Corilagin suppresses RANKL‐induced osteoclastogenesis and inhibits oestrogen deficiency‐induced bone loss via the NF‐κB and PI3K/AKT signalling pathways

Over‐activated osteoclastogenesis, which is initiated by inflammation, has been implicated in osteoporosis. Corilagin, a natural compound extracted from various medicinal herbaceous plants, such as Cinnamomum cassia, has antioxidant and anti‐inflammatory activities. We found that Corilagin suppressed osteoclast differentiation in a dose‐dependent manner, significantly decreased osteoclast‐related gene expression and impaired bone resorption by osteoclasts. Moreover, phosphorylation of members of the nuclear factor‐kappaB (NF‐κB) and PI3K/AKT signalling pathways was reduced by Corilagin. In a murine model of osteoporosis, Corilagin inhibited osteoclast functions in vivo and restored oestrogen deficiency‐induced bone loss. In conclusion, our findings suggested that Corilagin inhibited osteoclastogenesis by down‐regulating the NF‐κB and PI3K/AKT signalling pathways, thus showing its potential possibility for the treatment of osteoporosis.     

Asperpyrone A attenuates RANKL‐induced osteoclast formation through inhibiting NFATc1, Ca2+ signalling and oxidative stress

Imbalance of osteoblast and osteoclast in adult leads to a variety of bone‐related diseases, including osteoporosis. Thus, suppressing the activity of osteoclastic bone resorption becomes the main therapeutic strategy for osteoporosis. Asperpyrone A is a natural compound isolated from Aspergillus niger with various biological activities of antitumour, antimicrobial and antioxidant. The present study was designed to investigate the effects of Asperpyrone A on osteoclastogenesis and to explore its underlining mechanism. We found that Asperpyrone A inhibited RANKL‐induced osteoclastogenesis in a dose‐dependent manner when the concentration reached 1 µm, and with no cytotoxicity until the concentration reached to 10 µm. In addition, Asperpyrone A down‐regulated the mRNA and protein expression of NFATc1, c‐fos and V‐ATPase‐d2, as well as the mRNA expression of TRAcP and Ctsk. Furthermore, Asperpyrone A strongly attenuated the RNAKL‐induced intracellular Ca²⁺ oscillations and ROS (reactive oxygen species) production in the process of osteoclastogenesis and suppressed the activation of MAPK and NF‐κB signalling pathways. Collectively, Asperpyrone A attenuates RANKL‐induced osteoclast formation via suppressing NFATc1, Ca²⁺ signalling and oxidative stress, as well as MAPK and NF‐κB signalling pathways, indicating that this compound may become a potential candidate drug for the prevention or treatment of osteoporosis.     

A Lox/CHOP‐10 crosstalk governs osteogenic and adipogenic cell fate by MSCs

Accelerated marrow adipogenesis has been associated with ageing and osteoporosis and is thought to be because of an imbalance between adipogenic and osteogenic differentiation of mesenchymal stem cell (MSCs). We have previously found that lysyl oxidase (Lox) inhibition disrupts BMP4‐induced adipocytic lineage commitment and differentiation of MSCs. In this study, we found that lox inhibition dramatically up‐regulates BMP4‐induced expression of CCAAT/enhancer binding protein (C/EBP) homologous protein 10 (CHOP‐10), which then promotes BMP4‐induced osteogenesis of MSCs both in vitro and in vivo. Specifically, Lox inhibition or CHOP‐10 up‐regulation activated Wnt/β‐catenin signalling to enhance BMP4‐induced osteogenesis, with pro‐adipogenic p38 MAPK and Smad signalling suppressed. Together, we demonstrate that Lox/CHOP‐10 crosstalk regulates BMP4‐induced osteogenic and adipogenic fate determination of MSCs, presenting a promising therapeutic target for osteoporosis and other bone diseases.     

