RANKL-induced RAW264 Research Articles

Diterbutyl phthalate attenuates osteoarthritis in ACLT mice via suppressing ERK/c-fos/NFATc1 pathway, and subsequently inhibiting subchondral osteoclast fusion.

Osteoarthritis (OA) is the most common arthritis with a rapidly increasing prevalence. Disease progression is irreversible, and there is no curative therapy available. During OA onset, abnormal mechanical loading leads to excessive osteoclastogenesis and bone resorption in subchondral bone, causing a rapid subchondral bone turnover, cyst formation, sclerosis, and finally, articular cartilage degeneration. Moreover, osteoclast-mediated angiogenesis and sensory innervation in subchondral bone result in abnormal vascularization and OA pain. The traditional Chinese medicine Panax notoginseng (PN; Sanqi) has long been used in treatment of bone diseases including osteoporosis, bone fracture, and OA. In this study we established two-dimensional/bone marrow mononuclear cell/cell membrane chromatography/time of flight mass spectrometry (2D/BMMC/CMC/TOFMS) technique and discovered that diterbutyl phthalate (DP) was the active constituent in PN inhibiting osteoclastogenesis. Then we explored the therapeutic effect of DP in an OA mouse model with anterior cruciate ligament transaction (ACLT). After ACLT was conducted, the mice received DP (5 mg·kg-1·d-1, ip) for 8 weeks. Whole knee joint tissues of the right limb were harvested at weeks 2, 4, and 8 for analysis. We showed that DP administration impeded overactivated osteoclastogenesis in subchondral bone and ameliorated articular cartilage deterioration. DP administration blunted aberrant H-type vessel formation in subchondral bone marrow and alleviated OA pain assessed in Von Frey test and thermal plantar test. In RANKL-induced RAW264.7 cells in vitro, DP (20 μM) retarded osteoclastogenesis by suppressing osteoclast fusion through inhibition of the ERK/c-fos/NFATc1 pathway. DP treatment also downregulated the expression of dendritic cell-specific transmembrane protein (DC-STAMP)and d2 isoform of the vacuolar (H+) ATPase V0 domain (Atp6v0d2) in the cells. In conclusion, we demonstrate that DP prevents OA progression by inhibiting abnormal osteoclastogenesis and associated angiogenesis and neurogenesis in subchondral bone.

MicroRNA-151a-3p Functions in the Regulation of Osteoclast Differentiation: Significance to Postmenopausal Osteoporosis.

BackgroundStudies have found the pivotal role of miRNAs in the progression of postmenopausal osteoporosis (OP). However, the function of miRNAs in OP is unclear. This study aimed to explore the biological functions of microRNA-151a-3p in OP.MethodsRT-qPCR was employed to assess the expression of microRNA-151a-3p in serum isolated from OP patients and healthy controls. Dual-energy X-ray absorptiometry (DXA) was used to measure the bone mineral density (BMD) of the lumbar spine. The expression levels of c-Fos, NFATc1, and TRAP were tested by Western blot. Ovariectomized (OVX) rats were treated with antago microRNA-151a-3p or antago NC, and then serum and lumbar vertebrae were collected for ELISA and bone histomorphology analysis.ResultsThe expression of microRNA-151a-3p in postmenopausal women with osteoporosis was significantly up-regulated, and microRNA-151a-3p level was negatively correlated with BMD. During osteoclastogenesis, microRNA-151a-3p level was obviously increased. Overexpression of microRNA-151a-3p promoted the differentiation of RANKL-induced THP-1 and RAW264.7 cells into osteoclasts, whereas silencing of microRNA-151a-3p resulted in the opposite results. Silencing of microRNA-151a-3p in OVX rats altered osteoclastogenesis-related factors and raised BMD.ConclusionMicroRNA-151a-3p could partly regulate osteoporosis by promoting osteoclast differentiation, and miRNA-151a-3p could be a potential therapeutic target for postmenopausal osteoporosis.

Upregulation of mmu_circ_0001066 attenuates the inhibitory effect of bisphosphonates on osteoclastogenesis.

