1 Department of Cell and Molecular Biology, Karolinska Institute, SE-171 77
Stockholm, Sweden
2 Department of Neuroscience, Medical Nobel Institute, Karolinska Institute,
SE-171 77 Stockholm, Sweden
* Author for correspondence (e-mail: Urban.Lendahl{at}cmb.ki.se)
Accepted 28 August 2003
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: SMAD, Delta, Serrate, TGFß, skeletal muscle, -secretase inhibitor
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
It is an emerging concept that the response of a cell to extrinsic signals
relies not only on the effect of a particular signaling pathway, but on the
integration of signals from multiple pathways. This enables the cell to
respond to a more complex repertoire of signals, and to integrate this
information into the large number of physiological responses a cell can
elicit. Despite the importance of Notch signaling for proper cellular
differentiation in many tissues, little is known about the interaction of the
Notch signaling pathway with other major signaling pathways. To begin to
address this, we investigated the possibility of a signal integration between
the Notch and BMP signaling pathways. The underlying rationale for this was
that both Notch and BMP signaling block differentiation of certain cell types,
including myogenic cells (Kopan et al.,
1994; Takahashi et al.,
1994
). We wished to explore whether this differentiation block was
mediated by distinct mechanisms or through cross-talk between the two
pathways.
BMP is a member of the TGFß superfamily of ligands and can elicit a
large variety of cellular responses
(Attisano and Wrana, 2002). In
the case of BMP-mediated signaling, the ligand binds to a type II receptor,
which phosphorylates the type I receptor in a heterotetrameric receptor
complex at the plasma membrane (Attisano
and Wrana, 2002
). This leads to phosphorylation of the cytoplasmic
protein SMAD1, which is referred to as a receptor-regulated SMAD (R-SMAD).
SMAD1, together with another SMAD protein (co-SMAD), SMAD4, translocates to
the nucleus, where it controls the regulation of specific target genes
(Attisano and Wrana, 2002
;
Miyazawa et al., 2002
). SMADs
bind DNA with low affinity and are thought to recruit tissue-specific factors
to enhance DNA-binding and regulate cellular events. SMAD1 binds to GC-rich
stretches in promoter sequences (Kusanagi
et al., 2000
). SMAD proteins are composed of two conserved
domains, MH1 and MH2, which are separated by a linker sequence. SMADs have
been shown to bind a number of proteins in the nucleus, including general
transcription factors, co-activators and co-repressors
(Miyazawa et al., 2002
). The
combination of bound factors influences DNA-binding specificity and the
intensity of the transcriptional activation, which indicates that SMADs are
crucial for signal integration. BMP signaling shares some principle features
with Notch signaling, particularly that the transmission of the signal from
the exterior of the cell involves only a few intermediates and requires the
relocation of a signaling component from the cytoplasm to the nucleus.
Furthermore, some of the factors important in modulating SMAD signaling, such
as p300 and P/CAF, are also key proteins for regulating Notch signaling
(Janknecht et al., 1998
;
Moustakas et al., 2001
;
Wallberg et al., 2002
).
It has previously been demonstrated that both addition of BMP
(Katagiri et al., 1994) and
ligand induction of Notch (Kopan et al.,
1994
; Kuroda et al.,
1999
) cause a dramatic block in myotube formation in the myogenic
cell line C2C12. In this report, we have addressed whether BMP- and
Notch-mediated differentiation inhibition are distinct events, or whether they
are in some way linked.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
RNA extraction, cDNA synthesis and quantitative PCR
RNA was extracted using RNeasy Miniprep (Qiagen), according to the
manufacturer's instructions. For cDNA synthesis, 10 µl of RNA, together
with 1 µl oligodT and 1 µl 10 mM dNTPs, was incubated at 65°C for 5
minutes and then chilled on ice. This was followed by the addition of 4 µl
First Strand buffer, 2 µl 0.1 M DTT and 1 µl RNaseOut, and incubation at
42°C for 2 minutes. SuperscriptII (Invitrogen) was added and the reaction
further incubated at 42°C for 50 minutes. The reaction was stopped by heat
inactivation at 70°C for 15 minutes. Quantitative PCR was performed in
accordance with the manufacturer's instructions, using a LightCycler rapid
thermal cycler system (Applied Biosystems). A mastermix containing
nucleotides, Taq polymerase, SYBR Green and buffer was mixed with primers and
cDNA. A description of the primers used can be found online (see Data S2 at
http://dev.biologists.org/supplemental/).
