(Received for publication, May 5, 1997)
From the Leukemia inhibitory factor (LIF) induces a
variety of disparate biological responses in different cell types.
These responses are thought to be mediated through the functional LIF
receptor (LIFR), consisting of a heterodimeric complex of LIFR
A characteristic feature of cytokines such as leukemia inhibitory
factor (LIF)1 is their
ability to regulate a wide range of biological activities (1). The
diverse effects of LIF include both stimulation and inhibition of
cellular proliferation (2, 3) and activation of cell type-specific gene
expression (4). LIF also induces macrophage differentiation in M1
myeloid leukemia cells (5), whereas it elicits an opposite effect in
embryonic stem (ES) cells, maintaining these cells in an
undifferentiated, pluripotent state (6, 7).
In addition to functional pleiotropy, the biological actions of LIF and
related cytokines, such as interleukin (IL)-6, IL-11, oncostatin M
(OSM), cardiotrophin-1, and ciliary neurotrophic factor (CNTF), are
largely overlapping. The common activities of the LIF family of
cytokines have been attributed in part to the existence of multimeric
receptors, which share the affinity converting and signal transducing
subunit, gp130 (8-11). These receptors can be divided into three
distinct types (12). First, LIF, OSM and cardiotrophin-1 each use
receptors consisting of a heterodimeric complex of gp130 with the LIF
receptor The separate contributions of the LIFR In the present study, we investigated the relative potential of
LIFR M1 cells were maintained
in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal
bovine serum (FCS). Ba/F3 cells were cultured in RPMI 1640 medium
supplemented with 10% FCS and 10% WEHI-3B D Chimeric receptors were constructed by
cloning the HindIII-XbaI fragment of the murine
GCSFR (26) into pBLUESCRIPT (SK)+. A silent mutation was then
introduced at nucleotide +2045, to create a BamHI
restriction enzyme site near the start of the transmembrane region
(pBS/mGR(Bam)). BamHI sites were also introduced into murine gp130 at position +1851 and in human LIFR M1 and Ba/F3 cells were
transfected with plasmids expressing either full-length GCSFR,
GCSFR-LIFR HCK-hprt ES cells were obtained by transfection of parental
hprt To quantitate the
differentiation of transfected M1 clones in response to cytokine, 300 cells were cultured in 35-mm Petri dishes containing 1 ml of DMEM
supplemented with 20% FCS, 0.3% agar, and 0.1 ml of serial dilutions
of LIF or GCSF. Cultures were incubated at 37 °C in a humidified
incubator containing 10% CO2 for 7 days. The dishes were
then scored for the percentage of differentiated colonies, as judged by
colonies with a halo of dispersed cells. The total number of colonies
was also determined to assess the degree to which proliferation had
been extinguished by the addition of the cytokine.
The extent to which cytokine-mediated
signaling prevents ES cell differentiation was determined by both
morphology (as described previously; Refs. 31 and 32) and MTT staining
of undifferentiated, proliferating cells after HAT selection. For the
MTT assay, cells were seeded in quadruplicate cultures at 1500 cells/cm2 in gelatinized 24-well multiculture dishes (Nunc,
Kamstrup, Denmark). Cells were grown for 6 days at the indicated
concentration of GCSF in the absence of LIF, in medium supplemented
with 2 × HAT. At this time, >95% of the morphologically
differentiated cells had died. The cultures were then supplied with 0.5 mg/ml MTT and incubated for 3 h at 37 °C, after which the
aspirated cultures were air-dried. The reduced MTT dye was solubilized
in Me2SO, and the optical absorbance was measured at 560 nm
and expressed as a percentage of the maximal absorbance measured in
undifferentiated cultures maintained in 2.5 ng/ml LIF.
The survival/proliferation of Ba/F3
cells in response to cytokine was measured in Lux 60 microwell HL-A
plates (Nunc Inc., Roskilde, Denmark). Cells were washed three times in
DMEM containing 20% newborn calf serum and resuspended at a
concentration of 2 × 104 cells/ml in the same medium.
Aliquots of 10 µl of cell suspension were placed in the culture wells
with 5 µl of serial dilutions of 1 ng/ml IL-3 or 100 ng/ml GCSF.
After a 2-day culture at 37 °C in a humidified incubator containing
10% CO2, viable cells were counted using an inverted
microscope.