Medication-related osteonecrosis of the jaw (MRONJ) is the main adverse side effect of bisphosphonates (BPs), mainly owing to the inhibitory effect of BPs on osteoclastogenesis. CircRNAs were identified to be an important factor in regulating cellular processes. The aim of this study was to explore the effect of mmu_circ_0001066 on BP-inhibited osteoclastogenesis. The expression of MRONJ-related miRNA in RANKL-induced RAW264.7 cells treated with BP was analyzed using qRT-PCR analysis. Bioinformatics techniques were applied to screen potential circRNAs. Tartrate-resistant acid phosphatase (TRAP) staining and bone resorption assays were used to examine the effect of mmu_circ_0001066 on osteoclastogenesis. Bioinformatics analysis, luciferase reporter assays, and Western blotting assays were performed to investigate the underlying mechanism. Four MRONJ-related miRNAs were upregulated in BP-treated RAW264.7 cells, and the expression of mmu_circ_0001066 was negatively correlated with those of MRONJ-related miRNAs. Furthermore, the upregulation of mmu_circ_0001066 partially attenuated the inhibitory effect of BP on osteoclastogenesis in RAW264.7 cells. Mechanistically, upregulated miR-16 suppressed osteoclastogenesis and miR-16 inhibitor increased osteoclastogenesis. Furthermore, we have identified that miR-16 is a downstream effector of mmu_circ_0001066. Our results suggest that mmu_circ_0001066 played an important role in the BP-mediated suppression of osteoclastogenesis, which lays a foundation for identifying mmu_circ_0001066 as a potential biomarker for MRONJ.

The anti‐osteoclastic effect of rosemary ethanol extract is due to the down‐regulation of ROS level via NADPH oxidase1 and Nrf2 signal pathway by carnosic acid

Rosmarinus officinalis, commonly known as rosemary, is a woody, perennial herb which belongs to the mint family and wildly used in the fields of medicine and daily-use chemical industry. The anti-osteoclastic effect of rosemary ethanol extract (REE) was investigated using tartrate acid resistant phosphatase (TRAP) activity, bone resorption assay and western blot analysis in RANKL-induced RAW264.7 cell. REE dose-dependently inhibited TRAP-positive multinucleated osteoclasts formation and bone resorption between 1 µg/mL and 20 µg/mL. In addition, REE attenuated RANKL-induced cathepsin K and NFATc1 expression in a dose-dependent manner. The major bioactive constituents of REE are carnosol and carnosic acid(CA). The anti-osteoclastic effect of CA was stronger than that of carnosol and the potent suppressive effect of CA on ROS was shown by only carnosic acid. The suppressive effect of CA on ROS and O2• level was dose-dependently increased between 1 and 20 μM. Furthermore, CA attenuated the translocation of Nox1 activated factor, p47phox, to the membrane and abolished RANKL-induced ROS generation by activating Nrf2 and its transcriptional targets. These results indicate that the inhibitory effect of REE on RANKL-induced osteoclastogenesis may be attributed to the maintenance of redox homeostasis by CA through regulation of ROS production and scavenging.

The miR-155-5p inhibits osteoclast differentiation through targeting CXCR2 in orthodontic root resorption.

Root resorption is an unavoidable side effect of orthodontic tooth movement. The mechanism of root resorption is similar to bone resorption; the odontoclasts share similar characteristics with osteoclasts (OCs). MicroRNAs (miRNAs) such as miR-155-5p play an important role in OC differentiation, but the underlying molecular mechanism of miR-155-5p in this process is not fully understood. We found that the miR-155-5p seed sequences were complementary to a sequence conserved in the 3-untranslated region of CXCR2 mRNA. In this study, we explored the molecular mechanism underlying the effect of miR-155-5p on OC differentiation by targeting CXCR2. In this study, we divided the orthodontic patients into mild, moderate, and severe groups according to the severity of root resorption. The gingival crevicular fluid (GCF) of patients in different groups was collected, and the expression levels of dentin phosphoprotein (DPP) were detected by ELISA, and the expression levels of CXCR2 and miR-155-5p in GCF were detected by real-time quantitative PCR (qRT-PCR). The relationship between miR-155-5p and CXCR2 was verified by double luciferase. We analyzed changes of CXCR2 and miR-155-5p expression after transfection of miR-155-5p mimic and inhibitor into RAW264.7 cells induced by receptor activator of nuclear factor-κB ligand (RANKL) through qRT-PCR and western blotting. The effect of miR-155-5p on OC differentiation was evaluated by tartrate-resistant acid phosphatase (TRAP) staining. QRT-PCR and western blotting were used to analyze expression of the osteoclastic bone resorption-related enzymes carbonic anhydrase 2 (CA II), matrix metalloproteinase-9 (MMP-9), and cathepsin K. To further confirm the direct targeting effect of CXCR2 by miR-155-5p, we blocked CXCR2 using si-CXCR2 in RANKL-induced RAW264.7 cells. Dentin phosphoprotein levels were consistent with the trend of miR-155-5p changes, and the trend of CXCR2 expression was opposite to miR-155-5p changes. miR-155-5p can be directly targeted to act on CXCR2. The expression of miR-155-5p was significantly downregulated in differentiated OCs. MiR-155-5p inhibited OC differentiation, and downregulated CA II, MMP-9, and cathepsin K expression at the protein and mRNA levels. In summary, the results of this study suggested that miR-155-5p inhibited OC differentiation by targeting CXCR2, thus reducing root resorption in orthodontics. MiR-155-5p can be used as an effective target for avoiding or reducing the degree of root resorption in orthodontic treatment.

