All-trans-retinoic acid inhibits collapsin response mediator protein-2 transcriptional activity during SH-SY5Y neuroblastoma cell differentiation ´ ´ ´ ´ Lorena Fontan-Gabas1, Erik Oliemuller2, Juan Jose Martınez-Irujo1,2, Carlos de Miguel1 and Ana Rouzaut1,2 Department of Biochemistry, University of Navarra, Pamplona, Spain Division of Oncology, Center for Applied Medical Research, University of Navarra, Pamplona, Spain Keywords CRMP-2; gene regulation; neuroblastoma; promoter; retinoic acid Correspondence A Rouzaut, Center for Applied Medical ´ Research, University of Navarra, Av Pıo XII 55, 31008 Pamplona, Spain Fax: +34 948 19 47 18 Tel: +34 948 19 47 00 E-mail: arouzaut@unav.es (Received 14 July 2006, revised 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 functionally 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 frequent 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 alltrans-retinoic acid-induced differentiation of SH-SY5Y neuroblastoma cells All-trans-retinoic acid is considered to be a potential preventive and therapeutic agent, and has been extensively used to differentiate neuroblastoma cells in vitro Therefore, we first demonstrated that collapsin response mediator 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 expression of collapsin response mediator protein-2 after all-trans-retinoic acid exposure is associated with impaired Pax-3 and AP-2 binding to their consensus 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-transretinoic 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 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 dihydropyrimidinase-like 2, is a member of a family of cytoplasmic proteins [3] that were originally identified as 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 ´ ´ L Fontan-Gabas et al 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 heterodimers, allowing its polymerization in such a way that it seems crucial for axonal growth and for the determination 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, compromising 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 residues, 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 relevance 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 neurologic disorders, such as Alzheimer’s disease and schizophrenia [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 polarization is one of the main points in neuronal cell differentiation, 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 suppressors: CRMP-1 for lung cancer [17], and CRMP-5 in paraneoplastic disorders [18] Therefore, we considered it relevant to study the regulation of CRMP-2 gene expression during all-trans-retinoic acid (ATRA)-induced differentiation of SH-SY5Y neuroblastoma cells We opted for ATRA as differentiating agent because it has been widely employed in both Transcriptional regulation of CRMP-2 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 differentiation 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 established 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 treatment 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 proteins that participate as microtubule adaptors, stimulating 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 maintained for at least 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, SHSY5Y cells were treated with ATRA 10 lm for 48 h, and actinomycin D (ActD), an inhibitor of transcription, was added afterwards RNA samples were collected 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) FEBS Journal 274 (2007) 498–511 ª 2006 Foundation for Applied Medical Research (FIMA) Journal compilation ª 2006 FEBS 499 Transcriptional regulation of CRMP-2 ´ ´ L Fontan-Gabas et al A B C D Fig SH-SY5Y neuroblastoma cells experience changes in CRMP-2 gene expression during ATRA-induced differentiation (A) Neurite induction revealed SH-SY5Y neuroblastoma cell differentiation and days after exposure to 10 lM 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 lgỈmL)1 of ActD to SH-SY5Y neuroblastoma cells exposed to ATRA for 48 h RNA was extracted 0, 1, 3, 6, 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 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 exposure, whereas the reduction in protein content was slower and less pronounced (10–20%) ‘C’ corresponds to nontreated cells, and ‘T’ corresponds to cells treated with 10 lM ATRA Results represent the average from three independent experiments The statistical test used was Student’s t-test 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 transcription start site was previously identified by RACE 500 [22] It was located 282 bp upstream from the first base of the ATG translation initiating codon Therefore, 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 FEBS Journal 274 (2007) 498–511 ª 2006 Foundation for Applied Medical Research (FIMA) Journal compilation ª 2006 FEBS ´ ´ L Fontan-Gabas et al Transcriptional regulation of CRMP-2 Fig 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 + indicates the first transcribed nucleotide of the CRMP-2 mRNA FEBS Journal 274 (2007) 498–511 ª 2006 Foundation for Applied Medical Research (FIMA) Journal compilation ª 2006 FEBS 501 ´ ´ L Fontan-Gabas et al Transcriptional regulation of CRMP-2 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 species using blat software (http://genome.ucsc.edu), 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 functional assays [24] In order to further identify the minimal 5¢ region responsible for transcriptional regulation, we performed 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 nonsignificant 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 B C Fig 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 statistical 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 consensus-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 lg of anti-AP-2 serum (lane 3) or anti-Pax-3 serum (lane 7) SS, supershifted band in the presence of specific antibody 502 FEBS Journal 274 (2007) 