BMC Research Notes BioMed Central

Short Report Open Access Identification and classification of regulated by phosphatidylinositol 3-kinase- and TRKB-mediated signalling pathways during neuronal differentiation in two subtypes of the neuroblastoma cell line SH-SY5Y Yuichiro Nishida, Naoki Adati, Ritsuko Ozawa, Aasami Maeda, Yoshiyuki Sakaki and Tadayuki Takeda*

Address: Computational and Experimental Systems Biology Group, RIKEN Genomic Sciences Centre, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan Email: Yuichiro Nishida - [email protected]; Naoki Adati - [email protected]; Ritsuko Ozawa - [email protected]; Aasami Maeda - a- [email protected]; Yoshiyuki Sakaki - [email protected]; Tadayuki Takeda* - [email protected] * Corresponding author

Published: 28 October 2008 Received: 12 September 2008 Accepted: 28 October 2008 BMC Research Notes 2008, 1:95 doi:10.1186/1756-0500-1-95 This article is available from: http://www.biomedcentral.com/1756-0500/1/95 © 2008 Takeda et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract Background: SH-SY5Y cells exhibit a neuronal phenotype when treated with all-trans retinoic acid (RA), but the molecular mechanism of activation in the signalling pathway mediated by phosphatidylinositol 3-kinase (PI3K) is unclear. To investigate this mechanism, we compared the expression profiles in SK-N-SH cells and two subtypes of SH-SY5Y cells (SH-SY5Y-A and SH- SY5Y-E), each of which show a different phenotype during RA-mediated differentiation. Findings: SH-SY5Y-A cells differentiated in the presence of RA, whereas RA-treated SH-SY5Y-E cells required additional treatment with brain-derived neurotrophic factor (BDNF) for full differentiation. After exposing cells to a PI3K inhibitor, LY294002, we identified 386 genes and categorised these genes into two clusters dependent on the PI3K signalling pathway during RA- mediated differentiation in SH-SY5Y-A cells. Transcriptional regulation of the gene cluster, including 158 neural genes, was greatly reduced in SK-N-SH cells and partially impaired in SH-SY5Y- E cells, which is consistent with a defect in the neuronal phenotype of these cells. Additional stimulation with BDNF induced a set of neural genes that were down-regulated in RA-treated SH- SY5Y-E cells but were abundant in differentiated SH-SY5Y-A cells. Conclusion: We identified gene clusters controlled by PI3K- and TRKB-mediated signalling pathways during the differentiation of two subtypes of SH-SY5Y cells. The TRKB-mediated bypass pathway compensates for impaired neural function generated by defects in several signalling pathways, including PI3K in SH-SY5Y-E cells. Our expression profiling data will be useful for further elucidation of the -transcriptional network involving PI3K or TRKB.

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Background days and were then incubated with 50 ng/ml BDNF in D- SH-SY5Y cells are the third successive subclone of the SK- MEM/F12 without serum for 3 days. N-SH human neuroblastoma cell line [1]. These cells arrest in the G1 phase and exhibit a distinct neuronal phe- Total RNA preparation notype when treated with RA [2]. Morphological changes For microarray experiments, total cellular RNA was and expression of biochemical and functional neural extracted from cells at specific intervals using a RiboPure markers in SH-SY5Y cells treated with RA resemble those Kit (Ambion) in accordance with the manufacturer's of neurons. SH-SY5Y cells are thus used as a model system instructions. The quality of total RNA was assessed using for studying the molecular mechanisms involved in neu- an Agilent 2100 Bioanalyzer (Agilent Technologies). RNA ronal differentiation [3-5]. samples were prepared at least twice for each cell line and each time point, were then stored at -80°C. In SH-SY5Y cells, the PI3K/AKT signalling pathway acti- vated by RA is important for the regulation of neuronal Oligonucleotide microarray (GeneChip) analysis survival and differentiation [6]. In addition, RA promotes Microarray analysis was conducted according to the man- the activation of PI3K, leading to the activation of a Rho ufacturer's instructions (Affymetrix) and was performed at GTPase, RAC1, that is implicated in the activation of least twice in order to confirm the reproducibility of gene MAPKs, expression of neural markers and neurite out- expression profiles. growth in SH-SY5Y cells [7]. RA treatment of SH-SY5Y cells also induces expression of TRKB (NTRK2), but not of Computational analysis of microarray data TRKA (NTRK1), and mediates biological responsiveness Mean signal intensity for all probes was initially tuned to to receptors for the neurotrophins BDNF and NT-4/5 [8]. 500 as global scaling and individual signal intensities Additional treatment of SH-SY5Y cells with BDNF stimu- were evaluated by a detection call (present/marginal/ lates tyrosine phosphorylation of TRKB [9], followed by absent) using Affymetrix MicroArray Suite 5.0 software activation of the PI3K/AKT and Ras/MAPK pathways, and (Affymetrix). Absent or marginal detection was judged the promotion of cell survival and neurite outgrowth in based on a detected transcript being unreliable or suspi- serum-free medium [8,10]. cious. Signal intensities for all defective probes meeting this criterion were thus tuned to 100, the average signal Although the activation mechanisms of signalling path- intensity of these probes. As most probe sequences were ways stimulated by RA and the neurotrophin have been designed for the 3' regions of genes [11], each signal inten- extensively studied, the link between these pathways and sity was normalised using a normalisation factor of the downstream transcriptional network controlling the 30,000, the signal intensity of the probe (AFFX-HUM- expression of target genes required for differentiation of GAPDH/M33197_3_at) derived from the 3' region of SH-SY5Y cells remains unclear. To examine these mecha- GAPDH. All probes on the U133 Plus 2.0 Array were nisms, we compared the profiles in SK-N- mapped on the (NCBI Build 36.1) with SH cells and two subtypes of SH-SY5Y cells (SH-SY5Y-A BLAT [12], and probes with sequences that matched Ref- and SH-SY5Y-E), each of which display a different mor- Seq mapping data [13] were selected. We utilised 29,473 phology during RA-mediated differentiation. reliable target probes that mapped to their target genes using the Affymetrix formula annotation. Microarray data Methods related to this study are available from the GEO database Cell culture (accession number: GSE9169) [14]. A protocol including 15% FBS in the culture condition has been previously described [10] as a method for Results and discussion sequential treatment of SH-SY5Y cells with RA and BDNF, Phenotypic differences between two subtypes of SH-SY5Y although the present study used a D-MEM/F12 1:1 mix- We observed clear differences in the RA-induced neuronal ture medium, as recommended in the product informa- phenotype of the two SH-SY5Y subclones, SH-SY5Y-A and tion sheets. Briefly, random cultured cells from two clone SH-SY5Y-E, which were obtained from two different subtypes of SH-SY5Y and SK-N-SH were seeded on lam- bioresource centres (ATCC and ECACC, respectively). inin-coated culture dishes (BD Bioscience) for 1 day, and After 5 days of RA treatment, ECACC (SH-SY5Y-E) cells were then transferred to medium containing 10 μM RA in were slightly larger in size (Fig. 1A) and contained a signif- the presence or absence of LY294002 (10 μM) for 5 days. icant amount of neuroblastic (N-type) cells and a small For BDNF-induced sequential differentiation of SH-SY5Y- fraction of epithelial (S-type) cells (Fig. 1B). The appear- E cells, cells were washed with D-MEM/F12 twice after 5 ance of S-type cells was suppressed by RA-mediated differ- entiation on laminin, which promotes neurite outgrowth

