ARTICLE IN PRESS
Progress in Biophysics and Molecular Biology 96 (2008) 499–509
www.elsevier.com/locate/pbiomolbio
An overview of Notch3 function in vascular smooth muscle cells
Tao Wanga,, Martin Baronb, Dorothy Trumpa
a
Medical Genetics Research Group and Centre for Molecular Medicine, School of Clinical and Laboratory Sciences,
Faculty of Medicine and Human Sciences, The University of Manchester, UK
b
Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, The University of Manchester, UK
Available online 29 July 2007
Abstract
Proteins of the Notch family are cell surface receptors that transduce signals between neighbouring cells. The Notch
signalling pathway is highly evolutionarily conserved and critical for cell fate determination during embryonic
development, including many aspects of vascular development. The interaction of Notch receptors with ligands leads to
cleavage of the Notch intracellular domain (NICD) which then translocates to the nucleus and activates the transcription
factor CBF1/JBP-Jk, regulating downstream gene expression. To date four Notch receptors have been found in mammals.
Of these, Notch3 is predominantly expressed in adult arterial smooth muscle cells in human. NOTCH3 gene mutations
cause the autosomal dominant condition, cerebral autosomal dominant arteriopathy with subcortical infarcts and
leukoecephelopathy (CADASIL), an inherited early stroke syndrome leading to dementia due to systemic vascular
degeneration. This suggests that Notch3 plays a critical role in maintaining the phenotypic stability of vascular smooth
muscle cells (VSMCs). Recent publications indicate that Notch3 is involved in vascular injury and is a determinant of
VSMC survival, but its exact function is unknown. The molecular mechanisms underlying CADASIL pathology are
therefore intriguing. Investigation of CADASIL mutant Notch3 shows that the majority of mutations do not change
CBF1/JBP-Jk mediated classic Notch activation, so the pathological consequences of NOTCH3 mutations in CADASIL
patients can not be simply explained by loss- or gain-of-function in the classic Notch signalling pathway. This suggests that
a novel Notch3-mediated signalling pathway may be present in VSMCs, or cross-regulation of Notch3 to other signalling
pathway(s) may play a critical role on VSMCs survival. Alternatively, the mutant Notch3 may gain a novel or toxic
function in VSMCs. This review will focus on recent findings of Notch3 in vascular development and in regulating the
VSMC behaviour and phenotype, and will use findings on investigating the molecular pathology of the single gene disorder
CADASIL to understand the function of Notch3 in VSMCs.
r 2007 Elsevier Ltd. All rights reserved.
Keywords: Notch3; Vascular smooth muscle cells; CADASIL; Notch signalling; Vascular development
Contents
1.
2.
3.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Notch3 function in vascular development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Notch3 on phenotypic maintenance of vascular smooth muscle cells (VSMCs) . . . . . . . . . . . . . . . . . . . . . .
3.1. Notch3 and vascular injury . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Corresponding author. Tel.: +44 161 2751783; fax: +44 161 2751811.
E-mail address: tao.wang@manchester.ac.uk (T. Wang).
0079-6107/$ - see front matter r 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.pbiomolbio.2007.07.006
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4.
5.
3.2. Notch3 and VSMC survival and apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3. Notch3 in VSMC proliferation and migration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.4. Biomechanical force and Notch3 expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Notch3 and CADASIL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1. About CADASIL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2. The molecular mechanisms of CADASIL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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1. Introduction
Genes of the Notch family encode large single-pass transmembrane receptors that transduce signals between
neighbouring cells. The Notch signalling pathway is highly evolutionarily conserved and critical for cell fate
Fig. 1. The classic Notch signalling pathway. The Notch receptor locates at cell surface as a heterodimer following protease cleavage
(S1 cleavage) during protein maturation. The extracellular domain (NECD) associates non-covalently with a membrane-tethered
intracellular domain (NICD). The NECD contains up to 36 EGF-repeats and three cysteine-rich Notch/LIN-12 repeats (LNG domain);
the NICD has a RAM domain, six tandem ankyrin repeats and a praline-, glutamate-, serine, threonine-rich sequence (PEST domain). The
interaction between the DSL-ligand (Jagged, Delta) and the Notch receptor leads to two proteolytic cleavages: S2 cleavage is mediated by
the ADAM metalloproteinase family protein TNFa-converting enzyme, TACE; the subsequent S3 cleavage within the transmembrane
domain by presenilin-dependent g-secretase results in the release of the NICD. The NICD then translocates to the nucleus, where it
interacts with CBF1/RBP-Jk to activate transcription of target genes HES and HESR (or HRT).
