scholarly journals Role of UDP-Sugar Receptor P2Y14 in Murine Osteoblasts

2020 ◽  
Vol 21 (8) ◽  
pp. 2747 ◽  
Author(s):  
Nicholas Mikolajewicz ◽  
Svetlana V. Komarova
Keyword(s):  
Mechanical Stimulation ◽  
Calcium Signalling ◽  
Free Calcium ◽  
Osteoblastic Cell ◽  
Uridine Diphosphate ◽  
P2 Receptor ◽  
Mineral Density ◽  
Osteoblastic Cell Line ◽  
Camp Levels

The purinergic (P2) receptor P2Y14 is the only P2 receptor that is stimulated by uridine diphosphate (UDP)-sugars and its role in bone formation is unknown. We confirmed P2Y14 expression in primary murine osteoblasts (CB-Ob) and the C2C12-BMP2 osteoblastic cell line (C2-Ob). UDP-glucose (UDPG) had undiscernible effects on cAMP levels, however, induced dose-dependent elevations in the cytosolic free calcium concentration ([Ca2+]i) in CB-Ob, but not C2-Ob cells. To antagonize the P2Y14 function, we used the P2Y14 inhibitor PPTN or generated CRISPR-Cas9-mediated P2Y14 knockout C2-Ob clones (Y14KO). P2Y14 inhibition facilitated calcium signalling and altered basal cAMP levels in both models of osteoblasts. Importantly, P2Y14 inhibition augmented Ca2+ signalling in response to ATP, ADP and mechanical stimulation. P2Y14 knockout or inhibition reduced osteoblast proliferation and decreased ERK1/2 phosphorylation and increased AMPKα phosphorylation. During in vitro osteogenic differentiation, P2Y14 inhibition modulated the timing of osteogenic gene expression, collagen deposition, and mineralization, but did not significantly affect differentiation status by day 28. Of interest, while P2ry14-/- mice from the International Mouse Phenotyping Consortium were similar to wild-type controls in bone mineral density, their tibia length was significantly increased. We conclude that P2Y14 in osteoblasts reduces cell responsiveness to mechanical stimulation and mechanotransductive signalling and modulates osteoblast differentiation.

1,25(OH)2D3 blunts hormone-elevated cytosolic Ca2+ in osteoblast-like cells

1992 ◽  
Vol 263 (6) ◽  
pp. E1070-E1076
Author(s):  
J. Green ◽  
C. R. Kleeman ◽  
S. Schotland ◽  
L. H. Ye
Keyword(s):  
Vitamin D ◽  
Plasma Membrane ◽  
Bone Cell ◽  
Free Calcium ◽  
Osteoblastic Cell ◽  
Cell Physiology ◽  
Vitamin D Metabolites ◽  
Osteoblastic Cell Line ◽  
Calciotropic Hormones ◽  
Dihydroxyvitamin D

Cytosolic free calcium ([Ca2+]i) is an important regulator of bone cell physiology. We studied the interaction of vitamin D metabolites on the hormonal-activated Ca message system in the osteoblastic cell line UMR-106. The acute rise in [Ca2+]i induced by different calciotropic hormones [parathyroid hormone, prostaglandin E2 (PGE2)] was dose dependently blunted by 1,25-dihydroxyvitamin D [1,25(OH)2D3; half-maximal inhibitory concn approximately 5 x 10(-11) M] and was initially observed after 8 h of preincubation. The 1,25(OH)2D3 metabolite of vitamin D was two orders of magnitude more potent than 24,25(OH)2D3 and 25(OH)D3. To discern between an effect of 1,25(OH)2D3 on hormonal-induced Ca2+ entry through the plasma membrane channel vs. release of Ca2+ from internal stores, we suspended fura-2-loaded cells in Mn2+ rather than Ca2+ buffers. In cells preincubated with 1,25(OH)2D3, [Ca2+]i release (indicated by [Ca2+]i transient) was significantly blunted, whereas Mn2+ influx (indicating Ca2+ flux across the plasma membrane) was unaltered, suggesting a selective effect of 1,25(OH)2D3 on hormonal-activated release of Ca2+ from intracellular stores. 1,25(OH)2D3 also inhibited the PGE2-induced production of inositol 1,4,5-trisphosphate. We conclude that, in osteoblasts, chronic (hours) incubation with 1,25(OH)2D3 leads to attenuated stimulation of the [Ca2+]i transduction pathway by calciotropic hormones. This effect of 1,25(OH)2D3 may provide a cellular basis for the synergism between the effects of vitamin D and calciotropic hormones at the bone level.

