The 4-1BB ligand and 4-1BB expressed on osteoclast precursors enhance RANKL-induced osteoclastogenesis via bi-directional signaling

The 4-1BB is a costimulatory molecule similar to the receptor activator of NF-kappaB ligand (RANKL), both of which are key factors for the differentiation of osteoclasts and are expressed mainly by activated T cells. The 4-1BB shares common signaling pathways with RANK, suggesting a potential role in osteoclastogenesis. In this study, the role of 4-1BB and 4-1BB ligand (4-1BBL) in osteoclastogenesis was investigated using 4-1BB(-/-) and 4-1BB(+/+) mice. Osteoclast precursors normally express 4-1BB and 4-1BBL after exposure to RANKL, which was confirmed by semi-quantitative RT-PCR and flow cytometry. The 4-1BB(-/- )mice had a slightly increased bone mass accompanied by a reduced osteoclastogenic ability of 4-1BB(-/-) bone marrow-derived macrophages (BMM) ex vivo. In addition, 4-1BB(-/-) BMM demonstrated hypophosphorylation of JNK and p38 and decreased induction of c-Fos in response to RANKL stimulation. Retroviral transduction of wild-type as well as partial-length 4-1BB, which lacks TNF receptor-associated factor 2-binding sites for signaling, restored the osteoclastogenic ability of 4-1BB(-/-) BMM. Furthermore, both recombinant 4-1BB and 4-1BBL enhanced RANKL-induced osteoclastogenesis by 4-1BB(+/+) BMM and the induction of c-Fos and NFATc1.Together, these results indicate that 4-1BBL and 4-1BB expressed on osteoclast precursors enhance RANKL-induced osteoclastogenesis via bi-directional signaling, findings that may delineate the complex nature of the 4-1BBL and 4-1BB interaction.     

Suppression of puerarin on polymethylmethacrylate-induced lesion of peri-implant by inhibiting NF-κB activation in vitro and in vivo

Puerarin (PR), a natural isoflavone isolated from Chinese traditional plant pueraria lobata, has attracted considerable attention due to its important biological and pharmacological activities. However, its effects on lesion of peri-implant and related mechanism of action are still not clear, which require further investigation. In this study, we evaluated the effects of PR on polymethylmethacrylate (PMMA)-induced lesion of peri-implant in vitro and in vivo, and explored its possible mechanism of action. Our results indicated that PR could inhibit PMMA-induced osteoclastogenesis in RAW264.7 cells with a dose-dependent manner in vitro and effectively down-regulate mRNA and protein expressions of matrix metalloprotein 9 (MMP-9), tumor necrosis factor (TNF)-α, interleukin (IL)-6, and receptor activator of nuclear factor (NF)-κB (RANK), primarily via the suppression of NF-κB signaling. Furthermore, we found that PMMA induction could directly cause the phosphorylation of IκB and significantly promote the nuclear translocation of p65 in RAW264.7 cells. In other words, PR was able to dose-dependently attenuate the PMMA-induced nuclear translocation of p65 in RAW264.7 cells. In vivo, PR was observed to attenuate PMMA-induced osteoclastogenesis, osteolysis, mRNA expressions of receptor activator of nuclear factor (NF)-κB ligand (RANKL) and RANK, as well as protein levels of MMP-9, TNF-α, IL-6, and p65 in a murine calvarial osteolysis model. These findings suggested that PR might be a potential therapeutic drug to lesion of peri-implant, and provided new insights for understanding its possible mechanism.     

Effects of Inositol 1,4,5-triphosphate on Osteoclast Differentiation in RANKL-induced Osteoclastogenesis

The receptor activator of NF-κB ligand (RANKL) signal is an activator of tumor necrosis factor receptor-associated factor 6 (TRAF6), which leads to the activation of NF-κB and other signal transduction pathways essential for osteoclastogenesis, such as Ca(2+) signaling. However, the intracellular levels of inositol 1,4,5-trisphosphate (IP(3)) and IP(3)-mediated cellular function of RANKL during osteoclastogenesis are not known. In the present study, we determined the levels of IP(3) and evaluated IP(3)-mediated osteoclast differentiation and osteoclast activity by RANKL treatment of mouse leukemic macrophage cells (RAW 264.7) and mouse bone marrow-derived monocyte/macrophage precursor cells (BMMs). During osteoclastogenesis, the expression levels of Ca(2+) signaling proteins such as IP(3) receptors (IP(3)Rs), plasma membrane Ca(2+) ATPase, and sarco/endoplasmic reticulum Ca(2+) ATPase type2 did not change by RANKL treatment for up to 6 days in both cell types. At 24 h after RANKL treatment, a higher steady-state level of IP(3) was observed in RAW264.7 cells transfected with green fluorescent protein (GFP)-tagged pleckstrin homology (PH) domains of phospholipase C (PLC) δ, a probe specifically detecting intracellular IP(3) levels. In BMMs, the inhibition of PLC with U73122 [a specific inhibitor of phospholipase C (PLC)] and of IP(3)Rs with 2-aminoethoxydiphenyl borate (2APB; a non-specific inhibitor of IP(3)Rs) inhibited the generation of RANKL-induced multinucleated cells and decreased the bone-resorption rate in dentin slice, respectively. These results suggest that intracellular IP(3) levels and the IP(3)-mediated signaling pathway play an important role in RANKL-induced osteoclastogenesis.     

