1.The investigation of energy metabolism in osteoblasts and osteoclasts.
West China Journal of Stomatology 2021;39(5):501-509
The maintenance of bone homeostasis is critical for bone health. It is vulnerable to cause bone loss, even severely osteoporosis when the balance between bone formation and absorption is interrupted. Growing evidence has shown that energy metabolism disorders, such as abnormal glucose metabolism, irregular amino acid metabolism, and aberrant lipid metabolism, can damage bone homeostasis, causing or exacerbating bone mass loss and osteoporosis-related fractures. Here, we summarize the studies of energy metabolism in osteoblasts and osteoclasts and provide a better appreciation of how energy metabolism, especially glucose metabolism maintains bone homeostasis. With this knowledge, new avenues will be unraveled to understand and cue bone-related diseases such as osteoporosis.
Bone and Bones
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Energy Metabolism
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Osteoblasts
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Osteoclasts
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Osteogenesis
2.H2O2 Enhances Ca2+ Release from Osteoblast Internal Stores.
Soon Ho NAM ; Sang Yong JUNG ; Chang Moo YOO ; Ei Hwan AHN ; Chang Kook SUH
Yonsei Medical Journal 2002;43(2):229-235
The physiological activity of osteoblasts is known to be closely related to increased intracellular Ca2+ activity ([Ca2+]i) in osteoblasts. The cellular regulation of [Ca2+]i in osteoblasts is mediated by Ca2+ movements associated with Ca2+ release from intracellular Ca2+ stores, and transmembrane Ca2+ influx via Na+-Ca2+ exchanger, and Ca2+ ATPase. Reactive oxygen species, such as H2O2, play an important role in the regulation of cellular functions, and act as signaling molecules or toxins in cells. In this study, we investigated the effects of H2O2 on cellular Ca2+ regulation in osteoblasts by measuring intracellular Ca2+ activities using cellular calcium imaging techniques. Osteoblasts were isolated from the femurs and tibias of neonatal rats, and cultured for 7 days. The cultured osteoblasts were loaded with a Ca2+-sensitive fluorescent dye, Fura-2, and fluorescence images were monitored using a cooled CCD camera, and subsequently analyzed using image analyzing software. The results obtained are as follows: (1) The osteoblasts with lower basal Ca2+ activities yielded a transient Ca2+ increase, a Ca2+ spike, while osteoblasts with higher basal Ca2+ activities showed a continuous increase in [Ca2+]i leading to cell death. (2) Ca2+ spikes, generated after removing Na+ from superfusing solutions, were blocked by H2O2 and this was followed by a sustained increase in Ca2+ activity. (3) ATP- induced Ca2+ spikes were inhibited by pretreating with H2O2 and this was followed by a continuous increase of [Ca2+]i. When cells were pretreated with the exogenous nitric oxide (NO) donor S-Nitroso-N-acetylpenicilance (SNAP, 50 microM), treatments of ATP (1 mM) induced a Ca2+ spike-like increase, but [Ca2+]i did not return to the basal level. (4) The expression of inositol- 1,4,5-triphosphate receptor (IP3R) was enhanced by H2O2. Our results suggest that H2O2 modulates intracellular Ca2+ activity in osteoblasts by increasing Ca2+ release from the intracellular Ca2+ stores.
Animal
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Calcium/*metabolism
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Cells, Cultured
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Hydrogen Peroxide/*pharmacology
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Osteoblasts/*drug effects/*metabolism
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Oxidants/*pharmacology
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Rats
3.Intracellular localization and expression of importin 8 during osteoblast differentiation.
Bin LANG ; Xinping WANG ; Xiangxin CHE ; Ping WU ; Xiaoyuan XU
Journal of Southern Medical University 2013;33(4):516-519
OBJECTIVETo observe the variations of intracellular localization and expression of importin 8 (IPO8) during osteoblast differentiation.
METHODSAlizarin red staining, immunocytochemistry and real-time PCR were employed to examine the changes in the intracellular localization and expression of IPO8 mRNA during induced osteogenic differentiation of human osteoblast-like SaOS-2 cells.
RESULTSNumerous red mineralized nodules were observed on day 10 in the induced cells with alizarin red staining. Immunocytochemical staining showed that IPO8 immunoreactivity was the strongest in the perinuclear cytoplasm of the cells. On day 3 of osteoblast differentiation, IPO8 immunoreactivity in the cell nuclei became stronger. On day 7, IPO8 was located mainly in the nuclei, and on day 10 the cells were osteocyte-like and IPO8 was distributed in the cytoplasm. Real-time PCR showed a significantly increased expression of OPN mRNA during osteoblast differentiation, and the expression level of IPO8 mRNA was the highest on day 3 and declined on days 7 and 10.