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determination during embryonic development (Artavanis-Tsakonas et al., 1999; Baron, 2003; Baron et al.,
2002; Bray, 2006). In mammals, there are four Notch receptors (Notch1–4), and five classic DSL (Delta and
Serrate from Drosophila and Lag-2 from C. elegans) ligands: Jagged1–2, Delta-like1, 3 and 4. These ligands are
also transmembrane proteins. Notch receptors locate at the cell surface as a heterodimer due to proteolytic
cleavage at the S1 site during maturation. The large extracellular domain (ECD) associates non-covalently
with the membrane-tethered intracellular domain (ICD). Ligand binding leads to a second proteolytic
cleavage of Notch at the S2 site in the extracellular juxtamembrane region, and the ECD is shed (Fig. 1). The
membrane-tethered ICD becomes susceptible to S3 cleavage, releasing the ICD, which translocates to the
nucleus and activates the CSL (CBF1/Su(H)/LAG1) family of transcription factors (CBF1/RBP-Jk in
mammals) thus regulating expression of the target gene hairy-and-enhancer of split (HES) and HES-related
transcriptional factors (HESR or HRT) (Artavanis-Tsakonas et al., 1999; Bardot et al., 2005; Bray, 2006).
In the cardiovascular system, Notch signalling participates in multiple aspects of vascular development
including vasculogenesis, angiogenesis, differentiation and vascular remodelling of the vascular smooth
muscle cells (VSMC) (Alva and Iruela-Arispe, 2004; Iso et al., 2003). Mutations of the Notch ligand Jagged1
cause the congenital cardiovascular abnormalities in Alagille Syndrome (OMIM#118450). In addition,
increasing evidence suggests that abnormal Notch signalling contributes to the pathogenesis of adult-onset
human diseases. For example, mis-regulation of Notch signalling has been observed in a variety of common
human cancers (Haruki et al., 2005; Nicolas et al., 2003; Stylianou et al., 2006; Weng et al., 2004). Hence,
elements of the Notch pathway are now seen as potentially important therapeutic targets (Miele et al., 2006).
Notch3 is expressed mainly in human adult VSMCs (Joutel et al., 2000; Villa et al., 2001), and NOTCH3
mutations lead to the adult-onset genetic stroke syndrome CADASIL (cerebral autosomal dominant
arteriopathy with subcortical infarcts and leukoencephelopathy) which is characterized by systemic VSMC
degeneration (Joutel et al., 1996, 1997b). Recent studies have shown that Notch3 is involved in vascular injury
(Wang et al., 2002a, b) and is a determinant of VSMC survival (Campos et al., 2002; Sweeney et al., 2004), but
its function in VSMCs is still largely unknown. The molecular mechanisms underlying CADASIL pathology
are therefore intriguing. Considering the specific expression of Notch3 in VSMCs and its pathological link to
the genetic vascular disorder CADASIL, this review will focus on recent findings of Notch3 in regulating the
VSMC phenotype, and shall use findings on investigating the molecular pathology of the single gene disorder
CADASIL to shed light on the function of Notch3 in VSMCs.
2. Notch3 function in vascular development
The Notch3-null mice have been generated using different approaches (Kitamoto et al., 2005; Krebs et al.,
2003; Mitchell et al., 2001). Surprisingly these mice are all viable and fertile, unlike the Notch1 knock out
which proved lethal in utero as a result of defects in vascular development (Conlon et al., 1995; Krebs et al.,
2000; Swiatek et al., 1994). This indicates that Notch3 is not essential for embryonic development in mice.
However, subsequent analysing the Notch3 / mice, Domenga et al. (2004) found a series anomalies in the
arteries as summarised below, which suggests that Notch3 is required to generate functional arteries by
regulating differentiation and maturation of VSMCs.
