Tensile force on human macrophage cells promotes osteoclastogenesis through receptor activator of nuclear factor κB ligand induction

Little is known about the effects of tensile forces on osteoclastogenesis by human monocytes in the absence of mechanosensitive cells, including osteoblasts and fibroblasts. In this study we consider the effects of tensile force on osteoclastogenesis in human monocytes. The cells were treated with receptor activator of nuclear factor κB ligand (RANKL) to promote osteoclastogenesis. Then,expression and secretion of cathepsin K were examined. RANKL and the formation of osteoclasts during the osteoclast differentiation process under continual tensile stress were evaluated by Western blot. It was also found that ?100 kPa or lower induces RANKL-enhanced tartrate-resistant acid phosphatase activity in a dose-dependent manner. Furthermore, an increased tensile force raises the expression and secretion of cathepsin K elevated by RANKL, and is concurrent with the increase of TNF-receptor-associated factor 6 induction and nuclear factor κB activation. Overall, the current report demonstrates that tensile force reinforces RANKL-induced osteoclastogenesis by retarding osteoclast differentiation. The tensile force is able to modify every cell through dose-dependent in vitro RANKL-mediated osteoclastogenesis, affecting the fusion of preosteoclasts and function of osteoclasts. However, tensile force increased TNF-receptor-associated factor 6 expression. These results are in vitro findings and were obtained under a condition of tensile force. The current results help us to better understand the cellular roles of human macrophage populations in osteoclastogenesis as well as in alveolar bone remodeling when there is tensile stress.     

Effects of 17β-estradiol on adiponectin regulation of the expression of osteoprotegerin and receptor activator of nuclear factor-κB ligand

Adiponectin may exert a negative effect on bone metabolism by regulating osteoprotegerin (OPG) and receptor activator of nuclear factor-κB ligand (RANKL) expression. However, the action of adiponectin on bone may be influenced by estrogen in women. The present study was undertaken to investigate the effects of 17β-estradiol (E2) on adiponectin-regulated OPG and RANKL expression in human osteoblast. Human osteoblasts were treated with α-MEM containing 10μg/ml adiponectin alone or together with 10(-10) to 10(-8)M E2 for 12-48h. Cells were also treated with α-MEM containing 10μg/ml adiponectin together with 10(-8)M E2 plus p38 agonist-anisomycin or estrogen receptor (ER) antagonist ICI182780 for 48h. The effects of E2 were also investigated by knockdown of ERs or overexpression of p38 MAPK in osteoblasts. Further, we examined the effects of E2 on adiponectin-dependent osteoclastogenesis by the co-culture systems of osteoblast and CD14+ peripheral blood monocytes (PBMCs). Real-time quantitative PCR (RT-PCR) and ELISA were used to detect OPG/RANKL mRNA and their corresponding protein expression, Western Blot was used to analyze the phosphorylated p38 (p-p38) levels. The results showed that E2 blocked adiponectin-induced p38 phosphorylation, decreased adiponectin-regulated OPG/RANKL mRNA and protein expression in a dose- and time-dependent manner. ICI182780 or knockdown of ERs abolished the effects of E2 on adiponectin-dependent p38 phosphorylation and OPG/RANKL expression. Furthermore, anisomycin or overexpression of p38 also reserved the effects of E2 on adiponectin-dependent p38 phosphorylation and OPG/RANKL expression. E2 inhibited adiponectin-dependent osteoclastogenesis in the co-culture systems of osteoblast and CD14+ PBMCs, whereas anisomycin, ICI182780, knockdown of ERs and overexpression of p38 significantly reversed this response. In conclusions, our findings demonstrated, through blocking the activation of adiponectin-induced p38 MAPK, E2 suppressed the adiponectin-regulated OPG/RANKL expression and then inhibited osteoclastogenesis, which suggested that estrogen would suppress the effect of adiponectin on bone metabolism.     