Differentiation assay
C2C12 cells were seeded at high density on gelatin-coated glass,
transfected with the indicated constructs (2 µg/well in a 6-well plate) and
incubated in differentiation medium (2% horse serum) for 2-6 days. Satellite
cells were seeded onto pre-coated (Fibronectin) plates and subjected to
similar differentiation conditions.
-secretase inhibitor treatment and ligand stimulation
L-685,458 (Bachem) was added to the cells for 1-12 hours at a concentration
of 4 µM. For the differentiation assay, a concentration of 1 µM was used
and the compounds were added fresh everyday. BMP4 (R&D) was added to the
cells at a concentration of 25 or 50 ng/ml.
In vitro binding assays and western blot
GST-fusion proteins were produced in E. coli and purified on
glutathione-conjugated beads (Pharmacia). The fusion proteins were incubated
with cell lysates from cells transfected with the indicated plasmids
overnight. Immunoprecipitation and westerns blots are described in the
supplementary material available online (see Data S3 at
http://dev.biologists.org/supplemental/).
Immunocytochemistry
C2C12 cells were fixed for 1-3 minutes in 2% paraformaldehyde, blocked for
20 minutes in blocking solution (5% BSA, 0.3% Triton X-100 and 10% goat serum
in PBS) and incubated with primary antibody in blocking solution for 1 hour.
The cells were extensively washed in PBS and incubated in the dark with
secondary antibody for 40 minutes. Cells were then mounted in ProLong mounting
medium (Molecular Probes). Primary antibodies were rabbit anti-Myc (Santa
Cruz; diluted 1:20), rabbit anti-ß-gal (ICN; diluted 1:200) and/or mouse
anti-myosin heavy chain (MHC) (MF20, diluted 1:15; obtained from the
Developmental Studies Hybridoma Bank). Secondary antibodies were goat
anti-rabbit Alexa 488 and goat anti-mouse Alexa 546 (Molecular Probes).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
BMP4 increases expression of genes downstream of Notch in C2C12 cells
in a Notch-dependent manner
We next addressed whether BMP4 addition also induced expression of genes
immediately downstream of Notch. To this end, we analyzed changes in
expression levels of Hes1 and Hey1 following exposure to
BMP4, and/or addition of L-685,458, using quantitative PCR. L-685,458 was
added to the cells 12 hours before BMP4 to ensure that already cleaved Notch
ICD was degraded prior to BMP stimulation. Addition of only L-685,458 to C2C12
cells led to a small decrease in the levels of both Hes1 and
Hey1 mRNA (Fig. 2A,B),
presumably by blocking low level endogenous Notch signaling. Addition of BMP4
led to a 2.8- and 7-fold increase in the levels of Hes1 and
Hey1 mRNA, respectively (Fig.
2A,B). This increase was largely eliminated in cells
simultaneously treated with BMP4 and L-685,458
(Fig. 2A,B). These data
demonstrate that BMP4 increases the expression levels of the Hey1 and
Hes1 genes in a Notch-dependent manner. We also tested whether the
BMP induction could be observed at later stages in the differentiation
process. Hes1 and, in particular, Hey1 expression in C2C12
cells was elevated at 5 days of differentiation in response to BMP4, whereas
the expression levels under normal conditions were relatively similar
(Fig. 2C). To rule out the
possibility that L-685,458 affected BMP target genes by a more general
mechanism not related to cleavage of the Notch receptor, we performed
quantitative PCR on Runx2, which is a BMP4 target gene
(Tsuji et al., 1998) that is
known to be expressed in C2C12 cells. Runx2 expression was moderately
increased by BMP4, but L-685,458 did not alter Runx2 levels
(Fig. 2D). By contrast,
Hey1 expression was substantially increased and was blocked by
L-685,458 in the same experiment (Fig.
2D).
To determine whether BMP also regulated transcription factors important for
myogenesis, we analysed the effect of BMP4 stimulation on MyoD
expression. First, we established the protein expression profiles of MyoD and
MHC during C2C12 differentiation by western blot analysis
(Fig. 2E). In keeping with
previous data (Dedieu et al.,
2002), the expression of MyoD increased during the early
phases of differentiation and reached a maximum two days after induction of
differentiation (Fig. 2E). By
contrast, MHC expression was seen first at two days after induction of
differentiation and then increased to higher levels
(Fig. 2E). We therefore
analysed the regulation of MyoD by BMP4 after two days of
differentiation, i.e. when MyoD expression should be maximal.
