Gene microarray screening to identify glycosylation genes that are regulated during the formation of osteoclast cells
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE28937 | GEO | 2011/04/29
SECONDARY ACCESSION(S): PRJNA139893
REPOSITORIES: GEO
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Project description:Bone remodeling is a tightly regulated process that engages degradation and biogenesis of the bone matrix. The process is controlled by two major cell types, bone forming cells-osteoblasts and bone-degrading cells-osteoclasts. We are interested in the bone-resorption mechanism mediated by osteoclasts and wish to identify glycosylation genes that are regulated during the formation of osteoclast cells and determine the function of glycosylation and glycan-binding proteins in the osteoclastogenesis. We propose to examine the gene expression patterns that are altered during the osteoclastogenesis using mouse glyco-chips and RNA samples isolated from osteoclast precursors and mature osteoclasts prepared from mouse bone marrows. 6 chips are requested for the analysis. RNA preparations from mouse bone marrow MG2, MG4, MG6 (mature osteoclasts) and MG1, MG3, MG5 osteoclasts precursors (control) were sent to the Microarray Core (E). The RNA was amplified, labeled, and hybridized to the GLYCOv3 microarrays.
2011-04-29 | E-GEOD-28937 | biostudies-arrayexpress
Project description:Osteoclast derived from macrophage lineage cells are responsible for bone resorption and important in bone remodeling. Emerging of a new discipline-osteoimmunology highlights the important connection between immunology and bone biology. We had mouse osteoclast's RNA samples analyzed by the consortium’s gene microarray and identified a number of significant changes in expression of genes involved in glycan biosynthesis. Osteoclasts were treated with: control, Rankl (Receptor activator of nuclear factor kappa-B ligand (RANKL), tunicamycin, and both rankl and tunicamycin. RNA preparations from osteoclasts purified from mice: control (1,2,3), Rankl (1,2,3), tunicamycin (1,2,3), and both rankl and tunicamycin (1,2,3) were sent to the Microarray Core (E). The RNA was amplified, labeled, and hybridized to the GLYCOv3 microarrays.
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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.
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Project description:Osteoporosis and bone fractures affect millions of men and women worldwide and are often due to increased bone resorption (bone loss) mediated by osteoclasts. Here, we identify a novel role for the cytoplasmic protein ELMO1 as an important ‘signaling node’ controlling the bone resorption function of osteoclasts. Initially, we noted association of ELMO1 SNPs with bone abnormalities and altered bone density in humans. Experimentally, ELMO1 emerged as a promoter of bone loss wherein deletion of ELMO1 reversed osteoporosis / bone erosions in four in vivo mouse models: osteoprotegerin deficiency, ovariectomy, and two types of inflammatory arthritis. However, ELMO1 did not promote bone loss under homeostatic conditions. Mechanistic studies pointed to a larger ELMO1 signaling network that regulates osteoclast activity at several levels. First, transcriptomics coupled with CRISPR/Cas9 genetic deletion approaches identified new regulators of osteoclast function associated with Elmo1. Second, defining the ‘ELMO1 interactome’ in osteoclasts via proteomics revealed proteins linked to bone degradation. Third, ELMO1 affects the formation of the actin ring /sealing zone on bone-like surfaces and the distribution of osteoclast-specific proteases. Finally, a 3D structure-based inhibitory peptide targeting a highly conserved region of ELMO1 reduced bone resorption in wild type osteoclasts. Collectively, these data identify ELMO1 as a signaling hub that regulates osteoclast function and bone loss, with relevance to diseases such as osteoporosis and arthritis.
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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. 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.
2010-03-22 | E-GEOD-20850 | biostudies-arrayexpress
Project description:Osteoclasts are bone-resorbing cells that undergo extensive changes in morphology throughout their differentiation. Altered osteoclast differentiation and activity lead to changes in pathological bone resorption. The mammalian target of rapamycin (mTOR) is a kinase, and aberrant mTOR complex 1 (mTORC1) signaling is associated with altered bone homeostasis. The activation of mTORC1 is biphasically regulated during osteoclastogenesis; however, the mechanism behind mTORC1-mediated regulation of osteoclastogenesis and bone resorption is incompletely understood. Here, we found that MYC coordinates the dynamic regulation of mTORC1 activation during osteoclastogenesis. MYC-deficiency blocked the early activation of mTORC1 and also reversed the decreased activity of mTORC1 at the late stage of osteoclastogenesis. The suppression of mTORC1 activity by rapamycin in mature osteoclasts enhances bone resorption activity despite the indispensable role of high mTORC1 activation in osteoclast formation in both mouse and human cells. Mechanistically, MYC induces Growth arrest and DNA damage-inducible protein (GADD34) expression and suppresses mTORC1 activity at the late phase of osteoclastogenesis. Taken together, our findings identify a MYC-GADD34 axis as an upstream regulator of dynamic mTORC1 activation in osteoclastogenesis and highlight the interplay between MYC and mTORC1 pathways in determining osteoclast activity.
