Regulatory mechanism of NFATc1 in RANKL-induced osteoclast activation

NFATc1 is a master regulator of RANKL-induced osteoclast differentiation and herein we investigate the regulatory mechanism of NFATc1 in osteoclast activation. Inactivation of NFATc1 strongly attenuates RANKL-induced bone resorption and overexpression of a constitutively active form of NFATc1 in osteoclasts induces formation of actin rings and resorption pits on dentin slices. We demonstrate that NFATc1 binds directly to the promoter regions of its target genes and induces expression of various genes, including LTBP3, ClC7, cathepsin K, MMP9, and c-Src, which are key players in bone resorption. Thus, NFATc1 is essential for RANKL-induced osteoclast activation via up-regulation of osteoclast-activating genes.     

吴茱萸碱抑制破骨细胞分化延缓骨丢失的研究

目的探讨吴茱萸碱对破骨细胞分化与骨吸收功能的调控及对骨质疏松症的治疗作用。 方法取小鼠原代骨髓来源巨噬细胞分别给予0、10、20、50、100、200 μmol/L吴茱萸碱处理,CCK8检测细胞活力;然后利用原代骨髓来源巨噬细胞给予小鼠重组可溶性核因子κB受体活化因子配体与集落刺激因子行破骨细胞分化诱导,分别给予20与50 μmol/L吴茱萸碱干预。抗酒石酸酸性磷酸酶（TRAP）染色检测破骨细胞形成能力,荧光定量PCR分析破骨细胞分化相关基因表达,免疫荧光检测F肌动蛋白（F-actin）形成,扫描电镜观察破骨细胞骨吸收能力。7月龄C57BL/6小鼠灌胃给予100与200 mg/kg吴茱萸碱,给药3个月后Micro-CT检测小鼠骨密度与骨质量。采用单因素方差分析和t检验进行统计学分析。 结果CCK8结果显示,与对照组相比,给予10、20、50、100 μmol/L吴茱萸碱处理后细胞活力无明显变化,差异无统计学意义（P > 0.05）;而给予200 μmol/L吴茱萸碱的细胞活力下降（100.64±0.18比47.54±5.58）,差异具有统计学意义（P < 0.01）。与对照组相比,20 μmol/L吴茱萸碱的TRAP染色阳性细胞数[（200.57±28.35）比（142.29±19.21）个]、Trap （1.00±0.13比0.55±0.16）、组织蛋白酶K（Ctsk） （1.01±0.17比0.59±0.11）mRNA水平、骨吸收面积比（1.00±0.15比0.79±0.19）均减少,差异有统计学意义（P < 0.05）。与对照组相比,50 μmol/L吴茱萸碱的TRAP阳性细胞数[（200.57±28.35）比（112.71±12.18）个]、Trap （1.00±0.13比0.46±0.17）、Ctsk（1.01±0.17比0.49±0.12）、树突状细胞-特异性跨膜蛋白（DC- Stamp） （1.00±0.10比0.55±0.14）、c-Fos （1.01±0.10比0.58±0.14）、活化T细胞核因子c1 （Nfatc1） （1.00±0.10比0.59±0.14）、H+转运ATP酶v0亚基d2 （Atp6v0d2）的mRNA表达（1.00±0.10比0.59±0.18）、F-actin数量[（165.00± 18.50）比（98.33±21.15）个]和骨吸收面积比（1.00±0.15比0.62±0.10）均降低,差异有统计学意义（P < 0.05）。Micro-CT结果显示,与生理盐水组相比,100 mg/kg吴茱萸碱组小鼠骨密度有一定升高[（0.19±0.03）比（0.21±0.01）g/cm³],但差异无统计学意义（P > 0.05）;与生理盐水组相比,200 mg/kg吴茱萸碱组小鼠胫骨的骨密度[（0.19±0.03）比（0.23±0.01）g/cm³]、骨体积比[（9.79±1.39）﹪比（11.62±1.18）﹪]、骨小梁数量[（2.43±0.29）比（3.08±0.43）/mm]上升,骨小梁分离度[（0.44±0.06）比（0.27±0.05）mm]下降,差异具有统计学意义（P < 0.05）。 结论吴茱萸碱通过抑制破骨细胞分化与骨吸收功能延缓小鼠骨量丢失。     

Glutathione accelerates osteoclast differentiation and inflammatory bone destruction

