From the
Cardiotrophin-1 (CT-1) is a newly isolated cytokine that was
identified based on its ability to induce cardiac myocyte hypertrophy.
It is a member of the family of cytokines that includes interleukins-6
and -11, leukemia inhibitory factor (LIF), ciliary neurotrophic factor,
and oncostatin M. These cytokines induce a pleiotropic set of growth
and differentiation activities via receptors that use a common
signaling subunit, gp130. In this work we determine the activity of
CT-1 in six in vitro biological assays and examine the
composition of its cell surface receptor. We find that CT-1 is inactive
in stimulating the growth of the hybridoma cell line, B9 and inhibits
the growth of the mouse myeloid leukemia cell line, M1. CT-1 induces a
phenotypic switch in rat sympathetic neurons and promotes the survival
of rat dopaminergic and chick ciliary neurons. CT-1 also inhibits the
differentiation of mouse embryonic stem cells. CT-1 and LIF
cross-compete for binding to M1 cells, K
Cardiac muscle hypertrophy is an important adaptive response of
the heart to injury or to an increased demand for cardiac output
(1, 2, 3) . This hypertrophic response is
characterized by the reactivation of genes normally expressed during
fetal heart development and by the accumulation of sarcomeric proteins
in the absence of DNA replication or cell division
(4, 5, 6) . In the course of identifying factors
that mediate the various phases of cardiac hypertrophy, we recently
isolated by expression cloning a novel cytokine, cardiotrophin
(CT-1)
The biological effects
induced by IL-6 and related proteins are mediated by a family of
structurally similar cell surface receptors, the cytokine receptor
family, that includes the receptors for growth hormone and prolactin as
well as for many cytokines
(10, 11, 12, 13) . The IL-6 receptor
subfamily is composed of multisubunit complexes that share a common
signaling subunit, gp130
(14, 15, 16) . Some
members of the IL-6 cytokine family (IL-6 and IL-11) induce the
homodimerization of gp130
(17, 18) , while others (LIF,
OSM, and CNTF) induce gp130 heterodimer formation with the 190-kDa LIF
receptor
(19) . Following dimerization of the signaling
components, these receptors induce a number of intracellular signaling
events including activation of the transcription factor, NF-IL6,
probably via the Ras-microtubule-associated protein kinase cascade
(16) and activation of the Jak/STAT signaling pathway
(20) . The latter pathway includes the tyrosine phosphorylation
and activation of the intracellular tyrosine kinases, Jak1, Jak2, and
Tyk2
(21, 22, 23, 24) and of the
transcription factors, STAT1 and STAT3
(21, 25, 26) .
In this work, we show that
CT-1 is active in several in vitro biological assays where
cytokines of the IL-6 family have activity. We also show that CT-1 can
bind to and induce biological responses via the LIF receptor and its
signaling subunit, gp130.
Human IL-6 was from Genzyme, mouse LIF was from R& Systems
and Genentech manufacturing, and rat CNTF and glial cell line-derived
neurotrophic factor
(27) were produced by Genentech. Mouse CT-1
was expressed and purified as a fusion protein as described previously
(7) . This protein results in a 34-amino acid amino-terminal
extension that encodes a portion of the herpes simplex virus
glycoprotein D and a Factor Xa cleavage site
(7) . In some
cases, an alternative fusion protein was used that substitutes a
different site for the Factor Xa cleavage site
(7) giving the
amino acid sequence . . . DQLLEGGAAHY followed by the CT-1 sequence
MSQREGSL . . . CT-1 and LIF were iodinated by the IODO-BEAD (Pierce)
and lactoperoxidase
(28) methods to specific activities of
900-1100 Ci/mmol.
For the assay of the transmitter phenotype, newborn
rat sympathetic neurons were prepared as described previously
(32) . Superior cervical ganglia were dissociated with trypsin
(0.08%) and plated in serum-free F-12 medium containing nerve growth
factor and additives as described previously
(33) . Neurons were
plated at 30,000/well in 24-well plates precoated with polyornithine
and ECL cell attachment matrix (Promega) and allowed to grow for 10
days in the presence of indicated factors. Tyrosine hydroxylase and
choline acetyltransferase activities were assayed as described
previously
(34, 35) .
The survival of rat
dopaminergic neurons was assayed as described in Ref. 27. Ciliary
neuron survival assays were performed with neurons isolated from E8
chick embryos as described previously
(36) . Survival was
assessed by counting live neurons after staining with the vital dye MTT
(37) . The data were fit to the four-parameter equation
described above.