The antibody atliximab attenuates collagen-induced arthritis by neutralizing AIMP1, an inflammatory cytokine that enhances osteoclastogenesis

ARS-interacting multifunctional protein 1 (AIMP1) induces production of inflammatory cytokines from immune cells. Since osteoclastogenesis is promoted by positive regulation of inflammatory cytokines, whether AIMP1 could promote osteoclastogenesis was investigated. AIMP1 induced osteoclastogenesis and acted synergistically with RANKL to promote osteoclastogenesis. Down-regulation of CD23, an AIMP1 receptor, abolished AIMP1-mediated osteoclastogenesis. Enzyme-linked immunosorbent assays showed that the AIMP1 level was significantly higher in the peripheral blood (PB) and synovial fluid of rheumatoid arthritis patients than in normal PB. A monoclonal antibody (clone 15B3AF) that blocked the cytokine activity of AIMP1 inhibited the AIMP1-mediated production of inflammatory cytokines. Clone 15B3AF inhibited the AIMP1-mediated osteoclastogenesis in vitro. We then cloned the complementary determining regions of clone 15B3AF and generated a chimeric antibody (atliximab). In a collagen-induced arthritis mouse model (CIA), atliximab administration significantly attenuated disease severity and improved various histopathological parameters. Three-dimensional micro-computed tomography scanning confirmed that atliximab enhanced the joint structures in CIA mice. Furthermore, atliximab decreased the expression of inflammatory cytokines in the serum and inflamed joints of CIA mice. Taken together, our findings suggest that AIMP1 exacerbates RA by promoting inflammation and osteoclastogenesis and that atliximab could be developed as a therapeutic antibody to target inflammatory diseases, including RA.     

ANGPTL4在调控骨巨细胞瘤细胞增殖、血管生成和破骨分化作用的实验研究

目的探讨人血管生成素样蛋白4(ANGPTL4)基因沉默对骨巨细胞瘤单核基质细胞(GCTSC)增殖、血管生成以及破骨分化的作用。方法体外培养GCTSC细胞系,采用ANGPTL4 shRNA、Scrambled shRNA转染作为ANGPTL4 shRNA组和阴性对照组,另设置无处理的空白对照组。采用实时荧光定量PCR(QPCR)和Western blotting法检测ANGPTL4 mRNA和蛋白表达。MTT法和流式细胞术检测细胞增殖和凋亡。将GCTSC细胞分别与人脐静脉内皮细胞(HUVECs)、小鼠骨髓单核细胞(BMM)共培养,观察HUVECs成管能力和BMM破骨分化能力。结果与空白对照组和阴性对照组比较,ANGPTL4 shRNA组ANGPTL4 mRNA(0.174±0.045)和蛋白表达量(0.098±0.020)均明显降低;而ANGPTL4 shRNA组GCTSC细胞增殖活性降低,凋亡率升高(P<0.05)。与HUVECs共培养后,ANGPTL4 shRNA组闭合管数量[(57.35±17.24)%]减少(P<0.05);与BMM共培养后,ANGPTL4 shRNA组TRAP染色阳性的破骨细胞样多核巨细胞数量[(48.36±21.79)%]减少(P<0.05)。结论沉默骨巨细胞瘤GCTSC细胞ANGPTL4基因表达后,细胞增殖活性降低,凋亡率增加,并且对肿瘤血管生成和多核巨细胞破骨分化有一定的抑制作用。     

Icariin protects against titanium particle-induced osteolysis and inflammatory response in a mouse calvarial model

Periprosthetic osteolysis and subsequent aseptic loosening are common in implant failure, a complication with revision surgery being the only established treatment. Wear particle-induced inflammation and extensive osteoclastogenesis play critical roles in periprosthetic osteolysis. A recent approach in limiting osteolysis is therefore focused on inhibiting osteoclastic bone resorption. This study aimed to investigate the potential impact of icariin, the major ingredient of Epimedium, on titanium particle-induced osteolysis in a mouse calvarial model. Eighty-four male C57BL/J6 mice were divided randomly into four groups. Mice in the sham group underwent sham surgery only, whereas animals in the vehicle, low- and high-concentration icariin groups received titanium particles. Mice in the low- and high-concentration icariin groups were gavage-fed with icariin at 0.1 or 0.3 mg/g/day, respectively, until sacrifice. Mice in the sham and vehicle groups received phosphate-buffered saline daily. After 2 weeks, mouse calvariae were collected for micro-computed tomography, histomorphometry and molecular analysis. Icariin significantly reduced particle-induced bone resorption compared with the vehicle group. Icariin also prevented an increase in receptor activator of nuclear factor kappa B ligand/osteoprotegerin ratio and subsequently suppressed osteoclast formation in titanium particle-charged calvariae. In addition, immunohistochemical analysis and enzyme-linked immunosorbent assay showed icariin significantly reduced expression and secretion of tumor necrosis factor-α, interleukin-1β and interleukin-6 in the calvariae of titanium-stimulated mice. Collectively, these results suggest that icariin represents a potential treatment for titanium particle-induced osteolysis and could be developed as a new therapeutic candidate for the prevention and treatment of aseptic loosening.     

