The interplay between mechanoresponses and a broad range of fundamental biological processes, such as cell cycle progression, growth and differentiation, has been extensively investigated. However, metabolic regulation in mechanobiology remains largely unexplored. Here, we identified glucose transporter 1 (GLUT1)-the primary glucose transporter in various cells-as a novel mechanosensitive gene in orthodontic tooth movement (OTM). Using an in vivo rat OTM model, we demonstrated the specific induction of Glut1 proteins on the compressive side of a physically strained periodontal ligament. This transcriptional activation could be recapitulated in in vitro cultured human periodontal ligament cells (PDLCs), showing a time- and dose-dependent mechanoresponse. Importantly, application of GLUT1 specific inhibitor WZB117 greatly suppressed the efficiency of orthodontic tooth movement in a mouse OTM model, and this reduction was associated with a decline in osteoclastic activities. A mechanistic study suggested that GLUT1 inhibition affected the receptor activator for nuclear factor-κ B Ligand (RANKL)/osteoprotegerin (OPG) system by impairing compressive force-mediated RANKL upregulation. Consistently, pretreatment of PDLCs with WZB117 severely impeded the osteoclastic differentiation of co-cultured RAW264.7 cells. Further biochemical analysis indicated mutual regulation between GLUT1 and the MEK/ERK cascade to relay potential communication between glucose uptake and mechanical stress response. Together, these cross-species experiments revealed the transcriptional activation of GLUT1 as a novel and conserved linkage between metabolism and bone remodelling.
Animals ; Biomechanical Phenomena ; Blotting, Western ; Bone Remodeling ; drug effects ; Cells, Cultured ; Glucose Transporter Type 1 ; antagonists & inhibitors ; genetics ; Humans ; Hydroxybenzoates ; pharmacology ; Immunohistochemistry ; MAP Kinase Signaling System ; drug effects ; Mice ; Mice, Inbred C57BL ; Osteoprotegerin ; metabolism ; Periodontal Ligament ; cytology ; RANK Ligand ; metabolism ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction ; Tooth Movement Techniques ; Transcriptional Activation

The present study aimed at investigating the effects of Puerarin (PR), a major isoflavonoid isolated from the Chinese medicinal herb Puerariae radix, on bone metabolism and the underlying mechanism of action. The in vivo assay, female mice were ovariectomized (OVX), and the OVX mice were fed with a diet containing low, middle, and high doses of PR (2, 4, and 8 mg·d(-1), respectively) or 17β-estradiol (E2, 0.03 μg·d(-1)) for 4 weeks. In OVX mice, the uterine weight declined, and intake of PR at any dose did not affect uterine weight, compared with the control. The total femoral bone mineral density (BMD) was significantly reduced by OVX, which was reversed by intake of the diet with PR at any dose, especially at the low dose. In the in vitro assay, RAW264.7 cells were used for studying the direct effect of PR on the formation of osteoclasts. PR reduced the formation of tartrate resistant acid phosphatase (TRAP)-positive multi-nucleated cells in the RAW 264.7 cells induced by receptor activator for nuclear factor-κB Ligand (RANKL). MC3T3-E1 cells were used for studying the effects of PR on the expression of osteoprotegerin (OPG) and RANKL mRNA expression in osteoblasts. The expression of OPG mRNA and RANKL mRNA was detected by RT-PCR on Days of 5, 7, 10, and 12 after PR exposure. PR time-dependently enhanced the expression of OPG mRNA and reduced the expression of RANKL mRNA in MC3T3-E1 cells. In conclusion, our results suggest that PR can effectively prevent bone loss in OVX mice without any hyperplastic effect on the uterus, and the antiosteoporosis activity of PR may be related to its effects on the formation of osteoclasts and the expression of RANKL OPG in osteoblasts.
Animals ; Bone Density ; drug effects ; Drugs, Chinese Herbal ; administration & dosage ; Female ; Femur ; chemistry ; growth & development ; metabolism ; Humans ; Isoflavones ; administration & dosage ; Mice ; Osteoclasts ; drug effects ; metabolism ; Osteoporosis ; genetics ; metabolism ; physiopathology ; prevention & control ; Osteoprotegerin ; genetics ; metabolism ; Ovariectomy ; Pueraria ; chemistry ; RANK Ligand ; genetics ; metabolism

