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Integrative transcriptomic and proteomic analysis of osteocytic cells exposed to fluid flow reveals novel mechano-sensitive signaling pathways

ABSTRACT: 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE42874 | GEO | 2013/07/01

SECONDARY ACCESSION(S): PRJNA183689

REPOSITORIES: GEO

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Integrative transcriptomic and proteomic analysis of osteocytic cells exposed to fluid flow reveals novel mechano-sensitive signaling pathways

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-ÎºB inhibitors, MAP3 kinases, and Wnt/Î²-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.

2013-07-01 | E-GEOD-42874 | biostudies-arrayexpress

Mechano-sensitive Gene Expression with RNA-Seq: Revisiting the Osteocytic Cell Response to Fluid Flow

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.

2017-07-23 | GSE70667 | GEO

MDA-MB-231/1833 bone-metastatic breast cancer cell gene expression after conditioning in conditioned medium from HUVECs conditioned in conditioned medium from static and flow-stimulated MLO-Y4 osteocytes

Project description:Bone metastases is a common severe complication for breast cancer. We previously showed that conditioned medium (CM) from osteocytes stimulated with oscillatory fluid flow, mimicking bone mechanical loading during routine physical activities, reduced breast cancer cell extravasation across endothelial monolayers. Endothelial cells are situated at an ideal location to mediate signals between osteocytes in the bone matrix and metastasizing cancer cells in the blood vessels. Therefore, we used RNA sequencing to show that CM from endothelial cells conditioned in CM from flow-stimulated osteocytes significantly altered gene expression in bone-metastatic breast cancer cells. This This provides insights into the capability of bone-loading activity in preventing bone metastases.

2018-11-13 | E-MTAB-7176 | biostudies-arrayexpress

E-GEOD-42874

Project description:Integrative transcriptomic and proteomic analysis of osteocytic cells exposed to fluid flow reveals novel mechano-sensitive signaling pathways

2015-07-08 | E-GEOD-42874 | ExpressionAtlas

Mechanical loading and gene expression - Microarray study

Project description:Skeletal integrity in humans and animals is maintained by daily mechanical loading. It has been widely accepted that osteocytes function as mechanosensors. Many biochemical signaling molecules are involved in the response of osteocytes to mechanical stimulation. The aim of this study was to identify genes involved in the translation of mechanical stimuli into bone formation. The four-point bending model was used to induce a single period of mechanical loading (comprising 300 cycles (2 Hz) using a peak magnitude of 60 N) on the right tibia, while the contra lateral left tibia served as control. Six hours after loading, the effects of mechanical loading on gene-expression were determined with microarray analysis. Protein expression of differentially regulated genes was evaluated with immunohistochemistry. Nine genes were found to exhibit a significant differential gene expression in LOAD compared to control. MEPE, Garnl1, V2R2B, and QFG TN1 olfactory receptor were up-regulated, and creatine kinase (muscle form), fibrinogen-B beta-polypeptide, monoamine oxidase A, troponin-C and kinesin light chain-C were down-regulated. Validation with real-time RT-PCR analysis confirmed the up regulation of MEPE and the down-regulation of creatine kinase (muscle form) and troponin-C in the loaded tibia. Immunohistochemistry showed that the increase of MEPE protein expression was already detectable six hours after mechanical loading. In conclusion, these genes probably play a role during translation of mechanical stimuli six hours after mechanical loading. The modulation of MEPE expression may indicate a connection between bone mineralization and bone formation after mechanical stimulation. Two groups: LOAD vs contralateral control and SHAM vs contralateral control (n=5/group)

2013-11-21 | E-GEOD-50707 | biostudies-arrayexpress

Mus musculus

Project description:Integrative transcriptomic and proteomic analysis of osteocytic cells exposed to fluid flow reveals novel mechano-sensitive signaling pathways

| PRJNA183689 | ENA

In vitro and in vivo evidences of osteocytes involvement in myeloma-induced osteoclast formation and bone disease