Assays were performed
as described previously (33), using the high affinity
c-sis-inducible factor binding site m67 (34). Protein
extracts were prepared from M1 cells incubated with saline, 10 ng/ml
LIF, or 100 ng/ml GCSF for 10 min at 37 °C. For ES cells, cultures
consisting of approximately 8 × 107 undifferentiated
ES cells were starved overnight in ES cell medium free of serum and LIF
before being stimulated for 10 min at 37 °C with saline, 10 ng/ml
LIF, or 100 ng/ml GCSF. For certain experiments, protein samples were
preincubated with antibodies specific for either STAT1 (Transduction
Laboratories), STAT3 (Santa Cruz Biotechnology Inc., CA), or STAT5A
(specific for the C terminus, a gift from Dr. A. Mui, DNAX Research
Institute, Palo Alto, CA) as described (33).
Binding assays were performed
essentially as described (35). Approximately 2 × 106
cells in 40 µl of RPMI 1640 medium containing 20 mM
Hepes, pH 7.4, and 10% FCS were incubated for 3 h on ice with
varying amounts of 125I-GCSF in the presence or the absence
of 100-fold excess of unlabeled GCSF. Cell-associated and free
125I were then separated by rapid centrifugation of the
cell suspension through 200 µl of FCS. The amount of 125I
in the cell pellet and the supernatant was quantitated in a Stimulation of M1 myeloid cells with LIF or IL-6
induces macrophage differentiation and an inhibition of cellular
proliferation (5). M1 cells were transfected with either the
GCSFR-LIFR Table I.
Expression of transfected receptors and affinity of 125I-GCSF
binding
In previous studies, the degree of cellular
differentiation of ES cells in vitro had been quantitated
primarily by morphological inspection of individual cells and colonies.
To utilize a chemical selection protocol allowing for the survival of
undifferentiated ES cells only, we used the observation that the murine
hck gene undergoes transcriptional down-regulation following
the induction of ES cell differentiation in vitro (M.E.,
unpublished observation). Thus, HCK-hprt ES cell lines, which contain
an hprt minigene driven by an 865bp proximal hck promoter
fragment, are resistant to HAT selection if they remain in an
undifferentiated state. The proportion of undifferentiated,
proliferating cells in an ES cell culture can then be determined by
optical absorbance measurement of the reduced mitochondrial stain,
MTT.
Maintenance of the pluripotency of ES cells in vitro
normally requires signaling initiated by the LIF family of cytokines (6, 7, 37). ES cell lines were transfected with either full-length
GCSFR or the chimeric receptor constructs, and expression of the
receptors was confirmed by the ability of transfected cells to bind
125I-GCSF (Table I). In these cell lines, signaling through
the endogenous LIFR The potential of LIFR Considering the differing potential of LIFR
The molecular nature of the STAT complexes was further
investigated by the addition of antibodies specific for individual STAT
proteins. The pretreatment of M1 cell extracts with antibodies specific
for STAT1 supershifted the two minor lower bands to slower migrating
complexes, indicating that these bands contain STAT1 (Fig.
2B). Similarly, the upper two bands were supershifted by the
addition of an antibody recognizing STAT3 (Fig. 2B). The
most slowly migrating DNA complex was also affected by binding of an anti-STAT5A antibody, suggesting that this complex comprises a STAT3-STAT5A heterodimer. The pattern and composition of m67-binding complexes induced by either GCSF, via the introduced receptors, or LIF
stimulation, through endogenous LIF receptors, were identical for M1
cells expressing either GCSFR-gp130 or wild type GCSFR (Fig.