Oxymatrine exerts protective effects on osteoarthritis via modulating chondrocyte homoeostasis and suppressing osteoclastogenesis

Osteoarthritis (OA) is a common degenerative disease characterized by the progressive destruction both articular cartilage and the subchondral bone. The agents that can effectively suppress chondrocyte degradation and subchondral bone loss are crucial for the prevention and treatment of OA. Oxymatrine (OMT) is a natural compound with anti‐inflammatory and antitumour properties. We found that OMT exhibited a strong inhibitory effect on LPS‐induced chondrocyte inflammation and catabolism. To further support our results, fresh human cartilage explants were treated with LPS to establish an ex vivo degradation model, and the results revealed that OMT inhibited the catabolic events of LPS‐stimulated human cartilage and substantially attenuated the degradation of articular cartilage ex vivo. As subchondral bone remodelling is involved in OA progression, and osteoclasts are a unique cell type in bone resorption, we investigated the effects of OMT on osteoclastogenesis, and the results demonstrated that OMT suppresses RANKL‐induced osteoclastogenesis by suppressing the RANKL‐induced NFATc1 and c‐fos signalling pathway in vitro. Further, we found that the anti‐inflammatory and anti‐osteoclastic effects of oxymatrine are mediated via the inhibition of the NF‐κB and MAPK pathways. In animal studies, OMT suppressed the ACLT‐induced cartilage degradation, and TUNEL assays further confirmed the protective effect of OMT on chondrocyte apoptosis. MicroCT analysis revealed that OMT had an attenuating effect on ACLT‐induced subchondral bone loss in vivo. Taken together, these results show that OMT interferes with the vicious cycle associated with OA and may be a potential therapeutic agent for abnormal subchondral bone loss and cartilage degradation in osteoarthritis.     

Involvement of serum‐derived exosomes of elderly patients with bone loss in failure of bone remodeling via alteration of exosomal bone‐related proteins

Exosomes are secreted into the blood by various types of cells. These extracellular vesicles are involved in the contribution of exosomal proteins to osteoblastic or osteoclastic regulatory networks during the failure of bone remodeling, which results in age‐related bone loss. However, the molecular changes in serum‐derived exosomes (SDEs) from aged patients with low bone density and their functions in bone remodeling remain to be fully elucidated. We present a quantitative proteomics analysis of exosomes purified from the serum of the elderly patients with osteoporosis/osteopenia and normal volunteers; these data are available via Proteome Xchange with the identifier PXD006463. Overall, 1,371 proteins were identified with an overlap of 1,160 Gene IDs among the ExoCarta proteins. Bioinformatics analysis and in vitro studies suggested that protein changes in SDEs of osteoporosis patients are not only involved in suppressing the integrin‐mediated mechanosensation and activation of osteoblastic cells, but also trigger the differentiation and resorption of osteoclasts. In contrast, the main changes in SDEs of osteopenia patients facilitated both activation of osteoclasts and formation of new bone mass, which could result in a compensatory elevation in bone remodeling. While the SDEs from aged normal volunteers might play a protective role in bone health through facilitating adhesion of bone cells and suppressing aging‐associated oxidative stress. This information will be helpful in elucidating the pathophysiological functions of SDEs and aid in the development of senile osteoporosis diagnostics and therapeutics.     

Neobavaisoflavone inhibits osteoclastogenesis through blocking RANKL signalling‐mediated TRAF6 and c‐Src recruitment and NF‐κB,MAPK and Akt pathways

Psoralea corylifolia (P corylifolia) has been popularly applied in traditional Chinese medicine decoction for treating osteoporosis and promoting fracture healing since centuries ago. However, the bioactive natural components remain unknown. In this study, applying comprehensive two‐dimensional cell membrane chromatographic/C18 column/time‐of‐flight mass spectrometry (2D CMC/C18 column/TOFMS) system, neobavaisoflavone (NBIF), for the first time, was identified for the bioaffinity with RAW 264.7 cells membranes from the extracts of P corylifolia. Here, we revealed that NBIF inhibited RANKL‐mediated osteoclastogenesis in bone marrow monocytes (BMMCs) and RAW264.7 cells dose dependently at the early stage. Moreover, NBIF inhibited osteoclasts function demonstrated by actin ring formation assay and pit‐formation assay. With regard to the underlying molecular mechanism, co‐immunoprecipitation showed that both the interactions of RANK with TRAF6 and with c‐Src were disrupted. In addition, NBIF inhibited the phosphorylation of P50, P65, IκB in NF‐κB pathway, ERK, JNK, P38 in MAPKs pathway, AKT in Akt pathway, accompanied with a blockade of calcium oscillation and inactivation of nuclear translocation of nuclear factor of activated T cells cytoplasmic 1 (NFATc1). In vivo, NBIF inhibited osteoclastogenesis, promoted osteogenesis and ameliorated bone loss in ovariectomized mice. In summary, P corylifolia‐derived NBIF inhibited RANKL‐mediated osteoclastogenesis by suppressing the recruitment of TRAF6 and c‐Src to RANK, inactivating NF‐κB, MAPKs, and Akt signalling pathways and inhibiting calcium oscillation and NFATc1 translocation. NBIF might serve as a promising candidate for the treatment of osteoclast‐associated osteopenic diseases.     