Skeletal fluorosis is a chronically metabolic bone disease with extensive hyperostosis osteosclerosis caused by long time exposure to fluoride. Skeletal fluorosis brings about a series of abnormal changes of the extremity, such as joint pain, joint stiffness, bone deformity, etc. Differentiation and maturation of osteoblasts were regulated by osteoclasts via Sema4D/Plexin-B1 signaling pathway. Furthermore, the differentiation and maturation of osteoclasts are conducted by osteoblasts via RANKL/RANK/OPG pathway. Both of these processes form a feedback circuit which is a key link in skeletal fluorosis. In this study, an osteoblast-osteoclast co-culture model in vitro was developed to illustrate the mechanism of skeletal fluorosis. With the increase of fluoride concentration, the expression level of Sema4D was decreased and TGF-β1 was increased continuously. OPG/RANKL mRNA level, however, increased gradually. On the basis of that, the inhibition of Sema4D/Plexin-B1/RhoA/ROCK signaling pathway caused by fluoride promoted the level of TGF-β1 and activated the proliferation of osteoblasts. In addition, osteroprotegerin (OPG) secreted by osteoblasts was up-regulated by fluoride. The competitive combination of OPG and RANKL was strengthened and the combination of RANKL and RANK was hindered. And then the differentiation and maturation of osteoclasts were inhibited, and bone absorption was weakened, leading to skeletal fluorosis.
Animals ; Antigens, CD ; genetics ; metabolism ; Cell Proliferation ; drug effects ; Feedback, Physiological ; Fetus ; Fluorides ; pharmacology ; GTPase-Activating Proteins ; genetics ; metabolism ; Gene Expression Regulation, Developmental ; Osteoblasts ; drug effects ; metabolism ; pathology ; Osteoclasts ; drug effects ; metabolism ; pathology ; Osteogenesis ; drug effects ; genetics ; Osteoprotegerin ; genetics ; metabolism ; RANK Ligand ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Rats ; Receptor Activator of Nuclear Factor-kappa B ; genetics ; metabolism ; Receptors, Cell Surface ; genetics ; metabolism ; Semaphorins ; genetics ; metabolism ; Signal Transduction ; Transforming Growth Factor beta1 ; genetics ; metabolism ; rho-Associated Kinases ; genetics ; metabolism ; rhoA GTP-Binding Protein ; genetics ; metabolism

Osteoclast-like cells are known to inhibit arterial calcification. Receptor activator of NF-κB ligand (RANKL) is likely to act as an inducer of osteoclast-like cell differentiation. However, several studies have shown that RANKL promotes arterial calcification rather than inhibiting arterial calcification. The present study was conducted in order to investigate and elucidate this paradox. Firstly, RANKL was added into the media, and the monocyte precursor cells were cultured. Morphological observation and Tartrate resistant acid phosphatase (TRAP) staining were used to assess whether RANKL could induce the monocyte precursor cells to differentiate into osteoclast-like cells. During arterial calcification, in vivo and in vitro expression of RANKL and its inhibitor, osteoprotegerin (OPG), was detected by real-time PCR. The extent of osteoclast-like cell differentiation was also assessed. It was found RANKL could induce osteoclast-like cell differentiation. There was no in vivo or in vitro expression of osteoclast-like cells in the early stage of calcification. At that time, the ratio of RANKL to OPG was very low. In the late stage of calcification, a small amount of osteoclast-like cell expression coincided with a relatively high ratio of RANKL to OPG. According to the results, the ratio of RANKL to OPG was very low during most of the arterial calcification period. This made it possible for OPG to completely inhibit RANKL-induced osteoclast-like cell differentiation. This likely explains why RANKL had the ability to induce osteoclast-like cell differentiation but acted as a promoter of calcification instead.
Acid Phosphatase ; genetics ; metabolism ; Animals ; Aorta ; drug effects ; metabolism ; pathology ; Cell Differentiation ; Coculture Techniques ; Gene Expression Regulation ; Isoenzymes ; genetics ; metabolism ; Male ; Monocytes ; cytology ; drug effects ; metabolism ; Myocytes, Smooth Muscle ; drug effects ; metabolism ; pathology ; Osteoclasts ; drug effects ; metabolism ; pathology ; Osteoprotegerin ; genetics ; metabolism ; RANK Ligand ; genetics ; metabolism ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; Tartrate-Resistant Acid Phosphatase ; Vascular Calcification ; genetics ; metabolism ; pathology