2B). The same signaling molecules were activated by either LIF or GCSF stimulation of ES cells expressing GCSFR-gp130 (Fig. 2C). Furthermore, identical complexes were induced by GCSF
stimulation of ES cells signaling through GCSFR-LIFR It is clear from this study that despite a high degree of sequence
similarity, the signaling potential of the cytoplasmic domains of
LIFR The cell-specific differences in activity described here are unlikely
to be due to differences in the level of receptor expression, because
Scatchard analyses of binding isotherms indicated a similar level of
expression of the chimeric receptors in each cell type and a comparable
binding affinity for GCSF (Table I). Because none of the three cell
lines express detectable levels of endogenous full-length GCSFR, dimer
formation between the extracellular domains of endogenous and
introduced receptor subunits should not occur. Furthermore,
cooperativity between the cytoplasmic domains of introduced receptor
subunits and endogenous LIFR The observed differences in signaling potential of the homodimeric
cytoplasmic domains may be due to either quantitative or qualitative
differences in the signaling molecules activated. First, different
biological responses may require a different threshold of STAT
activation to be attained, with certain responses requiring a higher
level of activation. The activation of STAT molecules by homodimeric
LIFR Alternatively, different signaling intermediates may be activated by
the different receptor subunits. Activation of pathways necessary for
M1 cell differentiation and Ba/F3 proliferation may require gp130
cytoplasmic sequences, whereas both LIFR We thank L. Di Rago, S. Mifsud, and L. Paradiso for excellent technical assistance. The following are thanked
for generously providing reagents: Prof. S. Cory (pPGKPuropA), Dr. A. Mui (anti-STAT5A antibody), Prof. S. Nagata (human GCSFR cDNA
clone), AMGEN (rhGCSF), and Dr. J.-G. Zhang and C. McFarlane
(mGCSF).
Cooperative Research Centre for Cellular
Growth Factors and the Walter and Eliza Hall Institute for Medical
Research, the § Department of Medicine,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-chain (LIFR
) and gp130. The present study investigated the
relative capacity of the cytoplasmic domains of each receptor subunit
to signal particular responses in several cell types. To monitor the
signaling potential of LIFR
and gp130 individually, we constructed chimeric receptors by linking the extracellular domain of granulocyte colony-stimulating factor receptor (GCSFR) to the transmembrane and
cytoplasmic regions of either LIFR
or gp130. Both chimeric receptors
and the full-length GCSFR in expressed in M1 myeloid leukemic cells to
measure differentiation induction, in embryonic stem cells to measure
differentiation inhibition, and in Ba/F3 cells to measure cell
proliferation. Our results demonstrated that whereas GCSFR-gp130
receptor homodimer mediated a GCSF-induced signal in all three cell
types, the GCSFR-LIFR
receptor homodimer was only functional in
embryonic stem cells. These findings suggest that the signaling
potential of gp130 and LIFR
cytoplasmic domains may differ depending
upon the tissue and cellular response initiated.
-chain (LIFR
, sometimes known as LIFR
). In addition,
OSM has been shown to signal through an alternative receptor complex,
consisting of a heterodimer of a ligand-specific subunit (OSM receptor
-chain) and gp130 (13). Second, CNTF binds to a ligand-specific
subunit (CNTF receptor
-chain), which associates with a heterodimer
of LIFR
and gp130. In contrast, functional IL-6 and IL-11 receptors are formed by the association of ligand-bound
-chains with gp130 homodimers, with no involvement of LIFR
. Unlike the ligand-binding components of the IL-6, IL-11, and CNTF receptors, which do not contribute to intracellular signaling, LIFR
contains an extensive cytoplasmic domain with a structure similar to both gp130 and the GCSFR
(14).
and gp130 cytoplasmic domains
to LIF-induced signal transduction have not been investigated in
detail. Although it has been established that mutant LIFR
lacking a
cytoplasmic domain is inactive (15), its relative capacity compared
with gp130 for triggering diverse biological outcomes is less well
established. Both receptor subunits have the ability to associate with
and activate the Janus kinases Jak1, Jak2, and Tyk2 as well as several
other tyrosine kinases (16), suggesting that common signaling pathways
may be triggered by each receptor component (17). Despite their
molecular similarity, it is possible that the two receptor chains are
not functionally equivalent, with one subunit having a greater
potential to transduce particular responses. Differences in the signal
transduction pathways triggered by LIF-related cytokines have been
implicated by previous studies (18, 19), in which enforced expression
of the SCL transcription factor in M1 cells reduced the ability of
these cells to differentiate in response to LIF and OSM (signaling
through LIFR
-gp130 heterodimers) but not IL-6 (signaling through
gp130 homodimers).
