Persistent generalized low bone mineral density (BMD) has been reported in patients with adolescent idiopathic scoliosis (AIS). However, the exact mechanisms and causes of the low BMD in AIS patients are largely unknown. The purpose of this study was to examine the relationship between the receptor activator of NF-κB ligand (RANKL)/osteoprotegerin (OPG) levels in osteoblasts (OBs) from AIS patients with low BMD and with comparison made between the patients and controls. Twenty AIS patients and eight age-matched controls were included in the present study. The BMD of lumbar spine and proximal femur was measured in all subjects. OBs from the cancellous bone of each subject was harvested and primarily cultured. The mRNA and protein expression of RANKL and OPG in OBs was detected by RT-PCR and Western blotting. The results showed BMD was lower in AIS patients than in controls. A significantly higher mRNA and protein expression of RANKL was observed in OBs from AIS patients, while no significant difference was found in the expression of OPG between AIS patients and controls. As a result, RANKL/OPG ratio in patients with AIS was remarkably higher than controls. Our study preliminarily demonstrated expression of RANKL was higher in OBs from AIS patients with low BMD as compared with controls, suggesting the unbalanced RANKL/OPG ratio caused by an over-expression of RANKL in OBs may be responsible for the low BMD in AIS patients.
Adolescent ; Bone Density ; genetics ; Child ; Humans ; Osteoblasts ; metabolism ; RANK Ligand ; genetics ; Receptor Activator of Nuclear Factor-kappa B ; genetics ; metabolism ; Scoliosis ; genetics ; Young Adult

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

OBJECTIVE: To determine the effect of continuously compressive pressure (CCP) on the expression of receptor activator of nuclear factor kappa B ligand (RANKL) in human periodontal ligament cells (HPDLCs) and to investigate the role of RANKL in alveolar bone rebuilding during orthodontic tooth movement. METHODS: The primary HPDLCs were isolated from human periodontal ligament by explanting enzymatic digestion with trypsin and collagenase to establish a pressure model. Top-bottom axial pressures (1, 2, and 3 g/cm(2)) were laid on HPDLCs for 0.5, 1.5, 6, 12, 24, and 48 h, respectively. The RANKL expression was identified by the reverse transcription-polymerase chain reaction (RT-PCR) at the mRNA level. RESULTS: The expression of RANKL mRNA significantly increased in a time-dependent manner (P<0.01), so did the value of pressure, especially in the 2 g/cm(2) group (P<0.05). CONCLUSION CCP can up-regulate the expression of RANKL mRNA in human periodontal ligament cells.
Compressive Strength ; Humans ; Periodontal Ligament ; cytology ; metabolism ; RANK Ligand ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Receptor Activator of Nuclear Factor-kappa B ; genetics ; metabolism ; Stress, Mechanical

<b>OBJECTIVEb>To investigate the gene expression of osteoprotegerin (OPG) and osteoclast differentiation factor (ODF/TRANCE/RANKL), two new members of the TNF-receptor superfamily, in giant cell tumor (GCT); to discuss the molecular mechanism of extensive bone resorption caused by GCT.

<b>METHODSb>Using TRIzol reagent to prepare total RNA from GCT sample and normal bone tissue. By a first-strand complementary DNA (cDNA) synthesis kit, cDNA was synthesized from 2.0 micro g RNA according to the manufacturer's instructions. cDNA was then amplified by PCR. Amplification products were resolved by electrophoresis on a 1.5% agarose gel and stained with EB. The relative quantity of the PCR products were determined and the mRNA levels of OPG, ODF, M-CSF (cofactor of ODF), and RANK (receptor of ODF) were compared with that of the normal bone.

<b>RESULTSb>GCT contained highly expressed mRNA of ODF, OPG, M-CSF and RANK. There was mRNA expression of OPG, M-CSF and RANK and less expression of ODF in normal bone. The ODF mRNA and RANK mRNA in GCT were more abundant than that in normal bone. In GCT, the ratio of ODF mRNA exceeded OPG expression. But in normal bone, the OPG mRNA exceeded ODF expression.

<b>CONCLUSIONSb>The results suggest that GCT contains all signals including OPG, ODF, M-CSF and RANK that are essential for inducing osteoclastogenesis and promoting bone resorption.

Carrier Proteins ; genetics ; Gene Expression Regulation, Neoplastic ; Giant Cell Tumor of Bone ; genetics ; pathology ; Glycoproteins ; genetics ; Humans ; Membrane Glycoproteins ; genetics ; Osteoprotegerin ; RANK Ligand ; RNA, Messenger ; genetics ; metabolism ; Receptor Activator of Nuclear Factor-kappa B ; Receptors, Cytoplasmic and Nuclear ; genetics ; Receptors, Tumor Necrosis Factor

<b>OBJECTIVEb>To investigate the expression of receptor activator of nuclear factor-&kappa;B (RANK), its ligand RANKL, and osteoprotegerin, and observe the effects of αD3 on their expressions in male rats at different ages.

