A mammalian homolog of unc-53 is regulated by all-trans retinoic acid in neuroblastoma cells and embryos.
ABSTRACT: The vitamin A metabolite, all-trans retinoic acid (atRA), plays an important role in neuronal development, including neurite outgrowth. However, the genes that lie downstream of atRA and its receptors in neuronal cells are largely unknown. By using the human neuroblastoma cell line, SH-SY5Y, we have identified an atRA-responsive gene (RAINB1: retinoic acid inducible in neuroblastoma cells) that is induced within 4 h after exposure of SH-SY5Y cells to atRA. RAINB1 mRNA is highly expressed in the nervous system (10.5- to 11-kb transcript) in both developing embryos and adults. Its expression is perturbed in developing rat embryos exposed to excess or insufficient atRA. RAINB1 is present on chromosome 11 and is spread over 38 exons, resulting in a putative ORF of 2,429 amino acids. The RAINB1 protein shows high similarity to a gene in Caenorhabditis elegans, unc-53, that is required for axonal elongation of mechanosensory neurons, suggesting that these proteins are orthologs. Thus, RAINB1 may represent a critical downstream gene in atRA-mediated neurite outgrowth.
Project description:Neuron navigator 2 (Nav2) was first identified as an all-trans retinoic acid (atRA)-responsive gene in human neuroblastoma cells (retinoic acid-induced in neuroblastoma 1, RAINB1) that extend neurites after exposure to atRA. It is structurally related to the Caenorhabditis elegans unc-53 gene that is required for cell migration and axonal outgrowth. To gain insight into NAV2 function, the full-length human protein was expressed in C. elegans unc-53 mutants under the control of a mechanosensory neuron promoter. Transgene expression of NAV2 rescued the defects in unc-53 mutant mechanosensory neuron elongation, indicating that Nav2 is an ortholog of unc-53. Using a loss-of-function approach, we also show that Nav2 induction is essential for atRA to induce neurite outgrowth in SH-SY5Y cells. The NAV2 protein is located both in the cell body and along the length of the growing neurites of SH-SY5Y cells in a pattern that closely mimics that of neurofilament and microtubule proteins. Transfection of Nav2 deletion constructs in Cos-1 cells reveals a region of the protein (aa 837-1065) that directs localization with the microtubule cytoskeleton. Collectively, this work supports a role for NAV2 in neurite outgrowth and axonal elongation and suggests this protein may act by facilitating interactions between microtubules and other proteins such as neurofilaments that are key players in the formation and stability of growing neurites.
Project description:Recent data indicate that isomerisation to all-trans retinoic acid (ATRA) is the key mechanism underlying the favourable clinical properties of 13-cis retinoic acid (13cisRA) in the treatment of neuroblastoma. Retinoic acid (RA) metabolism is thought to contribute to resistance, and strategies to modulate this may increase the clinical efficacy of 13cisRA. The aim of this study was to test the hypothesis that retinoids, such as acitretin, which bind preferentially to cellular retinoic acid binding proteins (CRABPs), or specific inhibitors of the RA hydroxylase CYP26, such as R116010, can increase the intracellular availability of ATRA. Incubation of SH-SY5Y cells with acitretin (50 microM) or R116010 (1 or 10 microM) in combination with either 10 microM ATRA or 13cisRA induced a selective increase in intracellular levels of ATRA, while 13cisRA levels were unaffected. CRABP was induced in SH-SY5Y cells in response to RA. In contrast, acitretin had no significant effect on intracellular retinoid concentrations in those neuroblastoma cell lines that showed little or no induction of CRABP after RA treatment. Both ATRA and 13cisRA dramatically induced the expression of CYP26A1 in SH-SY5Y cells, and treatment with R116010, but not acitretin, potentiated the RA-induced expression of a reporter gene and CYP26A1. The response of neuroblastoma cells to R116010 was consistent with inhibition of CYP26, indicating that inhibition of RA metabolism may further optimise retinoid treatment in neuroblastoma.