498–511 ª 2006 Foundation for Applied Medical Research (FIMA) Journal compilation ª 2006 FEBS ´ ´ L Fontan-Gabas et al a tumor suppressor essential for mammalian development [25], and a neuron-restrictive silencer element recognized by a transcription factor known as the neuron-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 transcriptional repressors in the promoter of the semaphorin receptors [27], indicating possible coordinated regulation of genes involved in the same signaling pathway Finally, deletion of the most proximal fragment spanning bases ) 354 to ) 130 of the CRMP-2 promoter 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 responsible 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 factors were responsible for CRMP-2 transcriptional activity in SH-SY5Y neuroblastoma cells For this purpose, we analyzed the binding of the following transcriptional regulators: E2F, which participates in the control of cell cycle progression [28]; and Sp1, a ubiquitous transcription factor involved in constitutive gene expression [29] The involvement of these two transcription factors in CRMP-2 transcriptional regulation in TGW neuroblastoma cells was suggested by Kodama et al [30] We also chose to study the functionality 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 neuroblastoma cells after ATRA treatment [34]; and Pax-3, a member of the paired box (PAX) family of transcription 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 NeuroD1binding assays) of the CRMP-2 minimal promoter sequence were used as probes for electrophoretic mobility shift assays (EMSAs) in combination with nuclear extracts from SH-SY5Y cells (Fig 3B) DNprotein complexes were detected on the labeled ) 229 ⁄ ) 151 oligonucleotide (Fig 3B, lanes and 7) A Transcriptional regulation of CRMP-2 100 molar excess of unlabeled AP-2 consensus oligonucleotide (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 and 8, respectively) or a nonspecific competitor (Fig 3B, lane 5) did not inhibit the formation of proteinỈDNA 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 promoter (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 promoter region ) 130 ⁄ ) 63 (lane 11), but a NeuroD1 consensus 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 involvement 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 activity, 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 CRMP2 )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 oligonucleotide 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 activity (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 promoter 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 FEBS Journal 274 (2007) 498–511 ª 2006 Foundation for Applied Medical Research (FIMA) Journal compilation ª 2006 FEBS 503 Transcriptional regulation of CRMP-2 A B Fig 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 vectors 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 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 neuroblastoma 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 tyrosine 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 microtubulerelated 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 neuro504 ´ ´ L Fontan-Gabas et al blastoma cells [39,40] For this reason, we were interested 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 values 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 treatment 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 promoter decreased 24 and 48 h after treatment, whereas binding 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 ⁄ in CRMP-2 transcriptional regulation after glial cellderived neurotrophic factor exposure of neuroblastoma cells In fact, in their work, glial cell-derived neurotrophic 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 neuroblastoma 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- FEBS Journal 274 (2007) 498–511 ª 2006 Foundation for Applied Medical Research (FIMA) Journal compilation ª 2006 FEBS ´ ´ L Fontan-Gabas et al Transcriptional regulation of CRMP-2 A B C Fig Effects of ATRA treatment on CRMP-2 promoter activity and transcription factor binding (A) Exposure of SH-SY5Y cells for 48 h to 10 lM 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 experiments 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 aTreated versus nontreated bTreated 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 lM ATRA 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 distribution 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 cytoplasmic content This is not the first time that this has been FEBS Journal 274 (2007) 498–511 ª 2006 Foundation for Applied Medical Research (FIMA) Journal compilation ª 2006 FEBS 505 ´ ´ L Fontan-Gabas et al Transcriptional regulation of CRMP-2 A B C Fig 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 transcriptional 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 lM ATRA 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 accumulation 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 inducing cell differentiation through the inhibition of c-myc 506 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 h, and maintained for the whole differentiation process) (Fig 6C) In contrast, we found a similar reduction in the expression 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 FEBS Journal 274 (2007) 498–511 ª 2006 Foundation for Applied Medical Research (FIMA) Journal compilation ª 2006 FEBS ´ ´ L Fontan-Gabas et al being compromised, resulting in less transcription factor 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 transcription 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 transcriptional 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) conjugated 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 purchased from ATCC (CRL-2266) and cultured at 37 °C under 5% CO2 in DMEM: Ham’s F12 (1 : 1) medium supplemented with 10% (v ⁄ v) heat-inactivated fetal bovine serum, 100 mL)1 penicillin, 100 