At birth (P0.5), a stage in which the blood vessels were immature in mice, arterial vessels in Notch3 /
neonates were indistinguishable from those of control littermates. However, in adult the arteries of
Notch3 / mice showed structural abnormalities including enlargement of the vessels, thinner VSMC coat
and altered VSMC shape and size (Domenga et al., 2004). These arterial defects were observed in all organs
analysed, but were milder or absent in the major elastic arteries of the trunk, suggesting an important role of
Notch3 in small resistance arteries. Interestingly, the pathological changes in CADASIL patients are also
observed mainly in small arteries rather than the great vessels supporting the finding of functional importance
of Notch3 in the small arteries in the Notch3 / mice.
Maturation of arterial vessels occurs from birth to postnatal day 28 (P28) with VSMCs undergoing changes
in both morphology and orientation. This remodelling process is strongly impaired in the Notch3 / mice.
Instead, the Notch3 / VSMCs follow a venous rather than an arterial pattern of maturation, with features
such as elongated cell shape and clusters of poorly oriented cells around the lumen (Domenga et al., 2004).
Moreover, the VSMCs in these mice have a marked reduction in the cytoskeletal components characteristic of
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mature arterial smooth muscle cells, including dense plaques and dense bodies. The Notch3 / arteries have
smooth muscle cell-specific markers, including a-smooth muscle actin (a-SMA) and smooth muscle myosin
heavy chain (SMMHC) at birth, but by P7, the a-SMA- and SMMHC-positive cells were abnormally
aggregated. This suggests that the postnatal maturation of VSMCs that lends arterial vessels their final shape
is deficient in the Notch3 / mice (Domenga et al., 2004).
The Notch3-deficient mice also exhibit impaired arterial differentiation of VSMCs, indicated by a marked
reduction in expression of the arterial-specific marker smoothlin; and by a reduced ability to stimulate arterialspecific regulatory elements within the SM22a gene promoter when this was introduced in the Notch3 /
background (Domenga et al., 2004).
Neither attenuated proliferation nor increased apoptosis or cell death is found in the neonatal VSMCs, but
interestingly, over-expression of a constitutively active form of Notch3, i.e. the intracellular domain of Notch3
(N3ICD), in cultured VSMCs results in an increase of actin stress fibres and steady-state levels of polymerized
actin (Domenga et al., 2004). Together with the fact that the postnatal arterial maturation of VSMC does not
correlate with the temporal expression of the Notch ligands, including Jagged1 (Domenga et al., 2004), but
parallels the increase in arterial blood pressure which occurs during the first month after birth (Tiemann et al.,
2003), therefore Notch3 may act as a sensor or transducer that affords VSMC the ability to rearrange the actin
cytoskeleton in response to mechanical stretching of the vessel wall by the intraluminal blood pressure
(Domenga et al., 2004). Further experiments are needed to confirm this hypothesis and understand the
mechanism by which Notch acts in this process.
Functional analysis of the arteries of Notch3 / mice reveals no difference from controls in the increase in
resting arterial pressure in response to angiotensin II and phenylephrine stimulation. However, a strongly
impaired cerebral blood flow (CBF) and cerebrovascular resistance (CVR) in the Notch3 / mice was
observed, suggesting a deficiency in autoregulation of cerebral blood flow, which is important for maintenance
of constant blood flow during variations in systemic blood pressure (Domenga et al., 2004).
Interestingly, the expression of immediate downstream target genes of Notch signalling (including HES1,
HRT1-3, but not HES5) show no differences between Notch3 / mice and control littermates indicating that
the anomalies detected in the Notch3 / mice do not seem to involve previously identified Notch target genes
(Domenga et al., 2004). This suggests that Notch3-mediated signalling might occur via an alternative pathway
in addition to the classic Notch signalling pathway and this may play an important role in arterial VSMC
development.
The expression patterns of the Notch3 and Notch1 genes partially overlap during early mouse
embryogenesis (Fortini et al., 1993), so it is possible that Notch3 and Notch1 may have genetic redundancy
which might contribute towards the unchanged expression pattern of Notch-downstream genes. When double
homozygous mutant Notch3 / and Notch1 / are introduced into mice, the double mutant embryos
exhibit characteristics of Notch1 / single mutant embryos (Krebs et al., 2003), which die in early embryonic
development at E9.5 from defects in vascular development (Krebs et al., 2000; Swiatek et al., 1994). This
lack of synergism seems to suggest that the Notch3 gene does not have redundant function with the Notch1
gene during embryogenesis.
