RANKL synthesized by articular chondrocytes contributes to juxta-articular bone loss in chronic arthritis

Introduction

The receptor activator nuclear factor-kappaB ligand (RANKL) diffuses from articular cartilage to subchondral bone. However, the role of chondrocyte-synthesized RANKL in rheumatoid arthritis-associated juxta-articular bone loss has not yet been explored. This study aimed to determine whether RANKL produced by chondrocytes induces osteoclastogenesis and juxta-articular bone loss associated with chronic arthritis.

Methods

Chronic antigen-induced arthritis (AIA) was induced in New Zealand (NZ) rabbits. Osteoarthritis (OA) and control groups were simultaneously studied. Dual X-ray absorptiometry of subchondral knee bone was performed before sacrifice. Histological analysis and protein expression of RANKL and osteoprotegerin (OPG) were evaluated in joint tissues. Co-cultures of human OA articular chondrocytes with peripheral blood mononuclear cells (PBMCs) from healthy donors were stimulated with macrophage-colony stimulating factor (M-CSF) and prostaglandin E₂ (PGE₂), then further stained with tartrate-resistant acid phosphatase.

Results

Subchondral bone loss was confirmed in AIA rabbits when compared with controls. The expression of RANKL, OPG and RANKL/OPG ratio in cartilage were increased in AIA compared to control animals, although this pattern was not seen in synovium. Furthermore, RANKL expression and RANKL/OPG ratio were inversely related to subchondral bone mineral density. RANKL expression was observed throughout all cartilage zones of rabbits and was specially increased in the calcified cartilage of AIA animals. Co-cultures demonstrated that PGE₂-stimulated human chondrocytes, which produce RANKL, also induce osteoclasts differentiation from PBMCs.

Conclusions

Chondrocyte-synthesized RANKL may contribute to the development of juxta-articular osteoporosis associated with chronic arthritis, by enhancing osteoclastogenesis. These results point out a new mechanism of bone loss in patients with rheumatoid arthritis.     

Disturbance of the OPG/RANK/RANKL pathway and systemic inflammation in COPD patients with emphysema and osteoporosis

Background

Osteoporosis is one of the systemic features of COPD. A correlation between the emphysema phenotype of COPD and reduced bone mineral density (BMD) is suggested by some studies, however, the mechanisms underlying this relationship are unclear. Experimental studies indicate that IL-1β, IL-6 and TNF-α may play important roles in the etiology of both osteoporosis and emphysema. The OPG/RANK/RANKL system is an important regulator of bone metabolism, and participates in the development of post-menopausal osteoporosis. Whether the OPG/RANK/RANKL pathway is involved in the pathogenesis of osteoporosis in COPD has not been studied.

Methods

Eighty male patients (current or former smokers) completed a chest CT scan, pulmonary function test, dual x-ray absorptiometry measurements and questionnaires. Among these subjects, thirty patients with normal BMD and thirty patients with low BMD were selected randomly for measurement of IL-1β, IL-6, TNF-α (flow cytometry) and OPG/RANK/RANKL (ELISA). Twenty age-matched healthy volunteers were recruited as controls.

Results

Among these eighty patients, thirty-six had normal BMD and forty-four had low BMD. Age, BMI and CAT score showed significant differences between these two COPD groups (p < 0.05). The low-attenuation area (LAA%) in the lungs of COPD patients was negatively correlated with lumbar vertebral BMD (r = 0.741; p < 0.0001). Forward logistic regression analysis showed that only LAA% (p = 0.005) and BMI (p = 0.009) were selected as explanatory variables. The level of IL-1β was significantly higher in the COPD patients as compared to the normal controls (p < 0.05), but the difference between the two COPD groups did not reach significance. The levels of IL-6 and TNF-α among the three groups were significantly different (p < 0.05). The level of RANKL and the RANKL/OPG ratio were significantly higher in COPD patients with low BMD compared to those with normal BMD and the normal controls (p < 0.05), and correlated negatively with lumbar vertebral BMD, but positively with LAA%.

