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First published online 16 January 2008
doi: 10.1242/dev.006718
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1 Department of Dermatology, Columbia University, New York, NY 10032, USA.
2 Departments of Biochemistry and Molecular Biophysics, and Ophthalmology,
Columbia University, New York, NY 10032, USA.
3 Department of Genetics and Development, Columbia University, New York, NY
10032, USA.
* Author for correspondence (e-mail: amc65{at}columbia.edu)
Accepted 15 November 2007
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: P-cadherin, p63, Apical ectodermal ridge, Hair follicle placode
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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).
Recently, two members of the classical cadherin family, E-cadherin and
P-cadherin, encoded by the genes CDH1 and CDH3, have been
shown to be involved in hair follicle (HF) morphogenesis
(Jamora et al., 2003). HF
development begins with the formation of a placode, which is composed of
groups of rearranged epidermal cells, and subsequent formation of mesenchymal
condensate just beneath the placode. The interaction between these specific
structures further induces the downgrowth of the placode
(Schmidt-Ullrich and Paus,
2005). In the epidermis, E-cadherin is abundantly expressed in the
basal and suprabasal layers, while P-cadherin is expressed only weakly in the
basal layer (Hirai et al.,
1989). As the HF placode begins to form, the expression level of
E-cadherin markedly decreases, whereas that of P-cadherin becomes much
stronger in the placode than in the interfollicular epidermis
(Jamora et al., 2003). The
downregulation of E-cadherin, as well as the predominant expression of
P-cadherin, continues throughout HF development
(Muller-Rover et al., 1999).
This `cadherin switch' has been considered to be essential to change cell-cell
contacts and potentially facilitate cell movement
(Jamora et al., 2003).
The importance of P-cadherin in HF morphogenesis has been further
highlighted by recent evidence that human P-cadherin mutations are associated
with two congenital diseases affecting the HF. Recessively inherited mutations
in the human CDH3 gene were identified in affected individuals with
hypotrichosis with juvenile macular dystrophy (HJMD) characterized by
congenital sparse hair and early blindness due to macular dystrophy of the
retina (Sprecher et al., 2001;
Indelman et al., 2002;
Indelman et al., 2003;
Indelman et al., 2005;
Indelman et al., 2007). More
recently, mutations in the CDH3 were also reported to cause another
autosomal recessive human disease in two families: ectodermal dysplasia,
ectrodactyly and macular dystrophy (EEM syndrome)
(Kjaer et al., 2005). Patients
with EEM syndrome exhibit hair and eye symptoms similar to those with HJMD;
however, they show the additional phenotype of a severe split hand/foot
malformation (SHFM) or ectrodactyly.
Previous studies have clearly demonstrated the expression of P-cadherin in
the HF placode (Jamora et al.,
2003) and retinal pigmented epithelium (RPE) of the eye
(Xu et al., 2002) during mouse
embryogenesis, which corresponds to the sites of the human phenotype caused by
dysfunction of P-cadherin. To date, however, the precise expression of
P-cadherin during limb formation has not been determined. The development of
the HF and limb bud share some characteristic features. Several common
molecules such as Wnt, sonic hedgehog and bone morphogenic proteins contribute
to generate both structures (Duijf et al.,
2003). Morphologically, the apical ectodermal ridge (AER), which
is the thickened rim of ectoderm at the tip of limb bud, may correspond in
some ways to the HF placode. The AER mediates limb bud outgrowth via the
interaction with underlying mesenchyme
(Sanz-Ezquerro and Tickle,
2001).
Interestingly, the only other gene known to cause SHFM in humans is
p63. Mice that lack p63 have striking developmental defects and die
shortly after birth from dehydration. They lack stratified squamous epithelia
and associated appendages (Mills et al.,
1999; Yang et al.,
1999). They also display severe defects in limb and craniofacial
development, including cleft lip. Humans that possess dominant mutations in
p63 similarly have dramatic developmental defects, depending on the
location of the mutation within the p63 gene. One example is
ectrodactyly-ectodermal dysplasia-cleft lip/palate (EEC) syndrome. Most
patients who are affected with EEC have mutations that abolish the ability of
p63 to bind to DNA (Celli et al.,
1999). Other syndromes that result from mutations in p63 include
ankyloblepharon-ectodermal dysplasia-clefting (AEC) syndrome,
acro-dermato-ungual-lacrimal-tooth syndrome, non-syndromic SHFM and
limb-mammary syndrome (Rinne et al.,
2006). All of these syndromes are autosomal dominant and can
exhibit SHFM, with the exception of AEC.
