Human Cardiotrophin-1: Protein and Gene Structure, Biological and Binding Activities, and Chromosomal Localization

Human Cardiotrophin-1: Protein and Gene Structure, Biological and Binding Activities, and Chromosomal Localization


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HUMAN CARDIOTROPHIN-1: PROTEIN AND GENE STRUCTURE, BIOLOGICAL AND BINDING ACTIVITIES, AND CHROMOSOMAL LOCALIZATION Diane Pennica,1 Todd A. Swanson,1 Kenneth J. Shaw,1 Wun-Jing Kuang,1 Christa L. Gray,1 Barbara G. Beatty,2 William I. Wood1 Cardiotrophin-1 (CT-1) is a new member of the interleukin-6 cytokine family that was identified from a mouse embryoid body cDNA library by expression cloning. Mouse CT-1 induces features of hypertrophy in neonatal rat cardiac myocytes and binds to and activates the leukaemia inhibitory factor/gp130 receptor complex. In this work we report the isolation and characterization of cDNA and genomic clones encoding human CT-1. These clones encode a 201 amino acid protein that is 80% identical to the mouse protein. Human CT-1 produced by transfection of the cDNA clones into mammalian cells induces the hypertrophy of neonatal rat cardiac myocytes. Human and mouse CT-1 bind to the leukaemia inhibitory factor receptor on both human and mouse cell lines indicating a lack of species specificity. No binding to the human oncostatin M specific receptor was detected. A 1.7 kb CT-1 mRNA is expressed in adult human heart, skeletal muscle, ovary, colon, prostate and testis and in fetal kidney and lung. The coding region of CT-1 is contained on three exons and is located on human chromosome 16p11.1–16p11.2. © 1996 Academic Press Limited

Cardiotrophin-1 (CT-1) is a new member of the interleukin 6 (IL-6) family of cytokines that was identified by expression cloning from a mouse embryoid body cDNA library based on its ability to induce features of cardiac myocyte hypertrophy in vitro.1 Cardiac hypertrophy is an important adaptive response of the heart to an increased workload that 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.2–7 The IL-6 family of cytokines (which includes CT-1, leukaemia inhibitory factor (LIF), oncostatin M (OSM), ciliary neurotrophic factor (CNTF), IL-6 and IL-11) 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 is active in many of these systems as well.10 In the seven in vitro biological assays performed thus far, the activities of mouse CT-1 coincide with those of mouse LIF.10

From the 1Department of Molecular Biology, Genentech, Inc., 460 Point San Bruno Boulevard, South San Francisco, CA 94080, USA; 2Department of Pathology, The Hospital for Sick Children, 555 University Avenue, Toronto, Ont. M5G 1XB, Canada Correspondence to: W. I. Wood Received 6 June 1995; revised and accepted for publication 6 October 1995 © 1996 Academic Press Limited 1043-4666/96/03018317 $18.00/0 KEY WORDS: Cardiotrophin/cDNA CYTOKINE, Vol. 8, No. 3 (March), 1996: pp 183–189

CT-1 mRNA is expressed in several adult mouse tissues including heart, kidney, skeletal muscle and liver.1 The biological actions induced by cytokines of the IL-6 family are mediated by multisubunit, cell surface receptors that share a common signalling subunit, gp130.8,11,12 The subunits of these receptors are members of the cytokine/growth hormone receptor family that have conserved cysteine and tryptophan residues in the extracellular domain and that signal via the Jak/STAT signalling pathway.13,14 CT-1, LIF, OSM and CNTF induce the heterodimerization of gp130 and the 190 kDa LIF receptor,10,15 while IL-6 and IL-11 induce the homodimerization of gp130.16,17 The receptor signalling complexes for CNTF, IL-6 and IL-11 contain an additional ligand specific subunit that is apparently not required for CT-1, LIF or OSM binding.10 Recently, a separate OSM specific receptor has been identified that also forms a heterodimer with gp130.18 In this work we isolate and characterize cDNA and genomic clones encoding human CT-1 and express biologically active material. We also examine the binding of human CT-1 to the LIF receptor on human and mouse cell lines and to the human OSM specific receptor.