Stimulation by BMP4 reduced MyoD mRNA expression, whereas L-685,458
significantly increased the level of expression
(Fig. 2E). Simultaneous
treatment by BMP4 and L-685,458 reduced MyoD expression to the same
level as BMP4 alone (Fig. 2E).
This suggests that BMP4 and Notch signaling can suppress expression of the
myogenic transcription factor MyoD, but that the effect of BMP4 on
MyoD, in contrast to the regulation of Hes1 and
Hey1, may not depend on Notch signaling.
We next investigated the effect of BMP4 and L685,458 on Hes1 and Hey1 expression in satellite cells. Hey1 expression was induced by BMP4 in the absence, but not in the presence, of L-685,458 (Fig. 2F), whereas Hes1 expression was unaffected (Fig. 2G). To address the effect of BMP4 in the regulation of Hey1 in another, non-myogenic, primary cell type, we analyzed neural stem cells isolated from the lateral ventricle of the adult mouse brain and cultured as neurospheres. In these cells Hey1 expression was increased 3-fold following BMP4 stimulation (Fig. 2H). Collectively, these results indicate that Hey1 is a target gene for Notch and BMP stimulation in the three cell types tested.
BMP4-mediated induction of Hey1 is direct and requires
cell-cell contact
The fact that BMP4 was added to cells only one hour prior to analysis
suggests a direct upregulation of Hey1 expression that does not
require intermediate protein synthesis. To test this more thoroughly, we
stimulated cells with BMP4 in the presence of cycloheximide, which blocks
protein synthesis. Hey1 mRNA levels were upregulated in the presence
of BMP both in the absence and presence of cycloheximide
(Fig. 3A), in keeping with a
direct effect. To test whether normal ligand-induced Notch signaling was
required in the C2C12 cells for BMP to exert an effect on Hey1
expression, we tested the effect of culturing the C2C12 cells at various
densities. Cell populations at high density would be in direct cell-cell
contact and therefore could have active Notch signaling, whereas sparsely
seeded cells could not. At densities when the majority of C2C12 cells are in
contact (80 and 40% confluency) a robust increase in Hey1 expression
was observed in response to BMP4 (Fig.
3B). By contrast, when cells were grown at 20% density, i.e. when
there are few cell contacts, Hey1 expression did not increase after
BMP4 stimulation (Fig. 3B). Although this suggests that endogenous Notch signaling in C2C12 cells,
mediated through ligand activation, is important for the BMP effect, it
remained a possibility that BMP4 simply increased the amount of Notch ligand
to induce elevated Hey1 levels.
|
BMP and Notch 1 ICD synergistically activate a Hey1 promoter
reporter construct
As discussed above, Hey1 was upregulated by Notch and BMP in cells
of both muscle and neural origin, This observation, combined with the fact
that Hey1 has been suggested to be important for inhibition of muscle
development (Sun et al.,
2001), led us to concentrate on Hey1 to explore the
interplay between Notch and BMP in more detail. The Hey1 promoter
contains both CSL-binding sites and a GC-rich domain comprising six GCCGnCGC
sequences that are putative SMAD1 binding sites (see below)
(Kusanagi et al., 2000
). By
contrast, no such elements were found in the 0.4 kb of the Hes1
promoter that was functional in Notch response assays (data not shown).
We first tested the response of a 3 kb Hey1 promoter-luciferase
construct (Hey1-luc) to BMP4 and Notch stimulation in C2C12 cells. Addition of
BMP4 to Hey1-luc reporter transfected cells resulted in a 2-fold activation
(Fig. 4A). Transfection of a
BMP receptor-regulated SMAD, SMAD1, resulted in a similar increase
(Fig. 4A), and this could be
further increased by the addition of BMP4, suggesting that endogenous SMAD1
may be present in too small amounts in the cells to mediate a full BMP
response. Transfection of Notch 1 ICD into C2C12 cells led to a small
induction of the Hey1 reporter and addition of BMP4 further increased
this (Fig. 4B). To test whether
this increase could be enhanced with additional amounts of SMAD1, we
transfected SMAD1 together with Notch 1 ICD in the presence or absence of
BMP4. Introduction of SMAD1 together with Notch 1 ICD increased the
transactivation about 8-fold in the presence of BMP4
(Fig. 4C). In the next set of
experiments, we investigated the effects of Notch ICD and BMP4 signaling on
the Hey1 promoter in COS-7 cells. We chose COS-7 cells because the
Hey1 promoter can be robustly activated in this cell line
(Nakagawa et al., 2000) and
there is very little endogenous Notch signaling, which may provide an
opportunity to see more pronounced effects in reporter gene activation.