Cell cytoskeleton and proliferation study for the RANKL-induced RAW264.7 differentiation.

Although document studies (including ours) have been reported the achieved in vitro osteoclastic cellular model establishment from the RAW264.7 cell lineage, there was no study directly reported that American Type Culture Collection (ATCC) cell bank has various RAW264.7 cell lineages. Besides that, for our knowledge there was only one study compared the two different RAW264.7TIB‐71 and RAW264.7CRL‐2278 cell lineages for their osteoclastic differentiation, and they concluded that the RAW264.7CRL‐2278 demonstrated to generate much osteoclast than RAW264.7TIB‐71. However, on the contrary to their results we noticed the fusion of RAW264.7TIB‐71 in our previous studies was much compromising. Therefore, we try to explore the two cell lineages for their properties in osteoclastic differentiation with an in‐depth cellular cytoskeletal study. Our current study has showed that comparing to the RAW264.7CRL‐2278, RAW264.7TIB‐71 demonstrated a much higher efficacies for RANKL‐stimulated osteoclastic differentiation. Besides that, in our depth cytoskeletal studies, we found that the RANKL‐induced RAW264.7TIB‐71 cells could finally differentiate into mature osteoclasts. However, regardless the various pre‐treatment conditions, there was no mature osteoclast formed in RANKL‐induced RAW264.7CRL‐2278 cell lineage.

Aucubin slows the development of osteoporosis by inhibiting osteoclast differentiation via the nuclear factor erythroid 2-related factor 2-mediated antioxidation pathway

Context Osteoporosis (OP) is a metabolic disease. We have previously demonstrated that aucubin (AU) has anti-OP effects that are due to its promotion of the formation of osteoblasts. Objectives To investigate the mechanisms of anti-OP effects of AU. Materials and methods C57BL/6 mice were randomly divided into control group, 30 mg/kg Dex-induced OP group (OP model group, 15 μg/kg oestradiol-treated positive control group, 5 or 45 mg/kg AU-treated group), and 45 mg/kg AU-alone-treated group. The administration lasted for 7 weeks. Subsequently, 1, 2.5 and 5 µM AU were incubated with 50 ng/mL RANKL-induced RAW264.7 cells for 7 days to observe osteoclast differentiation. The effect of AU was evaluated by analysing tissue lesions, biochemical factor and protein expression. Results The LD50 of AU was greater than 45 mg/kg. AU increased the number of trabeculae and reduced the loss of chondrocytes in OP mice. Compared to OP mice, AU-treated mice exhibited decreased serum concentrations of TRAP5b (19.6% to 28.4%), IL-1 (12.2% to 12.6%), IL-6 (12.1%) and ROS (5.9% to 10.7%) and increased serum concentrations of SOD (14.6% to 19.4%) and CAT (17.2% to 27.4%). AU treatment of RANKL-exposed RAW264.7 cells decreased the numbers of multi-nuclear TRAP-positive cells, reversed the over-expression of TRAP5, NFATc1 and CTSK. Furthermore, AU increased the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and its downstream proteins in RANKL-exposed RAW264.7 cells. Conclusions AU slows the development of OP via Nrf2-mediated antioxidant pathways, indicating the potential use of AU in OP therapy and other types of OP research.