3. Notch3 on phenotypic maintenance of vascular smooth muscle cells (VSMCs)
In human adults, Notch3 is expressed only in arterial SMCs (Joutel et al., 2000; Villa et al., 2001). The
functional importance of Notch3 in adult VSMCs is indicated by the late-onset genetic vascular condition
CADASIL caused by NOTCH3 mutations (Joutel et al., 1996, 1997b), suggesting that Notch3 may have an
additional role in maintaining the homeoatasis of VSMCs. The adult VSMCs are not terminally differentiated
and are capable of changing phenotype in response to changes in local environmental cues, including growth
factors, mechanical forces, cell–cell and cell–matrix interactions, and various inflammatory mediators (KawaiKowase and Owens, 2007; Morrow et al., 2005a). The arterial smooth muscle cells maintain the ‘‘contractile’’
phenotype in normal physiological states as a result of the balanced regulation by the expression of contractile
and synthetic genes (Proweller et al., 2005). This phenotypic stability is therefore important in maintaining the
physiological function of arteries.
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3.1. Notch3 and vascular injury
It is well established that vascular injury is followed by release of the platelet-derived growth factor
(PDGF)-BB resulting in down-regulation of VSMC marker genes including a-SMA, SMMHC and SM22a,
and accelerated VSMC growth leading to vascular remodelling (Marmur et al., 1992). Balloon injury in rat
carotid arteries results in acute down-regulation of Notch3 within 1 week postinjury in both mRNA and
protein levels which returns to normal by day 7 (Wang et al., 2002b). Similar results were found by Campos
et al. (2002) who also showed an up-regulation of Notch3 after days 7–28. Angiotensin II is a potent
vasoconstrictor and is also involved in vascular injury (Hutchinson et al., 1999). An in vitro study of primary
rat VSMCs indicates that angiotensin II and PDGF treatment result in a sustained decreased in Notch3
expression, associated with up-regulation of the Notch ligand Jagged1 and decreased expression of the Notch
target gene HESR-1 (Campos et al., 2002) suggesting that Notch3 is the downstream mediator of growth
factors in vascular injury. However, Notch3 expression does not seem to be under the direct control of PDGF
as Notch3 expression pattern did not have significant change in PDGF-B deficient mouse embryos (Prakash
et al., 2002). The down-regulation of Notch3 by angiotensin II was mediated by ERK/MAPK signalling
pathway, as this effect was specifically blocked by ERK/MAPK pathway inhibitor U0216 (Wang et al.,
2002a).
3.2. Notch3 and VSMC survival and apoptosis
Since degeneration and eventual loss of VSMCs within the arterial wall are the main pathological changes in
CADASIL, Wang et al. (2002b) postulated that Notch3 signalling might be a critical determinant of VSMC
survival. A stable cell line expressing the constitutively active intracellular domain of Notch3 (N3ICD),
generated from rat embryonic VSMCs, was resistant to Fas ligand (FasL)-induced apoptosis. This antiapoptosis function of Notch3 was mediated by up-regulating c-FLIP, an apoptotic inhibitor that prevents
binding of caspase-8 to the Fas receptor complex. This activity of Notch3 was independent of CBF1/RBP-Jk
but through the ERK/MAPK pathway, as indicated that the steady-state protein expression of the
phosphorylated (active) form of ERK/MAPK, p42/p44, was significantly higher in the N3ICD stable cells;
and the elevated c-FLIP expression was blocked by ERK/MAPK inhibitor U0216 (Wang et al., 2002b).
However, in another study, Sweeney et al. (2004) demonstrated that Notch3 modulation of VSMC apoptosis
was CBF1/RBP-Jk-dependent, and occurred by endogenous Notch3 activation. Inhibition of CBF1/RBP-Jkmediated signalling in rat VSMCs significantly increased VSMC apoptosis. The anti-apoptotic role through
classic-Notch signalling pathway was supported by a separate study from Wang et al. (2003), in which they
found that the Notch target gene HRT-1 protected serum deprivation and FasL-induced apoptosis in VSMCs,
and furthermore, they found that this anti-apoptotic function was through activation of PI3K/Akt pathway.
