Hypoxic stress enhances osteoclast differentiation via increasing IGF2 production by non-osteoclastic cells

Development of bone depends on a continuous supply of bone-degrading osteoclasts. Although several factors such as cytokines and integrins have been shown to be important for osteoclast recruitment, their mechanism of action is poorly understood. In this study, we demonstrated the enhancement of osteoclast formation by hypoxia and investigated the molecular mechanisms involved. Primary mouse bone marrow cells were cultured in normoxic and hypoxic conditions, and RNA was prepared from each group of cells. Total RNAs were applied to a DNA microarray analysis and then RT-PCR was performed to confirm the microarray data. The most interesting finding of our microarray analysis was upregulation of insulin-like growth factor 2 (IGF2) and stromal cell-derived factor 1 (SDF1) under hypoxic conditions. RT-PCR analysis revealed that IGF2 expression was markedly upregulated in the non-osteoclastic cells. The addition of exogenous IGF2 increased the number of osteoclastic TRAP-positive multinuclear cells formed under normoxic conditions, whereas the addition of exogenous SDF1 did not change osteoclast formation. These results suggest that the upregulation of IGF2 derived from non-osteoclastic cells might be a crucial factor for osteoclast differentiation.     

Dermatan sulfate inhibits osteoclast formation by binding to receptor activator of NF-kappa B ligand

Dermatan sulfate (DS) is a major component of extracellular matrices in mammalian tissues. In the present study, DS demonstrated a high level of binding activity to receptor activator of NF-kappaB ligand (RANKL) and obstructed the binding of RANK to RANKL, determined using a quartz-crystal microbalance (QCM) technique. Further, when mouse bone marrow cells were cultured with RANKL and macrophage colony-stimulating factor, DS suppressed tartrate-resistant acid phosphatase-positive multinucleated cell formation in a dose-dependent manner. In addition, immunoblot analyses revealed that DS reduced the levels of phosphorylation of p38 mitogen-activated protein kinase and extracellular signal-regulated kinase protein in mouse osteoclast progenitor cells stimulated with RANKL. Together, these results indicate that DS regulates osteoclast formation through binding to RANKL and inhibition of signal transduction in osteoclast progenitor cells, suggesting that it has an important role in bone metabolism in pathological conditions.     

p44/42 MAPK activation is necessary for receptor activator of nuclear factor-kappaB ligand induction by high extracellular calcium

Although extracellular calcium (Ca(2+)(o)) has been suggested to modulate bone remodeling, the exact mechanism is unclear. This study was performed to explore the signaling pathways of high Ca(2+)(o) that are responsible for controlling the expression of receptor activator of NF-kappaB ligand (RANKL) in mouse osteoblastic cells. As previously reported, high Ca(2+)(o) increased RANKL expression. However, the G protein-coupled Ca(2+)(o)-sensing receptor (CaSR) was not detected in the primary cultured mouse osteoblastic cell. The inhibition of the pertussis-sensitive G protein, phospholipase C, protein kinase C, intracellular calcium mobilization, p38 MAPK, or phosphoinositide 3-kinase did not block RANKL induction caused by high Ca(2+)(o). In contrast, the inhibition of p44/42 MAPK pathway reduced the RANKL expression induced by high Ca(2+)(o). Moreover, high Ca(2+)(o) activated p44/42 MAPK and MEK1/2. These results suggest that RANKL induction by high Ca(2+)(o) might not be mediated by CaSR and its putative downstream signaling pathways, but the pathway employing p44/42 MAPK is involved in the high Ca(2+)(o)-induced RANKL expression in mouse osteoblastic cells.     

