Project description:Nerve growth factor (NGF) is a neurotrophin that plays an important role in regulating the survival, growth, and differentiation of sympathetic neurons. Many in vitro studies indicate that Egr transcription factors are coupled to NGF signaling and are essential signaling mediators of NGF-dependent differentiation of sympathetic neurons, such as neuroblastoma cells and pheochromocytoma cells. Mice that are deficient for both Egr1 and Egr3 have profound sympathetic nerve system defects, including abnormal neuron degeneration and impaired differentiation (unpublished observations). To further understand the role of Egr genes in sympathetic neuron development, it is necessary to examine the signal transduction pathways involved in NGF-mediated Egr-dependent gene regulation. The results will be helpful in understanding the pathobiology of those diseases related to aberrant sympathetic neuron differentiation, such as neuroblastoma and dysautonomias, and may provide new insights into therapies for these refractory diseases. To identify NGF-mediated Egr-dependent target genes in human SH-SY5Y/TrkA neuroblastoma cells: Many potential Egr target genes have been described over the years. However, very few have been characterized to be involved in NGF-mediated sympathetic neuron differentiation. In order to further understand the role of Egr genes in sympathetic neuron development, it is necessary to examine the signal transduction pathways involved in NGF-mediated Egr-dependent gene regulation. Egr1 and Egr3 are rapidly induced after NGF treatment and Egr1 is involved in activation of the differentiation marker gene NPY in SH-SY5Y/TrkA cells. Therefore, SH-SY5Y/TtrkA cells appear to be an excellent model system to study the role of Egr transcription factors in sympathetic neuron differentiation in vitro. A dominant negative Egr molecule that specifically blocks transcriptional activity mediated by Egr transcription factors will be used in this study to identify Egr-dependent target genes. Egr1 and Egr3 are rapidly induced after NGF treatment in human SH-SY5Y/TrkA neuroblastoma cells, which in turn differentiate into sympathetic-like neurons. We hypothesize that Egr transcription factors are involved in activating downstream signaling pathways during NGF mediated differentiation of SH-SY5Y/TrkA cells. Moreover, we hypothesize that by using a dominant negative Egr (dnEgr) molecule that blocks all Egr mediated gene transcription and Affymetrix microarray analysis, it will be possible to identify NGF-mediated Egr transcription dependent gene regulatory networks that may be involved in growth and differentiation of neuroblastoma. An unbiased approach to understanding these gene regulatory networks may lead to new insights relating to NGF signaling involved in neuronal growth and differentiation. Human neuroblastoma SH-SY5Y/TrkA cells will be infected with either dnEgr-expressing adenovirus (SH-SY5Y/TrkA-dnEgr) or with EGFP-expressing control adenovirus (SH-SY5Y/TrkA-EGFP). Equivalent infection efficiency and lack of viral toxicity will be verified by EGFP fluorescence microscopy 24 hours after infection and the cells will be treated with NGF (100 ng/ml). Total RNA will be extracted from SH-SY5Y/TrkA (uninfected), SH-SY5Y/TrkA-dnEgr, and SH-SY5Y/TrkA-EGFP cells treated with NGF for 0, 1 hour and 3 hours. Total RNA will be prepared from all of the samples and a portion subjected to real-time PCR analysis to ensure that NGF mediated Egr gene induction was not altered by the context of viral infection. Pilot experiments demonstrate that Egr genes are still induced in the context of viral infection greater than 100-fold. Egr1 mRNA peak expression is known to occur at 1 hour and decrease by 3 hours after NGF treatment in all of the samples. The peak expression of Egr target genes is expected to occur later than Egr1 peak expression since Egr1 proteins need to be expressed first to initiate the transcription of target promoters. Therefore, the RNA samples from SH-SY5Y/TrkA-dnEgr and SH-SY5Y/TrkA-EGFP treated with NGF for 3 hours will be used to probe Affymetrix high-density human genome U133 Plus 2.0 Arrays to identify differentially expressed genes. RNA amplification for probe synthesis should not be necessary since we will provide 10 ug of intact total RNA for each sample. We will provide three sets of samples to perform the comparative microarray analysis twice from different starting materials and a nine-way comparative analysis of the data will be performed. We expect that cells containing high levels of dnEgr will inhibit NGF mediated Egr-dependent target gene expression and that these gene networks should be identifiable when compared to EGFP infected cells that have normal Egr gene transcriptional activity. Experiment Overall Design: as above