Osteoclasts are multinucleated giant cell, which derived from mononuclear myeloid hematopoietic stem cells with the function of bone absorption. Osteoclasts plays a key role in bone metabolism, therefore the body is very strict to regulation of osteoclastogenesis. Mobilization and differentiation of osteoclast maturation is a complex and sophisticated multi-level regulatory processes. In the relevant regulatory mechanisms, OPG/RANKL/RANK system plays a pivotal role in the process of osteoclast differentiation and maturation. Recent studies revealed that immune cells and osteoclasts were closely connect with each other in the field of bone metabolism, also provide a new therapeutic target for the treatment of bone diseases. The apoptosis of osteoclasts in bone metabolism have been payed more attention,while its mechanism is still not clear, which need further research.
Animals ; Cell Differentiation ; Gene Expression Regulation ; Humans ; Osteoclasts ; cytology ; metabolism ; Osteoprotegerin ; genetics ; metabolism ; RANK Ligand ; metabolism ; Receptor Activator of Nuclear Factor-kappa B ; genetics ; metabolism

Effect of interleukin-6 receptor (IL-6R) antibody on polymethyl methacrylate (PMMA) bone cement-mediated expression of osteoprotegerin (OPG) and receptor activator of nuclear factor-kappaB ligand (RANKL) in synovial fibroblasts was investigated. Synovial tissue obtained from total knee arthroplasty was digested and cultured. Inverted microscope was employed to observe the synovial cells and immunocytochemistry (SABC method) staining was used to identify synovial fibroblasts. This experiment was divided into three groups according to different culture media: PMMA group (75 μg/mL PMMA bone cement particles), IL-6R antibody group (10 ng/mL IL-6R antibody+75 μg/mL PMMA bone cement particles), and control group (no IL-6R antibody or PMMA bone cement particles). Influence of IL-6R antibody and PMMA on proliferation of synovial fibroblasts was measured by cell counting kit-8 (CCK-8). ELISA method was used to measure OPG and RANKL levels in culture solution. Fluorescence quantitative real-time PCR (FQ-PCR) was used to detect the expression of OPG and RANKL mRNA. After three consecutive passages, more than 95% of the primary synovial cells became long spindle fibroblast-like cells. SABC staining results showed that the fibroblast-like cells were negative for anti-CD68 antibody and positive for anti-vimentin antibody, with brown madder stained. CCK-8 test demonstrated that the absorbance (A) value at 450 nm was significantly lower in IL-6R antibody group than in PMMA group and control group (P<0.01), but there was no statistically significant difference in A value at 450 nm between the control group and PMMA group (P>0.05). Results of ELISA indicated that the expression of OPG was significantly higher in IL-6R antibody group than in PMMA group and control group (P<0.01). The expression of RANKL was inhibited (P<0.05), and the ratio of OPG/RANKL was significantly increased in IL-6R antibody group as compared with PMMA group and control group. There was no significant difference in the expression of OPG between control group and PMMA group (P>0.05), but the expression of RANKL was higher in PMMA group than in control group (P<0.05), and there was a significant difference in the ratio of OPG/RANKL between them (P<0.05). Results of FQ-PCR revealed the expression of RANKL mRNA was significantly inhibited (P<0.01) and the expression of OPG mRNA was significantly increased (P<0.01) in IL-6R antibody group as compared with PMMA group and control group. The expression of RANKL mRNA was higher in PMMA group than in control group (P<0.05), but the expression of OPG mRNA had no significant difference between them (P>0.05). IL-6R antibody could significantly increase the expression of OPG, but inhibit the expression of RANKL, which might provide a theoretical basis of molecular biology for the prevention and treatment of aseptic loosening of prosthesis.
Antibodies ; administration & dosage ; immunology ; Bone Cements ; Fibroblasts ; immunology ; Gene Expression ; drug effects ; Humans ; Osteoprotegerin ; biosynthesis ; genetics ; Polymethyl Methacrylate ; administration & dosage ; Prostheses and Implants ; RANK Ligand ; biosynthesis ; genetics ; metabolism ; Receptors, Interleukin-6 ; immunology ; metabolism ; Synovial Fluid ; immunology ; metabolism

OBJECTIVETo study the effect of Erigeron Breviscapus (EB) at different concentrations and different intervention time points on the mRNA and protein expression of OPG/RANKL/RANK in MG63 osteoblast-like cells and RAW264. 7 pre-osteoclast cells cultured in vitro, thus exploring roles EB played in bone rebuilding and its mechanisms.

