Multinucleated Tartrate Resistant Acid Phosphatase Positive Cells with Macrophage and Osteoclast Phenotypes in Bone
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ABSTRACT:
ORGANISM(S): Rattus norvegicus
PROVIDER: GSE19950 | GEO | 2010/12/31
SECONDARY ACCESSION(S): PRJNA120187
REPOSITORIES: GEO
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Project description:Prothrombin (PT) and osteopontin (OPN) promotes adhesion of different TRAP-positive multinucleated cells isolated from rat long bone (Hu et al. Exp Cell Res. 2008; 314: 638-50). The PT-adhering cell could represent either an immature precursor to the OPN-adherent osteoclast or constitute a distinct multinucleated cell population with a unique role in bone. Herein, phenotypic differences between PT- and OPN- cells were investigated with microarray- expression analysis. Characteristic for PT-cells was expression of innate immune response genes and scavenger receptors whereas OPN-cells expressed typical osteoclast proteins such as collagenases implicated in bone degradation. Bone cells were extracted from neonatal Sprague Dawley rat long bones. The extracted cells were attached to PT or OPN, and thereby two cell populations were isolated. mRNA was extracted from these cells and their expression was compared with the Affymetrix GeneChip Rat Gene 1.0 ST Array.
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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.
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Project description:Multinucleated Tartrate Resistant Acid Phosphatase Positive Cells with Macrophage and Osteoclast Phenotypes in Bone
| PRJNA120187 | ENA
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).
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Project description:Osteoclast progenitors were collected from female Phlpp1 cKO mice or their control littermates and treated with RANKL and M-CSF. On day 4 of cultures when multinucleated cells were present, RNA was collected and RNA-Seq was performed.
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Project description:Osteoclasts are multinucleated, bone-resorbing cells. However, they also digest cartilage during skeletal maintenance, development and in degradative conditions including osteoarthritis, rheumatoid arthritis and primary bone sarcoma. This study explores the mechanisms behind the osteoclast–cartilage interaction. Human osteoclasts differentiated on acellular human cartilage expressed osteoclast marker genes (e.g. CTSK, MMP9) and proteins (TRAP, VNR), visibly damaged the cartilage surface and released glycosaminoglycan in a contact-dependent manner. Direct co-culture with chondrocytes during differentiation increased large osteoclast formation (p < 0.0001) except when co-cultured on dentine, when osteoclast formation was inhibited (p = 0.0002). Osteoclasts cultured on dentine inhibited basal cartilage degradation (p = 0.012). RNA-seq identified MMP8 overexpression in osteoclasts differentiated on cartilage versus dentine (8.89-fold, p = 0.0133), while MMP9 was the most highly expressed MMP. Both MMP8 and MMP9 were produced by osteoclasts in osteosarcoma tissue. This study suggests that bone-resident osteoclasts and chondrocytes exert mutually protective effects on their ‘native’ tissue. However, when osteoclasts contact non-native cartilage they cause degradation via MMPs. Understanding the role of osteoclasts in cartilage maintenance and degradation might identify new therapeutic approaches for pathologies characterized by cartilage degeneration.
2021-12-01 | GSE166535 | GEO
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.
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