CONCLUSIONThe intracellular localization and expression level of IPO8 undergo significant changes during osteogenesis, indicating its role in regulating osteoblast differentiation.
Cell Differentiation ; Cell Line ; Humans ; Osteoblasts ; cytology ; metabolism ; Osteogenesis ; beta Karyopherins ; metabolism
4.Biological effects of nano-hydroxyapatite/polyamide 66 on the osteoblast.
Ling YE ; Qin SU ; Xue-dong ZHOU
West China Journal of Stomatology 2005;23(2):142-144
OBJECTIVETo evaluate the biological effects of nano-hydroxyapatite/polyamide 66(nHA-PA66) on the growth and activity of osteoblast.
METHODSMTT assay was used to determine the growth of osteoblast, enzymatic measure was used to determine the activity of ALP and quantitative RT-PCR (QRT-PCR) to evaluate the changes of osteoclacin mRNA expression in osteoblasts treated by DMEM eluate of nHA-PA66.
RESULTSOsteoblasts of different test groups demonstrated relative proliferation rate ranging from 98% - 106% without dose-dependent effect. The ALP activity and osteocalcin mRNA expression were similar in test and control groups (P > 0.05).
CONCLUSIONnHA-PA66 has no negative effects on the osteoblast and its osteoblast-compatibility is proved.
Durapatite ; pharmacology ; Nylons ; pharmacology ; Osteoblasts ; drug effects ; Osteocalcin ; metabolism ; RNA, Messenger ; metabolism
5.Bone and Energy Metabolism.
Journal of Korean Diabetes 2013;14(4):174-177
Bone remodeling requires a large amount of energy, and is regulated by various hormones. Leptin, produced by adipocytes, is a well-known regulator of energy balance and is also involved in controlling bone mass through interaction with the central nervous system. Serotonin, downstream of leptin, is also emerging as a candidate for controlling energy balance and bone metabolism. Currently, bone is also considered to be an endocrine regulator of energy metabolism. Osteocalcin, secreted from osteoblasts, is known to be a key regulator of glucose and fat metabolism. In this review, we describe a novel concept that asserts that there exists a biological link between bone and energy metabolism, and we summarize what is currently known about the relationship between bone and energy metabolism.
Adipocytes
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Bone Remodeling
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Central Nervous System
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Energy Metabolism*
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Glucose
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Leptin
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Metabolism
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Neurotransmitter Agents
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Osteoblasts
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Osteocalcin
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Serotonin
6.Effect of polycystin2 on differentiation and maturation of osteoblasts promoted by low-frequency pulsed electromagnetic fields.
Yueying HE ; Mingjun YANG ; Zhuo CHEN ; Peng WEI ; Kun QIN ; Gaoqian XIE ; Keming CHEN
Chinese Journal of Biotechnology 2022;38(3):1159-1172
It is known that low-frequency pulsed electromagnetic fields (PEMFs) can promote the differentiation and maturation of rat calvarial osteoblasts (ROBs) cultured in vitro. However, the mechanism that how ROBs perceive the physical signals of PEMFs and initiate osteogenic differentiation remains unknown. In this study, we investigated the relationship between the promotion of osteogenic differentiation of ROBs by 0.6 mT 50 Hz PEMFs and the presence of polycystin2 (PC2) located on the primary cilia on the surface of ROBs. First, immunofluorescence staining was used to study whether PC2 is located in the primary cilia of ROBs, and then the changes of PC2 protein expression in ROBs upon treatment with PEMFs for different time were detected by Western blotting. Subsequently, we detected the expression of PC2 protein by Western blotting and the effect of PEMFs on the activity of alkaline phosphatase (ALP), as well as the expression of Runx-2, Bmp-2, Col-1 and Osx proteins and genes related to bone formation after pretreating ROBs with amiloride HCl (AMI), a PC2 blocker. Moreover, we detected the expression of genes related to bone formation after inhibiting the expression of PC2 in ROBs using RNA interference. The results showed that PC2 was localized on the primary cilia of ROBs, and PEMFs treatment increased the expression of PC2 protein. When PC2 was blocked by AMI, PEMFs could no longer increase PC2 protein expression and ALP activity, and the promotion effect of PEMFs on osteogenic related protein and gene expression was also offset. After inhibiting the expression of PC2 using RNA interference, PEMFs can no longer increase the expression of genes related to bone formation. The results showed that PC2, located on the surface of primary cilia of osteoblasts, plays an indispensable role in perceiving and transmitting the physical signals from PEMFs, and the promotion of osteogenic differentiation of ROBs by PEMFs depends on the existence of PC2. This study may help to elucidate the mechanism underlying the promotion of bone formation and osteoporosis treatment in low-frequency PEMFs.