Project description:BACKGROUND: Trans-activation response element (TAR) DNA binding protein of 43kDa (TDP-43) is causally related to the neurodegenerative diseases frontotemporal dementia and amyotrophic lateral sclerosis being the hallmark protein in the disease-characteristic neuropathological lesions and via genetic linkage. Histone deacetylase 6 (HDAC6) is an established target of the RNA-binding protein TDP-43. HDAC6 is an unusual cytosolic deacetylase enzyme, central for a variety of pivotal cellular functions including aggregating protein turnover, microtubular dynamics and filopodia formation. All these functions are important in the context of neurodegenerative proteinopathies involving TDP-43. We have previously shown in a human embryonic kidney cell line that TDP-43 knockdown significantly impairs the removal of a toxic, aggregating polyQ ataxin-3 fusion protein in an HDAC6-dependent manner. Here we investigated the influence of TDP-43 and its target HDAC6 on neurite outgrowth. RESULTS: Human neuroblastoma SH-SY5Y cells with stably silenced TDP-43 showed a significant reduction of neurite outgrowth induced by retinoic acid and brain-derived neurotrophic factor. Re-transfection with TDP-43 as well as HDAC6 rescued retinoic acid-induced neurite outgrowth. In addition, we show that silencing of HDAC6 alone is sufficient to reduce neurite outgrowth of in vitro differentiated SH-SY5Y cells. CONCLUSIONS: TDP-43 deficiency leads to impairment of neurite growth in an HDAC6-dependent manner, thereby contributing to neurodegenerative events in TDP-43 diseases.
Project description:The goal of this study was to determine and compare the effects of the secretome of mesenchymal stem cells (MSCs) isolated from human bone-marrow (BMSCs) and the Wharton jelly surrounding the vein and arteries of the umbilical cord (human umbilical cord perivascular cells (HUCPVCs)) on the survival and differentiation of a human neuroblastoma cell line (SH-SY5Y). For this purpose, SH-SY5Y cells were differentiated with conditioned media (CM) from the MSCs populations referred above. Retinoic acid cultured cells were used as control for neuronal differentiated SH-SY5Y cells. SH-SY5Y cells viability assessment revealed that the secretome of BMSCs and HUCPVCs, in the form of CM, was able to induce their survival. Moreover, immunocytochemical experiments showed that CM from both MSCs was capable of inducing neuronal differentiation of SH-SY5Y cells. Finally, neurite lengths assessment and quantitative real-time reverse-transcription polymerase chain reaction (RT-PCR) analysis demonstrated that CM from BMSCs and HUCPVCs differently induced neurite outgrowth and mRNA levels of neuronal markers exhibited by SH-SY5Y cells. Overall, our results show that the secretome of both BMSCs and HUCPVCs was capable of supporting SH-SY5Y cells survival and promoting their differentiation towards a neuronal phenotype.
Project description:BACKGROUND: Our recent results show that all-trans retinoic acid (ATRA), an active metabolite of vitamin A, induces COX-dependent hyperalgesia and allodynia in rats. This effect was mediated by retinoic acid receptors (RARs) and was associated with increased COX-2 expression in the spinal cord. Since ATRA also up-regulated COX-2 expression in SH-SY5Y human neuroblastoma cells, the current study was undertaken to analyze in these cells the mechanism through which ATRA increases COX activity. METHODS: Cultured SH-SY5Y neuroblastoma cells were treated with ATRA. COX expression and kinase activity were analyzed by western blot. Transcriptional mechanisms were analyzed by RT-PCR and promoter assays. Pharmacological inhibitors of kinase activity and pan-antagonists of RAR or RXR were used to assess the relevance of these signaling pathways. Production of prostaglandin E2 (PGE2) was quantified by enzyme immunoabsorbent assay. Statistical significance between individual groups was tested using the non-parametric unpaired Mann-Whitney U test. RESULTS: ATRA induced a significant increase of COX-2 expression in a dose- and time-dependent manner in SH-SY5Y human neuroblastoma cells, while COX-1 expression remained unchanged. Morphological features of differentiation were not observed in ATRA-treated cells. Up-regulation of COX-2 protein expression was followed by increased production of PGE2. ATRA also up-regulated COX-2 mRNA expression and increased the activity of a human COX-2 promoter construct. We next explored the participation of RARs and mitogen-activated peptide kinases (MAPK). Pre-incubation of SH-SY5Y human neuroblastoma cells with either RAR-pan-antagonist LE540 or MAP kinase kinase 1 (MEK-1) inhibitor PD98059 resulted in the abolition of ATRA-induced COX-2 promoter activity, COX-2 protein expression and PGE2 production whereas the retinoid X receptor pan-antagonist HX531, the p38 MAPK inhibitor SB203580 or the c-Jun kinase inhibitor SP600125 did not have any effect. The increase in RAR-beta expression and extracellular-regulated kinase 1/2(ERK1/2) phosphorylation in ATRA-incubated cells suggested that RARs and ERK1/2 were in fact activated by ATRA in SH-SY5Y human neuroblastoma cells. CONCLUSION: These results highlight the importance of RAR-dependent and kinase-dependent mechanisms for ATRA-induced COX-2 expression and activity.