, gp130, and GCSF receptor chains to signal cell type-specific responses. For this purpose, we expressed chimeric receptor constructs that comprised the extracellular domain of GCSFR and the transmembrane and cytoplasmic regions of either LIFR
or gp130. This approach enabled us to drive GCSF-dependent homodimerization of
chimeric receptor subunits independently of endogenous receptor chains. The validity of this strategy had been established by previous studies
(20, 21), including those demonstrating that human hepatoma cells
expressing similar chimeric receptors acquired GCSF responsiveness (15,
22). Our results demonstrate that the signaling potential of LIFR
,
gp130, and GCSFR homodimers varies in different cell types, with
LIFR
homodimers playing an active role in suppressing ES cell
differentiation but having a reduced potential to induce macrophage
differentiation compared with either gp130 or GCSFR homodimers.
Cell Culture and Cytokines
conditioned
medium as a source of IL-3. The ES cell lines, derived from the
parental line E14TG2a, which contains a null mutation in the
hypoxanthine-phosphoribosyl transferase gene (hprt
; Ref.
23), were passaged in ES cell medium (DMEM containing 15% FCS, 0.1 mM 2-mercaptoethanol, and 1000 units/ml LIF) in the absence
of feeder cells. Recombinant murine LIF was produced in Escherichia coli and purified as described previously (24). Purified recombinant human GCSF, used for all biological assays, was
the gift of AMGEN, and purified recombinant IL-3 was purchased from
PeproTech Inc. (Rocky Hill, NJ). Mouse GCSF was produced in
Pichia pastoris (25), purified, and then iodinated for use in binding assays.
(14) at position +2489 by
nucleotide substitution in the region immediately preceding the
transmembrane domain of each molecule. Expression constructs were
generated by annealing the BamHI site of the GCSFR
extracellular domain with the transmembrane/cytosolic domain of either
gp130 or LIFR
(construct 1) and ligating the chimeric receptor
cDNA into the SalI site of the expression vector
6P-IRESneo-BS, driven by a PGK promoter (27). An additional
GCSFR-LIFR
construct (construct 2) was generated by polymerase chain
reaction amplification of a fragment of the human LIFR
cDNA
containing the transmembrane and cytoplasmic regions. Polymerase chain
reaction primers contained an in-frame BamHI site at the 5
end (5
-ACGTGGATCCATCTGACGTGGGATTAATTATTGCCATT-3
) and an
XbaI site at the 3
end
(5
-AGCTTCTAGACTGTTAATCGTTTGGTTTG-3
). This fragment was then inserted
into BamHI-XbaI digested pBS/mGR(Bam). The
GCSFR-LIFR
fragment was released by digestion with
HindIII and XbaI, and the ends were filled in
with Klenow (Promega) and ligated into the expression vector pEF-BOS
(28) using BstXI adaptors (Invitrogen). Although the two
GCSFR-LIFR
constructs differed slightly, because construct 1 contained a Pro
Leu substitution at amino acid 599 and a deletion
of amino acid 601 at the 3
end of the GCSFR extracellular domain, no
difference between the constructs was observed in a colony assay in the
presence of GCSF following stable transfection of M1 cells. Similarly,
ES cells were transfected with both constructs, and expression of
either construct was found to confer a GCSF-responsive phenotype in
these cells. The sequence of all chimeric constructs was verified using
an ABI PRISM Dye Terminator Cycle Sequencing Kit (Perkin-Elmer) and a
model 373 Automated DNA Sequencer (Perkin-Elmer). cDNA encoding the
full-length human GCSFR (a gift from S. Nagata, Osaka Bioscience
Institute) was cloned into the XbaI site of the pEF-BOS
expression vector. The pHCK-hprt plasmid was created by substituting
the PGK promoter in the PGK-hprt expression cassette of pGEM-7Z with an
XhoI-BamHI fragment of the murine hck
promoter, encompassing the region between
645 and +240 relative to
the major transcription initiation site (29).
(construct 2), or GCSFR-gp130 by electroporation,
essentially as described (11). Receptor constructs were cotransfected
with the pPGKPuropA expression vector (kindly provided by Prof. S. Cory), and transfected cells were selected with 20 µg/ml puromycin
(Sigma). For all transfected cell lines, expression of the receptors
was determined by assessing the ability of cells to bind
125I-GCSF.
ES cells with the expression plasmid pHCK-hprt.
Cells were selected in HAT medium (100 µM hypoxanthine,
0.4 µM aminopterin, 16 µM thymidine).
