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J. Biol. Chem., Vol. 277, Issue 33, 29399-29405, August 16, 2002
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From the Departments of
Received for publication, April 17, 2002, and in revised form, May 30, 2002
We demonstrate a novel interaction of the
nephroblastoma overexpressed gene (NOV), a member of
the CCN gene family, with the Notch signaling pathway. NOV associates
with the epidermal growth factor-like repeats of Notch1 by the
CT (C-terminal cysteine knot) domain. The promoters of HES1
and HES5, which are the downstream transducers of Notch
signaling, were activated by NOV. Expressions of NOV and
Notch1 were concomitant in the presomitic mesoderm and
later in the myocytes and chondrocytes, suggesting their synergistic effects in mesenchymal cell differentiation. In C2/4 myogenic cells,
elevated expression of NOV led to down-regulation of MyoD and myogenin, resulting in inhibition of myotube formation. These results indicate that NOV-Notch1 association exerts a positive effect on Notch signaling and consequently suppresses myogenesis.
Nephroblastoma overexpressed gene
(NOV)1 was first
identified as an overexpressing gene in virus-induced avian
nephroblastoma (1), and its orthologues were later isolated from
Xenopus, rat, mouse, and human (1-6). NOV
belongs to the CCN gene
family,2 which is constituted
by connective tissue growth factor (CTGF), cysteine-rich 61 (Cyr61),
NOV, and other related genes (7-11).
NOV encodes a putative secretory protein with 343-357 amino
acids that contains four conserved modular domains with sequence
similarities to insulin-like growth factor-binding protein, von
Willebrand type C, and C-terminal cysteine knot (CT) domain (12). CCN
genes have not been found in invertebrates as yet, and each domain is
encoded by a separate exon, implying that the CCN genes might arise
from exon shuffling in the course of vertebrate evolution. These
modular structural domains are shared among the CCN family members,
except for WISP-2/rCOP-1, which lacks the CT
module (13), and WISP1v, which lacks the von Willebrand type C module (14). The CCN gene family and a number of related genes now
appear to constitute an emerging multigene superfamily.
Some CCN proteins have been demonstrated to possess a growth
factor-like activity and regulate cell growth and tissue formation. For
example, CTGF is a matrix-associated, heparin-binding protein that
mediates cell proliferation, migration, and adhesion. CTGF also
functions as a downstream mediator of TGF- We recently demonstrated that NOV is expressed in
Hensen's node, notochord, and the floor plate in early chick
embryogenesis (31). The expression of NOV is also detected
in the dermomyotome (28). Interestingly, the expression in the
dermomyotome coincides with that of transmembrane receptor,
Notch and its ligand, Delta. The Notch signaling
pathway plays a crucial role in cell proliferation, differentiation and
fate determination in various tissues. The expression of
Notch frequently overlaps with NOV expression.
For example, both genes are expressed in chondrocytes and are supposed to participate in cartilage formation (26, 32). NOV is also involved in vascular formation (33), and its role in response to injury
has been postulated, in which prominent up-regulation of NOV
was observed in smooth muscle cells after balloon catheter injury of
rat carotid artery (34). In this type of experimental model, remarkable
up-regulation of Notch and its ligands has also been
observed (35). In vitro studies revealed that both NOV and
Notch signaling mediate cell adhesion, suggesting their crucial roles
in tissue remodeling (34, 35). These observations encouraged us to test
the hypothesis that NOV may exert some of its function through the
Notch signaling pathway.
In this study, we show that NOV associates with the epidermal growth
factor (EGF)-like repeat region of Notch1 by its CT domain. We further
demonstrate that NOV-Notch association poses a positive effect on the
Notch signaling pathway and suppresses the differentiation of C2/4
myogenic cells. Possible mechanisms of Notch signal modification by
NOV-Notch association are discussed.
Genes--
Murine Notch1 was a gift from J. Nye (Northwestern University, Chicago). NICD
(Notch-d4) was a gift from E. Robey (University of
California, Berkeley). Delta1, Delta-d1, and
Serrate1 were previously described (36). The C terminus of
NOV was tagged with a Flag or an HA epitope by ligating the
EcoRI-StuI fragment of the original
NOV clone into pCMV-Tag4 (Stratagene) or
pTagHA (personal product), respectively. The restriction
digestion by StuI removes the C-terminal sequence encoding
12 amino acids that are out of the CT domain and show poor homologies
among species and the CCN family genes (see Fig. 2A).
NOV-d1, NOV-d2, NOV-d3, and NOV-d4 fragments were
amplified from the HA-tagged NOV by PCR and ligated to
pTagHA or pSecTag2 (Invitrogen) in such a manner as to have a signal sequence at their 5' ends and the HA epitope at
their 3' ends. The full-length Notch1 that was C-terminally tagged with a Flag epitope was described previously (36).
Notch-d1 was made by removing the ScaI fragment
from the full-length Notch1. Notch-d2 was made by
ligating the EcoRI-ScaI fragment of
Notch1 into the EcoRI-EcoRV site of
pCMV-Tag4. Notch-d3 was made by ligating the
ScaI fragment of Notch1 into the SmaI
site of pFLAG-CMV-1 (Sigma). Notch-d4 is the same
with that previously described as NICD (36). MyoD
was a gift from A. Koseki (Chiba University, Chiba, Japan).
Cell Culture and in Vitro Transfection--
C2/4 was provided by
S. Matsuda (Kyoto University, Kyoto, Japan). HEK293 or C2/4
cells were cultured in Dulbecco's modified Eagle's medium containing
10% fetal bovine serum (Sigma) and antibiotics. For induction of
differentiation, the culture medium was changed to Dulbecco's modified
Eagle's medium containing 2% horse serum (Sigma, lot 20K8408).