For the assay of embryonic stem cell
differentiation, passage 15 embryonic stem cells, ES.D3
(38) ,
were maintained in Dulbecco's modified Eagle's medium (Life
Technologies, Inc.) (high glucose, no sodium pyruvate), containing
23.83 g/liter HEPES, 500 mg/liter penicillin, 500 mg/liter
streptomycin, 4 g/liter L-glutamine, 1 g/liter gentamicin
sulfate, 1 mM 2-mercaptoethanol, 15% fetal bovine serum, and
1.2 megaunits/liter mouse LIF (Life Technologies, Inc.). Cells were
trypsinized, plated in duplicate at 1000 cells/well in 24-well tissue
culture plates in the above culture medium with or without LIF or CT-1,
and scored 9 days later. No change in colony numbers was observed
except in the no addition group where the cells had flattened and
differentiated.
Anti-gp130 antibody inhibition experiments were performed with a rat
anti-mouse gp130 monoclonal antibody (RX435)
Binding to neonatal rat cardiac myocytes
was performed as for M1 cells, but cells were isolated as described
previously
(7) and plated for 16 h. Assays were performed with
1 million cells in a volume of 100 µl.
Cross-linking was
performed with 10 million M1 cells in phosphate-buffered saline
containing 0.1% albumin, 7.2 nM
Binding to
the soluble LIF receptor and soluble gp130 was performed in a manner
similar to that described previously
(48) . Briefly, assays were
performed in 96-well Multiscreen-HV filtration plates with 0.45-µm
polyvinylidene difluoride membranes (Millipore) in phosphate-buffered
saline containing 0.1% bovine serum albumin and including 25 µl of
phosphate-buffered saline-washed Ni
We have previously shown that, like CT-1, some members of the
IL-6 cytokine family (LIF, OSM, and IL-11) induce cardiac myocyte
hypertrophy in vitro(7) . Since the members of this
family have a wide range of hematopoietic, neuronal, and developmental
activities
(9) , CT-1 was assayed for its activity in these
biological systems.
While inactive on dopaminergic neurons,
CNTF does promote the survival of ciliary neurons
(53) . CT-1
was tested for its activity in promoting the survival of chick ciliary
neurons (Fig. 2 C). While at maximal concentrations, CT-1
was as active as CNTF, the potency of CT-1 in promoting ciliary neuron
survival was about 1000-fold less than that of CNTF
(Fig. 2 C). Thus, CT-1 shares some neuronal activities
with the IL-6 family cytokines such as CNTF.
We have used in vitro hematopoietic, neuronal, and
developmental assays to show that CT-1 has a range of activities in
addition to the induction of cardiac myocyte hypertrophy for which it
was initially isolated
(7) . CT-1 is more potent than LIF in
inhibiting the growth of the myeloid leukemia cell line, M1. It induces
a phenotypic switch in sympathetic neurons; it promotes the survival of
dopaminergic neurons from the central nervous system and ciliary
neurons from the periphery; and it maintains the undifferentiated
phenotype of embryonic stem cells. CT-1 and LIF share a common activity
profile (both inhibit the growth of M1 cells, induce the switch in
sympathetic neuron phenotype, inhibit the differentiation of embryonic
stem cells, and induce cardiac myocyte hypertrophy
(7) ). CT-1
is active in assays where CNTF is active (both induce the switch in
sympathetic neuron phenotype
(63) , promote the survival of
ciliary neurons,
Alignments of the amino acid sequences of CT-1 and other
members of the IL-6 cytokine family show that while these cytokines
share biological activities and receptor subunits, they are only
distantly related in primary sequence (14-24% identity for the
mammalian proteins, Fig. 9 A). There is little
conservation of the cysteine residues and only a partial maintenance of
the exon-intron boundaries
(66, 67) . More sophisticated
analyses (including the crystal structure of LIF
(68) ) show
that these proteins share a common structural architecture of four
The presence or absence of the different
subunits of the IL-6 family receptors dictates the responsiveness of
various cells to the different cytokines
(12, 16) .
Thus, all responsive cells are believed to express gp130, B9 cells fail
to respond to LIF and CNTF because they lack LIF receptor, IL-6 is
inactive on embryonic stem cells because these cells lack the IL-6
receptor
CT-1 and LIF also cross-compete for binding to rat
cardiac myocytes. This finding is consistent with the hypothesis that
these two ligands act on these cells via the LIF receptor, as we have
established for M1 cells. The availability of primary cardiac myocytes
has limited our analysis of the CT-1 receptor in these cells.
While
LIF and OSM induce the heterodimerization of the same receptor
subunits, LIF receptor and gp130, the affinity of these two ligands for
the individual receptor components differs. LIF binds to the LIF
receptor ( K
Although CT-1 was isolated based on its ability to induce cardiac
myocyte hypertrophy
(7) , it clearly has a much wider range of
activities, as is found for the other cytokines of the IL-6 family
(9, 16) . The receptor data presented here predict that
CT-1 should mimic the many effects of LIF in vitro and in
vivo. Transgenic overexpression of IL-6 family cytokines often
results in dramatic and widespread consequences, a finding consistent
with their pleiotropic actions in vitro. Transgenic
overexpression of IL-6 leads to dramatic changes including
plasmacytosis
(79) , and overexpression of LIF leads to lethal
effects including weight loss and thymus atrophy
(80) . On the
other hand, as has been pointed out previously
(16) , these
cytokines show a functional redundancy such that there are relatively
minor effects upon the loss of function of one family member. Mice with
a targeted disruption of the IL-6 gene develop normally
(81) .
