Báo cáo khoa học: All-trans-retinoic acid inhibits collapsin response mediator protein-2 transcriptional activity during SH-SY5Y neuroblastoma cell differentiation pptx

All-trans-retinoic acid inhibits collapsin response mediator
protein-2 transcriptional activity during SH-SY5Y
neuroblastoma cell differentiation
Lorena Fonta
´
n-Gaba
´
s
1
, Erik Oliemuller
2
, Juan Jose
´
Martı
´
nez-Irujo
1,2
, Carlos de Miguel
1
and
Ana Rouzaut
1,2
1 Department of Biochemistry, University of Navarra, Pamplona, Spain
2 Division of Oncology, Center for Applied Medical Research, University of Navarra, Pamplona, Spain
Neural cells migrate and differentiate during brain
development in a highly regulated fashion [1]. During
this process, neurons orient their axons towards their
functional effectors in a coordinated way. The family
of semaphorin proteins (previously called collapsins)
play a significant role in axonal pathfinding, through
their action as chemorepellents [2]. Collapsin response

mediator protein-2 (CRMP-2), also known as dihydro-
pyrimidinase-like 2, is a member of a family of cyto-
plasmic proteins [3] that were originally identified as
Keywords
CRMP-2; gene regulation; neuroblastoma;
promoter; retinoic acid
Correspondence
A. Rouzaut, Center for Applied Medical
Research, University of Navarra, Av. Pı
´
o XII
55, 31008 Pamplona, Spain
Fax: +34 948 19 47 18
Tel: +34 948 19 47 00
E-mail:
(Received 14 July 2006, revised 9 October
2006, accepted 16 November 2006)
doi:10.1111/j.1742-4658.2006.05597.x
Neurons are highly polarized cells composed of two structurally and func-
tionally distinct parts, the axon and the dendrite. The establishment of this
asymmetric structure is a tightly regulated process. In fact, alterations in
the proteins involved in the configuration of the microtubule lattice are fre-
quent in neuro-oncologic diseases. One of these cytoplasmic mediators is
the protein known as collapsin response mediator protein-2, which interacts
with and promotes tubulin polymerization. In this study, we investigated
collapsin response mediator protein-2 transcriptional regulation during all-
trans-retinoic acid-induced differentiation of SH-SY5Y neuroblastoma cells.
All-trans-retinoic acid is considered to be a potential preventive and thera-
peutic agent, and has been extensively used to differentiate neuroblastoma
cells in vitro. Therefore, we first demonstrated that collapsin response medi-

ator protein-2 mRNA levels are downregulated during the differentiation
process. After completion of deletion construct analysis and mutagenesis
and mobility shift assays, we concluded that collapsin response mediator
protein-2 basal promoter activity is regulated by the transcription factors
AP-2 and Pax-3, whereas E2F, Sp1 and NeuroD1 seem not to participate
in its regulation. Furthermore, we finally established that reduced expres-
sion of collapsin response mediator protein-2 after all-trans-retinoic acid
exposure is associated with impaired Pax-3 and AP-2 binding to their con-
sensus sequences in the collapsin response mediator protein-2 promoter.
Decreased attachment of AP-2 is a consequence of its accumulation in the
cytoplasm. On the other hand, Pax-3 shows lower binding due to all-trans-
retinoic acid-mediated transcriptional repression. Unraveling the molecular
mechanisms behind the action of all-trans-retinoic acid on neuroblastoma
cells may well offer new perspectives for its clinical application.
Abbreviations
ActD, actinomycin D; ATRA, all-trans-retinoic acid; CRMP-2, collapsin response mediator protein-2; EMSA, electrophoretic mobility shift
assay.
498 FEBS Journal 274 (2007) 498–511 ª 2006 Foundation for Applied Medical Research (FIMA) Journal compilation ª 2006 FEBS
mediators of semaphorin-induced growth cone collapse
[4]. This protein is abundant in the distal part of the
rising axon [5], where it interacts with tubulin heterodi-
mers, allowing its polymerization in such a way that it
seems crucial for axonal growth and for the determin-
ation of axon–dendrite fate. Moreover, overexpression
of CRMP-2 induces the formation of multiple axons,
whereas a dominant negative mutant of CRMP-2
inhibits the formation of the primary axon, compromi-
sing normal cell development [6].
The molecular mechanisms through which CRMP-2
associates preferentially with tubulin heterodimers have

recently been described [7,8]. CRMP-2 is sequentially
phosphorylated at several serine and threonine resi-
dues, leading to a reduced affinity for tubulin dimers.
However, CRMP-2 can regulate axonal growth
through different mechanisms; the data available in the
literature point to a panoply of CRMP-2-interacting
proteins, such as Numb, a protein associated with
endocytosis and membrane recycling [9]. It has also
been shown that CRMP-2 can interact with actin in
a phosphorylation status-independent manner [10].
Therefore, CRMP-2 seems to be involved in neuronal
differentiation, modulating cytoskeletal organization
and endocytosis of neuronal cell adhesion molecules at
the growth cone.
As CRMP-2 activity is mainly controlled through
protein phosphorylation, it might well be assumed that
its transcriptional regulation is not of physiologic rele-
vance. Nevertheless, there are differences in its mRNA
expression levels in different tissues and developmental
stages [11,12]. Also, deregulated CRMP-2 mRNA
expression has been related to several important neuro-
logic disorders, such as Alzheimer’s disease and schizo-
phrenia [13,14]. Furthermore, differences in CRMP-2
mRNA and phosphorylation levels between normal
and neuroblastoma cells have been recently reported
by Tahimic et al. [15]. A relationship between altered
transcriptional regulation of CRMP-2 expression and
neuro-oncologic maladies seems possible, as cell polari-
zation is one of the main points in neuronal cell differ-
entiation, and involution towards dedifferentiated