Transfection was performed with total 1 µg of DNA in 12- or 24-well
culture vessels using LipofectAMINE 2000 (Invitrogen) per the
manufacturer's instruction. C2/4-NOV and C2/4-Mock were established by
Geneticin (Invitrogen) selection.
Baculovirus Expression System--
The HA-tagged NOV
fragment was ligated into pFastBac1 (Invitrogen). Bacmid
production and transfection to High Five cells were performed
per the manufacturer's instruction. NOV protein secreted in the
culture medium was concentrated using Microcon YM10 (Millipore).
Immunoprecipitation and Western Blot Analysis--
Cells were
passively lysed in TNTC buffer (100 mM Tris-Cl, pH 7.6, 150 mM NaCl, 1% Triton X-100, 1 mM
CaCl2 and Complete protease inhibitor (Roche
Molecular Biochemicals)) 24 h after transfection. In the
experiments to test calcium dependence, TNT buffer (100 mM
Tris-Cl, pH 7.6, 150 mM NaCl, 1% Triton X-100) containing
EGTA was also used. Cell lysate was pre-absorbed to CL4B-agarose beads and incubated with anti-Flag M2 affinity gel (Sigma) at 4 °C for 1 h. The beads were washed three times with lysis buffer. 8 or 10% SDS-PAGE was conducted with SDS gel loading buffer containing 100 mM dithiothreitol. The proteins were transferred to Hybond ECL nitrocellulose membrane (Amersham Pharmacia Biotech).
Immunodetection was performed as described previously (36). The
antibodies used were peroxidase conjugated anti-HA (clone 3F10, Roche
Molecular Biochemicals), peroxidase-conjugated anti-Flag (M2, Sigma),
anti-phosphotyrosine (clone PT-66, Sigma), anti-human smooth muscle
actin (clone 1A4, Dako), anti-porcine desmin (clone DE-R-11, Dako), and
anti-rat myogenin (clone F5D, Developmental Studies Hybridoma
Bank (DSHB)). Densitometric analysis was conducted on the scanned film
image using Photoshop5.0 (Adobe). In brief, the signal intensities were calculated by integrating the blackness (0-255) of each pixel of the
band images. The values of NOV co-precipitated with Notch were divided
by the values of Notch in the cell lysates. Note that the values are
merely relative indices without any physical meaning.
Northern Blot Analysis--
RNA was extracted from the C2/4
cells cultured in the differentiation-inducing medium using Trizol
(Invitrogen). Ten micrograms of total RNA were loaded to each lane.
Standard Northern blot procedures were adopted and hybridization was
performed using [32P]dCTP-labeled NOV,
Notch1, Delta1, or MyoD cDNA fragment.
Detection and imaging of the radioactive signals were conducted using
the image plate scanner BAS2500 (Fujifilm).
Luciferase Activity Assay--
The reporter plasmids of
HES1 and HES5 promoters, HES1-luc and
HES5-luc, were gifts from R. Kageyama (Kyoto
University, Japan). Luciferase activity was measured using the
dual-luciferase reporter assay system (Promega) and the Luminosensor
AB-2200 (Atto) per the manufacturers' instructions. All experiments
were performed in triplicate, and firefly luciferase activity was
normalized by co-transfected Renilla luciferase activity
(pRL-EF, a gift from Y. Mochida, Tokyo
Medical and Dental University, Japan).
In Situ Hybridization--
In situ hybridization
using digoxygenin-labeled RNA probe was performed as described
previously (31, 37).
NOV Associates with Notch1--
To examine the possibility that
NOV participates in the Notch signaling pathway, we investigated
whether NOV associates with Notch1 or its ligands, Delta1 and
Serrate1, using an immunoprecipitation assay. The C terminus of
NOV was tagged with an HA epitope, and the C terminus of
Notch1, Delta1 and Serrate1
were each tagged with a Flag epitope. Immunoprecipitation of
Notch1 from the protein extracts of HEK293 cells co-transfected with
NOV and Notch1 recovered a complex containing NOV
(Fig. 1A). NOV protein
was not recovered in the absence of Notch1 protein (Fig.
1A). The levels of NOV in the lysates were comparable. These
results indicate that NOV associates with Notch1. We also performed
immunoprecipitation of NOV from protein extracts of the cells
co-transfected with Flag-tagged NOV and HA-tagged
Notch1. Full-length Notch1 protein was recovered from the
immunoprecipitant of NOV, which confirmed the NOV-Notch1 association
(Fig. 1B). The small fragment that represents a cleaved
Notch1 product with the transmembrane and intracellular domain (Notch
(tm+ic)) was not co-immunoprecipitated with NOV, which implies that NOV
associates with the extracellular domain of Notch1 (Fig.
1B). The associations of NOV-Delta1 or NOV-Serrate1 were
also detected but at less than 50-fold the level of NOV-Notch1
(Fig. 1A).
The CT Domain of NOV Is Required for Association with
Notch1--
Like other CCN family members, NOV consists of four
modules, each of which appears to be involved in interaction with
different molecules. To determine which module engages in the
association with Notch1, we made a series of deletion constructs of
NOV and examined their association with Notch1 (Fig.
2A). Because the wild type NOV
has a secretory character, the signal sequence of IgG was appended to
the N-terminal deletion constructs (NOV-d1, NOV-d2, and NOV-d4). Immunoprecipitation analysis
revealed that NOV-d1, which lacks the first module, and NOV-d2, which
lacks the first and the second modules, associated with Notch1, whereas NOV-d3 and NOV-d4, both of which lack the fourth module, did not associate with Notch1 (Fig. 2B). These results indicate that
the CT domain is required for association with Notch1.