The role of serum osteoprotegerin and receptor–activator of nuclear factor-κB ligand in metabolic bone disease of women after obesity surgery

Metabolic bone disease may appear as a complication of obesity surgery. Because an imbalance in the osteoprotegerin and receptor–activator of nuclear factor-κB ligand system may underlie osteoporosis, we aimed to study this system in humans in the metabolic bone disease occurring after obesity surgery. In this study we included sixty women with a mean age of 47 ± 10 years studied 7 ± 2 years after bariatric surgery. The variables studied were bone mineral density, β-isomer of C-terminal telopeptide of type I collagen cross-links (a bone resorption marker), the bone formation markers osteocalcin and N-terminal propeptide of procollagen 1, serum osteoprotegerin and receptor–activator of nuclear factor-κB ligand. Serum osteoprotegerin inversely correlated with the bone remodeling markers osteocalcin, β-isomer of C-terminal telopeptide of type I collagen cross-links and N-terminal propeptide of procollagen 1. The osteoprotegerin and receptor–activator of nuclear factor-κB ligand ratio also correlated inversely with serum parathormone and osteocalcin. Bone mineral density at the lumbar spine was associated with age (β = ?0.235, P = 0.046), percentage of weight loss (β = 0.421, P = 0.001) and osteoprotegerin and receptor–activator of nuclear factor-κB ligand ratio (β = 0.259, P = 0.029) in stepwise multivariate analysis (R ² = 0.29, F = 7.49, P < 0.001). Bone mineral density at the hip site was associated only with percentage of weight loss (β = 0.464, P < 0.001) in stepwise multivariate regression (R ² = 0.21, F = 15.1, P < 0.001). These data show that the osteoprotegerin and receptor–activator of nuclear factor-κB ligand system is associated with bone markers and bone mineral density at the lumbar spine after obesity surgery.     

A novel role of l-serine (l-Ser) for the expression of nuclear factor of activated T cells (NFAT)2 in receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclastogenesis in vitro

Multinucleated cell formation is crucial for osteoclastogenesis, and the expression of nuclear factor of activated T cells (NFAT)2 (NFATc1) is essential for this process. We previously found, using mouse RAW264 cells, that culture at high cell density blocked progression to the multinucleated cell stage induced by stimulation with receptor activator of nuclear factor κB ligand (RANKL). Here, we have confirmed this finding in a bone marrow cell system and extended the analysis further. A high cell density appeared to cause a change in the composition of the culture medium accompanying downregulation of NFAT2 expression, and we identified l-serine (l-Ser) as essential for the expression of NFAT2 induced by RANKL. Namely, culture at high cell density caused a depletion of l-Ser in the medium. Consequently, l-Ser appeared to exert its effect at an early stage under the regular conditions used for inducing the expression of c-Fos, an upstream regulator of NFAT2. d-Ser, an enantiomer of l-Ser, showed no NFAT2-inducing activity. The expression of NFAT2, using a retrovirus vector, could compensate for the depletion of l-Ser and resume the progression to the multinucleated cell stage. These results demonstrate a novel role for l-Ser in RANKL-induced osteoclastogenesis in vitro.     

Effects of knockout receptor activator of NF-κB (RANK) in podocytes on proteinuria in mice

