Effect of chaperonin 60 kDa on gene expression during osteoclast differentiation of RAW 264.7 cells
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ABSTRACT:
PROVIDER: PRJNA944209 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR24144081.fastq.gz | Fastqsanger.gz | |||
| SRR24144082.fastq.gz | Fastqsanger.gz | |||
| SRR24144083.fastq.gz | Fastqsanger.gz | |||
| SRR24144084.fastq.gz | Fastqsanger.gz | |||
| SRR24144085.fastq.gz | Fastqsanger.gz |
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Project description:The goal of this study was to look at genes that were affected by 69-kDa and/or 82-kDa ChAT proteins in IMR32 cells Experiment Overall Design: The gene expression changes of IMR32 cells stably expressing either 69-kDa or 82-kDa ChAT proteins were anaylzed and compared to control IMR32 wild type cells. 3 biological replicates were anaylzed per condition (69-kDa ChAT expressing cells, 82-kDa ChAT expressing cells, or wild type IMR32 cells) for a total of 9 samples altogether.
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Project description:Osteoclasts (OCs) are bone-resorbing cells differentiated from macrophage/monocyte precursors in response to M-CSF and RANKL. In vitro models are principally based on primary bone marrow macrophages, but RAW 264.7 cells are frequently used because they are widely available, easy to culture, and more amenable to genetic manipulation than primary cells. Increasing evidence, however, has shown that the vastly different origins of these two cell types may have important effects on cell behavior. In particular, M-CSF is prerequisite for the differentiation of BMMs, by promoting survival and proliferation and priming the cells for RANKL induction. RAW 264.7 cells readily form OCs in the presence of RANKL, but M-CSF is not required. Based on these key differences, we sought to understand their functional implications and how it might affect osteoclast differentiation and related signaling pathways. Using a robust and high-throughput proteomics strategy, we quantified the global protein changes in OCs derived from bone marrow macrophages and RAW 264.7 cells at 1, 3, and 5 days of differentiation. Correlation analysis of the proteomes demonstrated low concordance between the two cell types (R2 ≈ 0.13). Bioinformatics analysis indicate that RANKL-dependent signaling was intact in RAW 264.7 cells, but biological processes known to be dependent on M-CSF were significantly different; including cell cycle control, cytoskeletal organization, and apoptosis. RAW 264.7 cells exhibited constitutive activation of Erk and Akt that was dependent on the activity of Abelson tyrosine kinase, and the timing of Erk and Akt activation was significantly different between BMMs and RAW 264.7 cells. Our findings provide the first evidence for major differences between BMMs and RAW 264.7 cells, indicating that careful consideration is needed when using the RAW 264.7 cell line when studying M-CSF-dependent signaling and functions.
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Project description:Microgravity leads to a 10-15% loss of bone mass in astronauts during space flight. Osteoclast is the multinucleated bone resorbing cell. In this study, we used NASA developed ground based Rotary Wall Vessel Bioreactor (RWV), Rotary Cell Culture System (RCCS) to simulate microgravity (μXg) conditions and demonstrated a significant increase (2-fold) in osteoclastogenesis compared to ground based control (Xg) mouse bone marrow cultures. We further determined the gene expression profiling of RAW 264.7 osteoclast progenitor cells in microgravity by agilent microarray analysis. Gene expression pattern was functional group clustered by transcriptome analysis using gene ontology tree machine (GOTM) for cell proliferation/survival, differentiation and function. We confirm the microgravity modulated gene expression critical for osteoclast differentiation by real-time RT-PCR and Western blot analysis in murine bone marrow cultures. We identify transcription factors such as c-Jun, c-Fos, PU-1 critical for osteoclast differentiation is up-regulated in microgravity conditions. In addition, microgravity resulted in 2.3 and 2.0-fold increase in the level of cathepsin K and MMP-9 matrix metalloproteinase expression in preosteoclast cells involved in the bone resorption process respectively. We also demonstrate a significant increase in the expression levels of M-CSF receptor, c-Fms and PLCγ2 and S100A8 molecules that play an important role in Ca2+ signaling essential for osteoclast function. Further, microgravity stimulated preosteoclast cells showed elevated cytosolic Ca2+ levels compared to ground based control cells. Thus, microgravity regulated gene expression profiling in preosteoclast cells provide new insights in to molecular mechanisms and therapeutic targets of osteoclast differentiation/activation responsible for bone loss and fracture risk in astronauts during space flight mission. Microgravity associated with space flight is a challenge for normal bone homeostasis. Astronauts experience 10-15% bone loss during a space flight mission. We aimed to determine the effect of simulated microgravity on osteoclast preosteoclasts cells. RAW264.7 cells (1.5 x 106 /ml) were loaded in RCCS with DMEM containing 10% FBS for 24 h. The cells were stimulated with RANKL (80ng/ml) for 24 h to obtain preosteoclasts in parallel with ground based control cells. Total RNA was isolated using RNAzol reagent (Biotecx Labs, Houston, TX) from control (Xg) and microgravity (μXg) subjected cells and hybridized with Agilent whole mouse genome 4x44K array system. Slides were washed and scanned on an Agilent G2565 microarray scanner. Data obtained were analyzed with Agilent feature extraction and GeneSpring GX v7.3.1 software packages (Genus biosystem, Inc. Northbrook, IL, USA).
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