NOV Binds to the EGF Motifs of Notch1--
To determine the region
of Notch1 involved in association with NOV, we created deletion
constructs of Notch1 and examined the association with NOV
(Fig. 3A). NOV associated with
Notch-d1, which lacks the 10th-36th EGF motifs (Fig. 3B).
NOV associated with Notch-d2, which comprises only the first to ninth
EGF motifs, and also with Notch-d3, which comprises only the 10th-36th
EGF motifs (Fig. 3B). The intracellular domain of Notch1
(Notch-d4) did not associate with NOV (Fig. 3B).
Densitometric analysis was performed to evaluate the amount of NOV
captured by each Notch product. As for Notch-d1, -d2, and -d3, the
amounts of the co-immunoprecipitated NOV were proportional to the
amounts of each Notch deletion product. On the other hand, the
full-length Notch1 captured NOV 10-fold more efficiently than those
artificially engineered Notch deletion products (Fig.
3B, bottom). These results indicate that NOV
tends to associate preferentially with the EGF repeat region of wild type Notch1.
NOV-Notch1 Association Is
Ca2+-independent--
Subsets of EGF domains contain
calcium ion-dependent EGF motifs (38-40). Notch has 36 EGF
motifs, of which 21 are potentially Ca2+-binding. We
examined whether the loss of Ca2+ alters the affinity of
NOV-Notch1 association. HEK293 cells co-transfected with NOV
and Notch1 were lysed with TNT lysis buffer, and
immunoprecipitation of Notch1 was performed. The immunoprecipitant was
divided into five aliquots, which were incubated for 30 min in
the TNT buffer or in TNT buffer containing 1 mM or 2 mM Ca2+ or 1 mM or 2 mM
EGTA, respectively. NOV protein that was kept captured on the beads
with Notch or was released in the supernatants was analyzed by
immunoblot. NOV·Notch1 complex was maintained in the buffer
without Ca2+, and EGTA treatment did not dissociate the
NOV·Notch1 complex, suggesting that NOV-Notch1 association is
independent of Ca2+ (Fig. 3C).
NOV Enhances HES1 and HES5 Promoter Activation via Notch Signaling
Pathway--
To investigate the biological activity of NOV on the
Notch signaling pathway, we examined the effect of NOV on the promoter activity of HES1 and HES5, which are downstream
target genes activated by the Notch signal (41, 42). We used a C2/4
cell line, a subclone of C2C12 mesenchymal cells, which retains only
myogenic potency (43, 44). Because C2/4 cells express Notch1
(see Fig. 6A), Notch signaling could occur and
regulate their differentiation. We co-transfected HES1-luc
or HES5-luc with different amounts of NOV. The
expression of NOV increased both HES1 and HES5
promoter activities in a dose-dependent manner (Fig.
4A). HES5 promoter activation was rather modest compared with HES1, but the
overall tendencies were similar. Co-transfection of the intracellular deleted form of Delta, which has a cell-autonomous dominant
negative effect on Notch signaling (36, 45-47), decreased these
HES promoter activities (Fig. 4B). NOV-d1 showed
similar activation of HES promoters, whereas NOV-d3 failed
to activate them (Fig. 4B). Administration of NOV protein
produced by a baculovirus system also led to the increase of
HES promoter activities (Fig. 4C). These results
suggest that NOV-Notch1 association through the CT domain of NOV
activates HES1 and HES5 promoters by facilitating
the Notch signaling pathway.
NOV Inhibits the Differentiation of C2/4 Myoblasts--
The
HES promoter assay indicated that NOV acts positively on the
Notch signaling pathway. In situ hybridization revealed that NOV, Notch1, and Delta1 are
co-expressed in the presomitic mesoderm (Fig.
5) although the expression patterns are
not identical. They are down-regulated with the onset of somite
segmentation, but expression is retained in the dermomyotome even in
late stage, implying their concomitant effects on myogenesis (26, 28, 33).
To elucidate the role of NOV in myogenesis, we analyzed the effects of
NOV on the differentiation of C2/4. C2/4 cells were stellate or
spindle-shaped when cultured in the serum-rich proliferating medium.
Under low serum conditions, C2/4 cells were induced to differentiate
and began to express the myogenic markers such as MyoD,
myogenin, smooth muscle actin (SMA), and desmin (Fig.
6A and data not shown). They
further transformed into fused multinuclear cells called myotubes.
Under the differentiation-inducing condition, the endogenous expression
of NOV and Notch1 were maintained but slightly
decreased (Fig. 6A). The expressions of Delta1
and MyoD were up-regulated with differentiation of C2/4
(Fig. 6A). We established the C2/4 cell lines stably
expressing NOV (C2/4-NOV) or transformed with an empty mock vector
(C2/4-Mock), and examined their differentiation ability. C2/4-NOV
exhibited a reduction in their ability to fuse into myotubes, and the
number of myotubes formed by C2/4-NOV was significantly reduced
compared with C2/4-Mock (Fig. 6, B and C). On day
4, the expression of SMA, MyoD, and myogenin was remarkably down-regulated in C2/4-NOV (Fig. 6D). Notch1
expression was not altered by NOV (Fig. 6D). These results
imply that NOV has an inhibitory effect on myogenic
differentiation.
Notch is a multimodular signal protein that has 36 EGF motifs and
Lin/Notch repeats in the extracellular region and CDC10/ankyrin repeats
and other motifs including nuclear localization signals in the
intracellular region. On ligand binding, the intracellular domain of
Notch is cleaved and translocates to the nucleus, where it associates
with the transcription repressor CBF1 and consequently up-regulates the
transcription factors such as HES1 and HES5 (42, 48-50). The Notch signaling system is supposed to be controlled by
various factors, most of which that have been identified thus far are
molecules that directly or indirectly interact with its intracellular
region and participate in the signal transduction (51). As for
molecules that modulate Notch signaling through interaction with the
extracellular region of Notch, only Drosophila Wingless and
Scabrous have currently been reported apart from the ligands (Delta,
Serrate) (52, 53). Because NOV appears to have no invertebrate
orthologues, its participation in Notch signaling is probably unique to
vertebrate development.
