The Experts below are selected from a list of 33 Experts worldwide ranked by ideXlab platform
Hiroshi Kamioka - One of the best experts on this subject based on the ideXlab platform.
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Screening of key candidate genes and pathways for osteocytes involved in the differential response to different types of mechanical stimulation using a bioinformatics analysis
Journal of Bone and Mineral Metabolism, 2019Co-Authors: Ziyi Wang, Yoshihito Ishihara, Takanori Ishikawa, Mitsuhiro Hoshijima, Naoya Odagaki, Ei Ei Hsu Hlaing, Hiroshi KamiokaAbstract:This study aimed to predict the key genes and pathways that are activated when different types of mechanical loading are applied to osteocytes. mRNA expression datasets (series number of GSE62128 and GSE42874) were obtained from Gene Expression Omnibus database (GEO). High gravity-treated osteocytic MLO-Y4 Cell-Line samples from GSE62128 (Set1), and fluid flow-treated MLO-Y4 samples from GSE42874 (Set2) were employed. After identifying the differentially expressed genes (DEGs), functional enrichment was performed. The common DEGs between Set1 and Set2 were considered as key DEGs, then a protein–protein interaction (PPI) network was constructed using the minimal nodes from all of the DEGs in Set1 and Set2, which linked most of the key DEGs. Several open source software programs were employed to process and analyze the original data. The bioinformatic results and the biological meaning were validated by in vitro experiments. High gravity and fluid flow induced opposite expression trends in the key DEGs. The hypoxia-related biological process and signaling pathway were the common functional enrichment terms among the DEGs from Set1, Set2 and the PPI network. The expression of almost all the key DEGs ( Pdk1 , Ccng2 , Eno2 , Egln1 , Higd1a , Slc5a3 and Mxi1 ) were mechano-sensitive. Eno2 was identified as the hub gene in the PPI network. Eno2 knockdown results in expression changes of some other key DEGs ( Pdk1 , Mxi1 and Higd1a ). Our findings indicated that the hypoxia response might have an important role in the differential responses of osteocytes to the different types of mechanical force.
Ziyi Wang - One of the best experts on this subject based on the ideXlab platform.
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Screening of key candidate genes and pathways for osteocytes involved in the differential response to different types of mechanical stimulation using a bioinformatics analysis
Journal of Bone and Mineral Metabolism, 2019Co-Authors: Ziyi Wang, Yoshihito Ishihara, Takanori Ishikawa, Mitsuhiro Hoshijima, Naoya Odagaki, Ei Ei Hsu Hlaing, Hiroshi KamiokaAbstract:This study aimed to predict the key genes and pathways that are activated when different types of mechanical loading are applied to osteocytes. mRNA expression datasets (series number of GSE62128 and GSE42874) were obtained from Gene Expression Omnibus database (GEO). High gravity-treated osteocytic MLO-Y4 Cell-Line samples from GSE62128 (Set1), and fluid flow-treated MLO-Y4 samples from GSE42874 (Set2) were employed. After identifying the differentially expressed genes (DEGs), functional enrichment was performed. The common DEGs between Set1 and Set2 were considered as key DEGs, then a protein–protein interaction (PPI) network was constructed using the minimal nodes from all of the DEGs in Set1 and Set2, which linked most of the key DEGs. Several open source software programs were employed to process and analyze the original data. The bioinformatic results and the biological meaning were validated by in vitro experiments. High gravity and fluid flow induced opposite expression trends in the key DEGs. The hypoxia-related biological process and signaling pathway were the common functional enrichment terms among the DEGs from Set1, Set2 and the PPI network. The expression of almost all the key DEGs ( Pdk1 , Ccng2 , Eno2 , Egln1 , Higd1a , Slc5a3 and Mxi1 ) were mechano-sensitive. Eno2 was identified as the hub gene in the PPI network. Eno2 knockdown results in expression changes of some other key DEGs ( Pdk1 , Mxi1 and Higd1a ). Our findings indicated that the hypoxia response might have an important role in the differential responses of osteocytes to the different types of mechanical force.
Maria Pia Rastaldi - One of the best experts on this subject based on the ideXlab platform.