Guizhi-Shaoyao-Zhimu decoction attenuates bone erosion in rats that have collagen-induced arthritis via modulating NF‐κB signalling to suppress osteoclastogenesis

Context Guizhi-Shaoyao-Zhimu decoction (GSZD) is commonly used to treat rheumatoid arthritis (RA), but its mechanism is unclear. Objective To investigate the effect of GSZD on bone erosion in type II collagen (CII)-induced arthritis (CIA) in rats and to identify the underlying mechanism. Materials and methods The CIA model was prepared in male Wistar rats by two subcutaneous injections of CII, 1 mg/mL. Fifty CIA rats were randomized equally into the control group given saline daily, the positive group given saline daily and methotrexate 0.75 mg/kg once a week, and three GSZD-treated groups gavaged daily with 800, 1600 and 3200 mg/kg of GSZD for 21 days. GSZD effects were assessed by paw volume, arthritic severity index and histopathology. Cytokine levels were determined by ELISA. The effects of GSZD on RAW264.7 cells were evaluated by receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis and bone resorption assay. Expression of IκB-α and p65 was measured by Western blotting. Major components of GSZD were identified by HPLC. Results Arthritis index score, paw volume and bone destruction score showed that GSZD improved inflammatory symptoms and reduced joint tissue erosion (p < 0.01). GSZD decreased RANKL, and the number of osteoclasts (OCs) in joint tissues (p < 0.01) and increased osteoprotegerin levels (p < 0.01). GSZD inhibited RANKL-induced RAW264.7 differentiation and reduced bone resorption by OCs. GSZD upregulated IκB (p < 0.01) and p65 (p < 0.01) in the cytoplasm and downregulated p65 (p < 0.01) in the cell nucleus. Conclusions Guizhi-Shaoyao-Zhimu decoction has an anti-RA effect, suggesting its possible use as a supplement and alternative drug therapy for RA.

Inhibitory Effect of (2R)-4-(4-hydroxyphenyl)-2-butanol 2-O-β-d-apiofuranosyl-(1→6)-β-d-glucopyranoside on RANKL-Induced Osteoclast Differentiation and ROS Generation in Macrophages.

In bone homeostasis, bone loss due to excessive osteoclasts and inflammation or osteolysis in the bone formation process cause bone diseases such as osteoporosis. Suppressing the accompanying oxidative stress such as ROS in this process is an important treatment strategy for bone disease. Therefore, in this study, the effect of (2R)-4-(4-hydroxyphenyl)-2-butanol 2-O-β-d-apiofuranosyl-(1→6)-β-d-glucopyranoside (BAG), an arylbutanoid glycoside isolated from Betula platyphylla var. japonica was investigated in RANKL-induced RAW264.7 cells and LPS-stimulated MC3E3-T1 cells. BAG inhibited the activity of TRAP, an important marker of osteoclast differentiation and F-actin ring formation, which has osteospecific structure. In addition, the protein and gene levels were suppressed of integrin β3 and CCL4, which play an important role in the osteoclast-induced bone resorption and migration of osteoclasts, and inhibited the production of ROS and restored the expression of antioxidant enzymes such as SOD and CAT lost by RANKL. The inhibitory effect of BAG on osteoclast differentiation and ROS production appears to be due to the inhibition of MAPKs phosphorylation and NF-κβ translocation, which play a major role in osteoclast differentiation. In addition, BAG inhibited ROS generated by LPS and effectively restores the mineralization of lost osteoblasts, thereby showing the effect of bone formation in the inflammatory situation accompanying bone loss by excessive osteoclasts, suggesting its potential as a new natural product-derived bone disease treatment.

Puerarin alleviates osteoporosis in the ovariectomy-induced mice by suppressing osteoclastogenesis via inhibition of TRAF6/ROS-dependent MAPK/NF-κB signaling pathways.

