Osteoclastic bone resorption through receptor tyrosine kinase and extracellular signal-regulated kinase signaling in mature osteoclasts

It has recently been suggested that signaling through receptor tyrosine kinases (RTKs) expressed on mature osteoclasts is involved in osteoclastic bone resorption. This study investigated the role and mechanism of two major RTKs expressed on mature osteoclasts, fibroblast growth factor receptor type 1 (FGFR1) and Tyro 3. Among the FGF receptors (FGFR1–4), only FGFR1 was detected on isolated mouse osteoclasts, while all FGFRs were identified on mouse osteoblasts. Tyro 3 was seen only in mature osteoclasts among bone cells. FGF-2 moderately stimulated pit formation by isolated rabbit osteoclasts at low concentrations (10^–12M), whereas at high concentrations (10^–9M) it strongly stimulated pit formation by unfractionated bone cells. Gas6, the ligand of Tyro 3, was expressed ubiquitously in bone cells and stimulated osteoclast function to form resorbed pits on a dentine slice. Both FGF-2 and Gas6 upregulated the phosphorylation of cellular proteins, including extracellular signal-regulated kinase (ERK), and increased the kinase activity of immunoprecipitated FGFR1 and Tyro 3, respectively, in mouse osteoclasts. The stimulation of these cytokines on mouse and rabbit osteoclast functions was abrogated by PD98059, a specific inhibitor of ERK. These results strongly suggest that these cytokines act directly on mature osteoclasts through the activation of RTKs and ERK, causing the stimulation of bone resorption.     

Effects of Ethyl Acetate Extract of Poncirus trifoliata Fruit for Glucocorticoid-Induced Osteoporosis

Poncirus trifoliata fruit (PTF) affects the digestive and cardiovascular systems, and kidney function. The authors studied the effects of ethyl acetate (EtOAc) extract of PTF on the activities of osteoblasts and in an animal model. The main compounds of the EtOAc extract, naringin and poncirin have been confi rmed by HPLC and NMR analysis. Effects of osteoblastic differentiation were mea-sured by alkaline phosphatase (ALP) activity, osteopontin (OPN) protein expression and osteoprotegerin (OPG) mRNA expression in MC3T3-E1 cells. Also, osteoclast differentiation was measured by multinucleated cells (MNCs) formation through tartrate resistance acid phosphatase (TRAP)-positive staining. Bone mineral density (BMD) was measured before and after treatment with EtOAc extract of PTF in prednisolone-induced osteoporotic mice. Dexamethasone (DEX) decreased OPN and OPG expression level in MC3T3-E1 cells and ALP activity was decreased by DEX dose-dependently. EtOAc extract of PTF recovered the levels of ALP activity, and the expression of OPN and OPG in MC3T3-E1 cells treated with DEX. In osteoclast differentiation, multinucleated TRAP-positive cell formation was significantly suppressed by the EtOAc extract of PTF. Total body BMD was restored by EtOAc extract of PTF in prednisolone-induced osteoporotic mice. In conclusion, EtOAc extract of PTF recovered DEX-mediated deteriorations in osteoblastic and osteoclastic functions, and increased BMD in glucocorticoid-induced osteoporosis.     

Water solution of onion crude powder inhibits RANKL-induced osteoclastogenesis through ERK, p38 and NF-κB pathways

Summary  Onion powder has been reported to decrease the ovariectomy-induced bone resorption of rats. However, the molecular mechanism of onion powder on the bone cells has not been reported. Here, we report that water solution of onion crude powder decreases the osteoclastogenesis from co-cultures of bone marrow stromal cells and macrophage cells. Additionally, water solution of onion crude powder inhibits the RANKL-induced ERK, p38 and NF-κB activation in macrophages. In summary, our data showed that onion powder may benefit bone through an anti-resorption effect on the osteoclasts. Introduction  A nutritional approach is important for both prevention and treatment of osteoporosis. Onion has been reported to decrease the ovariectomy-induced bone resorption. However, the functional effects of onion on the cultured osteoclasts and osteoblasts remain largely unknown. Here, we found that water solution of onion crude powder markedly inhibited the receptor activator of nuclear factor kappa B ligand (RANKL)-induced osteoclastogenesis through ERK, p38 and NF-κB pathways. Other studies were also designed to investigate the potential signaling pathways involved in onion-induced decrease in osteoclastogenesis. Methods  The osteoclastogenesis was examined using the TRAP staining method. The MAPKs and NF-κB pathways were measured using Western blot analysis. A transfection protocol was used to examine NF-κB activity. Results  Water solution of onion crude powder inhibited the RANKL plus M-CSF-induced osteoclastic differentiation from either bone marrow stromal cells or from RAW264.7 macrophage cells. Treatment of RAW264.7 macrophages with RANKL could induce the activation of ERK, p38 and NF-κB that was inhibited by water solution of onion crude powder. On the other hand, it did not affect the cell proliferation and differentiation of human cultured osteoblasts. Conclusions  Our data suggest that water solution of onion crude powder inhibits osteoclastogenesis from co-cultures of bone marrow stromal cells and macrophage cells via attenuation of RANKL-induced ERK, p38 and NF-κB activation. R.-S. Yang, and W.-M. Fu contributed equally to this study.     

