Gene expression analysis of differentiated primary murine bone marrow stem cells following OF-1 treatment
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
SUBMITTER: Julia Meier
PROVIDER: E-MTAB-4155 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Genetic deletion of Nfatc1 in mice results in profound osteoclast-poor osteopetrosis, a high bone mass state caused by a lack of osteoclast activity. We hypothesized that the family of NFATc1 regulated transcripts in the osteoclast would be enriched for genes associated with osteoclast function. We used microarrays profile gene expression in wild-type and NFATc1-deficient osteoclasts generated in vitro to identify NFATc1-dependent transcripts in osteoclasts. Bone marrow macrophages from wild-type and mice with an induced deficiency of NFATc1 (NFATc1 fl/fl MxCre+ mice where NFATc1 excision was induced by polyIC treatment) were cultured ex vivo in MCSF and RANKL for 3 days. 2 biological replicates were assayed for each genotype.
2012-08-18 | E-GEOD-37219 | biostudies-arrayexpress
Project description:Osteoclasts are multinucleated cells specialized in degrading the mineralized bone matrix. Osteoclast differentiation and function are tightly regulated, to prevent excessive or insufficient bone resorption. Several control mechanisms participate in modulating osteoclastogenesis, and an increasing number of reports describe the role of microRNAs (miRNAs) in this process. Disrupting the expression of specific miRNAs can result in alterations of osteoclast formation and bone homeostasis. We and others have previously characterized 9 miRNAs whose levels change during osteoclast differentiation, and identified some of the target genes that mediate their function. However, little is known about changes in the miRNA expression profile during osteoclastogenesis. In this study, we isolated a murine primary bone marrow population enriched for osteoclast precursors, and used the Agilent microarray platform to analyze the expression of mature miRNAs after 1, 3, and 5 days of RANKL-driven differentiation. 93 miRNAs showed greater than 2 fold-change during these early, middle, and late stages of osteoclastogenesis. Many of these miRNAs were detected for the first time in osteoclasts, and we validated the expression of selected miRNAs by quantitative RT-PCR. We identified clusters of differentially expressed miRNAs, and performed computational analyses to predict functional pathways that may be regulated by these miRNAs. Several miRNAs were predicted to regulate genes involved in cytoskeletal remodeling, a crucial mechanism for the migration of osteoclast precursors, their maturation, and bone resorbing activity. Our results suggest that clusters of miRNAs differentially expressed during the course of osteoclastogenesis converge on the regulation of several key functional pathways. Overall, this study identified miRNAs expressed during early, middle and late osteoclastogenesis, contributing to understanding the molecular mechanisms regulating this complex differentiation process. Mouse primary bone marrow cultures were enriched for osteoclast precursors by depletion of B220/CD45R+ and CD3+ cells (B and T lymphocytes, respectively). Cells were differentiated with M-CSF and RANKL, and miRNA expression was analyzed at days 1, 3, and 5. Four biological replicates for each time point were used.
2015-12-31 | E-GEOD-53017 | biostudies-arrayexpress
Project description:Interleukin-6 (IL-6) plays an important role in progressive bone loss in rheumatoid arthritis (RA). However, the molecular mechanisms through which IL-6 propels RA synovial fibroblasts (RASFs) to contribute to bone loss are not fully understood. In the present study, we investigated the effects of IL-6 and IL-6 receptor (IL-6/IL-6R induced trans-signaling in human RASFs. Treatment of human RASFs with IL-6 and IL-6 receptor (IL-6/IL-6R, 100 ng/ml each) alone or in combination with M-CSF (2 ng/ml) and RANKL (10 ng/ml) for 12 days resulted in the phenotypic changes to osteoclast-like features as determined by increase in TRAP staining and enhanced pit formation by ~3-fold in bone resorption assay in vitro. IL-6/IL-6R induced a dose-dependent increase in the expression of transcription factor Ets2 and enhanced its nuclear translocation in human RASFs. Interestingly, the untargeted proteomics analysis of the conditioned media from IL6/IL6R activated RASFs using Mass-Spectrometry (MS) technique and Trans-Proteomic Pipeline tool showed the production of 113 analytes unique to IL-6/IL-6R stimulation. Further analysis of the proteomics data using STRING database for pathway analysis showed the impact of IL-6/IL-6R on immunometabolic reprogramming of human RASFs towards osteoclast-like phenotype, which was partly mediated through Ets2. These preliminary studies identified a novel role of IL-6/IL-6R-mediated trans signaling in the metabolic reprogramming in RASFs that could potentially be targeted by inhibiting Ets2 to limit their role in bone resorption in RA.
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Project description:Osteoclasts are multinucleated giant cells generated by the fusion of precursors in response to stimulation with macrophage colony stimulating factor (MCSF) and receptor activator of NF-kB ligand (RANKL). These cells are the only cells capable of resorbing bone. Tartarate-resistant acid phosphatase is an enzyme secreted by osteoclasts that acts in bone resorption. Mice that are deficient for TRAP have shorter bones and their osteoclasts have decreased resorption capacity. In this project, we will isolate bone marrow macrophages from wild type and TRAP deficient mice, and differentiate the cells in osteoclasts in vitro. RNA will be extracted from macrophages and from macrophages stimulated with RANKL for both mouse lines (n=3/group) yielding 4 groups: Group 1 – macrophages from wild type mice, Group 2 – osteoclasts from wild type mice, Group 3 – macrophages from TRAP deficient mice, Group 4 – osteoclasts from TRAP deficient mice. The differential gene expression will be analyzed by RNAseq.
2024-02-02 | E-MTAB-11403 | biostudies-arrayexpress
Project description:Paget’s disease of bone (PDB) is a chronic focal skeletal disorder that affects 2-3% of the population over the age of 60. PDB is inherited as an autosomal dominant trait with genetic heterogeneity. SQSTM1/p62 UBA domain mutation (p62P392L) is widely identified in PDB and has been shown to increase osteoclastogenesis. Further, environmental factors such as paramyxovirus are implicated in PDB and MVNP has been shown to induce Pagetic phenotype in osteoclasts. However, the molecular mechanisms underlying p62P392L and MVNP stimulation of osteoclast differentiation in PDB are unclear. We therefore determined MVNP regulated gene expression profiling during osteoclast differentiation. We identified 8.4% of genes were upregulated (> 4-fold) in MVNP transduced cells. MVNP increased integrin β3 (63 fold), NFAT activating protein (6.5 fold), OSCAR (5.5 fold), TRAF5 (8.5 fold) mRNA expression compared to empty vector (EV) transduced cells. MVNP also elevated gene expression of cytokines/growth factors such as IL-17 (18 fold), IL-1F7 (10 fold), IL-17R (4.5 fold) and IL-11 (7 fold). Interestingly, MVNP transduced cells demonstrated high level expression of signal regulatory protein beta 1 (SIRPβ1) (353 fold). SIRPβ1 has been shown to interact with DAP 12, an ITAM containing adaptor protein which plays an important role in osteoclast differentiation. Real-time PCR analysis of total RNA isolated from normal human peripheral blood monocytes transduced with MVNP confirmed upregulation of SIRPβ1 mRNA expression in the absence of RANKL stimulation. In contrast, RANKL stimulation did not alter SIRPβ1 expression in these cells. Furthermore, bone marrow mononuclear cells derived from patients with PDB showed high levels of SIRPβ1 mRNA expression compared to normal subjects. Thus, MVNP regulated gene expression profiling during osteoclast differentiation provides new insights into molecular mechanisms and therapeutic targets to control elevated osteoclast activity in PDB. Total RNA isolated from normal human bone marrow mononuclear cells transduced with EV, MVNP retroviral expression vectors and stimulated with M-CSF and RANKL for 48 h were subjected to Agilent microarray (~26,000 genes) analysis. One replicate per treatment was hybridized.
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