©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cardiotrophin-1
BIOLOGICAL ACTIVITIES AND BINDING TO THE LEUKEMIA INHIBITORY FACTOR RECEPTOR/gp130 SIGNALING COMPLEX (*)

Diane Pennica , Kenneth J. Shaw , Todd A. Swanson , Mark W. Moore (1), David L. Shelton (2), Kimberly A. Zioncheck (3), Arnon Rosenthal (2), Tetsuya Taga (5), Nicholas F. Paoni (4), William I. Wood (§)

From the (1) Departments of Molecular Biology, Cell Genetics, (2) Neurobiology, (3) Immunology, and (4) Cardiovascular Biology, Genentech, Inc., South San Francisco, California 94080 and the (5) Institute for Molecular and Cellular Biology, Osaka University, Osaka 565, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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[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.


INTRODUCTION

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)() , 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) .

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.


MATERIALS AND METHODS

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.

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.

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.

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.

Anti-gp130 antibody inhibition experiments were performed with a rat anti-mouse gp130 monoclonal antibody (RX435)() 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.

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 nMI-labeled mouse CT-1 or 2.2 nMI-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.

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-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.


RESULTS

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.

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).

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.

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 nMI-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 nMI-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.




DISCUSSION

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,() 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.

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 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.

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 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) .

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 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.

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.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 415-225-1221; Fax: 415-225-6127.

The abbreviations used are: CT-1, cardiotrophin-1; IL, interleukin; LIF, leukemia inhibitory factor; CNTF, ciliary neurotrophic factor; OSM, oncostatin M; GPI, glycosyl-phosphatidylinositol; GPA, growth promoting activity; STAT, signal transducers and activators of transcription; SIE, c- sis-inducible element.

M. Saito, T. Kishimoto, and T. Taga, manuscript in preparation.

While CT-1 is active in promoting the survival of ciliary neurons, it is 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).


ACKNOWLEDGEMENTS

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.


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