The transcription factor p63 is primarily expressed in epithelia. It was
first identified as a member of the p53 family
(Augustin et al., 1998;
Yang et al., 1998;
Osada et al., 1998). p53 is a
tumor suppressor that is mutated in many human cancers. Although there is a
high degree of similarity in sequence and structure between p53 and p63, they
are functionally divergent. p63 can be expressed from two different promoters
and transcriptional start sites, generating the TA and 
N isoforms of
p63. Each isoform has three additional splice variants, designated 
,
β and 
(Yang et al.,
1998). All p63 proteins possess a DNA-binding domain, which is 60%
identical to that of p53. The TAp63 isoforms additionally have an N-terminal
transactivation region, which is only weakly homologous (22%) to that of p53.
The Np63 isoforms are truncated in this region. The diversity of
isoforms, splice variants and expression suggests complexity in the function
of p63.
The role of p63 during development is incompletely understood. The two p63
isoforms are differentially expressed in embryonic mouse epidermis. One model
is that the TAp63 isoform, which is expressed earlier in development, has a
role in the commitment to epithelial stratification, whereas the Np63
is involved in terminal differentiation
(Koster et al., 2004;
Koster et al., 2005). Although
the expression patterns of p63 in normal epithelia as well as in epithelial
tumors have been determined in part
(Nylander et al., 2000;
Nylander et al., 2002;
Di Como et al., 2002), the
targets of p63 regulation have remained largely elusive. Recently, however,
several studies using microarrays, chromatin immunoprecipitation and/or RNA
interference approaches have identified several candidate p63-target genes,
such as EGFR, ICAM3 and β4 integrin
(Carroll et al., 2006;
Barbieri et al., 2006;
Vigano et al., 2006;
Yang et al., 2006;
Testoni et al., 2006).
Interestingly, these studies have gradually disclosed that p63 regulates gene
expression programs involved in cell adhesion
(Carroll et al., 2006).
p63 and CDH3 are the only genes that have been known to be responsible for SHFM in humans. At present, the CDH3 mutations reported so far are limited, and the genes that control the expression of P-cadherin remain unknown. The phenotypic overlap led us to hypothesize that p63 might be a transcriptional regulator of CDH3. In this study, we identified pathogenic mutations in the CDH3 of five consanguineous Pakistani families affected with either HJMD or EEM syndrome. In addition, we determined the expression pattern of P-cadherin during hair and limb development in the mouse embryo. Finally, we show that P-cadherin is a direct transcriptional target gene of p63.
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MATERIALS AND METHODS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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), all
exons of CDH3 with adjacent sequences of exon-intron borders were
amplified by PCR. The PCR products were directly sequenced on an ABI Prism 310
Automated Sequencer, using the ABI Prism BigDye Terminator Cycle Sequencing
Ready Reaction Kit (PE Applied Biosystems).
Indirect immunofluorescence and whole-mount immunohistochemistry
Cryosections were prepared from C57BL/6J embryos at E9.5 (9.5 days post
coitus, day of plug considered 0.5 days), E10.5, E15.5 and E17.5. Sections
were fixed in cold methanol:acetone (1:1) for 7 minutes, followed by blocking
with 10% normal goat serum in PBS for 1 hour at room temperature. Primary
antibodies used were anti-P-cadherin rat monoclonal PCD-1 (1:100; ZYMED
Laboratories), anti-p63 rabbit polyclonal H-129 (1:200; Santa Cruz
Biotechnology), anti-N-cadherin rabbit polyclonal H-63 (1:200; Santa Cruz
Biotechnology) and mouse monoclonal anti-E-cadherin (1:200; BD Biosciences).