Taken together, the data indicate that Notch3 activation protects apoptosis and promotes VSMC survival, but
there is still a missing link between Notch3 activation and up-regulations of the survival signals. The
mechanisms described above may co-exist and function co-ordinately through network cross-talk or it
may all originate from RBP-Jk activation. Alternatively, the anti-apoptotic responses may be context and
stimuli-dependent.
3.3. Notch3 in VSMC proliferation and migration
Campos et al. (2002) described biphasic growth behaviour of an N3ICD stable cell line generated from rat
embryonic VSMCs in which the growth rate was retarded during the subconfluent phase and failed to
decelerate at postconfluence. The latter was associated with decreased expression of the cell-cycle inhibitor
p27KIP. Sweeney et al. (2004) found that Notch3 promotion of VSMC proliferation was CBF1/RBP-Jkdependent. While serum stimulates VSMC proliferation, it also up-regulates expression of Notch target genes
HRT-1 and HRT-2. Blocking RBP-Jk-mediated Notch signalling by RPMS-1 could reverse the serumstimulated VSMC proliferation, suggesting the involvement of endogenous classic Notch signalling in
promoting VSMC growth (Sweeney et al., 2004). It is not clear whether decreased p27KIP expression is a direct
downstream event of CBF1/RBP-Jk activation, and its contribution to the Notch3-induced VSMC
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proliferation remains to be determined. The serum-induced CBF1/RBP-Jk-dependant VSMC proliferation
seems to conflict with the notion that PDGF down-regulates both the Notch3 receptor and its target genes
HRT-1 and HRT-2 (Wang et al., 2002a). As components of serum are complicated containing more than just
growth factors it is difficult to compare these findings directly.It is not clear whether decreased p27KIP
expression is a direct downstream event of CBF1/RBP-Jk activation, and its contribution to the Notch3induced VSMC proliferation remains to be determined.
VSMC migration fundamentally contributes to the lesion formation in both atherosclerosis and restenosis
after angioplasty. Sweeney et al. (2004) found that constitutive activation of N3ICD inhibits VSMC
migration, and this effect was reversed following inhibition of CBF1/RBP-Jk-transcriptional activity. Notch3
activation also regulates adult VSMC differentiation. Expression of the constitutively active form of N3ICD
down-regulates SMC-specific a-actin, myosin, calponin and smoothlin in human aortic SMCs. This effect was
CBF1/RBP-Jk-dependent and mediated by Notch target gene HRT-1, -2 and -3 (Morrow et al., 2005a).
3.4. Biomechanical force and Notch3 expression
Recent data suggests that Notch3 signalling is involved in biomechanical force-induced VSMC phenotype
regulation (Morrow et al., 2005a, b). Cyclic circumferential strain (cyclic strain) affects the balance between
VSMC proliferation and apoptosis which participates in the local vascular reaction to hypertension and
stenosis of the vessel lumen. Morrow et al. demonstrated that rat VSMCs cultured under conditions of defined
equibiaxial cyclic strain exhibited a significant temporal and force-dependent reduction in Notch3 expression
and downstream signalling (Morrow et al., 2005a, b). Cyclic strain promotes SMC proliferation and induces
apoptosis and this was reversed by over-expression of N3ICD (Morrow et al., 2005b). Interestingly, cyclic
strain-induced Notch3 down-regulation was Gi-protein and MAPK-pathway-dependent as it was overcome
by pretreatment of cells with the Gi-protein inhibitor pertussis toxin and MAPK inhibitor PD98059,
respectively. This provided new evidence of the link between Notch signalling pathway and G-protein
signalling pathway.