Objective To investigate the role of RANK in injured podocyte and its effects on proteinuria. Methods PCR analysis was carried out to identify the genotypes of podocyte-specific RANK knockout mice. Six (6-8 weeks old) female RANK-/- mice were chosen as KO group. Six (6-8 weeks old) littermates and six (6-8 weeks old) C57/B6j mice were chosen as WT group and C57 group respectively. Each group was injected with LPS to create podocyte-injured proteinuria model. 24h urine was collected before and after injection. Urinary albumin, urine creatinine and UACR were detected by automatic biochemical analyzer. The mice were sacrificed after 48 h. Cortical kidney tissue samples were stained by PAS and collected for transmission electron microscopy to detect the foot process effacement. The number of podocytes was examined at different time points by immunohistochemistry .The expression of intergrin-β1, integrin-β3 and uPAR was detected by immunofluorescence and westernblot. Results (1) Comparing KO group, WT group and C57 group, it was found that there was no difference in weight, morphology, function of kidney and ACR. (2) After LPS injection, proteinuria and ACR were increased in three groups, but less significantly in KO group (23.70±9.90), compared to those in WT group (107.56±22.32) and C57 group (132.13±14.26) (P＜0.001). (3) In three groups, the number of podocytes was decreased after LPS injection and the decrease in the KO group was the slightest. (4) Compared to WT and C57 group, podocyte foot process effacement was less obvious in KO group after LPS injection in transmission electron microscopy. (5) Immunofluorescence results showed that after LPS injection, integrin-β1 was decreased in three groups, but most significantly in KO group. Integrin-β3 was increased in three groups, but less obvious in KO group. (6) Westernblot results showed consistency with immunofluorescence. The expression of uPAR protein was increased in three groups, with the increase in KO group being the slightest. Conclusions Podocyte-specific knockout of RANK reduces LPS-induced proteinuria, suggesting that RANK might be involved in the development of proteinuria in podocyt injury. Its mechanism is probably related with integrin-β1, integrin-β3 and uPAR.     

Effects of alpha-calcitonin gene-related peptide on osteoprotegerin and receptor activator of nuclear factor-κB ligand expression in MG-63 osteoblast-like cells exposed to polyethylene particles

Background  

Recent studies demonstrated an impact of the nervous system on particle-induced osteolysis, the major cause of aseptic loosening of joint replacements.     

Circulating osteoprotegerin and receptor activator of NF-κB ligand system in patients with β-thalassemia major

Osteoporosis represents an important cause of morbidity in patients with β-thalassemia major, and its etiology is multifactorial. Thus, the aim of this study was to characterize the possible role of the osteoprotegerin (OPG) and receptor activator of the NF-κB ligand (RANKL) system in thalassemia-related bone loss. Serum concentrations of OPG, soluble RANKL (s-RANKL), markers of bone turnover, and lumbar spine bone mineral density (BMD) were measured in random samples of males (n = 29; mean age ± SEM, 24.26 ± 1.29 years; range, 13–41 years) and females (n = 31; age, 24.59 ± 0.95 years; range, 12–34 years) with β-thalassemia major and in 30 healthy age-, height-, and weight-matched subjects. Thalassemic patients had significantly lower levels of OPG compared with controls (2.54 ± 0.12 vs. 3.25 ± 0.122, respectively; P < 0.05) and higher, albeit not statistically significantly, serum levels of s-RANKL (0.350 ± 0.03 vs. 0.295 ± 0.046, respectively; P < 0.05). s-RANKL correlated negatively with age (r = −0.3, P < 0.05), and OPG correlated positively with the duration of the interval between the onset of transfusions and chelation therapy (r = 0.52, P < 0.001). Regarding markers of bone metabolism, plasma values of osteocalcin correlated positively with s-RANKL (r = 0.40, P < 0.05) and negatively with OPG/s-RANKL ratio (r = −0.55, P < 0.01). In multiple regression analysis only cross-linked N-teleopeptide of type I collagen (NTX) significantly accounted for BMD. Although the OPG/RANKL system may have some clinical usefulness as a marker of bone turnover in β-thalassemia, conventional markers of bone turnover more accurately represent changes in the BMD of these patients.     

