Nuclear Factor Of Activated T-cells, Cytoplasmic 1 Expression Research Articles

Napyradiomycin B4 Suppresses RANKL-Induced Osteoclastogenesis and Prevents Alveolar Bone Destruction in Experimental Periodontitis.

The unique structure and beneficial biological properties of marine natural products have drawn interest in drug development. Here, we examined the therapeutic potential of napyradiomycin B4 isolated from marine-derived Streptomyces species for osteoclast-related skeletal diseases. Bone marrow-derived macrophages were treated with napyradiomycin B4 in an osteoclast-inducing medium, and osteoclast formation, osteoclast-specific gene expression, and nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) localization were evaluated using tartrate-resistant acid phosphatase staining, real-time PCR, and immunostaining, respectively. Phosphorylation levels of signaling proteins were assessed by immunoblot analysis to understand the molecular action of napyradiomycin B4. The in vivo efficacy of napyradiomycin B4 was examined under experimental periodontitis, and alveolar bone destruction was evaluated by microcomputed tomography (micro-CT) and histological analyses. Among the eight napyradiomycin derivatives screened, napyradiomycin B4 considerably inhibited osteoclastogenesis. Napyradiomycin B4 significantly suppressed the receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast formation and disrupted the expression of NFATc1 and its target genes. Mitogen-activated extracellular signal-regulated kinase (MEK) and extracellular signal-regulated kinase (ERK) phosphorylation levels were reduced by napyradiomycin B4 in response to RANKL. Under in vivo experimental periodontitis, napyradiomycin B4 significantly attenuated osteoclast formation and decreased the distance between the cementoenamel junction and alveolar bone crest. Our findings demonstrate the antiosteoclastogenic activity of napyradiomycin B4 by inhibiting the RANKL-induced MEK-ERK signaling pathway and its protective effect on alveolar bone destruction.

Inhibition of CD4 + T cells by fanchinoline via miR506-3p/NFATc1 in Sjögren's syndrome.

The hyperproliferation and hyperactivation of CD4 + T cells in salivary gland tissues are hallmarks of Sjögren's syndrome (SS). Fangchinoline (Fan) is extracted from the root of Stephania tetrandra Moore, which is used for treating rheumatic diseases in many studies. This study aimed to identify the mechanism underlying the inhibition of CD4 + T cells by Fan in the SS model NOD/ShiLtj mice. In vivo, Fan alleviated the dry mouth and lymphocyte infiltration in the salivary gland tissues of the NOD/ShiLtj mice and inhibited the number of CD4 + T cells in the infiltrating focus. In vitro, Fan's inhibitory effect on the proliferation of mouse primary CD4 + T cells was verified by CFSE and EdU tests. Furthermore, qRT-PCR and WB analysis confirmed that Fan could inhibit the expression of NFATc1 (Nuclear factor of activated T-cells, cytoplasmic 1) by upregulating miR-506-3p. Dual luciferase reporter gene assay suggested that miR-506-3p interacted with NFATc1. CFSE and EdU tests showed that Fan could inhibit the proliferation of CD4 + T cells through miR-506-3p/NFATc1. The key role of NFATc1 in the activation of CD4 + T cells and the high expression of NFATc1 in samples from SS patients suggested that NFATc1 might become a therapeutic target for SS. In vivo, 11R-VIVIT (NFATc1 inhibitor) alleviated SS-like symptoms. This study not only explained the new mechanism of Fan inhibiting proliferation of CD4 + T cells and alleviating SS-like symptoms but also provided NFATc1 as a potential target for the subsequent research and treatment of SS.

SIRT3-and FAK-mediated acetylation-phosphorylation crosstalk of NFATc1 regulates Nε-carboxymethyl-lysine-induced vascular calcification in diabetes mellitus

