Expression data from cultured macrophages and osteoclasts
ABSTRACT: Osteoclastogenesis is induced by the stimulation of RANKL. In the early stage of osteoclast differentiation, the osteoclast progenitor cells are primed by M-CSF, following a tightly controlled genetic program where specific sets of genes are up-regulated by RANKL. Some of them, for instance, control differentiation, cell-cell fusion and bone resorption. We used microarrays to detail the global program of gene expression underlying osteoclastogenesis and identified various up-regulated genes during this process. Macrophages and osteoclasts were cultured for RNA extraction and hybridization on Affymetrix microarrays. We sought to obtain homogeneous populations of macrophages and osteoclasts in order to increase the temporal resolution of expression profiles. To that end, mouse bone marrow cells were cultured in the presence of M-CSF for three days and harvested as macrophage and oseteoclast common progenitor cells. Then common progenitor cells were further cultured in the presence of M-CSF alone for macrophages and M-CSF plus RANKL for osteoclasts, respectively.
Project description:Osteoclastogenesis is induced by the stimulation of RANKL. In the early stage of osteoclast differentiation, the osteoclast progenitor cells are primed by M-CSF, following a tightly controlled genetic program where specific sets of genes are up-regulated by RANKL. Some of them, for instance, control differentiation, cell-cell fusion and bone resorption. We used microarrays to detail the global program of gene expression underlying osteoclastogenesis and identified various up-regulated genes during this process. Overall design: Macrophages and osteoclasts were cultured for RNA extraction and hybridization on Affymetrix microarrays. We sought to obtain homogeneous populations of macrophages and osteoclasts in order to increase the temporal resolution of expression profiles. To that end, mouse bone marrow cells were cultured in the presence of M-CSF for three days and harvested as macrophage and oseteoclast common progenitor cells. Then common progenitor cells were further cultured in the presence of M-CSF alone for macrophages and M-CSF plus RANKL for osteoclasts, respectively.
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
Project description:Osteoclast (OC) differentiation undergoes a two-step process: commitment of hematopoietic progenitor cells to tartrate-resistant acid phosphatase (TRAcP) positive OC precursors (OCPs), and fusion of OCPs into multinucleated OCs. In order to identify transcriptional profiles of genes in the transitional phase between OC commitment and fusion in OCG, Affymetrix® Mouse Gene 1.0 ST arrays were performed on total RNA extracted from mouse (SV129/BL6 ) monocytes and pre-osteoclasts (pre-OCs), primed with macrophage colony-stimulated factor (M-CSF) or M-CSF and soluble recombinant receptor activator of NF-кB ligand (sRANKL), respectively. The analysis identified 656 RANKL-up or down-regulated in the early stage of osteoclastogenesis. Monocytes isolated from mouse bone marrow were stimulated with M-CSF and soluble RANKL (m + r), or M-CSF alone (m).
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.
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.
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.
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.
Project description:Osteoclast differentiation and activation requires the presence of osteoblast-derived factors such as Macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor-kappaB ligand (RANKL). RANKL is influenced by osteotropic hormones such as calcitriol 1,25(OH)2D3 (vitamin D).The aim of this study is to evaluate the combined effect of vitamin D and M-CSF on murine bone marrow and on murine spleen cell, to define when the inhibitory effect of vitamin D plus M-CSF is maximum and what are the mechanisms involved in this inhibition. When murine bone marrow cells were cultured with 10-8 M vitamin D and 60 ng/ml M-CSF, there was a significant decrease in osteoclasts formation (3.13±3.44),compared to bone marrow cells cultured with vitamin D alone (80.86 ±45.02; p<0.05). Even when the amount of M-CSF added to the bone marrow cell culture was 1 ng/ml there was a 40% decrease in osteoclasts formation. Since bone marrow contains also stromal cells, we eliminate their influence by culturing murine spleen cells with 10-8 M vitamin D, 60 ng/ml M-CSF and RANKL 20 ng/ml. There was a significant decrease in osteoclasts formation (41±14.14) compared to spleen cells cultured with M-CSF and RANKL (215.67±46.44); p<0.05). These data lead to hypothesize that vitamin D and M-CSF together exert an inhibitory effect on osteoclast differentiation and development. To define the cellular mechanism involved in this inhibition we analyzed gene array data of bone marrow cells cultured with vitamin D and compare them with the cells cultured with vitamin D and M-CSF. Activation Toll like receptors after addition of vitamin D plus M-CSF to bone marrow cells appears to be involved in the inhibition of osteoclast differentiation. Keywords: parallel sample
Project description:C-C chemokine receptor 5 (CCR5) is a co-receptor of HIV. Its ligand, CCL5 significantly augmented the number of wild-type osteoclasts but not Ccr5-deficient cells, indicating that CCL5 enhanced RANKL-induced osteoclastogenesis through CCR5. To investigate the changes in the transcriptional signatures induced by CCL5 in osteoclastogenesis, cultured osteoclasts at pOC stages were incubated with or without rmCCL5 for 2 days, and then subjected for RNA sequencing. Overall design: mRNA profiles of primary bone marrow macrophages obtained from 7 weeks old C57BL6 mice were treated with recombinant CCL5, in triplicate, using Illumina Miseq.
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. Overall design: 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.