Receptor activator of nuclear factor‐kappa B ligand induces osteoclast formation in RAW 264.7 macrophage cells via augmented production of macrophage–colony‐stimulating factor

RAW 264.7 macrophage cells differentiate into osteoclast‐like cells in the presence of RANKL. Participation of M‐CSF in RANKL‐induced osteoclast formation of RAW 264.7 cells was examined. TRAP‐positive osteoclast‐like cells appeared in RAW 264.7 cells cultured in the presence of RANKL. RANKL‐induced osteoclast formation was markedly inhibited by anti‐M‐CSF antibody. RANKL augmented M‐CSF mRNA expression and M‐CSF production in RAW 264.7 cells. Further, anti‐M‐CSF antibody inhibited the expression of RANK, c‐fms, c‐fos and TRAP mRNA in RANKL‐stimulated RAW 264.7 cells. However, anti‐M‐CSF antibody did not affect the expression of DC‐STAMP in the stimulated cells. Therefore, RANKL was suggested to induce osteoclast formation in RAW 264.7 cells via augmented production of M‐CSF. The putative role of M‐CSF in RANKL‐induced osteoclast formation of RAW 264.7 cells is discussed.     

Bacterial lipopolysaccharide induces osteoclast formation in RAW 264.7 macrophage cells

Lipopolysaccharide (LPS) is a potent bone resorbing factor. The effect of LPS on osteoclast formation was examined by using murine RAW 264.7 macrophage cells. LPS-induced the formation of multinucleated giant cells (MGC) in RAW 264.7 cells 3 days after the exposure. MGCs were positive for tartrate-resistant acid phosphatase (TRAP) activity. Further, MGC formed resorption pits on calcium-phosphate thin film that is a substrate for osteoclasts. Therefore, LPS was suggested to induce osteoclast formation in RAW 264.7 cells. LPS-induced osteoclast formation was abolished by anti-tumor necrosis factor (TNF)-alpha antibody, but not antibodies to macrophage-colony stimulating factor (M-CSF) and receptor activator of nuclear factor (NF)-kappaB ligand (RANKL). TNF-alpha might play a critical role in LPS-induced osteoclast formation in RAW 264.7 cells. Inhibitors of NF-kappaB and stress activated protein kinase (SAPK/JNK) prevented the LPS-induced osteoclast formation. The detailed mechanism of LPS-induced osteoclast formation is discussed.     

MiRNA-199a-5p positively regulated RANKL-induced osteoclast differentiation by target Mafb protein

MicroRNAs are involved in osteoclast differentiation. Although miR-199a-5p plays an important role in many different systems and diseases, its function during osteoclastogenesis remains unclear. In this study, we investigated the function and the target gene of miR-199a-5p in osteoclast differentiation. The in vitro data showed that miR-199a-5p was significantly upregulated after the stimulation by receptor activator of nuclear factor kappa-B ligand in macrophages and RAW 264.7 cells. After transfection of miR-199a-5p mimic, the messenger RNA expression level of nuclear factor of activated T-cells cytoplasmic 1, tartrate-resistant acid phosphatase (TRAP), and receptor activator of nuclear factor kappa-B was significantly increased in RAW 264.7 cells and the number of TRAP-positive cells was also increased. MiR-199a-5p inhibitor showed the complete opposite outcome which brought additional proof to our finding. Overexpression of miR-199a-5p led to downregulation of Mafb protein. The luciferase activity was obviously repressed when WT-pGL3-Mafb and miR-199a-5p mimics were cotransfected into 293 T cells and the inhibitors cotransfected demonstrated reverse result. MiR-199a-5p overexpressed during osteoclast differentiation and positively regulated osteoclast formation in vitro by target Mafb.     

