The Experts below are selected from a list of 25320 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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Analysis of Ca^2+ response of Osteocyte network by three-dimensional time-lapse imaging in living bone
Journal of Bone and Mineral Metabolism, 2018Co-Authors: Tomoyo Tanaka, Junko Sunaga, Mana Hashimoto, Ryuta Osumi, Taiji Aadachi, Mitsuhiro Hoshijima, Naoya Odagaki, Takashi Nishida, Hiroshi KamiokaAbstract:Osteocytes form a three-dimensional (3D) cellular network within the mineralized bone matrix. The cellular network has important roles in mechanosensation and mechanotransduction related to bone homeostasis. We visualized the embedded Osteocyte network in chick calvariae and observed the flow-induced Ca^2+ signaling in Osteocytes using 3D time-lapse imaging. In response to the flow, intracellular Ca^2+ ([Ca^2+]_i) significantly increased in developmentally mature Osteocytes in comparison with young Osteocytes in the bone matrix. To investigate the differences in response between young and developmentally mature Osteocytes in detail, we evaluated the expression of Osteocyte-related genes using the Osteocyte-like cell line MLO-Y4, which was 3D-cultured within type I collagen gels. We found that the c - Fos , Cx43 , Panx3 , Col1a1 , and OCN mRNA levels significantly increased on day 15 in comparison with day 7. These findings indicate that developmentally mature Osteocytes are more responsive to mechanical stress than young Osteocytes and have important functions in bone formation and remodeling.
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Analysis of Ca2+ response of Osteocyte network by three-dimensional time-lapse imaging in living bone
Journal of Bone and Mineral Metabolism, 2017Co-Authors: Tomoyo Tanaka, Junko Sunaga, Mana Hashimoto, Ryuta Osumi, Taiji Aadachi, Mitsuhiro Hoshijima, Naoya Odagaki, Takashi Nishida, Hiroshi KamiokaAbstract:Osteocytes form a three-dimensional (3D) cellular network within the mineralized bone matrix. The cellular network has important roles in mechanosensation and mechanotransduction related to bone homeostasis. We visualized the embedded Osteocyte network in chick calvariae and observed the flow-induced Ca2+ signaling in Osteocytes using 3D time-lapse imaging. In response to the flow, intracellular Ca2+ ([Ca2+]i) significantly increased in developmentally mature Osteocytes in comparison with young Osteocytes in the bone matrix. To investigate the differences in response between young and developmentally mature Osteocytes in detail, we evaluated the expression of Osteocyte-related genes using the Osteocyte-like cell line MLO-Y4, which was 3D-cultured within type I collagen gels. We found that the c-Fos, Cx43, Panx3, Col1a1, and OCN mRNA levels significantly increased on day 15 in comparison with day 7. These findings indicate that developmentally mature Osteocytes are more responsive to mechanical stress than young Osteocytes and have important functions in bone formation and remodeling.
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Site-Dependence of Mechanosensitivity in Isolated Osteocytes
IFMBE Proceedings, 2009Co-Authors: Yuki Aonuma, Taiji Adachi, Mototsugu Tanaka, Masaki Hojo, Teruko Takano-yamamoto, Hiroshi KamiokaAbstract:It is proposed that Osteocytes embedded in mineralized bone matrix play a role in mechanosensory system during bone remodeling for mechanical adaptation. Various experimental and computer simulation approaches have revealed that the Osteocytes sense the mechanical stimulus such as deformation/force and microdamages in bone matrix generated by mechanical loading, and also still have continued discussion. In this study, for interest to morphological character of Osteocytes, we investigated site-dependence of mechanosensitivity in the Osteocytes through observation of calcium response to applied local deformation. We developed a quantitative method using a microneedle and microparticles to apply local deformation to an isolated chick Osteocyte, and analyzed intracellular calcium dynamics to the applied deformation. As the results, the applied local deformation induced calcium response at the vicinity of the stimulated point and caused its diffusive wave propagation to whole intracellular region in a single Osteocyte. Furthermore, the large deformation was necessary at the cell body to induce calcium response, while a relatively small deformation was sufficient at the cell process, suggesting that the mechanosensitivity at the cell process was higher than that at the cell body. These results suggest that the cell shape with processes contributes to the mechanism of cellular response to mechanical stimulus in Osteocytes, and that the site-dependent mechanosensitivity depends on cell shape in a single Osteocyte.
