Mus musculus
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA289315 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR2094290.fastq.gz | Fastqsanger.gz | |||
| SRR2094291.fastq.gz | Fastqsanger.gz | |||
| SRR2094292.fastq.gz | Fastqsanger.gz | |||
| SRR2094293.fastq.gz | Fastqsanger.gz | |||
| SRR2094288.fastq.gz | Fastqsanger.gz |
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Project description:Integrative transcriptomic and proteomic analysis of osteocytic cells exposed to fluid flow reveals novel mechano-sensitive signaling pathways
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Project description:Bone adaptation to mechanical loading is regulated via signal transduction by mechano-sensing osteocytes. Mineral-embedded osteocytes experience strain-induced interstitial fluid flow and fluid shear stress, and broad shifts in gene expression are key components in the signaling pathways that regulate bone turnover. RNA sequencing analysis, or RNA-Seq, enables more complete characterization of mechano-sensitive transcriptome regulation than previously possible. We hypothesized that RNA-Seq of osteocytic MLO-Y4 cells reveals both expected and novel gene transcript regulation in cells previously fluid flowed and analyzed using gene microarrays (Govey et al., J Biomech, 2014). MLO-Y4 cells were flowed for 2 h with 1 Pa oscillating fluid shear stress and post-incubated 2 h. RNA-Seq of original samples detected 58 fluid flow-regulated gene transcripts (p-corrected<0.05) versus 65 transcripts detected by microarray. However, RNA-Seq demonstrated greater dynamic range, with all 58 transcripts >1.5 fold-change whereas 10 of 65 met this cut-off by microarray. Analyses were complimentary in patterns of regulation, though only 6 transcripts were significant in both analyses: Cxcl5, Cxcl1, Zc3h12a, Ereg, Slc2a1, and Egln1. As part of a broad inflammatory response inferred by gene ontology analyses, we again observed greatest up-regulation of inflammatory C-X-C motif chemokines, and newly implicated HIF-1? and AMPK signaling pathways. Importantly, we detected both expected mechano-sensitive transcripts (e.g. Nos2, Ptgs2, Ccl7) and transcripts not previously identified as mechano-sensitive, e.g. Ccl2. We found RNA-Seq advantageous over microarrays because of its ability to analyze unbiased estimation of gene expression, informing our understanding of osteocyte signaling.
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Project description:Integrative transcriptomic and proteomic analysis of osteocytic cells exposed to fluid flow reveals novel mechano-sensitive signaling pathways
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Project description:Early Response Genes in Osteoblasts Exposed to Dynamic Fluid Flow
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Project description:Osteocytes, positioned within boneâs interstitial space, are subject to fluid flow upon whole bone loading. Such fluid flow is widely theorized to be a mechanical signal transduced by osteocytes, initiating a poorly understood cascade of signaling events mediating bone metabolism. The objective of this study was to utilize high-throughput approaches to examine the time course of flow-induced changes in osteocyte gene transcript and protein levels. Microarray analysis demonstrated fluid flow regulation of genes consistent with known anabolic loading responses, including Ptgs2, NF-κB inhibitors, MAP3 kinases, and Wnt/β-catenin pathway signaling molecules. However, two of the most highly up-regulated gene productsâCxcl1 and Cxcl2, confirmed by qPCRâhave not previously been reported to be responsive to fluid flow. Gene ontology analysis suggested a highly significant inflammatory and immune response, with cellular functions including trafficking, cell-to-cell signaling, and tissue development. Proteomic analysis of the same samples demonstrated greatest up-regulation of the ATP-producing enzyme NDK, calcium-binding Calcyclin, and G protein-coupled receptor kinase 6. An integrative pathway analysis merging fold changes in transcript and protein levels predicted signaling nodes not directly detected at the sampled time points, including STAT3 and c-Myc. These results extend our knowledge of the osteocytic response to fluid flow, most notably up-regulation of Cxcl1 and Cxcl2 as a possible paracrine agent for osteoblastic and osteoclastic recruitment. Osteocyte-like MLO-Y4 cells were subjected to 2 hours of 10 dyn/cm2 oscillating fluid flow in parallel-plate fluid flow chambers and harvested for analysis at 0, 2, 8, and 24 hours post-flow incubation. Parallel control samples from sham treated cells were also collected at each time point.
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Project description:Osteocytes, positioned within bone’s interstitial space, are subject to fluid flow upon whole bone loading. Such fluid flow is widely theorized to be a mechanical signal transduced by osteocytes, initiating a poorly understood cascade of signaling events mediating bone metabolism. The objective of this study was to utilize high-throughput approaches to examine the time course of flow-induced changes in osteocyte gene transcript and protein levels. Microarray analysis demonstrated fluid flow regulation of genes consistent with known anabolic loading responses, including Ptgs2, NF-κB inhibitors, MAP3 kinases, and Wnt/β-catenin pathway signaling molecules. However, two of the most highly up-regulated gene products—Cxcl1 and Cxcl2, confirmed by qPCR—have not previously been reported to be responsive to fluid flow. Gene ontology analysis suggested a highly significant inflammatory and immune response, with cellular functions including trafficking, cell-to-cell signaling, and tissue development. Proteomic analysis of the same samples demonstrated greatest up-regulation of the ATP-producing enzyme NDK, calcium-binding Calcyclin, and G protein-coupled receptor kinase 6. An integrative pathway analysis merging fold changes in transcript and protein levels predicted signaling nodes not directly detected at the sampled time points, including STAT3 and c-Myc. These results extend our knowledge of the osteocytic response to fluid flow, most notably up-regulation of Cxcl1 and Cxcl2 as a possible paracrine agent for osteoblastic and osteoclastic recruitment.
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