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MorphologicalFigure 1 changes in two subtypes of SH-SY5Y and SK-N-SH under differentiation conditions Morphological changes in two subtypes of SH-SY5Y and SK-N-SH under differentiation conditions. The panels show SH-SY5Y-E cells (A-C), SH-SY5Y-A cells (D-F) and SK-N-SH cells (G-I). The cells are nondifferentiated (A, D and G), treated for 5 days with 10 μM RA (B, E and H), and sequentially treated with BDNF in the absence of serum for 5 days in SH- SY5Y-E cells (C) or for 3 days in SH-SY5Y-A (F) and SK-N-SH (I) cells. Arrows indicate S-type cells. Phase contrast microscopy ×100. Scale bar represents 200 μm. Note that RA-treated SK-N-SH cells gradually die in the absence of serum (I).

of SH-SY5Y cells [15]. Full differentiation of RA-treated N-SH cells (> 80%) in the presence of RA (Fig. 1H). cells required sequential treatment with BDNF (Fig. 1C), Instead, these cells elongate and gradually undergo cell thus supporting previous findings [10]. Conversely, ATCC death with sequential BDNF treatment under serum-free (SH-SY5Y-A) cells easily differentiated into a neuronal conditions (Fig. 1I). phenotype with extended neurites and formed a neural network after 5 days of RA treatment (Fig. 1E), again sup- Identification of expressed genes by using microarray porting previous observations [16]. The neuronal pheno- analysis type of SH-SY5Y-A cells was further enhanced by In order to identify the genes required for progression of sequential treatment with RA and BDNF in serum-free neuronal differentiation in RA-treated neuroblastoma medium for 3 days (Fig. 1F). The original SK-N-SH human cells, we first performed a microarray analysis of global neuroblastoma cell line comprises two different cell types: gene expression profiles for the two subtype clones of SH- a large fraction of epithelial Schwann cells (S-type cells); SY5Y and SK-N-SH cells treated with RA for 5 days. We and a small number of neuroblast cells (N-type cells) [17]. also included a perturbation experiment using a potent No neural network is formed by the larger fraction of SK- PI3K inhibitor, LY294002, to impair RA-induced differen-