Chronic inflammation associated with bone tissues often destructs bones, which is essentially performed by osteoclasts in the presence of immunoregulatory molecules. Hence, regulating osteoclastogenesis is crucial to develop therapeutics for bone-destructive inflammatory diseases. It is believed that reactive oxygen species (ROS) are involved in receptor activator of NF-κB (RANK) ligand (RANKL)-induced osteoclast differentiation, and, therefore, glutathione (GSH), the most abundant endogenous antioxidant, suppresses osteoclast differentiation and bone resorption by RANKL. Interestingly, GSH also contributes to inflammatory responses, and the effects of GSH on osteoclast differentiation and bone destruction under inflammatory conditions have not yet been determined. Here, we investigated how GSH affects inflammatory cytokine-stimulated osteoclast differentiation in vitro and in a mouse model of inflammatory bone destruction. We found that GSH significantly promoted TNFα-stimulated osteoclast formation, while an inhibitor of GSH synthesis, buthionine sulfoximine, suppressed it. GSH facilitated the nuclear localisation of the nuclear factor of activated T cells c1 (NFATc1) protein, a master regulator of osteoclastogenesis, as well as the expression of osteoclast marker genes in a dose-dependent manner. N-acetylcysteine, a substrate of GSH synthesis, also stimulated osteoclast formation and NFATc1 nuclear localisation. GSH did not suppress cell death after osteoclast differentiation. In mouse calvaria injected with lipopolysaccharide, GSH treatment resulted in a fivefold increase in the osteolytic lesion area. These results indicate that GSH accelerates osteoclast differentiation and inflammatory bone destruction, suggesting GSH appears to be an important molecule in the mechanisms responsible for inflammatory bone destruction by osteoclasts.     

高浓度地塞米松通过下调LMP-1表达抑制大鼠成骨细胞的分化

为探讨地塞米松（dexamethasone,DEX)抑制成骨细胞分化的机制,观察了不同浓度DEX对体外培养大鼠成骨细胞的碱性磷酸酶活性,骨钙素（osteocalcin,OC)合成,I型胶原蛋白表达的影响。并用RT-PCR方法检测了成骨细胞中LIM矿化蛋白1mRNA的表达量,结果显示：低浓度（10^-9mol/L）的DEX能增强碱性磷酸酶的活性、OC的分泌和I型胶原蛋白的表达;而高浓度（10^-7mol/L)的DEX对它们则起抑制作用,并下调成骨细胞正调节因子LMP-1mRNA的表达,上述结果表明,低浓度的DEX促进成骨细胞的分化;高浓度的DEX则抑制成骨细胞的分化,其抑制作用可能是通过下调LMP-1mRNA的表达而实现的。     

MiR-7b directly targets DC-STAMP causing suppression of NFATc1 and c-Fos signaling during osteoclast fusion and differentiation

DC-STAMP is a key regulating molecule of osteoclastogenesis and osteoclast precursor (OCP) fusion. Emerging lines of evidence showed that microRNAs play crucial roles in bone metabolism and osteoclast differentiation, but no microRNA has yet been reported to be directly related to OCPs fusion. Through a microarray, we found that the expression of miR-7b in RAW264.7 cells was significantly decreased after induction with M-CSF and RANKL. The overexpression of miR-7b in RAW264.7 cells attenuated the number of TRAP-positive cells number and the formation of multinucleated cells, whereas the inhibition of miR-7b enhanced osteoclastogenesis. Through a dual luciferase reporter assay, we confirmed that miR-7b directly targets DC-STAMP. Other fusogenic molecules, such as CD47, ATP6v0d2, and OC-STAMP, were detected to be down-regulated in accordance with the inhibition of DC-STAMP. Because DC-STAMP also participates in osteoclast differentiation through the ITAM-ITIM network, multiple osteoclast-specific genes in the ITAM-ITIM network were detected to identify how DC-STAMP is involved in this process. The results showed that molecules associated with the ITAM-ITIM network, such as NFATc1 and OSCAR, which are crucial in osteoclastogenesis, were consistently altered due to DC-STAMP inhibition. These findings suggest that miR-7b inhibits osteoclastogenesis and cell-cell fusion by directly targeting DC-STAMP. In addition, the inhibition of DC-STAMP and its downstream signals changed the expression of other fusogenic genes and key regulating genes, such as Nfatc1, c-fos, Akt, Irf8, Mapk1, and Traf6. In conclusion, our findings indicate that miR-7b may be a potential therapeutic target for the treatment of osteoclast-related bone disorders.     