scholarly journals Tri-iodothyronine inhibits multilayer formation of the osteoblastic cell line, MC3T3-E1, by promoting apoptosis

1999 ◽  
Vol 160 (1) ◽  
pp. 57-65 ◽  
Author(s):  
F Varga ◽  
E Luegmayr ◽  
N Fratzl-Zelman ◽  
H Glantschnig ◽  
A Ellinger ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Chromatin Condensation ◽  
Osteoblastic Cell ◽  
Osteoblastic Cell Line ◽  
Inhibition Of Proliferation ◽  
Bone Maintenance ◽  
Cell Layers ◽  
Multilayer Formation ◽  
Culture Supernatants

Cell death through apoptosis is a well-known mechanism for maintaining homoeostasis in many developmental and pathological processes. We have recently presented evidence for the occurrence of apoptosis during the formation of bone-like tissue in vitro. MC3T3-E1 osteoblast-like cells in culture develop features of the osteoblastic phenotype and form many cell layers embedded in extracellular matrix which can mineralise. Tri-iodothyronine (T3), even though it enhances the expression of many osteoblastic features, attenuates the multilayer formation to about two layers. The aim of this study was to investigate how T3 prevents multilayer formation. MC3T3-E1 cells were seeded at different densities and cultured for up to 2 weeks. Thereafter we analysed proliferation rate and the distribution of the phases of the cell cycle and studied apoptosis. We found that T3 did not inhibit DNA synthesis. Analysis of the cell cycle phases showed an increase in the number of cells in G0/G1 with increasing cell density, but no significant effect of T3 treatment was found. Morphological investigations showed apoptotic features in both cell layers and culture supernatants. The cells exhibited typical plasma membrane blebbings, chromatin condensation, DNA fragmentation and phagocytosed apoptotic bodies. T3 treatment significantly increased the number of apoptotic cells. We conclude from our data that T3 inhibits multilayer formation of MC3T3-E1 cells by increasing the rate of apoptosis and not by inhibition of proliferation. Because apoptosis is a fundamental regulatory event during bone tissue differentiation, our findings emphasise the importance of thyroid hormones in bone maintenance and development.

In vitro effects of mechanical stimulation and photobiomodulation on osteoblastic cell function: A proof of concept study

2017 ◽  
Vol 27 (1) ◽  
pp. 29-41 ◽  
Author(s):  
Rochaya Chintavalakorn ◽  
Anak Khantachawana ◽  
Kwanchanok Viravaidya-Pasuwat ◽  
Peerapong Santiwong ◽  
Rudee Surarit
Keyword(s):  
Mechanical Stimulation ◽  
Cell Function ◽  
Osteoblastic Cell ◽  
Proof Of Concept ◽  
Concept Study ◽  
In Vitro Effects

scholarly journals Theβ-SiC Nanowires (~100 nm) Induce Apoptosis via Oxidative Stress in Mouse Osteoblastic Cell Line MC3T3-E1

2014 ◽  
Vol 2014 ◽  
pp. 1-9 ◽  
Author(s):  
Weili Xie ◽  
Qi Xie ◽  
Meishan Jin ◽  
Xiaoxiao Huang ◽  
Xiaodong Zhang ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Oxidative Dna Damage ◽  
Morphological Changes ◽  
Osteoblastic Cell ◽  
Osteoblastic Cell Line ◽  
Sic Nanowires ◽  
Tissue Replacement ◽  
Transmission Electron ◽  
First Time

Silicon carbide (SiC), a compound of silicon and carbon, with chemical formula SiC, the beta modification (β-SiC), with a zinc blende crystal structure (similar to diamond), is formed at temperature below1700∘C.β-SiC will be the most suitable ceramic material for the future hard tissue replacement, such as bone and tooth. Thein vitrocytotoxicity ofβ-SiC nanowires was investigated for the first time. Our results indicated that 100 nm long SiC nanowires could significantly induce the apoptosis in MC3T3-E1 cells, compared with 100 μm long SiC nanowires. And 100 nm long SiC nanowires increased oxidative stress in MC3T3-E1 cells, as determined by the concentrations of MDA (as a marker of lipid peroxidation) and 8-OHdG (indicator of oxidative DNA damage). Moreover, transmission electron microscopy (TEM) was performed to evaluate the morphological changes of MC3T3-E1 cells. After treatment with 100 nm long SiC nanowires, the mitochondria were swelled and disintegrated, and the production of ATP and the total oxygen uptake were also decreased significantly. Therefore,β-SiC nanowires may have limitations as medical material.

