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INTRODUCTION |
Cell fate determination by local cell-cell contact plays a pivotal
role in precise pattern formation during development of multicellular
organisms (1). The Notch receptors and their ligands are both cell
surface molecules conserved from worm through man, and involved in cell
fate determination of various cell lineages (1-3).
The mouse Notch1 (mNotch1) receptor is a transmembrane protein composed
of the 180-kDa extracellular and 90-kDa intracellular regions (4). Its
extracellular region contains 36 EGF1 repeats for ligand
binding and three lin-12/Notch repeats with unknown function. The
intracellular region of the Notch receptor contains the RAM domain
which interacts with a DNA-binding protein RBP-J (mammalian homologue
of Drosophila Suppresser of Hairless (Su(H)) (5-7), six
cdc10/ankyrin repeats, nuclear localization signals, OPA region, and
PEST sequence (4). Notch signal is triggered by interaction with its
ligand, the DSL family protein which includes Delta and Jagged/Serrate
in vertebrates (1, 8). All of them contain a DSL motif for binding to
Notch and tandem EGF repeats, and a short cytoplasmic domain (1, 8). There are two subfamilies of ligands: Delta and Serrate which are
expressed at different sites and time points during
Drosophila embryogenesis (9-13). Although
Serrate can compensate loss-of-function mutations of
Delta at least in part (14), it is not clear whether Delta and Serrate have identical functions in
Notch signaling. Fibroblasts expressing the mammalian homologue
(Jagged) of Serrate can inhibit differentiation of C2C12 myoblast cells
that express either endogenous Notch alone or both endogenous and
transgene-derived Notch (15, 16). However, the molecular mechanism
underlying this phenomenon is completely unknown. In addition, it is
not known whether mammalian Delta has a similar function.
The mechanism of signal transduction through the Notch receptor is
still unclear. Genetic studies of Drosophila have shown that
Notch interacts functionally with
Su(H) (17). Su(H) and RBP-J is shown to
physically interact with the RAM domain of Notch (7, 18-20). In
addition, knockout mice of Delta
/
(21),
Notch
/
(22, 23), and
RBP-J
/
(24) showed somewhat similar
phenotypes including defects in somite formation. Expression of the
truncated intracellular region (IC) of mNotch can induce
transactivation through the HES1 promoter carrying the RBP-J binding
motif (25) and suppress neurogenesis and myogenesis in mammalian
cultured cells (26, 27). More recently, the entire intracellular region
(RAMIC) as well as IC of mNotch was shown to suppress myogenic
differentiation by transactivation of genes that contain the RBP-J
binding motif in their promoters (28). Taken together with these
results, interaction of Notch with its ligand is likely to cause
proteolytic cleavage of Notch, resulting in the release of RAMIC or IC
that binds to RBP-J in the nucleus to activate genes involved in
differentiation suppression (7, 25, 29, 30). However, involvement of
RBP-J in Notch signaling was shown by using RAMIC or IC but not the
intact Notch receptor. It is critical to test whether Notch signaling
from cell surface also utilizes RBP-J because there are few reports that suggest the presence of RBP-J-independent Notch signaling pathways
(20, 31).
The muscle cell differentiation is determined by the MyoD family of
myogenic transcriptional regulators (MyoD, Myf-5, myogenin, and MRF4)
that belong to the basic helix loop helix type of DNA-binding proteins
(32). Myotube formation of C2C12 myoblast cells (33, 34) is frequently
used as a model to study regulation of myogenic differentiation. C2C12
cells generally express MyoD, myf-5, or both (35, 36). Upon
differentiation induction of C2C12 cells, a more downstream
transcription factor myogenin is up-regulated, followed by expression
of muscle structural genes such as myosin and muscle creatine kinase.
C2C12 cell differentiation is inhibited by expression of RAMIC or IC
(26, 28), which is accompanied by down-regulation of myogenin (31, 37).
However, it has been entirely unknown how Notch signaling blocks
myogenin expression and subsequently myogenic differentiation. The
facts that overexpression of a basic helix loop helix protein HES1 in
10T1/2 cells blocks differentiation into myotube (38) and that
transcriptional activity through the HES1 promoter is up-regulated by
Notch
E (25), RAMIC, or IC (28) led to speculation that Notch
signaling induces expression of HES1 which somehow suppresses
expression of myogenic transcription factors.