Effects of different magnitudes of mechanical strain on Osteoblasts in vitro

In addition to systemic and local factors, mechanical strain plays a crucial role in bone remodeling during growth, development, and fracture healing, and especially in orthodontic tooth movement. Although many papers have been published on the effects of mechanical stress on osteoblasts or osteoblastic cells, little is known about the effects of different magnitudes of mechanical strain on such cells. In the present study, we investigated how different magnitudes of cyclic tensile strain affected osteoblasts. MC3T3-E1 osteoblastic cells were subjected to 0%, 6%, 12% or 18% elongation for 24h using a Flexercell Strain Unit, and then the mRNA and protein expressions of osteoprotegerin (OPG) and receptor activator of nuclear factor-kappaB ligand (RANKL) were examined. The results showed that cyclic tensile strain induced a magnitude-dependent increase (0%, 6%, 12%, and 18%) in OPG synthesis and a concomitant decrease in RANKL mRNA expression and sRANKL release from the osteoblasts. Furthermore, the induction of OPG mRNA expression by stretching was inhibited by indomethacin or genistein, and the stretch-induced reduction of RANKL mRNA was inhibited by PD098059. These results indicate that different magnitudes of cyclic tensile strain influence the biological behavior of osteoblasts, which profoundly affects bone remodeling.     

Cepharanthine suppresses osteoclast formation by modulating the nuclear factor-κB and nuclear factor of activated T-cell signaling pathways

The increased activation of osteoclasts is the major manifestation of several lytic bone diseases, including osteoporosis, rheumatoid arthritis, aseptic loosening of orthopedic implants, Paget disease and malignant bone diseases. One important bone-protective therapy in these diseases focuses on the inhibition of osteoclast differentiation and resorptive function. Given that the deleterious side-effects of currently available drugs, it is beneficial to search for effective and safe medications from natural compounds. Cepharanthine (CEP) is a compound extracted from Stephania japonica and has been found to have antioxidant and anti-inflammatory effects. In this study, we found that CEP inhibited receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-induced osteoclast formation and bone-resorbing activities using osteoclastogenesis and bone resorption assay. By polymerase chain reaction, we also found that CEP inhibited the expression of osteoclast-differentiation marker genes including Ctsk, Calcr, Atp6v0d2, Mmp9 and Nfatc1. Mechanistic analyses including Western blot and luciferase reporter assay revealed that CEP inhibited RANKL-induced activation of NF-κB and nuclear factor of activated T-cell, which are essential for the formation of osteoclast. Collectively, these data suggested that CEP can potentially be used as an alternative therapy for preventing or treating osteolytic diseases.     

Inhibition of RANKL-mediated osteoclast differentiation by selective TRAF6 decoy peptides

RANK and RANKL are essential mediators of osteoclastogenesis. RANK interacts with members of the tumor necrosis factor receptor-associated factor (TRAF) family, of which TRAF6 is the critical signaling molecule. We identified a unique TRAF6-binding motif in RANK, which was subsequently co-crystallized with TRAF6 revealing distinct molecular interactions. A cell-permeable TRAF6 decoy peptide (T6DP) was shown to specifically target TRAF6 and inhibit RANKL-mediated signaling. In this study, we identified a core motif for binding to TRAF6 by generating a series of deletion mutants linked via palmitate as a means to internalize the peptide, thus making a smaller scaffold for intracellular delivery. The core motif of RKIPTEDEY inhibited RANKL-mediated osteoclastogenesis and bone resorption. In contrast, TRAF2/5 decoy peptides appeared to have no affect. Thus, disruption of the RANK-TRAF6 interaction may prove useful as a novel target for the development of a small molecule therapeutic agent for the treatment of bone-related diseases.     

Netrin-4 derived from murine vascular endothelial cells inhibits osteoclast differentiation in vitro and prevents bone loss in vivo

Bone is a highly vascularized organ, thus angiogenesis is a vital process during bone remodeling. However, the role of vascular systems in bone remodeling is not well recognized. Here we show that netrin-4 inhibits osteoclast differentiation in vitro and in vivo. Co-cultures of bone marrow macrophages with vascular endothelial cells markedly inhibited osteoclast differentiation. Adding a neutralizing antibody, or RNA interference against netrin-4, restored in vitro osteoclast differentiation. Administration of netrin-4 prevented bone loss in an osteoporosis mouse model by decreasing the osteoclast number. We propose that vascular endothelial cells interact with bone in suppressing bone through netrin-4.     