Transfection of Notch 1 ICD led to a 14-fold increase in activity, and this
increase was elevated to 52-fold when Notch 1 ICD was introduced in cells
exposed to BMP4 (Fig. 4D).
Transfection of Notch 1 ICD together with a constitutively active form of the
BMP type I receptor (Alk6CA) that mimics a ligand-activated BMP receptor
(Moren et al., 2000
) resulted
in a 20-fold increase in activity (Fig.
4D). Taken together, these data indicate that stimulation of the
BMP signaling pathway at three different levels, i.e. by ligand stimulation,
by the constitutively activated Alk6CA receptor or by SMAD1, leads to
increased Hey1 promoter activity. Moreover, BMP addition can
synergistically enhance the Notch ICD-mediated activation of the Hey1
promoter.
|
|
The GC-rich domain in the Hey1 promoter is partially
important for SMAD1 activation
To further test the notion that SMAD1 may act on the Hey1 promoter
in both a Notch-dependent and a Notch-independent manner, we analyzed
different forms of the Hey1 promoter for transcriptional activation.
The 3 kb Hey1 promoter used in the experiments described above
contains a 500 bp GC-rich domain within 600 bp of the transcriptional start
site (Fig. 6A). It has
previously been shown that the sequence GCCGnCGC is a low-affinity binding
site for SMAD1 (Kusanagi et al.,
2000) and, as discussed above, the GC-rich domain contains six
such sites (Maier and Gessler,
2000
). There are also two bona fide CSL-binding sites in the
promoter: one located within the GC-rich domain and another located
immediately before the first codon of the Hey1 gene
(Fig. 6A). To discover whether
the observed SMAD1-mediated effect on Hey1 promoter activation is
dependent on the GC-rich domain, we introduced a portion of the Hey1
promoter containing five of the six GCCGnCGC motifs in front of the luciferase
gene (referred to as GC-luciferase) (Fig.
6A). Transfection of the GC-luciferase construct into COS-7 cells
resulted in a low level (5-fold) of activation by SMAD1 in the absence of
exogenous BMP4 and a higher level (25-fold) if the cells were stimulated with
BMP4 (Fig. 6B). A low level of
induction was also observed with Notch 1 ICD alone
(Fig. 6B), suggesting that the
CSL-binding site in the GC-rich domain could bind Notch 1 ICD through CSL.
This view is supported by the fact that R218H CSL could reduce the effect of
Notch 1 ICD activation of the GC-luciferase construct. By contrast, R218H CSL
only moderately reduced the activation by SMAD1 and Notch 1 ICD in both the
absence and the presence of exogenous BMP4
(Fig. 6B).
|
SMAD1 increases transcription from promoters lacking SMAD1-binding
sites
The SMAD1-mediated activation of the Hey1-GC construct, from which
most of the potential SMAD1 binding region had been removed, may suggest that
SMAD1 exerts some effect without directly binding to DNA. To more stringently
test this idea, we assessed in two different ways whether SMAD1 could mediate
its effect on a promoter lacking SMAD1-binding sites. First, we used a
previously established system with which a Gal4-Notch 1 ICD fusion has been
shown to activate a reporter consisting of Gal4 binding sites and the
luciferase gene (Beatus et al.,
1999
). Co-transfection of SMAD1 and Gal4-Notch 1 ICD led to
increased transactivation, from 35-fold for Gal4-Notch 1 ICD alone, to
approximately 175-fold (Fig.
7A). Addition of BMP4 further enhanced this increase
(Fig. 7A). As the second
approach, a minimal CSL-binding promoter construct composed of six dimeric
CSL-binding sites (12xCSL-luc) (Kato
et al., 1997
) but lacking SMAD1-binding sites was used.
Transfection of Notch 1 ICD resulted in a robust activation of the
12xCSL-luc construct (3000-fold) in the absence and presence of
exogenous BMP (Fig. 7B), which
is in keeping with previous observations
(Kato et al., 1997
). SMAD1
alone did not increase transcription, but combined transfection of Notch 1 ICD
and SMAD1 in the presence of BMP4 generated a 6000-fold increase in
transcription, i.e. approximately twofold higher than for Notch 1 ICD alone
(Fig. 7B). A similar increase
was also observed in the absence of exogenous BMP4. This demonstrates that
SMAD1 can induce transcription from a Notch-responsive promoter that does not
contain SMAD1-binding sites.