<b>METHODSb>Wistar rats at 6 weeks, 6 months, and 24 months (n=15) were examined for mRNA expressions of RANK/RANKL and osteoprotegerin in the left proximal femur using RT-PCR and for their protein expressions in the right femur using immunohistochemistry. RANK/RANKL and osteoprotegerin expressions were also detected in another 15 rats aged 24 months following intragastric administration of 0.05 µg/kg αD3 (3 times a week for 10 weeks).

<b>RESULTSb>Compared with 6-week-old rats, 6-month- and 24-month-old rats showed a 6.2-fold and 7.3-fold increase of RANKL mRNA expression, respectively (P<0.05), and osteoprotegerin mRNA levels increased slightly with age. αD3 treatment resulted in significantly increased expression of RANK in 24-month-old rats with a lowered RANKL/osteoprotegerin ratio. RANKL and osteoprotegerin were co-localized in the osteoblasts and chondrocytes. αD3 treatment also caused an increased expression of osteoprotegerin mRNA in 24-month-old rats.

<b>CONCLUSIONb>The age-related increase of the ratio of RANKL/osteoprotegerin mRNA promotes osteoclast activity and bone turnover. αD3 has favorable effect on osteogenesis and suppress bone absorption in the femur possibly by reducing RANK expression and lowering RANKL/osteoprotegerin ratio.

Age Factors ; Animals ; Chondrocytes ; metabolism ; Femur ; metabolism ; Male ; Osteoblasts ; metabolism ; Osteoprotegerin ; genetics ; metabolism ; RANK Ligand ; genetics ; metabolism ; RNA, Messenger ; genetics ; Rats ; Rats, Wistar ; Receptor Activator of Nuclear Factor-kappa B ; genetics ; metabolism

<b>OBJECTIVEb>To study the effects of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on the expression of osteoprotegerin (OPG) and receptor activator of NF-kappaB ligand (RANKL) mRNA in mouse osteoblasts.

<b>METHODSb>Calvariae derived from CD-1 neonatal mouse (after born 24 h). Bone samples were processed by the collagenase/trypsin digestion method. Mouse osteoblasts were cultured in vitro. After 48 hours of addition of 1,25(OH)2D3 (0, 10(-8), 10(-9), 10(-11) mol/L) to the culture medium of mouse osteoblasts, the content of the OPG protein in culture medium was estimated with enzyme linked immunosorbent assay. Total RNA was prepared from mouse osteoblasts. mRNA expression of OPG and RANKL were detected by reverse transcription-polymerase chain reaction.

<b>RESULTSb>The mRNA expression of OPG in osteoblasts added with 1,25(OH)2D3 significantly decreased compared with the controls, which was markedly dose-dependent. OPG protein production in the medium decreased after treatment with 1,25(OH)2D3. In contrast, RANKL mRNA expression levels in osteoblasts significantly increased after 48 h of culture with 1,25(OH)2D3.

<b>CONCLUSIONb>1,25 (OH)2D3 can stimulate RANKL mRNA expression, but decrease OPG mRNA levels in vitro in mouse osteoblasts.

Animals ; Animals, Newborn ; Calcitriol ; pharmacology ; Carrier Proteins ; biosynthesis ; genetics ; Glycoproteins ; biosynthesis ; genetics ; physiology ; Ligands ; Membrane Glycoproteins ; biosynthesis ; genetics ; Mice ; NF-kappa B ; biosynthesis ; genetics ; Osteoclasts ; metabolism ; physiology ; Osteoprotegerin ; RANK Ligand ; RNA, Messenger ; biosynthesis ; genetics ; Receptor Activator of Nuclear Factor-kappa B ; Receptors, Cytoplasmic and Nuclear ; analysis ; biosynthesis ; genetics ; physiology ; Receptors, Tumor Necrosis Factor ; biosynthesis ; genetics ; Tumor Necrosis Factor-alpha ; biosynthesis ; genetics

<b>OBJECTIVEb>To 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.

<b>METHODSb>MG63 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.

<b>RESULTSb>Along 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).

<b>CONCLUSIONb>EB 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 OPG/RANKL/RANK system plays an important role in osteoclastogenesis and represents a great progress in bone biology. RANKL, which expresses on the surface of osteoblast/stromal cells and activated T cells, binds to RANK on the osteoclastic precursors or mature osteoclasts, and promotes osteoclastogenesis and bone resorption. While osteoprotegerin (OPG), which is expressed by osteoblasts/stromal cells, strongly inhibits bone resorption by binding to its ligand RANKL and thereby blocks the interaction between BANKL and RANK. A number of cytokines and hormones exert their effects on bone metabolism by regulating the OPG/RANKL ratio in the bone marrow microenvironment. RANK is also expressed on mammary epithelial cells and RANKL expression in these cells is induced by pregnancy hormones, RANKL and RANK are essential for the formation of the lactating mammary gland and the transmission of maternal calcium to neonates in mammalian species. Modulation of these systems provides a unique opportunity to develop novel therapeutics to inhibit bone loss in osteoporosis, rheumatoid arthritis, and bone metastasis of cancer. Further research should be focused on the cooperation of OPG/RANKL/RANK system with other signal pathways and the interactions among bone remodeling, immune system and endocrinology system. Currently, the development of OPG analogues or compounds which may stimulate OPG expression is becoming an attractive industry which may be profitable to both patients and manufacturers.
Animals ; Bone Resorption ; immunology ; metabolism ; Humans ; Osteoclasts ; cytology ; metabolism ; pathology ; Osteogenesis ; drug effects ; genetics ; immunology ; Osteoprotegerin ; metabolism ; physiology ; RANK Ligand ; metabolism ; physiology ; Receptor Activator of Nuclear Factor-kappa B ; metabolism ; pharmacology ; physiology ; T-Lymphocytes ; drug effects ; immunology

