STAT3 AS
oligonucleotide on the mitogenic response of 7-TD1 cells stimulated
with IL6. Fig. 6c shows that inhibition of STAT3 protein
production by STAT3 AS oligonucleotides had no effect on IL6-induced
DNA synthesis. By contrast, the IL6 effect was abolished in cultures
treated with the specific MAPK kinase (MEK) inhibitor PD098059,
consistent with activation of the Ras/MAPK pathway via the
membrane-most proximal SHP2 phosphotyrosine-binding sites in gp130 and
LIFR
. Taken together, our data obtained in ES and 7-TD1 cells
suggest a selective functional involvement of STAT3 in differentiation suppressing signal(s) generated by the heterodimeric LIFR
·gp130 complex or their respective homodimeric receptor counterparts.
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Fig. 6.
gp130-dependent signaling in ES
cells in the presence of STAT3-AS oligonucleotides. Untransfected
wild-type ES cell and ES cells (a and b)
expressing the chimeric G/gp130 receptor were cultured for 5 days in
the presence of 3 µM of the indicated phosphothioate AS
oligonucleotide in the presence of the indicated concentration of LIF
(wild-type cells) or GCSF (G/gp130 cells) as described under
"Experimental Procedures." The results for one representative cell
line are shown. a, level of STAT protein. Western blot
analysis of cultures incubated for 5 days with the indicated AS
oligonucleotide in the presence of 500 units/ml LIF (wild-type cells)
or 500 pM GCSF (G/gp130). Five hundred µg of total
lysates were immunoprecipitated and blotted with antisera specific for
either STAT3 or STAT5a. b, assessment of cellular
differentiation by morphology. At the end of the 5-day culture period,
the morphology of ES colonies was assessed as in described in the
legend to Fig. 1 in triplicate cultures. Data are from one
representative experiment, mean ± S.D. c, assessment
of mitogenic response of 7-TD1 cells. Cells were permeabilized with
streptolysin-O for 3 min in the presence of either 3 µM
STAT3 ( 3) or the random control (rd) phosphothioate AS
oligonucleotide and allowed to recover for 24 h in medium
supplemented with 3 µM AS oligonucleotide, 10% FCS, and
5 pM IL6. Cells were then starved of IL6 for 12 h in
medium containing 3 µM AS oligonucleotide and 0.5% FCS
before being inoculated in 24-well dishes at a concentration of 2 × 104 cells/well. Triplicate cultures were stimulated for
the next 24 h with the indicated concentration of IL6, FCS, or the
MEK kinase inhibitor PD098059 (PD) and pulsed for the last
4 h with [3H]thymidine (mean ± S.D.). The
amount of STAT protein was also determined by immunoblotting for STAT3
and STAT5a.
·gp130 receptor complex suppressed differentiation of more
than 90% of ES cell colonies expressing the various versions of the
GCSF-R constructs.
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Fig. 7.
Differentiation suppressing activity of
GCSF-R mutants in ES cells. a, assessment by cell
morphology. Transfected ES cells were cultured for 5 days with the
indicated concentration of GCSF. The proportion of morphologically
undifferentiated ES cell colonies was assessed as described in the
legend to Fig. 1 in triplicate culture dishes, mean ± S.D.
b, assessment by chemical selection. Transfected cells were
plated in 24-well plates and grown for 6 days in ES cell medium
supplemented with HAT and the indicated concentration of GCSF and an
MTT assay was carried out in quadruplicate cultures as described in the
legend to Fig. 1. The results for one representative cell line
transfected with the indicated receptor construct are shown, mean ± S.D.
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Fig. 8.
[3H]Thymidine incorporation
into Ba/F03 cells expressing mutant GCSF-R. Clonally derived
Ba/F03 cell lines expressing the indicated GCSF-R mutants were plated
in 96-well plates and stimulated for 48 h with 10 ng/ml GSF.
Triplicate cultures were labeled with [3H]thymidine for
the last 4 h and incorporated acid-precipitable radioactivity was
determined in a -scintillation counter. Similar results were
obtained with at least one more independently derived cell line for
each receptor construct, mean ± S.D.