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tiation of SH-SY5Y cells (data not shown) [6,7]. For full 3,690 genes (5,119 probes) differentiation of RA-treated SH-SY5Y-E cells, gene expres- sion profiles were also analysed following sequential BDNF treatment of cells for an additional 3 days [10]. Filteredby the difference in expression profiles We selected 3690 genes (5119 probes) for comparative between analysis of gene expression profiles. These genes were extracted by combining 3100 genes (3628 probes) SH-SY5Y-A + RA SH-SY5Y-A + RA derived from a sum of the top 1000 probes exhibiting sig- and and nificant expression patterns by ANOVA [18,19] under the SK-N-SH + RA SH-SY5Y-A + RA 5 culture conditions described above, with 1059 genes +LY294002 (1950 probes) identified as being more significant (Fig. 2). 2,517 genes 513 genes Classification of genes regulated by the PI3K signalling (3,268 probes) (603 probes) pathway We first compared the differential gene expression profiles of SH-SY5Y-A cells and SK-N-SH cells, because there is a 448 genes clear phenotypic difference between these cell lines under (Common tobothproduct sets) RA-treated conditions (Fig. 1). Two-factor ANOVA has previously been used for the statistical analysis of normal- Removed 29 genes ised data in order to determine differences in gene expres- (maximum intensity < 400) sion between cell lines and time points [20]. As summarised in Figure 2, we identified a gene cluster con- taining 2517 genes with significantly different expression 419 genes profiles (p < 0.001) between SH-SY5Y-A cells and SK-N- SH cells treated with RA for 5 days. We also identified 513 genes with expression levels that were significantly down- Removed 33 genes* or up-regulated when PI3K was inhibited in RA-treated (Cluster Other) SH-SY5Y-A cells. Interestingly, 448 of these genes (87.3%) were common in the product sets of gene clusters selected on the basis of differences in expression pattern between 386 genes SH-SY5Y-A and SK-N-SH. Moreover, expression behav- iours of the selected genes were almost identical to those in RA-treated SK-N-SH cells. By removing genes with con- Cluster A tradictory profiles or low expression levels, we finally Down-regulatedby LY294002 : 316 genes** identified gene clusters A and B, comprising 386 genes, (Low level expression in SK-N-SH) and a third "other" cluster with 33 genes regulated by the PI3K signalling pathway in RA-treated SH-SY5Y-A cells. Cluster B Up-regulatedby LY294002 : 71 genes Figures 3 and 4 show a heat map of expression profiles for (High level expression in SK-N-SH) all genes in the clusters. We calculated relative signal val- ues (0.000–1.000) of gene expression levels in each clus- * Showing discrepancies in their profiles ter under all culture conditions, as summarised in ** Including 2 TRKB variants Additional File 1. Cluster A comprises 316 genes with down-regulated expression in RA-treated SH-SY5Y-A cells ClassificationwayFigure 2 of genes regulated by the PI3K signalling path- cultured with LY294002 or extremely low-level expression Classification of genes regulated by the PI3K signal- in RA-treated SK-N-SH cells. Cluster B includes 71 genes ling pathway. A flow chart summary of how the genes with up-regulated expression in RA-treated SH-SY5Y-A (probes) were categorised into two clusters by statistical cells cultured with PI3K inhibitor or high-level expression analysis of the microarray data. Cluster A includes genes in SK-N-SH cells (Figs. 2, 3 and 4 ). Half of the genes in down-regulated by LY294002 in RA-treated SH-SY5Y-A cells cluster A showed average signal values < 0.1 in RA-treated and those maintained at a low level in RA-treated SK-N-SH cells. Conversely, cluster B contains genes up-regulated by SK-N-SH cells, too low for quantitative comparison LY294002 in SH-SY5Y-A cells and maintained at a high level (Additional File 1). To address the effect of a PI3K inhibi- in SK-N-SH cells. tor on gene expression in the two clusters, we compared

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0.0 0.5 1.0

Cluster A: Neural Functions (158 genes) Cluster A: Other Functions (158 genes) SY5Y-A+RA SK+RA SY5Y-E+RA SY5Y-A+RA SK+RA SY5Y-E+RA -LY +LY -LY +LY -LY +LY -LY +LY -LY +LY -LY +LY DCX SQLE HMP19 PTPRO PNMA2 ST3GAL6 KIF5C ACTR3B MAP2 C20orf103 SV2C ZNF300 NEFL RAB33A C6orf117 ZNF423 REEP1 CD24 ST8SIA2 CBS CRMP1 FAM59B INA LMO1 KIF1A FLJ25076 HAND1 C6orf159 TAGLN3 LOC441061 PTEN PRR6 PHF21B ARNTL SCG3 MYCN GDAP1 ALCAM ELAVL4 LOC201164 L1CAM HN1 PAK3 PGM2L1 RTN1 ENPP2 STMN2 ANKRD28 MAPT NMNAT2 SH3PXD2A PHACTR3 SLC8A3 AFF3 ANK2 AP3B2 GRIK2 ARL6 CACNA1B PAP2D RUFY3 ZNF195 SHANK2 ELAVL1 NAPB CAMTA1 ALK CXADR SATB1 TRIM24 SEZ6L JMJD1C MYT1L ESRRG PCDH17 BCOR MAP6 PLEKHA6 SYT13 BAMBI TPM3 TUB CSPG3 CD200 TSPAN2 PLCD4 NEF3 ZFP37 NRXN1 MLLT11 KNS2 DKFZP686A01247 PPEF1 MMD NDRG4 DUSP6 FSD1 RGS16 PRICKLE1 ATP6V0A2 GPR19 PDK1 CAMK2N1 C3orf32 IGSF4 GPR35 FOXC1 AQP1 CNNM2 C5orf16 ANKRD6 C6orf137 AGTPBP1 TTC9 ROR1 BCL2 LRP6 ARHGEF7 CHRFAM7A TRO CHRNA7 TSHZ3 PTPRN2 DPP6 MAML3 CORIN PLD5 CALCA MAPK10 OSBPL10 ABAT XPNPEP1 STXBP1 GNG2 PPP2R2C MAGED2 FYN LOC92196 JAKMIP1 FNDC5 ALDOC MMP28 KLF7 UBL3 OLFM3 SCD TRKB (-FL, -T-Shc) HOXD4 GRIA3 SCGN GNG3 CCBE1 NPTX1 PRKCH MAP4 PGM1 SLC30A3 PAPSS1 GRIA1 NEBL PRKAR2B B4GALNT1 BASP1 CCNA1 TUBB2A ENO3 DDC KIAA1458 HOOK1 SGK EML4 APC NEFH FAM43A ISL1 TRIB2 SLC38A1 DBC1 TUBA3 STARD4 PIK3R3 RGS13 DLX6 LHFPL2 DPYSL5 RAPGEF4 PRIMA1 FAM63B FEV KLHDC8B CKB PHTF1 NRCAM ZBED5 SHOX2 FKBP1B HTR1E MEIS2 UCHL1 VPS13D NBEA ADD3 EDG2 C7 DACH1 ARHGAP28 SNCA KIAA0644 GAP43 FAM13A1 ZIC1 SPRY1 D4S234E PPFIBP2 KCNH2 FLJ41603 BAALC TP53BP1 YWHAG DKK2 TUBA6 C20orf52 TUBB3 ECH1 K-ALPHA-1 MMEL1 ACOT7 HIVEP2 SMARCA4 C1orf76 HEY1 COLEC11 TLE1 ARMC9 SLITRK6 NEK3 ZNF179 MAFB LMO3 INSIG1 SEMA6A ARMCX1 TFAP2B WNT3 RPH3A LOC120376 SCG2 TSLP SCN3A IL20RA FGF11 ACAT2 NCALD RBMX IGSF11 DHCR24 PCDHAC2 UCK2 PCDHAC1 ZWILCH PCDHA8 METAP2 PCDHA7 SFRS2 PCDHA6 POP7 PCDHA5 OBFC2B PCDHA4 DNMT3A PCDHA3 PLCXD3 PCDHA2 ITGA9 PCDHA13 RCC2 PCDHA12 TBPL1 PCDHA11 RPP25 PCDHA10 KIAA1509 PCDHA1 C1orf19 PCDHA9 SEPHS1 SCG5 BCR C5orf13 UBE2E1 NGFRAP1 C1orf36 CALCB PIR NFIA SH3TC1 CRYM DCN HTR2B ASTN2 BAI3 GPR22 MPPED2 SEPT6 TRKB (-T1) PRSS2 FREM1 KIAA0125 PCDH18 C1orf106 CA10 CNKSR3 EFNB2 LOC283824 ACCN2 CREB3L2 0005 0 5 0 5 5 5 0 5 0005 0 5 0 5 5 5 0 5 day day