Background and aimsArterial calcification is the predictor of cardiovascular risk in diabetic patients. Nε-carboxymethyl-lysine (CML), a toxic metabolite, is associated with accelerated vascular calcification in diabetes mellitus (DM). However, the mechanism remains elusive. This study aims to explore the key regulators involved in CML-induced vascular calcification in DM. MethodsWe used Western blot and immuno-staining to test the expression and localization of nuclear factor of activated T cells, cytoplasmic 1 (NFATc1) in human samples, a diabetic apolipoprotein E-deficient (ApoE−/−) mouse model, and a vascular smooth muscle cells (VSMC) model. Further, we confirmed the regulator of NFATc1 phosphorylation and acetylation induced by CML. The role of NFATc1 in VSMCs calcification and osteogenic differentiation was explored in vivo and in vitro. ResultsIn diabetic patients, CML and NFATc1 levels increased in the severe calcified anterior tibial arteries. CML significantly promoted NFATc1 expression and nuclear translocation in VSMCs and mouse aorta. Knockdown of NFATc1 significantly inhibited CML-induced calcification. CML promoted NFATc1 acetylation at K549 by downregulating sirtuin 3 (SIRT3), which antagonized the focal adhesion kinase (FAK) induced NFATc1 phosphorylation at the Y270 site. FAK and SIRT3 affected the nuclear translocation of NFATc1 by regulating the acetylation-phosphorylation crosstalk. NFATc1 dephosphorylation mutant Y270F and deacetylation mutant K549R had opposite effects on VSMC calcification. SIRT3 overexpression and FAK inhibitor could reverse CML-promoted VSMC calcification. ConclusionsCML enhances vascular calcification in DM through NFATc1. In this process, CML increases NFATc1 acetylation by downregulating SIRT3 to antagonize FAK-induced NFATc1 phosphorylation.

NFATc1 marks articular cartilage progenitors and negatively determines articular chondrocyte differentiation.

The origin and differentiation mechanism of articular chondrocytes remain poorly understood. Broadly, the difference in developmental mechanisms of articular and growth-plate cartilage is still less elucidated. Here, we identified that the nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) is a crucial regulator of articular, but not growth-plate, chondrocyte differentiation during development. At the early stage of mouse knee development (embryonic day 13.5), NFATc1-expressing cells were mainly located in the flanking region of the joint interzone. With development, NFATc1-expressing cells generated almost all articular chondrocytes but not chondrocytes in limb growth-plate primordium. NFATc1-expressing cells displayed prominent capacities for colony formation and multipotent differentiation. Transcriptome analyses revealed a set of characteristic genes in NFATc1-enriched articular cartilage progenitors. Strikingly, the expression of NFATc1 was diminished with articular chondrocyte differentiation, and suppressing NFATc1 expression in articular cartilage progenitors was sufficient to induce spontaneous chondrogenesis while overexpressing NFATc1 suppresses chondrogenesis. Mechanistically, NFATc1 negatively regulated the transcriptional activity of the Col2a1 gene. Thus, our results reveal that NFATc1 characterizes articular, but not growth-plate, cartilage progenitors during development and negatively determines articular chondrocyte differentiation at least partly through regulating COL2A1 gene transcription.

Dental Implant Osseointegration Inhibition by Nicotine through Increasing nAChR, NFATc1 Expression, Osteoclast Numbers, and Decreasing Osteoblast Numbers.

The success of dental implants is determined by the osteointegration process. Many studies state that smoking cigarettes can inhibit osseointegration, but the inhibition mechanism is still unclear.The aim of this study was to identify and analyze the effect of nicotine on the inhibition of dental implant osseointegration through the expression of nicotinic acetylcholine receptor (nAChR), nuclear factor of activated T cells cytoplasmic 1 (NFATc1), osteoclast, and osteoblast numbers. This study is an experimental study of 16 New Zealand rabbits, randomized across two groups. Group 1 (eight rabbits) was a control group, and group 2 (eight rabbits) was a treatment group. The treatment group was given 2.5 mg/kg body weight/day of nicotine by injection 1 week before placement of the implant until the end of research. Observations were made in the first and the eighth week by measuring the number of osteoblast and osteoclast by immunohistology test and the expression of nAChR and NFATc1 by immunohistochemistry test. Data was analyzed using a one-way analysis of variance and Student's t-test. A p-value of < 0.05 was considered statistically significant. Significant differences were found between the control and treatment groups (p < 0.05). Results showed that nicotine increases the expression of nAChR and decreases the number of osteoblasts and the expression of BMP2 and osteocalcin. Nicotine inhibits the osseointegration of dental implants by increasing nAChR, NFATc1, osteoclast numbers, and decreasing osteoblast numbers.

Boric Acid Inhibits RANKL-Stimulated Osteoclastogenesis In Vitro and Attenuates LPS-Induced Bone Loss In Vivo.