Alkaline Phosphatase/metabolism*
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Animals
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Electromagnetic Fields
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Osteoblasts/metabolism*
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Osteogenesis/genetics*
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Rats
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TRPP Cation Channels/physiology*
7.Role of interaction between reactive oxygen species and ferroptosis pathway in methylglyoxal-induced injury in mouse embryonic osteoblasts.
Yuan Yi FENG ; Dong Mei YANG ; Xi Mei ZHI ; Hai Ou DENG ; Wei Jie ZHANG ; Rui Xue WANG ; Wen WU
Journal of Southern Medical University 2022;42(1):108-115
OBJECTIVE:
To explore the interaction between reactive oxygen species (ROS) and ferroptosis in methylglyoxalinduced injury of mouse embryonic osteoblasts (MC3T3-E1 cells).
METHODS:
MC3T3-E1 cells were treated with methylglyoxal to establish a cell model of diabetic osteoporosis. CCK-8 assay was used to detect the viability of MC3T3-E1 cells. Rhodamine 123 staining followed by photofluorography was used to examine mitochondrial membrane potential (MMP). The intracellular ROS level was detected by 2', 7'-dichlorodihydrofluorescein diacetate staining with photofluorograph. Alkaline phosphatase (ALP) activity in the cells was detected using an ALP kit, the number of mineralized nodules was determined with alizarin red S staining, and the level of iron ions was detected using a detection kit. The expression level of glutathione peroxidase 4 (GPX4, a marker protein that inhibits ferroptosis) in the osteoblasts was determined using Western blotting.
RESULTS:
Treatment of MC3T3-E1 cells with 0.6 mmol/L methylglyoxal for 24 h significantly inhibited the expression level of GPX4 (P < 0.001), increased intracellular iron ion concentration, decreased the cell viability, increased the loss of MMP and intracellular ROS level, decreased both ALP activity and the number of mineralized nodules in the cells (P < 0.001). Co-treatment of MC3T3-E1 cells with 2 mmol/L N-acetylcysteine (NAC, a ROS scavenger) and methylglyoxal significantly increased the expression level of GPX4 (P < 0.01); co-treatment with 4 mmo/L FER-1 (a ferroptosis inhibitor) and methylglyoxal obviously decreased the intracellular ROS level (P < 0.001). Co-treatment of the cells either with NAC and methylglyoxal or with FER-1 and methylglyoxal attenuated methylglyoxal-induced injuries in the osteoblasts (P < 0.001).
CONCLUSION
The interaction between ROS and ferroptosis pathway plays an important role in methylglyoxal-induced injury of mouse embryonic osteoblasts.
Animals
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Cell Survival
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Ferroptosis
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Mice
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Osteoblasts
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Pyruvaldehyde/metabolism*
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Reactive Oxygen Species/metabolism*
8.Exercise regulates bone metabolism via microRNAs.
Yu YUAN ; Lin-Zhen RAO ; Shi-Hua ZHANG ; Yang XU ; Ting-Ting LI ; Jun ZOU ; Xi-Quan WENG
Acta Physiologica Sinica 2023;75(3):429-438
It has been well documented that exercise can improve bone metabolism, promote bone growth and development, and alleviate bone loss. MicroRNAs (miRNAs) are widely involved in the proliferation and differentiation of bone marrow mesenchymal stem cells, osteoblasts, osteoclasts and other bone tissue cells, and regulation of balance between bone formation and bone resorption by targeting osteogenic factors or bone resorption factors. Thus miRNAs play an important role in the regulation of bone metabolism. Recently, regulation of miRNAs are shown to be one of the ways by which exercise or mechanical stress promotes the positive balance of bone metabolism. Exercise induces changes of miRNAs expression in bone tissue and regulates the expression of related osteogenic factors or bone resorption factors, to further strengthen the osteogenic effect of exercise. This review summarizes relevant studies on the mechanism whereby exercise regulates bone metabolism via miRNAs, providing a theoretical basis for osteoporosis prevention and treatment with exercise.
Humans
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MicroRNAs/metabolism*
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Osteogenesis/genetics*
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Cell Differentiation
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Osteoblasts
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Bone Resorption/metabolism*
9.Bone Marrow Osteoblasts Promotes the Proliferation Leukemia Stem Cell by Up-regulating Interleukin-1.
