Project description:Long noncoding RNAs (lncRNAs) have emerged as integral regulators of physiology and disease, but specific roles of lncRNAs in bone resorption remain largely unknown. Here, RNA-seq analysis indicated that lncRNA Nron was a candidate funtional regulators of bone resorption. Our further experimental analysis showed that Nron regulates bone resorption in mice. We further identified a functional motif of Nron. Consequently, delivery of Nron functional motif specifically in osteoclasts reversed bone loss without obvious side-effects in vivo. Together, our study indicate that Nron is a key bone resorption suppressor and reveal Nron functional motif might be a potential therapeutic strategy to combat excessive bone absorption in osteoporosis.
Project description:To investigate the role of HES1 in osteoclast differentiation and function, bone marrow-derived macrophages (BMMs) were derived from CtskCre/WT;RosaHes1 and control (Rosa[STOP]Hes1 ) mice and were differentiated into mature osteoclasts. We then performed gene expression profiling analysis using data obtained from RNA-seq of conrol and HES1-overexpressing osteoclasts.
Project description:Osteoclasts (OCs) are bone-resorbing cells differentiated from macrophage/monocyte precursors in response to M-CSF and RANKL. In vitro models are principally based on primary bone marrow macrophages, but RAW 264.7 cells are frequently used because they are widely available, easy to culture, and more amenable to genetic manipulation than primary cells. Increasing evidence, however, has shown that the vastly different origins of these two cell types may have important effects on cell behavior. In particular, M-CSF is prerequisite for the differentiation of BMMs, by promoting survival and proliferation and priming the cells for RANKL induction. RAW 264.7 cells readily form OCs in the presence of RANKL, but M-CSF is not required. Based on these key differences, we sought to understand their functional implications and how it might affect osteoclast differentiation and related signaling pathways. Using a robust and high-throughput proteomics strategy, we quantified the global protein changes in OCs derived from bone marrow macrophages and RAW 264.7 cells at 1, 3, and 5 days of differentiation. Correlation analysis of the proteomes demonstrated low concordance between the two cell types (R2 ≈ 0.13). Bioinformatics analysis indicate that RANKL-dependent signaling was intact in RAW 264.7 cells, but biological processes known to be dependent on M-CSF were significantly different; including cell cycle control, cytoskeletal organization, and apoptosis. RAW 264.7 cells exhibited constitutive activation of Erk and Akt that was dependent on the activity of Abelson tyrosine kinase, and the timing of Erk and Akt activation was significantly different between BMMs and RAW 264.7 cells. Our findings provide the first evidence for major differences between BMMs and RAW 264.7 cells, indicating that careful consideration is needed when using the RAW 264.7 cell line when studying M-CSF-dependent signaling and functions.
Project description:Increasing evidences are revealing the important biological functions of apoptotic bodies (ABs). Here we identified the proteomic profiles of ABs from osteoclasts of distinct differentiation stages and investigated the potential functions. ABs were isolated from apoptotic bone marrow macrophages (BMMs), pre-osteoclasts (pOCs) or mature osteoclasts (mOCs). Proteomic signature analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) followed by bioinformatic analysis showed that proteomic signatures of ABs exhibited high similarities with the parental cells. Functionally, pOC-ABs induced endothelial progenitor cell (EPC) differentiation and increased CD31hiEmcnhi endothelial cell formation in a murine bone defect model via PDGF-BB. mOC-ABs induced mesenchymal stem cell (MSC) osteogenic differentiation and promoted bone formation via RANKL reverse signaling. Together, our results mapped the detailed proteomic landscapes of osteoclast derived ABs and demonstrated that the biological functions of ABs are largely inherited from corresponding parental cells, suggesting a novel extended intercellular regulation after cell apoptosis.
Project description:To investigate the role of eosinophils on osteoclast differentiation from bone marrow-derived monocytes (BMMs), BMMs were cultivated in the presence or absence of eosinophils and eosinophil supernatant as compared with unstimulated control.
Project description:Flow cytometrically sorted bone marrow osteoclasts from tibia of 2-weeks-old C57Bl/6 mice were used for generating this dataset. VAO and BAO represent RANK+ 2 nucleated and RANK+ 4 (and >4) nucleated osteoclasts from bone marrow respectively.