From the Cooperative Research Centre for Cellular
Growth Factors and the Walter and Eliza Hall Institute of Medical
Research, Post Office Royal Melbourne Hospital, Victoria 3050, Australia, ¶ AMRAD Operations Pty Ltd, Richmond, Victoria 3121, Australia, the
Joint Protein Structure Laboratory of the Walter
and Eliza Hall Institute and the Ludwig Institute for Cancer Research,
Post Office Royal Melbourne Hospital, Victoria 3050, Australia, and the
** Tosoh Corporation, Ayase-shi, Kanagawa 252, Japan
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ABSTRACT |
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Leukemia inhibitory factor (LIF) is a
polyfunctional cytokine known to require at least two distinct receptor
components (LIF receptor -chain and gp130) in order to form a high
affinity, functional receptor complex. In this report, we present
evidence that there are two distinct truncated forms of gp130 in normal human urine and plasma: a large form with a molecular weight of approximately 100,000, which is similar to a previously described form
of soluble gp130 in human serum, and a previously undescribed small
form with a molecular weight of approximately 50,000. Using a panel of
monoclonal antibodies raised against the extracellular domain of human
gp130, we were able to show that the small form of the urinary gp130
probably contained only the hemopoietin domain. Both forms of gp130
bound LIF specifically and were capable of forming heterotrimeric
complexes with soluble human LIF receptor
-chain in the presence of
human LIF. In addition to the soluble forms of gp130, a soluble form of
LIF receptor
-chain was also detected in human urine and plasma.
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INTRODUCTION |
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Leukemia inhibitory factor
(LIF)1 is a polyfunctional
cytokine that can act on a wide range of cell types including
osteoblasts, hepatocytes, adipocytes, neurons, embryonal stem cells,
and megakaryocytes (1). LIF exerts its multiple biological functions
through a specific cell surface receptor system, which consists of at
least two membrane-bound glycoproteins, the LIF-binding chain (LIFR) and gp130. LIF binds first to LIFR
with low affinity (2) and then to
gp130 to form a high affinity functional receptor complex leading to
activation of downstream signal transduction pathways (3-6). Both
LIFR
and gp130 are members of the hemopoietin or cytokine type I
family of receptors (7, 8). The extracellular domains of members of
this receptor family share common structural features including
hemopoietin domains characterized by four conserved cysteine residues
and a WSXWS motif and three fibronectin type III (FN III)
modules (7, 8). The membrane-bound gp130 was initially defined as the
signal transducer of the interleukin-6 (IL-6) receptor system (9, 10)
and has been shown subsequently to also be a component of the
functional receptor complexes of ciliary neurotrophic factor (CNTF)
(4), oncostatin-M (OSM) (3, 11), cardiotrophin-1 (12, 13), and
interleukin (IL)-11 (IL-11) (14-16).
In addition to the cell membrane-anchored forms of LIFR and gp130,
it has been reported that naturally occurring soluble forms of these
receptor molecules are present in biological fluids and may act as
natural inhibitors of LIF activity (17-19). We and others have shown
previously that a soluble form of the mouse LIFR
with a molecular
weight (Mr) of approximately 90,000-150,000 occurs at high levels in normal mouse serum and is elevated
dramatically during pregnancy (17, 18). Recently, we have provided
evidence that the soluble form of mouse LIFR
probably arises from an
alternative splicing event of the LIFR
mRNA (20). Despite the
high levels of soluble LIFR
in mouse serum, its analogue was not
detected in human serum (17).
In contrast, a soluble form of gp130 with a Mr of 90,000-110,000 has been found in human serum (19). Although gp130 functions as the high affinity converting and signaling subunit in the receptor complexes for IL-6, LIF, OSM, CNTF, cardiotrophin-1, and IL-11, OSM was the only cytokine in this family initially demonstrated to bind to membrane-bound gp130 (3), and subsequently, it has been shown that OSM can bind directly to the soluble form of gp130 with low affinity (21, 22). We and others have recently shown that the soluble form of gp130 was able to bind not only directly and specifically to OSM but also to LIF (22, 23). Using biosensor technology, we were able to determine that the interaction between hLIF and soluble human gp130 was of low affinity, with an equilibrium dissociation constant of approximately 44 nM (23). This low affinity interaction could explain previous failures in detecting direct binding of LIF to the membrane-bound form of gp130.