FigureGene expression 3 profiles during RA-mediated differentiation Gene expression profiles during RA-mediated differentiation. Gene expression levels were examined at six time points (0 h, 6 h, 1 day, 2 days, 3 days and 5 days) after addition of RA to each neuroblastoma culture. Heat map representation of genes differentially expressed in the indicated cell lines stimulated with RA is shown in the presence or absence of a PI3K inhibitor. Genes are clustered according to hierarchical clustering with Pearson’s correlation. A colour-coded scale (green, down-regulation; red, up-regulation) for percentage expression is indicated at the top of the figure. Cluster A includes genes that were down-regulated by LY294002 in RA-treated SH-SY5Y-A cells. Genes in the cluster were further classified into two subgroups: neural genes; and genes related to other functions. Genes selected in Figure 6 are marked in blue on the right side of the heat map of cluster A.

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the average signal values of gene expression levels greater supplementation with an additional TRKB signalling than 0.179, the average signal value in the clusters, pathway is required for full differentiation of SH-SY5Y-E between RA-treated SK-N-SH cells in the presence and cells after down-regulation of the gene cluster required for absence of LY294002. In cluster A, the average signal val- neural events. ues of 32 of 77 (42%) gene expression levels did not dip below 75% of the average values when RA-treated SK-N- Human TRKB is alternatively spliced into at least 3 vari- SH cells were exposed to LY294002. Similarly, the average ants: TRKB-FL; TRKB-T1; and TRKB-T-Shc [25]. TRKB-FL is signal values in 35 of 65 (54%) gene expression levels in a tyrosine kinase-containing variant, whereas the intracel- cluster B did not exceed 125% of the average values by the lular tyrosine kinase domain is truncated in the other pro- addition of a PI3K inhibitor (Additional file 1). These teins [26,27]. Two typical gene expression profiles were findings are consistent with a substantial defect in tran- observed by microarray analysis in accordance with differ- scriptional regulation mediated by the PI3K signalling ent transcriptional regulation on the alternative promot- pathway in RA-treated SK-N-SH cells. ers (Fig. 5B; bottom panels) [28]. In SH-SY5Y-A cells, TRKB-FL and TRKB-T-Shc variants showed continuously We further categorised genes into two subgroups of neural induced over 5 days, whereas gene expres- functions and other functions (Figs. 3 and 4), on the basis sion of TRKB-T1 was initially induced but plateaued 1 day of (GO) annotation [21,22] and a com- after RA treatment. In SH-SY5Y-E cells, expression of all prehensive search of the literature [22-24]. Genes with TRKB variants, particularly TRKB-T1, was abruptly neural functions were defined when significant GO induced 3 days after RA treatment and peaked after 5 days term(s) and/or literal description(s) implicating involve- in the presence of RA (Fig. 5B; bottom panels), thus sup- ment in neural events were obtained by the gene annota- porting previous results [29]. These TRKB variants were tion. We also defined genes with other functions when also transcriptionally regulated by the PI3K signalling neither the GO term nor the literal description was related pathway (Fig. 5B; bottom panels), indicating clear cross- to neural events. GO annotation in two clusters yielded talk between the TRKB and PI3K signalling pathways. the identification of 114 genes with GO terms related to Conversely, induction of TRKB variants was not observed neural events (Additional File 2). Cluster A genes included in SK-N-SH cells at any time, providing a possible expla- 158 genes in the neural function subgroup and 158 genes nation for the defect in the neuronal phenotype of these in the other functions subgroup. Cluster B genes were sim- cells (Fig. 5B, bottom panels). ilarly classified, with 11 genes in the neural function sub- group and 60 genes in the other functions subgroup (Fig. Transcriptional compensation by an additional TRKB- 3 and 4). Neural functions were further divided into 9 mediated signalling pathway in SH-SY5Y-E functions, as summarised in Additional File 2. Additional BDNF treatment of SH-SY5Y cells stimulates tyrosine phosphorylation of TRKB [9], promoting neurite Differences in PI3K-mediated transcriptional regulation outgrowth in serum-free medium [8,10]. Supporting between two subtypes of SH-SY5Y these findings, additional exposure of RA-treated SH- To investigate whether the PI3K signalling pathway is suf- SY5Y-E cells to BDNF resulted in full differentiation and ficiently activated in RA-treated SH-SY5Y-E cells, we fur- induced 89 genes that were down-regulated or main- ther focused on the transcriptional regulation of the 386 tained at low expression levels with RA treatment only genes included in these clusters. Differences in expression (Figs. 5B, 6). These genes were further sorted into two profiles of RA-treated SH-SY5Y-E cells with and without gene pools: 54 genes from cluster A (shown in blue in Fig. LY294002 were compared to differences in expression 3; Fig. 6, left panel) and 35 genes regulated in a PI3K-inde- profiles of RA-treated SH-SY5Y-A cells with and without pendent manner (Fig. 6, right panel). As expected, both of LY294002 (Fig. 3 and 4). Neurite outgrowth induced in these pools contain many genes required for neural events RA-treated SH-SY5Y-E cells was also inhibited by (Fig. 6; Additional File 2), indicating that these genes are LY294002, thus suggesting that the PI3K signalling path- critical for TRKB-mediated differentiation of SH-SY5Y-E way is functional in SH-SY5Y-E cells. However, most of cells. These results also demonstrate that genes down-reg- the gene expression in the clusters was regulated in a PI3K- ulated either by an impaired PI3K signalling pathway or independent manner, and half of the neural genes were by another abrogated signalling pathway are transcrip- expressed at extremely low levels (Figs. 3, 4 and 5A), tionally compensated for via an additional TRKB-medi- whereas transcriptional inhibition by LY294002 exclu- ated signalling pathway, leading to full differentiation sively emerges after 2–3 days in the presence of RA (Fig. (Additional File 3). However, a significant number of 5A, 5B; bottom panels). These results suggest that the genes down-regulated or maintained at low expression PI3K signalling pathway is partially disrupted in RA- levels in RA-treated SH-SY5Y-E cells did not exceed the treated SH-SY5Y-E cells, with this disruption leading to threshold (Fig. 6 legend) following additional BDNF reduced differentiation. These results also suggest that treatment (Fig. 3; Additional File 1), thus suggesting that