phenotypes is one of the main events that contribute
to the settlement of some of these pathologies [16]. In
fact, members of the CRMP family of proteins have
been reported to act as tumor and metastasis suppres-
sors: CRMP-1 for lung cancer [17], and CRMP-5 in
paraneoplastic disorders [18].
Therefore, we considered it relevant to study the regu-
lation of CRMP-2 gene expression during all-trans-reti-
noic acid (ATRA)-induced differentiation of SH-SY5Y
neuroblastoma cells. We opted for ATRA as differenti-
ating agent because it has been widely employed in both
in vitro and in vivo studies on neuroblastoma and has
proven useful as a coadjuvant in chemotherapy [19].
In this article, we present data supporting reduced
CRMP-2 expression during neuroblastoma cell differ-
entiation. We provide strong experimental evidence for
the involvement of the Pax-3 and AP-2 transcription
factors in CRMP-2 transcriptional regulation. Both of
these have been widely reported to be implicated in
neuronal cell development. Finally, we have estab-
lished that CRMP-2 transcriptional repression during
this process is mediated through impaired binding of
AP-2 and Pax-3 to their consensus sequences.
Results and Discussion
ATRA downregulates CRMP-2 mRNA expression
CRMP-2 is a cytoskeleton-interacting protein that has
been linked to neuronal cell differentiation, as it binds
to tubulin heterodimers and promotes microtubule
assembly during this process [5]. It has been shown
that after ATRA treatment, SK-H-SH neuroblastoma

cells develop broad lamellipodia containing radial actin
fibers, reorganizing their cellular scaffold [20].
Interestingly, Butler et al. showed how ATRA treat-
ment of SH-SY5Y neuroblastoma cells caused great
modifications in microtubule distribution without
changing the expression levels of tubulin [21]. These
findings pointed to a possible regulation of the pro-
teins that participate as microtubule adaptors, stimula-
ting interest in the study of CRMP-2 transcriptional
regulation during this process.
Therefore, we started by using northern blot to
measure CRMP-2 mRNA expression levels during the
differentiation of SH-SY5Y neuroblastoma cells. As
shown in Fig. 1B, decreased levels of CRMP-2 mRNA
are seen 48 h after ATRA treatment and are main-
tained for at least 6 days.
As gene expression can be modulated through both
transcriptional and post-transcriptional mechanisms, we
sought to assess whether ATRA treatment affected
CRMP-2 gene expression at the promoter level or at the
level of its mRNA stability. To achieve this goal, SH-
SY5Y cells were treated with ATRA 10 lm for 48 h,
and actinomycin D (ActD), an inhibitor of transcrip-
tion, was added afterwards. RNA samples were collec-
ted at different time intervals after ActD treatment. As
shown in Fig. 1C, ATRA did not reduce the half-life of
CRMP-2 mRNA, indicating that it has no effect on
mRNA stability, but rather triggers its transcriptional
regulation. Western blot analyses revealed a reduction
in CMRP-2 protein levels, starting 72 h after ATRA

treatment and lasting for at least 15 days (Fig. 1D).
L. Fonta
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FEBS Journal 274 (2007) 498–511 ª 2006 Foundation for Applied Medical Research (FIMA) Journal compilation ª 2006 FEBS 499
Isolation and characterization of the regulatory
region of the human CRMP-2 gene
As a second step in our study, we aimed to evaluate
the presence of regulatory consensus sequences in the
human CRMP-2 promoter. The CRMP-2 transcrip-
tion start site was previously identified by RACE
[22]. It was located 282 bp upstream from the first
base of the ATG translation initiating codon. There-
fore, a sequence of 949 bp upstream from the first
transcribed base was introduced in the matinspector
program [23] for bioinformatic analysis. The results
of this examination are given in Fig. 2, and show the
presence of several cis-acting elements involved in
A
B
C
D
Fig. 1. SH-SY5Y neuroblastoma cells experience changes in CRMP-2 gene expression during ATRA-induced differentiation. (A) Neurite induc-
tion revealed SH-SY5Y neuroblastoma cell differentiation 2 and 6 days after exposure to 10 l
M ATRA. Scale bar 50 lm (B) Northern blot
analyses of CRMP-2 mRNA expression in differentiating cells. Total RNA was obtained at the indicated times, and 10 lg of each sample
was analyzed by northern blotting. The 18S ribosomal RNA gene was used as loading control. (C) CRMP-2 mRNA stability was measured by
adding 5 lgÆmL

)1
of ActD to SH-SY5Y neuroblastoma cells exposed to ATRA for 48 h. RNA was extracted 0, 1, 3, 6, 9 and 12 h afterwards,
and CRMP-2 mRNA levels were analyzed by northern blot. mRNA expression level was calculated as the percentage of the density of the
control sample at time 0 h (100%), and plotted as a function of time. (D) Western blot study of CRMP-2 protein levels in ATRA-treated
SH-SY5Y cells. Analysis by densitometry showed that CRMP-2 mRNA expression was decreased by approximately 30% after ATRA expo-
sure, whereas the reduction in protein content was slower and less pronounced (10–20%). ‘C’ corresponds to nontreated cells, and ‘T’ cor-
responds to cells treated with 10 l
M ATRA. Results represent the average from three independent experiments. The statistical test used
was Student’s t-test.
Transcriptional regulation of CRMP-2 L. Fonta
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500 FEBS Journal 274 (2007) 498–511 ª 2006 Foundation for Applied Medical Research (FIMA) Journal compilation ª 2006 FEBS
Fig. 2. Phylogenetic analyses show the existence of conserved regions in the CRMP-2 promoter. The CRMP-2 promoter regions from five
different mammals were aligned using
BLAT software. Conserved binding regions for several transcription factors involved in neurogenesis
and differentiation are highlighted in boxes. + 1 indicates the first transcribed nucleotide of the CRMP-2 mRNA.
L. Fonta
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FEBS Journal 274 (2007) 498–511 ª 2006 Foundation for Applied Medical Research (FIMA) Journal compilation ª 2006 FEBS 501
neuronal cell differentiation that could be implicated
in its transcriptional regulation, such as Sp1, AP-2,
E2F, Pax-3 and NeuroD1. Typical TATA and CAAT
boxes were also identified in this region. We also
searched for conserved regions between different spe-