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Application of retinoic acid to obtain osteocytes cultures from primary mouse osteoblasts.
Journal of Visualized Experiments, 2014Co-Authors: Deborah Mattinzoli, Piergiorgio Messa, Alessandro Corbelli, Masami Ikehata, Anna Mondini, Cristina Zennaro, Silvia Armelloni, Min Li, Laura Giardino, Maria Pia RastaldiAbstract:The need for osteocyte cultures is well known to the community of bone researchers; isolation of primary osteocytes is difficult and produces low Cell numbers. Therefore, the most widely used Cellular system is the osteocyte-like MLO-Y4 Cell Line. The method here described refers to the use of retinoic acid to generate a homogeneous population of ramified Cells with morphological and molecular osteocyte features. After isolation of osteoblasts from mouse calvaria, all-trans retinoic acid (ATRA) is added to Cell medium, and Cell monitoring is conducted daily under an inverted microscope. First morphological changes are detectable after 2 days of treatment and differentiation is generally complete in 5 days, with progressive development of dendrites, loss of the ability to produce extraCellular matrix, down-regulation of osteoblast markers and up-regulation of osteocyte-specific molecules. Daily Cell monitoring is needed because of the inherent variability of primary Cells, and the protocol can be adapted with minimal variation to Cells obtained from different mouse strains and applied to transgenic models. The method is easy to perform and does not require special instrumentation, it is highly reproducible, and rapidly generates a mature osteocyte population in complete absence of extraCellular matrix, allowing the use of these Cells for unlimited biological applications.
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A novel model of in vitro osteocytogenesis induced by retinoic acid treatment.
European Cells & Materials, 2012Co-Authors: Deborah Mattinzoli, Piergiorgio Messa, Alessandro Corbelli, Masami Ikehata, Cristina Zennaro, Silvia Armelloni, Min Li, Laura Giardino, Maria Pia RastaldiAbstract:: Despite recent research which more and more stresses the importance of osteocytes in regulating bone and systemic mineral metabolism, current molecular and functional knowledge of osteocyte properties are still incomplete, mostly due to limited availability of in vitro models. Osteocytes are terminally differentiated dendritic Cells, and therefore are not easy to obtain and maintain in primary cultures. As an alternative, osteocyte differentiation can be induced by progressive osteoblast embedding in mineralised extraCellular matrix. In this model, which is suitable for reproduction of bone development, the presence of calcified matrix prevents several Cell biological methods from being used. Therefore, the osteocyte-like MLO-Y4 Cell Line continues to be the most widely used Cellular system. Here we show that treatment of primary osteoblasts or MC3T3-E1 Cells with retinoic acid generates a homogeneous population of ramified Cells with osteocyte features, as confirmed by morphological and molecular analyses. The first morphological changes are detectable in primary Cells after 2 days of treatment, and in the Cell Line after 4 days of treatment. Differentiation is complete in 5 and 10 days, respectively, with progressive development of dendrites, loss of the ability to produce extraCellular matrix, down-regulation of osteoblast markers, and up-regulation of osteocyte-specific molecules, most notably among them sclerostin. Compared to other published protocols, our method has a number of advantages. It is easy to perform and does not require special instrumentation, it is highly reproducible, and rapidly generates a mature osteocyte population in the complete absence of extraCellular matrix, allowing the use of these Cells for unlimited biological applications.
Christopher R. Jacobs - One of the best experts on this subject based on the ideXlab platform.