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Fig. 9.
GCSF-dependent STAT3
phosphorylation and in ES cells expressing mutant GCSF-R.
Confluent cultures of ES cells, expressing the indicated GCSF-R
construct, were starved for 12 h of LIF in medium containing 1%
FCS before stimulating for 10 min with 20 ng/ml GCSF (G),
500 units/ml LIF (L), or saline (c). Cell lysates
were immunoprecipitated with anti-STAT3 antibodies, separated by
SDS-PAGE, transferred to nitrocellulose membranes, and blotted with
anti-phosphotyrosine antibodies (Tyr(P)). Blots were stripped and
reprobed to assess for equal amounts of STAT3. The results for one
representative cell line transfected with the indicated receptor
construct are shown.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
are required for regulation
of mitogenic activity while the membrane-distal region with an intact
Box 3 homology motif is critical for suppressing differentiation of ES
cells in vitro. This observation correlates with
ligand-dependent activation of Hck kinase and STAT3
phosphorylation and, unlike the transduction of mitogenic signal, is
selectively impaired in the presence of STAT3 AS oligonucleotides.
Therefore, our data support findings by Niwa et al. (32) who
suggested a critical role for STAT3 in maintaining the pluripotent
phenotype of ES cells in vitro.
130F and
G/LIFR
136 receptors, which retain the YXXV
motif, also promote growth of 7-TD1 cells. Phosphorylated SHP2 binds
Grb2 (13, 27, 37) which is constitutively associated with the
nucleotide exchange factor Sos thereby providing a direct link between
the YXXV motif and transient activation of the Ras/MAPK
pathway. By contrast, sustained activation of the Ras/MAPK pathway in
ES cells in response to LIF and IL6 (14) may also occur through direct
association of Shc with phosphorylated gp130 (38) or through
constitutive association of the two isoforms of Hck with gp130 (51). We
extend these findings here by identifying the carboxyl-terminal regions of gp130 and LIFR
as sites required for the induction of
ligand-dependent Hck activation. While the molecular nature
of this interaction remains to be established, the absence of
conspicuous cysteine residues in gp130 and LIFR
suggests a different
mechanism to that identified between the Src family kinase Lck and the
T-cell co-receptors CD4/CD8 (39). The present study suggests a
correlation between gp130/LIFR
-mediated Hck activation and
suppression of ES cell differentiation. Furthermore, GCSF-R dependent
activation of the Src-related kinase Lyn (40) and the observation that overexpression of constitutive active Lyn partially suppressed ES
cell differentiation,2
suggests that Src kinases may amplify signals that suppress ES cell differentiation.
130F
receptor induced growth of the plasmacytome cell line 7-TD1 but only
induced a transient mitogenic signal in the pro-B cell line Ba/F03
(27). In fact, STAT5 activation is required for maximal proliferation
in response to IL2 receptor activation (45) and we found in 7-TD1 cells
weak tyrosine phosphorylation of STAT5a with all truncated chimeric
gp130 and LIFR
constructs (data not shown).
, respectively,
abolish the ligand-dependent transduction of
differentiation promoting signals in M1 cells and the induction of
acute-phase proteins in hepatoma cells (5). However, the presence of a single YXXQ motif retained ligand-dependent
differentiation in M1 cells, but not
-fibrinogen expression in HepG2
cells (5). In response to IL6, STAT3 activation occurs rapidly and
transiently in ES and HepG2 cells but persists for more than 24 h
in M1 cells (26). The sustained nature of STAT3 activity may be
important in causing growth arrest and terminal differentiation, as in
the case of sustained MAPK activation in nerve growth factor-induced PC12 cell differentiation (47) and gp130-mediated prevention of ES cell
differentiation (14). Gp130 and LIFR
-dependent STAT3 activation requires at least one YXXQ consensus motif (51). Two of these motifs in gp130 (pY126RHQ and
pY173FKQ) mediate activation of STAT3 while the other two
motifs (pY265LPQ and pY275MPQ) are capable of
mediating the activation of STAT3 and STAT1 (48). Thus, substitution of
the tyrosine residue in the truncated G/gp130
130F
receptor blunts the ligand-dependent formation of the
SIF-A, SIF-B, and SIF-C DNA-binding complexes (Fig. 5 and Ref. 19). STAT1 appears to be a minor component of the total STAT proteins activated by gp130. It is therefore difficult to assess the role of
STAT1 in ES cell directly, because (as observed in M1 cells) STAT1
activity is weak and only detected when STAT3 is highly activated.