cies using blat software (), and
found that the CRMP-2 promoter sequence is
strongly conserved, especially in the region located
between ) 535 and + 91 from the first transcribed
base. Phylogenetically conserved regions serve as a
reliable predictor of regulatory elements, and the
detection of these significantly reduces the number of
candidate transcription factors to be tested in func-
tional assays [24].
In order to further identify the minimal 5¢ region
responsible for transcriptional regulation, we per-
formed four serial deletions of the CRMP-2 promoter
region and cloned them into the luciferase pGL3 basic
vector. Deletion constructs were transiently transfected
into SH-SY5Y neuroblastoma cells, and their relative
luciferase activity was assayed 48 h later. As can be
seen from Fig. 3A, deletion of the most distal part of
the promoter (from ) 941 to ) 768) led to a nonsignifi-
cant decrease in constitutive promoter activity. An
additional deletion of the region located between ) 768
and ) 354 of the CRMP-2 promoter resulted in an
increase in promoter activity. In fact, two putative
repressor elements were located in this region: HIC-1,
A
BC
Fig. 3. Characterization of the CRMP-2 minimal promoter region. (A) Deletion constructs of the CRMP-2 promoter were generated and used
in luciferase reporter gene assays 48 h after transfection. The relative luciferase values are shown as percentage of the mean ± SD activity
of each construct relative to CRMP-2 whole promoter (+ 91 to ) 949). Statistical significance was obtained by use of the Mann–Whitney sta-
tistical test. *P < 0.05; ns, not significant. Results represent averages from at least three independent experiments. (B) EMSA showing
DNA-binding activity of SH-SY5Y nuclear extracts. Oligonucleotides corresponding to the regions ) 229 ⁄ ) 151 (lanes 1–8) and ) 130 ⁄ ) 63

(lanes 9–13) from the human CRMP-2 promoter were labeled. A 100-fold molar excess of the unlabeled oligonucleotides containing the con-
sensus-binding sequence of AP-2 (lane 3), E2F (lane 4), Sp1 (lane 8) and Pax-3 (lane 11) or NeuroD1 (lane 12) was added for competition
experiments. (C) For the supershift assays, SH-SY5Y nuclear extracts were preincubated with 2 lg of anti-AP-2 serum (lane 3) or anti-Pax-3
serum (lane 7). SS, supershifted band in the presence of specific antibody.
Transcriptional regulation of CRMP-2 L. Fonta
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502 FEBS Journal 274 (2007) 498–511 ª 2006 Foundation for Applied Medical Research (FIMA) Journal compilation ª 2006 FEBS
a tumor suppressor essential for mammalian develop-
ment [25], and a neuron-restrictive silencer element
recognized by a transcription factor known as the neu-
ron-restrictive silencer factor, a zinc finger containing
a transcriptional repressor [26]. The neuron-restrictive
silencer element and neuron-restrictive silencer factor
have also been reported to function as direct transcrip-
tional repressors in the promoter of the semaphorin
receptors [27], indicating possible coordinated regula-
tion of genes involved in the same signaling pathway.
Finally, deletion of the most proximal fragment
spanning bases ) 354 to ) 130 of the CRMP-2 promo-
ter caused a sharp 90% decrease in promoter activity.
This region, which includes two Sp1, E2F and AP-2
potential binding sites, was considered to be respon-
sible for CRMP-2 minimal promoter activity.
Identification of functional AP-2 and Pax-3
elements within the CRMP-2 promoter
We further decided to clarify which transcription fac-
tors were responsible for CRMP-2 transcriptional

activity in SH-SY5Y neuroblastoma cells. For this pur-
pose, we analyzed the binding of the following tran-
scriptional regulators: E2F, which participates in the
control of cell cycle progression [28]; and Sp1, a ubi-
quitous transcription factor involved in constitutive
gene expression [29]. The involvement of these two
transcription factors in CRMP-2 transcriptional regu-
lation in TGW neuroblastoma cells was suggested by
Kodama et al. [30]. We also chose to study the func-
tionality of other transcription factor-binding sites,
based upon their relationship with the process of cell
differentiation, phylogenetic conservation, and highest
matrix similarity values. They were: AP-2, a retinoid
responsive factor that regulates the expression of many
mammalian genes during vertebrate development
[31,32]; NeuroD1, a transcription factor specifically
involved in neurogenesis [33], whose expression has
been reported to be increased in SH-SY5Y neurobla-
stoma cells after ATRA treatment [34]; and Pax-3, a
member of the paired box (PAX) family of transcrip-
tion factors implicated in neural crest differentiation
during embryogenesis [35,36].
Therefore, labeled oligonucleotides spanning the
regions ) 229 to ) 151 (for AP-2-, Sp1- and E2F-binding
assays) and ) 130 to ) 63 (for Pax-3- and NeuroD1-
binding assays) of the CRMP-2 minimal promoter
sequence were used as probes for electrophoretic mobil-
ity shift assays (EMSAs) in combination with nuclear
extracts from SH-SY5Y cells (Fig. 3B).
DNAÆprotein complexes were detected on the labeled