Cbfa1基因调节骨形成与骨吸收的研究进展

骨的形成与吸收是一个涉及众多的基因和生长因子的复杂过程,凡可使骨形成下降和骨吸收增加的因素都会促进骨质疏松的发生.Cbfa1是成骨细胞分化和成熟的特异性转录因子,研究发现Cbfa1可通过促进骨形成和抑制骨吸收,对骨质疏松的发生、发展起到抑制作用,因此,我们期望随着Cbfa1基因研究的深入将为防治骨质疏松提供新思路.     

淫羊蕾苷抗骨质疏松研究进展

淫羊藿苷是中草药淫羊藿的主要有效成分之一,近年来对其在细胞水平的抗骨质疏松作用研究日益增多.笔者从骨髓间充质干细胞、成骨细胞和破骨细胞三方面入手,综述了淫羊藿苷抗骨质疏松的研究进展,并提出了今后的研究思路.     

T-cell leukemia translocation-associated gene (TCTA) protein is required for human osteoclastogenesis

Synovial tissues of patients with rheumatoid arthritis (RA) include factors regulating bone resorption, such as receptor activator NF-κB ligand (RANKL), TNFα, IL-6, IL-17 and IFNγ. However, in addition to these cytokines, other factors expressed in synovial tissues may play a role in resorbing bone. Here, our objective was to identify novel proteins expressed in synovial tissues of RA that regulate human osteoclastogenesis. Proteins were purified from synovial tissues of patients with RA, using gel filtration chromatography, ion-exchange chromatography, reverse-aspect HPLC, and mass spectrometry. We evaluated the effects of the purified fractions on human osteoclastogenesis induced by RANKL and M-CSF. We determined the amino acid sequences showing inhibitory activity on human osteoclastogenesis. In addition, we synthesized novel peptides from the molecule including the amino acid sequences. Then, we evaluated the effects of the peptides and antibodies against the molecule on human osteoclastogenesis from monocytes and mature osteoclasts, and on pit formation by mature osteoclasts using Osteologic^R discs. We examined the effect of the peptide on the expression of both mRNA and protein of NFATc1. We also examined the effect of RANKL on the expression of mRNA of the molecule on osteoclasts and macrophages. We identified a small peptide including Gly-Gln-Asn (GQN) with inhibitory activity on human osteoclastogenesis. We then found that GQN is included in the amino acid sequence of the extra-cellular domain of TCTA protein, which is expressed ubiquitously in normal human tissues, but whose function has not been clarified. We designed novel peptides, including GQN, from the sequence of TCTA protein. One of these peptides (29-mer), but not a scrambled peptide for the 29-mer peptide, potently inhibited RANKL-induced human osteoclastogenesis. The peptide also inhibited pit formation of mature human osteoclasts and suppressed the formation of large osteoclasts in the culture of mature osteoclasts. Furthermore, polyclonal antibodies against TCTA protein suppressed the formation of large osteoclasts in the cultures of both monocytes and mature osteoclasts, supporting our hypothesis. Peptide A did not significantly inhibit the expression of both mRNA and protein of NFATc1 in osteoclasts. Our novel peptide and polyclonal antibodies against the peptide inhibited human osteoclastogenesis and the function of mature osteoclasts, preventing cellular fusion by TCTA protein and a putative counterpart molecule.     

IL-12 inhibits TNF-α induced osteoclastogenesis via a T cell-independent mechanism in vivo