Boron and boric acid (BA) can promote osteogenic differentiation and reduce bone resorption, which controls bone growth and maintenance of bone tissue. It has been reported that BA activates PERK-eIF2α signaling to induce cytoplasmic stress granules and cell senescence in human prostate DU-145 cells. However, whether BA can affect osteoclasts formation and LPS-induced inflammatory bone loss, and the role of the PERK-eIF2α pathway in the process, remains unknown. In vitro, RAW264.7 cells were pre-treated with boric acid (BA, 1, 10, 100μmol/L) for 4h, and then incubated with receptor activator of nuclear factor-kappaB ligand (RANKL, 50ng/mL) in the presence or absence of BA for 5days. CCK-8 and tartrate-resistant acid phosphatase (TRAP) were used to examine cell viability, osteoclastogenesis, and bone resorption; quantitative real-time PCR was performed to examine mRNA levels of c-Fos, nuclear factor of activated T cells, cytoplasmic 1 (NFATc1), TRAP, and cathepsin K; western blotting was used to examine protein expressions of glucose-regulated protein 78 (GRP78), protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), phosphorylated PERK (p-PERK), eukaryotic initiation factor 2α (eIF2α), and phosphorylated eIF2α (p-eIF2α). In vivo, lipopolysaccharide (LPS)-induced bone loss model in mice was established, and micro-computed tomography (micro-CT) scanning, bone biochemical analysis, and osteoclastogenic cytokines were detected to evaluate the effect of BA on LPS-induced bone loss. In our vitro results showed that BA treatment for 5days inhibited osteoclasts formation as well as osteoclastic bone resorption in a dose-dependent manner. The expression of osteoclasts marker genes c-Fos, NFATc1, TRAP, and cathepsin K were attenuated by BA. Immunoblotting analysis demonstrated that BA attenuated RANKL-induced PERK-eIF2α pathway activation. The in vivo data indicated that BA significantly prevented lipopolysaccharide (LPS)-induced bone loss. Our findings strongly suggest that BA may be a promising agent for the treatment of bone destructive diseases caused by excessive osteoclastogenesis.

Dieckol isolated from Eisenia bicyclis extract suppresses RANKL-induced osteoclastogenesis in murine RAW 264.7 cells

Objective: To demonstrate the effect of dieckol from Eisenia bicyclis on osteoclastogenesis using RAW 264.7 cells. Methods: Murine macrophage RAW 264.7 cells were subjected to dieckol treatment, followed by treatment with receptor activator of nuclear factor kappa-B ligand (RANKL) to induce osteoclastogenesis. Tartrate-resistant acid phosphatase (TRAP) activity was examined using a TRAP activity kit. Western blotting analysis was conducted to examine the level of osteoclast- related factors, including TRAP and calcitonin receptor (CTR), transcriptional factors, including c-Fos, c-Jun, and nuclear factor of activated T cells cytoplasmic 1 (NFATc1), nuclear factor kappa-B (NF-κB), extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK). Immunofluorescence staining was conducted to examine the expression of c-Fos, c-Jun, and NFATc1. Results: Among the four phlorotannin compounds present in Eisenia bicyclis, dieckol significantly hindered osteoclast differentiation and expression of RANKL-induced TRAP and CTR. In addition, dieckol downregulated the expression levels of c-Fos, c-Jun, NFATc1, ERK, and JNK, and suppressed NF-κB signaling. Conclusions: Dieckol can suppress RANKL-induced osteoclastogenesis. Therefore, it has therapeutic potential in treating osteoclastogenesis- associated diseases.

Long-Lasting Activity of ERK Kinase Depends on NFATc1 Induction and Is Involved in Cell Migration-Fusion in Murine Macrophages RAW264.7.