The targeted deletion of the LIF gene in mice leads to animals that are
outwardly normal, although they do exhibit a reduced growth rate, a
decrease in hematopoietic cells, and a failure of proper embryo
implantation
(82) . The targeted disruption of the CNTF gene
results in only small effects on muscle strength
(83) , and a
homozygous null allele of the CNTF gene has been found in 2.3% of
healthy individuals tested
(84) . Deletion of the CT-1 gene
alone and the breeding of mice with multiple cytokine disruptions
should help elucidate the specific and redundant roles played by the
members of this cytokine family.
We thank Teresa Woodruff for labeled CT-1 and LIF;
Christa Gray for DNA sequencing of the soluble receptor constructs;
Paula Jardieu, Joni Sutherland, and Kris Poulsen for help with the
in vitro assays; Kathy King and Jane Winer for the preparation
of cardiac myocytes; Tadamitsu Kishimoto and Mikiyoshi Saito for the
gp130 antibody; David Goeddel for reading the manuscript; and Wei Li
for help in purifying CT-1.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
[CT-1]
0.7 nM, and this binding is
inhibited by an anti-gp130 monoclonal antibody. Both ligands can be
specifically cross-linked to a protein on M1 cells with the mobility of
the LIF receptor (
200 kDa). In addition, CT-1 binds directly to a
purified, soluble form of the LIF receptor in solution
( K
2 nM). These data show
that CT-1 has a wide range of hematopoietic, neuronal, and
developmental activities and that it can act via the LIF receptor and
the gp130 signaling subunit.
(
)
, that induces cardiac myocyte
hypertrophy in vitro(7) . Amino acid sequence
similarity showed CT-1 to be a new member of the
IL-6/LIF/CNTF/OSM/IL-11 cytokine family. One member of this family,
LIF, a previously unrecognized inducer of cardiac myocyte hypertrophy,
was shown to be nearly as potent as CT-1 in inducing these effects
in vitro(7) . The IL-6 family of cytokines has a wide
range of growth and differentiation activities on many cell types
including those from the blood, liver, and nervous system
(8, 9) . CT-1 mRNA is widely (but not universally)
expressed in adult mouse tissues including heart, kidney, skeletal
muscle, and liver. Like CNTF, CT-1 lacks a conventional amino-terminal
secretion signal sequence; it is, however, found in the medium of
transfected mammalian cells
(7) .
Hematopoietic, Neuronal, and Developmental
Assays
Proliferation of the mouse hybridoma cell line, B9
(29) , was assayed by [H]thymidine
incorporation 84 h after the addition of cytokine as described
previously
(30) . Inhibition of the proliferation of the mouse
myeloblast cell line, M1 (T-22), was assayed by
[
H]thymidine incorporation 72 h after the
addition of cytokine as described previously
(31) . The data
were fit to the four-parameter equation, y = d - (( d - a)/(1 +
( x/ c))), where the parameter c is the
EC
.
Cell Binding and Cross-linking
Binding was
performed in RPMI 1640 containing 0.1% bovine serum albumin with
7.5-10 million M1 cells (TIB 192, ATCC) in a volume of 250 µl
for 2 h on ice with shaking. Reactions were layered on 250 µl of
RPMI containing 0.1% albumin and 20% sucrose and centrifuged at 4000
rpm for 1 min at 4 °C, and the cell pellet was counted. The data
were fit to a one-site binding model as described previously
(39) . Lines shown in the figures are from the curve fits.
(
)
or
a rat anti-gp120 control antibody (Genentech 6D8.1E9) in a volume of
150 µl. Reactions were incubated on ice for 2 h, centrifuged at
12,500 rpm, and washed with 1 ml of cold phosphate-buffered saline
containing 0.1% albumin. The data were fit to the four-parameter
equation described above.
I-labeled mouse
CT-1 or 2.2 nM
I-labeled mouse LIF with or
without a 100-fold molar excess of the unlabeled ligands in a volume of
250 µl. After 1 h at room temperature, 10 mM
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 5
mM N-hydroxysulfosuccinimide (Pierce) were added, and
the incubation continued for 30 min at room temperature. The samples
were then processed as described previously
(40) .