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Cluster B: Neural Functions (11 genes) Cluster B: Other Functions (60 genes) SY5Y-A+RA SK+RA SY5Y-E+RA SY5Y-A+RA SK+RA SY5Y-E+RA -LY +LY -LY +LY -LY +LY -LY +LY -LY +LY -LY +LY GAS1 CLU TFPI2 STAT3 KLF6 SPP1 PLXNA1 APLP2 TIMP1 CRLF1 VIL2 FLNB CD99 ID1 SFT2D1 FLNA VIM IQGAP1 SEMA3C THBS1 RDX PDLIM1 BCAP29 0005 0 5 0 5 5 5 0 5 ANXA2 METRNL day LOC653506 TPM1 FBN1 COL5A1 TMEM166 PRKCSH H2AFY CD59 HRH1 PPAPDC1A TAGLN DACT1 FN1 CYR61 NARG1 CCL2 GPC6 DOC1 FDXR RDH10 HIST3H2A DLC1 LTBP3 C10orf54 SMAD3 TBC1D17 PDCD6IP CEBPD ACP6 B3GAT3 HIBADH NPC2 C1QTNF1 TMEM129 HIST2H2AA3 HIST2H2AA4 SGSH CFI PDLIM5 FRMD6 PPIC IL13RA1 CRTAP LIFR KIAA0746 MGP 0005 0 5 0 5 5 5 0 5 day Cluster Other:Neural Functions (9 genes) Cluster Other: Other Functions (24 genes) SY5Y-A+RA SK+RA SY5Y-E+RA SY5Y-A+RA SK+RA SY5Y-E+RA -LY +LY -LY +LY -LY +LY -LY +LY -LY +LY -LY +LY CHRNA3 C20orf19 SNCAIP RPS15 DDIT4 RPL13 LAMA4 YPEL3 ARHGAP20 CG018 IGFBP5 CDH11 ENO1 AMPD3 CXCL12 KIAA1217 GREM1 ARF3 PGAM1 5 LOC643576 0005 0 5 0 5 5 5 0 LOC642969 day LDHA CGI-38 SLC22A18 UBE1L MAPK12 WARS C3orf28 MSRB2 HNRPA3 MKI67 CHEK2 MCAM 0005 0 5 0 5 5 5 0 5 day GeneFigure expression 4 profiles during RA-mediated differentiation (continued). Gene expression profiles during RA-mediated differentiation (continued). Cluster B includes genes that were up- regulated by LY294002 in SH-SY5Y-A cells. Cluster “Other” includes 33 genes with contradictory expression profiles between RA-treated SH-SY5Y-A cells with LY294002 and RA-treated SK-N-SH cells. Genes in each cluster were further classified into two subgroups: neural genes; and genes related to other functions. the TRKB-dependent signalling pathway only partially TRKB-FL. Further studies may be required to identify the compensates for an impaired PI3K signalling pathway. components and transcription factors involved in the The TRKB variants TRKB-FL and TRKB-T-Shc were induced TRKB and PI3K signalling pathways. and plateaued following sequential treatment of SH- SY5Y-E cells with RA and BDNF. Conversely, TRKB-T1 In conclusion, we identified gene clusters that are tran- expression, which was strongly induced by RA treatment scriptionally controlled by two different signalling path- of SH-SY5Y-E cells, was abruptly down-regulated after a ways mediated by PI3K and TRKB during the shift to serum-free medium with BDNF (Fig. 5B, bottom differentiation of two subtypes of SH-SY5Y cells. These panels), which supports the notion that the dominant- expression profiling data may prove useful in further elu- negative function of tyrosine kinase-deficient receptors cidating the molecular mechanisms regulating the pro- [30] is totally diminished by induction of functional moter activities of genes required for neuronal