) 229 ⁄ ) 151 oligonucleotide (Fig. 3B, lanes 2 and 7). A
100 molar excess of unlabeled AP-2 consensus oligonu-
cleotide (Fig. 3B, lane 3) abolished the formation of a
shifted band, pointing at the specificity of AP-2 binding.
On the other hand, a 100 molar excess of unlabeled E2F
and Sp1 consensus oligonucleotides (Fig. 3B, lanes 4
and 8, respectively) or a nonspecific competitor (Fig. 3B,
lane 5) did not inhibit the formation of proteinÆDNA
complexes. Moreover, supershift EMSAs employing an
AP-2-selective antibody demonstrated the direct binding
of AP-2 to its consensus sequence in the CRMP-2 pro-
moter (Fig. 3C, lane 3), whereas a nonspecific antibody
did not (Fig. 3C, lane 4).
To study putative Pax-3- and NeuroD1-binding sites
in the CRMP-2 promoter region, a 100 molar excess
of DNA oligonucleotides containing the binding sites
for Pax-3 or NeuroD1 were used as competitor probes.
As shown in Fig. 3B, Pax-3 consensus oligonucleotide
competed with protein binding to the CRMP-2 promo-
ter region ) 130 ⁄ ) 63 (lane 11), but a NeuroD1 con-
sensus oligonucleotide did not (lane 12). An excess of
a nonspecific competitor failed to compete with the
formation of the Pax-3 shifted band (lane 13).
Supershift assays of Pax-3 binding to the CRMP-2
promoter region were performed to confirm the involve-
ment of this transcription factor in the regulation of
CRMP-2 promoter basal activity (Fig. 3C, lane 7).
To gain further insights into the contribution of
each transcription factor to CRMP-2 promoter activ-
ity, point mutations were introduced in the AP-2 and

Pax-3 consensus-binding sites of the pGL3-CRMP-2
) 354 ⁄ + 91 minimal promoter, and their luciferase
reporter activities were tested in SH-SY5Y cells. To
test the suitability of the mutations AP-2 ⁄ ) 213,
AP-2 ⁄ ) 166 and Pax-3 ⁄ ) 113, labeled CRMP-
2 )229 ⁄ ) 151 or Pax-3 ⁄ ) 113M oligonucleotides were
tried in EMSA using the mutated sequences AP-2 ⁄
) 213M, AP-2 ⁄ )
166M or the ) 130 ⁄ ) 63 oligonucleo-
tide as cold probes (supplementary Fig. S1).
As can be seen in Fig. 4A, the CRMP-2 promoter
constructs harboring mutations for AP-2 ⁄ ) 213 and
Pax-3 ⁄ ) 133 elements showed reduced promoter activ-
ity (55% and 40%, respectively). Mutation of the
AP-2 element at ) 166 had no significant effect on the
constitutive promoter activity of the CRMP-2 promo-
ter construct (Fig. 4A).
To confirm the involvement of AP-2 and Pax-3 as
transcriptional activators of the CRMP-2 gene, AP-2
(pS-AP-2) and Pax-3 (pcDNA3-PAX-3) expression
vectors were cotransfected with the CRMP-2 promoter
in SH-SY5Y neuroblastoma cells. The reporter activity
of CRMP-2 promoter constructs pGL3-CRMP2
) 941 ⁄ + 91 and pGL3-CRMP2 ) 354 ⁄ + 91 increased
after transfection with 0.2 lg of the AP-2 and Pax-3
L. Fonta
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FEBS Journal 274 (2007) 498–511 ª 2006 Foundation for Applied Medical Research (FIMA) Journal compilation ª 2006 FEBS 503
expression vectors. No effect was observed when the
expression vectors were transfected along with the
AP-2 ⁄ ) 213M or Pax-3 ⁄ ) 113M mutated constructs
(Fig. 4B), providing more experimental evidence for
the involvement of AP-2 and Pax-3 in the regulation
of CRMP-2 expression.
These data indicate that AP-2 and Pax-3 bind to the
CRMP-2 minimal promoter region, whereas neither
E2F nor NeuroD1 or Sp1 seem to be involved in
CRMP-2 basal promoter activity in SH-SY5Y neurob-
lastoma cells. AP-2 and Pax-3 transcription factors
have been reported to be significantly involved in the
regulation of genes implicated in cell development and
cytoskeletal reorganization, e.g. the genes for the tyro-
sine kinase receptor gene c-kit and E-cadherin [37,38].
AP-2 and Pax-3 show impaired binding to the
CRMP-2 promoter after ATRA treatment of
SH-SY5Y cells
It has been previously reported that both mRNA levels
and phosphorylation status of several microtubule-
related proteins are critical for neurons to maintain
normal cytoskeletal architecture. In fact, the data
available in the literature show the regulation of these
proteins during the differentiation of SH-SY5Y neuro-
blastoma cells [39,40]. For this reason, we were interes-
ted in studying how CRMP-2 promoter activity could
be affected by ATRA treatment.
Luciferase reporter experiments demonstrated that
ATRA treatment caused a 50% reduction in CRMP-2

