Table Of ContentCLF/CLC
Greg Elson
NovImmune SA, 64 Avenue de la Roseraie, 1211 Geneva 4, Switzerland
correspondingauthortel:(cid:135)41(022)3823841,fax:(cid:135)41(022)3829817,e-mail:[email protected]
DOI: 10.1006/rwcy.2001.0608.
Chapter posted 5 November 2001
SUMMARY protein is to date known only to exist as a soluble
protein (i.e. lacking any putative transmembrane or
Cardiotrophin-likecytokine(CLC),identifiedinsilico membrane-anchoring domains). Due to its similarity
by sequence homology analysis, belongs to the IL-6 with another soluble type I cytokine receptor, the
family of cytokines. Although possessing a putative IL-12 p40 subunit, it was speculated that CLF made
signal peptide, the cytokine appears to be retained up part of a functional composite cytokine resem-
within the cell unless coexpressed with the soluble bling IL-12. This heterodimeric cytokineis composed
type I cytokine receptor cytokine-like factor (CLF). of two polypeptide chains, the aforementioned
CLF and CLC associate to form a stable secreted p40 subunit and a p35 subunit, sharing homology
complex in a manner similar to the formation of the withtheIL-6familyofcytokines.IL-12cantherefore
IL-12 heterodimer. The CLF/CLC composite cyto- be regarded as a composite factor consisting of
kine acts on cells expressing the tripartite ciliary a soluble cytokine receptor stably associated with its
neurotropic factor (CNTF) receptor complex and corresponding ligand.
therefore represents the second known ligand for this CLC is a recently identified member of the IL-6
particular receptor. The contrasting phenotype of cytokine family. Other members of this family include
mice deficient in either CNTF or the ligand-binding IL-6, IL-11, leukemia inhibitory factor (LIF), onco-
nonsignaling(cid:11) chain ofthe CNTF receptor complex, statin M (OSM), ciliary neurotropic factor (CNTF)
CNTFR(cid:11), has suggested for some time the existence and cardiotrophin 1 (CT-1). These cytokines are
of a second, developmentally important ligand grouped together based on the fact that they all
for the CNTF receptor. The fact that CLF-deficient recruit the signaling gp130 subunit in their respective
mice have a phenotype similar to those deficient receptor complexes. CLC was identified from human
in CNTFR(cid:11) goes a long way towards demonstrat- cDNA sequences based on its homology to this
ing this ligand to be CLF/CLC. Due to its well- family of cytokines, most notably CT-1 and CNTF.
establishedneuroprotective effects, CNTF has shown Recombinant CLC produced in bacteria induces
promise in certain clinical trials for the treatment of tyrosine phosphorylation of both gp130 and LIFR as
neurodegenerative disorders. As both CNTF and well as components of the Jak/STAT signaling
CLF/CLC have overlapping biological properties, it pathway in the SK-N-MC neuroblastoma cell line. In
would be of great interest to examine the effects of addition, the recombinant protein was found to have
the CLF/CLC complex in similar studies. certainbiologicalpropertiescommontotheIL-6cyto-
kine family, such as the ability to induce both serum
acute phase protein production and TF-1 cell proli-
feration as well as supporting the survival of chicken
BACKGROUND
embryo neurons (Shi et al., 1999; Senaldi et al., 1999).
Following transient expression in mammalian cells,
Discovery
CLC cannot be detected in the culture medium and
appears to be retained within the cell. Similar
CLF is a type I cytokine receptor homolog identified observations have been made for two other members
by EST database screening using conserved motifs of this cytokine family, CT-1 and CNTF. Although
from this family of receptors (Elson et al., 1998). The these two cytokines do not possess putative signal
CytokineReference Copyright#2001AcademicPress
2 Greg Elson
peptides, CLC clearly does, therefore suggesting that support the survival of neurons. As this cytokine
the protein can potentially enter into the classical signals via the tripartite CNTF receptor, one can
secretory pathway. In this respect, CLC would appear predict that its functions overlap with CNTF and
to be similar to IL-12 p35, itself having a putative therefore has the potential to function on a wide
signal peptide but being poorly secreted. These range of cells in the nervous system, as well as cells
observations suggested that CLC presented itself as a of muscular origin and ES cells (reviewed in Sleeman
candidate CLF-interacting subunit. This was con- et al., 2000).
firmed following cotransfection of the two proteins The phenotype of knockout mice for CNTF,
in mammalian cells. CLC secretion was found to be CNTFR(cid:11), and CLF also provide strong evidence
dependentonitscoexpressionwithCLF.Furthermore, that unlike CNTF, CLF/CLC is an important
co-immunoprecipitation experiments revealed that survival factor for neurons during development (see
CLF and CLC were present in the culture medium In vivo biological activities).
as a stable complex, suggesting that the two proteins
formasolublecompositecytokine(Elsonetal.,2000).