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Hey and Hes genes are important immediate downstream mediators of Notch
signaling in many organs, including muscle and the vascular system
(Donovan et al., 2002;
Jarriault et al., 1995
;
Shawber et al., 1996
;
Zhong et al., 2000
), and it is
noteworthy that the BMP4-induced differentiation block in C2C12 cells
coincides with increased expression of the two genes. The increase in both
Hes1 and Hey1 was substantially reduced by L-685,458 in
C2C12 cells, whereas satellite cells showed a BMP4-induced upregulation of
Hey1, which was abrogated by L-685,458. This may indicate that
Hey1 plays an important role in maintaining myogenic cells in an
undifferentiated state in response to the Notch-dependent BMP induction, but
more work is needed to specifically address this issue.
To begin to decipher in more detail how Notch and BMP4 signals are
integrated at a specific promoter, we analyzed the Hey1 promoter. The
BMP4 effect on Hey1 transcription appears to be caused by canonical
BMP signaling, as ligand stimulation, SMAD1 and a constitutively activated
form of the receptor (Alk6CA) can activate the Hey1 promoter. It
seems likely that this activation is mediated both through the binding of
SMAD1 to the promoter and through an interaction between SMAD1 and Notch ICD,
an interaction not dependent on SMAD1 DNA binding. Evidence for SMAD1 binding
to the Hey1 promoter comes from the promoter deletion experiments, in
which the GC-luciferase construct containing the GC-rich domain responded to
SMAD1 activation. Furthermore, the Hey1-GC and 12xCSL-luciferase
constructs, which either have one putative SMAD1 binding site or which lack
such sites, show little or no response to SMAD1 alone. Support for the view
that SMAD1 activates transcription in a non-DNA-binding mode, and presumably
through interaction with Notch ICD, comes from the experiments in which the
12xCSL-luciferase construct was not activated by SMAD1 alone, but in
which SMAD1 potentiated Notch ICD-induced activation 2-fold
(Fig. 7B). Similarly, the
activity of Gal4-Notch 1 ICD on an UAS-reporter gene construct was potentiated
by SMAD1.
An interaction between Notch 1 ICD and SMAD1 was demonstrated by
co-immunoprecipitation and GST-pulldown experiments. Co-immunoprecipitation
revealed a robust interaction between SMAD1 and Notch 1 ICD. By contrast, the
GST-pulldown experiments showed a weak interaction between Notch 1 ICD and
SMAD1, when compared with, for example, the interaction between Notch 1 ICD
and CSL. It should be noted that the GST-fused form of SMAD1 structurally
mimics the phosphorylated form of SMAD1, thus eliminating the need for
producing SMAD1 in mammalian cells to make it phosphorylated. The apparent
differences in the strength of the interaction between the
co-immunoprecipitation and GST-pulldown experiments raises the possibility
that SMAD1 and Notch 1 ICD interact directly, but that the interaction may be
stabilized by other Notch-interacting proteins, which may be more prominent
under the co-immunoprecipitation conditions than under the GST-pulldown
conditions. Precedence for such a stabilization comes from the Notch ICD and
CSL interaction, where Maml or SKIP serve to strengthen the interaction
(Wallberg et al., 2002;
Wu et al., 2000
;
Zhou et al., 2000
).
BMP signaling elicits a broad range of context-dependent cellular
responses, despite an apparent simplicity in terms of proteins directly
involved in the signaling cascade. The variety of different responses has, at
least in part, been attributed to the ability of SMADs to act as signaling
platforms through interactions with numerous proteins, such as p300/CBP, Runx2
and GATA3 (Bae et al., 2001;
Blokzijl et al., 2002
;
Janknecht et al., 1998
). The
outcome of the different interactions of SMADs with other factors is
context-dependent, where SMAD downstream genes are differentially regulated
depending on cell type and/or BMP concentration. The data presented here widen
the repertoire of BMP-inducible genes to include the Notch downstream genes
Hes1 and Hey1. The requirement for functional Notch
signaling in this process, as well as for relieving the C2C12 and satellite
cell differentiation block, brings a new facet to BMP signaling. Rather than
acting as a strict integration platform, SMADs can also facilitate Notch
signaling.