Regulation of osteoclastogenesis by three human RANKL isoforms expressed in NIH3T3 cells

Receptor activator of nuclear factor-kappaB ligand (RANKL) induces osteoclastogenesis by binding with the receptor, receptor activator of nuclear factor-kappaB in the presence of macrophage colony-stimulating factor. Three human RANKL isoforms, hRANKL1, hRANKL2, and hRANKL3, were identified. hRANKL1 was identical to previously reported RANKL and possessed intracellular, transmembrane, and extracellular domains, hRANKL2 did not have the intracellular domain, and hRANKL3 did not have the intracellular and transmembrane domains. When bone marrow macrophages were cultured with NIH3T3 cells expressing hRANKL1, osteoclasts were formed, but when cultured with NIH3T3 cells expressing hRANKL2 or hRANKL3, no tartrate resistant acid phosphatase-positive cell was observed. In the coculture system, coexpression of hRANKL3 with hRANKL1 significantly inhibited the formation of osteoclasts by hRANKL1, but coexpression of hRANKL2 with hRANKL1 did not affect the osteoclastogenesis by hRANKL1 significantly. These results suggest that the activity of osteoclastogenesis by hRANKL1 is regulated by the attenuator, hRANKL3.     

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.     

绝经后骨质疏松症TNF-α通过激活NF-κB促进RANKL诱导的破骨细胞形成

本研究检测了绝经后骨质疏松症妇女的肿瘤坏死因子-α(TNF-α)和雌激素水平,并探讨了TNF-α对破骨前体细胞RAW264.7中破骨细胞标志物核因子κB受体激活因子(nuclear factor kappa-B, RANK)、组织蛋白酶K (Cathepsin K, CTSK)和凝血酶受体激活肽(thrombin receptor activating peptide, TRAP)以及核因子-κB (NF-κB)亚基(p65)和NF-κB抑制蛋白(IκBα)的影响。研究结果表明,绝经后骨质疏松症患者的TNF-α水平显著升高,而雌二醇水平显著降低。核因子κB受体激活因子配体(receptor activator for NF-κBligand, RANKL)处理1周后,破骨前体细胞RAW264.7中破骨细胞标志物RANK、CTSK和TRAP的mRNA和蛋白高度表达。与RANKL对照组相比,TNF-α处理可上调RANK、CTSK和TRAP m RNA的表达。但是,仅TNF-α不能诱导培养的RAW264.7细胞分化为破骨细胞成。TNF-α以剂量依赖性方式诱导NF-κB亚基p65和IκBα磷酸化,而NF-κB抑制剂处理则有效降低了RANK和TRAP的表达。本研究结论表明,绝经后骨质疏松症中TNF-α通过激活NF-κB来促进RANKL诱导的破骨细胞形成。     

Adrm1 interacts with Atp6v0d2 and regulates osteoclast differentiation

Bone homeostasis is tightly regulated by matrix-producing osteoblasts and bone-resorbing osteoclasts. During osteoclast development, mononuclear preosteoclasts derived from myeloid cells fuse together to form multinucleated, giant cells. Previously, we reported that the d2 isoform of the vacuolar (H⁺) ATPase V0 domain (Atp6v0d2) plays an important role in osteoclast maturation and bone formation. To understand how Atp6v0d2 controls osteoclast maturation, we have performed a yeast two-hybrid screen using full-length Atp6v0d2 as the bait, and identified adhesion-regulating molecule 1 protein (Adrm1) as a potential functional partner of Atp6v0d2. The interaction between Atp6v0d2 and Adrm1 was confirmed in yeast and invivo using immunoprecipitation assays. We also show that Adrm1 is required for cell migration and osteoclast maturation.     

