CT-1

14 Pages·2.434 MB·English

by  Chien K.R.

CT-1
Kenneth R. Chien*
Department of Medicine, Center for Molecular Genetics, University of California, San Diego,
School of Medicine, 9500 Gilman Drive, La Jolla, CA 92093, USA
*corresponding author tel: [phone], fax: [phone], e-mail: [email protected]
DOI: 10.1006/rwcy.2000.06006.
SUMMARY cell hypertrophy assay led to the isolation of clones
encoding the novel cytokine, CT-1.
Althoughcardiotropin1(CT-1)wasisolatedusingan
Structure
in vitro assay system for cardiac cell hypertrophy, the
expression pattern of CT-1 and pleiotropic activities
suggestthatitmayhaveimportantfunctionsnotonly Sequence similarity data and structural considera-
in the cardiac context, but in extracardiac tissues as tions suggested that CT-1 is a novel member of the
well.TheanalysisofCT-1knockoutmicemaygiveus IL-6 family of cytokines. The members of the IL-6
further insights into its role in vivo. cytokine family are distantly related with regard to
their primary amino acid sequence (14–24% amino
acid identity), and are predicted to share a common
four helix bundle topology.
BACKGROUND
Main activities and
Discovery pathophysiological roles
Theinitial characterizationofCT-1 was based on the CT-1 activates a distinct form of cardiac muscle cell
development of an in vitro model system of cardiac hypertrophy from the phenotype seen after (cid:11)-
muscle cell hypertrophy (Pennica et al., 1995a). As a adrenergic stimulation through the shared signaling
basis for the isolation and characterization of novel subunit, gp130. In addition to its hypertrophic
hypertrophic stimuli, a miniaturized high-throughput activity, it also enhances survival of cardiomyocyte
hypertrophy assay system was developed in neonatal and different neuronal populations (see reviews by
rat myocardial cells. In the initial search for novel Pennica et al., 1996b; Wollert and Chien, 1997).
sourcesofcytokinesthatwouldactivatecardiacmyo-
cyte hypertrophy, an in vitro model of embryonic
GENE AND GENE REGULATION
stem (ES) cell cardiogenesis was utilized. These toti-
potent stem cells can differentiate into multicellular
Accession numbers
cystic embryoid bodies (EBs). Since these EBs spon-
taneously beat and display cardiac-specific markers,
it has been suggested that they may serve as a vital Mouse cDNA: GenBank U18366 (Pennica et al.,
sourceofnovelfactorsthatcaninduceahypertrophic 1995a)
responseinvitroand/orinvivo.Inordertoidentifythe Rat cDNA: DDBJ, EMBL, and GenBank D78591
hypertrophic factor elaborated by mouse EBs, 6–7- (Ishikawa et al., 1996)
day differentiated ES cells were used to prepare a Human cDNA: GenBank U43030 (Pennica et al.,
cDNA library in a mammalian expression vector. 1996a)
Transfectionofpoolsofthislibraryinto293cellsand 50 flanking region of the human CT-1 gene: EMBL
assay of the conditioned medium in the myocardial AJ002743 (Erdmann et al., 1998)
600 Kenneth R. Chien
Chromosome location (C2/C7, Sol8, and 129CB3) CT-1 mRNA was
detected by RT-PCR (Pennica et al., 1996c). Early
in murine embryogenesis, there is preferential expres-
The chromosomal location of the human CT-1 gene
sion of CT-1 in the heart, with negative expression in
was determined by fluorescence in situ hybridization
the other embryonic tissues (Sheng et al., 1996).
(FISH) and by hybridization to genomic DNA from
Later, CT-1 expression becomes more widespread.
somatic cell hybrid lines.
Northern blotting with adult RNA reveals wide-
By FISH, two spots indicative of CT-1 hybridiza-
spread expression in a variety of cardiac and non-
tion were found on the short arm of chromosome 16.
cardiacsystems(Pennicaetal.,1995a,1996a;Ishikawa
This hybridization was localized to the region
et al., 1996; see Table 1).
16p11.1–p11.2 (Pennica et al., 1996a).
