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 up-regulation of dynein IC2-GR-COX-1 signaling, which may be a novel therapeutic target for NF1.
Highlights
From the ‡Department of Tumor Genetics and Biology, Graduate school of Medical Sciences, Kumamoto University, 1-1-1, Honjo, Chuo-ku, Kumamoto 860-8556, Japan; §Bioinformatics Center, Institute for Chemical Research, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan; ¶Database Center for Life Science, Research Organization of Information and Systems, 2-11-16, Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
Integration of All Data Obtained from Proteomics and Transcriptomics—Data obtained from four time-course samples with or without Neurofibromatosis type 1 (NF1) knockdown using two differential proteome analyses and a transcriptomic analysis (123 proteins from 2D-difference gel electrophoresis (DIGE), 3239 proteins from 8-plex isobaric tagging for relative and absolute quantitation (iTRAQ), and 10,868 genes from DNA array) were combined by integrated protein and gene expression analysis chart (iPEACH) (Fig. 1B, supplemental Table S1)
We focused on a highly clustered area consisting of up-regulated proteins in this network (Fig. 3A, red circle), and a specific network consisting of COX-1, which was continuously up-regulated at all time points in iTRAQ analysis, dynein IC2, which was highly differentially expressed by 2D-DIGE, and glucocorticoid receptor (GR), which was detected as an upregulated gene in DNA array and a significant key molecule in the Gene Ontology (GO)-based pathway analysis
Summary
The extracted peptides were vacuum-dried and dissolved in 20 l of 0.1% (v/v) TFA in 2% (v/v) ACN These samples were desalted with a ZipTip C18 pipette tip (Millipore) and subjected to MALDIMS/MS analysis using the 4700 Proteomics analyzer (Applied Biosystems/AB SCIEX) or LC-ESI-MS/MS analysis using the API QSTAR Pulsar i time-of-flight mass spectrometer (Applied Biosystems/AB SCIEX) coupled with the UltiMate NanoLC system (Thermo Scientific Dionex). Data from MALDI or ESI analysis with the iTRAQ method and from ESI analysis with 2D-DIGE were analyzed using the ParagonTM algorithm [9] of ProteinPilot Version 4.1 (AB SCIEX), and the UniProt Rat proteome database (release2012_02, 37,104 entries) was searched. Input multi types of files for iPEACH are CEL files (DNA microarray), peptide summary and protein summary files from ProteinPilot (iTRAQ experiments using ESI and MALDI), and xml output files from Decyder 2D software (2D-DIGE experiment using pH3–11 and pH4 –7 gels). Preparation of Nuclear and Cytoplasmic Protein Extracts—PC12 cells were collected, and the nuclear and cytoplasmic proteins were obtained using the 2-D Sample Prep for Nuclear Proteins kit (Thermo Fisher scientific) according to the manufacturer’s instructions
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