In this study, we present evidence that there are two distinct
truncated forms of gp130 in normal human urine and plasma: a large form
with a Mr of approximately 100,000, which is
similar to that previously described (19), and a previously
undescribed small form with a Mr of
approximately 50,000. Both forms bound LIF specifically and were
capable of forming heterotrimeric complexes with soluble hLIFR in
the presence of hLIF. In addition to the soluble forms of gp130, a
soluble form of LIFR
was also detected in human urine and plasma.
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EXPERIMENTAL PROCEDURES |
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Reagents--
Escherichia coli-expressed hLIF (a gift
from Sandoz Pharmaceutical Co., Hanover, Switzerland) was
radioiodinated using a modified iodine monochloride method (24).
Anti-human gp130 monoclonal antibodies (mAbs), AM64, GPX22, and GPZ35,
which were raised against Chinese hamster ovary cell-expressed
extracellular domain of human gp130, were prepared as described
previously (10, 25). A goat anti-human LIFR polyclonal antibody
raised against the extracellular domain of human LIFR
was purchased
from R & D Systems.
Expression and Purification of Soluble Human LIFR and gp130 in
Pichia pastoris--
A soluble form of human gp130 (shgp130), which
consists of the Ig-like domain, hemopoietin domain, and three FN III
modules, was expressed in the methylotropic yeast P. pastoris with a FLAGTM epitope tag (DYKDDDDK) at its N
terminus and purified on an anti-FLAG M2 affinity column by elution
with FLAG peptide as described previously (23). A short form of soluble
human gp130 (sshgp130) was made identically as shgp130 except that the
construct lacked all three FN III modules. Protein quantitation for the
purified samples was performed by amino acid analysis. To make a
soluble form of hLIFR
, a cDNA encoding the hLIFR
(26) was
altered at its 5'-end to encode an XhoI site and an in-frame
12CA5 epitope (YPYDVPDYA) (27). The sequence at the N terminus of the
recombinant LIFR
was GAPYPYDVPDYA. The 3'-end was modified to encode
an XbaI site and a stop codon was introduced after position
536 (2) so that the recombinant LIFR
only contained the two
hemopoietin domains and the intervening Ig-like domain. The cDNA
was subsequently cloned into the yeast expression vector pPIC9 and
expressed in P. pastoris as described (23). The protein was
partially purified by gel filtration chromatography and quantified by
Scatchard analysis of hLIF binding isotherms (17).
Human Urine Collection-- Male and female normal human urine was collected from volunteers after informed consent, and 0.02% (v/v) Tween 20 and 0.02% (w/v) sodium azide were added. Small scale urine concentration was carried out using a Centriprep-10 (Amicon), and large scale concentration was performed using the Sartorius EasyFlow Device with a cellulose triacetate membrane (molecular weight cut-off of 20,000). Any precipitating materials in urine occurring before or after concentration were removed by centrifugation.
Gel Filtration Chromatography-- Concentrated human urine was applied to a Superdex 200 10/30 column (Amersham Pharmacia Biotech), previously equilibrated in 20 mM phosphate-buffered saline (0.15 M), pH 7.0, containing 0.02% (v/v) Tween 20 and 0.02% (w/v) sodium azide (PBS). Samples were eluted with PBS at a flow rate of 0.5 ml/min, and 0.5 ml fractions were collected.
Cross-linking Assay-- Aliquots of samples were incubated with 125I-hLIF in the absence or presence of unlabeled hLIF or antibodies in a final volume of 15 µl for at least 1 h at 4 °C. Then 5 µl of a 12 mM solution of the bifunctional cross-linker BS3 (Pierce) in 20 mM phosphate-buffered saline (pH 7.0) was added, and the mixtures were incubated for 30 min at 4 °C. Samples were mixed with 7 µl of 4-fold concentrated SDS sample buffer and analyzed by either 7.5 or 10% SDS-PAGE under nonreducing conditions. The gels were dried and visualized by either autoradiography or PhosphorImager analysis (Molecular Dynamics).