Here we report that Delta1-expressing myeloma cells can suppress
myogenesis of C2C12 cells first by up-regulation of HES1 mRNA,
followed by blocking expression of MyoD mRNA. Ligand-induced Notch
signaling was shown to involve RBP-J. It is, therefore, likely that the
ligand-induced Notch signaling transactivates the HES1 gene
that is regulated by RBP-J and involved in regulation of MyoD expression.
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MATERIALS AND METHODS |
Establishment of Delta1 Expressing Cell Line (D10
Cell)--
Total RNA from mouse embryo (day 11.5) was extracted with
TRIzol reagent (Life Technologies). Four pairs of specific primers were
used for PCR amplification of partial cDNA clones of mouse Delta1
(39): pair 1, 5'-GAATCTAGAATGGGCCGTCGGAGCGCGCTA-3' (forward) and
5'-GTCTGTGCGGCCGCTACTGT-3' (reverse); pair 2, 5'-ACAGTAGCGGCCGCACAGAC-3' (forward) and
5'-TCATGGCGCTCAGCTCACAGA-3' (reverse); pair 3, 5'-TCTGTGAGCTGAGCGCCATGA-3' (forward) and 5'-TCGCGCTGGCAATTGGCTAGGT-3'
(reverse); pair 4, 5'-ACCTAGCCAATTGCCAGCGCGA-3' (forward) and
5'-GCTGGATCCTCTAGATTAGCGACCCATTTGCTGTCCACCAGTCATGCTAGCCATCACCTCAGTCGCTAT-3' (reverse). The reverse primer of pair 4 contains a single gene 10 epitope tag (Novagen). The PCR products were cloned into the T-vector
(Promega) and overlapping partial cDNA clones were assembled in
pBluescript KS+. The assembled Delta1-gene 10 fragment was
subsequently subcloned into the plasmid pEF-BOS neo-derived from
pEF-BOS (40) and pMC1neo poly(A) (Stratagene) for expression in X63
myeloma cells. X63 cells were transfected with the pEF-BOS
neo/Delta1-gene 10 by electroporation and selecting in RPMI 1640 (JRH
Bioscience) containing 10% fetal bovine serum and neomycin
(0.3 mg/ml).
MNE-Rg4 Construction and Preparation--
A cDNA fragment
encoding the signal sequence of human immunoglobulin VH3-30 was
amplified with primers; 5'-ATGGAATTCACCCTGCAGCTCTGGGAGAGGAGC-3' (forward) and 5'-TTAGAGCTCACACTGGACACCTCTTAAAAGAGC-3' (reverse) (41). A
cDNA fragment encoding the extracellular region (EGF 11-12) of the
mouse Notch1 protein was amplified with primers, 5'-ATGGAGCTCGACACCACCCCTGTCAACGGCAAA-3' (forward) and
5'-CTCGGGATCCGCGCAGTGGCCATTGTGCAGACA-3' (reverse) (42). These fragments
were subcloned into the pUC19 vector containing human IgG1
constant region (43). The Notch-IgG1 fusion (MNE-Rg4)
construct was subsequently subcloned into the expression plasmid
pEF-BOS (MNE-Rg4/pEF-BOS) (40). MNE-Rg4/pEF-BOS plasmid was transfected
into COS7 cells using DEAE dextran. MNE-Rg4 chimeric protein was
purified from culture supernatants by RROSEP-A High Capacity (Bioprocessing).
Northern Blot Analysis--
Total RNA was extracted form
cultured cells using TRIzol reagent (Life Technologies). 15 or 30 µg
of total RNA were electrophoresed on a 1% agarose gel and transferred
to a nylon membrane (Hybond-N+, Amersham). cDNA probes
of MyoD (nucleotides 112 to 1162)(44), myogenin (nucleotides 513 to
1104) (45), MLC2 (nucleotides 66 to 553, GenBank U77943), and
glyceraldehyde-3-phosphate dehydrogenase (nucleotides 566 to 1016, GenBank U32599) were obtained by RT-PCR from appropriate cDNA
pools. HES-1 probe was a 1.4-kilobase pair EcoRI fragment
isolated from pSV-CMV-HES-1 (38). Hybridizations were done under
standard conditions (46).