The Notch and BMP signaling pathways are evolutionarily highly conserved
and influence differentiation processes in many organs. Skeletal muscle
differentiation from somites is an example where BMP and Notch could
co-operate in vivo. Absence of Notch results in enlarged clusters of
mesodermal cells that behave as muscle founder cells
(Corbin et al., 1991), and
mice devoid of Notch 1 have disorganized somites
(Conlon et al., 1995
).
Similarly, BMP4 acts in the lateral plate mesoderm to inhibit muscle formation
(Pourquie et al., 1995
;
Pourquie et al., 1996
). The
proposed co-operation between Notch and BMP signaling may also extend to cell
types other than muscle progenitors, such as neural stem cells. The
anti-neurogenic effects of the two signaling pathways may act together to keep
neural stem cells from differentiating into neurons.
In contrast to muscle and CNS development, in which Notch and BMP signaling
appear to act largely synergistically, neural crest development provides an
example of the two signaling pathways acting antagonistically. In neural crest
stem cells, BMP induces neuronal differentiation, but this is irreversibly
overridden by a short exposure of the cells to soluble Notch ligands
(Morrison et al., 2000). It
would be interesting to discover at what level Notch and BMP signaling
interact in this case to produce an antagonistic effect. One possibility is
that different SMAD combinations or the abundance of other SMAD-interacting
proteins influence the differentiation outcome. Although much work remains to
be done in order to understand these different interactions in vivo, this
report provides the first evidence for signal integration between the Notch
and BMP pathways, which may contribute to the ability of cells to decipher
complex extracellular cues into meaningful responses.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Artavanis-Tsakonas, S., Rand, M. D. and Lake, R. J.
(1999). Notch signaling: cell fate control and signal integration
in development. Science
284,770
-776.
Attisano, L. and Wrana, J. L. (2002). Signal
transduction by the TGF-beta superfamily. Science
296,1646
-1647.
Bae, S. C., Lee, K. S., Zhang, Y. W. and Ito, Y.
(2001). Intimate relationship between TGF-beta/BMP signaling and
runt domain transcription factor, PEBP2/CBF. J. Bone Joint Surg.
Am. 83-A Suppl. 1,S48
-S55.
Bains, W., Ponte, P., Blau, H. and Kedes, L. (1984). Cardiac actin is the major actin gene product in skeletal muscle cell differentiation in vitro. Mol. Cell. Biol. 4,1449 -1453.[Medline]
Beatus, P., Lundkvist, J., Oberg, C. and Lendahl, U.
(1999). The notch 3 intracellular domain represses notch
1-mediated activation through Hairy/Enhancer of split (HES) promoters.
Development 126,3925
-3935.
Berezovska, O., Jack, C., McLean, P., Aster, J. C., Hicks, C.,
Xia, W., Wolfe, M. S., Weinmaster, G., Selkoe, D. J. and Hyman, B.
T. (2000). Rapid Notch1 nuclear translocation after ligand
binding depends on presenilin-associated gamma-secretase activity.
Ann. NY Acad. Sci. 920,223
-226.
Blokzijl, A., ten Dijke, P. and Ibanez, C. F. (2002). Physical and functional interaction between GATA-3 and Smad3 allows TGF-beta regulation of GATA target genes. Curr. Biol. 12,35 -45.[CrossRef][Medline]
Bush, G., diSibio, G., Miyamoto, A., Denault, J. B., Leduc, R. and Weinmaster, G. (2001). Ligand-induced signaling in the absence of furin processing of Notch1. Dev. Biol. 229,494 -502.[CrossRef][Medline]
Chung, C. N., Hamaguchi, Y., Honjo, T. and Kawaichi, M. (1994). Site-directed mutagenesis study on DNA binding regions of the mouse homologue of Suppressor of Hairless, RBP-J kappa. Nucl. Acids Res. 22,2938 -2944.[Abstract]
Conlon, R. A., Reaume, A. G. and Rossant, J.
(1995). Notch1 is required for the coordinate segmentation of
somites. Development
121,1533
-1545.