The CT domain is predicted to form two twisted antiparallel pairs of
A cell aggregation assay indicated that the region containing the 11th
and 12th EGF motifs of Notch is the binding site of ligands (63, 64).
The 11th and 12th EGF motifs of Notch are predicted to be
calcium-binding EGF motifs, and the associations of Notch and its
ligands have been shown to be Ca2+-dependent
(64), implying that the EGF motif conformation, which is maintained by
an interdomain linkage in the presence of Ca2+, is
important for ligand binding (39, 40, 65). NOV-Notch association was
Ca2+-independent, implying that NOV binds to the
non-calcium-binding EGF modules of Notch and may not interfere with the
Delta (or Serrate)-Notch interaction. However, these two EGF motifs are not sufficient for Notch signal stimulation. In Drosophila,
loss of the remaining extracellular sequence leads to impaired
development, and several mutants with a single amino acid substitution
in the other EGF motifs showed lethal phenotypes due to aberrant Notch function (66-69). The role of the EGF motifs other than the 11th and
12th remains unknown, but because most of these mutations seem to alter
the structure of the EGF repeats, these facts suggest that interaction
with the ligands is not only mediated by local protein structure but
also affected by the whole conformation of the EGF repeats. A subtle
change in conformation may affect the ligand-receptor interaction and
modulate the signal receptivity, which would provide a rationale for
the effect of NOV on the Notch signaling pathway. Or it may be that NOV
enhances the receptivity of Notch by facilitating its multimerization.
This explanation seems plausible, considering that NOV can also form a
homodimer as well as associate with Notch. Further analysis will be
necessary to elucidate these hypotheses.
The Notch signaling system plays an essential role in determining cell
fate, and generally its signal keeps multipotent progenitor cells at an
uncommitted state. So-called lateral inhibition, which prevents an
ectodermal precursor cell from taking neural fate in
Drosophila, is a well known example. In vertebrates, cell
culture studies demonstrated that the expression of constitutively
active Notch inhibits the differentiation of several cell lines,
i.e. neural, hematopoietic, or myogenic cells (70-73).
These results were frequently interpreted to mean that Notch signaling
is essential to maintain the progenitor cells in an immature
proliferative state. This interpretation is supported by several lines
of evidence. However, in myogenesis, an in vivo study (74)
revealed that Notch expression in myoblasts is restricted to
the postmitotic cells and that Notch signaling did not inhibit their
exit from the cell cycle, implying that the role of Notch signaling in
myogenesis is not to maintain the progenitor cells but to participate
in later differentiation events. In the present study, we used C2/4 myogenic cells, a subclone of the C2C12 mesenchymal cell line (43, 44).
Notch signaling is crucial for the suppressive regulation of C2C12
differentiation, a process that is executed through two independent
Notch signaling cascades (73). One is a cell-type-specific CBF1-dependent pathway that transactivates HES1
and suppresses myogenic transcription factors such as MyoD.
The other signaling cascade is independent of CBF1 and regulates
general steps of differentiation. We demonstrated that elevated
expression of NOV inhibits the differentiation of C2/4, with
down-regulation of MyoD and myogenin, although the level of
Notch1 expression was not altered. Our results suggest that
the positive effect of NOV on Notch signaling promotes its inhibitory
effect on myogenic differentiation via the CBF1-dependent
Notch-HES1-MyoD pathway. Although HES1 and HES5 play similar roles in
neurogenesis (75, 76), it is not clear whether HES5 is required for
myogenesis as is HES1. Modest activation of HES5
promoter in C2/4 cells might implicate its relatively small
contribution to myogenesis.
Chen et al. (61) showed that CTGF and Cyr61 bind to integrin
and activates the focal adhesion kinase (FAK) signaling cascade. We
found that NOV can cause tyrosine phosphorylation of
FAK,4 and therefore there is
a possibility that NOV can partially substitute for the function of
CTGF and activate the FAK signaling cascade. FAK plays a central role
in notochord and somite morphogenesis, mediating their boundary
formation and maintenance. Recently, Henry et al. (77)
showed that Xenopus FAK is expressed in notochord and the
notochord-somite boundary and that phosphorylated FAK protein is seen
both at the notochord-somite boundary and at intersomitic boundaries.
Ectopic activation of Notch signaling by a constitutively activated
form of Su(H)/CBF1 mRNA injection
resulted in nonsegmental expression of FAK and disrupted the normal
somite segmentation (77), suggesting that the Notch signaling pathway
may regulate the FAK expression. These results implicate another
possible cross-talk between NOV and Notch in mediating somite
segmentation through FAK signaling cascade.
In conclusion, we have demonstrated a novel interaction between the CT
domain of NOV and the EGF repeats of Notch, which positively regulates
the Notch signaling pathway and suppresses myoblast differentiation.
Low affinity bindings of NOV with the other EGF motifs have also been
observed, which raises the question of whether NOV or the other CCN
members interact with many different EGF motif-containing molecules.
The results reported in this manuscript reinforce the hypothesis (30)
that a broad spectrum of possible interacting factors may explain the
general property of the CCN members, which exhibits various and
sometimes paradoxical functions depending on the cell types and conditions.
*
This research was supported in part by the Ministry of
Education, Science, Sports and Culture, Japan, a grant-in-aid
for Encouragement of Young Scientists (to K. S.), grants from the
Ligue Nationale contre le Cancer (Comités du Cher et de l'Indre)
(to B. P.), Association pour la Recherche contre le Cancer, and
Groupement des Entreprises Fran
**
To whom correspondence should be addressed: Dept. of Molecular
Pathology, Graduate School, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549. Tel.: 81-3-5803-5452; Fax:
81-3-5803-0188; E-mail: katsube.mpa@tmd.ac.jp.
Published, JBC Papers in Press, June 5, 2002, DOI 10.1074/jbc.M203727200
2
The International CCN Society has proposed to
rename NOV as ccn3 in pursuit of a unified CCN nomenclature.
3
B. Perbal and C. L. Li, unpublished results.
4
K. Sakamoto and K.-i. Katsube, unpublished data.
The abbreviations used are:
NOV, nephroblastoma
overexpressed;
EGF, epidermal growth factor;
CT domain, C-terminal
cysteine knot domain;
HES, hairy/enhancer of split;
CTGF, connective
tissue growth factor;
Cyr, cysteine rich;
WISP, Wnt1-inducible
signaling pathway protein;
TGF-
The Nephroblastoma Overexpressed Gene (NOV/ccn3) Protein
Associates with Notch1 Extracellular Domain and Inhibits Myoblast
Differentiation via Notch Signaling Pathway*
,,,
,, and**
Molecular Pathology and
¶ Oral Surgery, Graduate School of Tokyo Medical and Dental
University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan, the
§ Laboratory for Cell Function and Dynamics, Advanced
Technology Development Center, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako-city, Saitama 351-0198, Japan, and the
Laboratoire d'Oncologie Virale et Moléculaire, UFR de
Biochimie, Université Paris 7-D. Diderot, 75005 Paris,
France
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and stimulates proliferation and extracellular matrix synthesis of fibroblasts (15,
16). Compared with CTGF, the biological activity of NOV remains poorly
understood. Overexpression of NOV inhibits the proliferation
of chicken embryonic fibroblasts, suggesting its negative properties on
cell growth. On the contrary, human NOV protein stimulates the
proliferation of fibroblasts and also induces protein tyrosine
phosphorylation, implying its positive effect on cell proliferation
(5). N-terminal truncated form of NOV can transform chicken embryonic
fibroblasts by its oncogenic activity, and aberrant expression of NOV
is associated with the development of several tumors of different
origins including Wilms' tumor, renal cell carcinoma, neuroblastoma,
glioblastoma, adrenocortical carcinoma, and musculoskeletal tumors
(17-20). During normal development, NOV is expressed in a
wide variety of tissues. The major sites of NOV expression
include the notochord, central nervous system, kidney, adrenal cortex,
muscle, and cartilage (21-28). Although a growing body of evidence
suggests that NOV plays an important role in the development of various
tissues, the mechanisms of its function remain unclear. A search has
been undertaken for molecules that interact with NOV. Two proteins were
confirmed to physically associate with NOV. One is fibulin-1C, an
extracellular matrix protein that mediates cell adhesion, which
suggests the involvement of NOV in cell adhesion signaling (29). The
other is rpb7, a subunit of RNA polymerase II, implying that NOV might regulate transcription in nucleus (30). More recently, NOV was shown to
co-localize with connexin 43, suggesting that they might also interact
and influence the formation of gap junction (18). Although the
biological activities of NOV are attributed to interactions with these
molecules to some extent, two-hybrid system analysis has indicated that
other factors may interact with
NOV3 and would likely
regulate its downstream events.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (42K):
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Fig. 1.
NOV associates with Notch1. A,
HEK293 cells were co-transfected with C-terminal HA-tagged
NOV and C-terminal Flag-tagged Notch1,
Delta1, or Serrate1. Immunoprecipitation was
performed using anti-Flag antibody (IP:Flag). NOV was
co-precipitated with Notch1. NOV was co-precipitated also with Delta1
or Serrate1, but the amounts were very low. The large fragment of
Notch1 represents the full-length product (Notch
(fl)), and the small fragment represents the cleaved product
(Notch (tm+ic)) consisting of the transmembrane
and intracellular domain. WB, Western blot. B, to
confirm the NOV-Notch1 association, C-terminal Flag-tagged
NOV and C-terminal HA-tagged Notch1 were
transfected to HEK293 cells, and immunoprecipitation was performed with
anti-Flag antibody. The locations of these tags can be seen in Figs.
2A and 3A, respectively. Notch (fl) was
co-precipitated with NOV, whereas Notch (tm+ic) was not co-precipitated
with NOV, suggesting that NOV associates with the extracellular domain
of Notch1.
View larger version (61K):
[in a new window]
Fig. 2.
The C-terminal CT domain of NOV is required
for the association with Notch1. Deletion constructs of
NOV were created, and their ability to associate with Notch1
were examined. A, schematic illustration of the
NOV deletion constructs. C-terminal HA-tagged constructs
were created from wild type NOV
(Nov(wt)). The signal sequence of IgG was added
to the N-terminal deletion constructs (Nov-d1, Nov-d2,
Nov-d4). The numbers denote the positions of the amino
acids. IGFBP, insulin-like growth factor-binding
protein-like domain; VWC, von Willebrand type C factor-like
domain; TSP1, thrombospondin type 1-like domain;
CT, C-terminal cysteine knot domain. B, HEK293
cells were co-transfected with the HA-tagged NOV constructs
and Flag-tagged Notch1. Immunoprecipitation (IP)
was performed with anti-Flag M2 antibody. NOV-d1 and NOV-d2 associated
with Notch1 but NOV-d3 and NOV-d4 did not, suggesting that the CT
domain is required for association with Notch1. WB, Western
blot.
View larger version (29K):
[in a new window]
Fig. 3.
NOV associates with the EGF repeats of
Notch1. Deletion constructs of Notch1 were created, and
their abilities to associate with NOV were examined. A,
schematic illustration of the Notch1 deletion constructs.
Notch-d1 lacks the 10th-36th EGF motifs.
Notch-d2 consists of the N-terminal region that includes the
1st-9th EGF motifs. Notch-d3 consists of the 10th-36th EGF
motifs with a signal sequence. Notch-d4 consists of the
intracellular domain and has a constitutive activity of the Notch
signal transduction. The locations of the Flag tag are shown as
filled boxes in the illustration. EGF, epidermal
growth factor-like repeats; Lin, Lin/Notch repeats;
TM, transmembrane domain; Ank, CDC10/ankyrin
repeats. B, HEK293 cells were co-transfected with the
Flag-tagged Notch1 and the HA-tagged NOV
constructs. Immunoprecipitation was performed with anti-Flag M2
antibody (IP:Flag). NOV was co-precipitated with Notch,
Notch-d1, Notch-d2, and Notch-d3 but not with Notch-d4. The
numbers at the bottom of each lane are
the results of densitometric analysis and represent the relative amount
of NOV captured by each Notch protein. WB, Western blot.
C, NOV-Notch1 association is Ca2+-independent.
The cell lysate from HEK293 cells co-transfected with the HA-tagged
NOV and the Flag-tagged Notch1 were
immunoprecipitated by anti-Flag M2 antibody in the TNT buffer. The
immuoprecipitant was divided into five aliquots, which were
incubated for 30 min at room temperature with 40 µl of the TNT
buffer, the TNT buffer containing 1-2 mM
CaCl2, or EGTA. The protein associated with the beads and
the protein in the supernatants (sup) were blotted
separately and immunodetected with anti-HA antibody. The NOV·Notch1
complexes were stable in the buffer without Ca2+ and did
not dissociate even after EGTA treatment.
View larger version (23K):
[in a new window]
Fig. 4.
NOV enhances HES1 and
HES5 promoter activities through interaction with
Notch1. A, C2/4 cells were transfected with 0.1 µg of
HES1-luc or HES5-luc, 0.005 µg of
pRL-EF, and the indicated amounts of NOV. The
total amount of plasmid was equalized with the empty mock vector.
Twenty-four hours later, the cells were lysed and assayed for
luciferase activity. HES1 and HES5 promoter
activities were increased by NOV in a
dose-dependent manner. RLU, relative luciferase
unit. B, C2/4 cells were co-transfected with 0.1 µg of
HES1-luc or HES5-luc, 0.005 µg of
pRL-EF, and 0.2 µg of the indicated plasmids, and a
luciferase activity assay was performed. Transfection with
NOV or NOV-d1 resulted in HES1 and
HES5 activation (second and third bars
from left), whereas NOV-d3 did not show
this effect (fourth bars). Co-transfection with the dominant
negative form of Delta1
(Nov+dnDl) cancelled the effect of NOV
(fifth bars). C, C2/4 cells were transfected with
0.3 µg of HES1-luc or HES5-luc and 0.005 µg
pRL-EF. Twenty-four hours later, the culture medium was
changed to serum-free Dulbecco's modified Eagle's medium containing
different amounts of the NOV protein produced and secreted by
High Five cells. After 6 h of incubation, luciferase
activity assay was conducted. Enhancement of HES1 and
HES5 promoter activities was observed. RD,
relative density.
View larger version (124K):
[in a new window]
Fig. 5.
NOV,
Notch1, and Delta1 are co-expressed
in the presomitic mesoderm. In situ hybridization with
digoxygenin-labeled RNA probe was performed on day 3 chick embryos.
NOV exhibited strong expression in notochord and presomitic
mesoderm. Notch1 and Delta1 showed a similar
expression pattern in the presomitic mesoderm. Expression in the
notochord was unique to NOV and not observed in
Notch1 and Delta1. Original magnification,
×40.
View larger version (84K):
[in a new window]
Fig. 6.
NOV suppresses the differentiation of C2/4
cells. A, expression of NOV,
Notch1, Delta1, and MyoD in C2/4 cells
under differentiation-inducing condition. C2/4 cells were cultured in
serum-starved medium, and the total RNA was extracted on days 0 (8 h),
3, and 6 for Northern hybridization. NOV and
Notch1 were slightly down-regulated, whereas
Delta1 and MyoD were up-regulated. B,
suppression of myotube formation in C2/4 cells stably expressing NOV.
C2/4 cells stably expressing NOV (C2/4-Nov) or transfected
with an empty mock vector (C2/4-Mock) were generated and
induced to differentiate. The photographs were taken 6 days after
induction of differentiation. Original magnification, ×100.
C, myotube formation was suppressed in C2/4-NOV. The numbers
of myotubes were counted on 0 (8 h), 2, 4, 6, and 8 days after
induction of differentiation. D, expression of SMA,
myogenin, desmin, Notch1, and MyoD in C2/4-NOV
and C2/4-Mock 4 days after induction of differentiation. SMA, myogenin,
and desmin are Western results. Notch1 and MyoD are Northern results.
SMA, myogenin, and MyoD were down-regulated in C2/4-NOV
compared with C2/4-Mock.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-strands with three disulfide bonds (12, 54-56). Although its amino
acid sequences are poorly conserved, the location of their cysteine
residues is well conserved, and the common structure is shared by
several growth factors such as TGF-, NGF, PDGF, and von Willebrand
factor (12, 54). The CT domain is thought to mediate protein
dimerization, and NOV, unlike CTGF, was suggested to be dimerized
by this domain (29, 57). Because some of the receptor binding
properties of TGF-, NGF, and PDGF are within variable regions of the
CT domain, it is thought that the CT domain regulates both
dimerization and receptor binding (58, 59). Our results indicated that
the CT domain of NOV is necessary for association with Notch1. Another
example that the CT domain of NOV is used for association with other
proteins is fibulin-1C, an extracellular matrix protein that mediates
cell adhesion (29). Interestingly, the binding domain of fibulin-1C
with NOV also contains six EGF motifs. Furthermore, Delta1 and
Serrate1, which have 9 and 16 EGF motifs, respectively, showed a
capability to associate with NOV, although it was much weaker than
NOV-Notch1 association. CTGF, Cyr61, and NOV interact with integrin
complex (60, 61); recently Takagi et al. (62) pointed out
that the cysteine rich region of the integrin -subunits contains
EGF-like modules. These observations raise the possibility that CCN
family members including NOV may associate with a broad range of
proteins that contain EGF motifs.
FOOTNOTES
aises dans la Lutte
centre le Cancer (GEFLUC), and grants from the Japan Space Forum
Foundation (to K. K.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
ABBREVIATIONS
, transforming growth factor ;
CDC, cell division cycle;
CBF, core binding factor;
NGF, nerve growth
factor;
PDGF, platelet-derived growth factor;
FAK, focal adhesion
kinase;
SMA, smooth muscle actin;
HA, hemagglutinin.
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  M. Monet-Lepretre, B. Bardot, B. Lemaire, V. Domenga, O. Godin, M. Dichgans, E. Tournier-Lasserve, M. Cohen-Tannoudji, H. Chabriat, and A. Joutel Distinct phenotypic and functional features of CADASIL mutations in the Notch3 ligand binding domain Brain, June 1, 2009; 132(6): 1601 - 1612. [Abstract] [Full Text] [PDF]
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 H. Meng, X. Zhang, K. D. Hankenson, and M. M. Wang Thrombospondin 2 Potentiates Notch3/Jagged1 Signaling J. Biol. Chem., March 20, 2009; 284(12): 7866 - 7874. [Abstract] [Full Text] [PDF]
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 S. Tanabe, Y. Sato, T. Suzuki, K. Suzuki, T. Nagao, and T. Yamaguchi Gene Expression Profiling of Human Mesenchymal Stem Cells for Identification of Novel Markers in Early- and Late-Stage Cell Culture J. Biochem., September 1, 2008; 144(3): 399 - 408. [Abstract] [Full Text] [PDF]
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 N. Motohashi, A. Uezumi, E. Yada, S.-i. Fukada, K. Fukushima, K. Imaizumi, Y. Miyagoe-Suzuki, and S. Takeda Muscle CD31(-) CD45(-) Side Population Cells Promote Muscle Regeneration by Stimulating Proliferation and Migration of Myoblasts Am. J. Pathol., September 1, 2008; 173(3): 781 - 791. [Abstract] [Full Text] [PDF]
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 M. J. Hayes, D. Thomas, A. Emmons, T. J. Giordano, and C. G. Kleer Genetic Changes of Wnt Pathway Genes Are Common Events in Metaplastic Carcinomas of the Breast Clin. Cancer Res., July 1, 2008; 14(13): 4038 - 4044. [Abstract] [Full Text] [PDF]
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 K. Sakamoto, Y. Tamamura, K.-i. Katsube, and A. Yamaguchi Zfp64 participates in Notch signaling and regulates differentiation in mesenchymal cells J. Cell Sci., May 15, 2008; 121(10): 1613 - 1623. [Abstract] [Full Text] [PDF]
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W. Huang, Y. Zhang, S. Varambally, A. M. Chinnaiyan, M. Banerjee, S. D. Merajver, and C. G. Kleer Inhibition of CCN6 (Wnt-1-Induced Signaling Protein 3) Down-Regulates E-Cadherin in the Breast Epithelium through Induction of Snail and ZEB1 Am. J. Pathol., April 1, 2008; 172(4): 893 - 904. [Abstract] [Full Text] [PDF]
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S. Rydziel, L. Stadmeyer, S. Zanotti, D. Durant, A. Smerdel-Ramoya, and E. Canalis Nephroblastoma Overexpressed (Nov) Inhibits Osteoblastogenesis and Causes Osteopenia J. Biol. Chem., July 6, 2007; 282(27): 19762 - 19772. [Abstract] [Full Text] [PDF]
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 R. Gupta, D. Hong, F. Iborra, S. Sarno, and T. Enver NOV (CCN3) Functions as a Regulator of Human Hematopoietic Stem or Progenitor Cells Science, April 27, 2007; 316(5824): 590 - 593. [Abstract] [Full Text] [PDF]
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 M. Monet, V. Domenga, B. Lemaire, C. Souilhol, F. Langa, C. Babinet, T. Gridley, E. Tournier-Lasserve, M. Cohen-Tannoudji, and A. Joutel The archetypal R90C CADASIL-NOTCH3 mutation retains NOTCH3 function in vivo Hum. Mol. Genet., April 15, 2007; 16(8): 982 - 992. [Abstract] [Full Text] [PDF]
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X. Li, E. Calvo, M. Cool, P. Chrobak, D. G. Kay, and P. Jolicoeur Overexpression of Notch1 Ectodomain in Myeloid Cells Induces Vascular Malformations through a Paracrine Pathway Am. J. Pathol., January 1, 2007; 170(1): 399 - 415. [Abstract] [Full Text] [PDF]
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A. Leask and D. J. Abraham All in the CCN family: essential matricellular signaling modulators emerge from the bunker J. Cell Sci., December 1, 2006; 119(23): 4803 - 4810. [Abstract] [Full Text] [PDF]
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 M. Fukunaga-Kalabis, G. Martinez, Z.-J. Liu, J. Kalabis, P. Mrass, W. Weninger, S. M. Firth, N. Planque, B. Perbal, and M. Herlyn CCN3 controls 3D spatial localization of melanocytes in the human skin through DDR1 J. Cell Biol., November 20, 2006; 175(4): 563 - 569. [Abstract] [Full Text] [PDF]
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 L. McCallum, S. Price, N. Planque, B. Perbal, A. Pierce, A. D. Whetton, and A. E. Irvine A novel mechanism for BCR-ABL action: stimulated secretion of CCN3 is involved in growth and differentiation regulation Blood, September 1, 2006; 108(5): 1716 - 1723. [Abstract] [Full Text] [PDF]
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A. Miyamoto, R. Lau, P. W. Hein, J. M. Shipley, and G. Weinmaster Microfibrillar Proteins MAGP-1 and MAGP-2 Induce Notch1 Extracellular Domain Dissociation and Receptor Activation J. Biol. Chem., April 14, 2006; 281(15): 10089 - 10097. [Abstract] [Full Text] [PDF]
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 X. Luo, L. Ding, and N. Chegini CCNs, fibulin-1C and S100A4 expression in leiomyoma and myometrium: inverse association with TGF-{beta} and regulation by TGF-{beta} in leiomyoma and myometrial smooth muscle cells Mol. Hum. Reprod., April 1, 2006; 12(4): 245 - 256. [Abstract] [Full Text] [PDF]
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 Z.-Y. Tong and D. R Brigstock Intrinsic biological activity of the thrombospondin structural homology repeat in connective tissue growth factor. J. Endocrinol., March 1, 2006; 188(3): R1 - R8. [Abstract] [Full Text] [PDF]
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 L. Armstrong, G. Saretzki, H. Peters, I. Wappler, J. Evans, N. Hole, T. von Zglinicki, and M. Lako Overexpression of Telomerase Confers Growth Advantage, Stress Resistance, and Enhanced Differentiation of ESCs Toward the Hematopoietic Lineage Stem Cells, April 1, 2005; 23(4): 516 - 529. [Abstract] [Full Text] [PDF]
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C. G. Lin, C.-C. Chen, S.-J. Leu, T. M. Grzeszkiewicz, and L. F. Lau Integrin-dependent Functions of the Angiogenic Inducer NOV (CCN3): IMPLICATION IN WOUND HEALING J. Biol. Chem., March 4, 2005; 280(9): 8229 - 8237. [Abstract] [Full Text] [PDF]
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S.-J. Leu, N. Chen, C.-C. Chen, V. Todorovic, T. Bai, V. Juric, Y. Liu, G. Yan, S. C.-T. Lam, and L. F. Lau Targeted Mutagenesis of the Angiogenic Protein CCN1 (CYR61): SELECTIVE INACTIVATION OF INTEGRIN {alpha}6{beta}1-HEPARAN SULFATE PROTEOGLYCAN CORECEPTOR-MEDIATED CELLULAR FUNCTIONS J. Biol. Chem., October 15, 2004; 279(42): 44177 - 44187. [Abstract] [Full Text] [PDF]
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N. Zamurovic, D. Cappellen, D. Rohner, and M. Susa Coordinated Activation of Notch, Wnt, and Transforming Growth Factor-{beta} Signaling Pathways in Bone Morphogenic Protein 2-induced Osteogenesis: Notch TARGET GENE Hey1 INHIBITS MINERALIZATION AND Runx2 TRANSCRIPTIONAL ACTIVITY J. Biol. Chem., September 3, 2004; 279(36): 37704 - 37715. [Abstract] [Full Text] [PDF]
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D. M. French, R. J. Kaul, A. L. D'Souza, C. W. Crowley, M. Bao, G. D. Frantz, E. H. Filvaroff, and L. Desnoyers WISP-1 Is an Osteoblastic Regulator Expressed During Skeletal Development and Fracture Repair Am. J. Pathol., September 1, 2004; 165(3): 855 - 867. [Abstract] [Full Text] [PDF]
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A. Gellhaus, X. Dong, S. Propson, K. Maass, L. Klein-Hitpass, M. Kibschull, O. Traub, K. Willecke, B. Perbal, S. J. Lye, et al. Connexin43 Interacts with NOV: A POSSIBLE MECHANISM FOR NEGATIVE REGULATION OF CELL GROWTH IN CHORIOCARCINOMA CELLS J. Biol. Chem., August 27, 2004; 279(35): 36931 - 36942. [Abstract] [Full Text] [PDF]
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R. Gao and D. R. Brigstock Connective Tissue Growth Factor (CCN2) Induces Adhesion of Rat Activated Hepatic Stellate Cells by Binding of Its C-terminal Domain to Integrin {alpha}v{beta}3 and Heparan Sulfate Proteoglycan J. Biol. Chem., March 5, 2004; 279(10): 8848 - 8855. [Abstract] [Full Text] [PDF]
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 C.-C. Chang, J.-Y. Shih, Y.-M. Jeng, J.-L. Su, B.-Z. Lin, S.-T. Chen, Y.-P. Chau, P.-C. Yang, and M.-L. Kuo Connective Tissue Growth Factor and Its Role in Lung Adenocarcinoma Invasion and Metastasis J Natl Cancer Inst, March 3, 2004; 96(5): 364 - 375. [Abstract] [Full Text] [PDF]
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C. G. Lin, S.-J. Leu, N. Chen, C. M. Tebeau, S.-X. Lin, C.-Y. Yeung, and L. F. Lau CCN3 (NOV) Is a Novel Angiogenic Regulator of the CCN Protein Family J. Biol. Chem., June 20, 2003; 278(26): 24200 - 24208. [Abstract] [Full Text] [PDF]
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 B Perbal, D R Brigstock, and L F Lau Report on the second international workshop on the CCN family of genes Mol. Pathol., April 1, 2003; 56(2): 80 - 85. [Abstract] [Full Text] [PDF]
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