RANKL与骨重建

骨是一种动态更新的组织,它不断进行骨吸收（bone resorption）与骨形成（bone formation）的平衡,这个过程称之为骨重建（bone remodeling）．核因子κB受体活化因子配体（receptor activator of nuclear factor κB ligand,RANKL）是骨吸收和骨形成耦联的关键,具有诱导破骨细胞（osteoclast, OC）生成、活化,抑制破骨细胞凋亡的作用．RANKL最初发现于活化的T细胞,但骨重建过程中RANKL主要来源于骨细胞、成骨细胞和骨髓基质细胞．RANKL/核因子κB受体活化因子（receptor activator of nuclear factor κB,RANK）/骨保护素（osteoprotegerin, OPG）信号通路在成骨细胞调控破骨细胞生成的过程中起着重要的调节作用,是维持骨重建平衡的关键．本文就RANKL及其在骨中的分子作用机制作一综述.     

异丙酚对大鼠心肌缺血/再灌注损伤时NF-κB活化和细胞凋亡的影响

目的:观察异丙酚对大鼠心肌缺血/再灌注时核因子-κB(NF-κB)的活化和细胞凋亡的影响,以探讨异丙酚的心肌保护作用机制。方法:采用阻断大鼠左冠状动脉前降支30min,再灌注2h心肌缺血/再灌注损伤模型。60只SD大鼠随机分为假手术组(Sham)、缺血/再灌注组(I/R)和异丙酚3、6、12mg/(kg.h)组。光、电镜观察心肌组织的形态学变化。免疫组化染色分析心肌组织中NF-κB的核移位,Western blot检测心肌组织NF-κB和caspase-3的表达。原位末端标记(TUNEL)检测心肌细胞凋亡。结果:I/R组心肌纤维排列紊乱,心肌细胞水肿;线粒体膜肿胀,嵴排列紊乱甚至溶解消失。与I/R组相比,6,12mg/(kg·h)组异丙酚组心肌损伤明显减轻。与Sham组相比,I/R组NF-κB活化,明显从细胞浆移位于细胞核,表达量也显著增加(P0.05);心肌caspase-3表达增强(P0.01),心肌细胞凋亡指数升高(P0.05)。而异丙酚6mg/(kg·h)、12mg/(kg·h)组,NF-κB从细胞浆向细胞核的移位被明显限制,NF-κB的表达量也明显低于I/R组(P均0.05);心肌caspase-3表达减弱,心肌细胞凋亡指数减少(与I/R组相比,P0.05)。结论:异丙酚的心肌保护作用可能与其抑制NF-κB的活化,下调caspase-3的表达,从而抑制心肌细胞凋亡有关。     

Small GTPase Rho signaling is involved in beta1 integrin-mediated up-regulation of intercellular adhesion molecule 1 and receptor activator of nuclear factor kappaB ligand on osteoblasts and osteoclast maturation

We assessed the characteristics of human osteoblasts, focusing on small GTPase Rho signaling. Beta1 Integrin were highly expressed on osteoblasts. Engagement of beta1 integrins by type I collagen augmented expression of intercellular adhesion molecule 1 (ICAM-1) and receptor activator of nuclear factor kappaB ligand (RANKL) on osteoblasts. Rho was activated by beta1 stimulation in osteoblasts. Beta1 Integrin-induced up-regulation of ICAM-1 and RANKL was inhibited by transfection with adenoviruses encoding C3 transferase or pretreated with Y-27632, specific Rho and Rho-kinase inhibitors. Engagement of beta1 integrin on osteoblasts induced formation of tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cells (MNC) in a coculture system of osteoblasts and peripheral monocytes, but this action was completely abrogated by transfection of C3 transferase. Our results indicate the direct involvement of Rho-mediated signaling in beta1 integrin-induced up-regulation of ICAM-1 and RANKL and RANKL-dependent osteoclast maturation. Thus, Rho-mediated signaling in osteoblasts seems to introduce major biases to bone resorption.