4. Notch3 and CADASIL
4.1. About CADASIL
CADASIL, the single gene disorder caused by NOTCH3 gene mutations, is a useful model for
understanding Notch3 function in arterial SMCs. This is an adult-onset autosomal dominant vascular
disorder (Joutel et al., 1997a; Sharma et al., 2001) causing recurrent ischemic stroke, accompanied by migraine
and neurological defects, leading to dementia and premature death (OMIM#125310). The characteristic
pathological findings are progressive degeneration and eventual disappearance of VSMCs with the
accumulation of the extracellular domain of Notch3 protein (N3ECD) in the small arteries. CADASIL
arteriopathy is systemic affecting vessels in, for example, the skin (Joutel et al., 2001), the retina (Harju et al.,
2004) and the small coronary arteries (Lesnik Oberstein et al., 2003), but is more severe in the brain. In fact,
skin biopsy is used as a diagnostic test for CADASIL (Joutel et al., 2001). CADASIL is the most common
single gene disorder leading to ischaemic stroke, but is still underdiagnosed therefore its true prevalence is
unknown. There is no clear correlation between genotype and phenotype as the disease severity varies within
families.
4.2. The molecular mechanisms of CADASIL
It has been a decade since NOTCH3 was found to be the causative gene for CADASIL, but the molecular
mechanism underlying CADASIL pathology remains unclear. Data from Sweeney et al. (2004) indicate that
Notch3 regulates VSMCs growth, apoptosis, migration and differentiation via a CBF1/RBP-Jk-dependent
pathway, it is therefore plausible that the VSMC degeneration that occurs in CADASIL patients is due to
malfunction of this pathway. Numerous attempts to test this hypothesis using in vitro cell models have
compared the CBF1/RBP-Jk activity associated with the CADASIL mutants to that associated with wild type
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Notch3. No differences were detected for the majority of CADASIL mutants (Haritunians et al., 2002, 2005;
Joutel et al., 2000, 2004; Karlstrom et al., 2002; Low et al., 2006; Peters et al., 2004) (Table 1) which suggests
that CADASIL pathology is not associated with CBF1/RBP-Jk and there is an alternative Notch3-mediated
pathway that regulates the VSMC function and survival.
Notch3 protein has 2321 amino acid with a large ECD containing 34 epidermal growth factor (EGF)
repeats. Interestingly, around 70% of CADASIL mutations cluster in exons 3 and 4 (EGF 1–5) close to the
N-terminus of the protein (Joutel et al., 1997a) (Fig. 2). Two mutations (C428S and C455R) located in the
EGF-repeats 10/11, the predicted putative ligand binding site, have impaired ligand binding and signalling
(Joutel et al., 2004; Peters et al., 2004) and may represent a subset of the CADASIL mutants that differ
from the more N-terminal mutations. However, only five out of 75 reported missense mutations locate at
EGF-repeats 10/11, suggesting that the abnormal ligand Jagged/Delta binding is unlikely to be the major
mechanism underlying CADASIL pathology.
Seventy-five of the 81 reported CADASIL are missense mutations, and almost all of these loss or gain of a
cysteine residue resulting in odd numbers of cysteines in a given EGF-repeat. Normally, there are six cysteine
residues in each EGF-repeat forming three disulphide bonds. An odd number of cysteine residues will
inevitability disrupt the disulfide pairing and may lead to a conformational change of the domain, whilst the
resulting free cysteine may predispose towards oligomerisation on the cell surface that could interfere with
Notch trafficking. Notch3 protein accumulation in CADASIL arteries seems to support this prediction.
Table 1
Summary of in vitro cell expression study of mutant Notch3 proteins associated to CADASIL
Mutations
cDNA
species
Affect on RBP-Jk
activity
Affect on S1cleavage
R90C
Human
3
2
HEK293, SH-SY5Y Yes
No
No
R133C
Human
4
3
HEK293, SH-SY5Y, Yes
NIH3T3, A7r5
No
R142Ca
Mouse
4
3
HEK293
Reduced No
R171Ca
Mouse
4
4
HEK293
Yes
No
C183R
H184Ca
Human
Mouse
4
4
4
4
NIH3T3, A7r5
HEK293
Yes
Yes
No
No
C185R
C187Ra
Human
Rat
4
4
4
4
HEK293, SH-SY5Y Yes
HEK293
Yes
C212S
Human
4
5
HEK293, NIH3T3
Yes
No
Increased to
Jagged1, decreased
to Delta1, no change
to Delta4
No
No
C428S
R449C
C455R
C542Y
C544Ya
Human
Human
Human
Human
Mouse
8
9
9
11
11
10
11
11
13
13
HEK293, NIH3T3
HEK293, SH-SY5Y
NIH3T3, A7r5
HEK293, NIH3T3
HEK293
Yes
Yes
Yes
Reduced
Yes
Decreased
No
Decreased
Decreased
No
No
No
Delayed
No
–
R560Ca
Mouse
11
14
HEK293
Yes
No
–
R1006C
Human
19
26
HEK293, NIH3T3
Yes
No
No
a
Exons EGFrepeats
Expressing cells
CADASIL-like mutations in mouse or rat Notch3 cDNA.