It has been reported that TNF-α plays an important role in bone resorption in pathological conditions. IL-12, which is a T cell mediator, is also an important inflammatory cytokine. We previously reported that IL-12 induces apoptosis in bone marrow cells treated with TNF-α in vitro via an interaction between TNF-α-induced Fas and IL-12-induced Fas ligand (FasL), and that, as a result, osteoclastogenesis is inhibited. The purpose of this study was to investigate the effects of IL-12 on TNF-α-mediated osteoclastogenesis in vivo. We administered TNF-α with and without IL-12 into the supracalvaria in mice. The numbers of osteoclasts in the sutures in the calvaria were higher in mice administered TNF-α than in control mice not administered TNF-α. The numbers of osteoclasts in mice administered both TNF-α and IL-12 were lower than those in mice administered only TNF-α. Next, we determined the levels of mRNAs for cathepsin K and tartrate-resistant acid phosphatase (TRAP). mRNA levels were increased in mice administered TNF-α compared with control mice, but not in mice administered both TNF-α and IL-12. We also evaluated the amounts of tartrate-resistant acid phosphatase 5b (TRACP 5b) in mouse sera. The levels of TRACP 5b in mice administered TNF-α were higher than those in control mice. On the other hand, in mice administered both TNF-α and IL-12, the levels were lower than those in mice administered TNF-α alone. Fas and FasL expression levels were analyzed by real-time RT-PCR. The levels of Fas mRNA were increased in the calvaria of mice administered TNF-α compared with control mice, while those of FasL mRNAs were increased in the calvaria of mice administered IL-12. In TdT-mediated dUTP-biotin nick end-labeling (TUNEL) assays, many apoptotic cells were found in the sutures in the calvaria of mice administered both TNF-α and IL-12. IL-12 also inhibited TNF-α-induced osteoclastogenesis in mice whose T cells were blocked by anti-CD4 and anti-CD8 antibodies. These results suggest that IL-12 inhibits TNF-α-mediated osteoclastogenesis and induces apoptotic changes through an interaction between TNF-α-induced Fas and IL-12-induced FasL, in vivo, via a T cell-independent mechanism.     

Docetaxel inhibits bone resorption through suppression of osteoclast formation and function in different manners

Osteoclasts are formed from the monocyte-macrophage lineage in response to receptor activator of nuclear factor κB ligand (RANKL) expressed by osteoblasts. Bone is the most common site of breast cancer metastasis, and osteoclasts play roles in the metastasis. The taxane-derived compounds paclitaxel and docetaxel are used for the treatment of malignant diseases, including breast cancer. Here we explored the effects of docetaxel on osteoclastic bone resorption in mouse culture systems. Osteoclasts were formed within 6 days in cocultures of osteoblasts and bone marrow cells treated with 1,25-dihydroxyvitamin D₃ plus prostaglandin E₂. Docetaxel at 10⁻⁸ M inhibited osteoclast formation in the coculture when added for the entire culture period or for the first 3 days. Docetaxel, even at 10⁻⁶ M added for the final 3 days, failed to inhibit osteoclast formation. Osteoprotegerin, a decoy receptor of RANKL, completely inhibited osteoclast formation when added for the final 3 days. Docetaxel at 10⁻⁸ M inhibited the proliferation of osteoblasts and bone marrow cells. RANKL mRNA expression induced by 1,25-dihydroxyvitamin D₃ plus prostaglandin E₂ in osteoblasts was not affected by docetaxel even at 10⁻⁶ M. Docetaxel at 10⁻⁶ M, but not at 10⁻⁸ M, inhibited pit-forming activity of osteoclasts cultured on dentine. Actin ring formation and l-glutamate secretion by osteoclasts were also inhibited by docetaxel at 10⁻⁶ M. Thus, docetaxel inhibits bone resorption in two different manners: inhibition of osteoclast formation at 10⁻⁸ M and of osteoclast function at 10⁻⁶ M. These results suggest that taxanes have beneficial effects in the treatment of bone metastatic cancers. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The generation of osteoclasts from RAW 264.7 precursors in defined,serum-free conditions

Osteoclasts are the unique cell type capable of resorbing bone. The discovery of the TNF-ligand family member, RANKL, has allowed more reliable study of these important cells. The mouse monocytic cell line, RAW 264.7, has been shown to readily differentiate into osteoclasts upon exposure to recombinant RANKL. Unlike primary osteoclast precursors, there is no requirement for the addition of macrophage colony stimulating factor (M-CSF). However, to date, their differentiation has always been studied in the context of added foetal calf serum (FCS). FCS is a complex and largely undefined mixture of growth factors and matrix proteins, and varies between batches. For this reason, osteoclastogenesis would ideally be studied in the context of a defined, serum-free medium. RAW 264.7 cells were cultured in serum-replete α-MEM or serum-deprived medium (SDM) shown previously to support the growth of human osteoclasts in a co-culture with normal osteoblasts. In SDM, in the presence of recombinant RANKL, RAW 264.7 cells readily differentiated into tartrate resistant acid phosphatase (TRAP) positive multinucleated osteoclast-like cells, a process that was enhanced with the addition of 1α,25-dihydroxyvitamin D₃ (1,25D). While the osteoclasts grown in SDM were smaller in size compared with those derived in serum-replete media, their resorptive capacity was significantly increased as indicated by a twofold increase in average resorption pit size. In conclusion, we describe a defined model for studying osteoclast differentiation and activity in the absence of serum, which will be ideal for studying the role of agonistic and antagonistic molecules in this process.