DNA Binding Activity
Two hundred thousand M1 cells
were incubated in 1 ml of RPMI 1640 in 12-well dishes with ligand for
30 min at 37 C. After stimulation, the cells were collected by
centrifugation, suspended in 200 µl of homogenization buffer (10
mM HEPES (pH 7.2), 10 mM KCl, 0.1 mM EDTA,
0.1 mM EGTA, 1 mM dithiothreitol, 1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml
aprotinin) and incubated at 0 °C for 15 min. Cells were lysed by
the addition of Nonidet P-40 to 0.1%, and cell extracts were prepared
by incubation at 0 °C for 15 min, centrifugation at 100
g for 5 min, and retention of the supernatant. DNA binding
activity in the cell extracts was assayed by electrophoretic mobility
shift assay as described previously
(41) . Briefly, binding
reactions contained 10 mM Tris-HCl buffer (pH 7.5), 100
mM KCl, 5 mM MgCl
, 1 mM
dithiothreitol, 6.7% glycerol, 0.067 g/liter poly(dI-dC)(dI-dC), 0.5 ng
(25,000 cpm) of
P-SIE DNA (5`-CTAGAGTCGACATTTCCCGTAAATCT
and 5`-CTAGAGATTTACGGGAAATGTCGACT, high affinity m67
(42, 43) ), and 3 µl of cell extract in a final
volume of 15 µl. Some reactions included 100 ng of unlabeled SIE
DNA. The reactions were incubated 30 min at 22 °C and analyzed by
polyacrylamide gel electrophoresis and autoradiography.
Binding to Soluble LIF Receptor and Soluble
gp130
DNA encoding the extracellular domain of the mouse LIF
receptor (amino acids 1-826) and mouse gp130 (1-617) was
generated by PCR of M1 cell (above) mRNA and of a mouse lung cDNA
library (Clontech). These sequences were cloned with a C-terminal tag
encoding 6 histidine residues in the mammalian expression vector, pRK5
(44) , to give the plasmids, pRK5.mu.slifr and pRK5.mu.sgp130.
DNA sequencing of the coding regions confirmed that these plasmids
encode proteins that match the published amino acid sequence
(45, 46) with the exception of the substitution of
lysine for arginine at amino acid 326 of gp130, a change that was found
for three fragments from both sources. The plasmids were transfected
into human 293 cells
(7) , and the proteins isolated from 4-day
conditioned medium by Ni-nitrilo-triacetic
acid-agarose (Qiagen) affinity purification. Briefly, the conditioned
medium was concentrated
18-fold (Centriprep 10, Amicon), and the
tagged protein was purified by binding to the Ni
resin for 2 h at room temperature. Following two washes with
phosphate-buffered saline containing 5 mM imidazole, the
proteins were eluted with phosphate-buffered saline containing 200
mM imidazole and quantitated by colorimetric assay (Bio-Rad).
Analysis of the proteins by SDS-polyacrylamide gel electrophoresis
showed single bands of 120 kDa for the soluble LIF receptor and 85 kDa
for soluble gp130. Amino acid sequencing gave the expected
amino-terminal sequence for the soluble LIF receptor beginning at amino
acid 44
(45, 47) ; the amino terminus of gp130 is
expected to be blocked
(46, 47) , and amino-terminal
protein sequencing gave no sequence for soluble gp130.
-agarose (Qiagen)
in a final volume of 175 µl. Plates were incubated at room
temperature overnight with agitation. Following vacuum filtration and
one wash with 200 µl of cold phosphate-buffered saline, the
individual assay wells were cut from the plate and counted. The data
were analyzed as described above for M1 binding.
Hematopoietic Assays
IL-6 promotes the proliferation and
differentiation of B cells into antibody-producing cells following
antigen stimulation
(8) . In order to determine whether CT-1
could also mediate these effects, we tested CT-1 on the mouse hybridoma
cell line, B9
(29) . While IL-6 stimulates the proliferation of
B9 cells as indicated by an increase in
[H]thymidine incorporation, CT-1 and LIF were
inactive (Fig. 1 A), even at concentrations as high as 2
µM (data not shown). Thus, CT-1 does not mimic the
activity of IL-6 in promoting B cell expansion.
Figure 1:
Activity of CT-1 in hematopoietic cell
assays. The induction by the human ( h) or mouse ( m)
cytokines was performed as described under ``Materials and
Methods.'' A, stimulation of
[H]thymidine incorporation in the mouse hybridoma
cell line, B9, EC
[IL-6] = 0.13 (±
0.03) nM. B, inhibition of
[
H]thymidine incorporation in the mouse myeloid
leukemia cell line, M1, EC
[CT-1] =
0.0076 (± 0.0006) nM, EC
[LIF]
= 0.048 (± 0.004) nM.