Adiponectin inhibits osteoclastogenesis and bone resorption via APPL1-mediated suppression of Akt1

Adiponectin is an adipokine playing an important role in regulating energy homeostasis and insulin sensitivity. However, the effect of adiponectin on bone metabolism shows contradictory results according to different research studies. In this study femurs were isolated from genetically double-labeled mBSP9.0Luc/β-ACT-EGFP transgenic mice and were transplanted into adiponectin knock-out mice or wild type mice to investigate the effect of temporary exposure to adiponectin deficiency on bone growth and metabolism. We found that the growth of bone explants in adiponectin knock-out mice was significantly retarded. Histological analysis, microcomputed tomography analysis, and tartrate-resistant acid phosphatase staining revealed reduced trabecular bone volume, decreased cortical bone, and increased osteoclast number in bone explants in adiponectin knock-out mice. We then found that adiponectin inhibits RANKL-induced osteoclastogenesis from RAW264.7 cells and down-regulates RANKL-enhanced expressions of osteoclastogenic regulators including NFAT2, TRAF6, cathepsin K, and tartrate-resistant acid phosphatase. Adiponectin also increases osteoclast apoptosis and decreases survival/proliferation of osteoclast precursor cells. Using siRNA specifically targeting APPL1, the first identified adaptor protein of adiponectin signaling, we found that the inhibitory effect of adiponectin on osteoclasts was induced by APPL1-mediated down-regulation of Akt1 activity. In addition, overexpression of Akt1 successfully reversed adiponectin-induced inhibition in RANKL-stimulated osteoclast differentiation. In conclusion, adiponectin is important in maintaining the balance of energy metabolism, inflammatory responses, and bone formation.     

Puerarin inhibits the migration of osteoclast precursors and osteoclastogenesis by inhibiting MCP-1 production

ABSTRACT

Puerarin inhibits osteoclastogenesis and cells migration. This study aims to explore whether puerarin prevents osteoclastogenesis by inhibiting osteoclast precursors (OCPs) migration. The results showed that puerarin reduced MCP-1 production in OCPs, while inhibiting OCPs migration based on MCP-1. Puerarin reversed MCP-1-promoted osteoclastogenesis. CCR2 overexpression didn’t increase osteoclastogenesis with puerarin. Therefore, puerarin prevents OCPs migration by reducing MCP-1, whereby inhibiting osteoclastogenesis.     

Inhibition of matrix metalloproteinase-9 activity by doxycycline ameliorates RANK ligand-induced osteoclast differentiation in vitro and in vivo

Tetracycline antibiotics, including doxycycli\e (DOX), have been used to treat bone resorptive diseases, partially because of their activity to suppress osteoclastogenesis induced by receptor activator of nuclear factor kappa B ligand (RANKL). However, their precise inhibitory mechanism remains unclear. Therefore, the present study examined the effect of Dox on osteoclastogenesis signaling induced by RANKL, both in vitro and in vivo. Although Dox inhibited RANKL-induced osteoclastogenesis and down-modulated the mRNA expression of functional osteoclast markers, including tartrate-resistant acid phosphatase (TRAP) and cathepsin K, Dox neither affected RANKL-induced MAPKs phosphorylation nor NFATc1 gene expression in RAW264.7 murine monocytic cells. Gelatin zymography and Western blot analyses showed that Dox down-regulated the enzyme activity of RANKL-induced MMP-9, but without affecting its protein expression. Furthermore, MMP-9 enzyme inhibitor also attenuated both RANKL-induced osteoclastogenesis and up-regulation of TRAP and cathepsin K mRNA expression, indicating that MMP-9 enzyme action is engaged in the promotion of RANKL-induced osteoclastogenesis. Finally, Dox treatment abrogated RANKL-induced osteoclastogenesis and TRAP activity in mouse calvaria along with the suppression of MMP9 enzyme activity, again without affecting the expression of MMP9 protein. These findings suggested that Dox inhibits RANKL-induced osteoclastogenesis by its inhibitory effect on MMP-9 enzyme activity independent of the MAPK-NFATc1 signaling cascade.