Modulation of Myogenic Differentiation by Transfection--
The
full-length mouse RBP-J cDNA (47) was subcloned into the pCMX (48)
and pCMX-VP16 (49) vectors to generate pCMX-mRBP-J and
pCMX-VP16-mRBP-J, respectively. C2C12 cells were plated on coverslides,
transfected with either of the mRBP-J plasmids by lipofection, and
cultured in differentiation medium for 24 h. For MyoD rescue
assay, C2C12 cells were transfected with pH
APr-1 or pH
A-D(+) (45,
50) and co-cultured with D10 cells in differentiation medium for 4 days. The cells were fixed and permeabilized as described previously
(28). Cell monolayers were then incubated with anti-MyoD mouse
monoclonal antibody (51), anti-RBP-J rat monoclonal antibody (K0043)
that detects both mRBP-J and VP16-mRBP-J (52), and anti-myoglobin rabbit polyclonal antibody (Cappel). Fluorescein isothiocyanate-labeled anti-mouse IgG antibody (Cappel), TRITC-labeled anti-rat IgG antibody, and TRITC-labeled anti-rabbit IgG (Southern Biotechnology) were subsequently used for indirect fluorescence staining. Hoechst 33342 (Sigma) was used for nuclear staining. Slides were mounted in SlowFade
Light Antifade Kit Component A (Molecular Probes) and viewed with a
Zeiss Axiophot fluorescence microscope.
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RESULTS |
Delta1 Inhibits Myogenesis and Expression of MyoD mRNA of C2C12
Cells--
To examine the role of mouse Delta1 in differentiation
regulation, we first constructed a Delta1 transfectant (D10) of the mouse X63 myeloma cell line which expresses a full-length mouse Delta1-tagged C-terminal with the gene 10 epitope sequence. We also
produced a fusion protein consisting of EGF repeats 11 and 12 of
mNotch1 followed by the Fc portion of human IgG1 (MNE-Rg4) to monitor surface expression of the Delta1 protein on D10 cells (43,
53). Flow cytometric analysis showed that MNE-Rg4 bound strongly to the
cell surface of D10 cells but not to parental X63 cells (Fig.
1A), indicating that D10 cells
express a large number of Delta1 molecules which can bind to
mNotch1.

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Fig. 1.
Delta1-expressing cells prevent
differentiation of C2C12 cells. A, D10 cells and
parental X63 myeloma cells were incubated with MNE-Rg4 (a fusion
protein of the mNotch1 EGF repeats and the Fc portion of human
IgG1) for 1 h at 4 °C, and binding was detected
with fluorescein-conjugated antibodies against human IgG (Cappel).
After gently washing, the cells were analyzed on a FACScan (Becton
Dickinson). B-G, C2C12 cells were cultured for 4 days either
alone (B and C), together with -irradiated (6 Gy) X63 cells (D and E), or with -irradiated
D10 cells (F and G). Culture was done either in
growth medium (+15% fetal calf serum) (B, D, and
F) or in differentiation medium (+2% horse serum)
(C, E, and G). Cell culture and
differentiation induction were carried out as described (28).
Photomicrographs were taken after washing cells to remove the
nonadherent X63 or D10 cells. Magnification, × 50.
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When C2C12 cells were cultured in differentiation media in the presence
or absence of X63 cells, C2C12 cells differentiated normally and fused
into multinucleated myotubes (Fig. 1, C and E).
In contrast, C2C12 cells co-cultured with D10 cells showed drastic
reduction of myotube formation (Fig. 1G). C2C12 myoblasts have been shown to express endogenous Notch receptors
(16).2
To further explore the molecular mechanism for differentiation
suppression of C2C12 cells co-cultured with D10 cells, we examined expression of muscle lineage-specific genes such as MyoD, myogenin, and
myosin light chain 2 (MLC2) before and 24 h after differentiation induction. When C2C12 cells were co-cultured with X63 cells, high level
expression of MyoD mRNA was maintained before and after differentiation induction. Myf-5 was not detected in this particular C2C12 cell (data not shown). Expression of mRNAs for myogenin and
MLC2 was induced after differentiation induction (Fig.
