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, AffymetrixM-BM-. 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-M-PM-:B 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: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.
Project description:Osteoclasts are derived from the monocyte/macrophage lineage, but little is known about osteoclast precursors in circulation. Bone marrow cells were subdivided into three populations; RANKhighFmslow, RANKhighFmshigh and RANKlowFmshigh. GeneChip analysis confirmed that the expression levels of monocyte-macrophage markers such as Emr1 (F4/80), Itgam (CD11b) and Csf1 (c-Fms) were lower in the RANKhighFmslow than RANKlowFmshigh population. In contrast, cells in the RANKhighFmslow population expressed higher levels of osteoclast markers such as Car ll (carbonic anhydrase ll), Mmp9 (matrix metalloproteinase 9), Acp5 (acid phosphatase 5) and Tfrc (transferrin receptor). These results suggest that RANKhighFmslow cells express few of the phenotypes of monocytes, and their differentiation into osteoclasts occurs at a slightly more advanced stage than that of the RANKlowFmshigh population. RANKhighFmslow cells and RANKlowFmshigh cells were isolated from bone marrow in ddY mice by FACS (Fluorescent activated cell sorting). Differential expression levels of mRNA were determined by GeneChip analysis.
Project description:Osteoclasts are derived from the monocyte/macrophage lineage, but little is known about osteoclast precursors in circulation. Bone marrow cells were subdivided into three populations; RANKhighFmslow, RANKhighFmshigh and RANKlowFmshigh. GeneChip analysis confirmed that the expression levels of monocyte-macrophage markers such as Emr1 (F4/80), Itgam (CD11b) and Csf1 (c-Fms) were lower in the RANKhighFmslow than RANKlowFmshigh population. In contrast, cells in the RANKhighFmslow population expressed higher levels of osteoclast markers such as Car ll (carbonic anhydrase ll), Mmp9 (matrix metalloproteinase 9), Acp5 (acid phosphatase 5) and Tfrc (transferrin receptor). These results suggest that RANKhighFmslow cells express few of the phenotypes of monocytes, and their differentiation into osteoclasts occurs at a slightly more advanced stage than that of the RANKlowFmshigh population.
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.
Project description:It is unclear that osteoclast lineages can sense mechanical stimuli, mainly by lack of information about the presence or absence of mechano-sensing organelles in these cells. Primary cilium is a microtubule-based organelle, which can translate mechanical loading signals into biochemical and transcriptional responses. We first identified the presence of primary cilia in tartrate-resistant acid phosphatase (TRAP)-positive mononuclear cells (preosteoclasts) prior to form TRAP-positive multinuclear cells (osteoclasts), suggesting that preosteoclasts can sense mechanical stimuli. To understand a role of primary cilia in preosteoclasts, we generated osteoclast-specific Ift88 or Kif3a knockout Cathepsin K-Cre transgenic mice. Deletion of Ift88 or Kif3a reduced the number of cilia in preosteoclasts. Micro-CT analysis display that mice with conditional deletion of Ift88 or Kif3a in osteoclast lineages led to a decrease in femoral cortical bone tissue area and bone marrow area under exercise conditions, implying that disruption of primary cilia in preosteoclasts/osteoclasts narrowed the femoral cortical bone shape. In contrast, the trabecular and cortical bone mass was not altered in these mice. Mechanistic studies showed that shear stress suppressed osteoclastogenesis by c-fos degradation. Also, shear stress increased periostin levels in conditioned media from preosteoclasts, resulting in increased differentiation and bone nodule formation of osteoblasts through activation of LRP6/-catenin. All shear stress-induced changes were near completely blocked by knock-downs of Ift88 and Kif3a in preosteoclasts. These findings first suggest that preosteoclasts may sense mechanical stimuli with a primary cilium, and that mechanical stress-mediated primary cilia activation of preosteoclasts may play an important role in controlling bone modeling and shaping by modulating both bone resorption and formation.
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.
Project description:Macrophage multinucleation (MM) is essential for various biological processes such as osteoclast-mediated bone resorption and multinucleated giant cell-associated inflammatory reactions. Here we study the molecular pathways underlying multinucleation in the rat through an integrative approach combining MS-based quantitative phosphoproteomics and transcriptome (high throughput RNA-sequencing) to identify new regulators of MM. We show that a strong metabolic shift towards HIF1-mediated glycolysis occurs at transcriptomic level during MM, together with modifications in phosphorylation of over 50 proteins including several ARF GTPase activators and polyphosphate inositol phosphatases. These results will provide a new framework for the combined analysis of transcriptional and post-translational changes during macrophage multinucleation, prioritizing essential genes and revealing the sequential events leading to the multinucleation of macrophages.
Project description:The autosomal recessive immuno-osseus dysplasia spondyloenchondrodysplasia (SPENCD) is characterised by the variable combination of metaphyseal and vertebral bone lesions, immune dysfunction with features of both autoimmunity and immunodeficiency, and neurological involvement including developmental delay and spasticity with intracranial calcification and leukodystrophy. This transcription profiling study of blood compared four patients to two control subjects. A deficiency of ACP5 encoding tartrate resistant acid phosphatase (TRAP) was found to cause this skeletal dysplasia demonstrating a type I interferon signature with autoimmunity.
Project description:Osteoclast is the primary bone resorbing multinucleated cell. However, human osteooclastoma bone tumor derived osteoclat cellsare However, gene expression profile of giant osteoclat cells is unclear. Here, we performed gene expression profiling of enriched giant cells to elucidate the neoplastic effects and dynamic changes in gene expression responsible for abnormal hyperactive osteoclast formation in the tumor bone microenvironment.