RESULTS AND DISCUSSION Isolation of cDNA clones encoding human CT-1 cDNA clones of human CT-1 were isolated by 183

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screening a heart cDNA library with a mouse CT-1 probe.1 The DNA sequence of these clones encodes a protein of 201 amino acids (Fig. 1A) that is 80% identical with the 203 residue mouse CT-1 (Fig. 1B). Like the mouse protein,1 human CT-1 lacks a conventional, hydrophobic, N-terminal amino acid sequence indicative of a secretion signal.19 Human CT-1 has two cysteine residues and no potential N-linked glycosylation sites while the mouse protein has one cysteine and one potential glycosylation site. The 39 untranslated region

from both the human and mouse clones contain an Aluor B1-like repeat sequence. Activity of human CT-1 expressed in mammalian cells Mouse CT-1 was identified based on its ability to induce the hypertrophy of neonatal rat cardiac myocytes in vitro.1 In this assay, hypertrophy is assessed visually and a score of 3 is given in the absence of a



Figure 1.

CT-1 sequences.

(A) Human cDNA (GenBank accession number U43030) and protein sequences. Clone includes bases 1–1018, clone hu.CT1.h6 includes bases 47–1539, and clone hu.CT1.h2 includes bases 58–692. The sequence of clone hu.CT1.h2 diverges 59 of amino acid 9 at the position of the first intron. (B) Alignment of human and mouse CT-1 protein sequences. Triangles indicate the positions of two introns.

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hypertrophic factor; a score of 7 is given for maximal hypertrophy. In order to test the biological activity of human CT-1, the coding region from one human CT-1 cDNA clone was inserted into a mammalian expression vector and transfected into human 293 cells. Assay of the serum-free conditioned medium from this transfection showed that it induced cardiac myocyte hypertrophy (score 5 5.5 at a 1:20 dilution). No activity (score 5 3) was found for conditioned medium from vector transfected cells or from cells transfected with the CT1 cDNA insert cloned in the opposite orientation. Thus, human CT-1 is active in a rat cardiomyocyte hypertrophy assay. Despite the lack of a hydrophobic aminoterminal secretion sequence, both human and mouse1 CT-1 are found in the culture medium of transfected cells. Binding of human and mouse CT-1 to human and mouse cell lines Previous work has established that mouse CT-1 binds to the LIF receptor on mouse M1 cells (Kd ~ 0.7 nM) and competes fully with mouse LIF binding in this system.10 Additional studies with purified, soluble LIF receptor and gp130 have shown that mouse CT-1 binds directly to the LIF receptor subunit in the absence of other components.10 While these findings show that mouse CT-1 binds to the LIF receptor, they do not exclude the possibility that CT-1 can function via other receptor subunits that are as yet unidentified. In order to determine whether human CT-1 binds to the mouse LIF receptor, we tested for the competition of labelled LIF binding to M1 cells by conditioned media from 293 cells transfected with the human CT-1 expression vector. This conditioned medium competed for labelled LIF binding as did purified mouse and human LIF and mouse CT-1; conditioned medium from vector transfected cells failed to compete (Fig. 2A). Thus, human CT-1, which we have shown to be biologically active in a rat cell assay, binds to the LIF receptor on mouse cells. While both mouse and human LIF bind and activate the mouse LIF receptor, it has been shown previously that mouse LIF fails to bind to the human LIF receptor.20,21 We have used Hela cells to determine whether mouse and human CT-1 can bind the human LIF receptor found on these cells. Competition for the binding of labelled human LIF to Hela cells shows the expected species specificity for the human LIF receptor—human LIF competes for this binding while mouse LIF does not (Fig. 2B). Mouse CT-1 and conditioned medium from 293 cells transfected with the human CT1 expression vector also compete for human LIF binding (Fig. 2B). Conversely, the binding of labelled mouse CT-1 is completely competed by unlabelled human LIF (data not shown). Thus, both human and mouse CT-1 bind to the human LIF receptor, and CT-1 lacks the

