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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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Project description:Lymph nodes are vital for optimizing immune responses. Stress can induce several cellular and humoral immune responses. Acute restraint stress (RS) is a routinely used experimental procedure for studying psychological and/or physiological stress effects. Here, we determined the impact of RS on cervical lymph nodes in rats at the molecular and cellular levels. Stress was induced in male Sprague-Dawley rats by immobilization for 30, 60, and 120 min (RS30, RS60, and RS120, respectively) relative to a no-stress control (C) group. Expression of genes encoding chemokines CXCL1/CXCL2 (Cxcl1 and Cxcl2) and their receptor CXCR2 (Cxcr2) was analyzed at the mRNA level by reverse transcription-quantitative PCR (RT-qPCR) and microarray analyses. Immunohistochemistry and in situ hybridization were performed to determine the expression of these moieties along with the macrophage biomarker, CD68. Microarray analysis revealed that expression of 514 and 496 genes was upregulated and downregulated, respectively, in RS30. Cxcl1 and Cxcl2 expression showed a 23- and 13-fold increase, respectively, in RS30 relative to the C group. Expression of Cxcr2 was upregulated by approximately 1.6-fold in RS30 relative to the C group. Gene ontology analysis of three upregulated genes induced by RS30 suggested that they may be responsible for the cytokine network, inflammation, as well as leukocyte chemotaxis and migration. RT-qPCR analysis indicated that the mRNA levels of Cxcl1 and Cxcl2 significantly increased in RS30 but reverted to normal levels in RS60 and RS120. Cxcr2 mRNA level also increased significantly in RS30 and RS120 relative to the C group. RS-induced CXCL1-immunopositive cells corresponded to B/plasma cells, while CXCL2-immunopositive cells corresponded to endothelial cells of the high endothelial venules. Stress-induced CXCR2-immunopositive cells corresponded to macrophages. Psychological and/or physiological stress induces acute stress response and immunoreactive microenvironment in cervical lymph nodes, and the CXCL1/CXCL2-CXCR2 axis is pivotal in acute stress response.

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

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