Adiponectin inhibits induction of TNF-alpha/RANKL-stimulated NFATc1 via the AMPK signaling

We investigated here whether adiponectin can exhibit an inhibitory effect on tumor necrosis factor-alpha (TNF-alpha)- and receptor activator of nuclear factor-kappaB ligand (RANKL)-induced osteoclastogenesis by using RAW264 cell D clone with a high efficiency to form osteoclasts. Globular adiponectin (gAd) strongly inhibited TNF-alpha/RANKL-induced differentiation of osteoclasts by interfering with TNF receptor-associated factor 6 production and calcium signaling; consequently, the induction of nuclear factor of activated T cells c1 (NFATc1) was strongly inhibited. Moreover, we observed that inhibition of AMP-activated protein kinase abrogated gAd inhibition for TNF-alpha/RANKL-induced NFATc1 expression. Our data suggest that adiponectin acts as a potent regulator of bone resorption observed in diseases associated with cytokine activation.     

Daphnetin attenuates LPS-induced osteolysis and RANKL mediated osteoclastogenesis through suppression of ERK and NFATc1 pathways

Aseptic prosthetic loosening and periprosthetic infection resulting in inflammatory osteolysis is a leading complication of total joint arthroplasty (TJA). Excessive bone destruction around the bone and prosthesis interface plays a key role in the loosening prostheses leading to revision surgery. The bacterial endotoxins or implant-derived wear particles-induced inflammatory response is the major cause of the elevated osteoclast formation and activity. Thus, agents or compounds that can attenuate the inflammatory response and/or inhibit the elevated osteoclastogenesis and excessive bone resorption would provide a promising therapeutic avenue to prevent aseptic prosthetic loosening in TJA. Daphnetin (DAP), a natural coumarin derivative, is clinically used in Traditional Chinese Medicine for the treatment of rheumatoid arthritis due to its anti-inflammatory properties. In this study, we report for the first time that DAP could protect against lipopolysaccharide-induced inflammatory bone destruction in a murine calvarial osteolysis model in vivo. This protective effect of DAP can in part be attributed to its direct inhibitory effect on RANKL-induced osteoclast differentiation, fusion, and bone resorption in vitro. Biochemical analysis found that DAP inhibited the activation of the ERK and NFATc1 signaling cascades. Collectively, our findings suggest that DAP as a natural compound has potential for the treatment of inflammatory osteolysis.     

Helvolic acid attenuates osteoclast formation and function via suppressing RANKL-induced NFATc1 activation

Excessive osteoclast formation and function are considered as the main causes of bone lytic disorders such as osteoporosis and osteolysis. Therefore, the osteoclast is a potential therapeutic target for the treatment of osteoporosis or other osteoclast-related diseases. Helvolic acid (HA), a mycotoxin originally isolated from Aspergillus fumigatus , has been discovered as an effective broad-spectrum antibacterial agent and has a wide range of pharmacological properties. Herein, for the first time, HA was demonstrated to be capable of significantly inhibiting receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis and bone resorption in vitro by suppressing nuclear factor of activated T cells 1 (NFATc1) activation. This inhibition was followed by the dramatically decreased expression of NFATc1-targeted genes including Ctr (encoding calcitonin receptor), Acp5 (encoding tartrate-resistant acid phosphatase [TRAcP]), Ctsk (encoding cathepsin K), Atp6v0d2 (encoding the vacuolar H+ ATPase V0 subunit d2 [V-ATPase-d2]) and Mmp9 (encoding matrix metallopeptidase 9) which are osteoclastic-specific genes required for osteoclast formation and function. Mechanistically, HA was shown to greatly attenuate multiple upstream pathways including extracellular signal-regulated kinase (ERK) phosphorylation, c-Fos signaling, and intracellular Ca ²⁺ oscillation, but had little effect on nuclear factor-κB (NF-κB) activation. In addition, HA also diminished the RANKL-induced generation of intracellular reactive oxygen species. Taken together, our study indicated HA effectively suppressed RANKL-induced osteoclast formation and function. Thus, we propose that HA can be potentially used in the development of a novel drug for osteoclast-related bone diseases.     

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.     