Macrophages are mononuclear cells that become osteoclasts (OCs) in the presence of two cytokines, macrophage colony-stimulating factor (M-CSF), and receptor activator of NF-κB ligand (RANKL). RANKL binding to its specific receptor RANK leads to OCs differentiation mainly by nuclear factor of activated T-cells cytoplasmic 1 (NFATc1). In our previous study, the analysis of the protein network in NFATc1-knockdown cells, using the Ingenuity Pathway Analysis (IPA), showed a link between NFATc1 and Mitogen-activated protein kinase kinase (MEK)-extracellular receptor kinase (ERK) signaling pathway. Therefore, this study aimed to extend our knowledge of the relationship between NFATc1 and the ERK. Here, we demonstrate that delayed ERK1/2 phosphorylation in pre-OC RANKL-induced depends on NFATc1. Indeed, the knockdown of NFATc1 reduced the phosphorylation of ERK1/2 (60%) and the pharmacological inhibition of the ERK1/2 kinase activity impairs the expression of NFATc1 without preventing its translocation into the nucleus. Furthermore, silencing of NFATc1 significantly reduced RANKL-induced migration (p < 0.01), and most pre-OCs are still mononuclear after 48 h (80 ± 5%), despite the presence of actin rings. On the other hand, the inhibitors FR180204 and PD98059 significantly reduced RANKL-induced cell migration (p < 0.01), leading to a reduction in the number of multinucleated cells. Finally, we suggest that long-lasting ERK activity depends on NFATc1 induction and is likely linked to cell migration, fusion, and OC differentiation.

Concentration and Duration of Indoxyl Sulfate Exposure Affects Osteoclastogenesis by Regulating NFATc1 via Aryl Hydrocarbon Receptor.

Indoxyl sulfate (IS) is a chronic kidney disease (CKD)-specific renal osteodystrophy metabolite that affects the nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), a transcription factor promoting osteoclastogenesis. However, the mechanisms underlying the regulation of NFATc1 by IS remain unknown. It is intriguing that the Aryl hydrocarbon receptor (AhR) plays a key role in osteoclastogenesis, since IS is an endogenous AhR agonist. This study investigates the relationship between IS concentration and osteoclast differentiation in Raw 264.7 cells, and examines the effects of different IS concentrations on NFATc1 expression through AhR signaling. Our data suggest that both osteoclastogenesis and NFATc1 are affected by IS through AhR signaling in both dose- and time-dependent manners. Osteoclast differentiation increases with short-term, low-dose IS exposure and decreases with long-term, high-dose IS exposure. Different IS levels switch the role of AhR from that of a ligand-activated transcription factor to that of an E3 ubiquitin ligase. We found that the AhR nuclear translocator may play an important role in the regulation of these dual functions of AhR under IS treatment. Altogether, this study demonstrates that the IS/AhR/NFATc1 signaling axis plays a critical role in osteoclastogenesis, indicating a potential role of AhR in the pathology and abnormality of bone turnover in CKD patients.

Dehydrocostus lactone inhibits NFATc1 via regulation of IKK, JNK, and Nrf2, thereby attenuating osteoclastogenesis

Excessive and hyperactive osteoclast activity causes bone diseases such as osteoporosis and periodontitis. Thus, the regulation of osteoclast differentiation has clinical implications. We recently reported that dehydrocostus lactone (DL) inhibits osteoclast differentiation by regulating a nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), but the underlying mechanism remains to be elucidated. Here we demonstrated that DL inhibits NFATc1 by regulating nuclear factor-κB (NF-κB), activator protein-1 (AP-1), and nuclear factor-erythroid 2-related factor 2 (Nrf2). DL attenuated IκBα phosphorylation and p65 nuclear translocation as well as decreased the expression of NF-κB target genes and c-Fos. It also inhibited c-Jun N-terminal kinase (JNK) but not p38 or extracellular signal-regulated kinase. The reporter assay revealed that DL inhibits NF-κB and AP-1 activation. In addition, DL reduced reactive oxygen species either by scavenging them or by activating Nrf2. The DL inhibition of NFATc1 expression and osteoclast differentiation was less effective in Nrf2-deficient cells. Collectively, these results suggest that DL regulates NFATc1 by inhibiting NF-κB and AP-1 via down-regulation of IκB kinase and JNK as well as by activating Nrf2, and thereby attenuates osteoclast differentiation.

TGFβ1 Regulates Human RANKL-Induced Osteoclastogenesis via Suppression of NFATc1 Expression.