Caffeic acid 3,4-dihydroxy-phenethyl ester suppresses receptor activator of NF-κB ligand–induced osteoclastogenesis and prevents ovariectomy-induced bone loss through inhibition of mitogen-activated protein kinase/activator protein 1 and Ca2+–nuclear factor of activated T-cells cytoplasmic 1 signaling pathways

Receptor activator of NF-κB ligand (RANKL) stimulation leads to the activation of mitogen-activated protein kinase (MAPK)/AP-1 and Ca2+–nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) signaling pathways in osteoclastogenesis. Targeting these pathways has been an encouraging strategy for bone-related diseases, such as postmenopausal osteoporosis. In this study, we examined the effects of caffeic acid 3,4-dihydroxy-phenethyl ester (CADPE) on osteoclastogenesis. In mouse bone marrow monocytes (BMMs) and RAW264.7 cells, CADPE suppressed RANKL-induced osteoclast differentiation and actin-ring formation in a dose-dependent manner within non–growth inhibitory concentrations at the early stage, while CADPE had no effect on macrophage colony-stimulating factor (M-CSF)-induced proliferation and differentiation. At the molecular level, CADPE inhibited RANKL-induced phosphorylation of MAPKs, including extracellular signal-regulated kinases 1/2 (ERK1/2), p38, and c-Jun N-terminal kinase (JNK), without significantly affecting the NF-κB signaling pathway. CADPE abrogated RANKL-induced activator protein 1 (AP-1)/FBJ murine osteosarcoma viral oncogene homolog (c-Fos) nuclear translocation and activation. Overexpression of c-Fos prevented the inhibition by CADPE of osteoclast differentiation. Furthermore, CADPE suppressed RANKL-induced the tumor necrosis factor receptor associated factor 6 (TRAF6) interaction with c-src tyrosine kinase (c-Src), blocked RANKL-induced the phosphorylation of protein kinase B (AKT), and inhibited RANKL-induced Ca2+ oscillation. As a result, CADPE decreased osteoclastogenesis-related marker gene expression, including NFATc1, TRAP, cathepsin K, and c-Src. To test the effects of CADPE on osteoclast activity in vivo, we showed that CADPE prevented ovariectomy-induced bone loss by inhibiting osteoclast activity. Together, our data demonstrate that CADPE suppresses osteoclastogenesis and bone loss through inhibiting RANKL-induced MAPKs and Ca2+-NFATc1 signaling pathways. CADPE is a novel agent in the treatment of osteoclast-related diseases, such as osteoporosis.     

Regulation of synthesis of osteoprotegerin and soluble receptor activator of nuclear factor-κB ligand in normal human osteoblasts via the p38 mitogen-activated protein kinase pathway by the application of cyclic tensile strain

Mechanical stress is thought to play an important role in bone remodeling. However, the correlation between mechanical stress and bone remodeling is poorly understood. In this context, using a model of cyclic tensile strain (CTS) toward human osteoblasts, synthesis of osteoprotegerin (OPG) and soluble receptor activator of nuclear factor-κB ligand (sRANKL), and the activation of mitogen-activated protein kinases (MAPKs) were examined. The application of 7%, 0.25-Hz CTS once a day for 4 h for 3 successive days simultaneously caused an increase of OPG synthesis and a decrease of sRANKL release and RANKL mRNA expression in osteoblasts. As for MAPKs activation in osteoblasts with the application of CTS, p38 MAPK was activated 10–20 min after the application of CTS, but extracellular signal-regulated kinase (ERK1/2) and c-Jun NH₂-terminal kinase (JNK) were not activated by such application. Furthermore, when CTS was applied once a day for 4 h for 1, 2, or 3 successive days to osteoblasts, p38 MAPK activation was maintained during the 3-day period but ERK1/2 activation was downregulated from day to day, simultaneously. Then, when CTS was applied once a day for 4 h for 3 successive days to osteoblasts pretreated with the p38 MAPK inhibitor SB203580 for 1 h, OPG synthesis was dose-dependently suppressed and inhibition of sRANKL release and RANKL mRNA expression was abrogated. These results indicate that biological responses of OPG and sRANKL synthesis in osteoblasts to the application of CTS are regulated via the p38 MAPK pathway and suggest that CTS might modulate and regulate bone metabolism.     