GENE AND GENE REGULATION
Alternative names
CLF and CLC have gene structures typical of type I
cytokine receptors and IL-6 family cytokines respec-
CLC was independently identified as an IL-6 family tively. CLF is composed of nine exons (Elson et al.,
member by Senaldi et al. (1999), who called the 1998), whereas CLC contains three exons (Senaldi
protein novel neurotrophin-1/B cell stimulatory et al., 1999).
factor-3 (NNT-1/BSF-3). CLF is also known as
NR6 (Alexander et al., 1999).
Accession numbers
Structure The cDNAs encoding human and mouse CLF were
cloned in 1998 (Elson et al., 1998). The accession
CLF is a glycoprotein of approximately 60–65kDa, numbers are NM_004750 and NM_018827 for human
being typical of the type I cytokine receptors in that andmouserespectively.Expressedsequencetags(ESTs)
it contains an N-terminal Ig-like domain followed of bovine, rat and zebrafish origin whose derived
by a hallmark cytokine-binding domain, made up amino acid sequences share a high level of homology
oftwofibronectintypeIIIrepeats(Elsonetal.,1998). humanandmouseCLFcanalsobefoundinthepublic
Each repeat contains a highly conserved amino acid EST database (unpublished observations).
motif: the first has two cysteine doublets whilst the The human CLC cDNA sequence has accession
second has a Trp-Ser-X-Trp-Ser (W-S-X-W-S) box. number NM_013246 with the mouse cDNA being
These conserved residues are essential in maintaining NM_019952. No ESTs from species other than
the correct folding of these repeats, which are human or mouse with significant homology to CLC
implicated in ligand recognition (reviewed in Bravo can be identified to date in the public EST database
and Heath, 2000). At present only soluble forms of (unpublished observations).
CLF have been identified.
CLC is a glycoprotein of approximately 25kDa.
Chromosome location
The putative structure of CLC obtained from
modelingstudiesistypicalofthatoftheIL-6cytokine
CLF resides on human chromosome 19p12 whilst
family with a ‘long chain’ four helix bundle topo-
CLC is located on human chromosome 11q13.
logybeingpredicted(Plun-Favreauetal.,2001).These
four(cid:11)helixesare connected inanantiparallel‘up-up-
down-down’ arrangement (Bazan, 1991). Relevant linkages
Main activities and The arm of chromosome 19 where the CLF gene is
situated also contains other type I cytokine receptors,
pathophysiological roles
including the orphan receptor EBI3 (Devergne et al.,
1996)andtheIL-12receptor(cid:12)1subunit,althoughitis
Although only a limited amount of information is not known whether CLF is linked to any cytokine or
available on its functional activities, CLF/CLC can cytokine receptor genes. The CLC gene is situated in
CLF/CLC 3
proximity to that of CNTF (11q12) although the dis- Human CLC: NP_037378
tance between the twogeneshas notbeenascertained. Mouse CLC: NP_064336
Cells and tissues that express
Sequence
the gene
See Figure 1.
CLF expression is relatively widespread, with the
highest expression levels found in placenta, adult
brain,lung,stomachheart,andimmunetissuesaswell Description of protein
asinfetallung(Elsonetal.,1998andunpublishedob-
servations). CLC mRNA expression is also relatively CLF is a 60–65kDa secreted glycoprotein. Its core
widespread, with clear expression in immune tissue, polypeptide makes up approximately 39kDa with
placenta, adult heart, and adult lung coinciding with the remaining 20–25kDa accounted for by glyco-
that of CLF. Both CLF and CLC are expressed in sylation (CLF has six potential N-linked glycosyla-
murinefetalbrainandfetallungat18d.p.c.alongwith tion sites).
the receptor for CNTF (unpublished observations). CLC is a 25–30kDa glycoprotein whose core
CLFandCLCexpressioncoincidesinanumberof polypeptide makes up approximately 22.5kDa. CLC
cells lines, especially of tumor origin. Several has one N-linked glycosylation site.
glioblastoma and neuroblastoma cell lines as well as
certain breast and lung carcinomas were found to
express high levels of both CLF and CLC mRNAs Important homologies
(unpublished observations).