STAT3-dependent induction of M1 cell differentiation and
suppression of Ba/F03 cell apoptosis appears to be independent of the
number and position of these motifs in gp130 (18, 26, 27). In ES cells,
however, a single YXXQ motif outside of the Box 3 motif
(G/LIFR
136, G/mpl) failed to suppress
differentiation, while the single YXXQ motif within the Box
3 homology domain (G/gp130
156) partially retained the
activity in ES cells. These findings are reminiscent of
observations that only Box 3 containing receptors, but not c-Mpl,
induced transcriptional activation of acute-phase protein genes
(5).
, and gp130 is likely to contribute to the remarkably
similar pattern of biological activities transduced in a number of cell
systems (4, 5, 20, 29). However, GCSF-R-mediated STAT3 phosphorylation
and M1 cell differentiation is less tightly correlated with individual
tyrosine residues when compared with gp130 and LIFR
(29) and the
Y74F substitution of the only YXXQ motif in the human GCSF-R
did not have a significant effect on STAT3 phosphorylation and
GCSF-mediated inhibition of ES cell differentiation. By contrast, the
Y114LRC
FLRC substitution within the Box 3 sequence
decreased STAT3 phosphorylation and GCSF-mediated suppression of
differentiation. Thus, the regulation of ES cell differentiation by the
cytoplasmic domain of the Box 3-containing gp130, LIFR
, and GCSF-R
appears to depend largely on STAT3, however, the mechanisms by which
GCSF-R mediates STAT3 activation may be cell type-specific and more
complex than SH2 domain-dependent binding to the
YXXQ motif. In fact, GCSF-R-mediated STAT3 activation is not
observed in neutrophils (40), while STAT3 and STAT5 activation have
been observed with versions of the GCSF-R, the
-chain of the
granulocyte-macrophage CSF receptor, and c-Mpl which contain
substitutions of all cytoplasmic tyrosine residues (29, 49, 50).
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ACKNOWLEDGEMENTS |
---|
We thank M. Inglese, V. Murphy, and L. Paradiso for technical assistance, J. Stickland for figure production, Amgen for recombinant human GCSF, C. Lowell for the Hck antiserum, and R. Simpson for recombinant human IL6.
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FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed. Tel.: 61-3-9341-3155; Fax: 61-3-9341-3191; E-mail: matthias.ernst{at}ludwig.edu.au.
Present address: Dept. of Surgery, University of Melbourne,
P. O. Royal Melbourne Hospital, Victoria, 3050 Australia.
** Present address: The Walter and Eliza Institute for Medical Research, P. O. Royal Melbourne Hospital, Victoria, 3050, Australia.
2 M. Ernst, unpublished observation.
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ABBREVIATIONS |
---|
The abbreviations used are:
IL, interleukin;
AS, antisense;
ES, embryonic stem;
FCS, fetal calf serum;
GCSF, granulocyte
colony stimulating factor;
GCSF-R, granulocyte colony stimulating
factor receptor;
JAK, Janus kinase;
LIF, leukemia inhibitory factor;
LIFR, LIF receptor
-chain;
PAGE, polyacrylamide gel
electrophoresis;
SH2, Src homology 2;
STAT, signal transducer and
activator of transcription;
MTT, 3-(4,5-dimethlydiazol-2-yl)-2,5-diphenyltetrazolium bromide;
PIPES, 1,4,-piperazinediethanesulfonic acid;
MAPK, mitogen-activated protein
kinase;
HAT, hypoxanthine/aminopterin/thymidine.
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REFERENCES |
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