Relevant linkages
PROTEIN
The leukemia-inhibitory factor (LIF) and oncostatin
Sequence
M (OSM) genes have been mapped to chromosome
22q12, and the ciliary neurotropic factor (CNTF),
IL-6, and IL-11 genes are at 11q12, 7p21, and 19q13, See Figure 1.
showing that the human CT-1 gene is not linked to
other members of the IL-6 cytokine family.
Description of protein
Regulatory sites and corresponding
Sequence similarity data and structural considera-
transcription factors
tions suggested that CT-1 is a novel member of the
IL-6 family of cytokines. The members of the IL-6
The 50 flanking region of the human CT-1 gene has cytokine family are distantly related with regard to
been cloned. Databank research revealed several cis- their primary amino acid sequence (14–24% amino
active DNA elements (SP-1, CREB, C/EBP, AP-1- acid identity), and are predicted to share a common
likeandAP-2-like,andGATA)intheproximal1.1kb four helix bundle topology.
region (Erdmann et al., 1998).
Discussion of crystal structure
Cells and tissues that express
the gene
Analysis of the helices predicted for CT-1 based on
thesequencealignmentindicatesthattheyareamphi-
In undifferentiated myoblasts and differentiated pathic, as would be expected for a member of this
myotubes prepared from three different muscle lines family.
Table 1 Tissue distribution of RNA encoding CT-1
Mouse CT-1 mRNA (1.4kb) ++ Heart, skeletal muscle, liver, lung, kidney
+ Testis, brain
(cid:255) Spleen
Human CT-1 mRNA (1.7kb) ++ Heart, skeletal muscle, prostate, ovary
+ Lung, kidney, pancreas, thymus, testis, small intestine
(cid:255) Brain, placenta, liver, spleen, colon, peripheral blood leukocytes
Rat CT-1 mRNA (1.4kb) ++ Heart, skeletal muscle, lung, liver, stomach, urinary bladder
+ Brain, colon, testis
(cid:255) Spleen, thymus
CT-1 601
Figure 1 Alignment of mouse, human, and rat CT-1 protein sequences.
A
hCT-1 1 ...................MSRREGSLEDPQTDSSVSLLPHLEAKIRQTH
hLIF 1 MKVLAAGVVPLLLVLHWKHGAOSPLPITPVNATCAIRHPCHNNLMNQIRS
hCNTF 1 .............................MAFTEHSPLTPHRRDLCSRSI
hCT-1 32 SLAHLLTKYAEQLLQEYVQLQGDPFGLPSFSPPRLPVAGLSAPAPSHAGL
hLIF 51 QLAQLNGS-ANALFILYYTAQGEPF..PN.NLDKLCGPNVTDFPPFHANG
hCNTF 22 WLARKIRSDLTALTESYVKHQGLNKNINLDSADGMPVA....STDQWSEL
B C
hCT-1 82 PVHERLRLDAAALAALPPLLDAVCRRQAE.LNPRAPRLLRRLEDAARQAR
hLIF 97 TEKAKLVELYRIVVYLGTSLGNITRDQKI.LNPSALSLHSKLNATADILR
hCNTF 68 TEAERLQENLQAYRTFHVLLARLLEDQQVHFTPTEGDFHQAIHTLLLQVA
D
hCT-1 131 ALGAAVEALLAALGAANRGPRAEPPAATASAASATGVFPAKVLGLRVCGL
hLIF 146 GL...LSNVLCRLCSKYHVGHVD..VTYGPDTSGKDVFQKKKLGCQLLGK
hCNTF 118 AFAYQIEELMILL..EYKIPRNE.ADGMPINVGDGGLFEKKLWGLKVLQE
hCT-1 181 YREWLSRTEGDLGQLLPGGSA
hLIF 191 YKQIIAVLAQAF........................