scholarly journals Influence of substratum surface chemistry/energy and topography on the human fetal osteoblastic cell line hFOB 1.19: Phenotypic and genotypic responses observed in vitro☆

Biomaterials ◽  
2007 ◽  
Vol 28 (31) ◽  
pp. 4535-4550 ◽  
Author(s):  
Xiaomei Liu ◽  
Jung Yul Lim ◽  
Henry J. Donahue ◽  
Ravi Dhurjati ◽  
Andrea M. Mastro ◽  
...  
Keyword(s):  
Surface Chemistry ◽  
Cell Line ◽  
Osteoblastic Cell ◽  
Osteoblastic Cell Line

scholarly journals Forskolin and dexamethasone synergistically induce aromatase (CYP19) expression in the human osteoblastic cell line SV-HFO

2005 ◽  
Vol 152 (4) ◽  
pp. 619-624 ◽  
Author(s):  
Masatada Watanabe ◽  
Shuji Ohno ◽  
Shizuo Nakajin
Keyword(s):  
Oncostatin M ◽  
Osteoblastic Cell ◽  
Aromatase Activity ◽  
Gene Transcript ◽  
Rt Pcr ◽  
Mineral Density ◽  
Osteoblastic Cell Line ◽  
Human Osteoblastic Cell ◽  
Estrogen Concentration ◽  
Cyp19 Expression

Objective: Recent progress supports the importance of local estrogen secretion in human bone tissues to increase and maintain bone mineral density. In a previous study, we reported that the expression of aromatase (CYP19) is dexamethasone (Dex)-dependent and oncostatin M (OSM) increases the expression synergistically with Dex. In the present study, we examined the effects of forskolin (FSK) as another potential synergistic factor. Results: Co-administration of 100 nM Dex and 10 μM FSK increased aromatase activity 4-fold compared with Dex alone. The results of reverse transcriptase (RT)-PCR suggest that the amount of CYP19 gene transcript was also up-regulated by FSK synergistically with Dex, and that promoter I.4, which is not activated by FSK alone, is activated by FSK synergistically with Dex. The results of RT-PCR also suggest that promoter II, which responds to FSK, was not activated even in the presence of FSK in SV-HFO. The promoter I.4 sequence that was transfected into SV-HFO was activated by FSK synergistically with Dex. Conclusions: Synergistic up-regulation of aromatase activity, CYP19 gene transcript, and promoter I.4 activity were Dex-dependent and not up-regulated by FSK alone. The results of this work may form the basis of bone-specific estrogen-replacement therapy that increases the estrogen concentration in bone tissue only.

Factors governing the regulation of Sclerotinia sclerotiorum cutinase A and polygalacturonase 1 during different stages of infection

2012 ◽  
Vol 58 (5) ◽  
pp. 605-616 ◽  
Author(s):  
Zafer Dallal Bashi ◽  
S. Roger Rimmer ◽  
George G. Khachatourians ◽  
Dwayne D. Hegedus
Keyword(s):  
Sclerotinia Sclerotiorum ◽  
Galacturonic Acid ◽  
Hydrolytic Enzymes ◽  
Calcium Signalling ◽  
Solid Surfaces ◽  
Factors Affecting ◽  
Carbohydrate Starvation ◽  
Genes Encoding ◽  
Camp Levels

Sclerotinia sclerotiorum releases hydrolytic enzymes that sequentially degrade the plant cuticle, middle lamellae, and primary and secondary cell walls. The cuticle was found to be a barrier to S. sclerotiorum infection, as leaves stripped of epicuticular wax were more rapidly colonized. Consequently, the factors affecting the regulation of genes encoding polygalacturonase 1 (SsPG1) and a newly identified cutinase (SsCUTA) were examined. In vitro, SsCutA transcripts were detected within 1 h postinoculation of leaves, and expression was primarily governed by contact of mycelia with solid surfaces. Expression of SsPg1 was moderately induced by contact with solid surfaces including the leaf, and expression was restricted to the expanding margin of the lesion as the infection progressed. SsPg1 expression was induced by carbohydrate starvation but repressed by galacturonic acid. Glucose supported a basal level of SsPg1 expression but accentuated expression when provided to mycelia used to inoculate leaves. These observations were contrary to earlier reports indicating that glucose repressed SsPg1 expression while galacturonic acid induced expression. Pharmacological studies showed that disruption of calcium signalling affected SsCutA and SsPg1 expression and decreased S. sclerotiorum virulence, whereas elevated cAMP levels reduced virulence without affecting gene expression. The mechanisms involved in coordinating the expression of S. sclerotiorum hydrolytic enzymes throughout the various stages of the infection are discussed.