2). C2C12 cells co-cultured with D10
cells markedly decreased expression of MyoD mRNA in concomitant
inhibition of myogenin and MLC2 mRNAs induction. These results
indicate that the mouse Delta1 serves as the functional ligand for
Notch and their interaction inhibits myogenic differentiation of C2C12
cells by blocking expression of MyoD mRNA.

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Fig. 2.
The interaction of D10 cells inhibits
expression of the muscle regulatory and structural genes in C2C12
cells. C2C12 cells were co-cultured with -irradiated X63 or D10
cells in differentiation medium. After 24 h, total RNA was
extracted. Northern blots were performed with 15 or 30 µg of the
extracted RNA from the cultured cells. cDNA probes for MyoD (44),
myogenin (68), MLC2, and glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) were obtained by RT-PCR.
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Delta1 Inhibits Expression of MyoD mRNA by Up-regulating HES1
mRNA Expression in C2C12 Cells--
HES1 was suggested to be a
direct target of Notch signaling by co-transfection experiments (25)
and reported to inhibit MyoD-induced myogenesis of 10T1/2 cells (38).
We therefore tested whether HES1 mRNA was up-regulated in
differentiation-induced C2C12 cells by co-culture with D10 cells. HES1
mRNA was rapidly up-regulated about 2.5 times by co-culture with
D10 cells, followed by down-modulation of MyoD mRNA (Fig.
3, A-C). By contrast, the HES1
mRNA level was not changed by co-culture with X63 cells (Fig. 3,
A-C). A slight down-regulation of MyoD mRNA by X63 cells
appears to be caused by serum deprivation (54). These experiments
indicate that Delta1 inhibits myogenesis of C2C12 cells by suppressing MyoD mRNA expression with up-regulating expression of HES1
mRNA.

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Fig. 3.
The interaction with D10 cells up-regulates
HES1 mRNA expression and inhibits MyoD mRNA expression in C2C12
cells. A, C2C12 cells were co-cultured with D10 or X63
cells for indicated time in differentiation medium. Total RNA was
extracted, and Northern blots were performed with 30 µg of the
extracted RNA from the cultured cells at indicated time points. HES1
probe is a 1.4-kilobase pair EcoRI fragment excised from
pSV-CMV-HES1 (38). Relative quantitation of HES1 (B) and
MyoD (C) mRNA. Data shown an average values from four
independent experiments, with standard deviations. Northern blot bands
in A were quantitated using a BAS-1500 (Fuji Film),
normalized for glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) mRNA expression, and final values were expressed
as the relative level of the value at time 0.
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As the Delta1-induced decrease of MyoD mRNA rapidly followed the
up-regulation of HES1 mRNA, MyoD could be regulated by HES1 in
C2C12 cells as previously suggested (38). To determine whether induction of HES1 mRNA expression requires protein synthesis, C2C12
cells were co-cultured with D10 cells in differentiation medium in the
presence of 10 µM cycloheximide. The cycloheximide treatment blocked the decrease of MyoD mRNA expression but not the
increase of HES1 mRNA in C2C12 cells by co-culture with D10 cells
(Fig. 4). In fact, HES1 mRNA
expression was augmented about 16 times by co-culture with D10 cells in
the presence of cycloheximide. Furthermore, the block of protein
synthesis also stimulated the increase of HES1 mRNA (about 2.8 times) by co-culture with X63 cells (Fig. 4). These results indicate
that the HES1 gene is regulated not only directly by
activated Notch and RBP-J without synthesis of intermediate regulatory
molecules, but also negatively by the HES1 protein per se in
agreement with the previous report (55).

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Fig. 4.
Effects of cycloheximide treatment on the
Delta1-induced modulation of HES1 and MyoD mRNA expression.
C2C12 cells were co-cultured with X63 or D10 cells in differentiation
medium in the absence or presence of 10 µM cycloheximide
(CHX). HES1 and MyoD mRNA expression was analyzed by
Northern blot, as described in the legend to Fig. 3. Values were
normalized for glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) mRNA expression, and final values were expressed
as the relative level of the value at time 0. Similar results were
obtained in three additional experiments.