Zhi-Jie CAO ; Yi-Shuang LI ; Hui-Jun WANG ; Zhen-Ya XUE ; Shu-Ying CHEN ; Ke-Jing TANG ; Min WANG ; Qing RAO
Journal of Experimental Hematology 2022;30(5):1348-1353
OBJECTIVE:
To explore the extrinsic regulation mechanism of bone marrow microenvironment in leukemia cells, and investigate the promoting effect of osteoblast niche on the proliferation and self-renewal of leukemia stem cell by up-regulating the expression of interleukin-1 (IL-1) in leukemia cell.
METHODS:
The gene expression profiles on leukemia cells derived from AE9a mouse bone marrow endosteum and central bone marrow were determined by RNA sequencing and gene set enrichment analysis (GSEA). Quantitative real-time PCR (qRT-PCR) was used to detect the expression of IL-1 in AE9a mouse leukemia cells co-cultured with or without osteoblasts in vitro. In addition, qRT-PCR was also used to determine the expression of IL-1 in bone marrow mononuclear cell (BMMNC) from 43 patients with acute myeloid leukemia (AML). For leukemia cells co-cultured with osteoblasts or treated with IL-1β, colony forming ability of AE9a leukemia cells was determined by colony formation assay.
RESULTS:
In AE9a leukemia mouse, RNA-seq data and GSEA showed that the enrichment of the upregulated genes in leukemia cells located in endosteum fell into inflammatory response gene set, among them, IL-1α and IL-1β were significantly higher expressed in AE9a leukemia cells that located osteoblast niche (IL-1α: P<0.001, IL-1β:P<0.001). After AE9a leukemia cells were co-cultured with osteoblasts in vitro, the expression of IL-1α and IL-1β in leukemia cells were increased by 2.5 and 3.5 times respectively. In colony formation assay, the number of colonies was increased significantly after leukemia cells were co-cultured with osteoblasts (P<0.001). In addition, when AE9a leukemia cells were treated with IL-1β, the number of colonies was also increased significantly (P<0.01). In AML patients, BMMNC with high percentage of CD34 positive cells exhibited higher level of IL-1 expression.
CONCLUSION
Osteoblast niche can promote leukemia cell proliferation and self-renewal through up-regulating the expression of IL-1 in leukemia cells. In AML patients, the expression level of IL-1 was correlated to the percentage of CD34 positive cells in BMMNC.
Animals
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Antigens, CD34/metabolism*
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Bone Marrow/metabolism*
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Cell Proliferation
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Leukemia, Myeloid, Acute/metabolism*
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Mice
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Osteoblasts/metabolism*
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Stem Cells
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Tumor Microenvironment
10.Effect of alendronate on expressions of osteoprotegerin and receptor activator of nuclear factor κB ligand in mouse osteoblasts.
Wei DONG ; Mengchun QI ; Jiupeng DENG ; Hongwei CHEN ; Xiaojie FENG ; Nannan LIAO
Journal of Southern Medical University 2012;32(12):1695-1698
OBJECTIVETo investigate the effect of alendronate on the expressions of osteoprotegerin (OPG) and receptor activator of nuclear factor-κB ligand (RANKL) in mouse osteoblasts.
METHODSMouse calvarial osteoblasts cultured in vitro were identified by alkaline phosphatase (ALP) staining and immunofluorescence assay of OPG and RANKL expressions. The second passage of the osteoblasts were treated with different concentrations of alendronate (10(-4) to 10(-7) mol/L) for 48 h, and the changes in OPG and RANKL mRNA and protein expressions were examined using real-time PCR and Western blotting, respectively.
RESULTSThe isolated osteoblasts were positive for ALP and expressed OPG and RANKL. Real-time PCR and Western blotting showed that at the concentration of 1×10(-4) mol/L, alendronate caused an obvious down-regulation of OPG and RANKL expressions in the cells, whereas at lower concentrations, alendronate increased the expressions of both genes with the highest expressions occurring after treatment with 1×10(-5) mol/L.
CONCLUSIONHigh concentrations of alendronate (>1×10(-4) mol/L) decrease the expressions of OPG and RANKL, whereas low concentrations (1×10(-5) to 1×10(-7) mol/L) increase their expressions in mouse osteoblasts cultured in vitro.
Alendronate ; pharmacology ; Animals ; Cells, Cultured ; Mice ; Mice, Inbred BALB C ; Osteoblasts ; drug effects ; metabolism ; Osteoprotegerin ; metabolism ; RANK Ligand ; metabolism