Genetic background influences fluoride's effects on osteoclastogenesis

Excessive fluoride (F) can lead to abnormal bone biology. Numerous studies have focused on the anabolic action of F yet little is known regarding any action on osteoclastogenesis. Little is known regarding the influence of an individual's genetic background on the responses of bone cells to F. Four-week old C57BL/6J (B6) and C3H/HeJ (C3H) female mice were treated with NaF in the drinking water (0 ppm, 50 ppm and 100 ppm F ion) for 3 weeks. Bone marrow cells were harvested for osteoclastogenesis and hematopoietic colony-forming cell assays. Sera were analyzed for biochemical and bone markers. Femurs, tibiae, and lumbar vertebrae were subjected to microCT analysis. Tibiae and femurs were subjected to histology and biomechanical testing, respectively. The results demonstrated new actions of F on osteoclastogenesis and hematopoietic cell differentiation. Strain-specific responses were observed. The anabolic action of F was favored in B6 mice exhibiting dose-dependent increases in serum ALP activity (p < 0.001); in proximal tibia trabecular and vertebral BMD (tibia at 50&100 ppm, p = 0.001; vertebrae at 50 and 100 ppm, p = 0.023&0.019, respectively); and decrease in intact PTH and sRANKL (p = 0.045 and p < 0.001, respectively). F treatment in B6 mice also resulted in increased numbers of CFU-GEMM colonies (p = 0.025). Strain-specific accumulations in bone [F] were observed. For C3H mice, dose-dependent increases were observed in osteoclast potential (p < 0.001), in situ trabecular osteoclast number (p = 0.007), hematopoietic colony forming units (CFU-GEMM: p < 0.001, CFU-GM: p = 0.006, CFU-M: p < 0.001), and serum markers for osteoclastogenesis (intact PTH: p = 0.004, RANKL: p = 0.022, TRAP5b: p < 0.001). A concordant decrease in serum OPG (p = 0.005) was also observed. Fluoride treatment had no significant effects on bone morphology, BMD, and serum PYD cross-links in C3H suggesting a lack of significant bone resorption. Mechanical properties were also unaltered in C3H. In conclusion, short term F treatment at physiological levels has strain-specific effects in mice. The expected anabolic effects were observed in B6 and novel actions hallmarked by enhanced osteoclastogenesis shifts in hematopoietic cell differentiation in the C3H strain.     

Proinflammatory and osteoclastogenic effects of beta-tricalciumphosphate and hydroxyapatite particles on human mononuclear cells in vitro

Particulate wear debris can activate defence cells and osteoclasts at the bone–implant interface possibly leading to bone resorption and implant failure. Cellular responses and inflammatory effects have been reported for particulate hydroxyapatite (HA). However, the immunological effects of particulate beta-tricalciumphosphate (beta-TCP) have not been studied and the question of whether beta-TCP is more biocompatible in this regard as is HA remains to be determined. Therefore the present work investigates effects of endotoxin-free HA and beta-TCP particles of the same size (d₅₀ = 1 μm) and dose (SAR 10:1) on human peripheral blood mononuclear cells in vitro. The production of proinflammatory cytokines (TNF-alpha, IL-1beta, IL-8) and cytokines connected to osteoclast and dendritic cell differentiation (OPG, RANKL, M-CSF, GM-CSF) was determined by ELISA. After 6 and 18 h of incubation HA and beta-TCP caused a quite similar induction of TNF-alpha, IL-1beta and IL-8. Effects of particles on the production of M-CSF and OPG were not detectable. However, in sharp contrast to HA, beta-TCP caused less induction of GM-CSF and not any of RANKL, both known for promoting dendritic cells and osteoclastogenesis respectively. Therefore these in vitro data suggest that wear debris of beta-TCP poses lesser risk of the detrimental effects of osteoclast induction known from HA.     

Regulation of osteoblastogenesis and osteoclastogenesis by the other reproductive hormones, Activin and Inhibin

There is both cellular and physiological evidence demonstrating that both Activins and Inhibins regulate osteoblastogenesis and osteoclastogenesis, and regulate bone mass in vivo. Although Activins and Inhibins were initially isolated from the gonad, Activins are also produced and stored in bone, whereas Inhibins exert their regulation on bone cell differentiation and metabolism via endocrine effects. The accumulating data provide evidence that reproductive hormones, distinct from classical sex steroids, are important regulators of bone mass and bone strength. Given the well described dominant antagonism of Inhibin over Activin, as well as over BMPs and TGFβ, the gonadally derived Inhibins are important regulators of locally produced osteotrophic factors. Thus, the cycling Inhibins in females and diurnal changes in Inhibin B in males elicit temporal shifts in Inhibin levels (tone) that de-repress the pituitary. This fundamental action has the potential to de-repress locally stimulated changes in osteoblastogenesis and osteoclastogenesis, thereby altering bone metabolism.