Browsing by Subject "osteocyte"

Now showing 1 - 10 of 17
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    Chapter Six - Molecular signaling in bone cells: Regulation of cell differentiation and survival
    (Elsevier, 2019-02-04) Plotkin, Lilian I.; Bruzzaniti, Angela; Biomedical Sciences and Comprehensive Care, School of Dentistry
    The achievement of proper bone mass and architecture, and their maintenance throughout life requires the concerted actions of osteoblasts, the bone forming cells, and osteoclasts, the bone resorbing cells. The differentiation and activity of osteoblasts and osteoclasts are regulated by molecules produced by matrix-embedded osteocytes, as well as by cross-talk between osteoblasts and osteoclasts through secreted factors. In addition, it is likely that direct contact between osteoblast and osteoclast precursors, and the contact of these cells with osteocytes and cells in the bone marrow, also modulate bone cell differentiation and function. With the advancement of molecular and genetic tools, our comprehension of the intracellular signals activated in bone cells has evolved significantly, from early suggestions that osteoblasts and osteoclasts have common precursors and that osteocytes are inert cells in the bone matrix, to the very sophisticated understanding of a network of receptors, ligands, intracellular kinases/phosphatases, transcription factors, and cell-specific genes that are known today. These advances have allowed the design and FDA-approval of new therapies to preserve and increase bone mass and strength in a wide variety of pathological conditions, improving bone health from early childhood to the elderly. We have summarized here the current knowledge on selected intracellular signal pathways activated in osteoblasts, osteocytes, and osteoclasts.
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    Disruption of the Cx43/miR21 pathway leads to osteocyte apoptosis and increased osteoclastogenesis with aging
    (Wiley, 2017-03-01) Davis, Hannah M.; Pacheco-Costa, Rafael; Atkinson, Emily G.; Brun, Lucas R.; Gortazar, Arancha R.; Harris, Julia; Hiasa, Masahiro; Bolarinwa, Surajudeen A.; Yoneda, Toshiyuki; Ivan, Mircea; Bruzzaniti, Angela; Bellido, Teresita; Plotkin, Lilian I.; Department of Anatomy & Cell Biology, IU School of Medicine
    Skeletal aging results in apoptosis of osteocytes, cells embedded in bone that control the generation/function of bone forming and resorbing cells. Aging also decreases connexin43 (Cx43) expression in bone; and osteocytic Cx43 deletion partially mimics the skeletal phenotype of old mice. Particularly, aging and Cx43 deletion increase osteocyte apoptosis, and osteoclast number and bone resorption on endocortical bone surfaces. We examined herein the molecular signaling events responsible for osteocyte apoptosis and osteoclast recruitment triggered by aging and Cx43 deficiency. Cx43-silenced MLO-Y4 osteocytic (Cx43def) cells undergo spontaneous cell death in culture through caspase-3 activation and exhibit increased levels of apoptosis-related genes, and only transfection of Cx43 constructs able to form gap junction channels reverses Cx43def cell death. Cx43def cells and bones from old mice exhibit reduced levels of the pro-survival microRNA miR21 and, consistently, increased levels of the miR21 target phosphatase and tensin homolog (PTEN) and reduced phosphorylated Akt, whereas PTEN inhibition reduces Cx43def cell apoptosis. miR21 reduction is sufficient to induce apoptosis of Cx43-expressing cells and miR21 deletion in miR21fl/fl bones increases apoptosis-related gene expression, whereas a miR21 mimic prevents Cx43def cell apoptosis, demonstrating that miR21 lies downstream of Cx43. Cx43def cells release more osteoclastogenic cytokines [receptor activator of NFκB ligand (RANKL)/high-mobility group box-1 (HMGB1)], and caspase-3 inhibition prevents RANKL/HMGB1 release and the increased osteoclastogenesis induced by conditioned media from Cx43def cells, which is blocked by antagonizing HMGB1-RAGE interaction. These findings identify a novel Cx43/miR21/HMGB1/RANKL pathway involved in preventing osteocyte apoptosis that also controls osteoclast formation/recruitment and is impaired with aging.