Deficient invariant natural killer T cells had impaired regulation on osteoclastogenesis in myeloma bone disease

Recent research showed that invariant natural killer T (iNKT) cells take part in the regulation of osteoclastogenesis. While the role of iNKT cells in myeloma bone disease (MBD) remains unclear. In our study, the quantity of iNKT cells and the levels of cytokines produced by them were measured by flow cytometry. iNKT cells and osteoclasts were induced from peripheral blood mononuclear cells after activation by α‐GalCer or RANKL in vitro. Then, gene expressions and the levels of cytokines were determined by RT‐PCR and ELISA, respectively. The results showed that the quantity of iNKT and production of IFN‐γ by iNKT cells were significantly decreased in newly diagnosed MM (NDMM), and both negatively related with severity of bone disease. Then, the osteoclasts from healthy controls were cultured in vitro and were found to be down‐regulated after α‐GalCer‐stimulated, while there was no significant change with or without α‐GalCer in NDMM patients, indicating that the regulation of osteoclastogenesis by iNKT cells was impaired. Furthermore, the inhibition of osteoclastogenesis by iNKT cells was regulated by IFN‐γ production, which down‐regulated osteoclast‐associated genes. In conclusion, the role of α‐GalCer‐stimulated iNKT cells in regulation of osteoclastogenesis was impaired in MBD, as a result of iNKT cell dysfunction.     

Galangin suppresses RANKL-induced osteoclastogenesis via inhibiting MAPK and NF-κB signalling pathways

Osteoclasts play a critical role in osteoporosis; thus, inhibiting osteoclastogenesis is a therapeutic strategy for osteoporosis. Galangin, a natural bioflavonoid extracted from a traditional Chinese herb, possesses a variety of biological activities, including anti-inflammation and anti-oxidation. However, its effects on osteoporosis have not been elucidated. In this study, we found that galangin treatment dose-dependently decreased osteoclastogenesis in bone marrow–derived macrophages (BMMs). Moreover, during osteoclastogenesis, osteoclast-specific genes, such as tartrate-resistant acid phosphatase (TRAP), cathepsin K (CtsK), ATPase, H + transporting, lysosomal V0 subunit D2 (V-ATPase d2) and dendritic cell–specific transmembrane protein (DC-STAMP), were down-regulated by galangin treatment. Furthermore, the results of the pit formation assay and F-actin ring staining revealed impaired osteoclastic bone resorption in the galangin-treated group compared with that in the control group. Additionally, galangin treatment also inhibited the phosphorylation of p38 and ERK of MAPK signalling pathway, as well as downstream factors of NFATc1, C-Jun and C-Fos. Consistent with our in vitro results, galangin suppressed lipopolysaccharide (LPS)-induced bone resorption via inhibition of osteoclastogenesis. Taken together, our findings provide evidence that galangin is a promising natural compound for the treatment of osteoporosis and may be associated with the inhibition of MAPK and NF-κB signalling pathways.     

Cytisine attenuates bone loss of ovariectomy mouse by preventing RANKL‐induced osteoclastogenesis

Postmenopausal Osteoporosis (PMOP) is oestrogen withdrawal characterized of much production and activation by osteoclast in the elderly female. Cytisine is a quinolizidine alkaloid that comes from seeds or other plants of the Leguminosae (Fabaceae) family. Cytisine has been shown several potential pharmacological functions. However, its effects on PMOP remain unknown. This study designed to explore whether Cytisine is able to suppress RANKL‐induced osteoclastogenesis and prevent the bone loss induced by oestrogen deficiency in ovariectomized (OVX) mice. In this study, we investigated the effect of Cytisine on RAW 264.7 cells and bone marrow monocytes (BMMs) derived osteoclast culture system in vitro and observed the effect of Cytisine on ovariectomized (OVX) mice model to imitate postmenopausal osteoporosis in vivo. We found that Cytisine inhibited F‐actin ring formation and tartrate‐resistant acid phosphatase (TRAP) staining in dose‐dependent ways, as well as bone resorption by pit formation assays. For molecular mechanism, Cytisine suppressed RANK‐related trigger RANKL by phosphorylation JNK/ERK/p38‐MAPK, IκBα/p65‐NF‐κB, and PI3K/AKT axis and significantly inhibited these signalling pathways. However, the suppression of PI3K‐AKT‐NFATc1 axis was rescued by AKT activator SC79. Meanwhile, Cytisine inhibited RANKL‐induced RANK‐TRAF6 association and RANKL‐related gene and protein markers such as NFATc1, Cathepsin K, MMP‐9 and TRAP. Our study indicated that Cytisine could suppress bone loss in OVX mouse through inhibited osteoclastogenesis. All data provide the evidence that Cytisine may be a promising agent in the treatment of osteoclast‐related diseases such as osteoporosis.     