While IL-6
stimulates the growth of several B cell lymphomas, myelomas, and
plasmacytomas, it also has growth inhibitory effects on certain B
lymphoma and myeloid leukemia cells
(8) . IL-6 (as well as LIF
and OSM) inhibits the growth of the mouse myeloid leukemia cell line,
M1, and induces its differentiation into a macrophage-like phenotype
(8, 49) . On testing CT-1, we found that it was 6-fold
more potent than LIF in inhibiting the uptake of
[H]thymidine by M1 cells
(Fig. 1 B). Thus, CT-1 does share at least some of the
growth inhibitory activities of the IL-6 family cytokines.
Neuronal Assays
Members of the IL-6 cytokine
family modulate the phenotype and promote the survival of neuronal
cells
(50) . LIF and CNTF can induce a switch in the transmitter
phenotype of sympathetic neurons from noradrenergic to cholinergic, a
change that is accompanied by the induction of several neuropeptides
including substance P, somatostatin, and vasoactive intestinal
polypeptide
(51) . The ability of CT-1 to induce this switch in
the transmitter phenotype was determined with cultured rat sympathetic
neurons. CT-1 inhibited the tyrosine hydroxylase activity (a
noradrenergic marker) and stimulated somewhat the choline
acetyltransferase activity (a cholinergic marker) of these cells,
effects that paralleled the actions of LIF (Fig. 2 A).
Thus, CT-1 is active in modulating the phenotype of sympathetic
neurons.
Figure 2:
Activity of CT-1 in neuronal cell
assays. The induction by mouse ( m) or rat ( r)
cytokines was performed as described under ``Materials and
Methods.'' A, the switch in transmitter phenotype of rat
sympathetic neurons. Tyrosine hydroxylase ( TH) and choline
acetyltransferase ( ChAT) activities were determined in
duplicate. B, survival of rat dopaminergic neurons. Plotted
are the average and standard deviation of triplicate determinations.
C, survival of chick ciliary neurons, EC [CT-1] = 10 (± 8.2) nM,
EC
[CNTF] = 0.0074 (± 0.0049)
nM.
Parkinson's disease is caused by the degeneration of
dopaminergic neurons of the midbrain
(52) . While proteins of
the neurotrophin family (brain-derived neurotrophic factor and
neurotrophin-4/5) as well as of the transforming growth factor-
family (glial cell line-derived neurotrophic factor, transforming
growth factor-
2, and transforming growth factor-
3) promote
the survival of cultured dopaminergic neurons
(27) ; many other
growth factors and cytokines, including CNTF, do not. Unlike CNTF, CT-1
was found to promote the survival of rat dopaminergic neurons, although
it was not as potent as glial cell line-derived neurotrophic factor
(Fig. 2 B).
Embryonic Development Assay
The presence or
absence of soluble factors plays a key role during embryonic and fetal
development. For example, embryonic stem cells require the continuous
presence of soluble factors secreted by fibroblasts to maintain their
undifferentiated, pluripotent phenotype. LIF
(54, 55) ,
CNTF
(56) , and OSM
(57) (but not IL-6 without the
soluble IL-6 receptor
(58) ) can replace these
fibroblast-derived factors in maintaining the pluripotent phenotype of
embryonic stem cells in culture. CT-1 was also found to inhibit the
differentiation of mouse embryonic stem cells (Fig. 3); it was as
effective as LIF at the concentrations tested.
Figure 3:
Activity of CT-1 in embryonic stem cell
development. Mouse embryonic stem cells were cultured in the presence
of the mouse ( m) cytokines as described under ``Materials
and Methods.''
Thus, the data from
seven in vitro biological assays indicate that CT-1 is active
in assays where LIF is active and vice versa. These data also
show that CT-1 is active in assays where CNTF is active but that the
converse is not always the case and that CT-1 is inactive in IL-6
specific assays, assays in which LIF is also inactive. Since the
activity profiles of members of this cytokine family are determined by
the receptors expressed on target cell populations, these data are
consistent with the hypothesis that CT-1 binds and transduces its
biological effects via the LIF receptor.
CT-1 Binding to M1 Cells
In order to show directly
that CT-1 functions via the LIF receptor, binding was performed on M1
cells, where LIF binding has been previously characterized
(59) . Both CT-1 and LIF inhibit the growth of this cell line
(see above). Labeled CT-1 was specifically bound to M1 cells
(Fig. 4 A), and this binding was completely competed by
unlabeled LIF (Fig. 4 B). Similarly, labeled LIF binding
was competed by both unlabeled LIF and CT-1 (Fig. 4, C and D). These data suggest that CT-1 and LIF bind to the
same receptor on M1 cells. Scatchard analysis yields a single class of
binding sites in all cases; the binding parameters are similar
regardless of the labeled ligand ( K[CT-1]
0.7 nM, K
[LIF]
0.2 nM, and
1500
sites/cell).