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Osteocyte Shape Is Dependent on Actin Filaments and Osteocyte Processes Are Unique Actin‐Rich Projections
Journal of Bone and Mineral Research, 2009Co-Authors: Kayo Tanaka-kamioka, Hiroshi Kamioka, Hans Ris, Soo Siang LimAbstract:Osteocytes are derived from a select group of osteoblasts that have undergone a final differentiation. Due to their inaccessibility when embedded in the bone matrix, very little is known about the Osteocyte cytoskeleton. This study provides an extensive analysis of the Osteocyte cytoskeleton, based on the successful isolation of Osteocytes from 16-day embryonic chick calvariae. We used OB7.3, a chicken Osteocyte-specific monoclonal antibody, to confirm the osteocytic phenotype of the isolated cells and established culture conditions to promote growth of cells that most resemble Osteocytes in vivo. Immunofluorescence staining with antitubulin, antivimentin, and antiactin showed the relative distribution of the microtubules, intermediate filaments, and actin filaments in both Osteocyte cell body and processes. Field emission scanning electron microscopy revealed the three-dimensional relationships of the cytoskeletal elements and a unique organization of actin bundles that spanned the cell body and Osteocyte processes. When combined with drug studies, these experiments demonstrate that actin filaments are crucial for the maintenance of Osteocyte shape. Furthermore, we identified two actin-bundling proteins, alpha-actinin and fimbrin, in Osteocyte processes. The prominence and unique distribution of fimbrin in Osteocyte processes provides the possibility of its use as an intracellular marker to distinguish Osteocytes from osteoblasts.
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Osteocyte shape is dependent on actin filaments and Osteocyte processes are unique actin rich projections
Journal of Bone and Mineral Research, 2009Co-Authors: Kayo Tanakakamioka, Hiroshi Kamioka, Hans Ris, Soo Siang LimAbstract:Osteocytes are derived from a select group of osteoblasts that have undergone a final differentiation. Due to their inaccessibility when embedded in the bone matrix, very little is known about the Osteocyte cytoskeleton. This study provides an extensive analysis of the Osteocyte cytoskeleton, based on the successful isolation of Osteocytes from 16-day embryonic chick calvariae. We used OB7.3, a chicken Osteocyte-specific monoclonal antibody, to confirm the osteocytic phenotype of the isolated cells and established culture conditions to promote growth of cells that most resemble Osteocytes in vivo. Immunofluorescence staining with antitubulin, antivimentin, and antiactin showed the relative distribution of the microtubules, intermediate filaments, and actin filaments in both Osteocyte cell body and processes. Field emission scanning electron microscopy revealed the three-dimensional relationships of the cytoskeletal elements and a unique organization of actin bundles that spanned the cell body and Osteocyte processes. When combined with drug studies, these experiments demonstrate that actin filaments are crucial for the maintenance of Osteocyte shape. Furthermore, we identified two actin-bundling proteins, alpha-actinin and fimbrin, in Osteocyte processes. The prominence and unique distribution of fimbrin in Osteocyte processes provides the possibility of its use as an intracellular marker to distinguish Osteocytes from osteoblasts.
Teresita Bellido - One of the best experts on this subject based on the ideXlab platform.
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Basic Aspects of Osteocyte Function
Osteoporosis, 2020Co-Authors: Jesus Delgado-calle, Teresita BellidoAbstract:Exciting discoveries over the last years have propelled Osteocytes, originally considered passive and metabolically inactive bone cells, to the category of master regulators of bone homeostasis. Osteocytes differentiate from osteoblasts when they become surrounded by matrix during the process of bone formation. Osteoblast to Osteocyte differentiation is complex and involves profound modifications in gene expression that result in morphological changes and transform Osteocytes in dynamic and multifunctional cells. In addition to the traditional role of Osteocytes in the integration of mechanical signals, new osteocytic functions are emerging. Osteocytes are now considered a major source of molecules that coordinate the activity of osteoclasts and osteoblasts in response to both physical and hormonal cues. In addition, accumulating evidence supports the notion that dysregulation of Osteocyte function underlies the pathophysiology of several skeletal disorders, ranging from rare to common diseases such as osteoporosis. Further, the increased understanding of Osteocyte biology has led to the development of therapeutic approaches targeting Osteocytes and their derived factors. In this chapter, we summarize the current knowledge on Osteocyte biology and its different functions and discuss novel observations that support the role of Osteocytes as endocrine regulators of body composition and energy metabolism and as key players in the deleterious effects of cancer and diabetes on bone.
