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PROVIDER: PRJNA769960 | ENA |
REPOSITORIES: ENA
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Project description:Osteoclasts are absorptive cells and play a critical role in homeostatic bone remodeling and pathological bone resorption. Emerging evidence suggests an important role for epigenetic regulation of osteoclastogenesis. In this study, we investigated the role of DOT1L, which regulates gene expression epigenetically by histone H3K79 methylation during osteoclast formation. DOT1L and H3K79me2 levels were upregulated during osteoclast differentiation. Small molecule inhibitor- (EPZ5676 or EPZ004777) or short hairpin RNA-mediated reduction in DOT1L expression promoted osteoclast differentiation and resorption. DOT1L inhibition also increased osteoclast area and accelerated bone mass reduction in a mouse ovariectomy (OVX) model of osteoporosis. DOT1L inhibitors did not alter osteoblast differentiation in vitro and in vivo. Proteomics data, together with bioinformatics analysis, revealed that DOT1L inhibition altered reactive oxygen species (ROS) generation, autophagy activation, and cell fusion-related protein expression. ROS generation increased, and autophagy activation and cell migration ability enhancement were verified subsequently by flow cytometry and transwell migration assays. DOT1L inhibition increased NFATc1 nuclear translocation and NF-κB activation and strengthend osteoclast fusion and expression of resorption-related protein CD9, and MMP9 in osteoclasts derived from RAW264.7. Our findings support a new mechanism of DOT1L-mediated H3K79me2 epigenetic regulation of osteoclast differentiation, implicating DOT1L as a new therapeutic target for osteoclast dysregulation-induced disease.
2018-01-24 | PXD006448 | Pride
Project description:Similar temporal expression kinetics of transcription factors in human and mouse osteoclast differentiation evaluated by microarray We used the identical differentiation conditions to evaluate gene expression kinetics of mouse and human osteoclast differentiation by microarray
2018-04-10 | GSE107295 | GEO
Project description:Comparison of gene expression of the osteoclast precursor myeloid blast seeded on plastic and on bone, primed with M-CSF for 4 days and culture with M-CSF and RANKL for 1 day. Osteoclasts and macrophages share progenitors that must receive decisive lineage signals driving them into their respective differentiation routes. Macrophage colony stimulation factor M-CSF is a common factor; bone is likely the stimulus for osteoclast differentiation. To elucidate the effect of both, shared mouse bone marrow precursor myeloid blast was pre-cultured with M-CSF on plastic and on bone. M-CSF priming prior to stimulation with M-CSF and osteoclast differentiation factor RANKL resulted in a complete loss of osteoclastogenic potential without bone. This coincided with a steeply decreased expression of osteoclast genes TRACP and DC-STAMP, but an increased expression of the macrophage markers F4/80 and CD11b. Compellingly, M-CSF priming on bone accelerated the osteoclastogenic potential: M-CSF primed cells that had received only one day M-CSF and RANKL and were grown on bone already expressed an array of genes that are associated with osteoclast differentiation and these cells differentiated into osteoclasts within 2 days. This implies that adhesion to bone dictates the fate of osteoclast precursors. Common macrophage-osteoclast precursors may become insensitive to differentiate into osteoclasts and regain osteoclastogenesis when bound to bone or when in the vicinity of bone. Two conditions: Osteoclast precursors on plastic and on bone, n=4, dye swap
2014-06-02 | E-GEOD-58146 | biostudies-arrayexpress
Project description:Colony Stimulating Factor 1(CSF1) is known to promote osteoclast progenitor survival but its role in regulating osteoclast differentiation and mature osteoclast function are less well understood. Macrophages have the potential to differentiate into osteoclasts and are also considered as osteoclast precursors. The microarray screen was designed to identify potential CSF1 targets in macrophage and osteoclast lineage. Wild type C57/BL6 mice were treated with 0.2 ml 10 % thioglycolate medium (FTG) / mouse by intra-peritoneal injection. Mice were sacrificed one week after injection followed by intra-peritoneal lavage with 15ml PBS. Macrophages were plated at a density of 1.5×10^6/well in 6-well plates and cultured in ?-MEM supplemented with 10% FBS. At 80% confluence, cells were treated with 100ng/ml CSF1 for 12 hours and RNA extracted. Microarray analysis was performed using Affymatrix Mouse Genome 430 2.0 Arrays. Data were analyzed by Gene spring 6.2 software.