Caffeic acid phenethyl ester,an active component of honeybee propolis attenuates osteoclastogenesis and bone resorption via the suppression of RANKL‐induced NF‐κB and NFAT activity

Receptor activator NF‐κB ligand (RANKL)‐activated signaling is essential for osteoclast differentiation, activation and survival. Caffeic acid phenethyl ester (CAPE), a natural NF‐κB inhibitor from honeybee propolis has been shown to have anti‐tumor and anti‐inflammatory properties. In this study, we investigated the effect of CAPE on the regulation of RANKL‐induced osteoclastogenesis, bone resorption and signaling pathways. Low concentrations of CAPE (<1 µM) dose dependently inhibited RANKL‐induced osteoclastogenesis in RAW264.7 cell and bone marrow macrophage (BMM) cultures, as well as decreasing the capacity of human osteoclasts to resorb bone. CAPE inhibited both constitutive and RANKL‐induced NF‐κB and NFAT activation, concomitant with delayed IκBα degradation and inhibition of p65 nuclear translocation. At higher concentrations, CAPE induced apoptosis and caspase 3 activities of RAW264.7 and disrupts the microtubule network in osteoclast like (OCL) cells. Taken together, our findings demonstrate that inhibition of NF‐κB and NFAT activation by CAPE results in the attenuation of osteoclastogenesis and bone resorption, implying that CAPE is a potential treatment for osteolytic bone diseases. J. Cell. Physiol. 221: 642–649, 2009. © 2009 Wiley‐Liss, Inc.     

Dehydrocostus lactone attenuates osteoclastogenesis and osteoclast‐induced bone loss by modulating NF‐κB signalling pathway

Osteolysis is characterized by overactivated osteoclast formation and potent bone resorption. It is enhanced in many osteoclast‐related diseases including osteoporosis and periprosthetic osteolysis. The shortage of effective treatments for these pathological processes emphasizes the importance of screening and identifying potential regimens that could attenuate the formation and function of osteoclasts. Dehydrocostus lactone (DHE) is a natural sesquiterpene lactone containing anti‐inflammatory properties. Here, we showed that DHE suppressed receptor activator of nuclear factor‐κB ligand (RANKL)‐induced osteoclast formation and osteoclast marker gene expression. It also inhibited F‐actin ring formation and bone resorption in a dose‐dependent manner in vitro. Moreover, DHE inhibited the RANKL‐induced phosphorylation of NF‐κB, mitigated bone erosion in vivo in lipopolysaccharide‐induced inflammatory bone loss model and particle‐induced calvarial osteolysis model. Together, these results suggest that DHE reduces osteoclast‐related bone loss via the modulation of NF‐κB activation during osteoclastogenesis indicating that it might be a useful treatment for osteoclast‐related skeletal disorders.     

Guaiacol suppresses osteoclastogenesis by blocking interactions of RANK with TRAF6 and C‐Src and inhibiting NF‐κB,MAPK and AKT pathways

