Identification and classification of genes regulated by phosphatidylinositol 3-kinase- and TRKB-mediated signalling pathways during neuronal differentiation in two subtypes of the human neuroblastoma cell line SH-SY5Y
© Takeda et al; licensee BioMed Central Ltd. 2008
Received: 12 September 2008
Accepted: 28 October 2008
Published: 28 October 2008
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 gene 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.
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.
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 signal transduction-transcriptional network involving PI3K or TRKB.
SH-SY5Y cells are the third successive subclone of the SK-N-SH human neuroblastoma cell line . These cells arrest in the G1 phase and exhibit a distinct neuronal phenotype when treated with RA . Morphological changes and expression of biochemical and functional neural markers in SH-SY5Y cells treated with RA resemble those of neurons. SH-SY5Y cells are thus used as a model system for studying the molecular mechanisms involved in neuronal differentiation [3–5].
In SH-SY5Y cells, the PI3K/AKT signalling pathway activated by RA is important for the regulation of neuronal survival and differentiation . In addition, RA promotes the activation of PI3K, leading to the activation of a Rho GTPase, RAC1, that is implicated in the activation of MAPKs, expression of neural markers and neurite outgrowth in SH-SY5Y cells . RA treatment of SH-SY5Y cells also induces expression of TRKB (NTRK2), but not of TRKA (NTRK1), and mediates biological responsiveness to receptors for the neurotrophins BDNF and NT-4/5 . Additional treatment of SH-SY5Y cells with BDNF stimulates tyrosine phosphorylation of TRKB , followed by activation of the PI3K/AKT and Ras/MAPK pathways, and the promotion of cell survival and neurite outgrowth in serum-free medium [8, 10].
Although the activation mechanisms of signalling pathways stimulated by RA and the neurotrophin have been extensively studied, the link between these pathways and the downstream transcriptional network controlling the expression of target genes required for differentiation of SH-SY5Y cells remains unclear. To examine these mechanisms, we compared the gene expression profiles in SK-N-SH cells and two subtypes of SH-SY5Y cells (SH-SY5Y-A and SH-SY5Y-E), each of which display a different morphology during RA-mediated differentiation.
A protocol including 15% FBS in the culture condition has been previously described  as a method for sequential treatment of SH-SY5Y cells with RA and BDNF, although the present study used a D-MEM/F12 1:1 mixture medium, as recommended in the product information sheets. Briefly, random cultured cells from two clone subtypes of SH-SY5Y and SK-N-SH were seeded on laminin-coated culture dishes (BD Bioscience) for 1 day, and were then transferred to medium containing 10 μM RA in the presence or absence of LY294002 (10 μM) for 5 days. For BDNF-induced sequential differentiation of SH-SY5Y-E cells, cells were washed with D-MEM/F12 twice after 5 days and were then incubated with 50 ng/ml BDNF in D-MEM/F12 without serum for 3 days.
Total RNA preparation
For microarray experiments, total cellular RNA was extracted from cells at specific intervals using a RiboPure Kit (Ambion) in accordance with the manufacturer's instructions. The quality of total RNA was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies). RNA samples were prepared at least twice for each cell line and each time point, were then stored at -80°C.
Oligonucleotide microarray (GeneChip) analysis
Microarray analysis was conducted according to the manufacturer's instructions (Affymetrix) and was performed at least twice in order to confirm the reproducibility of gene expression profiles.
Computational analysis of microarray data
Mean signal intensity for all probes was initially tuned to 500 as global scaling and individual signal intensities were evaluated by a detection call (present/marginal/absent) using Affymetrix MicroArray Suite 5.0 software (Affymetrix). Absent or marginal detection was judged based on a detected transcript being unreliable or suspicious. Signal intensities for all defective probes meeting this criterion were thus tuned to 100, the average signal intensity of these probes. As most probe sequences were designed for the 3' regions of genes , each signal intensity was normalised using a normalisation factor of 30,000, the signal intensity of the probe (AFFX-HUMGAPDH/M33197_3_at) derived from the 3' region of GAPDH. All probes on the U133 Plus 2.0 Array were mapped on the human genome (NCBI Build 36.1) with BLAT , and probes with sequences that matched RefSeq mapping data  were selected. We utilised 29,473 reliable target probes that mapped to their target genes using the Affymetrix formula annotation. Microarray data related to this study are available from the GEO database (accession number: GSE9169) .