Conclusions

Radiographic emphysema is correlated with low BMD in current and former smokers with COPD. IL-1β, IL-6, TNF-α, and the osteoporosis-related protein system OPG/RANK/RANKL may have some synergetic effects on emphysema and bone loss in COPD.     

cAMP/PKA regulates osteogenesis, adipogenesis and ratio of RANKL/OPG mRNA expression in mesenchymal stem cells by suppressing leptin

Background

Mesenchymal stem cells (MSCs) are a pluripotent cell type that can differentiate into adipocytes, osteoblasts and other cells. The reciprocal relationship between adipogenesis and osteogenesis was previously demonstrated; however, the mechanisms remain largely unknown.

Methods and Findings

We report that activation of PKA by 3-isobutyl-1 methyl xanthine (IBMX) and forskolin enhances adipogenesis, the gene expression of PPARγ2 and LPL, and downregulates the gene expression of Runx2 and osteopontin, markers of osteogenesis. PKA activation also decreases the ratio of Receptor Activator of the NF-κB Ligand to Osteoprotegerin (RANKL/OPG) gene expression – the key factors of osteoclastogenesis. All these effects are mediated by the cAMP/PKA/CREB pathway by suppressing leptin, and may contribute to PKA stimulators-induced in vivo bone loss in developing zebrafish.

Conclusions

Using MSCs, the center of a newly proposed bone metabolic unit, we identified cAMP/PKA signaling, one of the many signaling pathways that regulate bone homeostasis via controlling cyto-differentiation of MSCs and altering RANKL/OPG gene expression.     

RANKL/OPG in primary cultures of osteoblasts from post-menopausal women. Differences between osteoporotic hip fractures and osteoarthritis

The OPG/RANKL/RANK system is important in the balance between bone formation and resorption.We used primary human osteoblasts (hOBs) cells to examine the impact of 17-β-estradiol (E2) or/and 1,25-dihydroxyvitamin D (1,25D) in OPG/RANKL system in 28 post-menopausal (PM) women; (a) with hip fracture (OP) or (b) with osteoarthritis (OA). The hOB from OP patients proliferated slower during the first stage, than the OA women (31.5 ± 2.6 and 21.4 ± 1.3 days, respectively, p < 0.05). The OP group secreted significantly higher OPG protein levels than the OA women (10.1 ± 2.6 and 4.4 ± 0.8 pmol/L, respectively, p < 0.05). The 1,25D and 1,25D+E2 induce an increase in RANKL and RANKL/OPG mRNA expression in OP patients above 200% (p < 0.05).HOBs from the osteoporotic hip initially proliferate slower but after reaching the first cellular confluence, the proliferation rate is equal in both groups. Furthermore, hOBs from hips with OP present a higher protein secretion of OPG, and higher RANKL and RANKL/OPG expression ratio in response to 1,25D and 1,25D+E2, than hOBs from OA women. All this could suggest that the greater bone loss that characterizes OP patients can be mediated due to differences in the secretion and expression of the RANKL/OPG system in response to different stimuli.     

The OPG/RANKL/RANK system in metabolic bone diseases

The OPG/RANKL/RANK cytokine system is essential for osteoclast biology. Various studies suggest that human metabolic bone diseases are related to alterations of this system. Here we summarize OPG/RANKL/RANK abnormalities in different forms of osteoporoses and hyperparathyroidism. Skeletal estrogen agonists (including 17beta-estradiol, raloxifene, and genistein) induce osteoblastic OPG production through estrogen receptor-alpha activation in vitro, while immune cells appear to over-express RANKL in estrogen deficiency in vivo. Of note, OPG administration can prevent bone loss associated with estrogen deficiency as observed in both animal models and a small clinical study. Glucocorticoids and immunosuppressants concurrently up-regulate RANKL and suppress OPG in osteoblastic cells in vitro, and glucocorticoids are among the most powerful drugs to suppress OPG serum levels in vivo. As for mechanisms of immobilization-induced bone loss, it appears that mechanical strain inhibits RANKL production through the ERK 1/2 MAP kinase pathway and up-regulates OPG production in vitro. Hence, lack of mechanical strainduring immobilization may favor an enhanced RANKL-to-OPG ratio leading to increased bone loss. As for hyperparathyroidism, chronic PTH exposure concurrently enhances RANKL production and suppresses OPG secretion through activation of osteoblastic protein kinase A in vitro which would favour increased osteoclastic activity. In sum, the capacity for OPG to antagonize the increases in bone loss seen in many rodent models of metabolic bone disease implicates RANKL/OPG imbalances as the likely etiology and supports the potential role for a RANKL antagonist as a therapeutic intervention in these settings.     