2013-04-30 | E-GEOD-46390 | biostudies-arrayexpress
Project description:Osteoclast differentiation is crucial for bone absorption and osteoclast is involved in bone destruction in rheumatoid arthritis. The aim of this study was to investigate the inhibitory effect of MJ2 on osteoclast differentiation and to elucidate its mechanism. Murine macrophage cell line, Raw 264.7 cells and collagen-induced arthritis mouse model were used for in vitro and in vivo study, respectively. MJ2-treated cells significantly inhibited osteoclast differentiation and decreased arthritic score. Surface proteins (SP) extracted from MJ2 also showed inhibitory effect on osteoclast differentiation by upregulating lipocalin 2 (lcn2) expression. Specifically, heat shock protein 60 (hsp60) in SP was revealed as an active component of MJ2. Hsp60 inhibited binding of receptor activator of nuclear factor-κB ligand (RANKL) to receptor activator of nuclear factor κB (RANK). In conclusion, MJ2 inhibited osteoclast formation and differentiation through lcn2 and RANKL-binding property and the effective component of MJ2 might be hsp60 present in surface layer.
2022-09-18 | GSE213113 | GEO
Project description:Purpose: Among the diverse cytokines involved in osteoclast differentiation, IL-3 has been shown to inhibit RANKL-induced osteoclastogenesis. However, the mechanism underlying IL-3-mediated inhibition of osteoclast differentiation is not fully understood. In the present study, we demonstrate that IL-3 activation of STAT5 inhibits RANKL-induced osteoclastogenesis through the induction of Id genes. Methods: To investigate the effect of STAT5 on osteoclast differentiation and IL-3-mediated inhibition of osteoclast differentiation, bone marrow derived macrophages isolated from STAT5 wild-type (Stat5fl/fl) or STAT5 cKO (STAT5;MX1-Cre) were differentiated to osteoclast in the presence of M-CSF and RANKL with or without IL-3; and bone marrow derived macrophges from STAT5 wild-type and STAT5 cKO were overexpressed with PMX-FIG (control) or STAT5A1*6 (constitutively active form of STAT5A) and differentiated to osteoclast. To analyze bone phenotype, femurs and tibiae of 16 week-old STAT5 wild-type and STAT5 cKO were subjected to micro CT analysis and histomorphometry, respectively. Results: Overexpression of STAT5 inhibited RANKL-induced osteoclastogenesis. However, RANKL did not regulate either expression or activation of STAT5 during osteoclast differentiation. STAT5 deficiency prevented IL-3-mediated inhibition of osteoclastogenesis, suggesting that STAT5 plays an important role in IL-3-mediated inhibition of osteoclast differentiation. In addition, IL-3-induced STAT5 activation upregulated expression of the Id1 and Id2 genes, which are negative regulators of osteoclastogenesis. Overexpression of ID1 or ID2 in STAT5-deficient cells reversed osteoclast development recovered from IL-3-mediated inhibition. Moreover, micro-computed tomography and histomorphometric analysis revealed that STAT5 conditional knockout mice showed reduced bone mass, with an increased number of osteoclasts. Furthermore, IL-3 inhibited RANKL-induced osteoclast differentiation less effectively in STAT5 conditional knockout mice than in wild-type mice in a RANKL injection model. Conclusion: Taken together, our results suggest that STAT5 is a key transcription factor for IL-3-mediated inhibition of RANKL-induced osteoclastogenesis through Id gene expression. Examination of 4 different combination of osteoclast differentiation condition of bone marrow derived macrophages.
2016-08-03 | E-GEOD-76988 | biostudies-arrayexpress
Project description:Osteoclast differentiation is a dynamic differentiation process, which is accompanied by dramatic changes in metabolic status as well as in gene expression. Recent findings have revealed an essential connection between metabolic reprogramming and dynamic gene expression changes during osteoclast differentiation. However, the upstream regulatory mechanisms that drive these metabolic changes in osteoclastogenesis remain to be elucidated. We demonstrate that induced deletion of a tumor suppressor gene, Folliculin (Flcn), in mouse osteoclast precursors causes severe osteoporosis in 3 weeks through excess osteoclastogenesis. Flcn deficient osteoclast precursors (Raw264.7 cells) reveal cell autonomous accelerated osteoclastogenesis. For the purpose of elucidating the molecular mechanism of accelerated osteoclastogenesis in Flcn deficient Raw264.7 cells, we performed DNA microarray analysis.