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    Dose analysis of photobiomodulation therapy on osteoblast, osteoclast, and osteocyte
    (SPIE, 2018-07) Na, Sungsoo; TruongVo, ThucNhi; Jiang, Feifei; Joll, Jeffery E.; Guo, Yunxia; Utreja, Achint; Chen, Jie; Biomedical Engineering, School of Engineering and Technology
    The objective of this study was to evaluate the effects of varying light doses on the viability and cellular activity of osteoblasts, osteocytes, and osteoclasts. A light application device was developed to apply 940-nm wavelength light from light-emitting diodes on three cultured cells, MC3T3-E1, MLO-A5, and RANKL-treated RAW264.7 cells. The doses (energy density) on cells were 0, 1, 5, and 7.5  J  /  cm2. The corresponding light power densities at the cell site were 0, 1.67, 8.33, and 12.5  mW  /  cm2, respectively, and the duration was 10 min. The results showed that the three cell types respond differently to light and their responses were dose dependent. Low-dose treatment (1  J  /  cm2) enhanced osteoblast proliferation, osteoclast differentiation, and osteoclastic bone resorption activity. Osteocyte proliferation was not affected by both low- and high-dose (5  J  /  cm2) treatments. While 1  J  /  cm2 did not affect viability of all three cell types, 5  J  /  cm2 significantly decreased viability of osteocytes and osteoclasts. Osteoblast viability was negatively impacted by the higher dose (7.5  J  /  cm2). The findings suggest that optimal doses exist for osteoblast and osteoclast, which can stimulate cell activities, and there is a safe dose range for each type of cell tested.
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    High glucose alters the secretome of mechanically stimulated osteocyte-like cells affecting osteoclast precursor recruitment and differentiation
    (Wiley, 2017-12) Maycas, Marta; Portolés, Maria Teresa; Matesanz, María Concepción; Buendía, Irene; Linares, Javier; Feito, María José; Arcos, Daniel; Vallet-Regí, María; Plotkin, Lilian; Esbrit, Pedro; Gortázar, Arancha R.; Anatomy and Cell Biology, School of Medicine
    Diabetes mellitus (DM) induces bone deterioration, while mechanical stimulation promotes osteocyte-driven bone formation. We aimed to evaluate the interaction of acute exposure (24 h) to high glucose (HG) with both the pro-survival effect conferred to osteocytic MLO-Y4 cells and osteoblastic MC3T3-E1 cells by mechanical stimulation and the interaction of these cells with osteoclast precursor RAW264.7 cells. We found that 24 h of HG (25 mM) pre-exposure prevented both cell survival and ERK and β-catenin nuclear translocation upon mechanical stimulation by fluid flow (FF) (10 min) in both MLO-Y4 and MC3T3-E1 cells. However, migration of RAW 264.7 cells was inhibited by MLO-Y4 cell-conditioned medium (CM), but not by MC3T3-E1 cell-CM, with HG or FF. This inhibitory effect was associated with consistent changes in VEGF, RANTES, MIP-1α, MIP-1β MCP-1, and GM-CSF in MLO-Y4 cell-CM. RAW264.7 proliferation was inhibited by MLO-Y4 CM under static or HG conditions, but it increased by FF-CM with or without HG. In addition, both FF and HG abrogated the capacity of RAW 264.7 cells to differentiate into osteoclasts, but in a different manner. Thus, HG-CM in static condition allowed formation of osteoclast-like cells, which were unable to resorb hydroxyapatite. In contrast, FF-CM prevented osteoclastogenesis even in HG condition. Moreover, HG did not affect basal RANKL or IL-6 secretion or their inhibition induced by FF in MLO-Y4 cells. In conclusion, this in vitro study demonstrates that HG exerts disparate effects on osteocyte mechanotransduction, and provides a novel mechanism by which DM disturbs skeletal metabolism through altered osteocyte-osteoclast communication.