Osteoclasts are multinucleated giant cells responsible for bone resorption. Various mediators involved in osteoclast differentiation have been investigated as possible therapeutic targets for osteoporosis and rheumatoid arthritis (RA). Although transforming growth factor beta1 (TGFβ1) has been described as one such multifunctional cytokine essential for bone remodeling, its effect on osteoclastogenesis remains controversial. Therefore, we sought to examine the effect of TGFβ1 on osteoclast generation induced by receptor activator of nuclear factor (NF)-κB ligand (RANKL) in humans. Peripheral blood monocytes, isolated using magnetic bead sorting, were cultured with macrophage-colony stimulating factor (M-CSF) or RANKL with or without TGFβ1. Tartrate-resistant acid phosphatase (TRAP) staining, as well as bone resorption assays, revealed that TGFβ1 suppressed RANKL-mediated human osteoclast development. Real-time reverse transcription PCR and Western blotting revealed that TGFβ1 reduced the gene and protein expression of nuclear factor of activated T cells, cytoplasmic 1 (NFATc1), the master regulator of osteoclast differentiation, respectively. Luciferase assays indicated that TGFβ1 inhibited the NF-κB p65-stimulated promoter activity of NFATc1. Immunofluorescence analysis demonstrated that TGFβ1 abrogated RANKL-induced nuclear translocation of p65. Thus, TGFβ1 regulates human RANKL-induced osteoclastogenesis via downregulation of NFATc1 by blocking nuclear translocation of NF-κB, suggesting that TGFβ1 may be a potential therapeutic target for RA.

FUN14 domain-containing 1 promotes breast cancer proliferation and migration by activating calcium-NFATC1-BMI1 axis

BackgroundFUN14 domain-containing 1 (FUNDC1), as a novel member of mitochondria-associated endoplasmic reticulum (ER) membranes associates with mitochondrial division and mitophagy. However, the expression profile and functional roles of FUNDC1 remain largely unclear in human cancer biology, including breast cancer (BC).MethodsImmunohistochemistry and western blot analysis were used to determine the expression of FUNDC1 and BMI1 polycomb ring finger oncogene (BMI1). CCK8, cell counting and transwell assays were used to analyze cell proliferation, migration and invasion, respectively. Luciferase reporter and chromatin immunoprecipitation (ChIP) assays were used to detect the transcriptional regulation of Nuclear factor of activated T-cells, cytoplasmic 1 (NFATC1). The prognostic merit of NFATC1 expression was assessed by Kaplan-Meier assay.FindingsImmunohistochemistry revealed strong immunostaining for FUNDC1 in cytoplasmic and nuclear membrane distribution in BC tissues as compared with normal breast epithelium. Kaplan–Meier survival analysis showed worse outcome for BC patients with high FUNDC1 expression. In vitro assay of gain- and loss-of-function of FUNDC1 suggested that FUNDC1 could stimulate BC cell proliferation, migration and invasion. Furthermore, elevated FUNDC1 level promoted Ca2+ cytosol influx from ER and extracellular, as well as NFATC1 nuclear translocation and activity. Nuclear NFATC1 bound to the BMI1 gene promoter and transcriptionally upregulated its expression. Notably, BMI1 overexpression could rescue the loss of function of FUNDC1. Co-expression of FUNDC1 and BMI1 in BC patients predicted worse prognosis than without either expression.InterpretationFUNDC1 might promote BC progression by activating the Ca2+–NFATC1–BMI1 axis. This pathway may be promising for developing multiple targets for BC therapy.

Rosiglitazone suppresses RANKL-induced NFATc1 autoamplification by disrupting the physical interaction between NFATc1 and PPARγ.

Receptor activator of nuclear factor‐κB ligand (RANKL) is required for initiation of osteoclastogenesis, with the signaling pathway including the NF‐kB, c‐Fos, and nuclear factor of activated T cells, cytoplasmic 1 (NFATc1) transcription factors. Because NFATc1 expression is autoamplified, we investigated the molecular mechanism by which peroxisome proliferator‐activated receptor gamma (PPARγ) activation by the thiazolidinedione drug rosiglitazone decreases NFATc1 expression during RANKL stimulation. Western blotting demonstrated that rosiglitazone attenuated the increase in NFATc1 protein level induced by RANKL without affecting that of PPARγ. Immunofluorescence data indicated that rosiglitazone tended to suppress RANKL‐induced NFATc1 nuclear translocation, partly by reducing calcineurin activity, as reflected by the observed decrease in nuclear NFATc1 abundance. On coimmunoprecipitation, the intensity of the physical interaction between NFATc1 and PPARγ was unexpectedly higher in the RANKL‐stimulated group than in the control, but rosiglitazone reduced this to basal levels. Furthermore, RANKL failed to elevate mRNA expression of NFATc1 after PPARγ knockdown. ChIP assay indicated that rosiglitazone significantly reduced the binding of NFATc1 to its own promoter despite RANKL stimulation. These findings suggest that PPARγ activation by rosiglitazone blocks NFATc1 from binding to its own promoter, thereby reducing RANKL‐induced NFATc1 autoamplification.