promoter activity compared with nontreated SH-SY5Y
cells (Fig. 5A). These results were in agreement with
the decrease observed in CRMP-2 mRNA expression
levels shown in Fig. 1B. We also noticed that the val-
ues for the reporter activity of AP-2 ⁄ ) 213 mutated
construct were the same as those obtained from
ATRA-treated cells, raising the issue of ATRA treat-
ment affecting AP-2 transactivation of the CRMP-2
promoter (Fig. 5A).
Therefore, to determine whether there was alteration
of the binding activity of AP-2 and Pax-3 to their
consensus sites after ATRA treatment, we performed
EMSAs using SH-SY5Y nuclear protein extracts
obtained 24 and 48 h after ATRA exposure. As shown
in Fig. 5B, AP-2 DNA binding to the CRMP-2 promo-
ter decreased 24 and 48 h after treatment, whereas bind-
ing of Pax-3 to its consensus sequence was reduced only
48 h after treatment. Pax-3 is a transcription factor that
is finely regulated during nervous system development
[41]. The fall in Pax-3 expression seems to be a necessary
prerequisite for the onset of morphologic differentiation
and ⁄ or for cessation of cell proliferation [42].
Our results are different from those published by
Kodama et al. [30], who pointed out the involvement of
the transcription factors E2F, Sp1 and GATA1 ⁄ 2in
CRMP-2 transcriptional regulation after glial cell-
derived neurotrophic factor exposure of neuroblastoma
cells. In fact, in their work, glial cell-derived neuro-
trophic factor treatment increased CRMP-2 mRNA
expression levels, whereas we found a reduction in

CRMP-2 transcriptional activity after ATRA treatment.
This apparent discrepancy can be explained by the use
of different cell lines and stimuli, and by the fact that
glial cell-derived neurotrophic factor has been reported
to induce cell proliferation and inhibit ATRA-induced
neuritogenic and growth inhibitory effects in neurobla-
stoma cells [43]. It seems reasonable that the expression
of a tubulin adaptor protein in actively proliferating
cells would be increased, whereas the level should
decrease in those cells that have their cellular scaffold
‘fixed’, as is the case with differentiated cells.
Two different mechanisms to explain the
impaired transcription factor binding to CRMP-2
promoter
Having demonstrated decreased AP-2 and Pax-3 DNA
binding to their consensus sequences in ATRA-differ-
A
B
Fig. 4. Mutation of AP-2- and Pax-3-binding sites in the CRMP-2
promoter impaired its transcriptional activity. (A) pGL-3-CRMP-2
minimal promoter constructs harboring mutations in their
AP-2 () 213) or Pax-3 () 113) consensus-binding sites showed
decreased luciferase reporter activity in SH-SY5Y cells. The mean
luciferase activities from three separate transfection experiments
are expressed as percentage relative to the minimal promoter
region (+ 91 to ) 354). Statistical significance was obtained by use
of the Mann–Whitney statistical test. ns, not significant; *P < 0.05;
**P < 0.01. (B) Cotransfection of AP-2a or Pax-3 expression vec-
tors with the pGL3-CRMP-2 minimal promoter resulted in increased
reporter activity, whereas the use of their corresponding mutated

promoter constructs did not induce any change.
Transcriptional regulation of CRMP-2 L. Fonta
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504 FEBS Journal 274 (2007) 498–511 ª 2006 Foundation for Applied Medical Research (FIMA) Journal compilation ª 2006 FEBS
entiated neuroblastoma cells, we sought to identify
whether these changes were caused by alterations in
their expression or in their cellular availability.
The study of AP-2a protein levels demonstrated, as
reported by others [44], an increase in the total AP-2
content in the cell in response to ATRA treatment, but
this increase was not statistically significant (Fig. 6A).
Therefore, this could not explain its reduced binding to
the CRMP-2 promoter seen with EMSAs (Fig. 5B). We
therefore searched for changes in its subcellular distribu-
tion after ATRA exposure, and found a significant
decrease in the nuclear content of AP-2a protein
(Fig. 6B) that would explain the reduced AP-2a DNA
binding to the CRMP-2 promoter after the treatment
(Fig. 5B), and therefore the increase in AP-2a cytoplas-
mic content. This is not the first time that this has been
A
BC
Fig. 5. Effects of ATRA treatment on CRMP-2 promoter activity and transcription factor binding. (A) Exposure of SH-SY5Y cells for 48 h to
10 l
M ATRA reduced the transcriptional activity of all the CRMP-2 promoter constructs tested except for those mutated in the AP-2 () 213)
or Pax-3 () 113) consensus-binding sites. Results are given as the mean ± SD luciferase activities from three separate transfection experi-
ments expressed as the percentage relative to the minimal promoter region (+ 91 to ) 354) in control cells. ns, not significant; **P < 0.01;

*P < 0.05.
a
Treated versus nontreated.
b
Treated versus pGL3-CRMP-2-354 ⁄ + 91 (ATRA). Statistical significance was obtained by use of the
Mann–Whitney test. (B) EMSA assay illustrating a reduction in AP-2 DNA binding to the ) 229 ⁄ ) 151 region of the CRMP-2 promoter after
ATRA treatment. (C) Pax-3 EMSA assays demonstrate that 48 h of ATRA exposure leads to a smaller amount of Pax-3 DNA being bound to
its consensus sequence in the ) 130 ⁄ ) 63 region of the CRMP-2 promoter. ‘C’ corresponds to nontreated cells, and ‘T’ corresponds to cells
treated with 10 l
M ATRA.
L. Fonta
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n-Gaba
´
s et al. Transcriptional regulation of CRMP-2
FEBS Journal 274 (2007) 498–511 ª 2006 Foundation for Applied Medical Research (FIMA) Journal compilation ª 2006 FEBS 505
reported; for example, Popa et al. [45] demonstrated a
loss of AP-2 transcriptional activity in differentiated
keratinocytes, associated with a reduction in its binding
to DNA. This failure was caused by increased accumu-
lation of phosphorylated AP-2 in the cytoplasm.
On the other hand, mRNA expression levels of Pax-3
were reduced 48 h after ATRA treatment (Fig. 6C).
This decrease could be responsible for the lower
amounts of protein bound to the CRMP-2 promoter.
We tried to measure Pax-3 protein content by western
blot, using the same antibody employed for the EMSA,
but were not successful.
There are reports in the literature of ATRA indu-
cing cell differentiation through the inhibition of c-myc