METHODSMG63 osteoblast-like cells and RAW264.7 pre-osteoclast cells were cultured in vitro. The 3rd passage cells were divided into the control group and different experimental groups. Total RNA and protein were respectively isolated from cells treated with different concentrations of EB (0, 0.001, 0.01, 0.1, and 1.0 mg/mL) for 48 h. Meanwhile, the protein was extracted from 0 and 1 mg/mL EB groups at 12, 24, and 48 h respectively. Expression of OPG mRNA and RANKL mRNA in MG63 osteoblast-like cells, and expression of RANK mRNA in RAW264.7 pre-osteoclast cells were detected by semi-quantitative RT-PCR. Expression of OPG protein and RANKL protein in MG63 osteoblast-like cells, and expression of RANK protein in RAW264. 7 pre-osteoclast cells were detected by Western blot.

RESULTSAlong with increased EB concentration, expression of OPG mRNA and protein in MG63 osteoblast-like cells was gradually lowered (P < 0.05) after 48-h intervention of EB, the expression of RANKL mRNA and protein in MG63 osteoblast-like gradually increased (P < 0.05); the expression of RANK mRNA in RAW264.7 pre-osteoclast cells increased (P < 0.05). But the expression of RANK mRNA was slightly lower in the 0.1 mg/mL EB group than in the 0.01 mg/mL EB group, and the expression of RANK protein in RAW264.7 pre-osteoclast cells gradually increased (P < 0.05). After treatment with 1 mg/mL EB for 12, 24, 48 h, the expression of OPG protein in MG63 osteoblast-like cells gradually decreased as time went by (P < 0.05), and the expression of RANKL protein in MG63 osteoblast-like and RANK protein in RAW264.7 pre-osteoclast cells gradually increased (P < 0.05). The expression of RANKL protein in RAW264.7 pre-osteoclast cells increased as time went by (P < 0.05).

CONCLUSIONEB could inhibit the expression of OPG in osteoblasts in a dose- and time-dependent manner, promote the expression of RANKL in osteoblasts and the secretion of RANK in pre-osteoclast, indicating EB might play roles in promoting bone resorption.

Animals ; Cell Differentiation ; Cell Line ; Drugs, Chinese Herbal ; pharmacology ; Erigeron ; Humans ; Mice ; Osteoblasts ; drug effects ; metabolism ; Osteoclasts ; drug effects ; metabolism ; Osteoprotegerin ; metabolism ; RANK Ligand ; metabolism ; RNA, Messenger ; genetics ; Receptor Activator of Nuclear Factor-kappa B ; metabolism

The present research was aimed to investigate the effect of 50Hz, 1. 8mT of sinusoidal electromagnetic fields(SEMFs)on femur tissue cultivation in vitro. The rat femur tissue was isolated from SD rats by method of enzyme digestion, and randomly divided into two groups: SEMFs group and control. The femur tissue of SEMFs groups were exposured under 50Hz 1. 8mT of SEMFs for 1. 5h/time/d, but those in the control groups were without SEMFs treatment. The correlative gene was detected by the Real-time RT-PCR that after SEMFs treatment for 0 (first times treatments is 0 days), 1, 2, 3, 4 and 5d. The alkaline phosphatase (ALP) activity was measured after SEMFs treatment for 3, 6, 9 and 12d respectively. The calcium content was detected after SEMFs treatment for 3, 6, 9 and 12d. The results showed that the OPG and Collagen-1 mRNA expression level was kept at a relatively stable level by SEMFs in the SEMFs group significantly. The Runx-2 mRNA expression level was significantly increased after the SEMFs treatment for 1d and 5d. The ALP activity of femur tissue was significantly increased alter SEMFs treatment for 3d and 9d. The calcium content was higher than untreated groups after SEMFs treatment for 6, 9 and 12d. The SEMFs promoted OPG, Collagen-1, Runx-2 mRNA expression level, ALP activity and calcium content. The result indicated that SEMFs increased the metabolism activity of femur tissues.
Alkaline Phosphatase ; metabolism ; Animals ; Calcium ; metabolism ; Collagen Type I ; genetics ; metabolism ; Core Binding Factor Alpha 1 Subunit ; genetics ; metabolism ; Electromagnetic Fields ; Female ; Femur ; growth & development ; Male ; Osteoprotegerin ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Tissue Culture Techniques ; methods