Affinity Chromatography-- The rhLIF affinity column was prepared by covalently coupling 1 mg of E. coli-derived rhLIF to 1 ml of Affi-Gel 10 (Bio-Rad) according to the manufacturer's instructions. Normal human urine samples were concentrated 100-fold as described above and incubated with 1 ml of hLIF-Affi-Gel 10 resin for 3-5 h at 4 °C. After unbound proteins were removed by centrifugation, the hLIF affinity beads were washed with 16 × 1 ml of PBS followed by additional washes with 8 × 0.3 ml of 10-fold diluted Actisep elution medium (Sterogenes Bioseparations, CA). The bound protein was then eluted with 10 × 0.3 ml of undiluted Actisep elution medium. The affinity column eluates were buffer-exchanged into PBS using NAP-5 columns (Amersham Pharmacia Biotech). Using the same procedures, LIF-binding proteins were enriched and partially purified on the hLIF affinity column from 50 ml of an outdated normal human plasma sample (obtained from the Royal Melbourne Hospital Blood Bank). Aliquots of buffer-exchanged fractions were analyzed for their ability to bind to 125I-hLIF using the cross-linking protocol described above.
Immunoprecipitation--
Aliquots (20 µl) of the hLIF affinity
column eluate, 0.2 µg/ml shLIFR, or 2 µg/ml shgp130 were
incubated with 125I-hLIF (800,000 cpm) in the absence or
presence of 50 µg/ml unlabeled hLIF in a final volume of 50 µl for
at least 1 h at 4 °C. Then 10 µl of a 12 mM
BS3 solution was added, and the mixtures were incubated for
30 min at 4 °C. After adding 1 M Tris-HCl buffer (pH
7.5) to a final concentration of 50 mM, the cross-linking
reactions were incubated for 40 min at room temperature. The
cross-linked samples were then mixed with an anti-human LIFR
polyclonal antibody at a concentration of 50 µg/ml. After a 30-min
incubation at 4 °C, the mixtures were added to 30 µl of 50% (v/v)
protein G-Sepharose gel slurry (Amersham Pharmacia Biotech) previously
equilibrated in PBS and incubated for 30 min at 4 °C. The samples
were centrifuged, and the protein G-Sepharose beads were washed with
4 × 0.5 ml of PBS. For elution, the beads were mixed with 30 µl
of 2× concentrated SDS sample buffer. The supernatants were then
analyzed by 7.5% SDS-PAGE under nonreducing conditions.
Protein Estimation-- Protein concentrations of pure recombinant soluble receptors were determined by amino acid analysis on a Beckman 6300 high performance amino acid analyzer equipped with a model 7000 data analyzer (Beckman).
Inhibition of STAT-3 Phosphorylation in M1 Myeloid Cells-- M1 cells (~107 cells/sample) were stimulated for 5 min at 37 °C with either 1 ng of hLIF or saline together with either soluble hLIF receptor or soluble hgp130 and then lysed in 50 mM Tris-HCl (pH 7.5) containing 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 2 mM NaF, 1 mM Na3VO4, and proteinase inhibitors. After pelleting insoluble material and protein standardization, approximately 100 µg of total cellular proteins were subjected to 4-15% acrylamide SDS-PAGE under reducing conditions and then transferred to a prewetted polyvinylidene difluoride membrane (PVDF-Plus, Micron Separations Inc.). After blocking, the membrane was incubated with an anti-phospho-STAT-3 polyclonal antibody (New England Biolabs), followed by incubation with a goat anti-rabbit Ig polyclonal antibody conjugated with horseradish peroxidase (DAKO, Denmark). The phosphorylated STAT-3 protein was visualized by radiography using the ECL system (Amersham Pharmacia Biotech). To check the quantity of protein loading, the same membranes were stripped with 0.1 M glycine-HCl, pH 3.0, for 30-60 min and washed three times in PBS, 0.1% Tween 20 before reprobing with a rabbit polyclonal antibody to STAT-3 (K-15, Santa Cruz Biotechnology, Inc.).