and N-myc expression [46]. Pax-3 transcription factor
expression is regulated by N-myc during neural cell
development [47]. Therefore, we looked for a link
between the expression of c-myc, N-myc and Pax-3 at
the mRNA level in neuroblastoma cell differentiation.
ATRA treatment induced a fast decrease in the
mRNA level of the c-myc proto-oncogene as measured
by semiquantitative PCR (as early as 6 h, and main-
tained for the whole differentiation process) (Fig. 6C).
In contrast, we found a similar reduction in the expres-
sion levels of the N-myc and Pax-3 genes 48 h after
ATRA treatment. Therefore, it seemed reasonable to
propose that, as ATRA treatment is mediating N-myc
gene inhibition, Pax-3 mRNA expression is probably
A
C
B
Fig. 6. ATRA treatment alters AP-2 subcellular distribution and Pax-3 expression. (A) Western blot analyses of total AP-2 protein expression
levels in control (‘C’) and ATRA-treated (‘T’) SH-SY5Y cells. (B) ATRA treatment alters AP-2 subcellular distribution. Western blot analysis of
nuclear and cytoplasmic protein extracts purified from ATRA-treated cells demonstrated significantly reduced levels of AP-2 in the nuclear
fraction, whereas it was increased in the cytoplasm. (C) Effect of ATRA treatment on the mRNA expression levels of Pax-3 and its transcrip-
tional regulators c-myc and N-myc, as measured by semiquantitative PCR. The b-actin gene was amplified as loading control. Analysis by
densitometry showed that Pax-3, CRMP-2 and N-myc mRNA levels decreased significantly (P<0.05) in differentiating cells, along with a
major descent in c-myc expression (P<0.001). The statistical test used was Student’s t-test. **P < 0.01; *P < 0.05. ‘C’ corresponds to
nontreated cells, and ‘T’ corresponds to cells treated with 10 l
M ATRA.
Transcriptional regulation of CRMP-2 L. Fonta
´
n-Gaba
´

s et al.
506 FEBS Journal 274 (2007) 498–511 ª 2006 Foundation for Applied Medical Research (FIMA) Journal compilation ª 2006 FEBS
being compromised, resulting in less transcription fac-
tor being available to modulate CRMP-2 promoter
activity.
In conclusion, we have demonstrated that the
human CRMP-2 promoter contains AP-2 and Pax-3
functional elements in its minimal promoter. These
two transcription factors seem to regulate basal
CRMP-2 promoter activity and expression. ATRA
treatment induces a decrease in CRMP-2 expression
during the differentiation of neuroblastoma cells
through impaired binding of the aforementioned tran-
scription factors to their consensus sequences in the
CRMP-2 promoter. We finally demonstrated that the
reduced binding of Pax-3 is due to a decreased tran-
scriptional rate, whereas loss of specific DNA-bound
AP-2 complexes results from a reduced level of AP-2
in the nucleus.
Given that the family of collapsin response mediator
proteins has been reported to be mainly regulated by
post-translational mechanisms, the notion of them being
also regulated at the transcriptional level gives us
another example of a coordinated cell response at both
the mRNA synthesis and protein activation stages.
Experimental procedures
Materials
ATRA and ActD were purchased from Sigma (St Louis,
MO, USA). The pSP(RSV)-NN and pSP(RSV)-AP-2a
expression vectors were kindly provided by T. Williams

(University of Colorado Health Sciences Center, Denver,
CO, USA). Rabbit anti-(human Pax-3) serum and the
pcDNA3-Pax-3 expression vector were generously provided
by F J Rauscher III (The Wistar Institute, Philadelphia,
PA, USA). Antibody to CRMP-2 (C4G) was a generous
gift of Y. Ihara (Faculty of Medicine, University of Tokyo,
Japan). Rabbit anti-(human AP-2a) serum and secondary
goat anti-(mouse IgG) and goat anti-(rabbit IgG) conju-
gated to horseradish peroxidase were purchased from Santa
Cruz Biotechnology (Santa Cruz, CA, USA). Anti-b-actin
serum (A5441: monoclonal) was purchased from Sigma.
Cell culture
The human neuroblastoma cell line SH-SY5Y was pur-
chased from ATCC (CRL-2266) and cultured at 37 °C
under 5% CO
2
in DMEM: Ham’s F12 (1 : 1) medium sup-
plemented with 10% (v ⁄ v) heat-inactivated fetal bovine
serum, 100 UÆmL
)1
penicillin, 100 lgÆmL
)1
streptomycin,
250 ngÆmL
)1
fungizone and nonessential amino acids, all
from Invitrogen (Paisley, UK). Cells were plated at 200
cellsÆmm
)2
for all the experiments.