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Delta1 Up-regulates RBP-J-mediated Transcriptional Activity--
Although Notch signaling by overexpression of Notch IC or
RAMIC has been shown to activate transcription mediated by RBP-J, it
has not been shown whether ligand-induced Notch signaling also transactivates RBP-J. We therefore examined whether Notch signaling in
C2C12 cells triggered by co-culture with D10 cells up-regulates RBP-J-mediated transcription. To monitor RBP-J-mediated transcription, we used the Tp1 and HES1 promoters that carry RBP-J-binding sites and
are transactivated by overexpression of the mNotch1 RAMIC (25, 28, 56,
57). C2C12 cells were transfected with Tp1-luciferase (pGa981-6) or
the HES1-luciferase reporter plasmid (ptk-HES1) and co-cultured with
either X63 or D10 cells. C2C12 cells co-cultured with D10 cells showed
that transcription from the HES1 promoter was severalfold enhanced as
compared with that of X63 cells (Fig. 5A). A small level enhancement
of the HES1 promoter activity may be due to negative autoregulation by
endogenous HES1 in C2C12 cells, because the HES1 promoter contains the
N-box for HES1 binding (55). To avoid this complication, we used the
Tp1 promoter which contained only the RBP-J-binding site. C2C12 cells
co-cultured with increasing numbers of D10 cells showed markedly
enhanced transcriptional activities through the Tp1 promoter in
parallel with the number of the D10 cells added (Fig. 5B).
By contrast, C2C12 cells co-cultured with X63 cells showed negligible
levels of transcriptional activity through the Tp1 promoter.
Essentially the same results were obtained regardless of the culture
media used, i.e. for differentiation or growth.

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Fig. 5.
Transcriptional activation of RBP-J-binding
motif-containing promoter constructs in C2C12 cells by co-culture with
D10 cells. C2C12 cells were transfected by lipofection with 500 ng
of reporter plasmid ptk-HES1 (HES1-luc) (A), and
pGa50-7 (luc) or pGa981-6 (Tp1-luc)
(B) and 200 ng of pCMX-lacZ as internal control for
transfection efficiency as described previously (69). Reporter plasmid
ptk-HES1 contained the 87 to 51 promoter fragment of the
HES1 gene (55). After transfection, C2C12 cells were
co-cultured in growth medium with -irradiated X63 cells or D10 cells
for 24 h. Essentially the same results were obtained by using
differentiation medium. Luciferase and -galactosidase assays were
done as described previously (70). C, the effects of RBP-J
on transactivation by the co-culture with D10 cells. C2C12 cells were
transfected with pGa981-6 and increasing amounts of pCMX-mRBP-J. All
experiments were repeated at least three times, and the averages of
more than three independent experiments with standard deviations are
shown as bars.
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To further confirm that RBP-J is involved in transactivation through
the Tp1 promoter by ligand-induced Notch signaling, excess amounts of
RBP-J constructs were co-transfected with the Tp1 reporter plasmid into
C2C12 cells because excess amounts of RBP-J have been shown to inhibit
transcriptional activity through the Tp1 promoter by RAMIC (28, 57). In
fact, excess RBP-J significantly reduced the transcriptional activity
through the Tp1 promoter in C2C12 cells co-cultured with D10 cells
(Fig. 5C). Excess RBP-J reduced marginally the basal
transactivation activity of the Tp1 promoter in C2C12 cells co-cultured
with X63 cells. These results indicate that interaction of Delta1 on
D10 cells with the receptor Notch on C2C12 cells leads to
RBP-J-mediated transactivation of the HES1 and Tp1 promoters.
Activated RBP-J Decreases MyoD Expression in C2C12 Cells--
A
fusion protein of human RBP-J with a viral transactivation domain VP16,
hRBP-J-VP16 (58), has been shown to markedly suppress myogenesis of
C2C12 cells (28). We showed above that ligand-dependent Notch signaling suppressed MyoD mRNA expression and enhanced
RBP-J-dependent transcription of HES1 mRNA in C2C12
cells (Figs. 1, 2, and 5). To examine whether inhibition of MyoD
expression by Notch-Delta interaction is mediated through RBP-J, we
transfected the active form of mouse RBP-J, VP16-mRBP-J into C2C12
cells, and measured expression of MyoD (green) and VP16-mRBP-J (red) by
two-color immunocytostaining (Fig. 6).