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    High mobility group box 1 protein regulates osteoclastogenesis through direct actions on osteocytes and osteoclasts in vitro
    (Wiley, 2019-05-20) Davis, Hannah M.; Valdez, Sinai; Gomez, Leland; Malicky, Peter; White, Fletcher A.; Subler, Mark A.; Windle, Jolene J.; Bidwell, Joseph P.; Bruzzaniti, Angela; Plotkin, Lilian I.; Anatomy and Cell Biology, School of Medicine
    Old age and Cx43 deletion in osteocytes are associated with increased osteocyte apoptosis and osteoclastogenesis. We previously demonstrated that apoptotic osteocytes release elevated concentrations of the pro-inflammatory cytokine, high mobility group box1 protein (HMGB1) and apoptotic osteocyte conditioned media (CM) promotes osteoclast differentiation. Further, prevention of osteocyte apoptosis blocks osteoclast differentiation and attenuates the extracellular release of HMGB1 and RANKL. Moreover, sequestration of HMGB1, in turn, reduces RANKL production/release by MLO-Y4 osteocytic cells silenced for Cx43 (Cx43def), highlighting the possibility that HMGB1 promotes apoptotic osteocyte-induced osteoclastogenesis. However, the role of HMGB1 signaling in osteocytes has not been well studied. Further, the mechanisms underlying its release and the receptor(s) responsible for its actions is not clear. We now report that a neutralizing HMGB1 antibody reduces osteoclast formation in RANKL/MCSF treated bone marrow cells (BMC). In bone marrow macrophages (BMMs), TLR4 inhibition with LPS-RS, but not RAGE inhibition with Azeliragon attenuated osteoclast differentiation. Further, inhibition of RAGE but not of TLR4 in osteoclast precursors reduced osteoclast number, suggesting that HGMB1 produced by osteoclasts directly effects differentiation by activating TLR4 in BMMs and RAGE in pre-osteoclasts. Our findings also suggest that increased osteoclastogenesis induced by apoptotic osteocytes CM is not mediated through HMGB1/RAGE activation and that direct HMGB1 actions in osteocytes stimulate pro-osteoclastogenic signal release from Cx43def osteocytes. Based on these findings, we propose that HMGB1 exerts dual effects on osteoclasts, directly by inducing differentiation through TLR4 and RAGE activation and indirectly by increasing pro-osteoclastogenic cytokine secretion from osteocytes.
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    The Importance of Biologically Active Vitamin D for Mineralization by Osteocytes After Parathyroidectomy for Renal Hyperparathyroidism
    (ASBMR, 2019-11) Yajima, Aiji; Tsuchiya, Ken; Burr, David B.; Wallace, Joseph M.; Damrath, John D.; Inaba, Masaaki; Tominaga, Yoshihiro; Satoh, Shigeru; Nakayama, Takashi; Tanizawa, Tatsuhiko; Ogawa, Hajime; Ito, Akemi; Nitta, Kosaku; Anatomy and Cell Biology, School of Medicine
    Hypomineralized matrix is a factor determining bone mineral density. Increased perilacunar hypomineralized bone area is caused by reduced mineralization by osteocytes. The importance of vitamin D in the mineralization by osteocytes was investigated in hemodialysis patients who underwent total parathyroidectomy (PTX) with immediate autotransplantation of diffuse hyperplastic parathyroid tissue. No previous reports on this subject exist. The study was conducted in 19 patients with renal hyperparathyroidism treated with PTX. In 15 patients, the serum calcium levels were maintained by subsequent administration of alfacalcidol (2.0 μg/day), i.v. calcium gluconate, and oral calcium carbonate for 4 weeks after PTX (group I). This was followed in a subset of 4 patients in group I by a reduced dose of 0.5 μg/day until 1 year following PTX; this was defined as group II. In the remaining 4 patients, who were not in group I, the serum calcium (Ca) levels were maintained without subsequent administration of alfacalcidol (group III). Transiliac bone biopsy specimens were obtained in all groups before and 3 or 4 weeks after PTX to evaluate the change of the hypomineralized bone area. In addition, patients from group II underwent a third bone biopsy 1 year following PTX. A significant decrease of perilacunar hypomineralized bone area was observed 3 or 4 weeks after PTX in all group I and II patients. The area was increased again in the group II patients 1 year following PTX. In group III patients, an increase of the hypomineralized bone area was observed 4 weeks after PTX. The maintenance of a proper dose of vitamin D is necessary for mineralization by osteocytes, which is important to increase bone mineral density after PTX for renal hyperparathyroidism.