After the incubation with primary antibodies, FITC and/or Alexa-Fluor-labeled
secondary antibodies (Jackson ImmunoResearch) were applied to the sections.
Nuclei were counterstained with DAPI. The images were processed with Adobe
Photoshop (version 8.0) software. Whole-mount immunohistochemistry was
performed as previously described (Yoshida
et al., 1996) with some modifications. C57BL/6J embryos were
incubated with anti-P-cadherin antibody PCD-1 (1:100), and subsequently with 1
µg/ml HRP-conjugated anti-rat-IgG antibody. Antibody binding was visualized
with diaminobenzidine substrate.
p63-expression vectors
Expression constructs encoding c-myc-tagged mouse TAp63,
TAp63, Np63 and Np63 were kindly provided
by Drs Dennis Roop and Maranke Koster (University of Colorado, Aurora,
Colorado, USA). To generate the mutant (R280C) TAp63, the upstream
coding region of TAp63 was PCR-amplified using the forward primer
(5'-AAAAGGATCCCTCGCAGAGCACCCAGACAAG-3') and the reverse primer
(5'-ATGATTAAAATTGGACATCTGTTCA-3') and wild-type
TAp63 expression construct as a template. Note that the mutation of
interest was introduced into the reverse primer as underlined. The remaining
coding region of TAp63 was also amplified by PCR using the forward
primer (5'-AATTTTAATCATCGTTACTCTGGAAAC-3') and the reverse primer
(5'-AAAACTCGAGCTATGGGTACACGGAGTGGT-3'). The two amplified
fragments were then ligated at the MseI restriction enzyme site,
which was subcloned in frame into the BamHI and XhoI sites
of pCMV-Tag3 vector (Stratagene).
Transient transfections and quantitative PCR analysis
HEK293 (human embryonic kidney epithelial) cells were plated in 12-well
plates the day before transfection. Expression plasmids of TAp63,
TAp63, Np63 and Np63 (1.6 µg each) were
transfected with Lipofectamine 2000 (Invitrogen) according to the
manufacturer's recommendations. Empty vector was also transfected as a
control. Twenty-four hours after transfection, total RNA was isolated from the
cells using the RNeasy Minikit (Qiagen). Total RNA (2 µg) was
reverse-transcribed using random primers and SuperScript III (Invitrogen).
Real-time PCR was performed on an ABI 7300 (Applied Biosystems). PCR reactions
were performed using ABI SYBR Green PCR Master Mix, 300 nM primers, 200 ng
cDNA at the following consecutive steps: (1) 50°C for 2 minutes; (2)
95°C for 10 minutes; (3) 40 cycles of 95°C for 15 seconds and 60°C
for 1 minute. The samples were run in triplicate and normalized to an internal
control (GAPDH) using the accompanying software. The following
primers were used: CDH3 (forward
5'-CGAAGAGGACCAGGACTATGA-3', reverse
5'-GTCTGTGTTAGCCGCCTTCA-3'), GAPDH (forward
5'-TCACCAGGGCTGCTTTTAACTC-3', reverse
5'-GGGTGGAATCATATTGGAACATG-3'). The transfection and real-time PCR
experiments were performed in triplicate. Statistical analysis was performed
using the ANOVA procedure, followed by the Dunnett's t-test.
P<0.05 was considered significant.
Promoter analyses and reporter gene assays
The whole genome rVISTA tool
(http://genome.lbl.gov/vista/index.shtml)
evaluates conservation of genomes between pairs of species (specified as mouse
and humans) and pinpoints transcription-factor-binding sites that are
over-represented in upstream regions of genes of interest. PCR-amplified
CDH3 promoter regions were cloned into the NheI and
XhoI sites of the pGL3 basic luciferase reporter vector (Promega). To
generate the mutated constructs, mutations of interest were introduced into
the forward primers. HeLa (human cervical cell carcinoma) cells were seeded in
a 12-well plate and the next day transfected with the appropriate plasmids
using Lipofectamine 2000 (Invitrogen). A constitutive β-galactosidase
reporter was used for normalization of transfection efficiency. The cells were
lysed 24 hours after transfection and the signals were assayed using the
appropriate substrates for luciferase (Steady-Glo Luciferase Assay System) and
β-galactosidase (Promega) on a 20/20n luminometer (Turner
Biosystems) for luciferase and a Model 680 microplate reader (BioRad) for
β-galactosidase.