GAP-43 ameliorates Podocyte injury by decreasing nuclear NFATc1 expression

Podocyte injury is sufficient to cause glomerulosclerosis and proteinuria, eventually leading to kidney failure. Previous studies found that podocytes and neurons had similar biological characteristics. Growth-associated protein-43 (GAP-43) is a growth cone protein in neurons, and a marker of axonal and synaptic growth. However, it is not known whether GAP-43 is expressed in podocytes. Compared with normal glomerular podocytes, GAP-43 was significantly reduced in patients with glomerular diseases. GAP-43 also significantly reduced in lipopolysaccharide (LPS)-treated podocytes. We found that the decreased expression of nephrin, the cell marker of the podocyte, was significantly recovered with GAP-43 overexpression. In contrast, the migration ability in LPS-treated podocyte was reduction after GAP-43 overexpressing. Moreover, overexpression of GAP-43 attenuated podocyte apoptosis by up-regulating the ratio of Bcl-2/Bax with LPS treatment. Finally, Plaue and Rcan1 which are downstream target gene of NFATc1 decreased with overexpression of GAP-43 podocytes. We concluded that GAP-43 attenuated podocyte injury by inhibiting calcineurin/NFATc1 signaling. The findings may provide a promising treatment for podocyte injury-related diseases.     

Diosmetin inhibits osteoclast formation and differentiation and prevents LPS-induced osteolysis in mice

Osteolytic bone diseases are closely linked to the over-activation of osteoclasts and enhancement of bone resorption. It has become a major health issue in orthopedic practice worldwide. Inhibition of osteoclasts is proposed to be the main treatment for osteolytic disorders. Diosmetin (DIO) is a natural flavonoid with properties of antioxidant, anti-infection, and antishock. The effect of DIO on osteoclast differentiation is poorly understood. In this study project, we found that DIO could inhibit osteoclastic formation induced by receptor activator of nuclear factor kappa-B ligand (RANKL) in a dose-dependent manner. The expression of the osteoclast differentiation marker genes, cathepsin K, nuclear factor of activated T-cells 1 (NFATc1), Acp5, Ctr, Atp6v0d2, and Mmp9 were also decreased by the treatment of DIO. In addition, DIO attenuated the formation of actin ring and the ability of bone resorption. Further, the western blotting showed that DIO inhibits the phosphorylation of the mitogen-activated protein kinases signaling pathway induced by RANKL, accompanied by the downregulation of NFATc1 and c-Fos expression. We also found that DIO could reduce the accumulation of reactive oxygen species (ROS) induced by RANKL. In vivo, the study revealed that DIO can significantly reduce LPS-induced osteolysis in mice. Collectively, our study shows that DIO can inhibit osteoclast formation and activation, and could serve as a potential therapeutic drug for osteolytic bone diseases.     

Secretory clusterin inhibits osteoclastogenesis by attenuating M-CSF-dependent osteoclast precursor cell proliferation

Secretory clusterin (sCLU)/apolipoprotein J is a multifunctional glycoprotein that is ubiquitously expressed in various tissues. Reduced sCLU in the joints of patients with bone erosive disease is associated with disease activity; however, its exact role has yet to be elucidated. Here, we report that CLU is expressed and secreted during osteoclastogenesis in mouse bone marrow-derived macrophages (BMMs) that are treated with receptor activator of nuclear factor kappa-B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). CLU-deficient BMMs obtained from CLU^−/− mice exhibited no significant alterations in OC differentiation in comparison with BMMs obtained from wild-type mice. In contrast, exogenous sCLU treatment significantly inhibited OC formation in both BMMs and OC precursor cultures. The inhibitory effect of sCLU was more prominent in BMMs than OC precursor cultures. Interestingly, treating BMMs with sCLU decreased the proliferative effects elicited by M-CSF and suppressed M-CSF-induced ERK activation of OC precursor cells without causing apoptotic cell death. This study provides the first evidence that sCLU reduces OC formation by inhibiting the actions of M-CSF, thereby suggesting its protective role in bone erosion.     