Estimation of Soluble Human LIFR Concentration in
Plasma--
To quantify the soluble LIFR
, an aliquot of an outdated
normal human plasma sample (obtained from the Royal Melbourne Hospital Blood Bank) was first precleared with protein G-Sepharose beads at a
ratio of 1:0.2 (v/v) for 1 h at 4 °C. The protein G-absorbed plasma was then incubated in the presence or absence of 2 µg of a
goat anti-human LIFR
polyclonal antibody for 1 h at 4 °C,
followed by the addition of 25 µl of protein G-Sepharose beads and a
2-h incubation at 4 °C. Immunoprecipitation of recombinant shLIFR
at various concentrations was performed in parallel except that the
preabsorption step with protein G beads was not included. The
immunocomplexes were washed with 3 × 1 ml of PBS containing 0.02% (v/v) Tween 20 and eluted from the protein G beads by boiling in
SDS sample buffer under reducing conditions for 5 min before being
subjected to 7.5% acrylamide SDS-PAGE. The Western blotting was
performed as described above except that the anti-human LIFR
polyclonal antibody and a rabbit anti-goat Ig polyclonal antibody conjugated with horseradish peroxidase (DAKO, Denmark) were used as the
first and second antibodies, respectively.
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RESULTS |
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Detection of LIF-binding Proteins in Normal Human Urine-- Human urine samples, collected from six healthy individuals (H1-H6) were concentrated and tested for soluble LIF-binding proteins by chemical cross-linking. Analysis of the cross-linking products by SDS-PAGE (Fig. 1) indicated that 125I-hLIF was cross-linked specifically to two species of proteins in all six samples with Mr of approximately 100,000 (here referred to as the "large form") and 50,000 (here referred to as the "small form") after subtraction for the Mr of the bound unglycosylated hLIF, respectively. The levels of the two hLIF-binding proteins varied in the six samples. This variation was likely to be due to the differences in protein content of these samples (data not shown).
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The Two Species of the Urinary hLIF-binding Proteins Are Not Precomplexed-- To examine whether the two hLIF-binding proteins were part of a preformed complex in urine, concentrated human urine was fractionated on a Superdex 200 gel filtration column as shown in Fig. 2A, and fractions were then analyzed for 125I-hLIF binding by chemical cross-linking. Analysis of column fractions 21, 23, 25, 27, 29, and 31 by SDS-PAGE (Fig. 2B) after cross-linking showed that the two hLIF-binding proteins were completely separated from each other according to their sizes, suggesting that they do not exist in a preformed complex in human urine. The Mr estimates of the two proteins by gel filtration were consistent with those obtained above. Also, it can be seen from Fig. 2B that there was a downward trend in Mr for both the large and small forms of the LIF-binding proteins across the fractions being assayed. This may be due to differential glycosylation of the two proteins.
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Identification of the Urinary LIF-binding Proteins as Soluble gp130
and LIFR--
In mouse and human serum, the presence of soluble
forms of the LIF receptor components, mouse LIFR
and human gp130,
respectively, has been described (17-19). It has also been
demonstrated that LIF can bind to gp130 directly (22, 23), although the
affinity was relatively low (23, 28). To examine whether the detected 125I-hLIF binding activity in human urine was due to the
presence of these reported proteins, we first performed competitive
cross-linking experiments, as shown in Fig.
3, in which 125I-hLIF was
mixed with increasing concentrations of unlabeled hLIF prior to
cross-linking to a partially purified urinary LIF-binding protein
sample (Fig. 3A). This was then compared with the same cross-linking to (a) a recombinant form of soluble human
gp130 (shgp130; Fig. 3B), which consists of the Ig-like
domain, hemopoietin domain, and three FN III modules with a
FLAGTM epitope tag at the N terminus, (b) a
recombinant short form of soluble human gp130 (sshgp130; Fig.
3C) identical to shgp130 except that the construct lacked
the FN III modules, or (c) a recombinant form of soluble
human LIFR
(shLIFR
; Fig. 3D). Densitometric analyses
of these data (not shown) revealed that half-maximal inhibition of
125I-hLIF cross-linking to both the large and small forms
of the two urinary binding proteins occurred at approximately 750 ng/ml unlabeled hLIF, similar to the hLIF concentrations required to inhibit
50% of the cross-linking to both shgp130 and sshgp130, whereas an
approximately 3-fold smaller amount of hLIF was required to achieve the
same inhibition for shLIFR
. These results indicated that the
relative binding affinities of 125I-hLIF to the two forms
of urinary binding proteins were similar to those of
125I-hLIF to recombinant shgp130 and sshgp130, suggesting
that the LIF binding activity in human urine might be due to the
presence of soluble forms of gp130 with different truncations at the
C-terminal ends.