Chromatin immunoprecipitation (ChIP)
HEK293 cells were seeded at 1x106 per 60 mm dish and
transfected 24 hours later with 8 µg TAp63 or TAp63
expression vector, or empty vector as described above. After 24 hours, the
cells were fixed in 1% formaldehyde for 10 minutes and the Chromatin
Immunoprecipitation Kit (Upstate) was used per the manufacturer's
recommendations. In addition, HaCaT (human epidermal keratinocytes) cells were
cultured in a 100 mm dish and used for in vivo ChIP. An anti-p63 antibody
mouse monoclonal 4A4 (Santa Cruz Biotechnology) was used for
immunoprecipitation. Cell lysates were also precipitated with normal mouse IgG
as a negative control. PCR was performed using Platinum Taq DNA Polymerase
High Fidelity (Invitrogen). The amplification conditions for each PCR were
94°C for 2 minutes, followed by 33 cycles of 94°C for 30 seconds,
55°C for 30 seconds and 68°C for 30 seconds, with a final extension at
68°C for 7 minutes. p21 is a bona fide target of p63 and was used
as a positive control (Shimada et al.,
1999). The following primers were used: p21 promoter
(-1519 to -1245; forward 5'-GCAGTGGGGCTTAGAGTGGGG-3', reverse
5'-CAGGCTTGGAGCAGCTACAATTAC-3')
(Osada et al., 2005),
CDH3 promoter-region 1 (-2246 to -2000; forward
5'-TGCCCGAGAAGGAGGCCGGAAATG-3', reverse
5'-GCTTTTTATGCCCGAGGGGAGGT-3'), CDH3 promoter-region 2
(+6 to +227; forward 5'-TGTAGCCGCGTGTGGGAGGA,reverse
5'-TCTCGCAGTTCTG GGTTAGAG GAG). A 248 bp fragment spanning exon 3 of the
CDH3 gene was also amplified as a negative control using the primers
described previously (Kjaer et al.,
2005).
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Identification of three novel and one recurrent mutation in the CDH3 gene
Using genomic DNA from members of the five Pakistani families, we performed
mutation analysis and identified pathogenic mutations in the CDH3
gene in all five families. First, we found a novel homozygous mutation 490insA
in exon 5 of CDH3 in affected individuals of Family 1
(Fig. 1B). Mismatch
allele-specific PCR showed that 50 unrelated healthy control individuals did
not possess the mutation (data not shown). Next, affected individuals in both
Families 2 and 3 were homozygous for a novel mutation Ivs10-1G>T in
CDH3 (Fig. 1C). This
mutation abolished a BpmI restriction enzyme site, which was used to
exclude the mutation from 50 control individuals (data not shown). Affected
individuals in Family 4 were found to carry a novel homozygous substitution
353A>G in CDH3, which results in a substitution of glutamic acid
by glycine at amino acid 118 (Fig.
1D). Multiple amino-acid sequence alignments showed that the
glutamic acid at position 118 is completely conserved among P-cadherin of
other species, as well as other human classical cadherins
(Fig. 1D). In addition, the
mutation generated a BstNI restriction enzyme site, which was used to
exclude the possibility that it represents a common polymorphism (data not
shown). Finally, patients in Family 5 possessed a homozygous transition
965A>T (N322I) (Fig. 1E),
which was previously detected in a Danish family with EEM syndrome
(Kjaer et al., 2005).
P-cadherin is expressed in the hair follicle placode and the AER of limb bud, overlapping with p63
In order to determine the precise expression pattern of P-cadherin in the
developing HF, eye and limb bud, we performed immunostaining using
cryosections of whole mouse embryos. At E15.5, P-cadherin was strongly
expressed in the HF placode, wheareas it was only weakly expressed at basal
layer of the interfolliclular epidermis
(Fig. 2A). In addition,
P-cadherin expression was detected in the RPE of the developing eye at E17.5
(Fig. 2B). These results are
consistent with previous studies by others
(Jamora et al., 2003;
Xu et al., 2002). In forelimb
bud at E9.5 (Fig. 2C) and E10.5
(Fig. 2D), P-cadherin was
strongly expressed throughout the AER. The positive signal was also detected
in dorsal and ventral surface ectoderm around the AER, but the expression was
more prominent in the AER (Fig.