lgỈmL)1 streptomycin, 250 ngỈmL)1 fungizone and nonessential amino acids, all from Invitrogen (Paisley, UK) Cells were plated at 200 cellsỈmm)2 for all the experiments Transcriptional regulation of CRMP-2 RNA extraction SH-SY5Y cells were serum-starved for 24 h and then treated 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 membrane (Amersham Biosciences, Uppsala, Sweden), UV crosslinked, and prehybridized at 42 °C in · 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, · Denhart’s solution and 0.1 mgỈmL)1 salmon sperm DNA for h at 42 °C Hybridization was carried out overnight at 42 °C in prehybridization solution with · Denhart’s solution ⁄ 12.5% dextran sulfate, using a final concentration of 106 c.p.m labeled probe per milliliter Radiolabeling of specific probes was carried out with [a-32P]dCTP (specific activity 3000 CiỈmmol)1) by random priming using the Klenow fragment of DNA polymerase I (Bioline, London, UK) After hybridization, blots were washed three times with · NaCl ⁄ Cit ⁄ 0.1% SDS for 15 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 kb pGL3-CRMP-2 construct 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-CRMP2 ⁄ ) 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 Stratagene (Cedar Creek, TX, USA) The pGL3-CRMP-2 promo- FEBS Journal 274 (2007) 498–511 ª 2006 Foundation for Applied Medical Research (FIMA) Journal compilation ª 2006 FEBS 507 ´ ´ L Fontan-Gabas et al Transcriptional regulation of CRMP-2 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 sixwell 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 standardize for transfection efficiency, were transfected using FuGene6 (Roche, Mannheim, Germany) into SH-SY5Y neuroblastoma cells ATRA treatment was performed 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 System 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, serumstarved for 24 h, and treated with 10 lm ATRA for 24 or 48 h Briefly, cells were washed twice with cold NaCl ⁄ Pi, 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 pGL3CRMP-2 ) 354 ⁄ + 91 construct and labeled with [a-32P]dCTP (specific activity 3000 CiỈmmol)1) Nuclear extracts (5 lg) in 10 mm Tris ⁄ HCl (pH 7.4) containing 50 mm NaCl, 4% glycerol, 0.5 mm dithiothreitol, mm MgCl2, 0.5 mm EDTA and lg poly(dI-dC) were incubated in the presence or absence of competitor oligonucleotides at the indicated concentrations for 15 at room temperature The following oligonucleotides were used: Pax3 (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 32P-labeled DNA probe (20–30 000 c.p.m.) was added to the DNprotein complexes and incubated for a further 15 at room temperature 508 For the supershift assays, nuclear extracts were incubated for 15 at room temperature with 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 polyacrylamide gel in 0.5 · TBE buffer (1 · TBE is 8.9 mm Tris, 110 mm boric acid and mm EDTA, pH 8.3) After the electrophoresis, the gel was dried, autoradiographed, and analyzed using imagemaster software (Amersham Biosciences) RT-PCR analysis Total RNA (2 lg) was reverse transcribed with hexanucleotides (5 lm) and 200 units of the Moloney murine leukemia virus reverse transcriptase (M-MLV) (Promega) at 37 °C for h PCR amplification was performed with BioTaq DNA polymerase (Bioline), 1.5 mm MgCl2, lm each sense and antisense primer, and lL of cDNA as template Cycling conditions were as follows: at 95 °C, followed by 30 cycles of 95 °C for 45 s, melting temperature for 45 s, 72 °C for min, and a final step of 72 °C for Primer sequences and the Tm used for the PCR reactions are summarized 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 ⁄ Pi 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, mm phenylmethanesulfonyl fluoride, protease inhibitor cocktail (Roche)] Cell debris was cleared by centrifugation (10 000 g, 10 at °C, Eppendorf 5415R centrifuge, rotor F45-24-11; Eppendorf, Madrid, Spain), and the protein 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 °C Incubations with primary antibodies were performed in TBS-T ⁄ 5% dried milk powder (1 : 10 000 dilution for C4G antibody, : 2000 dilution for b-actin antibody, : 200 dilution for the AP-2a anti-serum) Protein membranes were washed three times and incubated for h at room temperature with a horseradish peroxidase-conjugated secondary antibody (Amersham Biosciences) at a final dilution of : 5000 in 5% dried milk powder ⁄ TBS-T Immunoreactive bands were visualized using ECL Plus (Amersham Biosiences) as developing reagent FEBS Journal 274 (2007) 498–511 ª 2006 Foundation for Applied Medical Research (FIMA) Journal compilation ª 2006 FEBS ´ ´ L Fontan-Gabas et al Acknowledgements This work was supported by the Spanish Ministry of Science and Technology (PM-1999-0044) LF was supported by a predoctoral fellowship from the Govern´ ment of Navarra, Spain and Asociacion de Amigos de la Universidad de Navarra, Spain We would like to thank F J Rauscher III (The Wistar Institute, Philadelphia, 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 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for the relative quantitation of transcripts by semiquantitative RTPCR This material is available as part of the online article from http://www.blackwell-synergy.com 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 corresponding author for the article Supplementary material The following supplementary material is available online: FEBS Journal 274 (2007) 498–511 ª 2006 Foundation for Applied Medical Research (FIMA) Journal compilation ª 2006 FEBS 511 ... expression during all-trans-retinoic acid (ATRA)-induced differentiation of SH-SY5Y neuroblastoma cells We opted for ATRA as differentiating agent because it has been widely employed in both Transcriptional. .. 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... CRMP-2 transcriptional activity in SH-SY5Y neuroblastoma cells For this purpose, we analyzed the binding of the following transcriptional regulators: E2F, which participates in the control of cell