CLF shares significant homology with other
members of the type I cytokine receptor family,
PROTEIN
especially gp130 and other subunits involved in
receptor complexes for the IL-6 family of cytokines.
Accession numbers
CLC is most homologous to CNTF and CT-1, and
with these cytokines defines a subfamily within
Human CLF: NP_004741 the IL-6 cytokine family. Most strikingly, both CLF
Mouse CLF: NP_061297 andCLC areextremelyhomologousbetweenspecies
Figure 1 (a) Amino acid sequence for CLF represented by a human versus
mouse alignment. hCLF, human CLF; mCLF, mouse CLF. (b) Amino acid
sequence for CLC represented by a human versus mouse alignment. hCLC,
human CLC; mCLC, mouse CLC.
4 Greg Elson
(96% homology between human and mouse for Eliciting and inhibitory stimuli,
both proteins). The functional receptor for the
including exogenous and
CLF/CLC complex, CNTFR, is also very highly
homologous between species, suggesting that this endogenous stimuli
receptor/ligand pair is strongly conserved through-
out mammalian evolution. CLFmRNAexpressioncanbeupregulatedinhuman
primary fibroblast cells treated with TNF(cid:11), IL-6, or
IFN(cid:13) (Elson et al., 1998).
Posttranslational modifications
RECEPTOR UTILIZATION
Both CLF and CLC are modified by N-linked
glycosylation. In the case of CLF, it is not known
The CLF/CLC composite cytokine binds to the
whether glycosylation plays a role in its function.
tripartite CNTF cell surface receptor, comprising the
CLF has been produced and is glycosylated and
ligandbindingsubunit,CNTFR(cid:11)andtwo(cid:12)signaling
secretedfrominsectcells,althoughitsactivityhasnot
subunits, gp130 and LIFR. Target cell specificity is
been tested.
dictated by CNTFR(cid:11), which has a restricted expres-
Recombinant CLC has been shown to be active
sion pattern, being mainly confined to neural and
when derived from both mammalian cells (Elson
skeletalmusclecells.WhereasCLFisrequiredforthe
et al., 2000) and Escherichia coli (Shi et al., 1999;
secretion of CLC, it appears that its presence is not
Senaldi et al., 1999), suggesting that glycosylation
essential for receptor activation. CLC purified from
is not required for the protein to be functional.
transfected mammalian cell lysates has been found to
Itmay,however,playaroleinthecontrolofsecretion
be active on cells expressing the tripartite CNTF
of the protein, as has been shown for IL-12 p35
receptor in the absence of CLF, and with a similar
(Carra et al., 2000).
potency to the CLF/CLC complex (unpublished
observations). These data suggest that CLF does not
participate in the high-affinity interaction between
CLC and the CNTF receptor (Figure 2).
CELLULAR SOURCES AND
TISSUE EXPRESSION
IN VITRO ACTIVITIES
Cellular sources that produce
In vitro findings
Data are only available on cell lines expressing
mRNA for CLF and CLC (see Cells and tissues that Only a limited amount of data has been generated on
express the gene). theactivitiesofCLF/CLCtodate.Theresultsindicate
Figure 2 Schematic representation of the secretion and target cell receptor
complex of CLF/CLC.
CLF/CLC 5
that CLF/CLC activities overlap with those of CNTF to a suckling defect (Alexander et al., 1999). This
in vitro. CLF/CLC induces proliferation of Ba/F3 physical defect is similar to the one displayed by
cells transfected with cDNAs expressing the three CNTFR(cid:11)-deficient mice, which themselves show
subunits of the functional CNTF receptor, induced profound motor neuron deficits (DeChiara et al.,
IL-6 production in KB cells transfected with cDNA 1995). This observation, and the fact that CNTF
encoding CNTFR(cid:11), induces tyrosine phosphorylation knockout mice exhibit a phenotype only during
of gp130, LIFR, and STAT3 in the SK-N-GP adulthood (Masu et al., 1994), suggests that CLF/
neuronal stoma cell line and supports the survival of CLC plays an important role in fetal nervous system
culturedratembryonicmotorneurons.CLF/CLCwas development. The generation of CLC knockout mice
consistently found to be inactive on cells expressing and an analysis of their motor neuron populations,
gp130 and LIFR but lacking CNTFR(cid:11) (Elson et al., along with those of CLF-deficient mice, will be
2000). Like CNTF, recombinant CLC produced in required to confirm this.