2C,D). At E15.5, P-cadherin was ubiquitously expressed at the
epithelial cell junctions of the forelimb digits
(Fig. 2E,F). We also performed
whole-mount immunohistochemistry using E9.5 embryos, which precisely confirmed
the expression of P-cadherin at the AER of forelimb buds
(Fig. 2G,H).
Double immunostaining with antibodies for P-cadherin and p63 clearly demonstrated overlapping expression of the two proteins in the HF placode (Fig. 3A,B). In addition, p63 was strongly expressed throughout the nuclei of the forelimb bud epithelium, especially in the AER (Fig. 3C,D), which overlaps with P-cadherin expression (Fig. 3E-H).
In addition to P-cadherin, we analyzed the expression of two other classical cadherin members in the developing mouse limb bud. At E9.5, E-cadherin was markedly expressed in the AER of forelimb bud (Fig. 4A), and its expression almost completely overlapped with that of P-cadherin (Fig. 4B,C). The strong expression of E-cadherin was similarly detected throughout the AER at E10.5 (Fig. 4D,E). N-cadherin expression was widely detected in the mesenchyme, but not in the overlying epithelium (Fig. 4F). In particular, the expression of N-cadherin was prominent just beneath the AER and never overlapped with E-cadherin expression (Fig. 4G,H).
p63 induces the expression of the P-cadherin (CDH3) gene
In order to investigate the role of p63 isoforms in regulating the
expression of CDH3, we tested the ability of p63 to induce
CDH3 expression. HEK293 cells were transfected with TAp63,
TAp63, Np63, Np63 or empty vector, and the
relative CDH3-transcript endogenous levels were examined by real-time
PCR. Cells transfected with TAp63 isoforms showed significant increase in the
expression of CDH3 (Fig.
5). In addition, cells transfected with Np63 isoforms also
exhibited a moderate, albeit statistically significant, increase
(P<0.05) in CDH3 expression
(Fig. 5).
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and
Np63. As shown in Fig.
6B, both TAp63 and Np63 markedly upregulated
luciferase activity (more than 25-fold relative to control).
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). We
analyzed the sequence of Regions 1 and 2 and found five and two sites,
respectively, that show high homology (7/10 to 9/10) with the sequence
`RRRCWWGYYY', which could be regarded as potential p63-binding sites
(Fig. 6C,D). Indeed, both
regions were sufficient to show a significant increase in transcription by p63
when they were truncated to 
230 bp fragments containing these potential
p63-binding sites (Fig. 6E). By
contrast, we noted that p53 did not induce transactivation of the
CDH3 in any of the constructs analyzed (data not shown). Of the five
potential p63-binding sites in Region 1, the downstream two sites partially
overlapped with each other at -2088/-2070
(Fig. 6C). We focused on these
successive binding sites and further analyzed them in detail.
In order to test each binding site, we compared the activities of the
CDH3-2106/-2004-Wild, CDH3-2106/-2004-M1 (both sites
were mutated), CDH3-2106/-2004-M2 (only the upstream site was
mutated), and CDH3-2106/-2004-M3 (only the downstream site was
mutated) constructs in the presence of TAp63
(Fig. 6F). All three mutated
constructs showed markedly decreased luciferase activity compared with
CDH3-2106/-2004-Wild construct, indicating that both sites are
required for the transactivation by TA-p63
(Fig. 6F). In addition, we also
introduced mutations into the putative binding site at +31/40 of Region 2 and
compared the induction of transcription by TAp63 between
CDH3+1/+124-Wild and CDH3+1/+124-Mutant constructs
(Fig. 6G). Significant
reduction in luciferase activity was detected using the
CDH3+1/+124-Mutant construct
(Fig. 6G).
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(R280C) mutant fails to transactivate CDH3 expression; Ianakiev et al.,
2000). In particular, missense mutations at codon 279 or 280 have
been shown to cause SHFM at a high frequency
(Rinne et al., 2006).