Project description:The involvement of osteocytes in multiple myeloma (MM)-induced osteoclast formation and the occurrence of bone lesions are still unknown. Osteocytes regulate bone remodeling at least in part through the cell death and apoptosis triggering osteoclast recruitment and formation. In this study, firstly we shown that MM cells increased osteocyte death and affect their transcriptional profile evaluated by microarray analysis up-regulating osteoclastogenic cytokines as interleukin (IL)-11. Consistently we show that the conditioned media of human pre-osteocytes co-cultured with MM cells significantly increased osteoclastogenesis. To translate into a clinical perspective such in vitro evidences, we then performed histological analysis on bone biopsies obtained from MM patients, MGUS and healthy controls. We found a significant reduction in the number of viable osteocytes in MM patients as compared to controls. A significant negative correlation between the number of viable osteocytes and that of osteoclasts was also demonstrated. Moreover, as regards the skeletal involvement, we found that MM patients with bone lesions have a significant lower number of viable osteocyte than those without. Overall, our data suggest a role of osteocytic cell death in MM-induced osteoclast formation in vitro and MM bone disease in vivo in MM patients.

2012-05-22 | GSE27372 | GEO

Transcriptome and secretome analysis of mechanically stretched osteocytic cells reveals activation of signalling pathways implicated in bone regeneration and DNA-repair systems

Project description:Prescribed exercise has the potential to ameliorate tissue repair or regeneration and therefore is gaining increasing importance in regenerative rehabilitation. Osteocytes are an appealing type of bone cell to target by regenerative rehabilitation protocols due to their mechanosensitive and secretory nature. Making use of tandem liquid chromatography-mass spectrometry (LC-MSMS) as well as curated bioinformatics, we examined the secretome of mouse and human osteocytic bone cells cultured under cyclic tension delivered by a computer-controlled bioreactor. Quantitative SWATH MS analysis of the secretome secreted by mechanically stimulated cells revealed differential expression of 13 out of 759 secreted factors in mouse and 16 out of 276 in human osteocytic cells. GO enrichment analysis suggested that response to oxidative stress was the most predominant biological process in mouse and cholesterol or lipoprotein-related in human cells. Ossification and bone remodelling and manganese transport were 2 overrepresented process networks common to both mouse and human osteocytic cells

2021-06-08 | PXD020774 | Pride

Murine Bone Marrow Stromal Cell Response to Granulocyte Colony-Stimulating Factor

Project description:Neutrophil homeostasis is maintained, in part, by the regulated release of neutrophils from the bone marrow. Constitutive expression of the chemokine CXCL12 by bone marrow stromal cells provides a key retention signal for neutrophils in the bone marrow through activation of its receptor CXCR4. Herein, we show that the ELR chemokines CXCL1 and CXCL2 are constitutively expressed by bone marrow endothelial cells and osteoblasts, and CXCL2 expression is induced in endothelial cells during granulocyte colony-stimulating factor (G-CSF)-induced neutrophil mobilization. Neutrophils lacking CXCR2, the receptor for CXCL1 and CXCL2, are preferentially retained in the bone marrow, reproducing a myelokathexis phenotype. Transient disruption of CXCR4 failed to mobilize CXCR2 neutrophils. However, doubly deficient neutrophils (CXCR2-/- CXCR4-/-) displayed constitutive mobilization, showing that CXCR4 plays a dominant role. Collectively, these data suggest that CXCR2 signaling is a second chemokine axis that interacts antagonistically with CXCR4 to regulate neutrophil release from the bone marrow. We used gene expression microarrays to determine the changes in osteoblasts and bone marrow endothelial cells after G-CSF treatment. 3 untreated and G-CSF-treated osteoblast samples and 4 untreated and G-CSF-treated endothelial samples.

2010-07-14 | E-GEOD-22925 | biostudies-arrayexpress

Mechanical loading and gene expression - Microarray study

2013-11-21 | GSE50707 | GEO

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