Expression of RANKL/OPG during bone remodeling in vivo

The interaction between receptor activator of nuclear factor κB ligand (RANKL) and osteoprotegerin (OPG) plays a dominant role in osteoclastogenesis. As both proteins are produced by osteoblast lineage cells, they are considered to represent a key link between bone formation and resorption. In this study, we investigated the expression of RANKL and OPG during bone remodeling in vivo to determine the relationship between osteoclastogenic stimulation and osteoblastic differentiation.Total RNA was prepared from rat femurs after marrow ablation on days 0, 3, 6, and 9. The temporal activation patterns of osteoblast-related genes (procollagen α1 (I), alkaline phosphatase, osteopontin, and osteocalcin) were examined by Northern blot analysis. An appreciable increase in the expression of these osteoblast markers was observed on day 3. The peak increase in gene expression was observed on day 6 followed by a slight reduction by day 9. Real-time PCR analysis showed that the OPG mRNA expression was markedly upregulated on day 6 and slightly decreased on day 9. In contrast, RANKL mRNA expression was increased by more than 20-fold on day 9. The RANKL/OPG ratio, an index of osteoclastogenic stimulation, peaked on day 9. Histological analysis showed that RANKL and OPG immunoreactivity were predominantly associated with bone marrow cells. The expression of bone formation markers was activated in the bone formation phase, followed by the stimulation of RANKL/OPG expression in the bone resorption phase, which confirmed that these molecules are key factors linking bone formation to resorption during bone remodeling.     

Azilsartan Increases Levels of IL-10, Down-Regulates MMP-2, MMP-9, RANKL/RANK,Cathepsin K and Up-Regulates OPG in an Experimental Periodontitis Model

Aims

The aim of this study was to evaluate the effects of azilsartan (AZT) on bone loss, inflammation, and the expression of matrix metallo proteinases (MMPs), receptor activator of nuclear factor κB ligand (RANKL), receptor activator of nuclear factor κB (RANK), osteoprotegerin (OPG), cyclooxygenase-2 (COX-2), and cathepsin K in periodontal tissue in a rat model of ligature-induced periodontitis.

Materials and Methods

Male Wistar albino rats were randomly divided into 5 groups of 10 rats each: (1) nonligated, water; (2) ligated, water; (3) ligated, 1 mg/kg AZT; (4) ligated, 5 mg/kg AZT; and (5) ligated, 10 mg/kg AZT. All groups were treated with saline or AZT for 10 days. Periodontal tissues were analyzed by histopathology and immunohistochemical detection of MMP-2, MMP-9, COX-2, RANKL, RANK, OPG, and cathepsin K. Levels of IL-1β, IL-10, TNF-α, myeloperoxidase (MPO), and glutathione (GSH) were determined by ELISA.

Results

Treatment with 5 mg/kg AZT resulted in reduced MPO (p<0.05) and IL-1β (p<0.05), increased levels of IL-10 (p<0.05), and reduced expression of MMP-2, MMP-9, COX-2, RANK, RANKL, cathepsin K, and increased expression of OPG.

Conclusions

These findings reveal that AZT increases anti-inflammatory cytokines and GSH and decreases bone loss in ligature-induced periodontitis in rats.     