hCNTF 165 LSQWTVRSIHDLRFISSHQRGIPARGSHYIANNKKM
Important homologies CELLULAR SOURCES AND
TISSUE EXPRESSION
TheaminoacidsequenceofCT-1hassomesimilarity
with that of LIF (24% identity) and CNTF (19% Cellular sources that produce
identity). These proteins are members of a family
including OSM, IL-6, and IL-11. Although these
Using antibodies directed against a CT-1 fusion
cytokines share biological activities and receptor
protein,ithasbeenshownthatCT-1ispredominantly
subunits, alignment of the amino acid sequence of
expressed in the primitive mouse heart tube at E8.5,
CT-1 and other members of the IL-6 cytokine family
while other tissues display a background level of ex-
reveals that they are only distantly related in primary
pression(Figure2).Withintheheart,CT-1isexpressed
sequence (15–25% identity). There is little conserva-
exclusively in the atrial and ventricular muscle seg-
tion of the cysteine residues and only a partial
ments of the heart tube, while the endocardium
maintenance of the exon–intron boundaries, but they
remains negative.Until E10.5,thehearttube remains
are predicted to have similar tertiary structures con-
the dominant site of CT-1 expression. This unique
taining four amphipathic helices. CT-1, like CNTF,
expression pattern cannot simply be explained by the
lacks a hydrophobic N-terminal secretion signal se-
fact that the heart is one of the first organs to form
quence. The individual family members are more
duringmammalianembryogenesis,sinceseveralother
conservedacrossspecies(41–88%aminoacididentify
embryonic structures, such as the neural tube, noto-
from mouse to human).
chord, and somites, are essentially negative. At later
developmental stages (post-E11.5), the myocardium
continues to express CT-1 at relatively high levels,
Posttranslational modifications
whereas most of the other organs, such as brain,
kidney,andlung,displayrelativelylowlevelsofCT-1
Purified recombinant CT-1 produced in human 293 expression (Sheng et al., 1996) (Table 2). Thus, in
cells migrated with an apparent molecular weight of contrast to other members of the IL-6 subfamily,
30kDa in western blots. It corresponds to a glycosyl- CT-1 appears to be expressed in a relatively cardiac-
ated form of the 22kDa polypeptide (Pennica et al., restricted manner at a relatively early stage of
1996c). mouse cardiogenesis. Although LIF and CT-1 share
602 Kenneth R. Chien
Figure 2 Expression of CT-1 during mouse cardiogenesis. (A) Immunofluorescence with an anti-CT-1
antibody in an E9.0 embryonic mouse heart. (B) Immunofluorescence with an anti-CT-1 antibody in an
E14.0 embryonic mouse heart. HT, heart; V, ventricle; OT, outflow tract; AV, atrioventricular cushion;
A, atrium.
CT-1 603
Table 2 Tissue distribution of immunohistochemically detectable aCT-1 in later stages of mouse organogenesis
Tissue Localization E11.5 E13.5 E15.5
Central nervous system Brain (cid:255) +/(cid:255) +
Pituitary (cid:255) (cid:255) (cid:255)
Choroid plexus ND (cid:255) +
Peripheral nervous system Spinal cord (cid:255) (cid:255) +
Dorsal root ganglia ++ ++ +++
Thymus gland (cid:255) (cid:255) (cid:255)
Esophagus ND + ++
Cartilage ++ ++ +++
Tongue + +++ +++
Heart Atrial +++ +++ +++
Ventricle +++ +++ +++
Cushion tissues (cid:255) (cid:255) (cid:255)
Outflow tract (cid:255) (cid:255) (cid:255)
Arterial vasculature Smooth muscle + + +
Skeletal muscle + ++ +++
Adrenal ND ++ +++
Kidney ND ND +
Liver (cid:255) + +++
Skin + ++ +++
Lung Epithelial cells (cid:255) (cid:255) (cid:255)
Smooth muscle cells +/(cid:255) + +
Intestine Mucosal epithelium (cid:255) (cid:255) +/(cid:255)
Smooth muscle cells + + +
Testes ND ND +
E(cid:136)Embryoageindays;ND(cid:136)notdetermined.
overlapping in vitro biological effect on cardiac hypertrophic phenotype observed following G
muscle cells, LIF is expressed at high levels only in protein-dependent stimulation with (cid:11)-adrenergic
the uterus and weakly in other tissues, including the agonist (phenylephrine, Phe), endothelin-1 (ET-1),
myocardium. and angiotensin II (AngII) (Wollert et al., 1996). On
For a cellular source, CT-1 is detected in the asingle-celllevel,heterotrimericGprotein-dependent
conditioned medium of EB and the differentiated pathways induce a form of hypertrophy with a rela-
C2/C7 myotubes (Pennica et al., 1996c). tively of new myofibrils in parallel. In contrast, CT-1
inducesanincreaseinmyocytesizecharacterizedbya
marked increase in cell length, but little or no change
IN VITRO ACTIVITIES
in cell width.