Project description:All-trans retinoic acid (atRA) is required for nervous system development, including the developing hindbrain region. Neuron navigator 2 (Nav2) was first identified as an atRA-responsive gene in human neuroblastoma cells (retinoic acid-induced in neuroblastoma 1, Rainb1), and is required for atRA-mediated neurite outgrowth. In this paper, we explore the importance of Nav2 in nervous system development and function in vivo.Nav2 hypomorphic homozygous mutants show decreased survival starting at birth. Nav2 mutant embryos show an overall reduction in nerve fiber density, as well as specific defects in cranial nerves IX (glossopharyngeal) and X (vagus). Nav2 hypomorphic mutant adult mice also display a blunted baroreceptor response compared to wild-type controls.Nav2 functions in mammalian nervous system development, and is required for normal cranial nerve development and blood pressure regulation in the adult.
Project description:Retinoic acid- (RA-) triggered neuroblastoma cell lines are widely used cell modules of neuronal differentiation in neurodegenerative disease studies, but the gene regulatory mechanism underlying differentiation is unclear now. In this study, system biological analysis was performed on public microarray data from three neuroblastoma cell lines (SK-N-SH, SH-SY5Y-A, and SH-SY5Y-E) to explore the potential molecular processes of all-trans retinoic acid- (ATRA-) triggered differentiation. RT-qPCR, functional genomics analysis, western blotting, chromatin immunoprecipitation (ChIP), and homologous sequence analysis were further performed to validate the gene regulation processes and identify the RA response element in a specific gene. The potential disturbed biological pathways (111 functional GO terms in 14 interactive functional groups) and gene regulatory network (10 regulators and 71 regulated genes) in neuroblastoma differentiation were obtained. 15 of the 71 regulated genes are neuronal projection-related. Among them, NTRK2 is the only one that was dramatically upregulated in the RT-qPCR test that we performed on ATRA-treated SH-SY5Y-A cells. We further found that the overexpression of the NTRK2 gene can trigger differentiation-like changes in SH-SY5Y-A cells. Functional genomic analysis and western blotting assay suggested that, in neuroblastoma cells, ATRA may directly regulate the NTRK2 gene by activating the RA receptor (RAR) that binds in its promoter region. A novel RA response DNA element in the NTRK2 gene was then identified by bioinformatics analysis and chromatin immunoprecipitation (ChIP) assay. The novel element is sequence conservation and position variation among different species. Our study systematically provided the potential regulatory information of ATRA-triggered neuroblastoma differentiation, and in the NTRK2 gene, we identified a novel RA response DNA element, which may contribute to the differentiation in a human-specific manner.
Project description:MicroRNAs (miRNAs) are a class of small noncoding RNAs that regulate gene expression at the posttranscriptional level. Research on miRNAs has highlighted their importance in neural development, but the specific functions of neurally enriched miRNAs remain poorly understood. We report here the expression profile of miRNAs during neuronal differentiation in the human neuroblastoma cell line SH-SY5Y. Six miRNAs were significantly upregulated during differentiation induced by all-trans-retinoic acid and brain-derived neurotrophic factor. We demonstrated that the ectopic expression of either miR-124a or miR-125b increases the percentage of differentiated SH-SY5Y cells with neurite outgrowth. Subsequently, we focused our functional analysis on miR-125b and demonstrated the important role of this miRNA in both the spontaneous and induced differentiations of SH-SH5Y cells. miR-125b is also upregulated during the differentiation of human neural progenitor ReNcell VM cells, and miR-125b ectopic expression significantly promotes the neurite outgrowth of these cells. To identify the targets of miR-125b regulation, we profiled the global changes in gene expression following miR-125b ectopic expression in SH-SY5Y cells. miR-125b represses 164 genes that contain the seed match sequence of the miRNA and/or that are predicted to be direct targets of miR-125b by conventional methods. Pathway analysis suggests that a subset of miR-125b-repressed targets antagonizes neuronal genes in several neurogenic pathways, thereby mediating the positive effect of miR-125b on neuronal differentiation. We have further validated the binding of miR-125b to the miRNA response elements of 10 selected mRNA targets. Together, we report here for the first time the important role of miR-125b in human neuronal differentiation.