scholarly journals 3D Cocultures of Osteoblasts and Staphylococcus aureus on Biomimetic Bone Scaffolds as a Tool to Investigate the Host–Pathogen Interface in Osteomyelitis

Pathogens ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 837
Author(s):  
Raffaella Parente ◽  
Valentina Possetti ◽  
Maria Lucia Schiavone ◽  
Elisabetta Campodoni ◽  
Ciro Menale ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Opportunistic Pathogen ◽  
Bone Architecture ◽  
Osteoblastic Cell ◽  
Bone Homeostasis ◽  
Osteoblastic Cell Line ◽  
Host Pathogen

Osteomyelitis (OM) is an infectious disease of the bone primarily caused by the opportunistic pathogen Staphylococcus aureus (SA). This Gram-positive bacterium has evolved a number of strategies to evade the immune response and subvert bone homeostasis, yet the underlying mechanisms remain poorly understood. OM has been modeled in vitro to challenge pathogenetic hypotheses in controlled conditions, thus providing guidance and support to animal experimentation. In this regard, traditional 2D models of OM inherently lack the spatial complexity of bone architecture. Three-dimensional models of the disease overcome this limitation; however, they poorly reproduce composition and texture of the natural bone. Here, we developed a new 3D model of OM based on cocultures of SA and murine osteoblastic MC3T3-E1 cells on magnesium-doped hydroxyapatite/collagen I (MgHA/Col) scaffolds that closely recapitulate the bone extracellular matrix. In this model, matrix-dependent effects were observed in proliferation, gene transcription, protein expression, and cell–matrix interactions both of the osteoblastic cell line and of bacterium. Additionally, these had distinct metabolic and gene expression profiles, compared to conventional 2D settings, when grown on MgHA/Col scaffolds in separate monocultures. Our study points to MgHA/Col scaffolds as biocompatible and bioactive matrices and provides a novel and close-to-physiology tool to address the pathogenetic mechanisms of OM at the host–pathogen interface.

scholarly journals Anti-Osteoporotic Effects of Combined Extract of Lycii Radicis Cortex and Achyranthes japonica in Osteoblast and Osteoclast Cells and Ovariectomized Mice

Nutrients ◽  
2019 ◽  
Vol 11 (11) ◽  
pp. 2716 ◽  
Author(s):  
Eunkuk Park ◽  
Jeonghyun Kim ◽  
Subin Yeo ◽  
Eunguk Lim ◽  
Chun Whan Choi ◽  
...  
Keyword(s):  
Bone Loss ◽  
Osteoclast Differentiation ◽  
Osteoporotic Bone ◽  
Osteoblastic Cell ◽  
Mouse Bone Marrow ◽  
Osteoblastic Cell Line ◽  
Ovariectomized Mice ◽  
Achyranthes Japonica

Osteoporosis is characterized by low bone density and quality with high risk of bone fracture. Here, we investigated anti-osteoporotic effects of natural plants (Lycii Radicis Cortex (LRC) and Achyranthes japonica (AJ)) in osteoblast and osteoclast cells in vitro and ovariectomized mice in vivo. Combined LRC and AJ enhanced osteoblast differentiation and mineralized bone-forming osteoblasts by the up-regulation of bone metabolic markers (Alpl, Runx2 and Bglap) in the osteoblastic cell line MC3T3-E1. However, LRC and AJ inhibited osteoclast differentiation of monocytes isolated from mouse bone marrow. In vivo experiments showed that treatment of LRC+AJ extract prevented OVX-induced trabecular bone loss and osteoclastogenesis in an osteoporotic animal model. These results suggest that LRC+AJ extract may be a good therapeutic agent for the treatment and prevention of osteoporotic bone loss.

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