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Ternary Complex Formation of both the Large and Small Forms of
gp130 with shLIFR in the Presence of hLIF--
Both the large and
small forms of the urinary gp130 as well as recombinant shgp130 and
sshgp130 were analyzed in cross-linking experiments for their ability
to form a ternary complex with shLIFR
in the presence of hLIF. As
shown in Fig. 6, cross-linking of 125I-hLIF to the purified large form or recombinant shgp130
in the presence of shLIFR
generated extra higher
Mr species (Fig. 6, lanes 7 and
3, respectively), the Mr of which
could be accounted for by summing up the Mr of
all three cross-linking components, suggesting the formation of a
tripartite complex. The inability of the purified large form
LIF-binding fraction by itself (Fig. 6, lane 6) to yield a
higher Mr complex corresponding to the ternary complex was probably due to insufficient LIFR
in the sample. When an
extra amount of recombinant shLIFR
was added to the cross-linking mixture of this sample, the formation of a ternary complex became detectable (Fig. 6, lane 7). This was consistent with the
above observation that the appearance of an extra high
Mr cross-linking species occurred only in the
hLIF affinity column eluate containing the most LIF-binding activity.
As above, when the cross-linking was performed with the small form of
the urinary gp130 and recombinant sshgp130, both proteins were shown to
be capable of forming ternary complexes with shLIFR
in the presence
of hLIF (Fig. 6, lanes 9 and 5,
respectively).
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Evidence for a Soluble LIFR in Human Plasma--
It appeared
that the soluble LIFR
in human urine was of low abundance, since the
LIF binding activity was hardly detectable in unconcentrated urine
(data not shown). This may be the reason that we were unable to detect
the soluble LIFR
in human serum in our earlier study, which was
based on detecting binding activity for LIF (17). To examine this
possibility, a human plasma sample (50 ml) was loaded onto a hLIF
affinity column (see "Experimental Procedures" for details), and
the column eluates were then analyzed in cross-linking experiments for
the presence of LIF binding activity. As shown in Fig.
8A, SDS-PAGE analysis of the
cross-linked products of 125I-hLIF to the five column
fractions (eluates 1-5) revealed that there were at least three
LIF-binding proteins present in human plasma with
Mr ranging from 50,000 to 130,000 (after
subtraction for the Mr of the bound
125I-hLIF). Judging by the sizes of their cross-linking
products with 125I-hLIF, we assumed that two of these
proteins, with Mr of approximately 100,000 and
50,000, respectively, corresponded to the large and small forms of
gp130 detected in human urine. In two of the eluates (eluates 2 and 3),
there was an additional minor band (Fig. 8A, lanes
4 and 7, indicated by the open arrow), which
was similar to the extra higher Mr species
previously observed in the hLIF affinity column eluate (Fig. 5,
lane 8). Upon the addition of the anti-hLIFR
antibody
prior to cross-linking, this minor band disappeared and was replaced by
an increase in intensity of the band migrating at the top of the
separating gel, indicated by the solid arrow (Fig.
8A, compare lanes 4 and 6 and
lanes 7 and 9), suggesting that it corresponded
to the cross-linked complex between 125I-hLIF and the
soluble LIFR
. There was also a similar Mr
complex migrating at the top of the separating gel in the affinity
column eluates, which predominantly occurred in eluate 1 (Fig.
8A, lane 1). The intensity of this complex was
decreased upon the addition of unlabeled hLIF (compare lanes
1 and 2) but unaffected by the addition of the
anti-hLIFR
antibody (compare lanes 1 and 3), probably indicating the presence of some aggregated form of gp130 in
the sample. It is also worth noting that there was relatively more of
the small form of gp130 in urine than in plasma.