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    Linking Osteocyte Oxygen Sensing and Biomineralization via FGG23: Implications for Chronic Kidney Disease
    (2022-05) Noonan, Megan L.; White, Kenneth E.; Kota, Janaiah; Graham, Brett H.; Thompson, William R.
    FGF23 is an osteocyte produced hormone necessary for maintaining systemic phosphate handling, and thus bone structure and function in both rare and common disorders such as chronic kidney disease (CKD). FGF23 is a critical factor in CKD, with elevated levels causing alterations in mineral metabolism and increased odds for mortality. However, the mechanisms directing the production of key modulators of skeletal homeostasis and biomineralization within osteocytes, and how this is altered in chronic kidney disease, remain unclear. The experimental focus of this dissertation was to dissect the molecular systems and role of oxygen sensing in the regulated production of FGF23. In CKD, up to 75% of patients have anemia and concomitant marked elevations in FGF23, increasing mortality odds. Anemia is a potent driver of FGF23 secretion, therefore, current and emerging therapies, including recombinant EPO and the hypoxia inducible factorprolyl hydroxylase inhibitors (HIF-PHI) FG-4592 and BAY 85-3934, were used to improve anemia in the adenine diet-induced mouse model of CKD. In the mice with CKD, iFGF23 was markedly elevated in control mice but was attenuated by 65-85% after delivery of EPO or HIF-PHI, with no changes in serum phosphate. This was associated with improved systemic iron utilization and reductions in mRNA markers of renal fibrosis. In osteocyte-like cell cultures treated with HIF-PHI, integrative RNAseq and ATACseq analysis identified candidate genes upregulated in response to mimicked hypoxia, concomitant with elevated Fgf23 expression. These genes were found to be downregulated in CKD bone, therefore, knock-out cells were generated using CRISPR/Cas9 technology. These cells were found to be functionally similar to in vivo conditional knockout models that have enhanced bone mass and elevated FGF23. Taken together, these results further define novel factors involved in the regulation of FGF23 and identify new therapeutic targets.
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    Modeling and simulation of flow–osteocyte interaction in a lacuno-canalicular network
    (AIP, 2023-09) Barber, Jared; Manring, Isaac; Boileau, Sophie; Zhu, Luoding; Mathematical Sciences, School of Science
    Osteocytes are bone cells that can sense mechanical cues (stress and strain) and respond by releasing biochemical signals that direct bone remodeling. This process is called mechanotransduction which, in osteocytes, is not well understood yet because in vivo studies have proven difficult due to the complexity and inaccessibility of the flow–osteocyte lacuna-canaliculi system. While in silico studies (modeling and simulation) have become powerful, currently computational studies for the system often omit the fluid–structure interaction (FSI) between the cell and the surrounding fluids. To investigate the role of FSI in osteocyte mechanotransduction, we introduce a two-dimensional coarse-grained yet integrative model for flow–osteocyte interaction in a lacuno-canalicular network. The model uses the lattice Boltzmann immersed boundary framework to incorporate the flexible osteocyte (membrane, cytoskeleton, and cytosol), its processes, the interstitial fluid, and the rigid extracellular matrix that encases the system. One major result of our model is that the stress and strain tend to attain their local maxima near the regions where the processes meet the membrane of the main body.