To characterize the effects of gp130-dependent
In vitro findings
stimulation on the myofibrillar cytoarchitecture,
cardiomyocytesweredual-stainedforthick((cid:11)myosin
Cardiomyocyte: CT-1 Induced Myocardial Cell
heavy chain) and thin (F-actin) myofilaments, and
Hypertrophy
viewedbyconfocallasermicroscopy.Cardiomyocytes
We have provided clear evidence that CT-1-induced stimulated with CT-1 and LIF displayed a high
hypertrophic phenotype is distinct from the degree of myofibrillar organization: myofibrils were
Figure3 Sarcomericorganization.Neonatalratventricularcardiomyocyteswereplatedwith1nMCT-1,1nMLIF,100mMphenylephrine(Phe)andnoaddition(Cont.).
Cells were labeled with rhodamine phalloidine.
CT-1 605
organized in a strictly sarcomeric pattern, oriented The transfection of a MAP kinase kinase 1 (MEK1)
along the longitudinal cell axis, and extended to the dominant negative mutant cDNA into myocardial
cell periphery (Figure 3). Importantly, the increase cells blocked the antiapoptotic effects of CT-1,
in cell size and length was not accompanied by a indicating a requirement of the MAP kinase pathway
changeintheaveragesarcomerelength,stronglysug- for the survival effect of CT-1. A MEK-specific
gesting that the cell elongation in response to CT-1 inhibitor (PD098059) is capable of blocking the acti-
results from an addition of new sarcomeric units in vationofMAPkinase,aswellasthesurvivaleffectof
series. CT-1. In contrast, this inhibitor did not block the
On a molecular level, gp130-dependent stimulation activationofSTAT3,nordidithaveanyeffectonthe
and (cid:11)-adrenergic stimulation result in distinct pat- hypertrophic response elicited following stimulation
terns of embryonic gene myosin light chain 2v of CT-1. Therefore, CT-1 promotes cardiac myocyte
(MLC2v), and immediate-early gene expression. The survival via the activation of an anti-apoptotic sig-
reactivation of an embryonic pattern of gene expres- naling pathway that requires MAP kinases, whereas
sion is a central feature of cardiomyocyte hypertro- the hypertrophy induced by CT-1 may be mediated
phy. Members of the embryonic gene program, such by alternative pathways, e.g. JAK kinase/STAT or
as atrial natriuretic peptide (ANP) and skeletal (cid:11)- MEK kinase/c-Jun N-terminal protein kinase.
actin, are abundantly expressed in the ventricular With regard to the downstream CT-1 inducing the
myocardium during embryonic development, but target that mediates antiapoptotic events, the treat-
their expression is downregulated shortly after birth. ment of cultured neonatal cardiomyocytes with CT-1
Stimulation of cardiomyocytes with CT-1 induced induces the enhanced synthesis of the heat shock
ANP and brain natriuretic peptide (BNP) gene proteins hsp70 and hsp90, with hsp70 levels being
expression (Kuwahara et al., 1998). However, in con- enhanced 3-fold and hsp90 levels being enhanced
trastto(cid:11)-adrenergicstimulation,CT-1didnotinduce 7-fold. Such CT-1-treated cells are protected against
skeletal (cid:11)-actin expression. Growth factors, signaling subsequent exposure to severe thermal or ischemic
through G protein-coupled receptors, including (cid:11)- stress (Stephanou et al., 1998).