Establishment of a new murine model of hypercalcemia with anorexia by overexpression of soluble receptor activator of NF-κB ligand using an adenovirus vector

Hypercalcemia is a significant complication of certain human malignancies that is primarily caused by the release of calcium from bone due to marked bone resorption by osteoclast activation. Osteoclast differentiation and activation is mediated by receptor activator of NF-κB ligand (RANKL). Transgenic mice overexpressing murine soluble RANKL (sRANKL) that we generated previously exhibited severe osteoporosis accompanied with enhanced osteoclastogenesis, but never exhibited hypercalcemia. To analyze the relationship between serum concentration of sRANKL and hypercalcemia and generate a simple and quick hypercalcemia model, an adenovirus vector harboring murine sRANKL cDNA (Ad-sRANKL) was injected i.p. into male C57BL/6 mice. Sera were collected to measure the levels of sRANKL, calcium and biochemical markers of bone turnover. Food intake and body weight were measured every 3 or 4 days. All the mice were killed 2 weeks after the injection, and femurs were collected to measure bone structure and bone mineral density (BMD). Serum sRANKL and calcium increased, peaking on day 7. Food intake and body weight significantly declined on day 7. These results indicated that the mice had anorexia as a symptom of hypercalcemia. Increases in bone resorption and formation markers with a marked decrease in BMD were observed on day 14. These results reflect accelerated bone formation following activation of osteoclasts, indicating coupling between bone formation and resorption. In conclusion, a new murine model of hypercalcemia with anorexia was established by overexpressing sRANKL. This model would be useful for studies of hypercalcemia and coupling between bone formation and resorption.     

Activation of nuclear factor κB at the onset of ossification of the spinal ligaments

We examined the correlation between the activation of nuclear factor κB (NFκB), stimulated by environmental factors involving cytokines and growth factors in ligament cells, and the onset of ossification of the spinal ligaments (OSL) or diffuse idiopathic skeletal hyperostosis (DISH). Aseptic samples were taken carefully from non-ossified sites during surgery (75 patients). We carried out preliminary hematoxylin and eosin and toluidine blue staining, using five portions of each specimen, and excluded samples containing chondrocytic, osteoblastic, or inflammatory cells (n = 25). We used specimens from the remaining 50 patients (35 men and 15 women, ranging in age from 45–81 years); average age, 59.5 years (18 nuchal ligament specimens, and 32 yellow ligament specimens). OSL or DISH had occurred in 25 patients, 20 patients were in the non-OSL group (8 with cervical spondylotic myelopathy, and 12 with lumbar canal stenosis), and the remaining 5 samples were collected from patients with injury. For culture study, we used portions of the 14 largest samples from the above 50 patients. We extracted nuclear proteins and cytoplasmic proteins from non-ossified spinal ligaments in 50 patients and detected p65RelA/NFκB by Western blotting. Tumor necrosis factor-α (TNF α), interleukin 1β (IL-1β), platelet-derived growth factor BB (PDGF-BB) and transforming growth factor-β1 (TGF-β1) in cytoplasm were quantified by enzyme-linked immunosorbent assays (ELISA). Cultured cells from the 14 samples were then stimulated with 10, 100, 250, or 500 ng/ml of recombinant human (rh)PDGF-B or TGFβ1. A control experiment was performed without rhPDGF-BB or TGFβ1 stimulation. Alkaline phosphatase (ALP) activity was standardized by the DNA content of the cells. The number of NFκB-positive samples was significantly higher in patients with OSL or DISH than in non-OSL patients. This tendency was obvious in the case of OSL or DISH with non-insulin-dependent diabetes mellitus (NIDDM). In OSL and in DISH patients, significantly greater amounts of PDGF-BB and TGFβ1 were seen in ligament cells than in non-OSL patients (P < 0.05). There was a positive correlation between the detection of p65RelA/NFκB band and the content of PDGF-BB and TGFβ1 in ligament cells (P < 0.05). ALP activity tended to be higher in cells in the OSL group not receiving any other treatment. Our results indicate the possibility that NFκB, stimulated by environmental factors involving PDGF-BB and TGFβ1 in ligament cells, influences the osteoblastic differentiation of undifferentiated mesenchymal cells. Received: March 27, 2000 / Accepted: June 22, 2000     

Effect of interleukin-17 on nitric oxide production and osteoclastic bone resorption: is there dependency on nuclear factor-kappaB and receptor activator of nuclear factor kappaB (RANK)/RANK ligand signaling?