Project description:Nerve growth factor (NGF) is a neurotrophin that plays an important role in regulating the survival, growth, and differentiation of sympathetic neurons. Many in vitro studies indicate that Egr transcription factors are coupled to NGF signaling and are essential signaling mediators of NGF-dependent differentiation of sympathetic neurons, such as neuroblastoma cells and pheochromocytoma cells. Mice that are deficient for both Egr1 and Egr3 have profound sympathetic nerve system defects, including abnormal neuron degeneration and impaired differentiation (unpublished observations). To further understand the role of Egr genes in sympathetic neuron development, it is necessary to examine the signal transduction pathways involved in NGF-mediated Egr-dependent gene regulation. The results will be helpful in understanding the pathobiology of those diseases related to aberrant sympathetic neuron differentiation, such as neuroblastoma and dysautonomias, and may provide new insights into therapies for these refractory diseases. To identify NGF-mediated Egr-dependent target genes in human SH-SY5Y/TrkA neuroblastoma cells: Many potential Egr target genes have been described over the years. However, very few have been characterized to be involved in NGF-mediated sympathetic neuron differentiation. In order to further understand the role of Egr genes in sympathetic neuron development, it is necessary to examine the signal transduction pathways involved in NGF-mediated Egr-dependent gene regulation. Egr1 and Egr3 are rapidly induced after NGF treatment and Egr1 is involved in activation of the differentiation marker gene NPY in SH-SY5Y/TrkA cells. Therefore, SH-SY5Y/TtrkA cells appear to be an excellent model system to study the role of Egr transcription factors in sympathetic neuron differentiation in vitro. A dominant negative Egr molecule that specifically blocks transcriptional activity mediated by Egr transcription factors will be used in this study to identify Egr-dependent target genes. Egr1 and Egr3 are rapidly induced after NGF treatment in human SH-SY5Y/TrkA neuroblastoma cells, which in turn differentiate into sympathetic-like neurons. We hypothesize that Egr transcription factors are involved in activating downstream signaling pathways during NGF mediated differentiation of SH-SY5Y/TrkA cells. Moreover, we hypothesize that by using a dominant negative Egr (dnEgr) molecule that blocks all Egr mediated gene transcription and Affymetrix microarray analysis, it will be possible to identify NGF-mediated Egr transcription dependent gene regulatory networks that may be involved in growth and differentiation of neuroblastoma. An unbiased approach to understanding these gene regulatory networks may lead to new insights relating to NGF signaling involved in neuronal growth and differentiation. Human neuroblastoma SH-SY5Y/TrkA cells will be infected with either dnEgr-expressing adenovirus (SH-SY5Y/TrkA-dnEgr) or with EGFP-expressing control adenovirus (SH-SY5Y/TrkA-EGFP). Equivalent infection efficiency and lack of viral toxicity will be verified by EGFP fluorescence microscopy 24 hours after infection and the cells will be treated with NGF (100 ng/ml). Total RNA will be extracted from SH-SY5Y/TrkA (uninfected), SH-SY5Y/TrkA-dnEgr, and SH-SY5Y/TrkA-EGFP cells treated with NGF for 0, 1 hour and 3 hours. Total RNA will be prepared from all of the samples and a portion subjected to real-time PCR analysis to ensure that NGF mediated Egr gene induction was not altered by the context of viral infection. Pilot experiments demonstrate that Egr genes are still induced in the context of viral infection greater than 100-fold. Egr1 mRNA peak expression is known to occur at 1 hour and decrease by 3 hours after NGF treatment in all of the samples. The peak expression of Egr target genes is expected to occur later than Egr1 peak expression since Egr1 proteins need to be expressed first to initiate the transcription of target promoters. Therefore, the RNA samples from SH-SY5Y/TrkA-dnEgr and SH-SY5Y/TrkA-EGFP treated with NGF for 3 hours will be used to probe Affymetrix high-density human genome U133 Plus 2.0 Arrays to identify differentially expressed genes. RNA amplification for probe synthesis should not be necessary since we will provide 10 ug of intact total RNA for each sample. We will provide three sets of samples to perform the comparative microarray analysis twice from different starting materials and a nine-way comparative analysis of the data will be performed. We expect that cells containing high levels of dnEgr will inhibit NGF mediated Egr-dependent target gene expression and that these gene networks should be identifiable when compared to EGFP infected cells that have normal Egr gene transcriptional activity. Keywords: time-course

Dataset Information

Identification of novel target genes of nerve growth factor (NGF) in human mastocytoma cell line (HMC-1 (V560G c-Kit)) by transcriptome analysis

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