adrenergic agonists, ET-1, and AngII, induce ANP,
BNP, and skeletal (cid:11)-actin in a coordinate fashion. A
HepG2 and H35 (Hepatocyte-derived Cell Line)
recent study compared the expression pattern of
distinct members of the embryonic gene program in CT-1 elicits a dose-dependent induction of protein
pressure overload versus volume overload hypertro- synthesis in primary rathepatocytes, with effective
phy in vivo in the rat myocardium. As shown prev- concentrations ranging from 0.1 to 100ng/mL
iously, pressure overload resulted in the coordinate (Richards et al., 1996). Production of a number of
induction of ANP and skeletal (cid:11)-actin. However, acute-phase proteins, including haptoglobin, fibrino-
volume overload hypertrophy was associated with a gen, (cid:11) -acid glycoprotein, (cid:11) -macroglobulin, (cid:11) -
1 2 1
selective increase in ANP expression, and no induc- cysteine proteinase inhibitor ((cid:11) -CPI), (cid:11) -proteinase
1 1
tionofskeletal(cid:11)-actin,suggestingthattheregulation inhibitor((cid:11) -Pi),wasmarkedlyincreasedat48and72
1
of distinct embryonic genes in vivo is related to the hours of cytokine stimulation (Peters et al., 1995). In
hypertrophicstimulus.Thepatternofembryonicgene rat H35 cells, CT-1 stimulated (cid:11) -Pi and (cid:11) -CPI
1 1
expression inducedby CT-1 incardiomyocyteculture protein production and upregulated (cid:11) -CPI mRNA
1
therefore resembles the pattern observed in volume levels with similar potency. These results show that
overload hypertrophy. CT-1isastrongacute-phasemediatorforrathepato-
cytes in vitro.
Cardiomyocyte: CT-1 Promotes Cardiac Neuronal Cell
Myocyte Survival
TheabilityofCT-1toinducethephenotypicswitchin
Recent studies have demonstrated that CT-1 is also neuronsfromnoradrenergictocholinergic–achange
able markedly to promote the survival of either that is accompanied by the induction of several neu-
embryonic or neonatal cardiac myocytes at subnano- ropeptides, including substance P, somatostatin, and
molar concentrations (Sheng et al., 1997) (Figure 4). vasoactive intestinal polypeptide in the transmitter
To explore the potential downstream pathways that phenotype – was determined with cultured rat sym-
might be responsible for this effect, we documented pathetic neurons. CT-1 inhibited the tyrosine hydro-
that CT-1 activated both signal transducer and xylase activity (a noradrenergic marker) and slightly
activator of transcription 3 (STAT3)- and mitogen- stimulated the choline acetyltransferase activity (a
activatedprotein (MAP) kinase-dependentpathways. cholinergic marker) of these cells, effects that
Figure4 InhibitionofapoptosisincardiacmyocytesbyCT-1after5daysofserumdeprivation.A–C,inthepresenceofCT-1;D–F,intheabsenceofCT-1.AandD,
stainedwithMLC-2Vantibody.BandE,TUNEL-stainedmyocytes.CandF,nuclearstainingwithHoescht33258dye.Arrowsshowcellswithevidenceofapoptosis,
including chromatin condensation and nuclear fragmentation.
CT-1 607
paralleled the actions of LIF. Thus, CT-1 is active in Regulatory molecules: inhibitors
modulating the phenotype of sympathetic neurons
and enhancers
(Pennica et al., 1995b).
Using the possibilities for long-term analysis,
motoneurons were cultured in the presence and The introduction of mutations into human LIF that
absence of CT-1 for periods up to 16 days in vitro reduced the affinity for gp130 while retaining affinity
(Pennica et al., 1996c). In the presence of CT-1, for LIFR has generated antagonists for LIF. In the
motoneurons developed rapidly in culture and after recent study by Vernallis et al. (1997), a LIF antag-
3 days had developed long axons and multipolar onistthatwasfreeofdetectableagonisticactivitywas
morphology. After longer periods in the presence of tested for antagonism against the family of LIFR
CT-1, morphological development of motoneurons ligands. On cells that express LIFR and gp130, all
was even more pronounced. At 9–11 days of culture, LIFR ligands including CT-1 were antagonized.
surviving neurons showed a highly multipolar mor- Ligand-triggered tyrosine phosphorylation of both
phology, with axon-like processes often several milli- LIFR and gp130 was blocked by the antagonist. The
meters in length and tapering, and displaying thick antagonist is therefore likely to work by preventing
dendrite-likeprocesseswithmanysecondarybranches. receptor oligomerization.
The theoretical age of E14 motoneurons cultured
for 11 days was postnatal day 4; their morphology
suggests that many aspects of their maturation
Bioassays used
occurred normally in culture in the presence of CT-1.