Figure 2. Competition for the binding of human LIF to mouse M1 or human Hela cells. 125

I-human LIF was bound in duplicate to (A) M1 cells (5 million cells per reaction) or (B) Hela cells (2.5 million cells per reaction) in the presence of the indicated competitors as described in Materials and Methods. CM, conditioned medium.

species specificity of binding found for LIF. The affinity of mouse CT-1 for the human LIF receptor was determined (Fig. 3). A single binding component was observed with an affinity (Kd ~ 0.75 nM), about equal to that for the mouse LIF receptor.10 Oncostatin M also binds and functions via the LIF receptor;22 however, for this cytokine, an additional, OSM specific, receptor has been identified and cloned from the human lung cell line, WI-26 VA4.18 In order to determine whether CT-1 binds to this OSM-specific receptor, we examined the competition of various ligands for the binding of labelled human OSM to WI-26 VA4 cells (Fig. 4). Unlabelled human OSM competes for this binding while human LIF does not, confirming the specificity of this OSM receptor. Both purified mouse CT-1 and conditioned medium from 293 cells

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Figure 3. Binding of mouse CT-1 to human Hela cells. Duplicate assays containing 0.23 nM 125I-mouse CT-1 and 9 million cells were performed as described in Materials and Methods. Shown is the competition and a Scatchard (insert) plot of the data. Kd 5 0.75 (60.15) nM, 860 (6130) sites/cell.

transfected with the human CT-1 cDNA expression vector failed to compete for labelled OSM binding (Fig. 4). Parallel, control reactions with human CT-1 conditioned medium showed complete competition for labelled OSM binding to M1 cells (data not shown). Thus, CT-1 is not a ligand for the OSM specific receptor. Expression of CT-1 mRNA in human tissues A 1.7 kb mRNA encoding CT-1 was found in about half of the adult tissues examined (Fig. 5). Higher levels were found in RNA from heart, skeletal muscle, prostate and ovary. Lower levels were observed from lung, kidney, pancreas, thymus, testis and small intestine. Little or no expression was detected in the brain, placenta, liver, spleen, colon or peripheral blood leuko-

Figure 4. Competition for the binding of human OSM to human WI-26 cells. 125

I-human OSM was bound in duplicate to WI-26 VA4 cells (2.4 million cells per reaction) in the presence of the indicated competitors as described in Materials and Methods. CM, conditioned medium.

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cytes. This pattern is similar to that found previously in mouse tissues1 with the exception of expression in the liver, which is at best weakly positive in the human samples and strongly positive in the mouse. A strong, 1.7 kb band was also found in human fetal lung and kidney (Fig. 5). The high levels of CT-1 expression in the heart coupled with its actions on cardiac cells in vitro has led us to begin to examine the role CT-1 may play in vivo both during cardiac development and in conditions of cardiac overload. Experiments are in progress to determine the levels of CT-1 expression in animal models of heart failure and to delete the gene by homologous recombination. The expression of CT-1 in several tissues and its functioning via the LIF receptor (which is widely expressed) indicates that CT-1 may well have important functions beyond its possible role in the heart. Isolation of CT-1 genomic clones and chromosomal localization Genomic clones encoding human CT-1 were isolated from a λ library, and the regions encoding CT-1 were sequenced (GenBank accession number U43031–33) to determine the exon structure of the gene (Figure 6A). The coding region of CT-1 is contained on three exons that extend over 6–7 kbp; the coding sequence matches that of the cDNA. All of the exon/intron junctions are bounded by the expected AG/GT sequences.23 The locations of the introns in the CT-1 coding sequence are similar to those found for LIF24 and OSM,25 while IL-626 and IL-1127 contain two additional introns in the C-terminal half of the protein (Fig. 6B). CNTF28 lacks the 59 intron found in the coding regions of the other genes. While the six cytokines that signal via gp130 containing receptors are predicted

Figure 5.

Expression of CT-1 mRNA in human tissues.

Blots containing poly A1 RNA from the indicated tissues were hybridized as described in Materials and Methods. (A) Hybridization with a CT-1 probe, (B) rehybridization of the same blots with an actin probe.

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Figure 6. (A) Map of the human genomic clone containing the CT-1 gene. Shown is the Not I insert of clone pBSSK1.HuCT1.7G. The filled boxes indicate the coding regions for CT-1; the open boxes indicate the non-translated regions. (B) Diagram of the cytokines of the IL6 family. Filled boxes indicate the mature proteins; open boxes indicate the signal sequences. C indicates the cysteine residues; triangles indicate the locations of the introns.

Figure 7.

to have similar protein structures with four alpha helices1,29 their amino acid sequences are only 14–23% identical10 (data not shown) and the cysteine residues are not conserved (Fig. 6B). The chromosomal location of the human CT-1 gene was determined by fluorescence in situ hybridization (FISH) and by hybridization to genomic DNA from somatic cell hybrid lines. By FISH, two spots indicative of CT-1 hybridization were found on the short arm of chromosome 16 just above the centromere (Fig. 7A). This hybridization was localized to the region 16p11.1– 16p11.2 (Figs 7B, C). Genomic blot hybridization to DNA from 18 rodent-human somatic cell hybrid lines showed complete concordance with the presence or absence of human chromosome 16 (data not shown). Further analysis with eight lines containing portions of chromosome 16 showed that the hybridization can be localized to the 16p11-q13 region (Fig. 7D), a finding consistent with the FISH results. Thus, the human CT-1 gene is located at 16p11.1–16p11.2. The LIF and OSM genes have been mapped to chromosome 22q12,30 and the CNTF,31 IL-632 and IL-1127 genes are at 11q12, 7p21 and 19q13, showing that the human CT-1 gene is not linked to other members of the IL-6 cytokine family.

Localization of the human CT-1 gene to 16p11.1–16p11.2.

(A) Localization by FISH to normal human chromosomes. Images of the DAPI stained chromosomes (blue) and CT-1 fluorescence (FITC) hybridization (yellow) were obtained as described in Materials and Methods. The arrow indicates the location of the two CT-1 spots on the p arm of chromosome 16. (B) Enlarged view of chromosome 16. (C) Schematic ideogram of chromosome 16. (D) Localization by hybridization to genomic DNA isolated from rodent-human cell hybrids. Vertical lines show the extent of human chromosome 16 DNA present in the indicated cell hybrids.

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In conclusion, the human and mouse CT-1 cDNA clones encode a protein of about 200 amino acids that is a member of the IL-6 cytokine family. CT-1 binds and induces some if not most of its effects via the LIF receptor;10 however, unlike LIF, human and mouse CT-1 bind with a lack of species specificity. The CT-1 coding region is contained on three exons. The human gene is located on chromosome 16p11.1–16p11.2 and is not linked to other members of the IL-6 cytokine family.

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Isolation of clones containing the human CT-1 gene Human genomic clones containing the CT-1 gene were isolated from a genomic library (David Goeddel, Tularik, Inc.) in LambdaGEM-12 (Promega) using the probe and conditions described above for the cDNA cloning. The Not I insert of one positive clone was subcloned into a plasmid vector (clone pBSSK1.HuCT1.7G), mapped, and the coding regions sequenced.

Chromosomal localization

MATERIALS AND METHODS Isolation of cDNA clones Clones encoding human CT-1 were isolated by screening a λgt10 human heart cDNA library (Clontech) with a 180 bp mouse CT-1 probe extending from 19 bp 59 of amino acid 1 through amino acid 50,1 in 20% formamide, 5 3 SSC at 42oC with a final wash at 0.2 3 SSC at 55oC.33 Eleven clones were isolated from 1 million screened. The EcoR1 inserts of several of these clones were subcloned into plasmid vectors and their DNA sequence determined by dideoxy DNA sequencing.34

Expression and assay of recombinant human CT-1 To express human CT-1, the coding region from plasmid was cloned into the mammalian expression vector pRK535 to give the plasmid This plasmid, as well as the vector, pRK5, and a control plasmid with the CT-1 coding region in the reverse orientation were transfected into human 293 cells and the cells were maintained in serum-free medium for 4 days. This medium was clarified by centrifugation and assayed for cardiac myocyte hypertrophy as performed previously for mouse CT-1.1

Binding assays Mouse CT-1,1 human LIF (R & D Systems), and human OSM (R & D Systems) were iodinated with IODO-BEAD (Pierce) or lactoperoxidase methods to a specific activity of 1000–1500 Ci/mmol as previously described.10 Binding to M1 (ATCC, TIB 192), Hela (Verna Gibbs, VA Hospital San Francisco), and WI-26 VA4 (ATCC, CCL-95.1) cells was performed for 2 h at 4oC and analyzed as described.10 For the Hela cell binding, conditioned medium from the 293 cell transfections above was concentrated 10-fold (Centriprep 10, Amicon) and added at a 3-fold dilution to the binding assays. For the binding to WI-26, cells the conditioned medium was used without concentration.

Expression of CT-1 mRNA in human tissues Blots of poly A1 RNA (2 µg/lane) from human tissues (Clontech) were hybridized with a 215 bp human CT-1 probe (31 bp 59 of amino acid 1 through residue 61) and washed at 0.1 3 SSC at 55oC. Blots were rehybridized with a 75 bp synthetic probe36 from the human β actin gene.

FISH was performed as described37,38 with normal human lymphocyte metaphase chromosomes counterstained with propidium iodide and 49,6-diamidin-2-phenylindol-dihydrochloride (DAPI). Biotinylated probe was prepared by nick translation of the 21 kbp insert of the CT-1 genomic clone pBSSK1.HuCT1.7G (above) and hybridized in the presence of two competitor DNA fragments that contain highly repeated sequence (the 59 3 kbp EcoRI fragment and the central 3.8 kbp XbaI/SacI fragment of the HuCT1.7G insert, Fig. 6A). Hybridization was detected with avidin-fluorescein isothiocyanate (FITC), followed by biotinylated anti-avidin antibody and avidin-FITC. Images of metaphase preparations were captured by a thermoelectrically cooled charge coupled camera (Photometrics, Tucson, AZ). Separate images of DAPI banded chromosomes39 and of FITC targeted chromosomes were obtained and merged electronically using image analysis software (courtesy of Tim Rand and David Ward, Yale University, New Haven, CT) and pseudo coloured blue (DAPI) and yellow (FITC) as described.38 The band assignment was determined by measuring the fractional chromosome length and by analysing the banding pattern generated by the DAPI counterstained image.40,41 Localization using hybrid cell lines was performed with 5 µg of human-rodent somatic cell hybrid DNA (Coriell Cell Repositories mapping panel 1 and chromosome 16 lines as indicated). These DNAs were digested with Sac I, electrophoresed on a 0.8% agarose gel, transferred to nitrocellulose filters,33 and hybridized with the probe and conditions described above for the RNA blots.

Acknowledgements We thank Kathy King for the hypertrophy assays and Zong Mei Zhang and Teresa Scheidl for assistance with the FISH analysis. This work was supported by the Canadian Genome Analysis and Technology Program (CGAT) and by Genentech, Inc.

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