Therefore, we generated a construct expressing TAp63 with the common
SHFM mutation R280C, and tested whether this mutation affects the
CDH3 expression. The mutant TAp63 did not activate the
reporter activity in either Region 1 (Fig.
7A) or Region 2 (Fig.
7B). Furthermore, in both regions, wild-type TAp63-induced
transactivation of the CDH3 reporter was markedly inhibited by the
mutant TAp63 in a dose-dependent manner
(Fig. 7A,B).
p63 binds directly to two regions in the CDH3 promoter
We investigated the ability of p63 to bind to the CDH3 promoter
using ChIP assays. First, we overexpressed both TAp63 and TAp63
isoforms in HEK293 cells, and precipitated the DNA-protein complexes with
anti-p63 4A4 antibody. The precipitated DNA fragments were PCR-amplified. In
the cells in which TAp63 and TAp63 were induced, anti-p63
antibody clearly immunoprecipitated both Region 1 and Region 2 of the
CDH3 promoter, which contain putative p63-binding sites, whereas it
did not immunoprecipitate the CDH3 coding region
(Fig. 8A). Finally, we
performed in vivo ChIP using HaCaT cells that predominantly express
Np63 isoform (Vigano et al.,
2006). Similarly, both Region 1 and Region 2 were PCR-amplified
from the sample precipitated with anti-p63 antibody
(Fig. 8B). These results
demonstrate that both TAp63 and Np63 isoforms directly bind to two
distinct regions within the CDH3 promoter.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Kjaer et al., 2005). In this
study, we identified five consanguineous Pakistani families (Families 1-5)
affected with either HJMD or EEM syndrome and found mutations in the
CDH3 in all five families. The mutation 490insA identified in Family
1 with HJMD is a novel insertion mutation that results in a frameshift at
codon 164 and premature termination codon (PTC) at codon 171
(Fig. 1B). The mutation
Ivs10-1G>T found in Families 2 and 3 with HJMD is a novel splice site
mutation that is predicted to cause out of frame skipping of exon 11, 146 bp
in size, and generate a PTC within exon 12
(Fig. 1C). It is possible that
truncated proteins lacking most of the essential domains could be generated
from both the 490insA and the Ivs10-1G>T mutant alleles. Alternatively, the
aberrant CDH3-transcripts containing the PTC might be largely
degraded due to nonsense-mediated messenger RNA decay
(Maquat, 1996;
Frischmeyer and Dietz, 1999).
E118G is a novel missense mutation detected in Family 4 with EEM syndrome
showing severe SHFM (Fig. 1D).
The glutamic acid at position 118 (E118), which corresponds to E11 in the
mature protein, is a highly conserved amino acid residue within the EC1 domain
of P-cadherin (Fig. 1D,
Fig. 9). The side chain of E11
coordinates calcium in the EC1-EC2 calcium-binding site
(Fig. 9), and without this side
chain, the structure of the calcium-binding site will be compromised. Calcium
binding rigidifies cadherin ectodomains, which is thought to geometrically
constrain cadherins so as to interact preferentially with cadherins from
apposing cells rather than the same cell
(Boggon et al., 2002;
Patel et al., 2006). The
mutation E118G may thus enable the interaction of mutant proteins on the same,
rather than opposing, cell surfaces, thereby negating adhesive function. The
mutation N322I found in Family 5 with EEM syndrome is a non-conservative amino
acid change in the EC2-EC3 calcium-binding site
(Fig. 9), which was previously
identified in a Danish family with EEM syndrome
(Kjaer et al., 2005). It is
noteworthy that the affected individuals in the Danish family exhibited more
severe hand involvement (Kjaer et al.,
2005) than those in Family 5
(Fig. 1A). In addition, the
severity is significantly different between two affected individuals in Family
5 (Fig. 1A). These data
indicate that the N322I shows variation in severity between different families
and even between members in a family, even though the same organs (hair,
retina and hand/foot) are involved in both families.
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), while
it led to EEM syndrome in another family
(Kjaer et al., 2005), which
suggests that an identical mutation can give rise to different phenotypes
between families (Fig. 9).
Nevertheless, in HJMD families, all affected individuals have hair and eye
involvement only (Sprecher et al.,
2001; Indelman et al.,
2002; Indelman et al.,
2003; Indelman et al.,
2005; Indelman et al.,
2007). Similarly, in EEM families, all affected individuals show
not only hair and eye phenotype but also hand/foot involvement
(Kjaer et al., 2005). An
obvious genotype-phenotype correlation between CDH3 mutations has yet
to emerge.
Balanced expression of classical cadherin members might be crucial for limb bud development
To our knowledge, the expression of P-cadherin in the limb bud has not been
previously reported. Here, we clearly demonstrated that P-cadherin expression
is upregulated in the AER of the limb bud in the developing mouse embryo
(Fig. 2C-H). Our data suggest
that P-cadherin plays an important role in outgrowth of limb bud, perhaps akin
to its role in the downgrowth of the HF placode. In addition to P-cadherin, we
also examined the expression of N- and E-cadherins in the limb bud. Like
previous reports (Yajima et al.,
2002), we showed that N-cadherin expression is detected in
mesenchyme underlying the AER (Fig.
4F-H). Surprisingly, E-cadherin was strongly expressed in the AER,
overlapping with P-cadherin (Fig.
4A-E), suggesting that the E-cadherin to P-cadherin switch does
not occur in the AER of the developing mouse embryo as it does in the HF
placode.
The identification of mutations in the human CDH3 gene underscores
the involvement of P-cadherin in the human HF, eye and limb development.
Despite these findings in humans, it is somewhat surprising that
P-cadherin-knockout mice, which are on a C57BL/6 background, show only
precocious mammary gland development, whereas hair, eye and limb anomalies are
not observed in these animals (Radice et
al., 1997). These differences may be due to the influence of some
modifier genes and/or genetic background in the animal models. The strong
expression of E-cadherin in the AER raises the possibility that E-cadherin
could play a role as a modifier and compensate for the P-cadherin deficiency
in the mouse model. In addition, as it is known that the sensitivity to
teratogen-induced limb malformations is variable among different mouse strains
(Shimizu et al., 2007), there
is the possibility that P-cadherin deficiency might cause more severe
anomalies, including hair and limb phenotypes on different mouse strains.
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) and mouse
(Mills et al., 1999;
Yang et al., 1999). In
particular, SHFM caused by human p63 mutations is phenotypically similar to
that in affected individuals with CDH3 mutations. Our expression
studies, as well as the phenotypic similarity, prompted us to postulate that
P-cadherin could be a direct transcriptional target of p63. In order to test
this hypothesis, we first examined whether P-cadherin expression is affected
when p63 is overexpressed in 293 cells. We found that both TAp63 and
Np63 isoforms upregulated the expression of the CDH3 mRNA
(Fig. 5). Osada et al. have
recently shown that a total of 129 genes were activated more than four-fold
when TAp63 was ectopically expressed in 293 cells, and CDH3
was found among these genes (8.1-fold upregulation)
(Osada et al., 2005).
Subsequently, we focused on p63 isoforms and performed promoter
assays using the luciferase reporter gene. We identified two distinct regions
(Regions 1 and 2), which were significantly responsive in reporter gene
expression analysis by both TAp63 and Np63
(Fig. 6A,B,E). The reporter
gene activity in the longest construct containing both regions was lower than
that of a shorter construct with either region
(Fig. 6B). A suppressor element
might be present in the region between Regions 1 and 2, thereby repressing the
expression of P-cadherin. Furthermore, a mutant (R280C) TAp63 inhibited
the transactivation by the wild-type TAp63 in a dose-dependent manner
in both regions (Fig. 7A,B),
indicating that the mutant TAp63 would cause a dominant-negative effect
to the wild-type TAp63 and thereby reduce P-cadherin expression.
Regions 1 and 2 have a total of seven potential p63-binding sites
(Fig. 6C,D). Even though they
show high homology with the canonical p53-binding sequence (RRRCWWGYYY), p53
did not activate the luciferase expression in either region (data not shown),
indicating that these binding sites are specific to p63. Four of seven binding
sites possess a CCTG core sequence (Fig.
6C,D), which has been found in the promoter regions of recently
identified p63-target genes as well (Yan
and Chen, 2006; Romano et al.,
2006). We generated mutated constructs in which mutations were
introduced to abolish the core sequence, and showed significant reduction of
transactivation compared with wild-type constructs
(Fig. 6F,G). These data
indicate that the CCTG core sequence represents a p63-specific element in
addition to the recently identified CGTG core sequence
(Osada et al., 2005). Finally,
we performed a ChIP assay using HEK293 cells in which either TAp63 or
TAp63 was induced, and demonstrated that TAp63 isoforms bind to both
Regions 1 and 2 of the CDH3 promoter containing potential p63-binding
sites (Fig. 8A). Furthermore,
we also performed in vivo ChIP using HaCaT cells in which endogenous
Np63 is strongly expressed, and clearly showed that p63,
especially Np63, binds to the CDH3 promoter in vivo
(Fig. 8B). Collectively, our
data indicate that P-cadherin is a direct transcriptional target of p63.
The interaction between p63 and P-cadherin gene is crucial for HF and limb bud development in humans
Using large-scale genomic approaches, a number of potential p63-target
genes have been predicted by several groups
(Carroll et al., 2006;
Barbieri et al., 2006;
Vigano et al., 2006;
Yang et al., 2006;
Testoni et al., 2006). Of
these, Carroll et al. showed the upregulation of CDH3 expression when
TAp63 was overexpressed in MCF-10A (normal human breast epithelial)
cells (Carroll et al., 2006).
In addition, Yang et al. performed ChIP using an anti-p63 antibody and ME180
(human cervical carcinoma) cells, which was followed by microarray studies
(Yang et al., 2006). As a
result, approximately 5800 p63-target sites were identified in the whole
genome. One of these sites corresponded to intron 2 of the CDH3,
which is not conserved between human and mouse, and is distant from the
transcription start site. These data, together with Osada et al.
(Osada et al., 2005),
suggested a potential relationship between p63 and the CDH3. For the
first time, definitive evidence to prove that the CDH3 is a direct
target of p63 is provided in this study, and our results are consistent with
the recent report that implicates p63 as a key regulator of broader cell
adhesion gene expression programs (Carroll
et al., 2006). Recently, Senoo et al. showed that one role of p63
is to control programmed cell death and maintain the proliferative potential
of embryonic and adult stem cells (Senoo
et al., 2007). p63 might prevent cell death through regulating the
expression of some adhesion molecules that are essential for epithelial
integrity and maintenance of proliferation
(Blanpain and Fuchs, 2007).
P-cadherin might be involved in this mechanism.
During mouse embryogenesis, TAp63 isoforms have been shown to be expressed
much more strongly than Np63 isoforms
(Koster et al., 2004;
Radoja et al., 2007), whereas
Np63 isoforms are predominantly expressed in the basal layer of the
epidermis and govern basal-epidermal gene expression (Laurikkaka et al., 2006;
Candi et al., 2006). Recently,
a TAp63-specific knockout mouse, in which Np63 isoforms are expressed
normally, has been reported to show neither hair nor limb anomalies
(Suh et al., 2006). In our
study, we showed that not only TAp63, but also Np63 isoforms, bind to
the CDH3 promoter and activate CDH3 expression. We conclude
that TAp63 isoforms, together with Np63, play an essential role in
maintaining the P-cadherin expression in the AER during limb bud outgrowth, as
well as in the HF placode. As p63 is more broadly expressed than P-cadherin,
not only p63 but also other factors are likely to play a role in regulating
P-cadherin expression in the HF placode and the AER. Nevertheless, the
phenotypic similarities caused by p63 and P-cadherin mutations, as well as our
results, strongly suggest that the interaction between p63 and P-cadherin gene
is crucial for HF and limb bud development in humans, whereas it does not
appear to be essential for either in mice. We have defined a functional
relationship between p63 and P-cadherin, and also determined their role in
failure of the HF placode and the AER that can lead to sparse hair and SHFM
when either gene is mutated in humans.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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