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Inhibitory Action of shLIFR and shgp130 on LIF Biological
Activities--
LIF was originally defined by its ability to induce
macrophage differentiation in the M1 myeloid leukemic cell line (1). One of the earliest detectable actions of LIF on M1 cells is the induction of tyrosine phosphorylation and activation of the signal transducer and activator of transcription, STAT-3 (29). Because of the
small amounts of recombinant receptors available, we decided to test
the inhibitory action of the soluble receptors on hLIF-induced STAT-3
activation in M1 cells, which occurs rapidly. Fig.
9 shows that shLIFR
and mixtures of
shLIFR
and shgp130 were able to significantly inhibit tyrosine
phosphorylation of STAT-3 but that, at the concentrations used, neither
form of shgp130 alone nor a mixture of soluble human urinary LIFR
and gp130 could inhibit hLIF-induced STAT-3 phosphorylation.
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DISCUSSION |
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There are many examples of receptors in the hemopoietin receptor
family that are found in soluble forms in body fluids (30, 31). In the
IL-6 receptor subfamily, these include IL-6 receptor -chain
(IL-6R
) in human urine (32) and serum (33), LIFR
in mouse serum
(17, 18), CNTF receptor
-chain in human cerebrospinal fluid (34),
and gp130 in human serum (19). Soluble receptors have been implicated
in both enhancing and reducing the biological effects of their cognate
ligands. For example, the complex of soluble IL-6R
and IL-6 is
capable of interacting with cell surface gp130 to trigger a variety of
biological responses (9), while a naturally occurring soluble form of
gp130 in human serum has been implied to serve as a negative regulator
in vivo of the signaling mediated by the IL-6·soluble
IL-6R
complex (19), and soluble LIFR
in mouse serum also acts
exclusively as an inhibitor of LIF signaling (17, 18).
Here, we describe the presence in normal human urine and plasma of a
soluble form of LIFR and two distinct truncated forms of soluble
gp130 (the large and small forms) that can bind hLIF specifically. To
our knowledge, this is the first report describing a naturally
occurring soluble form of LIFR
in human biological fluids as well as
describing a small form of soluble gp130 capable of binding LIF
directly and specifically. The large form of the urinary soluble gp130
with a Mr of approximately 100,000 was similar to the previously described soluble form of gp130 in serum with a
Mr of 90,000-110,000 (19). The previous
detection of the soluble gp130 in serum was facilitated by the
anti-human gp130 mAb AM64 (19). We have demonstrated in this study that
mAb (AM64) could not recognize the soluble form of gp130 lacking FN III
modules. This may be one of the reasons for the earlier inability (19) to detect the small form of gp130, which appeared to be also present in
human plasma. Based on the similar sizes of the cross-linked complexes
between 125I-hLIF and sshgp130 and between
125I-hLIF and the small form of urinary gp130, as well as
the results obtained from the analyses with the anti-gp130 mAbs, we
concluded that the small form of the urinary gp130 was likely to
contain only the hemopoietin domain and was missing all or almost all of the three FN III modules.
Recently, an alternatively spliced mRNA encoding a soluble form of
human gp130 was described from blood mononuclear cells (35). This
transcript would encode a form of gp130 truncated within one base pair
of the transmembrane domain and would correspond to the long form
described here and elsewhere (19). Whether the short form is encoded by
a separate transcript or by posttranslational processing of the longer
form is currently unclear. It is of interest, however, that three
alternate transcripts encoding soluble human LIFR have been
described from liver, placenta, and choriocarcinoma cell line (36). One
possibility may be that the FN III domains of the long form allow it to
be sequestered at tissue sites and that proteolytic cleavage to
generate the short form could serve as an additional control point to
regulate circulatory levels of bioactive forms of soluble gp130.
Further experiments are required to address this issue.
The recombinant shLIFR containing the two hemopoietin domains and
the intervening Ig-like domain, but lacking all three FN III modules,
was shown to be sufficient for hLIF binding, a finding consistent with
previous results (28). In the presence of hLIF, this shLIFR
was also
capable of forming ternary complexes with both the recombinant gp130
lacking all three FN III modules (sshgp130) and the naturally occurring
small form of gp130, probably also lacking all three FN III modules.