Results and discussion
Phenotypic differences between two subtypes of SH-SY5Y
Identification of expressed genes by using microarray analysis
In order to identify the genes required for progression of neuronal differentiation in RA-treated neuroblastoma cells, we first performed a microarray analysis of global gene expression profiles for the two subtype clones of SH-SY5Y and SK-N-SH cells treated with RA for 5 days. We also included a perturbation experiment using a potent PI3K inhibitor, LY294002, to impair RA-induced differentiation of SH-SY5Y cells (data not shown) [6, 7]. For full differentiation of RA-treated SH-SY5Y-E cells, gene expression profiles were also analysed following sequential BDNF treatment of cells for an additional 3 days .
Classification of genes regulated by the PI3K signalling pathway
We first compared the differential gene expression profiles of SH-SY5Y-A cells and SK-N-SH cells, because there is a clear phenotypic difference between these cell lines under RA-treated conditions (Fig. 1). Two-factor ANOVA has previously been used for the statistical analysis of normalised data in order to determine differences in gene expression between cell lines and time points . As summarised in Figure 2, we identified a gene cluster containing 2517 genes with significantly different expression 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- or up-regulated when PI3K was inhibited in RA-treated 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 SH-SY5Y-A and SK-N-SH. Moreover, expression behaviours of the selected genes were almost identical to those in RA-treated SK-N-SH cells. By removing genes with contradictory profiles or low expression levels, we finally identified gene clusters A and B, comprising 386 genes, and a third "other" cluster with 33 genes regulated by the PI3K signalling pathway in RA-treated SH-SY5Y-A cells.
We further categorised genes into two subgroups of neural functions and other functions (Figs. 3 and 4), on the basis of Gene Ontology (GO) annotation [21, 22] and a comprehensive search of the literature [22–24]. Genes with neural functions were defined when significant GO term(s) and/or literal description(s) implicating involvement in neural events were obtained by the gene annotation. We also defined genes with other functions when neither the GO term nor the literal description was related to neural events. GO annotation in two clusters yielded the identification of 114 genes with GO terms related to neural events (Additional File 2). Cluster A genes included 158 genes in the neural function subgroup and 158 genes in the other functions subgroup. Cluster B genes were similarly classified, with 11 genes in the neural function subgroup and 60 genes in the other functions subgroup (Fig. 3 and 4). Neural functions were further divided into 9 functions, as summarised in Additional File 2.
Differences in PI3K-mediated transcriptional regulation between two subtypes of SH-SY5Y
Human TRKB is alternatively spliced into at least 3 variants: TRKB-FL; TRKB-T1; and TRKB-T-Shc . TRKB-FL is a tyrosine kinase-containing variant, whereas the intracellular tyrosine kinase domain is truncated in the other proteins [26, 27]. Two typical gene expression profiles were observed by microarray analysis in accordance with different transcriptional regulation on the alternative promoters (Fig. 5B; bottom panels) . In SH-SY5Y-A cells, TRKB-FL and TRKB-T-Shc variants showed continuously induced transcription over 5 days, whereas gene expression of TRKB-T1 was initially induced but plateaued 1 day after RA treatment. In SH-SY5Y-E cells, expression of all TRKB variants, particularly TRKB-T1, was abruptly induced 3 days after RA treatment and peaked after 5 days in the presence of RA (Fig. 5B; bottom panels), thus supporting previous results . These TRKB variants were also transcriptionally regulated by the PI3K signalling pathway (Fig. 5B; bottom panels), indicating clear cross-talk between the TRKB and PI3K signalling pathways. Conversely, induction of TRKB variants was not observed in SK-N-SH cells at any time, providing a possible explanation for the defect in the neuronal phenotype of these cells (Fig. 5B, bottom panels).
