Integrated Proteomics Identified Novel Activation of Dynein IC2-GR-COX-1 Signaling in Neurofibromatosis Type I (NF1) Disease Model Cells

Abstract

Neurofibromatosis type 1 (NF1) tumor suppressor gene product, neurofibromin, functions in part as a Ras-GAP, and though its loss is implicated in the neuronal abnormality of NF1 patients, its precise cellular function remains unclear. To study the molecular mechanism of NF1 pathogenesis, we prepared NF1 gene knockdown (KD) PC12 cells, as a NF1 disease model, and analyzed their molecular (gene and protein) expression profiles with a unique integrated proteomics approach, comprising iTRAQ, 2D-DIGE, and DNA microarrays, using an integrated protein and gene expression analysis chart (iPEACH). In NF1-KD PC12 cells showing abnormal neuronal differentiation after NGF treatment, of 3198 molecules quantitatively identified and listed in iPEACH, 97 molecules continuously up-or down-regulated over time were extracted. Pathway and network analysis further revealed overrepresentation of calcium signaling and transcriptional regulation by glucocorticoid receptor (GR) in the up-regulated protein set, whereas nerve system development was overrepresented in the down-regulated protein set. The novel up-regulated network we discovered, "dynein IC2-GR-COX-1 signaling," was then examined in NF1-KD cells. Validation studies confirmed that NF1 knockdown induces altered splicing and phosphorylation patterns of dynein IC2 isomers, up-regulation and accumulation of nuclear GR, and increased COX-1 expression in NGF-treated cells. Moreover, the neurite retraction phenotype observed in NF1-KD cells was significantly recovered by knockdown of the dynein IC2-C isoform and COX-1. In addition, dynein IC2 siRNA significantly inhibited nuclear translocation and accumulation of GR and up-regulation of COX-1 expression. These results suggest that dynein IC2 up-regulates GR nuclear translocation and accumulation, and subsequently causes increased COX-1 expression, in this NF1 disease model. Our integrated proteomics strategy, which combines multiple approaches, demonstrates that NF1-related neural abnormalities are, in part, caused by upregulation of dynein IC2-GR-COX-1 signaling, which may be a novel therapeutic target for NF1. Neurofibromatosis type 1 (NF1)1 is an autosomal dominantly inherited disorder with an estimated prevalence of 1 in 3000 people (1). The hallmarks of NF1 include development of benign tumors of the peripheral nervous system and increased risk of malignancies. The phenotype of NF1 is highly variable, with several organ systems affected including the skin, bones, irises, and central and peripheral nervous systems. The effects on the nervous system are manifested in multiple neurofibroma, gliomas, and learning disabilities.The NF1 gene is located on chromosome 17q11.2 and encodes a large protein of 2818 amino acids, neurofibromin (2). Because the great majority of NF1 gene mutations frequently found in NF1 patients disturb the expression of intact neurofibromin, functional disruption of neurofibromin is potentially relevant to the expression of some or all of the abnormalities that occur in NF1 patients (3). A region centered 1...

Translationally Controlled Tumor Protein Is a Novel Biological Target for Neurofibromatosis Type 1-associated Tumors

“…Here, we report the up-regulation of TCTP in neurofibromin-deficient PC12 cells and Schwann cells and its regulation of cellular viability. Interestingly, our proteomic data also revealed that eEF1A and eEF1B␤ were up-regulated in neurofibromin-deficient PC12 cells after NGF stimulation (6). Because major components of the translational machinery are frequently up-regulated in human tumors, including neurofibromatosis-associated tumors (17), it is reasonable to assume that the up-regulation of both TCTP and eEF1A/B␤ contributes to the hyperactivation of protein synthesis in neurofibromas.…”

“…In our previous studies, using an integrated proteomics approach in neurofibromin-deficient PC12 cells (6), translationally controlled tumor protein (TCTP) was identified as an antiapoptotic factor uniquely regulated in response to NGF stimulation in PC12 cells (7). TCTP has been found in many eukaryotes, reported as multifunctional, and implicated in diverse processes, including growth, apoptosis, survival, development, protein synthesis, and transcription regulation (8).…”

“…Collectively, these data suggest that the up-regulation of TCTP was enhanced by the activation of MAPK and PI3K-AKT in response to NF1 deficiency. For all four spots, TCTP was significantly up-regulated by NF1 knockdown (6). The mean of the intensities at 0 h (before NGF stimulation) in control cells was used as the standard (ratio ϭ 1).…”

Integrative Network Analysis Combined with Quantitative Phosphoproteomics Reveals Transforming Growth Factor-beta Receptor type-2 (TGFBR2) as a Novel Regulator of Glioblastoma Stem Cell Properties

“…Pathway Analysis-The statistical extraction of canonical pathways was conducted through the use of KeyMolnet (KM Data, Tokyo, Japan) (28). The list of the regulated phosphoproteins was imported into KeyMolnet and the "Interrelation search" algorithm was used to generate the network of the molecular interactions between the related proteins.…”

Neuroproteomics — LC-MS Quantitative Approaches

“…In neuroproteomics, these approaches have been extensively used to characterize the differential proteomes of neuronal disorders, drug responses or brain regions. Many studies have been performed using these techniques in areas such as neurodegenerative disorders, as the study of the putamen proteome of an MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) monkey model of Parkinson's disease [101] or in the serum of Parkinson's disease patients [102], or even in the analysis of synaptossomes from cortical brain tissues from Alzheimer's disease patients [103]; in neuropharmacoproteomics, as in the examples of a study of protein quantitative alterations induced by antidepressants in the hippocampus of mice [104]; also in addiction as in the evaluation of the effects of administration of plasminogen activator after ischemic injury in mice [105] or the alterations upon chronic exposure to cocaine [106]; in neuropsychiatric and other CNS disorders, such as schizophrenia, with the study of protein expression in the thalamus and CSF of patients [107] and a study of neurofibromin knockdown PC12 cell line as a model of neurofibrimatosis [108]; or even in studies of neuronal function such as memory formation in hippocampus [109].…”