The purpose of this study was to determine whether osteoprotegerin (OPG) could affect osteoclat differentiation and activation under serum-free conditions. Both duck embryo bone marrow cells and RAW264.7 cells were incubated with macrophage colony stimulatory factor (M-CSF) and receptor activator for nuclear factor kappaB ligand (RANKL) in serum-free medium to promote osteoclastogenesis. During cultivation, 0, 10, 20, 50, and 100 ng/mL OPG were added to various groups of cells. Osteoclast differentiation and activation were monitored via tartrate-resistant acid phosphatase (TRAP) staining, filamentous-actin rings analysis, and a bone resorption assay. Furthermore, the expression osteoclast-related genes, such as TRAP and receptor activator for nuclear factor kappaB (RANK), that was influenced by OPG in RAW264.7 cells was examined using real-time polymerase chain reaction. In summary, findings from the present study suggested that M-CSF with RANKL can promote osteoclast differentiation and activation, and enhance the expression of TRAP and RANK mRNA in osteoclasts. In contrast, OPG inhibited these activities under serum-free conditions.
Acid Phosphatase/genetics/metabolism ; Animals ; Avian Proteins/*pharmacology ; Bone Marrow Cells/drug effects/*metabolism ; Cells, Cultured ; Ducks ; Embryo, Nonmammalian/drug effects/metabolism ; Isoenzymes/genetics/metabolism ; Macrophage Colony-Stimulating Factor/metabolism ; Osteoclasts/cytology/*drug effects/*metabolism ; Osteoprotegerin/*pharmacology ; RANK Ligand/metabolism ; Real-Time Polymerase Chain Reaction ; Receptor Activator of Nuclear Factor-kappa B/genetics/metabolism

<b>OBJECTIVEb>To study the mechanism of Huogu I formula (I) in treating osteonecrosis of femoral head.

<b>METHODSb>Forty-eight healthy female Leghorn chickens were randomly divided into control group, model group and Huogu I group, and each group consisted of 16 chickens. At the meantime of model establishment, chickens of the Huogu I group were administrated with decoction, while the model and control group with distilled water by gavage. At the 8th and 16th week after medication, blood samples were obtained for blood lipid detection while both sides of femoral head were harvested for the rest of examinations. Specifically, expressions of bone morphogenetic protein-2 (BMP2), transforming growth factor beta1 (TGFβ(1)), Smad4 and Smad7 were evaluated by immunohistochemistry, while expression of osteoprotegerin/receptor activator of nuclear factor kappaB ligand (OPG/RANKL) mRNA was detected by in situ hybridization.

<b>RESULTSb>Compared with the control group, serum levels of total cholesterol (TC), triglyceride (TG) and low-density lipoprotein cholesterol (LDL-C) in the model group rose significantly. Positive cell counting of BMP2, TGFβ(1), Smad4 and OPG in femoral head of the model group dropped prominently. Positive cell counting of Smad7 and RANKL increased dramatically. In contrast with the model group, levels of TC, TG and LDL-C in Huogu I group reduced significantly. Positive cell counting of BMP2, TGFβ(1), Smad4 and OPG in femoral head of the Huogu I group increased prominently. Indices of Smad7 and RANKL both decreased significantly. Especially at the 8th week, these variations were more significant.

<b>CONCLUSIONb>Huogu I formula is effective in promoting repair of necrotic femoral head by regulating the expressions of BMP2, TGFβ(1), Smads and OPG/RANKL of osteoclast in femoral head.

Animals ; Bone Morphogenetic Protein 2 ; metabolism ; Bone Regeneration ; drug effects ; physiology ; Chickens ; Chondrocytes ; metabolism ; Disease Models, Animal ; Drugs, Chinese Herbal ; pharmacology ; Female ; Femur Head Necrosis ; chemically induced ; drug therapy ; metabolism ; Lipid Metabolism ; physiology ; Osteocytes ; metabolism ; Osteoprotegerin ; genetics ; metabolism ; Receptor Activator of Nuclear Factor-kappa B ; genetics ; metabolism ; Smad4 Protein ; metabolism ; Smad7 Protein ; metabolism ; Steroids ; pharmacology ; Transforming Growth Factor beta1 ; metabolism