Transcriptional compensation by an additional TRKB-mediated signalling pathway in SH-SY5Y-E
In conclusion, we identified gene clusters that are transcriptionally controlled by two different signalling pathways mediated by PI3K and TRKB during the differentiation of two subtypes of SH-SY5Y cells. These expression profiling data may prove useful in further elucidating the molecular mechanisms regulating the promoter activities of genes required for neuronal differentiation. These promoter activities are mediated by an upstream signal transduction-transcriptional network including PI3K and/or TRKB.
all-trans retinoic acid
brain-derived neurotrophic factor
- TRKA (NTRK1):
Tropomyosin-related kinase A
- TRKB (NTRK2):
Tropomyosin-related kinase B
mitogen-activated protein kinase
American Type Culture Collection
European Collection of Cell Cultures
Dulbecco's modified Eagle's medium/nutrient mixture F12
Fetal Bovine Serum
analysis of variance.
We thank Drs. Todd Taylor and Igor Kurochkin for critical reading of the manuscript, helpful suggestions and comments. We also thank Yuko Sano, Emi Abe, Maki Kobayashi and Nagisa Nakata for providing technical support and Dr. Takeshi Nagashima for his technical advice on the GO analysis. Evaluations of GeneChip probes were carried out by the Super Computer System, Human Genome Center, Institute of Medical Science, University of Tokyo. This work was supported by the in-house-budget of RIKEN (J53-40030).
- Ross RA, Spengler BA, Biedler JL: Coordinate morphological and biochemical interconversion of human neuroblastoma cells. J Natl Cancer Inst. 1983, 71: 741-747.PubMedGoogle Scholar
- Pahlman S, Ruusala A-I, Abrahmsson L, Mattsson MEK, Esscher T: Retinoic acid-induced differentiation of cultured human neuroblastoma cells: a comparison with phorbolester-induced differentiation. Cell Differ. 1984, 14: 135-144. 10.1016/0045-6039(84)90038-1.View ArticlePubMedGoogle Scholar
- Pahlman S, Hoehner JC, Nanberg E, Hedborg F, Fagerstrom S, Gestblom C, Johansson I, Larsson U, Lavenius E, Ortoft E, Soderholm H: Differentiation and survival influences of growth factors in human neuroblastoma. Eur J Cancer. 1995, 31A: 453-458. 10.1016/0959-8049(95)00033-F.View ArticlePubMedGoogle Scholar
- Singh US, Pan J, Kao Y-L, Joshi S, Young KL, Baker KM: Tissue transglutaminase mediates activation of RhoA and MAP kinase pathways during retinoic acid-induced neuronal differentiation of SH-SY5Y cells. J Biol Chem. 2003, 278: 391-399. 10.1074/jbc.M206361200.View ArticlePubMedGoogle Scholar
- Tucholski J, Lesort M, Johnson GVW: Tissue transglutaminase is essential for neurite outgrowth in human neuroblastoma SH-SY5Y cells. Neuroscience. 2001, 102: 481-491. 10.1016/S0306-4522(00)00482-6.View ArticlePubMedGoogle Scholar
- Lopez-Carballo G, Moreno L, Masia S, Perez P, Barettino D: Activation of the phosphatidylinositol 3-kinase/Akt signaling pathway by retinoic acid is required for neural differentiation of SH-SY5Y human neuroblastoma cells. J Biol Chem. 2002, 277: 25297-25304. 10.1074/jbc.M201869200.View ArticlePubMedGoogle Scholar
- Pan J, Kao Y-L, Joshi S, Jeetendran S, DiPette D, Singh US: Activation of Rac1 by phosphatidylinositol 3-kinase in vivo: role in activation of mitogen-activated protein kinase (MAPK) pathways and retinoic acid-induced neuronal differentiation of SH-SY5Y cells. J Neurochem. 2005, 93: 571-583. 10.1111/j.1471-4159.2005.03106.x.View ArticlePubMedGoogle Scholar
- Encinas M, Iglesias M, Llecha N, Comella JX: Extracellular-regulated kinases and phosphatidylinositol 3-kinase are involved in brain-derived neurotrophic factor-mediated survival and neuritogenesis of the neuroblastoma cell line SH-SY5Y. J Neurochem. 1999, 73: 1409-1421. 10.1046/j.1471-4159.1999.0731409.x.View ArticlePubMedGoogle Scholar
- Kaplan DR, Matsumoto K, Lucarelli E, Thiele CJ: Induction of TrkB by retinoic acid mediates biologic responsiveness to BDNF and differentiation of human neuroblastoma cells. Neuron. 1993, 11: 321-331. 10.1016/0896-6273(93)90187-V.View ArticlePubMedGoogle Scholar
- Encinas M, Iglesias M, Liu Y, Wang H, Muhaisen A, Cena V, Gallego C, Comella JX: Sequential treatment of SH-SY5Y cells with retinoic acid and brain-derived neurotrophic factor gives rise to fully differentiated, neurotrophic factor-dependent human neuron-like cells. J Neurochem. 2000, 75: 991-1003. 10.1046/j.1471-4159.2000.0750991.x.View ArticlePubMedGoogle Scholar
- Affymetrix Expression Analysis Technical Support. [http://www.affymetrix.com/support/]
- Kent WJ: BLAT-the BLAST-like alignment tool. Genome Res. 2002, 12: 656-664.PubMed CentralView ArticlePubMedGoogle Scholar
- UCSC Genome Bioinformatics. [http://genome.ucsc.edu/]
- GEO Database. [http://www.ncbi.nlm.nih.gov/geo/]
- Hynds DL, Snow DM: Fibronectin and laminin elicit differential behaviors from SH-SY5Y growth cones contacting inhibitory chondroitin sulfate proteoglycans. J Neurosci Res. 2001, 66: 630-642. 10.1002/jnr.10020.View ArticlePubMedGoogle Scholar
- Tieu K, Zuo DM, Yu PH: Differential effects of staurosporine and retinoic acid on the vulnerability of the SH-SY5Y Neuroblastoma cells: involvement of Bcl-2 and p53 proteins. J Neurosci Res. 1999, 58: 426-435. 10.1002/(SICI)1097-4547(19991101)58:3<426::AID-JNR8>3.0.CO;2-F.View ArticlePubMedGoogle Scholar
- Biedler JL, Helson L, Spengler BA: Morphology and growth tumorigenicity, and cytogenetics of human neuroblastoma cell in continuous culture. Cancer Res. 1973, 33: 2643-2652.PubMedGoogle Scholar
- Draghici S: Data Analysis Tools for DNA Microarrays. 2003, Washington DC, Chapman & Hall/CrcView ArticleGoogle Scholar
- Saeed AI, Sharov V, White J, Li J, Liang W, Bhagabati N, Braisted J, Klapa M, Currier T, Thiagarajan M, Sturn A, Snuffin M, Rezantsev A, Popov D, Ryltsov A, Kostukovich E, Borisovsky I, Liu Z, Vinsavich A, Trush V, Quackenbush J: TM4: a free, open-source system for microarray data management and analysis. Biotechniques. 2003, 34: 374-378.PubMedGoogle Scholar
- Mense SM, Sengupta A, Zhou M, Lan C, Bentsman G, Volsky DJ, Zhang L: Gene expression profiling reveals the profound upregulation of hypoxia-responsive genes in primary human astrocytes. Physiol Genomics. 2006, 25: 435-449. 10.1152/physiolgenomics.00315.2005.View ArticlePubMedGoogle Scholar
- The GO Ontology. [http://www.geneontology.org/]
- NCBI, Entrez Gene. [http://www.ncbi.nlm.nih.gov/sites/entrez?db=gene]
- NCBI, Pub Med. [http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed]
- Gene Cards. [http://www.genecards.org/]
- 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 Commun. 2002, 290: 1054-1065. 10.1006/bbrc.2001.6301.View ArticlePubMedGoogle Scholar
- Klein R, Conway D, Parada LF, Barbacid M: The trkB tyrosine protein kinase gene codes for a second neurogenic receptor that lacks the catalytic kinase domain. Cell. 1990, 61: 647-656. 10.1016/0092-8674(90)90476-U.View ArticlePubMedGoogle Scholar
- 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.PubMed CentralView ArticlePubMedGoogle Scholar
- 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.View ArticlePubMedGoogle Scholar
- 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. 10.1128/MCB.24.7.2662-2672.2004.PubMed CentralView ArticlePubMedGoogle Scholar
- Eide FF, Vining ER, Eide BL, Zang K, Wang X-Y, Reichardt LF: Naturally occurring truncated trkB receptors have dominant inhibitory effects on brain-derived neurotrophic factor signaling. J Neurosci. 1996, 16: 3123-3129.PubMed CentralPubMedGoogle Scholar
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