Figure 4:
Binding and cross-competition of CT-1 and
LIF to mouse M1 cells. Assays contained 0.047 nMI-labeled mouse CT-1 (
I-mCT-1) and
unlabeled mouse ( m) CT-1 ( A), or unlabeled LIF
( B); or 0.042 nM
I-labeled mouse LIF
(
I-mLIF) and unlabeled CT-1 ( C), or LIF
( D). Shown are competition and Scatchard ( inset)
plots of the data. For the labeled CT-1 binding, K [CT-1] = 0.61 (± 0.11) nM, 1500
(± 220) sites/cell; K [LIF] = 0.19
(± 0.05) nM, 1800 (± 150) sites/cell. For
labeled LIF binding, K [CT-1] = 0.83
(± 0.13) nM, 1300 (± 80) sites/cell; K [LIF] = 0.26 (± 0.10) nM, 1200
(± 300) sites/cell.
Cross-linking of CT-1 on M1 Cells
To analyze the
protein(s) that bind CT-1 on the cell surface, labeled CT-1 and LIF
were bound to M1 cells and chemically cross-linked, and the solubilized
proteins were analyzed by SDS-gel electrophoresis (Fig. 5). Both
ligands gave one specific band with a mobility of 200 kDa, and in
both cases this cross-linked band was competed by either unlabeled
ligand. Thus, CT-1 and LIF interact with a protein of the same size on
the surface of M1 cells; this protein has a mobility expected for the
LIF receptor
(19, 60) .
Figure 5:
Cross-linking of CT-1 and LIF to M1 Cells.
I-labeled mouse CT-1 (
I-mCT-1) or
I-mouse LIF (
I-mLIF) were bound
and cross-linked to M1 cells in the absence ( None) or presence
of a 100-fold excess of the indicated mouse ( m) cytokine, and
the reaction products analyzed by SDS-gel electrophoresis. The mobility
of molecular weight standards is indicated.
Inhibition of CT-1 Binding to M1 Cells by an Anti-gp130
Monoclonal Antibody
In order to show that gp130, the common
signaling subunit shared by all receptors for ligands of the IL-6
cytokine family, is a part of the receptor binding complex for CT-1, we
determined the effect of an anti-gp130 monoclonal antibody on CT-1
binding (Fig. 6 A). This neutralizing antibody inhibited
over 80% of the specific CT-1 binding to M1 cells; no inhibition was
found with comparable concentrations of a control antibody. These data
indicate that gp130 is a component of the CT-1 receptor complex.
Figure 6:
A, inhibition of CT-1 binding to M1 cells
by an anti-gp130 monoclonal antibody. Assays contained 0.12 nMI-labeled mouse CT-1 and antibodies as indicated. For the
anti-gp130 antibody, EC
= 44 (± 8)
nM. B, electrophoretic mobility shift of the DNA
element SIE induced by CT-1 binding to M1 cells. M1 cells were
incubated without (-) or with (+) 5 nM mouse
( m) CT-1 or LIF and lysed, and the cell extract was assayed
for binding to the DNA element SIE as described under ``Materials
and Methods.'' Binding specificity was determined by the addition
of unlabeled SIE DNA ( Cold Oligo). The specific DNA complex is
indicated ( arrow).
CT-1 Induction of DNA Binding Activity in M1
Cells
To show that CT-1 induces intracellular signaling events
like those found for other cytokines that signal via gp130
(21, 22, 23, 24, 25, 26) ,
we performed DNA mobility shift assays with cell extracts from M1 cells
(Fig. 6 B). CT-1, like LIF, induced a shift in the
mobility of the DNA element, SIE. Addition of the unlabeled element
showed that the shifted band was specific. Thus, CT-1 induces the
activation of a DNA binding activity like that expected for signaling
via gp130 and activation of the Jak/STAT pathway.
CT-1 Binding to Cardiac Myocytes
The binding of
labeled CT-1 and LIF was also determined for rat cardiac myocytes, the
cells used for the original assay and isolation of CT-1
(7) .
Both ligands specifically bound and cross-competed for binding to these
cells (Fig. 7), as was the case for M1 cells (Fig. 4).
These data suggest that CT-1 and LIF bind and induce cardiac myocyte
hypertrophy via the LIF receptor.
Figure 7:
Binding and cross-competition of CT-1 and
LIF to rat primary cardiac myocytes. Duplicate assays contained either
0.047 nMI-labeled mouse CT-1
(
I-mCT-1) or 0.042 nM
I-labeled mouse LIF (
I-mLIF) and
unlabeled mouse ( m) CT-1 or LIF as
indicated.
CT-1 Binding to the Soluble LIF Receptor
In order
to clarify whether CT-1 can bind directly to the LIF receptor or gp130
without the need for an additional membrane-bound component (as is the
case for CNTF), we performed binding experiments with purified, soluble
forms of the mouse LIF receptor and gp130 expressed as their
extracellular domains containing a carboxyl-terminal histidine tag.
Such experiments have recently shown that OSM binds directly to soluble
gp130 ( K 44 nM for the
human proteins)
(61) . On the other hand, LIF binds directly to
the LIF binding protein, a naturally occurring soluble form of the LIF
receptor ( K
2 nM for the
mouse proteins)
(48, 62) . The soluble mouse LIF
receptor and gp130 were expressed in mammalian cells, purified by
Ni
chelate chromatography, and judged to be at least
90% pure by SDS-gel electrophoresis (data not shown). Binding
experiments with labeled CT-1 show that it specifically binds to the
soluble LIF receptor (Fig. 8 A), as does labeled LIF
(data not shown). CT-1 failed to bind to soluble gp130 at gp130
concentrations as high as 350 nM (Fig. 8 B). The
binding of CT-1 to the soluble LIF receptor was enhanced by the
addition of soluble gp130 (Fig. 8 C), suggesting that
CT-1, soluble LIF receptor, and soluble gp130 form a tripartite complex
as would be expected for the CT-1 activation of the LIF receptor
complex. Competition binding experiments show that CT-1 binds to the
soluble LIF receptor with a reasonable affinity,
K
= 1.9 nM
(Fig. 8 D). This affinity is about the same as that found
for the binding of LIF ( K
= 1.5
nM, data not shown) and is the same as that found previously
for LIF binding to the naturally occurring form of the soluble LIF
receptor ( K
= 1-4
nM(48) ). These data demonstrate that CT-1 interacts
directly with the soluble LIF receptor without the need for an
additional binding component. The results suggest that CT-1 (like LIF)
binds first with a relatively low affinity to the LIF receptor on the
cell membrane and then forms a heterotrimeric complex with a higher
apparent affinity upon interaction with gp130.
Figure 8:
Binding of CT-1 to purified, soluble LIF
receptor and gp130. A-C, percent binding of
I-labeled mouse CT-1 (0.089 nM) to soluble mouse
LIF receptor ( smLIFR) and soluble mouse gp130
( smgp130) in the absence (-) or presence (+) of 164
nM unlabeled mouse CT-1 ( mCT-1). A, binding
to increasing concentrations of soluble LIF receptor alone; B,
binding to increasing concentrations of soluble gp130 alone;
C, binding at one soluble LIF receptor concentration with
increasing concentrations of soluble gp130. Plotted is the average and
half of the difference of duplicate determinations. The results for
0.84 nM soluble LIF receptor are shown twice for clarity.
D, competition binding of
I-labeled mouse CT-1
(0.089 nM) to the soluble LIF receptor (2.8 nM) with
increasing concentrations of unlabeled CT-1. K [CT-1] = 1.9 (± 0.2)
nM.
(
)
and inhibit the
differentiation of embryonic stem cells
(56) . On the other
hand, CT-1 is active in several assays where CNTF is inactive
(inhibition of M1 cell growth (CNTF activity requires the inclusion of
soluble CNTF receptor
(64) ), promotion of dopaminergic neuron
survival, and induction of cardiac myocyte hypertrophy
(7) ).
CT-1 is inactive, as are LIF and CNTF
(64, 65) , in the
stimulation B9 cell growth, an assay that is relatively specific for
IL-6.
helices
(7, 67) . The individual family members
are more related across species. The human and mouse sequences for
CT-1, LIF, CNTF, or IL-11 are 79-88% identical
(Fig. 9 A); the IL-6 homologues are 41% identical. Some
uncertainty remains as to whether the chick protein, identified as GPA,
is the avian homologue of CNTF or another family member for which no
mammalian homologue has yet been identified
(69, 70) .
CT-1 does not appear to be the mammalian homologue of GPA, as chicken
GPA is more similar in amino sequence to mouse CNTF than to mouse CT-1
(46% versus 26% identity, Fig. 9 A). On the other hand,
there are similarities among CT-1, CNTF, and GPA (all lack a
conventional amino-terminal, secretion signal sequence). Interestingly,
CT-1 and GPA appear to be secreted from cells while CNTF is not
(7, 69, 71, 72) .
Figure 9:
Similarity of IL-6 family ligands and
subunit structure of their receptors. A, percent amino acid
identity of the mature form of the IL-6 family ligands; ( m)
mouse, ( h) human, ( c) chicken. The bottomrow gives the percent identity of the cytokine to its
human homologue. Shown in boldface are the percentages greater
than 40%. B, diagram of the IL-6 family receptors. The subunit
stoichiometry of the various complexes is not known in most cases,
although recent work has led to a conclusion that the IL-6 receptor
complex is a hexamer containing two IL-6 molecules, two IL-6 receptors,
and two gp130 signaling subunits (73).