Project description:We report here the expression profile of microRNAs in human neuronal differentiation in the neuroblastoma cell line SH-SY5Y. Six microRNAs were significantly upregulated during differentiation induced by all-trans¬-retinoic acid and brain-derived neurotrophic factor. We demonstrated that ectopic expression of either miR-124a or miR-125b increases the percentage of differentiated SH-SY5Y cells with neurite outgrowth. Subsequently, we focused our functional analysis on miR-125b and demonstrated the important role of this miRNA in both spontaneous and induced differentiation of SH-SH5Y cells, based on neurite outgrowth and neuronal marker expression. In human neural progenitor ReNcell VM cells, miR-125b is also upregulated during differentiation and miR-125b ectopic expression significantly promotes neurite outgrowth. To identify the targets of miR-125b regulation, we profiled the global changes in gene expression following miR-125b ectopic expression in SH-SY5Y cells. miR-125b represses 164 genes that contain the seed match sequence of the microRNA and/or predicted to be direct targets of miR-125b by conventional methods. Pathway analysis suggests that a subset of miR-125b-repressed targets antagonize neuronal genes in several neurogenic pathways, thereby mediating the positive effect of miR-125b on neuronal differentiation. We have further validated the binding of miR-125b to the microRNA response elements of ten selected targets. Together, we report here for the first time the important role of miR-125b in human neuronal differentiation. Keywords: mir125-OE/mir-scrambled control comparison & mir125-KD/mir-scrambled control comparsion Overall design: In this study we sought to understand the role of miRNAs in differentiation of human neural cells using simple in vitro models, human neuroblastoma SH-SY5Y cells and human neural progenitor ReNcell VM cells. When sequentially treated with all-trans-retinoic acid (RA) and brain-derived neurotrophic factor (BDNF), SH-SY5Y cells give rise to fully differentiated neuron-like cells (8). These differentiated SH-SY5Y cells are withdrawn from the cell cycle, express various neuronal markers, and exhibit carbachol-evoked noradrenaline release (8). Moreover, as no glial cell is derived by this process, it is a robust and homogenous model system for investigating neuronal differentiation (8). Using microarrays and Northern blots, we identified a group of miRNAs that are significantly upregulated in differentiated SH-SY5Y cells. We further showed that one of these miRNAs, miR-125b, significantly enhances differentiation and neuronal morphogenesis of SH-SY5Y cells. In addition, this miRNA also promotes neurite outgrowth in human neural progenitor ReNcell VM cells.
Project description:Painful human intervertebral discs (IVDs) exhibit nerve growth deep into the IVD. Current treatments for discogenic back pain do not address the underlying mechanisms propagating pain and are often highly invasive or only offer temporary symptom relief. The notochord produces factors during development that pattern the spine and inhibit the growth of dorsal root ganglion (DRG) axons into the IVD. We hypothesize that notochordal cell (NC)-conditioned medium (NCCM) includes soluble factors capable of inhibiting neurite growth and may represent a future therapeutic target.To test if NCCM can inhibit neurite growth and determine if NC-derived glycosaminoglycans (GAGs) are necessary candidates for this inhibition.Human neuroblastoma (SH-SY5Y) cells and rat DRG cells were treated with NCCM in two-dimensional culture in vitro, and digestion and mechanistic studies determined if specific GAGs were responsible for inhibitory effects.Notochordal cell-conditioned medium was generated from porcine nucleus pulposus tissue that was cultured in Dulbecco's modified eagle's medium for 4 days. A dose study was performed using SH-SY5Y cells that were seeded in basal medium for 24 hours and neurite outgrowth and cell viability were assessed after treatment with basal media or NCCM (10% and 100%) for 48 hours. Glycosaminoglycans from NCCM were characterized using multiple digestions and liquid chromatography mass spectroscopy (LC-MS). Neurite growth was assessed on both SH-SY5Y and DRG cells after treatment with NCCM with and without GAG digestion.Notochordal cell-conditioned medium significantly inhibited the neurite outgrowth from SH-SY5Y cells compared with basal controls without dose or cytotoxic effects; % of neurite expressing cells were 39.0±2.9%, 27.3±3.6%, and 30.2±2.7% and mean neurite length was 60.3±3.5, 50.8±2.4, 53.2±3.7 ?m for basal, 10% NCCM, and 100% NCCM, respectively. Digestions and LC-MS determined that chondroitin-6-sulfate was the major GAG chain in NCCM. Neurite growth from SH-SY5Y and DRG cells was not inhibited when cells were treated with NCCM with digested chondroitin sulfate (CS).Soluble factors derived from NCCM were capable of inhibiting neurite outgrowth in multiple neural cell types without any negative effects on cell viability. Cleavage of GAGs via digestion was necessary to reverse the neurite inhibition capacity of NCCM. We conclude that intact GAGs such as CS secreted from NCs are potential candidates that could be useful to reduce neurite growth in painful IVDs.