Corbin, V., Michelson, A. M., Abmayr, S. M., Neel, V., Alcamo, E., Maniatis, T. and Young, M. W. (1991). A role for the Drosophila neurogenic genes in mesoderm differentiation. Cell 67,311 -323.[Medline]
Dedieu, S., Mazeres, G., Cottin, P. and Brustis, J. J. (2002). Involvement of myogenic regulator factors during fusion in the cell line C2C12. Int. J. Dev. Biol. 46,235 -241.[Medline]
Donovan, J., Kordylewska, A., Jan, Y. N. and Utset, M. F. (2002). Tetralogy of fallot and other congenital heart defects in Hey2 mutant mice. Curr. Biol. 12,1605 -1610.[Medline]
Ebinu, J. O. and Yankner, B. A. (2002). A RIP tide in neuronal signal transduction. Neuron 34,499 -502.[Medline]
Frisen, J. and Lendahl, U. (2001). Oh no, Notch again! BioEssays 23,3 -7.[CrossRef][Medline]
Furukawa, T., Maruyama, S., Kawaichi, M. and Honjo, T. (1992). The Drosophila homolog of the immunoglobulin recombination signal-binding protein regulates peripheral nervous system development. Cell 69,1191 -1197.[Medline]
Haass, C. and Steiner, H. (2002). Alzheimer disease gamma-secretase: a complex story of GxGD-type presenilin proteases. Trends Cell Biol. 12,556 -562.[CrossRef][Medline]
Iso, T., Sartorelli, V., Chung, G., Shichinohe, T., Kedes, L.
and Hamamori, Y. (2001). HERP, a new primary target of
Notch regulated by ligand binding. Mol. Cell. Biol.
21,6071
-6079.
Janknecht, R., Wells, N. J. and Hunter, T.
(1998). TGF-beta-stimulated cooperation of smad proteins with the
coactivators CBP/p300. Genes Dev.
12,2114
-2119.
Jarriault, S., Brou, C., Logeat, F., Schroeter, E. H., Kopan, R. and Israel, A. (1995). Signalling downstream of activated mammalian Notch. Nature 377,355 -358.[CrossRef][Medline]
Karlstrom, H., Bergman, A., Lendahl, U., Naslund, J. and
Lundkvist, J. (2002). A sensitive and quantitative assay for
measuring cleavage of presenilin substrates. J. Biol.
Chem. 277,6763
-6766.
Katagiri, T., Yamaguchi, A., Komaki, M., Abe, E., Takahashi, N., Ikeda, T., Rosen, V., Wozney, J. M., Fujisawa-Sehara, A. and Suda, T. (1994). Bone morphogenetic protein-2 converts the differentiation pathway of C2C12 myoblasts into the osteoblast lineage. J. Cell Biol. 127,1755 -1766.[Abstract]
Kato, H., Taniguchi, Y., Kurooka, H., Minoguchi, S., Sakai, T.,
Nomura-Okazaki, S., Tamura, K. and Honjo, T. (1997).
Involvement of RBP-J in biological functions of mouse Notch1 and its
derivatives. Development
124,4133
-4141.
Kopan, R., Nye, J. S. and Weintraub, H. (1994).
The intracellular domain of mouse Notch: a constitutively activated repressor
of myogenesis directed at the basic helix-loop-helix region of MyoD.
Development 120,2385
-2396.
Kuroda, K., Tani, S., Tamura, K., Minoguchi, S., Kurooka, H. and
Honjo, T. (1999). Delta-induced Notch signaling
mediated by RBP-J inhibits MyoD expression and myogenesis. J. Biol.
Chem. 274,7238
-7244.
Kusanagi, K., Inoue, H., Ishidou, Y., Mishima, H. K., Kawabata,
M. and Miyazono, K. (2000). Characterization of a bone
morphogenetic protein-responsive Smad-binding element. Mol. Biol.
Cell 11,555
-565.
Maier, M. M. and Gessler, M. (2000). Comparative analysis of the human and mouse Hey1 promoter: Hey genes are new Notch target genes. Biochem. Biophys. Res. Commun. 275,652 -660.[CrossRef][Medline]
Miyazawa, K., Shinozaki, M., Hara, T., Furuya, T. and Miyazono,
K. (2002). Two major Smad pathways in TGF-beta superfamily
signalling. Genes Cells
7,1191
-1204.