Eurycoma longifolia, a promising suppressor of RANKL-induced differentiation and activation of osteoclasts: An in vitro mechanistic evaluation

BackgroundEurycoma longifolia (E. longifolia) has gained remarkable recognition due to its promising efficacy of stimulating bone formation in androgen-deficient osteoporosis. Numerous in vivo studies have explored the effects of E. longifolia on osteoporosis; however, the in vitro cellular mechanism was not discovered yet. ObjectivesThe present study was aimed to investigate the effect of E. longifolia on the proliferation, differentiation and maturation of osteoclasts and the translational mechanism of inhibition of osteoclastogenesis using RAW 264.7 cells as an in vitro osteoclastic model. Materials and methodsHaving assessed cytotoxicity, the cell viability, cell proliferation rate and osteoclastic differentiation capacity of E. longifolia was investigated by evaluating the tartrate-resistant acid phosphatase (TRAP) activity in receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-induced osteoclasts. Taken together, the time-mannered expression of osteoclast-related protein biomarkers such as matrix metallopeptidase-9 (MMP-9), cathepsin-K, TRAP, nuclear factor of activated T-cells cytoplasmic 1 (NFATc1), superoxide (free radicals) generation and superoxide dismutase activity were also measured to comprehend the mechanism of osteoclastogenesis. ResultsE. longifolia did not show significant effects on cytotoxicity and cell proliferation of RAW 264.7 cells; however, a significant inhibition of cells differentiation and maturation of osteoclasts was observed. Moreover, a significant down-regulation of RANKL-induced TRAP activity and expression of MMP-9, cathepsin-K, TRAP, NFATc1 and generation of superoxide and enhanced superoxide dismutase activity was observed in E. longifolia treated cell cultures. ConclusionWe anticipated that E. longifolia that enhances bone regeneration on the one hand and suppresses osteoclast’s maturation on the other hand may have great therapeutic value in treating osteoporosis and other bone-erosive diseases such as rheumatoid arthritis and metastasis associated with bone loss.

The study of curcumin modulates nuclear factor-kappa B signaling pathway to inhibit wear particle inducted osteolysis in vitro

Objective To investigate the effect of curcumin on osteolysis through regulating nuclear factor-kappa B (NF-κB) signal pathway, by establishing the model of wear particle induced osteolysis in vitro. Methods Wear particles induced cellular model of osteolysis was build by co-culturing ultra high molecular weight polyethylene (UHMWPE) with mouse monocytes/macrophages RAW264.7 cells, intervented by adding curcumin. Four groups were involved: A: blank group; B: polyethylene particle induced group; C: polyethylene particle induced + low concentration of curcumin treatment group; D: poly-ethylene particle induced + high concentration of curcumin. Proteins were extracted from each group to detect the expression of the key protein inhibitor of NF-κB (IκB)and phosphorylated IκB (p-IκB) in the NF-κB pathway by using Western blotting. The same grouping method was used to detect the expression of transcription factor c-Fos and nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) by using real time polymerase chain reaction (Realtime-PCR). Results (1) Western blotting results: in group B, the grave value ratio of IκBα and β-actin (IκBα/β-actin) was 0.029±0.004, which was lower than the other three groups (P=0.001) (group A: 0.321±0.014, group C: 0.352±0.019, group D: 0.554±0.009). Comparing group C with group D, the P value was 0.001; Group B’s p-IκBα/β-actin was 1.450±0.081, which was higher than other three groups (P=0.001) (group A: 0.593±0.041, group C: 0.480±0.023, group D: 0.511±0.024). The comparison between group C and group D had no statistical significance (P=0.163). (2) Real-time PCR results: expression of c-Fos (1.243±0.040) and NFATc1 (1.255±0.009) was the highest. Group C’s c-Fos expression (0.868±0.032) was higher than group D’s (0.814±0.009) (P=0.015). Group C’s NFATc1 expression (0.956±0.016) was higher than group D’s (0.624±0.009) (P=0.001). Conclusion Curcumin can inhibit the occurrence of periprosthetic osteolysis by inhibiting the activation of NF-κB signal pathway in vitro. Key words: Curcumin; Wear particles; Aseptic loosening; Osteolysis

Dihydroartemisinin represses osteoclastogenesis of bone marrow macrophages through reduced NFATc1 expression and impaired phosphorylation of IκBα.