There are few overlaps of green (MyoD) and red (VP16-mRBP-J) staining
(Fig. 6, I-K), showing that MyoD expression was suppressed
in VP16-mRBP-J positive cells. The frequency of MyoD+ cells
was markedly reduced by VP16-mRBP-J expression as compared with control
cells transfected with the vector alone (Table
I). In contrast, there are many overlaps
of green and red staining (Fig. 6, E-G) and no reduction of
MyoD expressing cells by mRBP-J expression, showing that MyoD
expression was not inhibited in wild type mRBP-J positive cells (Table
I). These results indicate that the activated RBP-J also suppresses
MyoD expression in C2C12 cells.

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Fig. 6.
The active form of RBP-J (VP16-RBP-J)
inhibits MyoD expression in C2C12 cells. C2C12 cells were
transfected with pCMX-N (mock vector) (A-D), pCMX-mRBP-J
(E-H), or pCMX-VP16-mRBP-J (I-L). After
transfection, C2C12 cells were cultured in differentiation medium for
24 h and then immunocytostaining was carried out to monitor
expression of the MyoD (green, A, E, and I), and
mRBP-J or VP16-mRBP-J (red, B, F, and G). The two
images were superimposed (C, G, and K). Nuclei
were stained with Hoechst 33342 (blue, D, H, and
L). MyoD expression is inhibited by VP16-mRBP-J
(I-K) but not by mRBP-J (E-G).
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Table I
VP16-RBP-J inhibits expression of MyoD in C2C12 cells
Staining was done 24 h after differentiation induction as
described in the legend to Fig. 6.
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To further confirm that Delta1-induced inhibition of MyoD expression is
involved in myogenic suppression, we constitutively expressed MyoD in
C2C12 cells and co-cultured with D10 cells. To monitor rescue of
myogenic suppression, two-color immunocytostaining experiments that
detect expression of both MyoD and myoglobin were carried out (Fig.
7). C2C12 cells expressing MyoD
differentiated into myoglobin expressing cells despite co-culture with
D10 cells (Fig. 7, E-G). These data indicate that
down-regulation of MyoD is the major cause in Delta1-induced myogenic
suppression of C2C12 cells.

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Fig. 7.
The constitutive expression of MyoD rescues
the Delta1-induced myogenic suppression of C2C12 cells. C2C12
cells were transfected with pH APr-1 (mock vector)
(A-D) or pH A-D(+) (mouse MyoD)
(E-H) (45, 50). After transfection, C2C12 cells were
co-cultured with D10 cells in differentiation medium for 4 days and
then immunocytostaining was carried out to monitor expression of the
MyoD (green, A and E) and myoglobin (red,
B and F) (28). The two images were superimposed
(C and G). Nuclei were stained with Hoechst 33342 (blue, D and H). The Delta1-induced myogenic
suppression is rescued by the constitutive expression of MyoD
(E-G).
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DISCUSSION |
The skeletal muscle cell differentiation is controlled by basic
helix loop helix transcriptional regulators (MyoD, Myf-5, myogenin, and
MRF4) that belong to the MyoD family (32). Previous experiments showed
that truncated mNotch1 (IC) inhibits MyoD- or
Myf-5-dependent transcriptional activity of
E-box-containing promoters (26) and that ligand-induced Notch signaling
down-regulates expression of myogenin (15, 31). However, a direct
target of ligand-induced Notch signaling was unknown. In this study, we
have demonstrated that Delta1-induced Notch signaling mediated by RBP-J
induces directly expression of HES1. Furthermore, the Delta1-induced
Notch signaling or the activated form of mRBP-J (VP16-mRBP-J) inhibited
expression of MyoD in C2C12 cells and their myogenic differentiation.
Taken together with the previous report that HES1 down-regulates
MyoD-dependent transcriptional activity of E-box-containing
promoters and inhibits the MyoD-induced myogenic conversion of 10T1/2
cells (38), Delta-Notch interaction is likely to induce HES1 which then
down-regulates MyoD, resulting in inhibition of myogenesis.