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Osteocytes and Skeletal Pathophysiology
Current Molecular Biology Reports, 2015Co-Authors: Jesus Delgado-calle, Teresita BellidoAbstract:For many years, Osteocytes have been the forgotten bone cells and considered as inactive spectators buried in the bone matrix. We now know that Osteocytes detect and respond to mechanical and hormonal stimuli to coordinate bone resorption and bone formation. Osteocytes are currently considered a major source of molecules that regulate the activity of osteoclasts and osteoblasts, such as RANKL and sclerostin, and genetic and pharmacological manipulations of either molecule markedly affect bone homeostasis. Besides playing a role in physiological bone homeostasis, accumulating evidence supports the notion that dysregulation of Osteocyte function and alteration of Osteocyte lifespan underlies the pathophysiology of skeletal disorders characterized by loss bone mass and increased bone fragility, as well as the damaging effects of cancer in bone. In this review, we highlight some of these investigations and discuss novel observations that demonstrate that Osteocytes, far from being passive cells entombed in the bone, are critical for bone function and maintenance.
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inhibition of Osteocyte apoptosis prevents the increase in osteocytic receptor activator of nuclear factor κb ligand rankl but does not stop bone resorption or the loss of bone induced by unloading
Journal of Biological Chemistry, 2015Co-Authors: Lilian I Plotkin, Keith W Condon, Matthew R Allen, Teresita Bellido, Hannah M. Davis, Arancha R Gortazar, Hugo Gabilondo, Marta MaycasAbstract:Abstract Apoptosis of Osteocytes and osteoblasts precedes bone resorption and bone loss with reduced mechanical stimulation, and receptor activator of NF-κB ligand (RANKL) expression is increased with unloading in mice. Because Osteocytes are major RANKL producers, we hypothesized that apoptotic Osteocytes signal to neighboring Osteocytes to increase RANKL expression, which, in turn, increases osteoclastogenesis and bone resorption. The traditional bisphosphonate (BP) alendronate (Aln) or IG9402, a BP analog that does not inhibit resorption, prevented the increase in Osteocyte apoptosis and osteocytic RANKL expression. The BPs also inhibited osteoblast apoptosis but did not prevent the increase in osteoblastic RANKL. Unloaded mice exhibited high serum levels of the bone resorption marker C-telopeptide fragments of type I collagen (CTX), elevated osteoclastogenesis, and increased osteoclasts in bone. Aln, but not IG9402, prevented all of these effects. In addition, Aln prevented the reduction in spinal and femoral bone mineral density, spinal bone volume/tissue volume, trabecular thickness, mechanical strength, and material strength induced by unloading. Although IG9402 did not prevent the loss of bone mass, it partially prevented the loss of strength, suggesting a contribution of Osteocyte viability to strength independent of bone mass. These results demonstrate that Osteocyte apoptosis leads to increased osteocytic RANKL. However, blockade of these events is not sufficient to restrain osteoclast formation, inhibit resorption, or stop bone loss induced by skeletal unloading.
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Effects of PTH on Osteocyte function
Bone, 2012Co-Authors: Teresita Bellido, Vaibhav Saini, Paola Divieti PajevicAbstract:Osteocytes are ideally positioned to detect and respond to mechanical and hormonal stimuli and to coordinate the function of osteoblasts and osteoclasts. However, evidence supporting the involvement of Osteocytes in specific aspects of skeletal biology has been limited mainly due to the lack of suitable experimental approaches. Few crucial advances in the field in the past several years have markedly increased our understanding of the function of Osteocytes. The development of osteocytic cell lines initiated a plethora of in vitro studies that have provided insights into the unique biology of Osteocytes and continue to generate novel hypotheses. Genetic approaches using promoter fragments that direct gene expression to Osteocytes allowed the generation of mice with gain or loss of function of particular genes revealing their role in Osteocyte function. Furthermore, evidence that Sost/sclerostin is expressed primarily in Osteocytes and inhibits bone formation by osteoblasts, fueled research attempting to identify regulators of this gene as well as other Osteocyte products that impact the function of osteoblasts and osteoclasts. The discovery that parathyroid hormone (PTH), a central regulator of bone homeostasis, inhibits sclerostin expression generated a cascade of studies that revealed that Osteocytes are crucial target cells of the actions of PTH. This review highlights these investigations and discusses their significance for advancing our understanding of the mechanisms by which Osteocytes regulate bone homeostasis and for developing therapies for bone diseases targeting Osteocytes.
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cell autonomous requirement of connexin 43 for Osteocyte survival consequences for endocortical resorption and periosteal bone formation
Journal of Bone and Mineral Research, 2012Co-Authors: Nicoletta Bivi, Keith W Condon, Matthew R Allen, Nathan R Farlow, Giovanni Passeri, Lucas R Brun, Yumie Rhee, Teresita Bellido, Lilian I PlotkinAbstract:Connexin 43 (Cx43) mediates Osteocyte communication with other cells and with the extracellular milieu and regulates osteoblastic cell signaling and gene expression. We now report that mice lacking Cx43 in osteoblasts/Osteocytes or only in Osteocytes (Cx43ΔOt mice) exhibit increased Osteocyte apoptosis, endocortical resorption and periosteal bone formation, resulting in higher marrow cavity and total tissue areas measured at the femoral mid-diaphysis. Blockade of resorption reversed the increased marrow cavity but not total tissue area, demonstrating that endocortical resorption and periosteal apposition are independently regulated. Anatomical mapping of apoptotic Osteocytes, osteocytic protein expression, and resorption and formation, suggests that Cx43 controls osteoclast and osteoblast activity by regulating osteoprotegerin and sclerostin levels, respectively, in Osteocytes located in specific areas of the cortex. Whereas empty lacunae and living Osteocytes lacking osteoprotegerin were distributed throughout cortical bone in Cx43ΔOt mice, apoptotic Osteocytes were preferentially located in areas containing osteoclasts, suggesting that osteoclast recruitment requires active signaling from dying Osteocytes. Furthermore, Cx43 deletion in cultured osteocytic cells resulted in increased apoptosis and decreased osteoprotegerin expression. Thus, Cx43 is essential in a cell-autonomous fashion in vivo and in vitro for Osteocyte survival and for controlling the expression of osteocytic genes that affect osteoclast and osteoblast function.
Soo Siang Lim - One of the best experts on this subject based on the ideXlab platform.
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Osteocyte Shape Is Dependent on Actin Filaments and Osteocyte Processes Are Unique Actin‐Rich Projections
Journal of Bone and Mineral Research, 2009Co-Authors: Kayo Tanaka-kamioka, Hiroshi Kamioka, Hans Ris, Soo Siang LimAbstract:Osteocytes are derived from a select group of osteoblasts that have undergone a final differentiation. Due to their inaccessibility when embedded in the bone matrix, very little is known about the Osteocyte cytoskeleton. This study provides an extensive analysis of the Osteocyte cytoskeleton, based on the successful isolation of Osteocytes from 16-day embryonic chick calvariae. We used OB7.3, a chicken Osteocyte-specific monoclonal antibody, to confirm the osteocytic phenotype of the isolated cells and established culture conditions to promote growth of cells that most resemble Osteocytes in vivo. Immunofluorescence staining with antitubulin, antivimentin, and antiactin showed the relative distribution of the microtubules, intermediate filaments, and actin filaments in both Osteocyte cell body and processes. Field emission scanning electron microscopy revealed the three-dimensional relationships of the cytoskeletal elements and a unique organization of actin bundles that spanned the cell body and Osteocyte processes. When combined with drug studies, these experiments demonstrate that actin filaments are crucial for the maintenance of Osteocyte shape. Furthermore, we identified two actin-bundling proteins, alpha-actinin and fimbrin, in Osteocyte processes. The prominence and unique distribution of fimbrin in Osteocyte processes provides the possibility of its use as an intracellular marker to distinguish Osteocytes from osteoblasts.
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Osteocyte shape is dependent on actin filaments and Osteocyte processes are unique actin rich projections
Journal of Bone and Mineral Research, 2009Co-Authors: Kayo Tanakakamioka, Hiroshi Kamioka, Hans Ris, Soo Siang LimAbstract:Osteocytes are derived from a select group of osteoblasts that have undergone a final differentiation. Due to their inaccessibility when embedded in the bone matrix, very little is known about the Osteocyte cytoskeleton. This study provides an extensive analysis of the Osteocyte cytoskeleton, based on the successful isolation of Osteocytes from 16-day embryonic chick calvariae. We used OB7.3, a chicken Osteocyte-specific monoclonal antibody, to confirm the osteocytic phenotype of the isolated cells and established culture conditions to promote growth of cells that most resemble Osteocytes in vivo. Immunofluorescence staining with antitubulin, antivimentin, and antiactin showed the relative distribution of the microtubules, intermediate filaments, and actin filaments in both Osteocyte cell body and processes. Field emission scanning electron microscopy revealed the three-dimensional relationships of the cytoskeletal elements and a unique organization of actin bundles that spanned the cell body and Osteocyte processes. When combined with drug studies, these experiments demonstrate that actin filaments are crucial for the maintenance of Osteocyte shape. Furthermore, we identified two actin-bundling proteins, alpha-actinin and fimbrin, in Osteocyte processes. The prominence and unique distribution of fimbrin in Osteocyte processes provides the possibility of its use as an intracellular marker to distinguish Osteocytes from osteoblasts.
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Primary cultures of chick Osteocytes retain functional gap junctions between Osteocytes and between Osteocytes and osteoblasts.
Microscopy and Microanalysis, 2007Co-Authors: Hiroshi Kamioka, Hans Ris, Teruko Takano-yamamoto, Yoshihito Ishihara, Sakhr A. Murshid, Yasuyo Sugawara, Soo Siang LimAbstract:The inaccessibility of Osteocytes due to their embedment in the calcified bone matrix in vivo has precluded direct demonstration that Osteocytes use gap junctions as a means of intercellular communication. In this article, we report successfully isolating primary cultures of Osteocytes from chick calvaria, and, using anti-connexin 43 immunocytochemistry, demonstrate gap junction distribution to be comparable to that found in vivo. Next, we demonstrate the functionality of the gap junctions by (1) dye coupling studies that showed the spread of microinjected Lucifer Yellow from osteoblast to Osteocyte and between adjacent Osteocytes and (2) analysis of fluorescence replacement after photobleaching (FRAP), in which photobleaching of cells loaded with a membrane-permeable dye resulted in rapid recovery of fluorescence into the photobleached Osteocyte, within 5 min postbleaching. This FRAP effect did not occur when cells were treated with a gap junction blocker (18 alpha-glycyrrhetinic acid), but replacement of fluorescence into the photobleached cell resumed when it was removed. These studies demonstrate that gap junctions are responsible for intercellular communication between adjacent Osteocytes and between osteoblasts and Osteocytes. This role is consistent with the ability of Osteocytes to respond to and transmit signals over long distances while embedded in a calcified matrix.
S E Harris - One of the best experts on this subject based on the ideXlab platform.
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parathyroid hormone induces bone cell motility and loss of mature Osteocyte phenotype through l calcium channel dependent and independent mechanisms
PLOS ONE, 2015Co-Authors: Matthew Prideaux, Sarah L. Dallas, Ning Zhao, Erica D Johnsrud, Patricia A Veno, Dayong Guo, Yuji Mishina, S E HarrisAbstract:Parathyroid Hormone (PTH) can exert both anabolic and catabolic effects on the skeleton, potentially through expression of the PTH type1 receptor (PTH1R), which is highly expressed in Osteocytes. To determine the cellular and molecular mechanisms responsible, we examined the effects of PTH on osteoblast to Osteocyte differentiation using primary Osteocytes and the IDG-SW3 murine cell line, which differentiate from osteoblast to Osteocyte-like cells in vitro and express GFP under control of the dentin matrix 1 (Dmp1) promoter. PTH treatment resulted in an increase in some osteoblast and early Osteocyte markers and a decrease in mature Osteocyte marker expression. The gene expression profile of PTH-treated Day 28 IDG-SW3 cells was similar to PTH treated primary Osteocytes. PTH treatment induced striking changes in the morphology of the Dmp1-GFP positive cells in IDG-SW3 cultures and primary cells from Dmp1-GFP transgenic mice. The cells changed from a more dendritic to an elongated morphology and showed increased cell motility. E11/gp38 has been shown to be important for cell migration, however, deletion of the E11/gp38/podoplanin gene had no effect on PTH-induced motility. The effects of PTH on motility were reproduced using cAMP, but not with protein kinase A (PKA), exchange proteins activated by cAMP (Epac), protein kinase C (PKC) or phosphatidylinositol-4,5-bisphosphonate 3-kinase (Pi3K) agonists nor were they blocked by their antagonists. However, the effects of PTH were mediated through calcium signaling, specifically through L-type channels normally expressed in osteoblasts but decreased in Osteocytes. PTH was shown to increase expression of this channel, but decrease the T-type channel that is normally more highly expressed in Osteocytes. Inhibition of L-type calcium channel activity attenuated the effects of PTH on cell morphology and motility but did not prevent the downregulation of mature Osteocyte marker expression. Taken together, these results show that PTH induces loss of the mature Osteocyte phenotype and promotes the motility of these cells. These two effects are mediated through different mechanisms. The loss of phenotype effect is independent and the cell motility effect is dependent on calcium signaling.
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in vitro and in vivo approaches to study Osteocyte biology
Bone, 2013Co-Authors: Ivo Kalajzic, Brya G Matthews, Elena Torreggiani, Marie A Harris, Paola Divieti Pajevic, S E HarrisAbstract:Abstract Osteocytes, the most abundant cell population of the bone lineage, have been a major focus in the bone research field in recent years. This population of cells that resides within mineralized matrix is now thought to be the mechanosensory cell in bone and plays major roles in the regulation of bone formation and resorption. Studies of Osteocytes had been impaired by their location, resulting in numerous attempts to isolate primary Osteocytes and to generate cell lines representative of the osteocytic phenotype. Progress has been achieved in recent years by utilizing in vivo genetic technology and generation of Osteocyte directed transgenic and gene deficiency mouse models. We will provide an overview of the current in vitro and in vivo models utilized to study Osteocyte biology. We discuss generation of Osteocyte-like cell lines and isolation of primary Osteocytes and summarize studies that have utilized these cellular models to understand the functional role of Osteocytes. Approaches that attempt to selectively identify and isolate Osteocytes using fluorescent protein reporters driven by regulatory elements of genes that are highly expressed in Osteocytes will be discussed. In addition, recent in vivo studies utilizing overexpression or conditional deletion of various genes using dentin matrix protein (Dmp1) directed Cre recombinase are outlined. In conclusion, evaluation of the benefits and deficiencies of currently used cell lines/genetic models in understanding Osteocyte biology underlines the current progress in this field. The future efforts will be directed towards developing novel in vitro and in vivo models that would additionally facilitate in understanding the multiple roles of Osteocytes. This article is part of a Special Issue entitled "The Osteocyte".
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e11 gp38 selective expression in Osteocytes regulation by mechanical strain and role in dendrite elongation
Molecular and Cellular Biology, 2006Co-Authors: Keqi Zhang, Marie A Harris, S E Harris, Cielo Arraganadjemia, S P Kotha, Mark Dallas, Shujie Zhao, Jia Q Feng, Lynda F OnewaldAbstract:Within mineralized bone, Osteocytes form dendritic processes that travel through canaliculi to make contact with other Osteocytes and cells on the bone surface. This three-dimensional syncytium is thought to be necessary to maintain viability, cell-to-cell communication, and mechanosensation. E11/gp38 is the earliest Osteocyte-selective protein to be expressed as the osteoblast differentiates into an osteoid cell or Osteocyte, first appearing on the forming dendritic processes of these cells. Bone extracts contain large amounts of E11, but immunostaining only shows its presence in early Osteocytes compared to more deeply embedded cells, suggesting epitope masking by mineral. Freshly isolated primary osteoblasts are negative for E11 expression but begin to express this protein in culture, and expression increases with time, suggesting differentiation into the Osteocyte phenotype. Osteoblast-like cell lines 2T3 and Oct-1 also show increased expression of E11 with differentiation and mineralization. E11 is highly expressed in MLO-Y4 Osteocyte-like cells compared to osteoblast cell lines and primary osteoblasts. Differentiated, mineralized 2T3 cells and MLO-Y4 cells subjected to fluid flow shear stress show an increase in mRNA for E11. MLO-Y4 cells show an increase in dendricity and elongation of dendrites in response to shear stress that is blocked by small interfering RNA specific to E11. In vivo, E11 expression is also increased by a mechanical load, not only in Osteocytes near the bone surface but also in Osteocytes more deeply embedded in bone. Maximal expression is observed not in regions of maximal strain but in a region of potential bone remodeling, suggesting that dendrite elongation may be occurring during this process. These data suggest that Osteocytes may be able to extend their cellular processes after embedment in mineralized matrix and have implications for osteocytic modification of their microenvironment.
Lynda F Bonewald - One of the best experts on this subject based on the ideXlab platform.
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skeletal muscle secreted factors prevent glucocorticoid induced Osteocyte apoptosis through activation of β catenin
European Cells & Materials, 2012Co-Authors: Katharina Jähn, Nuria Laracastillo, Leticia Brotto, Mark L Johnson, Marco Brotto, Lynda F BonewaldAbstract:It is a widely held belief that the sole effect of muscle on bone is through mechanical loading. However, as the two tissues are intimately associated, we hypothesized that muscle myokines may have positive effects on bone. We found that factors produced by muscle will protect Osteocytes from undergoing cell death induced by dexamethasone (dex), a glucocorticoid known to induce Osteocyte apoptosis thereby compromising their capacity to regulate bone remodeling. Both the trypan blue exclusion assay for cell death and nuclear fragmentation assay for apoptosis were used. MLO-Y4 Osteocytes, primary Osteocytes, and MC3T3 osteoblastic cells were protected against dex-induced apoptosis by C2C12 myotube conditioned media (MT-CM) or by CM from ex vivo electrically stimulated, intact extensor digitorum longus (EDL) or soleus muscle derived from 4 month-old mice. C2C12 MT-CM, but not undifferentiated myoblast CM prevented dex-induced cell apoptosis and was potent down to 0.1 % CM. The CM from EDL muscle electrically stimulated tetanically at 80 Hz was more potent (10 fold) in prevention of dex-induced Osteocyte death than CM from soleus muscle stimulated at the same frequency or CM from EDL stimulated at 1 Hz. This suggests that electrical stimulation increases production of factors that preserve Osteocyte viability and that type II fibers are greater producers than type I fibers. The muscle factor(s) appears to protect Osteocytes from cell death through activation of the Wnt/β-catenin pathway, as MT-CM induces β-catenin nuclear translocation and β-catenin siRNA abrogated the positive effects of MT-CM on dex-induced apoptosis. We conclude that muscle cells naturally secrete factor(s) that preserve Osteocyte viability.
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isolation and culture of primary Osteocytes from the long bones of skeletally mature and aged mice
BioTechniques, 2012Co-Authors: Amber Rath Stern, Katharina Jähn, Matthew M Stern, Mark E Van Dyke, Matthew Prideaux, Lynda F BonewaldAbstract:The purpose of this work was to establish a methodology to enable the isolation and study of Osteocytes from skeletally mature young (4-month-old) and old (22-month-old) mice. The location of Osteocytes deep within bone is ideal for their function as mechanosensors. However, this location makes the observation and study of Osteocytes in vivo technically difficult. Osteocytes were isolated from murine long bones through a process of extended collagenase digestions combined with EDTA-based decalcification. A tissue homogenizer was used to reduce the remaining bone fragments to a suspension of bone particles, which were placed in culture to yield an outgrowth of Osteocyte-like cells. All of the cells obtained from this outgrowth that displayed an Osteocyte-like morphology stained positive for the Osteocyte marker E11/GP38. The Osteocyte phenotype was further confirmed by a lack of staining for alkaline phosphatase and the absence of collagen1a1 expression. The outgrowth of Osteocytes also expressed additiona...
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Identification of Osteocyte‐selective proteins
Proteomics, 2010Co-Authors: Dayong Guo, Patricia A Veno, Andrew Keightley, Jill R. Guthrie, Stephen E. Harris, Lynda F BonewaldAbstract:Since little is known regarding Osteocytes, cells embedded within the mineralized bone matrix, a proteomics approach was used to discover proteins more highly expressed in Osteocytes than in osteoblasts to determine Osteocyte-specific function. Two proteomic profiles obtained by two different proteomic approaches using total cell lysates from the Osteocyte cell line MLO-Y4 and the osteoblast cell line MC3T3 revealed unique differences. Three protein clusters, one related to glycolysis (Phosphoglycerate kinase 1, fructose-bisphosphate aldolase A, hypoxia up-regulated 1 [ORP150], triosephosphate isomerase), one to protein folding (Mitochondrial Stress-70 protein, ORP150, Endoplasmin), and one to actin cytoskeleton regulation (Macrophage-capping protein [CapG], destrin, forms of lamin A and vimentin) were identified. Higher protein expression of ORP-150, Cap G, and destrin in MLO-Y4 cells compared with MC3T3 cells was validated by gene expression, Western blotting, and in vivo expression. These proteins were shown to be selective in Osteocytes in vivo using immuno-staining of mouse ulnae. Destrin was most highly expressed in embedding osteoid Osteocytes, GapG in embedded Osteocytes, and ORP150 in deeply embedded Osteocytes. In summary, the proteomic approach has yielded important information regarding molecular mechanisms used by Osteocytes for embedding in matrix, the formation of dendritic processes, and protection within a hypoxic environment.
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establishment of an Osteocyte like cell line mlo y4
Journal of Bone and Mineral Research, 2010Co-Authors: Yoichi Kato, Jolene J Windle, Barbara A Koop, Gregory R Mundy, Lynda F BonewaldAbstract:Although Osteocytes are the most abundant cells in bone, their functional role remains unclear. In part, this is due to lack of availability of Osteocyte cell lines which can be studied in vitro. Since others have shown that cell lines can be readily developed from transgenic mice in which the SV40 large T-antigen oncogene is expressed under the control of a promoter which targets the cells of interest, we used this approach to develop an Osteocyte cell line. We chose as a promoter osteocalcin, whose expression is essentially limited to bone cells and which is expressed more abundantly in Osteocytes than in osteoblasts. From these transgenic mice, we isolated cells from the long bones using sequential collagenase digestion and maintained these cells on collagen-coated surfaces which are optimal for Osteocyte maintenance and growth. We describe here the properties of a cell line cloned from these cultures, called MLO-Y4 (for murine long bone Osteocyte Y4). The properties of MLO-Y4 cells are very similar to primary Osteocytes. Like primary Osteocytes and unlike primary osteoblasts, the cell line produces large amounts of osteocalcin but low amounts of alkaline phosphatase. The cells produce extensive, complex dendritic processes and are positive for T-antigen, for osteopontin, for the neural antigen CD44, and for connexin 43, a protein found in gap junctions. This cell line also produces very small amounts of type I collagen mRNA compared with primary osteoblasts. MLO-Y4 cells lack detectable mRNA for osteoblast-specific factor 2, which appears to be a positive marker for osteoblasts but may be a negative marker for Osteocytes. This newly established cell line should prove useful for studying the effects of mechanical stress on Osteocyte function and for determining the means whereby Osteocytes communicate with other bone cells such as osteoblasts and osteoclasts.
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Dynamics of the transition from osteoblast to Osteocyte
Annals of the New York Academy of Sciences, 2010Co-Authors: Sarah L. Dallas, Lynda F BonewaldAbstract:Osteocytes are derived from osteoblasts and make up over 90% of the cells in bone. However, the mechanisms that control the differentiation of osteoblasts into Osteocytes embedded in bone matrix are not well understood. With the recent developments of transgenic models for manipulating gene expression in Osteocytes and of transgenic mice carrying lineage reporters for osteoblasts and Osteocytes, unprecedented new insights are becoming possible. In this article we review recent advances, such as comparative gene and protein expression studies, that are delineating the changes in gene and protein expression that accompany Osteocyte differentiation. We also review recent studies in which time-lapse dynamic imaging approaches have been used to visualize osteoblast and Osteocyte populations within bone. These approaches reveal the key role of cell motility in bone cell function and highlight the dynamic nature of mineralized tissues. Changes in motile properties of the cell may be key in the transition from osteoblast to Osteocyte, as reflected in the altered expression of many molecules involved in cytoskeletal function.