Moren, A., Itoh, S., Moustakas, A., Dijke, P. and Heldin, C. H. (2000). Functional consequences of tumorigenic missense mutations in the amino-terminal domain of Smad4. Oncogene 19,4396 -4404.[CrossRef][Medline]
Morrison, S. J., Perez, S. E., Qiao, Z., Verdi, J. M., Hicks, C., Weinmaster, G. and Anderson, D. J. (2000). Transient Notch activation initiates an irreversible switch from neurogenesis to gliogenesis by neural crest stem cells. Cell 101,499 -510.[Medline]
Moustakas, A., Souchelnytskyi, S. and Heldin, C. H. (2001). Smad regulation in TGF-beta signal transduction. J. Cell Sci. 114,4359 -4369.[Medline]
Nakagawa, O., McFadden, D. G., Nakagawa, M., Yanagisawa, H., Hu,
T., Srivastava, D. and Olson, E. N. (2000). Members of
the HRT family of basic helix-loop-helix proteins act as transcriptional
repressors downstream of Notch signaling. Proc. Natl. Acad. Sci.
USA 97,13655
-13660.
Nofziger, D., Miyamoto, A., Lyons, K. M. and Weinmaster, G.
(1999). Notch signaling imposes two distinct blocks in the
differentiation of C2C12 myoblasts. Development
126,1689
-1702.
Pourquie, O., Coltey, M., Breant, C. and Le Douarin, N. M. (1995). Control of somite patterning by signals from the lateral plate. Proc. Natl. Acad. Sci. USA 92,3219 -3223.[Abstract]
Pourquie, O., Fan, C. M., Coltey, M., Hirsinger, E., Watanabe, Y., Breant, C., Francis-West, P., Brickell, P., Tessier-Lavigne, M. and Le Douarin, N. M. (1996). Lateral and axial signals involved in avian somite patterning: a role for BMP4. Cell 84,461 -471.[Medline]
Rusconi, J. C. and Corbin, V. (1998). Evidence for a novel Notch pathway required for muscle precursor selection in Drosophila. Mech. Dev. 79, 39-50.[CrossRef][Medline]
Shawber, C., Nofziger, D., Hsieh, J. J., Lindsell, C., Bogler,
O., Hayward, D. and Weinmaster, G. (1996). Notch
signaling inhibits muscle cell differentiation through a CBF1-independent
pathway. Development
122,3765
-3773.
Sun, J., Kamei, C. N., Layne, M. D., Jain, M. K., Liao, J. K.,
Lee, M. E. and Chin, M. T. (2001). Regulation of myogenic
terminal differentiation by the hairy-related transcription factor CHF2.
J. Biol. Chem. 276,18591
-18596.
Takahashi, N., Ikeda, T., Rosen, V., Wozney, J. M., Fujisawa-Sehara, A. and Suda, T. (1994). Bone morphogenetic protein-2 converts the differentiation pathway of C2C12 myoblasts into the osteoblast lineage. J. Cell Biol. 127,1755 -1766.[Abstract]
Tsuji, K., Ito, Y. and Noda, M. (1998). Expression of the PEBP2alphaA/AML3/CBFA1 gene is regulated by BMP4/7 heterodimer and its overexpression suppresses type I collagen and osteocalcin gene expression in osteoblastic and nonosteoblastic mesenchymal cells. Bone 22,87 -92.[CrossRef][Medline]
Wallberg, A. E., Pedersen, K., Lendahl, U. and Roeder, R. G.
(2002). p300 and PCAF act cooperatively to mediate
transcriptional activation from chromatin templates by notch intracellular
domains in vitro. Mol. Cell. Biol.
22,7812
-7819.
Wettstein, D. A., Turner, D. L. and Kintner, C.
(1997). The Xenopus homolog of Drosophila Suppressor of Hairless
mediates Notch signaling during primary neurogenesis.
Development 124,693
-702.
Wu, L., Aster, J. C., Blacklow, S. C., Lake, R., Artavanis-Tsakonas, S. and Griffin, J. D. (2000). MAML1, a human homologue of Drosophila mastermind, is a transcriptional co-activator for NOTCH receptors. Nat. Genet. 26,484 -489.[CrossRef][Medline]
Zhong, T. P., Rosenberg, M., Mohideen, M. A., Weinstein, B. and
Fishman, M. C. (2000). gridlock, an HLH gene required
for assembly of the aorta in zebrafish. Science
287,1820
-1824.
Zhou, S., Fujimuro, M., Hsieh, J. J., Chen, L., Miyamoto, A.,
Weinmaster, G. and Hayward, S. D. (2000). SKIP, a
CBF1-associated protein, interacts with the ankyrin repeat domain of NotchIC
to facilitate NotchIC function. Mol. Cell. Biol.
20,2400
-2410.