Direct effects of osteoprotegerin on human bone cell metabolism

Purpose

Osteoprotegerin (OPG) affects bone metabolism by intercepting the RANK-RANKL interaction which prevents osteoclastic differentiation and consequently reduces bone resorption. Different bone phenotypes of mice overexpressing OPG and of mice with knockdown of receptor activator of NF-κB (RANK) or RANK-ligand (RANKL) suggest that the mechanism of action of the OPG-RANKL-RANK system in regulating bone remodeling is not completely understood. Furthermore, OPG increases bone mass and density independently from reduced osteoclastogenesis which is consistent with the possibility that OPG may directly affect bone metabolism beyond its known role as decoy receptor for RANKL.

Methods

We treated primary human osteoblastic cells with OPG and inhibitory anti-RANKL antibodies and measured cellular ALP activity, in vitro mineralization, vitronectin receptor protein expression and ERK phosphorylation. We also analyzed the mRNA co-expression of ALP and OPG ex vivo in bone biopsies from acute and old stable vertebral fractures.

Results

OPG directly increased ALP activity and in vitro mineralization of HOC, enhanced expression of the vitronectin receptor thereby increasing adherence of HOC to vitronectin and stimulated ERK phosphorylation. All OPG-mediated effects could be prevented by RANKL antibodies or RANKL-siRNA transfection and MAPK inhibitor PD98059 reduced the stimulatory effect of OPG on integrin αv expression. In acutely fractured vertebrae OPG and ALP mRNA expression was significantly increased compared to stable vertebral fractures. In conclusion, OPG exerts direct osteoanabolic effects on HOC metabolism via RANKL in addition to its well described role as decoy receptor for RANKL.     

Receptor activator of NF-kB (RANK) expression in primary tumors associates with bone metastasis occurrence in breast cancer patients

Background

Receptor activator of NFkB (RANK), its ligand (RANKL) and the decoy receptor of RANKL (osteoprotegerin, OPG) play a pivotal role in bone remodeling by regulating osteoclasts formation and activity. RANKL stimulates migration of RANK-expressing tumor cells in vitro, conversely inhibited by OPG.

Materials and Methods

We examined mRNA expression levels of RANKL/RANK/OPG in a publicly available microarray dataset of 295 primary breast cancer patients. We next analyzed RANK expression by immunohistochemistry in an independent series of 93 primary breast cancer specimens and investigated a possible association with clinicopathological parameters, bone recurrence and survival.

Results

Microarray analysis showed that lower RANK and high OPG mRNA levels correlate with longer overall survival (P = 0.0078 and 0.0335, respectively) and disease-free survival (P = 0.059 and 0.0402, respectively). Immunohistochemical analysis of RANK showed a positive correlation with the development of bone metastases (P = 0.023) and a shorter skeletal disease-free survival (SDFS, P = 0.037). Specifically, univariate analysis of survival showed that “RANK-negative” and “RANK-positive” patients had a SDFS of 105.7 months (95% CI: 73.9–124.4) and 58.9 months (95% CI: 34.7–68.5), respectively. RANK protein expression was also associated with accelerated bone metastasis formation in a multivariate analysis (P = 0.029).

Conclusions

This is the first demonstration of the role of RANK expression in primary tumors as a predictive marker of bone metastasis occurrence and SDFS in a large population of breast cancer patients.     