Osteoclasts are multinucleated bone resorbing cells whose differentiation is regulated by several important signaling pathways. Several lines of evidence indicate that dihydroartemisinin (DHA), an anti-malarial drug, inhibits osteoclast differentiation with little cytotoxicity. However, the detailed inhibitory mechanisms of DHA on osteoclastogenesis from native cells remain to be elucidated. In this study, we investigated the effects of DHA on the differentiation of bone marrow-derived macrophages into osteoclasts. DHA inhibited receptor activator of nuclear factor κ-B ligand (RANKL)-induced osteoclast formation and its bone resorbing activity. Mechanistically, DHA treatment markedly abolished phosphorylation of IκBα, and slightly affected a p38 MAPK dependent pathway. Moreover, DHA treatment induced down-regulation of nuclear factor of activated T cells cytoplasmic-1 (NFATc1), a master regulator for osteoclast differentiation and its target proteins, such as Src and cathepsin K. These results indicate that DHA represses RANKL-induced osteoclastogenesis of bone marrow macrophages through reduced NFATc1 expression and impaired phosphorylation of IκBα.

Inhibitory effects of sodium pentosan polysulfate on formation and function of osteoclasts derived from canine bone marrow

BackgroundSodium pentosan polysulfate (NaPPS) was testified as a chondroprotective drug in with a detailed rationale of the disease-modifying activity. This study was undertaken to determine whether anti-osteoarthritis drug, NaPPS inhibited osteoclasts (OC) differentiation and function. Canine bone marrow mononuclear cells (n = 6) were differentiated to OC by maintaining with receptor activator of nuclear factor kappa B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) for up to 7 days with the treatment of NaPPS at concentration of 0, 0.2, 1 and 5 μg/mL. Differentiation and function of OC were accessed using tartrate-resistant acid phosphate (TRAP) staining and bone resorption assay, while monitoring actin ring formation. Invasion and colocalization patterns of fluorescence-labeled NaPPS with transcribed gene in OC were monitored. Gene expression of OC for cathepsin K (CTK), matrix metallopeptidase-9 (MMP-9), nuclear factor of activated T-cells cytoplasmic 1 (NFATc1), c-Fos, activator protein-1(AP-1) and carbonic anhydrase II was examined using real-time PCR.ResultsSignificant inhibition of OC differentiation was evident at NaPPS concentration of 1 and 5 μg/mL (p < 0.05). In the presence of 0.2 to 5 μg/mL NaPPS, bone resorption was attenuated (p < 0.05), while 1 and 5 μg/mL NaPPS achieved significant reduction of actin ring formation. Intriguingly, fluorescence-labeled NaPPS invaded in to cytoplasm and nucleus while colocalizing with actively transcribed gene. Gene expression of CTK, MMP-9 and NFATc1 were significantly inhibited at 1 and 5 μg/mL (p < 0.05) of NaPPS whereas inhibition of c-Fos and AP-1 was identified only at concentration of 5 μg/mL (p < 0.05).ConclusionsTaken together, all the results suggest that NaPPS is a novel inhibitor of RANKL and M-CSF-induced CTK, MMP-9, NFATc1, c-Fos, AP-1 upregulation, OC differentiation and bone resorption which might be a beneficial for treatment of inflammatory joint diseases and other bone diseases associated with excessive bone resorption.

Molecular mechanisms of the inhibitory effects of jiangu granule‑containing serum on RANKL‑induced osteoclastogenesis.