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A ABAT PCDHA9 BCL2 KIF5C y y y y t t t t i i i i s s s s n n n n e e e e t t t t n n n n I I I I 2000 4000 6000 500 1500 2500 1000 3000 5000 0 1000 3000 5000 0 1d 2d 3d 4d 5d 0 1d 2d 3d 4d 5d 0 1d 2d 3d 4d 5d 0 1d 2d 3d 4d 5d +RA +RA +RA +RA Time Time Time Time

B MAP2 NFASC y y t t i i s s n n e e t t n n I I 2000 4000 6000 8000 200 600 1000

0 1d 2d 3d 4d 5d/0 1d 2d 3d 0 1d 2d 3d 4d 5d/01d 2d 3d +RA +BDNF +RA +BDNF Time Time TRKB-FL TRKB-T1 TRKB-T-Shc y y t t y i i t i s s s n n n e e t t e t n n I I n I 200 600 1000 1400 0 2000 4000 6000 8000 0 500 1500 2500 0 1d 2d 3d 4d 5d/0 1d 2d 3d 0 1d 2d 3d 4d 5d/0 1d 2d 3d 0 1d 2d 3d 4d 5d/0 1d 2d 3d +RA +BDNF +RA +BDNF +RA +BDNF Time Time Time

SH-SY5Y-A SH-SY5Y-A+LY SK-N-SH SK-N-SH+LY SH-SY5Y-E SH-SY5Y-E+LY TypicalFigure gene5 expression profiles of genes identified in this study. Typical gene expression profiles of genes identified in this study.A) Typical gene expression profiles in RA-treated SH- SY5Y-E cells. ABAT, which is partially regulated by the PI3K signalling pathway, and PCDHA9, BCL2 and KIF5C, which are independently regulated by the PI3K signalling pathway, are included in cluster A. B) Typical expression profiles of genes such as MAP2 (left upper panel), NFASC (right upper panel) and TRKB variants (bottom panels), which were categorised as depend- ent on the PI3K signalling pathway in RA-treated SH-SY5Y-A cells. differentiation. These promoter activities are mediated by Eagle's medium/nutrient mixture F12; FBS: Fetal Bovine an upstream signal transduction-transcriptional network Serum; ANOVA: analysis of variance. including PI3K and/or TRKB. Competing interests Abbreviations The authors declare that they have no competing interests. RA: all-trans retinoic acid; PI3K: phosphatidylinositol 3- kinase; BDNF: brain-derived neurotrophic factor; TRKA Authors' contributions (NTRK1): Tropomyosin-related kinase A; TRKB (NTRK2): TT, YN and NA designed the research plan and AM per- Tropomyosin-related kinase B; MAPK: mitogen-activated formed morphological analysis of the neuroblastoma kinase; NT-4/5: neurotrophin-4/5; ATCC: Ameri- cells. NA and RO conducted the microarray analysis. TT can Type Culture Collection; ECACC: European Collec- evaluated the results and YN performed statistical analysis tion of Cell Cultures; D-MEM/F12: Dulbecco's modified of the microarray data. YS and TT supported the work. TT, YN and NA wrote the manuscript.

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SH-SY5Y-A SH-SY5Y-E SK-N-SH +RA +RA +LY +RA +BDNF +BDNF 54 genes TUBB3 EML4 NEFH DPYSL5 SQLE TUBA6 0.0 0.5 1.0 ACOT7 ZNF300 HOOK1 DUSP6 TAGLN3 IS L1 MAP2 PLD5 J MJD1C AGTPBP1 ANKRD6 SH-SY5Y-A SH-SY5Y-E SK-N-SH TRKB (-T-Shc) SEZ6L +RA +RA+LY +RA +BDNF +BDNF 35 genes ALK ZDHHC21 ELAVL4 UBE2D3 PGM2L1 MXI1 PTPRN2 MAPK8 STARD4 RGS7 ESRRG NFASC TUBB2A KCNQ2 TRIB2 TTC3 PPP2R2C TSPAN7 CHRNA7 RIMBP2 CHRFAM7A LOC196394 KIAA1458 NCAM1 SLC8A3 HPCAL1 SH3PXD2A IRF6 REEP1 RAPGEF5 C6orf117 CREB5 KLF7 TMSL8 PAK3 GTSE1 PAP2D NPTX2 MAPT YBX1 CAMTA1 GLDC PNMA2 ARG2 KIF5C WDR68 MAP6 PHOX2B KNS2 GDAP1L1 CXADR GNAZ TSPAN2 GPC2 RTN1 WHSC1 CAMK2N1 ACTL6B IGS F4 TNRC4 HAND1 J PH3 SCG3 KIF1B PHF21B GABRB3 GDAP1 PGBD5 SATB1 PDZD4 0 5 0 5 0 5 0 1 3 031 0 5 0 5 0 5 0 1 3 0 1 3 day day