RNA extraction
SH-SY5Y cells were serum-starved for 24 h and then trea-
ted with 10 lm ATRA for different time periods. Total
RNA was isolated using TRIzol (Invitrogen), following the
manufacturer’s instructions.
Northern blot
Total RNA (10 lg) was resolved by electrophoresis in a
1% formaldehyde ⁄ agarose gel, transferred to a nylon mem-
brane (Amersham Biosciences, Uppsala, Sweden), UV
crosslinked, and prehybridized at 42 °Cin5· NaCl ⁄ Cit
(1 · NaCl ⁄ Cit: 150 mm NaCl, 0.15 mm sodium citrate,
pH 7.0), 50% formamide, 50.4 mm phosphate buffer
(pH 6.5), 0.1% SDS, 5 · Denhart’s solution and
0.1 mgÆmL
)1
salmon sperm DNA for 8 h at 42 °C. Hybrid-
ization was carried out overnight at 42 °C in prehybridiza-
tion solution with 1 · Denhart’s solution ⁄ 12.5% dextran
sulfate, using a final concentration of 10
6
c.p.m. labeled
probe per milliliter. Radiolabeling of specific probes was
carried out with [a-
32
P]dCTP (specific activity 3000
CiÆmmol
)1
) by random priming using the Klenow frag-
ment of DNA polymerase I (Bioline, London, UK).
After hybridization, blots were washed three times with

2 · NaCl ⁄ Cit ⁄ 0.1% SDS for 15 min at 65 °C. Blots were
autoradiographed with intensifying screens. Quantification
of the mRNA levels in the autoradiograms was performed
using imagemaster software (Amersham Biosciences).
Promoter–luciferase constructs and site-directed
mutagenesis
The region from the human CRMP-2 promoter spanning
bases between ) 941 and + 91 from the transcription start
site was PCR amplified from human genomic DNA. The
primers used for the amplification reaction were as follows:
CRMP-2 sense, 5¢-CCATTCCTCCGCCCTACTAAGTT-3¢;
and CRMP-2 antisense, 5¢-TTCTTCCTCTCCTCCAACA
CAGC-3¢. The PCR product was ligated into the pGEMT
easy vector (Promega, Madison, WI, USA), sequenced, and
subcloned into the SmaI and SacI sites of the luciferase
reporter vector pGL3-basic (Promega). Constructs harboring
5¢ serial deletions derived from the 1 kb pGL3-CRMP-2 con-
struct were obtained either by restriction enzyme digestion
(construct pGL3-CRMP-2 ) 768 ⁄ + 91 digested with PstI),
or by PCR (constructs pGL3-CRMP-2 ) 354 ⁄ + 91 and
pGL3-CRMP-2 ) 130 ⁄ + 91) using primers KpnI-CRMP-
2 ⁄ ) 354, 5¢-CTGGTACCGCGACGACCACCCCTCCAT
TG-3¢, and KpnI-CRMP-2 ⁄ ) 130, 5¢-CTGGTACCATCG
CTGCTCGTCTCTCTCG-3¢, as forward primers, and
CRMP-2 antisense as the reverse primer.
Site-directed mutations were generated by PCR using the
QuickChange Site-Directed Mutagenesis Kit from Strata-
gene (Cedar Creek, TX, USA). The pGL3-CRMP-2 promo-
L. Fonta
´

n-Gaba
´
s et al. Transcriptional regulation of CRMP-2
FEBS Journal 274 (2007) 498–511 ª 2006 Foundation for Applied Medical Research (FIMA) Journal compilation ª 2006 FEBS 507
ter construct ) 354 ⁄ + 91 was used as template with the
following mutated primers: AP-2 ⁄ ) 213M,5¢-GCGCC
GCCTGCTGTACCCATCCGTTCACTGCCGC-3¢;AP-2⁄
) 166M, 5¢-CGCCGCGCCCCGCCTCACCGGCCTAAATT
TG-3¢; and Pax-3 ⁄ ) 113M,5¢-GGCCAATCGCTGCTCG
ATACTCTCGAAGCGGATGGC-3¢. All the constructs
were sequenced in an ALF Express Sequencer (Pharmacia
LKB, Uppsala, Sweden).
Transient transfection and luciferase assay
SH-SY5Y cells were plated as previously described in six-
well plates and serum-starved for 24 h before transfection.
One microgram of each of the pGL3-CRMP-2 reporter
constructs and 50 ng of the pRL-SV40, used to standard-
ize for transfection efficiency, were transfected using
FuGene6 (Roche, Mannheim, Germany) into SH-SY5Y
neuroblastoma cells. ATRA treatment was performed 4 h
after transfection. Cells were harvested 48 h after each
treatment and assayed for firefly and Renilla luciferase
activities using the Dual-Luciferase Reporter Assay Sys-
tem from Promega. Transfection assays were performed
as at least three independent experiments, with three
replicates each.
Preparation of nuclear extracts
SH-SY5Y cells were grown on 100 mm dishes, serum-
starved for 24 h, and treated with 10 lm ATRA for 24 or
48 h. Briefly, cells were washed twice with cold NaCl ⁄ P

i
,
and nuclear proteins were extracted as previously described
[48].
EMSA
Double-stranded oligonucleotide probes spanning the bases
) 229 ⁄ ) 151 or ) 130 ⁄ ) 63 from the CRMP-2 promoter
were obtained by enzymatic digestion from the pGL3-
CRMP-2 ) 354 ⁄ + 91 construct and labeled with
[a-
32
P]dCTP (specific activity 3000 CiÆmmol
)1
).
Nuclear extracts (5 lg) in 10 mm Tris ⁄ HCl (pH 7.4) con-
taining 50 mm NaCl, 4% glycerol, 0.5 mm dithiothreitol,
1mm MgCl
2
, 0.5 mm EDTA and 1 lg poly(dI-dC) were
incubated in the presence or absence of competitor oligonu-
cleotides at the indicated concentrations for 15 min at room
temperature. The following oligonucleotides were used: Pax-
3(5¢-GGGGGAGACTCGGTCCCGCTTATCTCCGGCT
GTGC-3¢) and NeuroD1 (5¢-ACGTTCTGGCCATCTG
CTGATCCTACGT-3¢), which had been previously used
with human nuclear extracts [49,50]. Commercially available
oligonucleotides were: AP-2a (Promega) and Sp1 and E2F
(Santa Cruz Biotechnology). The
32
P-labeled DNA probe