Osteoclast inhibitory peptide-1 (OIP-1) inhibits measles virus nucleocapsid protein stimulated osteoclast formation/activity

Paget's disease (PD) of bone is characterized by increased activity of large abnormal osteoclasts (OCLs) which contain paramyxoviral nuclear and cytoplasmic inclusions. MVNP gene expression has been shown to induce pagetic phenotype in OCLs. We previously characterized the osteoclast inhibitory peptide-1 (OIP-1/hSca) which inhibits OCL formation/bone resorption. OIP-1 is a glycophosphatidylinositol (GPI)-linked membrane protein containing a 79 amino acid extra cellular peptide and a 32 amino acid carboxy terminal GPI-linked peptide (c-peptide) which is critical for OCL inhibition. In this study, we demonstrate that OIP-1 c-peptide significantly decreased (43%) osteoclast differentiation of peripheral blood mononuclear cells from patients with PD. Also, OIP-1 treatment to normal human bone marrow mononuclear cells transduced with the MVNP inhibited (41%) osteoclast precursor (CFU-GM) growth in methyl-cellulose cultures. We further tested if OIP-1 overexpression in the OCL lineage in transgenic mice inhibits MVNP stimulated OCL formation. MVNP transduction and RANKL stimulation of OIP-1 mouse bone marrow cells showed a significant decrease (43%) in OCL formation and inhibition (38%) of bone resorption area compared to wild-type mice. Western blot analysis identified that OIP-1 decreased (3.5-fold) MVNP induced TRAF2 expression during OCL differentiation. MVNP or OIP-1 expression did not affect TRAF6 levels. Furthermore, OIP-1 expression resulted in a significant inhibition of MVNP stimulated ASK1, Rac1, c-Fos, p-JNK, and NFATc1 expression during OCL differentiation. These results suggest that OIP-1 inhibits MVNP induced pagetic OCL formation/activity through suppression of RANK signaling. Thus, OIP-1 may have therapeutic utility against excess bone resorption in patients with PD.     

一种新的人CNA1基因转录本的克隆和功能鉴定

钙调神经磷酸酶（calcineurin,CN）是唯一依赖于Ca2+和钙调蛋白（calmodulin,CaM）的丝氨酸/苏氨酸型蛋白磷酸酶,由1个催化亚基CNA和1个调节亚基CNB组成. CNA 有3种亚型,最常见的是由CNA1基因编码的α亚型（CNAα）. 在克隆CNA1基因cDNA的过程中,发现了1种新的人CNA1转录本-CNAα4. 与CNA1基因的其它转录本相比,CNAα4缺失第2外显子,其编码蛋白质由454个氨基酸组成,具有比其它3种CNAα亚型更短的磷酸酶催化结构域. CNAα4具有与CNAα1相似的CaM亲和力,但是其激活活化T细胞核因子（nuclear factor of activated T cells,NFAT）的活性明显强于CNAα1,提示CNAα4所缺失的氨基酸序列（Ala20 Thr86）并非CNA催化结构域所必需,相反,Ala²⁰-Thr⁸⁶缺失可能有助于其酶活性中心与NFAT的结合并发挥作用.     

Parthenolide inhibits osteoclast differentiation and bone resorbing activity by down-regulation of NFATc1 induction and c-Fos stability,during RANKL-mediated osteoclastogenesis

Parthenolide, a natural product derived from Feverfew, prevents septic shock and inflammation. We aimed to identify the effects of parthenolide on the RANKL (receptor activator of NF-κB ligand)-induced differentiation and bone resorbing activity of osteoclasts. In this study, parthenolide dose-dependently inhibited RANKL-mediated osteoclast differentiation in BMMs, without any evidence of cytotoxicity and the phosphorylation of p38, ERK, and IκB, as well as IκB degradation by RANKL treatment. Parthenolide suppressed the expression of NFATc1, OSCAR, TRAP, DC-STAMP, and cathepsin K in RANKL-treated BMMs. Furthermore, parthenolide down-regulated the stability of c-Fos protein, but could not suppress the expression of c-Fos. Overexpression of NFATc1 and c-Fos in BMMs reversed the inhibitory effect of parthenolide on RANKL-mediated osteoclast differentiation. Parthenolide also inhibited the bone resorbing activity of mature osteoclasts. Parthenolide inhibits the differentiation and bone-resolving activity of osteoclast by RANKL, suggesting its potential therapeutic value for bone destructive disorders associated with osteoclast-mediated bone resorption. [BMB Reports 2014; 47(8): 451-456]     

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.