巴戟天及其糖提取物对于体外培养成骨细胞OPG/RANKL基因系统表达的影响

目的:探讨巴戟天及多糖提取物对成骨细胞骨保护素(OPG)/核因子κB受体活化因子配体(RANKL)基因系统表达的影响。方法:取2~3天的SD大鼠5只分离原代成骨细胞,再取8周龄SD大鼠35只随机分为七组,对照组不进行处理,三组给予10 g/L、50 g/L、100 g/L巴戟天水灌胃,其余三组分别给予10 g/L、50 g/L、100 g/L巴戟天多糖灌胃,72 h后采用采用ELISA法测定培养液中OPG、RANKL及骨钙素的含量,采用MTT法检测不同浓度巴戟天水及多糖提取物对大鼠成骨细胞增殖的影响,采用荧光定量PCR检测OPG和RANKL mRNA表达情况;通过Westernblot检测OPG和RANKL蛋白表达水平。结果:巴戟天水及多糖提取物组A570nm、ALP活性、骨钙素含量、OPG/RANKL mRNA表达量、OPG和RANKL蛋白表达阳性密度均高于对照组(P0.05);A 570 nm、ALP活性、骨钙素含量、OPG/RANKL mRNA表达量、OPG和RANKL蛋白表达阳性密度均高于同等剂量的水提取物各组(P0.05);巴戟天多糖组中随着多糖剂量的升高A 570 nm、ALP活性、骨钙素含量、OPG/RANKL mRNA表达量、OPG和RANKL蛋白表达阳性密度,差异比较有统计学意义(P0.05)。结论:巴戟天水及多糖提取物均能促进体外培养成骨细胞的增殖,提高成骨细胞活性。     

Functions of RANKL/RANK/OPG in bone modeling and remodeling

The discovery of the RANKL/RANK/OPG system in the mid 1990s for the regulation of bone resorption has led to major advances in our understanding of how bone modeling and remodeling are regulated. It had been known for many years before this discovery that osteoblastic stromal cells regulated osteoclast formation, but it had not been anticipated that they would do this through expression of members of the TNF superfamily: receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG), or that these cytokines and signaling through receptor activator of NF-κB (RANK) would have extensive functions beyond regulation of bone remodeling. RANKL/RANK signaling regulates osteoclast formation, activation and survival in normal bone modeling and remodeling and in a variety of pathologic conditions characterized by increased bone turnover. OPG protects bone from excessive resorption by binding to RANKL and preventing it from binding to RANK. Thus, the relative concentration of RANKL and OPG in bone is a major determinant of bone mass and strength. Here, we review our current understanding of the role of the RANKL/RANK/OPG system in bone modeling and remodeling.     

Detection of osteoprotegerin (OPG) and its ligand (RANKL) mRNA and protein in femur and tibia of the rat

Osteoprotegerin (OPG) and the receptor activator of nuclear factor (NF)-kB ligand (RANKL) are key regulators of osteoclastogenesis. The present study had the main aim of showing the localization of OPG and RANKL mRNA and protein in serial sections of the rat femurs and tibiae by immunohistochemistry (IHC) and in situ hybridization (ISH). The main results were: (1) OPG and RANKL mRNA and protein were co-localized in the same cell types, (2) maturative/hypertrophic chondrocytes, osteoblasts, lining cells, periosteal cells and early osteocytes were stained by both IHC and ISH, (3) OPG and RANKL proteins were mainly located in Golgi areas, and the ISH reaction was especially visible in active osteoblasts, (4) immunolabeling was often concentrated into cytoplasmic vacuoles of otherwise negative proliferative chondrocytes; IHC and ISH labeling increased from proliferative to maturative/hypertrophic chondrocytes, (5) the newly laid down bone matrix, cartilage-bone interfaces, cement lines, and trabecular borders showed light OPG and RANKL immunolabeling, (6) about 70% of secondary metaphyseal bone osteocytes showed OPG and RANKL protein expression; most of them were ISH-negative, (7) osteoclasts were mostly unstained by IHC and variably labeled by ISH. The co-expression of OPG and RANKL in the same bone cell types confirms their strictly coupled action in the regulation of bone metabolism.     