These findings were in agreement with a previous mutagenesis study of
gp130, which demonstrated that only the membrane distal half of gp130,
consisting of the Ig-like domain and hemopoietin domain, was
responsible for the formation of a ternary complex with IL-6 and the
IL-6R
(37). The exact roles of the three FN III modules in ternary
complex formation of these hemopoietin receptors still remain to be
determined. The ternary complexes of shLIFR
with both forms of the
recombinant and the urinary gp130 in the presence of hLIF appeared to
be heterotrimeric, which agreed with our previous finding that hLIF
could form a cross-species heterotrimeric complex with soluble mouse
LIFR
and human gp130 in solution (23) but differed from the
IL-6·IL-6R
·gp130 and CNTF·CNTF receptor
-chain·gp130·LIFR
complexes, which were hexameric
(38-40).
At the concentrations used, the shLIFR displayed significant
inhibition of hLIF-induced STAT-3 phosphorylation in M1 cells, but both
forms of soluble gp130 were ineffective. Moreover, no significant
increase in inhibition was observed when shLIFR
was mixed with
either form of gp130.
These data are in agreement with previous data indicating that
shLIFR acts exclusively as a LIF antagonist (17, 18) but contrast
with previous reports that soluble gp130 inhibited the biological
actions of IL-6 on Kaposi's sarcoma cells (41) as well as the
proliferative actions of LIF and OSM on TF-1 erythroleukemia cells
(22). The reason for these differences is probably the use in the
latter study of a dimeric form of soluble gp130 at considerably higher
concentrations than those used here (approximately 6 µg/ml compared
with 0.45 µg/ml). This is probably also the reason that the soluble
human urinary LIFR
and gp130 failed to inhibit STAT-3
phosphorylation in M1 cells stimulated by hLIF (Fig. 9). Nevertheless,
taken together, all of these observations suggest that shLIFR
and
shgp130 in serum and urine would serve to act as inhibitors of LIF,
OSM, IL-6, CNTF, and IL-11 action. Although the concentrations of these
receptors in normal serum appear insufficient to inhibit these
cytokines, it is likely that some biological responses may elevate the
concentrations of the receptors to a level that is able to suppress the
action of these proinflammatory cytokines. For example, the
concentration of shLIFR
detected in normal human plasma (~10
ng/ml) was only 5-fold lower than the dose that gave significant
inhibition of LIF-stimulated STAT-3 phosphorylation in M1 cells.
Consequently, it will be of some interest to determine stimuli that
result in increased secretion and circulatory levels of soluble
hLIFR
and gp130.
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ACKNOWLEDGEMENTS |
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We thank Sandoz for E. coli-derived rhLIF, Tetsuya Taga for providing the gp130 cDNA clone, James Eddes for amino acid analysis, Simon Olding for preparing the figures, and Denis Quilici and Thomas Nikolaou for assistance with the densitometric data analysis.
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FOOTNOTES |
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* This work was supported by the Australian Federal Government Cooperative Research Centres Program; the National Health and Medical Research Council, Canberra, Australia; and National Institutes of Health Grant CA-22556.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.
§ Present address: Molecular Genetics and Development Group, Institute of Reproduction and Development, Monash Medical Centre, Clayton, Victoria 3168, Australia.
To whom correspondence should be addressed: The Walter and
Eliza Hall Institute of Medical Research, Post Office Royal Melbourne Hospital, Victoria 3050, Australia. Tel.: 61-3-9345-2559; Fax: 61-3-9345-2616; E-mail: nicola{at}wehi.edu.au.
1
The abbreviations used are: LIF, leukemia
inhibitory factor; hLIF, human LIF; LIFR, LIF receptor
-chain;
hLIFR
, human LIFR
; shLIFR
, soluble human LIFR
; shgp130,
soluble human gp130; sshgp130, short form of soluble human gp130; FN
III, fibronectin type III; STAT-3, signal transducer and activator of
transcription 3; mAb, monoclonal antibody; PAGE, polyacrylamide gel
electrophoresis; PBS, phosphate-buffered saline; CNTF, ciliary
neurotrophic factor; OSM, oncostatin-M; IL, interleukin; IL-6R
,
interleukin 6 receptor
-chain; BS3,
bis(sulfosuccinimidyl)suberate.
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REFERENCES |
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