This study is to investigate effects of genistein on rat femoral bone metabolic in vitro. Rat femoral tissues was isolated and randomly divided into two groups including control group and genistein (1 x 10(-5) mol x(-1)) group. Determinations of alkaline phosphatase (ALP) activity, calcium content and osteoprotegerin (OPG), type I-collagen (Collagen-I), RANKL, Runx-2 and bone morphogenetic protein (BMP-2) mRNA expression were done by real-time PCR. The results showed that 1 x 10(-5) mol x L(-1) genistein could increase the activity of ALP and contents of Ca, regulate bone metabolism activity of OPG, RANKL, BMP-2, Collagen-I and Runx-2 mRNA expression level. Genistein can significantly modulate bone metabolism related gene expression level of rat femoral tissue in vitro, and can increase calcium content and the activity of ALP.
Alkaline Phosphatase ; metabolism ; Animals ; Bone Morphogenetic Protein 2 ; genetics ; metabolism ; Calcium ; metabolism ; Collagen Type I ; genetics ; metabolism ; Core Binding Factor Alpha 1 Subunit ; genetics ; metabolism ; Enzyme Activation ; drug effects ; Femur ; metabolism ; Gene Expression Regulation ; Genistein ; pharmacology ; Osteoprotegerin ; genetics ; metabolism ; Phytoestrogens ; pharmacology ; RANK Ligand ; genetics ; metabolism ; RNA, Messenger ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Real-Time Polymerase Chain Reaction

OBJECTIVETo observe the effect of treadmill exercise on the mRNA expression of osteoprotegerin, RANKL and RUNX2 in bone tissue of ovariectomized rat,and to investigate the mechanism of treadmill exercise for the preventing and treating postmenopausal osteoporosis.

METHODSThirty healthy adult SD rats which average weight was (270 +/- 10) g were randomly divided into 3 groups: sham-operation group, ovariectomized group and treadmill exercise group. Each group had 10 rats. After anesthesia, ovariectomized group and treadmill exercise treated group were operated by bilateral ovarian resection, sham group by sham operation. Sham-operation group and ovarian rats were normal breed. Rats in the exercise group were treated by running on treadmill for animal at 1 week after the operation. Running speed was 18 meters per minutes lasting 45 minutes in one day, and worked six days per week for 11 weeks. All rats were killed after 12 weeks. After decalcification, left femur head was made to paraffin slice and observed by inverted phase contrast microscope for histological examination. Total RNA were extracted from the right femur heads and the mRNA expression of OPG,RANKL and RUNX2 were examined by real time PCR.

RESULTSThe femur heads of ovariectomized group showed thin trabecular bone and less bone cells, meanwhile trabecular bone of exercise group looked thicker in histological examination. The mRNA expression of OPG (0.131 +/- 0.080), RANKL (8.013 +/- 3.550) and RUNX2 (3.245 +/- 5.090) was seen in the ovariectomized group. Meanwhile different results were found as the mRNA expression of OPG (0.566 +/- 0.260), RANKL (5.232 +/- 3.670) and RUNX2 (2.753 +/- 3.680) in the group of treadmill exercise. In compared with ovariectomized group, the mRNA expression of OPG in treadmill exercise increased and the result had statistical significance,whereas mRNA expression of RNAKL and RUNX2 decreased but the results had no statistical significance.

CONCLUSIONThe effect of exercise treating postmenopausal osteoporosis is tightly correlated with the up-regulation of expression of OPG. This provides a new train of thought of postmenopausal osteoporosis treatment for the future study.

Adult ; Animals ; Bone Density ; Core Binding Factor Alpha 1 Subunit ; genetics ; metabolism ; Exercise Test ; Exercise Therapy ; Female ; Femur ; metabolism ; Humans ; Osteoporosis, Postmenopausal ; genetics ; metabolism ; physiopathology ; therapy ; Osteoprotegerin ; genetics ; metabolism ; Ovariectomy ; RANK Ligand ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley