Project description:To identify the microRNAs that are involved in osteoclastogenesis, microRNA expression profiles in mouse bone marrow macrophages (BMMs) stimulated with RANKL (BMOc) were compared with that of control untreated BMMs. These results provide insights into the mechanisms to regulate osteoclastogenesis and bone resorption activities in osteoclasts by microRNA. BMMs were cultured with 20 ng/ml M-CSF in the presence or absence of 50 ng/ml RANKL for 24 hours. Cells were collected for total RNA isolation, and were subjected to microRNA array analysis.

2013-07-10 | E-GEOD-48629 | biostudies-arrayexpress

Microarray studies on genes stimulated by RANKL in bone marrow macrophages in a cyclooxygenase-2-dependent manner

Project description:Overall goal was to look for the differentially expressed genes between receptor activator of nuclear factor kappa-B ligand (RANKL)-stimulated wild type (WT) bone marrow macrophages (BMMs) and the BMMs in which the expression or the enzyme activity of cyclooxygenase-2 (Cox2), the major enzyme for prostaglandin E2 (PGE2) synthesis, is absent. For this purpose, two separate microarrays (experiment 1 and 2) were done. In both the experiments, Serum Amyloid A3 (Saa3) was one of the most highly differentially expressed gene. For experiment 1, BMMs were isolated from the bone marrow of Cox2 knockout (KO) and WT mice. Out of the three sample groups, two were treated with 30 ng/ml each of macrophage-colony stimulating factor (M-CSF) and RANKL, while the third group was treated with M-CSF only, for 1 day. Two of the sample groups had n=3 biological replicates (i.e. samples), while the third one had n=4 biological replicates. The differential gene expression comparisons among the sample groups were done as (A) WT+MCSF+RANKL_d1 versus Cox2KO+MCSF+RANKL_d1 and (B) WT+MCSF+RANKL_d1 versus WT+MCSF_d1. For experiment 2 also, BMMs were obtained from Cox2 KO and WT mice. All the four sample groups were treated with M-CSF and RANKL for 3 days. All the four sample groups had n=4 biological replicates. One of the WT sample group was also treated with an inhibitor of Cox2 activity,NS398 (0.1 Âµm) and one of the KO sample group was also treated with the product of Cox2 enzyme, PGE2 (1 Âµm). The differential gene expression comparisons among the sample groups were done as (A) WT+MCSF+RANKL_d3 versus Cox2KO+MCSF+RANKL_d3; (B) WT+MCSF+RANKL_d3 versus WT+MCSF+RANKL+NS398_d3 and (C) Cox2KO+MCSF+RANKL+PGE2_d3 versus Cox2KO+MCSF+RANKL_d3.

2016-01-07 | E-GEOD-72047 | biostudies-arrayexpress

Targeting Lipid raft-related stomatin strategy for challenging osteoporosis

Project description:Mouse bone marrow macrophages, with or without the addition of RANKL, are being investigated for their interactions with proteins that associate with the stomatin protein.

2025-03-13 | PXD061779 |

Comparative characterization of osteoclasts derived from murine bone marrow macrophages and RAW 264.7 cells using quantitative proteomics

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.

2018-11-27 | PXD009610 | Pride

RNAseq from macrophages and osteoclasts from wild-type and TRAP deficient bone cell reporter mice

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

Osteocytic Wnt generates RANKL for osteoclastogenesis via TGFβ signaling

Project description:Wnt signaling is generally believed to inhibit osteoclast differentiation (OcD) by upregulating its target Opg expression; however, we unexpectedly found that Wnt mice with activation of Wnt/β-catenin signaling in osteocytes have higher OcD and bone resorption while generating more bone (PNAS 2015,112: E478). Here, we show cellular and molecular mechanisms for this unpredictable function of osteocytic Wnt on OcD. Isolated osteocytes (daCOt) of the Wnt mice display higher expression of RANKL and Opg with increased RANKL/Opg ratio, promoting OcD of bone marrow monocytes/macrophages to enhance bone resorption. RANKL-siRNA knocks down RANKL to reduce this OcD. In daCOt transcriptome analysis, the enrichment of KEGG pathways highlights the largest correlation of OcD with TGFβ signaling, which is activated by highly expressed Tgfb1 and Tgfb2, identified by heat-map analysis, and verified in a dose-dependent manner in Wnt agonist C91-treated wild-type osteocytes. Moreover, TGFβ signaling increases RANKL expression in dose and time-dependent manners, respectively with higher RANKL/Opg ratio and OcD. Conversely, TGFβ signaling inhibition reverses these effects. Importantly, mice with osteocytic deletion of the only TGFβRII decreases osteoclast number by 48.2% to 3.0/mm in Oc.N/B.Pm with markedly reduced expression of RANKL both at protein and mRNA levels in the osteocytes of cancellous bone in distal femurs. Furthermore, luciferase assay with 3kb-RANKL promoter region reveals that TGFβ signaling activates RANKL transcription. In summary, osteocytic Wnt promotes OcD mainly by activating TGFβ signaling, which activates RANKL gene transcription, opening up a novel avenue for the treatment or application of osteoclast or bone resorption-related diseases.

2025-12-02 | GSE301026 | GEO

Mus musculus

Project description:Microarray analysis of interferon stimulated bone-marrow derived macrophages Transcriptomes

| PRJNA352223 | ENA