As is shown
diagramatically in Fig. 9 B, the receptors for cytokines
of the IL-6 family are composed of related subunits, some of which are
cytokine-specific and some of which are shared
(14, 15, 16, 18) . All of the receptors
in this family have in common the transmembrane signaling subunit,
gp130. The binding of IL-6 to the 80-kDa IL-6 receptor subunit
leads to the dimerization of gp130 as the first step in signal
transduction. Similarly, the binding of IL-11 to the IL-11 receptor
also leads to gp130 dimerization. LIF, OSM, and CNTF induce the
heterodimerization of gp130 with another signaling subunit, the LIF
receptor. LIF and OSM bind directly to the LIF receptor or gp130 and
induce dimerization without a ligand-specific
subunit, while CNTF
binds first to the GPI-linked CNTF receptor. While the formation of
receptor complexes containing homo- or heterodimers of gp130 is
believed to be an essential signaling event, the exact stoichiometry of
the subunits in the complex is not known in most cases. For the IL-6
receptor, a recent report concludes that the signaling complex is a
hexamer containing two 20-kDa ligands, two 80-kDa IL-6 receptors, and
two 130-kDa gp130 molecules
(73) . The ligand-induced
dimerization of gp130 or gp130 and LIF receptor leads to the tyrosine
phosphorylation and activation of associated tyrosine kinases of the
Jak family (Jak1, Jak2, and Tyk2) followed by the activation of
transcription factors of the STAT family (STAT1 and STAT3)
(21, 22, 23, 24, 25, 26) .
Activation of the Jak-STAT pathway is probably one of the key steps in
the signal transduction mechanism for most if not all of the actions of
the IL-6 family cytokines.
subunit, LIF is active on M1 cells because both gp130
and LIF receptor are present, while CNTF is inactive due to a lack of
CNTF receptor
, etc. The profile of CT-1 activities reported here
suggests that this cytokine can function via the LIF receptor. In order
to establish directly that this is the case, we first show that CT-1
and LIF completely cross-compete for binding to M1 cells, a cell line
where LIF binding has been previously well characterized,
K
[LIF] = 0.1-0.2
nM(59, 74) . Regardless of which ligand is
used as the label or competitor, we find an affinity for CT-1,
K
0.7 nM that is
3-4-fold less than that found for LIF, K
0.2 nM. Secondly, cross-linking data show that
CT-1 and LIF specifically interact with a protein of
200 kDa, a
protein about the size expected for the LIF receptor
(19, 60) . Third, we show that an anti-gp130 monoclonal
antibody specifically inhibits the binding of labeled CT-1 to M1 cells,
showing that gp130 is a component of the CT-1 receptor complex. Fourth,
CT-1 induces the activation of a DNA binding activity, an intracellular
signaling event induced by LIF and other members of the IL-6 cytokine
family in the course of activation of the Jak-STAT pathway
(21, 23, 25, 26) . These data
demonstrate that CT-1 can bind to and activate the LIF receptor
complex. This finding does not exclude the possibility that some cells
have an additional CT-1-specific receptor or receptor subunit that
forms a heterodimer with gp130, as has been reported for OSM
(75) .
2 nM(60) ) but does not interact with gp130 in the absence of the
LIF receptor. Conversely, OSM binds to gp130 ( K
1 nM(76) ) but does not bind to the LIF
receptor alone
(60) . Soluble forms of these two receptor
subunits, consisting of their extracellular domains, are found in the
circulation
(62, 77) . The soluble LIF binding protein
binds LIF with a K
2 nM
(for the mouse proteins)
(48) , while a recombinant form of
soluble gp130 binds OSM with a K
44
nM (for the human proteins)
(61) . Here we show that
CT-1 binds to the soluble LIF receptor with about the same affinity as
LIF ( K
2 nM, for the mouse
proteins) and in the absence of other proteins. CT-1 does not bind to
soluble mouse gp130 even at high concentrations. The addition of
soluble gp130 does increase the binding of CT-1 to the soluble LIF
receptor, however, presumably by the formation of a heterotrimeric
complex. The concentration of soluble gp130 required for this effect
(
100 nM), while high by solution binding standards, is
readily attainable on the surface of a cell. For example, 500 molecules
of gp130 expressed on the surface of a cell of 10-µm diameter would
have an effective concentration of
300 nM in a
100-Å shell surrounding the cell, see Ref. 78. Thus, these
results indicate that CT-1 binds to the LIF receptor in the same manner
as LIF, by first binding with low affinity to the LIF receptor subunit,
an interaction that does not require additional components, and second
by recruiting gp130 to form a high affinity signaling complex.
1000-fold less potent than CNTF (Fig.
2 C). Perhaps, this reduced potency is due to a greater species
specificity of mouse CT-1 relative to rat CNTF for the chicken ciliary
neuron assay. Differential species specificity has been proposed as the
basis for the lack of activity of LIF in this system (70).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.