Mechanical stimulation effects on functional end effectors in osteoblastic MG-63 cells

Receptor activator of Nf-kappaB ligand (RANKL) and osteoprotegerin (OPG) have been implicated in bone metabolism. Specifically, the balance of these factors in conjunction with receptor activator of Nf-kappaB (RANK) is believed to be key in determining the rate of osteoclastogenesis and the net outcome of bone formation/resorption. While it is well accepted that mechanical loading in vivo affects bone formation/resorption and that alterations in the responsiveness of bone cells to mechanical loading have been implicated in metabolic bone diseases, the effect of in vitro mechanical loading on osteoblastic production of OPG and RANKL has not been extensively studied. Thus, in the current study, we developed an in vitro model to load human osteoblasts and studied levels of OPG, RANKL, PGE(2) and macrophage colony stimulating factor (M-CSF). We hypothesized that stimulating osteoblastic cells would increase the release of soluble OPG relative to RANKL favoring a bone-forming (and resorption-inhibiting) event. To accomplish this, we developed a small-scale loading machine that imparts via bending, well-defined substrate deformation to bone cells cultured on artificial substrates. Following 2h of loading and a 1h incubation period, media was collected and levels of soluble OPG, RANKL, PGE(2) and M-CSF were quantified using ELISA and western blotting. We found that mechanical loading significantly increased soluble OPG levels relative to RANKL at this 3h time point. Levels of soluble and cellular RANKL detected were not significantly affected by mechanical stimulation. The relative shift in abundance of OPG over RANKL associated with applied mechanical stimulation suggests the soluble OPG:RANKL ratio may be important in load-induced coupling mechanisms of bone cells.     

胰岛素对2型糖尿病骨质疏松大鼠血清与骨OPG、RANKL表达的影响

目的:探讨胰岛素对2型糖尿病骨质疏松大鼠血清及骨OPG(osteoprotegerin)、RANKL(OPG receptor activator nuclear factork B)表达水平的影响。方法:以高脂高糖饲料喂养4周同时饮用3%果糖水导致胰岛素抵抗小鼠,再以小剂量链脲佐菌素(30mg/kg)腹腔注射1次,2周后诱导建立2型糖尿病小鼠模型。对照组动物则给予正常饲料及饮用水进行喂养。模型建立成功后,对模型2组大鼠进行胰岛素治疗,分别采用OPG和RANKLelisa试剂盒对正常动物模型和糖尿病动物模型血清和骨组织中OPG,RANKL含量进行比较分析,采用血糖分析仪对不同组动物的血糖进行比较分析,采用骨密度分析仪对动物的骨密度进行分析,了解高血糖对于骨密度及血清,骨组织中OPG,RANKL含量的影响以及胰岛素对高血糖骨质疏松造成的结果的影响。结果:相较于正常组大鼠,模型组大鼠血清及髂骨中OPG、血糖、糖化血红蛋白、髂骨密度表达显著下调(P0.05),而RANKL表达显著上调(P0.05),胰岛素处理的模型大鼠血清及骨中OPG含量较模型组大鼠显著升高,血清及骨组织中RANKL表达显著下调(P0.05)。结论:胰岛素能够显著降低2型糖尿病骨质疏松大鼠血清及骨组织中RANKL的表达,显著上调OPG的表达。     

The relationship between osteoclastogenic and anti-osteoclastogenic pro-inflammatory cytokines differs in human osteoporotic and osteoarthritic bone tissues

Background

Pro-inflammatory cytokines possess osteoclastogenic or anti-osteoclastogenic activities. They influence osteoclasts directly or via the receptor activator of nuclear factor κB (RANK), RANK ligand (RANKL) and osteoprotegerin (OPG) system. Recent evidence suggests that inflammation may play a role in osteoporosis (OP) and osteoarthritis (OA). We aimed therefore to determine whether there is a difference between both groups: first, in the expression of the osteoclastogenic and anti-osteoclastogenic cytokines, second, in correlation of these cytokines with bone mineral density (BMD) and levels of bone turnover markers (BTM) and third, in correlation between the expression of these cytokines and osteoclast specific genes and RANK/RANKL/OPG genes.

Methods

Human bone samples from 54 age and sex matched patients with OP or OA were collected during hip arthroplasty surgery. The expression of 25 genes encoding pro-inflammatory cytokines, their receptors, osteoclast specific genes and RANK/RANKL/OPG genes was measured using quantitative real-time PCR. Total hip, femoral neck and lumbar spine BMD and BTM in blood samples were measured. The comparison between OP and OA was assessed using Student''s t-test or Mann-Whitney U test and correlations between gene expression, BMD and BTM were determined using nonparametric correlation.