E.coli supports the survival of cultured chick embryo
motorneurons andinduces gp130,LIFR, andSTAT3 Transgenic overexpression
phosphorylation in SK-N-MC neuroblastoma cells,
but more surprisingly induced the growth of M1
No data are available for CLF/CLC, although
myeloidleukemiacells(Senaldietal.,1999).Thislatter
injection in mice of recombinant CLC produced in
property cannotbeattributedtoCNTF. In a separate
E. coli led to an increase in the circulating levels of
study, recombinant CLC from E. coli was shown to
the acute phase protein SAA, and potentiated the
induce gp130 and STAT phosphorylation in SK-N-
induction of both serum cortiocosterone and IL-6
MC. CLC also activated TF-1 reporter cell lines (Shi
levels by IL-1. In addition, prolonged treatment
et al., 1999). CNTF is typically inactive on TF-1 cells.
withCLCresultedinan8%reductioninbodyweight
The reason for the differences in activity between
and caused important differences in secondary
recombinant CLC produced in E. coli and CNTF has
lymphoid organs, in particular on B cell populations.
yet to be determined.
Endogenous inhibitors and
Regulatory molecules: Inhibitors
enhancers
and enhancers
Although no in vivo data exist, in vitro data suggest
No natural inhibitors or enhancers have been that a naturally occurring soluble form of CNTFR(cid:11)
reported for CLF/CLC. Curiously, unlike for isunlikelytoactasanenhancerofCLF/CLCactivity
CNTF, soluble CNTFR(cid:11) is unable to confer CLF/ (Plun-Favreau et al., 2001). Naturally occurring
CLC responsiveness to cells expressing gp130 and soluble forms of gp130 and LIFR are potential
LIFRbutlackingmembrane-boundCNTFR(cid:11)(Plun- inhibitors of CLF/CLC activity, although this
Favreauetal.,2001).Thereasonforthisisatpresent remains to be demonstrated.
unclear.
IN THERAPY
IN VIVO BIOLOGICAL
ACTIVITIES OF LIGANDS IN Due to its overlapping functional properties with
CNTF,CLF/CLChasthepotentialtobeoftherapeu-
ANIMAL MODELS
tic benefit for several diseases. As a result of its well-
characterizedneuroprotectiveeffects,CNTFhasbeen
Normal physiological roles
tested in preclinical and clinical studies for neuro-
degenerative disorders such as amyotrophic lateral
Although no formal data exist at present, it is sclerosis and Huntington’s disease. Further, it is
suspected that CLF/CLC functions as a motor currently being clinically tested in obesity due to its
neuron survival factor during fetal development. abilitytoinduceweightloss(seeCNTF,Intherapy).
Knockout mouse phenotypes ACKNOWLEDGEMENTS
Although no data are available on CLC knockout Dr Hugues Gascan and the INSERM E 9928 unit in
mice, those deficient in CLF die soon after birth due Angers, France as well as Dr Jean-Franc¸ois Gauchat
6 Greg Elson
attheCentred’ImmunologiePierreFabre,St. Julien- Gauchat, J. F. (1998). Cytokine-like factor-1, a novel soluble
en-Genevois, France for their significant contribu- protein,shareshomologywithmembersofthecytokinetypeI
receptorfamily.J.Immunol.161,1371–1379.
tion towards the characterization of the CLF/CLC
Elson,G.C.,Lelievre,E.,Guillet,C.,Chevalier,S.,Plun-Favreau,
heterodimer.
H., Froger, J., Suard, I., de Coignac, A. B., Delneste, Y.,
Bonnefoy, J. Y., Gauchat, J. F., and Gascan, H. (2000). CLF
associateswithCLCtoformafunctionalheteromericligandfor
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