Postmenopausal osteoporosis (PMOP) is characterized by increased bone loss due to enhanced osteoclastogenesis and bone resorption. A Chinese herbal formula, jiangugranule (JG), exhibited great efficacy in the clinical treatment of PMOP. However, the molecular mechanisms underlying the therapeutic effects remain unclear. The present study aimed to examine the effects of JG‑containing serum on receptor activator of nuclear factor‑κB (NF‑κB) ligand (RANKL)‑induced osteoclastogenesis. Osteoclast precursor RAW264.7 cells were cultured and treated with JG‑containing serum in the presence of RANKL. Following 6days of culture, the cells were stained with tartrate‑resistant acid phosphatase and the rate of differentiation was calculated. In addition, cells were treated with JG‑containing serum for 24, 48 and 96h and total RNA and proteins were extracted for reverse transcription‑quantitative polymerase chain reaction and western blot analysis to detect mRNA and protein expression, respectively, of key molecules in the RANK/RANKL signaling pathway, including RANK, tumor necrosis factor receptor‑associated factor 6, NF‑κB (p50 and p52 subunits), c‑Fos and nuclear factor of activated T cells, cytoplasmic 1 (NFATc1). The results revealed that JG‑containing serum inhibited RANKL‑induced osteoclastogenesis and reduced mRNA and protein expression of RANK, c‑Fos and NFATc1. The results suggested that JG may regulate osteoclast differentiation through the RANK/RANKL signaling pathway, which may be a possible mechanism for the therapeutic effects of JG on PMOP.

Effects of deficiency of Kelch-like ECH-associated protein 1 on skeletal organization: a mechanism for diminished nuclear factor of activated T cells cytoplasmic 1 during osteoclastogenesis.

Kelch-like ECH-associated protein 1 (Keap1) binds to nuclear factor E2 p45-related factor 2 (Nrf2), a transcription factor for antioxidant enzymes, to suppress Nrf2 activation. The role of oxidative stress in many diseases supports the possibility that processes that are associated with Nrf2 activation might offer therapeutic potential. Nrf2 deficiency induces osteoclastogenesis, which is responsible for bone loss, by activating receptor activator of NF-κB ligand (RANKL)-mediated signaling; however, the effects of Keap1 deficiency remain unclear. By using Keap1-deficient newborn mice, we observed that talus and calcaneus bone formation was partially retarded and that osteoclast number was reduced in vivo without severe gross abnormalities. In addition, Keap1-deficient macrophages were unable to differentiate into osteoclasts in vitrovia attenuation of RANKL-mediated signaling and expression of nuclear factor of activated T cells cytoplasmic 1 (NFATc1), a key transcription factor that is involved in osteoclastogenesis. Furthermore, Keap1 deficiency up-regulated the expression of Mafb, a negative regulator of NFATc1. RANKL-induced mitochondrial gene expression is required for down-regulation of IFN regulatory factor 8 (IRF-8), a negative transcriptional regulator of NFATc1. Our results indicate that Keap1 deficiency down-regulated peroxisome proliferator-activated receptor-γ coactivator 1β and mitochondrial gene expression and up-regulated Irf8 expression. These results suggest that the Keap1/Nrf2 axis plays a critical role in NFATc1 expression and osteoclastogenic progression.-Sakai, E., Morita, M., Ohuchi, M., Kido, M. A., Fukuma, Y., Nishishita, K., Okamoto, K., Itoh, K., Yamamoto, M., Tsukuba, T. Effects of deficiency of Kelch-like ECH-associated protein 1 on skeletal organization: a mechanism for diminished nuclear factor of activated T cells cytoplasmic 1 during osteoclastogenesis.

Dectin-1 signaling inhibits osteoclastogenesis via IL-33-induced inhibition of NFATc1.

Abnormal osteoclast activation contributes to osteolytic bone diseases (OBDs). It was reported that curdlan, an agonist of dectin-1, inhibits osteoclastogenesis. However, the underlying mechanisms are not fully elucidated. In this study, we found that curdlan potently inhibited RANKL-induced osteoclast differentiation and the resultant bone resorption. Curdlan inhibited the expression of nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), the key transcriptional factor for osteoclastogenesis. Notably, dectin-1 activation increased the expression of MafB, an inhibitor of NFATc1, and IL-33 in osteoclast precursors. Mechanistic studies revealed that IL-33 enhanced the expression of MafB in osteoclast precursors and inhibited osteoclast precursors to differentiate into mature osteoclasts. Furthermore, blocking ST2, the IL-33 receptor, partially abrogated curdlan-induced inhibition of NFATc1 expression and osteoclast differentiation. Thus, our study has provided new insights into the mechanisms of dectin-1-induced inhibition of osteoclastogenesis and may provide new targets for the therapy of OBDs.