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Project description:Enhanced osteoclastogenesis and osteoclast activity contribute to the development of osteoporosis, which is characterized by increased bone resorption and inadequate bone formation. As novel anti-osteoporotic therapeutics are needed, understanding the genetic regulation of human osteoclastogenesis could help identify potential treatment targets. This study aimed to provide an overview of the transcriptional reprogramming during human osteoclast differentiation. Osteoclasts were differentiated from CD14+-monocytes from eight female donors. RNA-sequencing during differentiation demonstrated 8980 differentially expressed genes grouped into eight temporal patterns conserved across donors. These patterns showed distinct molecular functions, associated with postmenopausal osteoporosis susceptibility genes based on RNA from iliac crest biopsies, and bone mineral density SNPs. Network analyses showed mutual dependencies between the temporal expression patterns and provides insight into subtype-specific transcriptional networks. Donor specific expression patterns identified genes at monocyte stage, such as filamin B (FLNB) and oxidized low density lipoprotein receptor 1 (OLR1, encoding LOX-1), that are predictive for the resorptive activity of mature osteoclasts. Differentially expressed G-protein coupled receptors showed strong expression during osteoclast differentiation and associated with bone mineral density SNPs, implying a pivotal role in osteoclast differentiation and activity. The regulatory effects of three differentially expressed G-protein coupled receptors were exemplified by in vitro pharmacological modulation of complement 5A receptor 1 (C5AR1), somatostatin receptor 2 (SSTR2), and free fatty acid receptor 4 (FFAR4/GPR120). Activating C5AR1 enhanced osteoclast formation, while activating SSTR2 decreased resorptive activity of mature osteoclasts, and activating FFAR4 decreased both number and resorptive activity of mature osteoclasts. In conclusion, we report the transcriptional reprogramming during human osteoclast differentiation and identified SSTR2 and FFAR4 as anti-resorptive G-protein coupled receptors as well as FLNB and LOX-1 as potential molecular markers of osteoclast activity. These data can help future investigations to identify molecular regulators of osteoclast differentiation and activity and provide the basis for novel anti-osteoporotic targets.
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Project description:Osteoporosis and bone fractures affect millions of men and women worldwide and are often due to increased bone resorption (bone loss) mediated by osteoclasts. Here, we identify a novel role for the cytoplasmic protein ELMO1 as an important ‘signaling node’ controlling the bone resorption function of osteoclasts. Initially, we noted association of ELMO1 SNPs with bone abnormalities and altered bone density in humans. Experimentally, ELMO1 emerged as a promoter of bone loss wherein deletion of ELMO1 reversed osteoporosis / bone erosions in four in vivo mouse models: osteoprotegerin deficiency, ovariectomy, and two types of inflammatory arthritis. However, ELMO1 did not promote bone loss under homeostatic conditions. Mechanistic studies pointed to a larger ELMO1 signaling network that regulates osteoclast activity at several levels. First, transcriptomics coupled with CRISPR/Cas9 genetic deletion approaches identified new regulators of osteoclast function associated with Elmo1, including cathepsin G and myeloperoxidase. Second, defining the ‘ELMO1 interactome’ in osteoclasts via proteomics revealed membrane proteins and v-ATPases required for bone degradation. Third, ELMO1 affects the formation of the actin ring /sealing zone on bone-like surfaces and the distribution of osteoclast-specific proteases. Finally, a 3D structure-based inhibitory peptide targeting a highly conserved region of ELMO1 reduced bone resorption in wild type osteoclasts. Collectively, these data identify ELMO1 as a signaling hub that regulates osteoclast function and bone loss, with relevance to diseases such as osteoporosis and arthritis.
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2013-07-10 | E-GEOD-48629 | biostudies-arrayexpress