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A ROLE FOR THE PRIMARY CILIUM IN PARACRINE SIGNALING BETWEEN MECHANICALLY STIMULATED OSTEOCYTES AND MESENCHYMAL STEM CellS
Biochemical and Biophysical Research Communications, 2011Co-Authors: David A. Hoey, Daniel J. Kelly, Christopher R. JacobsAbstract:Bone turnover is a mechanically regulated process, coordinated in part by the network of mechanosensitive osteocytes residing within the tissue. The recruitment and bone forming activity of the mesenchymal derived osteoblast is determined by numerous factors including mechanical loading. It is therefore somewhat surprising that although mechanically regulated signaling between the coordinating osteocytes and mesenchymal stem Cells (MSCs) should exist, to date it has not been directly demonstrated. In this study, conditioned media from mechanically stimulated osteocytes (MLO-Y4 Cell Line) was collected and added to MSCs (C3H10T1/2 Cell Line). The addition of mechanically stimulated osteocyte conditioned media resulted in a significant upregulation of the osteogenic genes OPN and COX-2 in MSCs compared to statically cultured conditioned media, demonstrating a novel paracrine signaling mechanism between the two Cell types. The same mechanically conditioned media did not alter gene expression in osteoblasts (MC3T3 Cell Line), and mechanically stimulated osteoblast conditioned media did not alter gene expression in MSCs demonstrating that this signaling is unique to osteocytes and MSCs. Finally, the upregulation in osteogenic genes in MSCs was not observed if primary cilia formation was inhibited prior to mechanical stimulation of the osteocyte. In summary, the results of this study indicate that soluble factors secreted by osteocytes in response to mechanical stimulation can enhance osteogenic gene expression in MSCs demonstrating a novel, unique signaling mechanism and introduces a role for the primary cilium in flow mediated paracrine signaling in bone thereby highlighting the cilium as a potential target for therapeutics aimed at enhancing bone formation.
Anthony R. Green - One of the best experts on this subject based on the ideXlab platform.
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A Role for SCL in Developing Bone? the SCL +19 Core Enhancer Targets Expression to Cells of Developing Bone, Osteocytes, Chondrocytes and Bone Lining Cells.
Blood, 2004Co-Authors: Lev Silberstein, John E. Pimanda, Sandie Piltz, Liz Delaney, Scott Oldham, Asha R. Kallianpur, Massimo Dominici, Stephen J. Brandt, Berthold Göttgens, Anthony R. GreenAbstract:The transcription factor SCL/TAL1 is essential for haematopoiesis and vascular development in the embryo. It is also expressed in the developing skeletal and central nervous systems, but lethality of SCL null mice has precluded detailed analysis of the role of this gene in these tissues.We have previously demonstrated that the SCL +18/19 enhancer directs reporter gene expression to haemangioblasts, endothelium and haematopoietic progenitors. Using human placental alkaLine phosphatase as a reporter gene, we now report that the +19 core enhancer also directs expression to osteocytes, articular chondrocytes and bone lining Cells in adult mice. In E14.5 embryos, the transgene is expressed in Cells within the cartilaginous template of future bone and in the perichondrium. The pattern of expression of the transgene resembles that of endogenous SCL RNA and protein expression in age matched embryos. At E16.5, transgene-positive Cells are seen within ossification centres. Activity of the enhancer during osteogenesis was observed both in bones formed through intra-membranous (e.g. cranial vault) and endochondral ossification (limbs and axial skeleton). We found that the murine pre-osteoblast Cell Line, MC3T3, expresses endogenous SCL. Stable transfection of these Cells with luciferase reporter constructs that include the +19 core enhancer produce a 5-fold increase in luciferase activity. There is a progressive reduction in SCL RNA expression in these Cells during osteogenic differentiation. This is consistent with a previous report that expression of SCL protein is down-regulated in skeletal tissues of more advanced stage embryos, and with our finding that the MLO-Y4 Cell Line, which phenotypically and functionally resembles mature osteocytes, does not express SCL. We addressed the issue of SCL function in bone formation using in-vitro differentiation of embryonic stem (ES) Cells. Wild type and SCL −/− J1 ES Cells did not differ in their ability to form bone nodules. These data demonstrate that SCL is not required for bone specification, but do not exclude a role for SCL in regulating bone formation in vivo. The SCL +19 core enhancer directs expression to haematopoietic progenitors and endothelium together with their putative precursors, haematopoietic endothelium and haemangioblasts. Our demonstration that the +19 core enhancer also targets expression to osteogenic Cells is consistent with recent data suggesting that haematopoietic and osteogenic Cells share a common precursor (Olmsted-Davis, EA et al PNAS 2003, Dominici M et al PNAS 2004) and that osteoblastic bone lining Cells are intimately associated with haematopoietic stem Cells (Calvi LM et al Nature 2003; Zhang J et al Nature 2003).