Individual resistant colonies were expanded and tested for HAT
sensitivity after a 5-day induction of cellular differentiation in the
absence of LIF. ES cells expressing chimeric constructs were obtained
by electroporation of HCK-hprt cells with the 6P-IRESneo-BS-based expression constructs (Bio-Rad Gene Pulser; 270 V, 500 microfarad), followed by selection for 7 days in 175 µg/ml Geneticin (Life Technologies, Inc.). ES cells were transfected with the GCSFR-LIFR construct 1. ES cell lines expressing the full-length GCSFR were obtained by co-electroporation with the plasmid PGKneo (30). Several
independently derived M1, Ba/F3, and ES cloned cell lines expressing
each receptor construct were selected for further analysis.
-counter. Scatchard analysis of saturation binding isotherms were
performed using the computer program LIGAND (36).
Roles of LIFR, gp130, and GCSFR Cytoplasmic Domains in M1 Cell
Differentiation
, GCSFR-gp130, or wild type GCSFR constructs, and the
expression of these receptors was confirmed by the ability of the
transfected cells to bind 125I-GCSF (Table
I). The capacity of the transfected cells
to differentiate in response to GCSF was assessed by semi-solid agar
colony assays. Untransfected parental M1 cells failed to respond to
GCSF, and all cell lines expressing the chimeric receptors responded
normally to LIF (data not shown). M1 cells expressing either
full-length GCSFR (Fig. 1A) or
GCSFR-gp130 (Fig. 1C) responded to GCSF in a similar manner,
with complete differentiation and clonal extinction at higher
concentrations of cytokine. In contrast, we were unable to detect a
GCSF-induced response by cells expressing GCSFR-LIFR
receptors,
because neither differentiation nor clonal suppression of M1 cells
expressing these receptors was evident (Fig. 1B). The
expression of characteristic macrophage markers, including Fc
receptor types I and II, Mac-1, and the macrophage colony-stimulating factor receptor, was also assessed in these cells in an effort to
determine whether any aspects of differentiation were induced in
response to GCSF. Flow cytometric analysis demonstrated that none of
these markers were up-regulated by GCSF stimulation of these cells
(data not shown). Furthermore, unlike cells expressing GCSFR, no change
in expression of the flk-2 receptor was detected in cells
expressing GCSFR-LIFR
in response to GCSF, as assessed by
flk-2 ligand binding assays (data not shown). Thus, by all criteria examined, no evidence for the induction of differentiation through GCSFR-LIFR
receptors in M1 cells could be demonstrated. Collectively, these data suggest that homodimerization of GCSFR or
gp130 cytoplasmic domains, but not LIFR
cytoplasmic domains, is
sufficient to induce macrophage differentiation in M1 cells.
Receptor
construct
M1 cells
ES
cells
Ba/F3 cells
No. of receptors/cell
Binding
affinity
No. of receptors/cell
Binding affinity
No. of
receptors/cell
Binding affinity
pM
pM
pM
GCSFR wild
type
9359
163
NDa
ND
4524
97
GCSFR-LIFR
3030
110
1060
280
963
303
GCSFR-gp130
2600
1100
850
430
763
350
a
ND, not determined.
Fig. 1.
Activity of chimeric receptors in M1, ES, and
Ba/F3 cells. GCSFR (A, D, G, and
J), GCSFR-LIFR (B, E, H,
and K), and GCSFR-gp130 (C, F,
I, and L) receptors were transfected into either M1, ES, or Ba/F3 cells, and their ability to transduce a
GCSF-dependent signal was assessed in a variety of assays.
Two independently derived clones of each transfection were examined in
each assay. A-C, GCSF-induced differentiation of
transfected M1 lines was assessed by soft agar colony assays. The
percentage of differentiated colonies () was scored after a 7-day
culture in the indicated concentration of GCSF. The number of colonies
present in each dish was determined and expressed as a percentage of
the total number of colonies formed in the absence of factor (
).