FigureExpression 6 profiles of genes induced by sequential treatment with BDNF in SH-SY5Y-E cells. Expression profiles of genes induced by sequential treatment with BDNF in SH-SY5Y-E cells. Heat map represen- tation is shown for genes that are down-regulated or maintained at a low level in the presence of RA but are induced by addi- tional treatment with BDNF in SH-SY5Y-E cells. RNA sampling was performed at five time points (0 h, 6 h, 1 day, 2 days and 3 days) after cells were transferred to serum-free medium containing BDNF. To identify the gene expression induced by BDNF, we focused on genes with expression levels that were higher in SH-SY5Y-A cells during differentiation, rather than those in SH-SY5Y-E or SK-N-SH cells. We also selected genes with expression levels in SH-SY5Y-A cells treated with RA for 5 days that exceeded 66.7% of that on day 0, while excluding genes with expression levels that exceeded the 66.7% threshold in SH- SY5Y-E cells treated with RA for 5 days. We selected genes with expression levels that underwent a >1.5-fold change and exceeded the 66.7% threshold when SH-SY5Y-E cells were treated sequentially with BDNF for 3 days. Finally, genes exhibiting similar up-regulation by BDNF in SH-SY5Y-E cells and SK-N-SH cells were also excluded. Genes regulated by the PI3K signal- ling pathway in RA-treated SH-SY5Y-A cells (left panel) and genes not regulated by the PI3K signalling pathway in SH-SY5Y-A cells (right panel) are shown. The relative alteration of gene expression in these gene pools is summarised in Additional file 1. Genes involved in neural processes are shown in red, and those expressed strongly in brain are shown in purple. Genes encod- ing transcription factors are underlined.