Cell
surface
location
References
Low et al. (2006),
Joutel et al. (2000,
2004)
No (Low et al., Low et al. (2006),
2006); Delayed Peters et al. (2004)
(Peters et al.,
2004)
Reduced
Karlstrom et al.
(2002)
–
Haritunians et al.
(2002)
Delayed
Peters et al. (2004)
–
Haritunians et al.
(2002)
No
Low et al. (2006)
Reduced
Haritunians et al.
(2005)
Joutel et al. (2000,
2004)
Joutel et al. (2004)
Low et al. (2006)
Peters et al. (2004)
Joutel et al. (2004)
Haritunians et al.
(2002)
Haritunians et al.
(2002)
Joutel et al. (2004)
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Fig. 2. Schematic diagram of NOTCH3 mutations identified in CADASIL patients. The positions of CADASIL mutations in Notch3
protein are summarised. CADASIL mutations are only present in the EGF-domains and clustered in the N-terminus of the Notch3
protein. The purple bars indicate EGF-repeats 1–34, and two green bars indicate EGF-repeats 10/11 that are essential for ligand binding.
Data were collected from the Human Gene Mutation Database Cardiff (HGMD). Each dot indicates a recurrence mutation.
Interestingly, Joutel et al. (2000) found that only the extracellular domain of Notch3 accumulated in the
arteries from the CADASIL patients. It is not clear why the N3ICD do not accumulate together with
the N3ECD. There could be abnormal clearance of mutant N3ECD following ligand-induced release of the
N3ICD; or the mutant Notch3 undergoes abnormal processing and somehow releases the N3ECD
independent of ligand binding; alternatively, there may be an unidentified pathway present for Notch3 protein
trafficking and activation. Recent work has emphasised the importance of endocytic trafficking in both downregulation and activation in Drosophila Notch (Le Borgne et al., 2005; Wilkin and Baron, 2005; Wilkin et al.,
2004). Most CADASIL mutations are close to the N-terminus of the Notch protein, and away from the
putative ligand binding region (EGF10–11) (Fig. 2). Although the functions of this N-terminal region are
largely unknown, the Drosophila CADASIL-like notchoid3 Notch allele results in impaired endocytosis and
abnormal Notch accumulation (Bardot et al., 2005). This suggests that the N-terminal region may regulate the
intracellular trafficking of Notch. Very few publications have addressed the Notch3 protein trafficking with
the only published data focused on the Notch3 protein processing during its maturation. Using cell surface
biotinylation assay, it seems that almost all mutant receptors analysed were correctly located to the cell surface
(Table 1), suggesting CADASIL mutation in Notch3 did not alter the receptor maturation (Haritunians et al.,
2002, 2005; Joutel et al., 2000, 2004; Karlstrom et al., 2002; Peters et al., 2004). Comparing the ratio of
S1-cleaved N3ECD and full-length between wild type and mutant Notch3, a reduced or delayed S1-processing
was observed (Haritunians et al., 2005; Karlstrom et al., 2002; Peters et al., 2004); however, this has not been
confirmed by other studies (Joutel et al., 2004; Low et al., 2006). In cells over-expressing Notch3, a variable
ratio is observed even in the wild type, so it is difficult to draw accurate conclusions form these experiments.