In this study, we investigated the mechanisms by which puerarin alleviates osteoclast-related loss of bone mass in ovariectomy (OVX)-induced osteoporosis model mice. Puerarin-treated OVX mice exhibited higher bone density, fewer tartrate-resistant acid phosphatase (TRAcP)-positive osteoclasts, and levels of lower reactive oxygen species (ROS) within bone tissues than vehicle-treated OVX mice. Puerarin suppressed in vitro osteoclast differentiation, hydroxyapatite resorption activity, and expression of osteoclastogenesis-related genes, such as NFATc1, MMP9, CTSK, Acp5 and c-Fos, in RANKL-induced bone marrow macrophages (BMMs) and RAW264.7 cells. It also reduced intracellular ROS levels by suppressing expression of TRAF6 and NADPH oxidase 1 (NOX1) and increasing expression of antioxidant enzymes such as heme oxygenase-1 (HO-1). Puerarin inhibited TRAF6/ROS-dependent activation of the MAPK and NF-κB signaling pathways in RANKL-induced RAW264.7 cells, and these effects were partially reversed by HO-1 silencing or TRAF6 overexpression. These findings suggest puerarin alleviates loss of bone mass in the OVX-model mice by suppressing osteoclastogenesis via inhibition of the TRAF6/ROS-dependent MAPK/NF-κB signaling pathway.

Inhibitory Effect of Cudratrixanthone U on RANKL-Induced Osteoclast Differentiation and Function in Macrophages and BMM Cells.

Cudratrixanthone U (CTU) is a prenylated xanthone compound isolated from Maclura tricuspidata Bureau (Moraceae). Prenylated xanthones have been reported to exhibit a variety of biological activities. However, the effects of prenylated xanthone on osteoclast differentiation and function are still unclear. Excessive bone resorption by osteoclasts is considered a major cause of diseases such as osteoporosis. Accordingly, suppression of excessive osteoclast formation and function is one of strategies for treating osteoclast related bone diseases. In this study, CTU inhibited osteoclast differentiation and function in RAW264.7 macrophages and BMM cells induced by receptor activator of nuclear factor-κB ligand (RANKL). CTU regulated the formation of TRAF6-TAK1 complex in RANKL-induced RAW264.7 macrophages and BMM cells. Osteoclast-specific genes including those encoding matrix metallopeptidase 9 (MMP-9), dendritic cell-specific transmembrane proteins (DC-STAMP), cathepsin K (CTSK) and chemokine CC motif ligand 4 (CCL4) play an important role in bone resorption and migration, and were effectively regulated by CTU. These results suggest that CTU is a potential therapeutic agent in osteoporosis.

Transcriptome Analysis Reveals the Negative Effect of 16 T High Static Magnetic Field on Osteoclastogenesis of RAW264.7 Cells.

The magnetic field is the most common element in the universe, and high static magnetic field (HiSMF) has been reported to act as an inhibited factor for osteoclasts differentiation. Although many studies have indicated the negative role of HiSMF on osteoclastogenesis of RANKL-induced RAW264.7 cells, the molecular mechanism is still elusive. In this study, the HiSMF-retarded cycle and weakened differentiation of RAW264.7 cells was identified. Through RNA-seq analysis, RANKL-induced RAW264.7 cells under HiSMF were analysed, and a total number of 197 differentially expressed genes (DEGs) were discovered. Gene ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that regulators of cell cycle and cell division such as Bub1b, Rbl1, Ube2c, Kif11, and Nusap1 were highly expressed, and CtsK, the marker gene of osteoclastogenesis was downregulated in HiSMF group. In addition, pathways related to DNA replication, cell cycle, and metabolic pathways were significantly inhibited in the HiSMF group compared to the Control group. Collectively, this study describes the negative changes occurring throughout osteoclastogenesis under 16 T HiSMF treatment from the morphological and molecular perspectives. Our study provides information that may be utilized in improving magnetotherapy on bone disease.

Wedelolactone exerts double protection for bones through enhancing osteoblastogenesis but inhibiting osteoclastogenesis

Bone homeostasis is maintained by osteoclast-mediated bone destruction and osteoblast-mediated bone formation, which are two tightly coupled and controlled processes. Double protection for bones through enhancing osteoblastogenesis but inhibiting osteoclastogenesis becomes a promising strategy for treating osteoporosis. However, there are little small molecular compounds found to regulate bone remodeling balance. We recently examined the effect of wedelolactone, a natural product from Ecliptae herba , on osteoblastogenesis and osteoclastogenesis in vitro and in vivo. Our results revealed that wedelolactone stimulated osteoblast differentiation and bone mineralization in mouse bone marrow mesenchymal stem cells (BMSC). At the same concentration range, wedelolactone inhibited RANKL-induced preosteoclastic RAW264.7 actin-ring formation and bone resorption pits. The dual functional role of wedelolactone was mediated through inducing Wnt/GSK-3β/β-catenin pathway in BMSC and inhibiting NF-κB/c-fos/NFATc1 pathway in preosteoclastic RAW264.7 cells. Thus, we found that wedelolacone simultaneously regulated bone resorption and bone formation, and could therefore develop a new class of dual-action therapeutic agents for osteoporosis.