Angelica sinensis (AS; Dang Gui), a traditional Chinese herb, has for centuries been used for the treatment of bone diseases, including osteoporosis and osteonecrosis. However, the effective ingredient and underlying mechanisms remain elusive. Here, we identified guaiacol as the active component of AS by two‐dimensional cell membrane chromatography/C18 column/time‐of‐flight mass spectrometry (2D CMC/C18 column/TOFMS). Guaiacol suppressed osteoclastogenesis and osteoclast function in bone marrow monocytes (BMMCs) and RAW264.7 cells in vitro in a dose‐dependent manner. Co‐immunoprecipitation indicated that guaiacol blocked RANK‐TRAF6 association and RANK‐C‐Src association. Moreover, guaiacol prevented phosphorylation of p65, p50, IκB (NF‐κB pathway), ERK, JNK, c‐fos, p38 (MAPK pathway) and Akt (AKT pathway), and reduced the expression levels of Cathepsin K, CTR, MMP‐9 and TRAP. Guaiacol also suppressed the expression of nuclear factor of activated T‐cells cytoplasmic 1(NFATc1) and the RANKL‐induced Ca²⁺ oscillation. In vivo, it ameliorated ovariectomy‐induced bone loss by suppressing excessive osteoclastogenesis. Taken together, our findings suggest that guaiacol inhibits RANKL‐induced osteoclastogenesis by blocking the interactions of RANK with TRAF6 and C‐Src, and by suppressing the NF‐κB, MAPK and AKT signalling pathways. Therefore, this compound shows therapeutic potential for osteoclastogenesis‐related bone diseases, including postmenopausal osteoporosis.     

ZEB1‐mediated vasculogenic mimicry formation associates with epithelial–mesenchymal transition and cancer stem cell phenotypes in prostate cancer

The zinc finger E‐box‐binding homeobox 1 (ZEB1) induced the epithelial–mesenchymal transition (EMT) and altered ZEB1 expression could lead to aggressive and cancer stem cell (CSC) phenotypes in various cancers. Tissue specimens from 96 prostate cancer patients were collected for immunohistochemistry and CD34/periodic acid–Schiff double staining. Prostate cancer cells were subjected to ZEB1 knockdown or overexpression and assessment of the effects on vasculogenic mimicry formation in vitro and in vivo. The underlying molecular events of ZEB1‐induced vasculogenic mimicry formation in prostate cancer were then explored. The data showed that the presence of VM and high ZEB1 expression was associated with higher Gleason score, TNM stage, and lymph node and distant metastases as well as with the expression of vimentin and CD133 in prostate cancer tissues. Furthermore, ZEB1 was required for VM formation and altered expression of EMT‐related and CSC‐associated proteins in prostate cancer cells in vitro and in vivo. ZEB1 also facilitated tumour cell migration, invasion and clonogenicity. In addition, the effects of ZEB1 in prostate cancer cells were mediated by Src signalling; that is PP2, a specific inhibitor of the Src signalling, dose dependently reduced the p‐Src⁵²⁷ level but not p‐Src⁴¹⁶ level, while ZEB1 knockdown also down‐regulated the level of p‐Src⁵²⁷ in PC3 and DU‐145 cells. PP2 treatment also significantly reduced the expression of VE‐cadherin, vimentin and CD133 in these prostate cancer cells. Src signalling mediated the effects of ZEB1 on VM formation and gene expression.     

Tn antigen promotes human colorectal cancer metastasis via H‐Ras mediated epithelial‐mesenchymal transition activation

Tn antigen is a truncated O‐glycan, frequently detected in colorectal cancer (CRC), but its precise role in CRC metastasis is not well addressed. Here we investigated the effects of Core 1 β3Gal‐T specific molecular chaperone (Cosmc) deletion‐mediated Tn antigen exposure on CRC metastasis and its underlying mechanism. We first used CRISPR/Cas9 technology to knockout Cosmc, which is required for normal O‐glycosylation, and thereby obtained Tn‐positive CRC cells. We then investigated the biological consequences of Tn antigen expression in CRC. The results showed that Tn‐positive cells exhibited an enhanced metastatic capability both in vitro and in vivo. A further analysis indicated that Tn antigen expression induced typical activation of epithelial‐mesenchymal transition (EMT). Mechanistically, we found that H‐Ras, which is known to drive EMT, was markedly up‐regulated in Tn‐positive cells, whereas knockdown of H‐Ras suppressed Tn antigen induced activation of EMT. Furthermore, we confirmed that LS174T cells (Tn‐positive) transfected with wild‐type Cosmc, thus expressing no Tn antigen, had down‐regulation of H‐Ras expression and subsequent inhibition of EMT process. In addition, analysis of 438 samples in TCGA cohort demonstrated that Cosmc expression was reversely correlated with H‐Ras, underscoring the significance of Tn antigen‐H‐Ras signalling in CRC patients. These data demonstrated that Cosmc deletion‐mediated Tn antigen exposure promotes CRC metastasis, which is possibly mediated by H‐Ras‐induced EMT activation.