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Additional material References 1. Ross RA, Spengler BA, Biedler JL: Coordinate morphological and biochemical interconversion of human neuroblastoma cells. Additional file 1 J Natl Cancer Inst 1983, 71:741-747. 2. Pahlman S, Ruusala A-I, Abrahmsson L, Mattsson MEK, Esscher T: Relative change (0.000–1.000) in gene expression profiles under all cul- Retinoic acid-induced differentiation of cultured human neu- ture conditions. A relative minimal value (0.000) and a relative maximal roblastoma cells: a comparison with phorbolester-induced value (1.000) were calculated for each gene by using minimal and - differentiation. Cell Differ 1984, 14:135-144. imal signal values in a series of gene expression profiles under all culture 3. Pahlman S, Hoehner JC, Nanberg E, Hedborg F, Fagerstrom S, Gest- conditions. The signal value on day 0, average signal value and maximal blom C, Johansson I, Larsson U, Lavenius E, Ortoft E, Soderholm H: Differentiation and survival influences of growth factors in signal value (or minimal signal value) was calculated for each gene human neuroblastoma. Eur J Cancer 1995, 31A:453-458. expression profile in all clusters and are shown together with the expres- 4. Singh US, Pan J, Kao Y-L, Joshi S, Young KL, Baker KM: Tissue trans- sion patterns such as up-regulation (up), down-regulation (down) or con- glutaminase mediates activation of RhoA and MAP kinase stant expression (const) on the basis of the average signal value, as pathways during retinoic acid-induced neuronal differentia- compared with ± 25% fluctuation values of the signal value on day 0. tion of SH-SY5Y cells. J Biol Chem 2003, 278:391-399. Click here for file 5. Tucholski J, Lesort M, Johnson GVW: Tissue transglutaminase is essential for neurite outgrowth in human neuroblastoma [http://www.biomedcentral.com/content/supplementary/1756- SH-SY5Y cells. Neuroscience 2001, 102:481-491. 0500-1-95-S1.xls] 6. Lopez-Carballo G, Moreno L, Masia S, Perez P, Barettino D: Activa- tion of the phosphatidylinositol 3-kinase/Akt signaling path- Additional file 2 way by retinoic acid is required for neural differentiation of SH-SY5Y human neuroblastoma cells. J Biol Chem 2002, Gene functions for neural genes in each cluster. Genes in the clusters were 277:25297-25304. first analysed and categorised using Gene Ontology (GO) annotation 7. Pan J, Kao Y-L, Joshi S, Jeetendran S, DiPette D, Singh US: Activation (downloaded in April 2007) [21]. The gene-GO association was obtained of Rac1 by phosphatidylinositol 3-kinase in vivo: role in acti- from the NCBI Gene [22]. A comprehensive search of the litera- vation of mitogen-activated (MAPK) path- ture was further performed using the "Summary" and "GeneRIFs" sec- ways and retinoic acid-induced neuronal differentiation of SH-SY5Y cells. J Neurochem 2005, 93:571-583. tions in NCBI, Entrez Gene [22], NCBI, Pub Med [23] and the 8. Encinas M, Iglesias M, Llecha N, Comella JX: Extracellular-regu- "Research Articles" section in Gene Cards [24]. The 9 categories for gene lated kinases and phosphatidylinositol 3-kinase are involved function are: -based process, cell adhesion, axon guidance, in brain-derived neurotrophic factor-mediated survival and synaptic events, ion channel, neurite outgrowth, and receptor neuritogenesis of the neuroblastoma cell line SH-SY5Y. J binding, signal transduction and others. Neurochem 1999, 73:1409-1421. Click here for file 9. Kaplan DR, Matsumoto K, Lucarelli E, Thiele CJ: Induction of TrkB by retinoic acid mediates biologic responsiveness to BDNF [http://www.biomedcentral.com/content/supplementary/1756- and differentiation of human neuroblastoma cells. Neuron 0500-1-95-S2.xls] 1993, 11:321-331. 10. Encinas M, Iglesias M, Liu Y, Wang H, Muhaisen A, Cena V, Gallego C, Additional file 3 Comella JX: Sequential treatment of SH-SY5Y cells with retinoic acid and brain-derived neurotrophic factor gives rise A possible molecular mechanism required for transcriptional regulation to fully differentiated, neurotrophic factor-dependent mediated by two different signalling pathways. Genes up-regulated during human neuron-like cells. J Neurochem 2000, 75:991-1003. RA-mediated differentiation in SH-SY5Y-A cells were classified into 11. Affymetrix Expression Analysis Technical Support [http:// groups I, II and III. Group I and group II are controlled by the PI3K sig- www.affymetrix.com/support/] nalling pathway, whereas group III is regulated by PI3K-independent 12. Kent WJ: BLAT-the BLAST-like alignment tool. Genome Res pathway(s). In RA-treated SH-SY5Y-E cells, most of the genes involved in 2002, 12:656-664. 13. UCSC Genome Bioinformatics [http://genome.ucsc.edu/] RA-mediated differentiation are regulated in a PI3K-independent man- 14. GEO Database [http://www.ncbi.nlm.nih.gov/geo/] ner. When down-regulated by the impaired PI3K signalling pathway and 15. Hynds DL, Snow DM: Fibronectin and laminin elicit differential by a defect in another signalling pathway, these genes are transcriptionally behaviors from SH-SY5Y growth cones contacting inhibitory compensated for by an additional TRKB-mediated signalling pathway, chondroitin sulfate proteoglycans. J Neurosci Res 2001, leading to full differentiation. 66:630-642. 16. Tieu K, Zuo DM, Yu PH: Differential effects of staurosporine Click here for file and retinoic acid on the vulnerability of the SH-SY5Y Neu- [http://www.biomedcentral.com/content/supplementary/1756- roblastoma cells: involvement of Bcl-2 and . J 0500-1-95-S3.pdf] Neurosci Res 1999, 58:426-435. 17. Biedler JL, Helson L, Spengler BA: Morphology and growth tum- origenicity, and cytogenetics of human neuroblastoma cell in continuous culture. Cancer Res 1973, 33:2643-2652. 18. Draghici S: Data Analysis Tools for DNA Microarrays Washington DC, Acknowledgements Chapman & Hall/Crc; 2003. 19. Saeed AI, Sharov V, White J, Li J, Liang W, Bhagabati N, Braisted J, We thank Drs. Todd Taylor and Igor Kurochkin for critical reading of the Klapa M, Currier T, Thiagarajan M, Sturn A, Snuffin M, Rezantsev A, manuscript, helpful suggestions and comments. We also thank Yuko Sano, Popov D, Ryltsov A, Kostukovich E, Borisovsky I, Liu Z, Vinsavich A, Emi Abe, Maki Kobayashi and Nagisa Nakata for providing technical support Trush V, Quackenbush J: TM4: a free, open-source system for and Dr. Takeshi Nagashima for his technical advice on the GO analysis. microarray data management and analysis. Biotechniques 2003, 34:374-378. Evaluations of GeneChip probes were carried out by the Super Computer 20. Mense SM, Sengupta A, Zhou M, Lan C, Bentsman G, Volsky DJ, System, Human Genome Center, Institute of Medical Science, University of Zhang L: Gene expression profiling reveals the profound Tokyo. This work was supported by the in-house-budget of RIKEN (J53- upregulation of hypoxia-responsive genes in primary human 40030). astrocytes. Physiol 2006, 25:435-449. 21. The GO Ontology [http://www.geneontology.org/]

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22. NCBI, Entrez Gene [http://www.ncbi.nlm.nih.gov/sites/ent rez?db=gene] 23. NCBI, Pub Med [http://www.ncbi.nlm.nih.gov/sites/ent rez?db=pubmed] 24. Gene Cards [http://www.genecards.org/] 25. Stoilov P, Castren E, Stamm S: Analysis of human TrkB gene genomic organization reveals novel TrkB isoforms, unusual gene length, and splicing mechanism. Biochem Biophys Res Com- mun 2002, 290:1054-1065. 26. Klein R, Conway D, Parada LF, Barbacid M: The trkB tyrosine pro- tein kinase gene codes for a second neurogenic receptor that lacks the catalytic kinase domain. Cell 1990, 61:647-656. 27. Middlemas DS, Lindberg RA, Hunter T: trkB, a neural receptor protein-tyrosine kinase: evidence for a full-length and two truncated receptors. Mol Cell Biol 1991, 11:143-153. 28. Barettino D, Pombo PMG, Espliguero G, Rodrigez-Pena A: The mouse neurotrophin receptor trkB gene is transcribed from two different promoters. Biochim Biophys Acta 1999, 1446:24-34. 29. Liu Y, Encinas M, Comella JX, Aldea M, Gallgo C: Basic Helix-Loop- Helix proteins bind to TrkB and p27Cip1 promoters linking differentiation and cell cycle arrest in neuroblastoma cells. Mol Cell Biol 2004, 24:2662-2672. 30. Eide FF, Vining ER, Eide BL, Zang K, Wang X-Y, Reichardt LF: Natu- rally occurring truncated trkB receptors have dominant inhibitory effects on brain-derived neurotrophic factor sign- aling. J Neurosci 1996, 16:3123-3129.

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