Insixindependentexperimentscountedbetween9and
In brief, ventricular cardiac myocytes were isolated
16 days of culture, the fraction of motoneurons sur-
fromneonatalratsbycollagenasedigestionandPercoll
viving in the presence of CT-1 was 43%(cid:6)1%. The
gradient purification. These cells were suspended at
corresponding value for cultures without trophic
75cells/mL in Dulbecco’s modified Eagle’s medium/
factor was 6%(cid:6)2%. In the same experiment, glial
Ham’s nutrient mixture F-12 supplemented with
cell line-derived neurotrophic factor (GDNF), the
mostpotentsurvivalfactorformotoneuronsinshort- transferrin, insulin, aprotinin, L-glutamine, penicillin,
and streptomycin and were plated in aliquots of
term culture, maintained only 24%(cid:6)6% of moto-
200mL in a 96-well plate that had been previously
neurons that initially developed in culture.
coated with supplemented DMEM/F-12 containing
Unlike CNTF, CT-1 was found to promote the
4% fetal bovine serum for 8 hours at 37(cid:14)C. After
survivalofratdopaminergicneurons,althoughitwas
culture for 24 hours, test substances (ex. CT-1) were
not as potent as GDNF (Pennica et al., 1995b).
added, and the cells were cultured for an additional
Interestingly, the synergic effect of CT-1 and
48 hours. The cells were then stained with crystal
GDNF was reported. Study on the survival of puri-
violet, and the hypertrophy was scored visually.
fied embryonic day 14.5 rat motoneurons in culture
For historical reasons, a score of 3 is given to cells
indicatesGDNFfromtheSchwanncelllineandCT-1
incubated without a hypertrophy factor; a score of
from a muscle cell line in this synergy. Their expres-
7 is for maximal hypertrophy, such as that induced
sion in the environment of the motoneuron is com-
by 0.1mM phenylephrine. The activity of CT-1 can
partmentalized: GDNF transcripts are expressed
be detected with 0.1nM (Pennica et al., 1995a)
principally in Schwann cell lines, whereas CT-1
(Table 3).
mRNA is present in myotubes. Blocking antibodies
to GDNF inhibit the trophic activity of Schwann cell
line-conditioned media by 75%, whereas CT-1
antibodies diminish the myotube-derived activity by
IN VIVO BIOLOGICAL
46%. CT-1 and GDNF actsynergistically toenhance
motoneuron survival in vitro. GDNF and CT-1, ACTIVITIES OF LIGANDS IN
therefore, are major components of the trophic sup-
ANIMAL MODELS
port provided by the Schwann and muscle cells,
respectively (Arce et al., 1998).
Normal physiological roles
Others
TheeffectsofchronicadministrationofCT-1tomice
CT-1 inhibits the growth of a mouse myeloid (0.5 or 2mg by intraperitoneal injection, twice a day
leukemia cell line, M1 and the differentiation of for 14 days) were previously reported (Jin et al.,
mouse embryonic stem cells (Pennica et al., 1995b). 1996).
608 Kenneth R. Chien
Table 3 Hypertrophy score
Test cytokine Concentration (nM) Hypertrophy
scorea
EB conditioned mediumb 3c 7
Unconditioned medium 3c 3
None 0 3
CT-1 fusion 0.05 6
0.1 5
0.25 6
0.5 6.5
1.0 7
Mouse LIF 0.05 4
0.25 5.5
2.5 6
Human IL-11 0.1 3.5
0.2 4.5
0.5 4.5
1.0 4.5
2.0 5.5
Human OSM 6.25 4.5
12.5 4.5
25 5
50 6
Mouse IL-6 50 3.5
100 3.5
Rat CNTF 25 4
100 4
Endothelin 1 5
10 5
100 5
Angiotensin II 10 3
100 3
1000 3
aAscoreof3isnohypertrophy;7ismaximalhypertrophy.
bConditionedmediumof6-to7-dayembryoidbodies.
cFoldconcentrationofthemedium.
General Observations
Effects of CT-1 on the Heart
There was no difference in body weight before and
after treatment. Mice injected with CT-1 did not A dose-dependent increase in both the heart weight
exhibit behavioral changes. andventricularweighttobodyratioswasobservedin