Hells protects mitochondrial integrity via Nr2f2 suppression during osteoclast differentiation
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE303340 | GEO | 2025/08/01
REPOSITORIES: GEO
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Project description:Bone remodeling is characterized by the sequential, local tethering of osteoclasts and osteoblasts, and is key to the maintenance of bone integrity. While bone matrix-mobilized growth factors, such as TGF-β, are proposed to regulate remodeling, no in vivo evidence exists that an osteoclast-produced molecule is the enigmatic coupling factor. We have identified Cthrc1, a protein secreted by mature bone-resorbing osteoclasts, that targets stromal cells so as to stimulate osteogenesis. The expression of Cthrc1 is robustly induced when mature osteoclasts are placed on dentin or hydroxyapatite, and also by increasing extracellular calcium. Cthrc1 expression in bone increases in a high turnover state, such as that which is induced by RANKL injections in vivo, whereas it decreases with aging or following alendronate treatment, conditions associated with suppressed bone turnover. The targeted deletion of the Cthrc1 gene eliminates Cthrc1 expression in bone, whereas its deficiency in osteoblasts does not exert any significant effect. Osteoclast-specific deletion of the Cthrc1 gene results in osteopenia due to reduced bone formation: it also impairs the coupling process following resorption induced by RANKL injections, with a resultant impairment of bone mass recovery. Thus, Cthrc1 is an osteoclast-secreted “coupling factor” that regulates bone remodeling and hence, skeletal integrity. Total bone marrow cells were prepared from the femurs and tibias of 8-10-week-old C57BL/6 mice and cultured in the presence of M-CSF (100ng/ml) for 3 days as described previously (Takeshita et al., 2000 JBMR 15:1477-1488). Cells were harvested with 0.02% EDTA/PBS and used as bone marrow macrophages (BMMs). These BMMs were cultured in the presence of M-CSF (100 ng/ml) and RANKL (100ng/ml) for 2 days. TRAP positive mononuclear cells were harvested and used as pre-osteoclasts (pOC). These pOC cells were further cultured in the presence of M-CSF and RANKL for 2 days in normal plastic plate or on dentin slices. After 2 days, multinucleated TRAP positive mature osteoclasts were generated as mature osteoclasts on plate (mOCp) and mature resorbing osteoclasts on dentin (mOCd), respectively. RNAs were extracted from four different stages of osteoclast lineage cells; BMMs, pOC, mOCp and mOCd, and used for microarray analysis.
2015-04-01 | E-GEOD-45656 | biostudies-arrayexpress
Project description:Irf8 functions as negative regulator of osteoclastogenesis. Genetic deletion of Irf8 in mice results in osteoporosis, a low bone mass state caused by increased osteoclast activity. However, the effects of Irf8 ablation on genome wide regulation of osteoclast-specific genes is unknown. We performed RNA-seq analysis of Irf8-/- and Irf8+/+ bone marrow macrophages (BMMs) prior to (day 0) and after RANKL treatment (day 4 and day 8). Additionally, we have identified a novel IRF8 mutation in humans, that is associated with increased osteoclast activity and susceptibility to Multiple Idiopathic Cervical Root Resorption (MICRR). To gain insights into the functional consequences of human IRF8 mutation (hIRF8G388S) on osteoclastogenesis, we performed RNA-seq analysis of Irf8-/- BMMs transduced with hIRF8WT, hIRF8G388S, and a larger deletion construct hIRF8379-389, followed by RANKL treatment. Comparative studies in Irf8-/- and hIRF8G388S cells show that IRF8 deficiency promotes significant enhancement of osteoclast-specific transcripts.
2019-06-01 | GSE115497 | GEO
Project description:Bone marrow derived macrophages (BMM) from Notch2tm1.1Ecan, harboring a NOTCH2 gain-of-function mutation, and control mice were cultured with macrophage colony stimulating factor (M-CSF) and receptor activator of NF-kB ligand (RANKL). Bulk RNA-Seq revealed enhanced cell metabolism, aerobic respiration, and mitochondrial function, all associated with osteoclastogenesis, in Notch2tm1.1Ecan cells. Single cell RNA-Seq data of BMMs treated with M-CSF or M-CSF and RANKL for 3 days identified 11 well-defined cellular clusters. There was an increased number of cells expressing gene markers associated with the osteoclast and with related clusters in Notch2tm1.1Ecan than in control BMMs.
2024-01-03 | GSE248138 | GEO
Project description:Bone marrow derived macrophages (BMM) from Notch2tm1.1Ecan, harboring a NOTCH2 gain-of-function mutation, and control mice were cultured with macrophage colony stimulating factor (M-CSF) and receptor activator of NF-kB ligand (RANKL). Bulk RNA-Seq revealed enhanced cell metabolism, aerobic respiration, and mitochondrial function, all associated with osteoclastogenesis, in Notch2tm1.1Ecan cells. Single cell RNA-Seq data of BMMs treated with M-CSF or M-CSF and RANKL for 3 days identified 11 well-defined cellular clusters. There was an increased number of cells expressing gene markers associated with the osteoclast and with related clusters in Notch2tm1.1Ecan than in control BMMs.
2024-01-03 | GSE248137 | GEO
Project description:Bone undergoes a constant and continuous remodeling process which is tightly regulated by the coordinated and the sequential action of bone-resorbing osteoclasts and bone-forming osteoblasts. Recent studies have shown that histone demethylases are implicated in osteoblastogenesis. However, little is known about the role of histone demethylases in osteoclast formation. Here, we identify KDM4B as an epigenetic regulator of osteoclast differentiation.Our biochemical analysis revealed that KDM4B physically and functionally associates with CCAR1 and Med complex. Using chromatin immunoprecipitation (ChIP) with sequencing, we revealed that KDM4B-CCAR1-Med1 is localized at the promoters of several osteoclast-related genes after RANKL stimulation. We demonstrated that KDM4B-CCAR1-MED1 axis is required for p65 localization at the promoters of osteoclast-related genes through a direct interaction between KDM4B and p65 and changes in chromatin structures (euchromatinization). Finally, small-molecular inhibition of KDM4B impedes bone loss in the ovariectomized mouse model. Taken together, our findings establish KDM4B as a critical regulator of osteoclastogenesis, providing a potential therapeutic target of osteoporosis.
2021-06-30 | GSE154125 | 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: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
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2013-07-10 | E-GEOD-48629 | 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:Glucocorticoid-induced osteonecrosis of the femoral head (GIONFH) is a refractory joint disease caused by prolonged and excessive use of glucocorticoids (GCs). Excessive osteoclast activation has been implicated in the pathological bone erosion of GIONFH. Our previous study found that dexamethasone (10-8M) stimulation significantly enhances osteoclast formation in vitro. To identify therapeutic targets for GIONFH by characterizing glucocorticoid-driven transcriptional alterations in osteoclastogenesis, we performed RNA sequencing on osteoclast precursor cells (OPCs) under RANKL (25ng/ml) and Dex condition (treated with RANKL (25ng/ml) and Dex(10-8M) ) for 24 hours.
2025-05-30 | GSE298117 | GEO