Results

The results demonstrated a higher expression of interleukin (IL)-6 and IL-1α in OP, and interferon (IFN)-γ in OA (p < 0.0005). Negative correlations of total hip BMD with tumor necrosis factor-α (TNF-α) in OA and with RANKL/RANK in OP were found (p < 0.05). Significant correlations with BTM were shown for IL-1α and IFN-γ in OP (rho = 0.608 and -0.634) and for TNF-α, IL-6 and transforming growth factor-β1 (TGF-β1) in OA (rho = 0.591, -0.521 and 0.636). Results showed OP specific negative correlations (IFN-γ with ITGB3, IFN-β1 with CTSK, tartrate resistant acid phosphatase (TRAP), CALCR, RANK, RANKL, IL-1α with CTSK, OPG, IL-17A with CALCR) and positive (TGF-β1 with CTSK, TRAP, RANK), and OA specific negative (IL-1α with osteoclast associated immunoglobulin-like receptor (OSCAR), TNF-α with RANK, RANKL, OPG) and positive (IL-6 with RANK, RANKL, OPG) correlations.

Conclusions

Our results demonstrate that the relationship between osteoclastogenic and anti-osteoclastogenic pro-inflammatory cytokines differs in human OP and OA bone and could present an important factor for characteristics of OP and OA bone phenotypes.     

Serum osteoprotegerin and RANKL are not specifically altered in women with postmenopausal osteoporosis treated with teriparatide or risedronate: a randomized, controlled trial.

Risedronate and teriparatide have opposite actions on the osteoblast-osteoclast dipole and are expected to influence the RANK/RANKL/osteoprotegerin (OPG) system. We aimed to evaluate changes in serum OPG and RANKL after risedronate or teriparatide administration in postmenopausal osteoporotic women. Seventy-four postmenopausal Caucasian women (age 64.1+/-1.0 years) were studied. Women with osteopenia served as controls (group 1, n=30). Women with osteoporosis were randomly assigned to either risedronate 35 mg once weekly (group 2, n=21) or teriparatide 20 microg once daily (group 3, n=23) for six months. Blood samples for serum RANKL, OPG, N-terminal propeptide of type 1 collagen (P1NP), and C-terminal telopeptide of type 1 collagen (CTx) were obtained before treatment and three and six months after treatment. P1NP and CTx levels remained unchanged in group 1, decreased in group 2 (p<0.001), and increased in group 3 women (p<0.001) throughout the treatment. OPG levels remained unchanged while RANKL decreased gradually in all groups (p<0.001). There was no correlation between OPG or RANKL and P1NP or CTx. Our data suggest that neither antiresorptive nor osteoanabolic therapy causes specific alterations of serum OPG/RANKL levels; therefore, these cytokines cannot substitute for the established markers of bone turnover in the monitoring of response to osteoporosis treatment.     

Mechanical stress regulates osteogenic differentiation and RANKL/OPG ratio in periodontal ligament stem cells by the Wnt/β-catenin pathway