(20–30 000 c.p.m.) was added to the DNAÆprotein complexes
and incubated for a further 15 min at room temperature.
For the supershift assays, nuclear extracts were incubated
for 15 min at room temperature with 2 lg of anti-AP-2a or
anti-Pax-3 prior to the addition of the labeled probe, as
previously described [48].
All samples were resolved on a 5% nondenaturing poly-
acrylamide gel in 0.5 · TBE buffer (1 · TBE is 8.9 mm
Tris, 110 mm boric acid and 2 mm EDTA, pH 8.3). After
the electrophoresis, the gel was dried, autoradiographed,
and analyzed using imagemaster software (Amersham Bio-
sciences).
RT-PCR analysis
Total RNA (2 lg) was reverse transcribed with hexanucleo-
tides (5 lm) and 200 units of the Moloney murine leukemia
virus reverse transcriptase (M-MLV) (Promega) at 37 °C
for 1 h.
PCR amplification was performed with BioTaq DNA
polymerase (Bioline), 1.5 mm MgCl
2
,1lm each sense and
antisense primer, and 1 lL of cDNA as template. Cycling
conditions were as follows: 2 min at 95 °C, followed by 30
cycles of 95 °C for 45 s, melting temperature for 45 s,
72 °C for 1 min, and a final step of 72 °C for 2 min. Primer
sequences and the T
m
used for the PCR reactions are sum-
marized in supplementary Table S1. All PCR products were
analyzed on a 2.5% agarose gel and stained with ethidium

bromide.
Western blot analysis
Cells were washed twice with cold NaCl ⁄ P
i
and lysed in
RIPA buffer [50 mm Tris ⁄ HCl, pH 8, 150 mm NaCl, 0.1%
SDS, 1% Triton X-100, 0.5% sodium deoxycholate, 1 mm
phenylmethanesulfonyl fluoride, protease inhibitor cocktail
(Roche)]. Cell debris was cleared by centrifugation
(10 000 g, 10 min at 4 °C, Eppendorf 5415R centrifuge,
rotor F45-24-11; Eppendorf, Madrid, Spain), and the pro-
tein concentration of each sample was determined using the
Pierce (Rockford, IL, USA) BCA protein Assay kit.
Twenty micrograms of each protein sample was resolved
by SDS ⁄ PAGE, transferred onto a nitrocellulose membrane
(Bio-Rad, Hercules, CA, USA), and blocked with 5% dried
powder milk ⁄ TBS-T (25 mm Tris ⁄ HCl, pH 7.4, 200 mm
NaCl, 0.1% Tween-20) overnight at 4 °C. Incubations with
primary antibodies were performed in TBS-T ⁄ 5% dried
milk powder (1 : 10 000 dilution for C4G antibody,
1 : 2000 dilution for b-actin antibody, 1 : 200 dilution for
the AP-2a anti-serum). Protein membranes were washed
three times and incubated for 1 h at room temperature with
a horseradish peroxidase-conjugated secondary antibody
(Amersham Biosciences) at a final dilution of 1 : 5000 in
5% dried milk powder ⁄ TBS-T. Immunoreactive bands were
visualized using ECL Plus (Amersham Biosiences) as devel-
oping reagent.
Transcriptional regulation of CRMP-2 L. Fonta
´

n-Gaba
´
s et al.
508 FEBS Journal 274 (2007) 498–511 ª 2006 Foundation for Applied Medical Research (FIMA) Journal compilation ª 2006 FEBS
Acknowledgements
This work was supported by the Spanish Ministry of
Science and Technology (PM-1999-0044). LF was sup-
ported by a predoctoral fellowship from the Govern-
ment of Navarra, Spain and Asociacio
´
n de Amigos de
la Universidad de Navarra, Spain. We would like to
thank F. J. Rauscher III (The Wistar Institute, Phil-
adelphia, PA, USA) and Trevor Williams (University
of Colorado Health Sciences Center, Denver, CO,
USA) for their kindness in providing us with the anti-
(human Pax-3) serum and the expression vectors for
pSP(RSV)-NN and pSP(RSV)-AP-2a, respectively. We
are also grateful to Yasuo Ihara from the Faculty of
Medicine in the University of Tokyo for providing the
anti-CRMP-2 serum.
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Supplementary material
The following supplementary material is available
online:
Fig. S1. Confirmation of the suitability of the Pax-3
and AP-2 mutated oligonucleotides. In order to confirm
the efficacy of the point mutations introduced into the
Pax-3 and AP-2 consensus binding sites, the mutated
oligonucleotides were used as cold probes (lanes 4, 5
and 11) in gel shift experiments performed on regions
–229 ⁄ –151 and –130 ⁄ –63 of the hCRMP-2 promoter. As
expected, they did not produce any shifted band, beha-
ving as nonspecific oligonucleotides.
Table S1. Primers and conditions used for the relative
quantitation of transcripts by semiquantitative RT-

PCR.
This material is available as part of the online article
from
Please note: Blackwell Publishing is not responsible
for the content or functionality of any supplementary
materials supplied by the authors. Any queries (other
than missing material) should be directed to the corres-
ponding author for the article.
L. Fonta
´
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s et al. Transcriptional regulation of CRMP-2
FEBS Journal 274 (2007) 498–511 ª 2006 Foundation for Applied Medical Research (FIMA) Journal compilation ª 2006 FEBS 511