2018-07-30 | GSE115084 | GEO
Project description:Comparison of gene expression of the osteoclast precursor myeloid blast seeded on plastic and on bone, primed with M-CSF for 4 days and culture with M-CSF and RANKL for 1 day. Osteoclasts and macrophages share progenitors that must receive decisive lineage signals driving them into their respective differentiation routes. Macrophage colony stimulation factor M-CSF is a common factor; bone is likely the stimulus for osteoclast differentiation. To elucidate the effect of both, shared mouse bone marrow precursor myeloid blast was pre-cultured with M-CSF on plastic and on bone. M-CSF priming prior to stimulation with M-CSF and osteoclast differentiation factor RANKL resulted in a complete loss of osteoclastogenic potential without bone. This coincided with a steeply decreased expression of osteoclast genes TRACP and DC-STAMP, but an increased expression of the macrophage markers F4/80 and CD11b. Compellingly, M-CSF priming on bone accelerated the osteoclastogenic potential: M-CSF primed cells that had received only one day M-CSF and RANKL and were grown on bone already expressed an array of genes that are associated with osteoclast differentiation and these cells differentiated into osteoclasts within 2 days. This implies that adhesion to bone dictates the fate of osteoclast precursors. Common macrophage-osteoclast precursors may become insensitive to differentiate into osteoclasts and regain osteoclastogenesis when bound to bone or when in the vicinity of bone.
2014-06-02 | GSE58146 | GEO
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:Microgravity leads to a 10-15% loss of bone mass in astronauts during space flight. Osteoclast is the multinucleated bone resorbing cell. In this study, we used NASA developed ground based Rotary Wall Vessel Bioreactor (RWV), Rotary Cell Culture System (RCCS) to simulate microgravity (μXg) conditions and demonstrated a significant increase (2-fold) in osteoclastogenesis compared to ground based control (Xg) mouse bone marrow cultures. We further determined the gene expression profiling of RAW 264.7 osteoclast progenitor cells in microgravity by agilent microarray analysis. Gene expression pattern was functional group clustered by transcriptome analysis using gene ontology tree machine (GOTM) for cell proliferation/survival, differentiation and function. We confirm the microgravity modulated gene expression critical for osteoclast differentiation by real-time RT-PCR and Western blot analysis in murine bone marrow cultures. We identify transcription factors such as c-Jun, c-Fos, PU-1 critical for osteoclast differentiation is up-regulated in microgravity conditions. In addition, microgravity resulted in 2.3 and 2.0-fold increase in the level of cathepsin K and MMP-9 matrix metalloproteinase expression in preosteoclast cells involved in the bone resorption process respectively. We also demonstrate a significant increase in the expression levels of M-CSF receptor, c-Fms and PLCγ2 and S100A8 molecules that play an important role in Ca2+ signaling essential for osteoclast function. Further, microgravity stimulated preosteoclast cells showed elevated cytosolic Ca2+ levels compared to ground based control cells. Thus, microgravity regulated gene expression profiling in preosteoclast cells provide new insights in to molecular mechanisms and therapeutic targets of osteoclast differentiation/activation responsible for bone loss and fracture risk in astronauts during space flight mission. Microgravity associated with space flight is a challenge for normal bone homeostasis. Astronauts experience 10-15% bone loss during a space flight mission. We aimed to determine the effect of simulated microgravity on osteoclast preosteoclasts cells. RAW264.7 cells (1.5 x 106 /ml) were loaded in RCCS with DMEM containing 10% FBS for 24 h. The cells were stimulated with RANKL (80ng/ml) for 24 h to obtain preosteoclasts in parallel with ground based control cells. Total RNA was isolated using RNAzol reagent (Biotecx Labs, Houston, TX) from control (Xg) and microgravity (μXg) subjected cells and hybridized with Agilent whole mouse genome 4x44K array system. Slides were washed and scanned on an Agilent G2565 microarray scanner. Data obtained were analyzed with Agilent feature extraction and GeneSpring GX v7.3.1 software packages (Genus biosystem, Inc. Northbrook, IL, USA).
2010-04-07 | E-GEOD-21093 | biostudies-arrayexpress