D-I, transfected ES cells were cultured in the indicated
concentration of GCSF in the absence of LIF. D-F, after a
5-day culture, the proportion of undifferentiated colonies was
calculated by scoring the morphology of 300 randomly selected colonies
in triplicate culture dishes. The proportion of ES colonies remaining
undifferentiated after culture for 5 days in 32 pM LIF is
also indicated (
). G-I, MTT assay. After a 6-day culture
in medium containing 2 × HAT, cultures were incubated for 3 h with MTT dye. 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 LIF. Results from
both ES cell assays represent the mean of three independent
experiments. J-L, transfected Ba/F3 cells were cultured at
200 cells/well in the presence of indicated dilutions of 100 ng/ml GCSF
(
) or 1 ng/ml IL-3 (
). After a 2-day culture, the number of
viable cells in each well was determined. All wells containing greater
than 200 viable cells after the 2-day culture were scored as
demonstrating a maximal response (>200).
[View Larger Version of this Image (30K GIF file)]
, gp130, and GCSFR Cytoplasmic Domains in ES Cell
Differentiation
-gp130 heterodimer was normal, because culture in
LIF maintained the cells in an undifferentiated state (Fig. 1,
D, E, and F). The capacity of GCSF to
substitute for LIF and retard differentiation of the transfected cells
was assessed. The dose-response curve obtained with GCSF stimulation
was similar for both the cell morphology (Fig. 1, D,
E, and F) and MTT (Fig. 1, G,
H, and I) assays, with a half-maximal effect at
approximately 63 pM GCSF. Thus, signaling through
homodimerized cytoplasmic domains of LIFR
, gp130, or GCSFR
maintained up to 80% of all ES cell colonies in an undifferentiated
state (Fig. 1, D-I). In contrast, parental ES cells did not
respond to GCSF in either assay, indicating the absence of endogenous
GCSFR expression in untransfected cells (data not shown). These data
suggest that homodimerization of the cytoplasmic domains of either
LIFR
or gp130 is sufficient to mediate a signal that maintains the
pluripotentiality of ES cells in vitro. Futhermore, these
data indicate that in addition to the LIFR, the GCSFR can transmit a
signal that maintains the undifferentiated state of ES cells.
, gp130, and GCSFR Cytoplasmic Domains in
Triggering Proliferation
, gp130, and
GCSFR cytoplasmic domains to signal a mitogenic response was determined
after introduction of the receptor constructs into the
IL-3-dependent cell line, Ba/F3. Transfected cells were
analyzed in a proliferation assay. As shown in Fig. 1 (J,
K, and L), the full-length GCSFR was able to
transduce a proliferative/survival signal comparable to that elicited
through the endogenous IL-3 receptor. Ba/F3 cells expressing GCSFR-gp130 showed a weaker proliferative or survival response to GCSF,
whereas no GCSF-dependent proliferation or survival was observed for cells expressing the GCSFR-LIFR
chimera (Fig. 1, J, K, and L). The reduced ability of
gp130 homodimers to generate a GCSF response parallels the transient
response of Ba/F3 cells expressing gp130 to stimulation by IL-6 and
soluble IL-6 receptor (38). This may be due to reduced expression of
LIFR
-gp130 signaling intermediates in Ba/F3 cells compared with M1
or ES cells, both of which express endogenous LIF receptors. This
hypothesis is supported by preliminary results in the
IL-6-dependent plasmacytoma cell line, 7TD1, that suggest
that the GCSFR-LIFR
is active in these cells (data not shown).
, gp130,
and GCSFR cytoplasmic domains to mediate cell-type specific responses, we were interested to compare the activation pattern of the Jak/STAT signaling pathway in response to receptor dimerization. To characterize the STAT complexes induced in response to GCSF, extracts of M1 and ES
cells transfected with various receptor constructs were examined in
electrophoretic mobility shift assays, using the high affinity
c-sis-inducible factor binding sequence, m67, as a probe (34). All DNA-protein interactions were specifically competed by an
excess of unlabeled m67 probe (data not shown). Three main complexes
were induced in M1 cells stimulated with LIF through the activation of
the endogenous LIFR (Fig. 2A).
A similar pattern was observed upon GCSF stimulation of M1 cells
expressing GCSFR-gp130 (Fig. 2A) and for GCSF stimulation of
M1 cells expressing wild type GCSFR (Fig. 2B). In contrast,
no m67-binding complexes were formed in response to GCSF in M1 cells
expressing GCSFR-LIFR
nor in parental M1 cells (Fig. 2A).