Interleukin-17 (IL-17) is a proinflammatory cytokine produced exclusively by activated memory T cells and has recently been found to stimulate osteoclastic resorption. Like other proinflammatory cytokines, IL-17 may affect osteoclastic bone resorption indirectly via osteoblasts, possibly by mechanisms previously reported for chondrocytes that respond in very similarly to osteoblasts. As in chondrocytes, but only in combination with tumor necrosis factor-alpha (TNF-alpha), IL-17 induced nitric oxide (NO) production in osteoblastic cells and fetal mouse metatarsals by a nuclear factor-kappaB (NF-kappaB)-dependent mechanism. This effect was associated with elevated mRNA levels of the NF-kappaB isoforms RelA and p50. In fetal mouse metatarsals, IL-17 stimulated osteoclastic bone resorption only in combination with TNF-alpha. The pathway by which the cytokine combination exerts this effect was examined using inhibitors of NO synthesis and NF-kappaB activation. Although both inhibitors used abolished NO production, they did not prevent the stimulatory effect of the cytokine combination on osteoclastic resorption. In contrast, the inhibitors slightly increased osteoclastic resorption, suggesting a suppressive rather than stimulatory effect of NO on cytokine-induced bone resorption. In addition, we showed that IL-17 + TNF-alpha stimulated osteoclastic resorption independent of NF-kappaB signaling. To further examine the pathway by which osteoclastic resorption was stimulated, we used osteoprotegerin, a specific inhibitor of the receptor activator of NF-kappaB (RANK)/receptor activator of the NF-kappaB ligand (RANKL) pathway. Osteoprotegerin partially inhibited IL-17 + TNF-alpha-stimulated osteoclastic resorption only at the high concentration of 1000 ng/mL, whereas it completely blocked parathyroid hormone-related peptide-stimulated resorption at 300 ng/mL. In conclusion, IL-17 stimulated NO production by an NF-kappaB-dependent pathway in osteoblastic cells and fetal mouse metatarsals only in combination with TNF-alpha. Neither NO production nor NF-kappaB signaling, and only partly the RANK/RANKL pathway, were involved in the stimulatory effect of the cytokine combination on osteoblastic bone resorption in these long bones, suggesting the existence of other pathways by which osteoclastic resorption can be stimulated.     

Advances in research on nuclear factor - kappa B( NF - κB) and diabetic nephropathy

糖尿病肾病是糖尿病最普遍的并发症,其通常导致终末期肾衰和很高的死亡率.核因子-kappa B(nuclear factor-kappa B,NF-κB)是包含二聚体转录因子的家族其能调控包括炎症和细胞增殖在内的许多基因的表达.NF-KB可能通过高糖、高脂,肾素血管紧张素醛固酮系统、肾脏血流动力学和遗传等多种途径参与糖尿病肾损伤的发生发展.靶向于NF-κB介导的肾脏损伤的治疗被认为是干预糖尿病肾病的有效手段.     

Phosphate, oxidative stress, and nuclear factor-κB activation in vascular calcification

Phosphate-induced vascular calcification, characterized by induction of osteogenic programs, mineral vesicle release, and apoptosis, is prevalent in patients with kidney disease. Zhao et al. provide a mechanistic link between phosphate-induced calcification and increased mitochondrial membrane potential, increased mitochondrial reactive oxygen species, activation of the nuclear factor-κB pathway, and subsequent expression of osteogenic genes and vascular mineralization. This link clarifies the intracellular mechanism of vascular calcification and may allow exploration of antioxidants as therapeutic agents for vascular calcification.