The mammalian ligands of Notch can be divided into two groups, Delta
and Serrate/Jagged (1, 8). Previously only Jagged was shown to function
as a ligand of Notch (15, 16). This is the first report that Delta1 can
interact with Notch and deliver a differentiation suppression signal in
mammalian cultured cells. We have shown that Delta1 can bind to Notch1
(Fig. 1A), but this observation does not exclude the
possibility that Delta1 interacts also with other Notch family members
(Notch2, Notch3, and Notch4). It is not clear that Delta and Jagged
have the identical function including preference among the Notch family members.
Su(H) has been shown to be involved in Notch signaling by extensive
genetic studies in Drosophila. RBP-J has been shown to mediate differentiation suppression activity of RAMIC and IC (28). Furthermore, Epstein-Barr virus nuclear antigen 2, which physically associates with RBP-J, can also suppress differentiation of C2C12 cells
(57). However, involvement of RBP-J in ligand-induced Notch signaling
has not been demonstrated. In this study we showed that RBP-J is
involved in Delta1-induced Notch signaling because (a) it
activated the transactivation activity of the HES1 and Tp1 promoters
containing RBP-J-binding motifs, (b) this activity was
blocked by excess amounts of RBP-J, and finally (c) the
activated form of RBP-J inhibited both MyoD transcription and
differentiation of C2C12 cells into myotubes (28) just like
Delta1-induced Notch signaling. This conclusion is supported by the
gene targeting results: Delta1
/
(21),
Notch1
/
(22, 23), and
RBP-J
/
(24) mutant mice all affected somite formation.
Although RBP-J has been suggested to be a transcriptional repressor
(59), overexpression of RBP-J per se did not affect the MyoD
expression in C2C12 cells (Fig. 6; Table I). The 7.0-kilobase pair
upstream region of the mouse MyoD gene (60) does not contain RBP-J-binding sites.2 In addition, the cycloheximide
treatment blocked the Notch signaling induced MyoD suppression in C2C12
cells (Fig. 4). It is, therefore, unlikely that the MyoD
gene is the direct target of Notch/RBP-J signaling. Since VP16-mRBP-J
has a strong transcriptional activity, at least one molecule that
inhibits the MyoD expression would be induced by Notch/RBP-J signaling
in C2C12 cells. Negative regulators of myogenesis, such as the Id and
HES families, inhibit the transcriptional activity of the MyoD family
(38, 61). HES1 belongs to the basic helix loop helix protein family
whose members have been shown to antagonize the function of other basic
helix loop helix proteins such as MyoD (38). Since RAMIC or IC of
mNotch1 acts as a transcriptional activator of the HES1 promoter that
contains the RBP-J-binding sites in HeLa (25), and COS7 (28) and C2C12 (data not shown) cells, HES1 is assumed to be responsible for blocking
myogenesis by Notch1 signaling (25, 62, 63). Ligand-induced Notch
signaling enhances HES1 promoter activity and up-regulates HES1
mRNA expression quickly and transiently in C2C12 cells, which is
followed by the reduction of MyoD mRNA expression (Figs. 3 and 4).
We also confirmed that co-culture with mouse Jagged1-expressing cells
up-regulates HES1 mRNA expression and subsequently reduces MyoD
mRNA expression in C2C12 cells.2 Since Delta-induced
HES1 mRNA augmentation is not blocked but rather stimulated by the
cycloheximide treatment, the HES1 gene is at least one of
the primary target genes in the Notch/RBP-J signaling pathway in C2C12
cells. The results also support the previous report that the
HES1 gene is negatively autoregulated (55). A small and
transient up-regulation of HES1 can strongly down-regulate MyoD
expression because the MyoD gene is positively autoregulated
(64).
The conclusion that HES1 is the direct target of Notch/RBP-J signaling
does not agree with the previous findings that HES1 expression is
unaltered in mouse embryos with the RBP-J
/
genotype (65) and that knockout mice with the
HES1
/
genotype are affected in neurogenesis
but not in myogenesis (66). An explanation to reconcile these results
would be that direct target genes other than HES1 may be involved in
the Notch/RBP-J signaling pathway. Finally, the RBP-J protein is
ubiquitously expressed (67), commonly used by the Notch family members
(56),2 and directly and uniquely targeted by Notch
signaling (24, 65). Thus RBP-J may function as a master protein in cell
fate determination by Notch signaling.