The increased intracellular aggregates were also found for the mutant Notch3 in two studies (Karlstrom et al.,
2002; Low et al., 2006). As the aggregates were also seen for the wild type Notch3, although in a reduced level,
it may not reflect the real situation in vivo. It seems that there is not convincing evidence indicating an
abnormality of Notch3 receptor processing during its maturation in CADASIL, but data for the mature
receptor trafficking are lacking. Clarifying the Notch3 trafficking pathway is essential for understanding the
pathological change of CADASIL, as well as the function of Notch3 in human VSMCs.
It is not clear whether CADASIL pathology occurs as an indirect consequence of the abnormal
accumulation of the Notch3 protein, as a direct consequence of perturbed Notch signal regulation, or due to a
combination of both. The accumulated Notch3 protein may be responsible for the disease pathology through
damaging VSMCs anchorage and accelerating VSMC degeneration or it may be only a read-out of an earlier
causative defect. In the transgenic CADASIL mouse model that expresses mutant Notch3 R90C, lost
anchorage and functional impairment of VSMCs were observed before obvious GOM deposition and Notch3
accumulation (Ruchoux et al., 2003), suggesting an early event may exist that contributes to the later
pathology of CADASIL, for example, abnormal cell–cell and cell–matrix interactions and apoptosis.
However, there is no apoptotic cell been observed in the arterial SMCs in the transgenic mice (Domenga et al.,
2004), and further investigation is needed to clarify this.
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507
Notch receptor function has been shown to be regulated by O-linked glycosylation catalysed by protein
O-fucosyltransferase and Fringe (Bruckner et al., 2000; Moloney et al., 2000). It has been shown that CADASIL
mutants do not affect the addition of O-fucose but do impair carbohydrate chain elongation by Fringe, at least
in a purified Notch3 fragment containing the first five EGF-repeats (Arboleda-Velasquez et al., 2005). Given
that glycosylation of the Notch receptor is essential for its ligand binding and that the majority of CADASIL
mutants do not interfere with ligand Jagged1/Delta binding and subsequent CBF1/RBP-Jk-mediated
signalling, the contribution of Fringe in CADASIL pathology remains intriguing.
Two CADASIL transgenic mice models were generated expressing mutant Notch3 R90C and R142C,
respectively (Lundkvist et al., 2005; Ruchoux et al., 2003). The R90 mice developed characteristic CADASIL
pathology including GOM deposition and Notch3 accumulation, although this was more obvious at the tail
arteries rather than in the brain; while the R142 mice did not show any CADASIL-like phenotype. The reason
for the latter was not clear. The possible explanation favours the dependency of the CADASIL phenotype on
species or type of mutations (Lundkvist et al., 2005). A functional study on the R90C mice showed a reduced
response of cerebral arteries to vasodilators and increased vasoresistance during hypertension, suggesting an
impaired autoregulation of cerebral arteries in CADASIL (Lacombe et al., 2005). Furthermore, in vitro study
of the tail arteries in the R90C mice showed normal contraction to phenylephrine and normal endotheliumdependent relaxation to acetylcholine but impaired response to flow-induced dilation and pressure-induced
myogenic tone, indicating an early defect in mechanotransduction in CADASIL arteries (Dubroca et al.,
2005). The Drosophila Abruptex alleles has been described to be a useful CADASIL model in which the
mutations were all in EGF-repeat domain that cause loss or gain of a cysteine (Fryxell et al., 2001). However,
the Abruptex region is in the EGF-repeats 22–29 while the CADASIL mutations are cluster in the very
N-terminus of the EGF domain. The Drosophila notchoid3 Notch allele could be a better model for CADASIL
as it carries a mutation with cysteine to phenylalanine substitution in the EGF-repeat 2 (Bardot et al., 2005).
5. Conclusion
The functional importance of Notch3 signalling in human VSMCs has recently been recognised. Notch3
participates in artery maturation and specification; responses to vascular injury; regulating VSMC growth and
apoptosis. Although these functions of Notch3 have been shown to be CBF1/RBP-Jk-dependent, evidences
also suggest that there are CBF1/RBP-Jk-independent pathways involving cross-talk to other signalling
pathways. The co-ordinate regulations of the Notch3-mediated pathways in phenotypic maintenance of
VSMCs are largely unknown. The late-onset genetic systemic arteriopathy caused by NOTCH3 gene
mutations has provided a useful model for study Notch3 function and malfunction in VSMCs.
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