Hence, activation of m67-binding complexes correlated with the
expression of active cell surface receptors, suggesting that STAT
molecules may act as downstream effectors of the response.
Fig. 2.
Analysis of DNA-binding complexes induced by
cytokine stimulation of transfected cells. A,
electrophoretic mobility shift assays of transfected M1 cells
stimulated with either saline (), LIF (L), or GCSF
(G). M1-P refers to the untransfected parental M1
cell line. DNA-protein complexes are indicated by arrows.
Extracts from M1 cells (B) and ES cells (C) were
analyzed in electrophoretic mobility shift assays in the presence (+)
or the absence (
) of the indicated antibodies and stimuli. The
composition of the STAT complexes is indicated at the left
of the figure.
[View Larger Version of this Image (44K GIF file)]
(Fig.
2C). The level of STAT activation induced by this chimeric
receptor was comparatively weaker, despite its comparable capacity to
retard differentiation (Fig. 1, D-I).
, gp130, and GCSFR differ depending on the cell type as well as
the type of biological response initiated. Differences in the signal
transduction pathways triggered by gp130 homodimers and gp130-LIFR
heterodimers have previously been suggested by studies in which
overexpression of SCL in M1 cells inhibited the response to LIF and
OSM, which use gp130-LIFR
heterodimers, but did not affect signaling
through the IL-6 receptor, which uses gp130 homodimers (18, 19).
LIFR
alone may not be sufficient for transducing a differentiative
signal in M1 cells nor for induction of a proliferation/survival signal
in Ba/F3 cells, because homodimerized LIFR
cytoplasmic domains were
unable to signal these responses independently of gp130. However,
homodimers of either LIFR
or gp130 cytoplasmic domains were able to
deliver the signal to block differentiation in ES cells. In addition, a
previous report has demonstrated that either of these cytoplasmic
domains can induce an acute phase response in hepatoma cells and
mediate signaling in neuronal cells when activated as homodimers
(22).
or gp130 is unlikely, given that the
GCSFR-LIFR
receptor is unable to signal in M1 cells, which contain
both endogenous LIFR
and gp130.
cytoplasmic domains may fail to reach the required threshold in
M1 or Ba/F3 cells for a biological response to occur, perhaps due to a
reduced efficiency of the LIFR
chain to activate STATs or a reduced
pool size of signaling intermediates. The number of available STAT
molecules in ES cells may be greater than that in either M1 or Ba/F3
cells. Although the LIFR
chain may be less efficient at activating
STATs, the increased availability of STATs, coupled with a lower
threshold requirement for biological activity in these cells, could
explain the ability of the GCSFR-LIFR
receptor to signal in ES
cells.
and gp130 may be able to
interact with the specific cytoplasmic intermediates required for
signal transduction in ES, hepatoma, and neuronal cells. Verification
of this model would require the identification of the relevant
signaling pathways. In either case, the results described here clearly
show that LIFR
homodimers are less efficient than either gp130 or
GCSFR homodimers in signaling biological responses. Further
investigation of the signaling molecules activated by LIFR
and gp130
is needed to fully document the different signaling potentials of these
receptor subunits.
*
This work was supported by the Anti-Cancer Council of
Victoria, Melbourne, Australia; AMRAD Operations Pty. Ltd., Melbourne, Australia; the National Health and Medical Research Council, Canberra, Australia; the J. D. and L. Harris Trust; National Institutes of
Health Grant Grant CA-22556; and the Australian Federal Government Cooperative Research Centres Program.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.
Supported by a Queen Elizabeth II Postdoctoral Fellowship from
the Australian Research Council.
**
To whom correspondence should be addressed. Tel.: 61-3-9347-3155;
Fax: 61-3-9347-1938; E-mail; ernst{at}licre.ludwig.edu.au.
1
The abbreviations used are: LIF, leukemia
inhibitory factor; LIFR, LIF receptor; LIFR, LIFR
-chain; ES,
embryonic stem; IL, interleukin; OSM, oncostatin M; CNTF, ciliary
neurotrophic factor; GCSF, granulocyte colony-stimulating factor;
GCSFR, GCSF receptor; DMEM, Dulbecco's modified Eagle's medium; FCS,
fetal calf serum; MTT,
3-[4,5-dimethyldiazol-2-yl]-2,5-diphenyltetrazolium bromide.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.