BackgroundThe balance between osteoblastic and osteoclastic activity is critical in orthodontic tooth movement (OTM). Mesenchymal stem cells (MSCs) play an important role in maintaining bone homeostasis, and periodontal ligament stem cells (PDLSCs) are tissue-specific MSCs in the periodontal ligament. However, whether PDLSCs are required for periodontal tissue remodeling during OTM is not fully understood.MethodsHere, we used PDGFRα and Nestin to trace PDLSCs during OTM in rats. We treat human PDLSCs with 100 kpa static pressure for 1 h or 12 h in vitro, and examined the phenotypic changes and expression of RANKL and OPG in these cells.ResultsIn vivo, we found that positive signals of PDGFRα and Nestin in the PDL gradually increased and then decreased on the pressure side to which pressure was applied. In vitro, the osteogenic differentiation of PDLSCs was significantly increased after force treatment for 1 h relative to 12 h. In contrast, the expression ratio of RANKL/OPG was reduced at 1 h and significantly increased at 12 h. Furthermore, we found that the Wnt/β-catenin pathway was dynamically activated in the PDL and in PDLSCs after mechanical stimulation. Importantly, the canonical Wnt pathway inhibitor DKK1 blocked the osteogenesis effect and rescued the ratio of RANKL/OPG in PDLSCs under force treatment for 1 h.ConclusionsOur findings reveal that PDLSCs participate in OTM and that the Wnt/β-catenin pathway maintains bone homeostasis during tooth movement by regulating the balance between osteoblastic and osteoclastic activity.General significanceWe describe a novel potential mechanism related to tooth movement.     

Regulation of RANKL by biomechanical loading in fibrochondrocytes of meniscus

OBJECTIVE: We sought to determine whether fibrochondrocytes from menisci express receptor activator of NF-kappaB (RANK), its ligand (RANKL), or osteoprotegerin (OPG) and, if so, whether their expression is modulated by dynamic mechanical loading under inflammatory and normal conditions. METHODS: Fibrochondrocytes from rat menisci were subjected to cyclic tensile strain (CTS) at various magnitudes and frequencies in the presence or absence of interleukin (IL)-1beta for up to 24 h. In order to determine whether a possible regulatory effect of mechanical loading on RANKL and its receptors under inflamed conditions is sustained, cells were stimulated with IL-1beta for 24 h while being subjected to CTS only for the initial 4 and 8h, respectively. Regulation of RANKL, RANK, and OPG expression and synthesis were determined by semiquantitative and real-time PCR, Western blotting, and immunofluorescence. RESULT: Fibrochondrocytes constitutively expressed low levels of RANKL and RANK but marked levels of OPG. IL-1beta upregulated expression and synthesis of RANKL and RANK significantly (p<0.05), whereas expression of OPG was unaffected following 4 and 24 h. When fibrochondrocytes were simultaneously subjected to CTS and IL-1beta, expression of RANKL and RANK was significantly (p<0.05) downregulated as compared to that of IL-1beta-stimulated unstretched cells. The inhibitory effect of CTS on the IL-1beta-induced upregulation of RANKL and RANK was sustained as well as magnitude and frequency dependent. CONCLUSIONS: Our study provides evidence that RANKL and its receptors are expressed in fibrochondrocytes from meniscus. These data also demonstrate that dynamic mechanical loading can modify the expression of RANKL and RANK in inflammatory conditions.     

OPG/RANKL/RANK系统与骨破坏性疾病

近年来发现的OPG/RANKL/RANK系统在破骨细胞生成中起着至关重要的作用,是骨骼生理研究领域的重大进展。成骨细胞、骨髓基质细胞、激活的T淋巴细胞表达RANKL,与破骨细胞前体细胞或成熟破骨细胞表面上的RANK结合后,促进破骨细胞的分化及骨吸收活性。成骨细胞及骨髓基质细胞分泌表达OPG可与RANKL竞争性结合,从而阻断RANKL与RANK之间的相互作用。体内多种激素或因子通过影响骨髓微环境内的OPG/RANKL比率来调节骨代谢。此外,乳腺上皮细胞表达有RANK,孕期在性激素的诱导下可表达RANKL,OPG/RANKL/RANK系统在孕期乳腺发育以及母体向胎儿的钙转运过程中发挥重要作用。阻断RANKL/RANK通路有望给骨质疏松、类风湿关节炎及癌症骨转移等骨破坏性疾病的治疗开辟新的途径。进一步研究应了解OPG/RANKL/RANK系统与其它信号传导途径的关系,重视骨骼、免疫及内分泌系统之间的相互作用。目前,开发与OPG功能相似或促进其表达的合成药物有可能成为具有良好经济效益和社会效益的产业。