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    Modeling and simulation of interstitial fluid flow around an osteocyte in a lacuno-canalicular network
    (AIP, 2022-04-01) Zhu (祝罗丁), Luoding; Barber, Jared; Zigon , Robert; Na (나성수), Sungsoo; Yokota (横田博樹), Hiroki; Mathematical Sciences, School of Science
    Experiments have shown that external mechanical loading plays an important role in bone development and remodeling. In fact, recent research has provided evidence that osteocytes can sense such loading and respond by releasing biochemical signals (mechanotransduction, MT) that initiate bone degradation or growth. Many aspects on MT remain unclear, especially at the cellular level. Because of the extreme hardness of the bone matrix and complexity of the microenvironment that an osteocyte lives in, in vivo studies are difficult; in contrast, modeling and simulation are viable approaches. Although many computational studies have been carried out, the complex geometry that can involve 60+ irregular canaliculi is often simplified to a select few straight tubes or channels. In addition, the pericellular matrix (PCM) is usually not considered. To better understand the effects of these frequently neglected aspects, we use the lattice Boltzmann equations to model the fluid flow over an osteocyte in a lacuno-canalicular network in two dimensions. We focus on the influences of the number/geometry of the canaliculi and the effects of the PCM on the fluid wall shear stress (WSS) and normal stress (WNS) on an osteocyte surface. We consider 16, 32, and 64 canaliculi using one randomly generated geometry for each of the 16 and 32 canaliculi cases and three geometries for the 64 canaliculi case. We also consider 0%, 5%, 10%, 20%, and 40% pericellular matrix density. Numerical results on the WSS and WNS distributions and on the velocity field are visualized, compared, and analyzed. Our major results are as follows: (1) the fluid flow generates significantly greater force on the surface of the osteocyte if the model includes the pericellular matrix (PCM); (2) in the absence of PCM, the average magnitudes of the stresses on the osteocyte surface are not significantly altered by the number and geometry of the canaliculi despite some quantitative influence of the latter on overall variation and distribution of those stresses; and (3) the dimensionless stress (stress after non-dimensionalization) on the osteocyte surface scales approximately as the reciprocal of the Reynolds number and increasing PCM density in the canaliculi reduces the range of Reynolds number values for which the scaling law holds.
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    Molecular Mechanisms Underlying Osteocyte Apoptosis and the Associated Osteoclastogenesis in CX43-Deficiency and Aging
    (2019-06) Davis, Hannah Marie; Plotkin, Lillian I.; Bidwell, Joseph P.; Allen, Matthew R.; Bruzzaniti, Angela
    Old age is associated with increased bone fragility and risk of fracture as a result of skeletal alterations, including low bone density and cortical thinning. Further, apoptotic osteocytes accumulate in old mice and humans. We have previously shown that mice lacking osteocytic connexin (Cx) 43 (Cx43ΔOt) exhibit a phenotype similar to that of the aging skeleton, with elevated osteocyte apoptosis and an associated increase in osteoclastogenesis. These findings suggest that osteocyte apoptosis results in the release of factors that recruit osteoclasts to bone surfaces close to areas that contain apoptotic osteocytes. However, the specific chemotactic signals, the events mediating their release, and the mechanisms of their action remain unknown. Consistent with this notion, we also found that HMGB1 released by Cx43-deficient (Cx43def) MLO-Y4 osteocytic cells enhances osteoclastogenesis in part by increasing osteocytic RANKL, which promotes osteoclastogenesis, and, at the same time, directly stimulating osteoclastogenesis. Further, expression of the pro-survival microRNA (miR), miR21, is low in Cx43def cells and bones from old female mice, and low miR21 levels increase osteocyte apoptosis. However, surprisingly, mice lacking miR21 (miR21ΔOt) have decreased osteoclast number and activity even under conditions of elevated osteocyte apoptosis; suggesting that osteocytic miR21 may mediate osteoclast precursor recruitment/survival induced by apoptotic osteocytes. However, whether HMGB1/miR21 are released by osteocytes, and if the HMGB1 receptors, receptor for advanced glycation end products (RAGE) and/or tolllike receptor (TLR4) are involved in osteoclast recruitment in Cx43ΔOt and old mice is unknown. The overall objectives of this series of studies were to elucidate the mechanisms