Stattic inhibits RANKL-mediated osteoclastogenesis by suppressing activation of STAT3 and NF-κB pathways

Tofacitinib, a small molecule JAK inhibitor, has been widely used to reduce inflammation and inhibit progression of bone destruction in rheumatoid arthritis. STAT3, a downstream signaling molecule of JAK, plays a key role in the activation of signaling in response to inflammatory cytokines. Thus, targeting STAT3 may be an inspiring strategy for treating osteoclast-related diseases such as rheumatoid arthritis. In this study, we first investigated the effects of Stattic, a STAT3 inhibitor, on receptor activator of NF-κB ligand (RANKL)-mediated osteoclastogenesis. Stattic inhibited osteoclast differentiation and bone resorption in RANKL-induced RAW264.7 cells in a dose-dependent manner. Stattic also suppressed RANKL-induced upregulation of osteoclast-related genes tartrate-resistant acid phosphatase, matrix metalloproteinase 9, cathepsin K, RANK, tumor necrosis factor receptor-associated factor 6, and osteoclast-associated receptor in RAW264.7 cells. Moreover, Stattic exhibited an inhibitory effect on cell proliferation and cell cycle progression at higher dosages. At the molecular level, Stattic inhibited RANKL-induced activation of STAT3 and NF-κB pathways, without significantly affecting MAPK signaling. In addition, Stattic inhibited RANKL-induced expression of osteoclast-related transcription factors c-Fos and NFATc1. Importantly, Stattic also prevented bone loss caused by ovariectomy. Together, our data confirm that Stattic restricts osteoclastogenesis and bone loss by disturbing RANKL-induced STAT3 and NF-κB signaling. Thus, Stattic represents a novel type of osteoclast inhibitor that could be useful for conditions such as osteoporosis and rheumatoid arthritis.

Establish mouse osteoblast -osteoclast cell co-culture system in a Transwell chamber

To establish osteoblast-osteoclast cell co-culture system in a Transwell chamber, and detect cell viability of osteoblasts and osteoclasts in system. Osteoblast MC3T3-E1 and mouse monocytes RAW264.7 were cultivated in vitro. RANKL-induced mouse RAW264.7 monocytes differentiated into mature osteoclasts, osteoblast-osteoclast cell co-culture system was established in Transwell chamber. Cell activity of osteoblasts and osteoclasts were detected by CCK-8 experimenting, Alizarin Red staining, TRAP staining. The expression of OPG, ALP, RANKL, TGF-b1 gene and RANKL protein in osteoblast MC3T3-E1 were detected by PCR, Western-Blot methods. Also, the expression of RANK, NF-κB in gene and protein level in osteoclast were measured through the same method respectively. The co-culture system of Mouse MC3T3-E1 cells and RAW264.7 cell were established in Transwell chamber. Co-culture system affected cell division activities of osteoblasts and osteoclasts. Differentiation of osteoblasts were increased, while differentiation of osteoclast division were slight decreased under microscope observation. OPG (0.65±0.08) and ALP (0.16±0.01) gene expression of co-culture system were less than single culture OPG(1.00±0.08) and ALP (1.01±0.16); TGF-b1(4.42±0.21) and RANKL(4.12±1.04) of osteoblasts in co-culture system were higher than TGF-b1(1.00±0.10) and RANKL(1.00±0.09) under single culture. However, gene expression of RANK(0.63±0.06) and NF-κB(0.64±0.08) in co-culture system were decreased than RANK(1.00±0.08) and NF-κB(1.00±0.09), in single culture, and had significant differences. Similarly, protein expression of OPG(0.43±0.05) and NF-κB(0.59±0.05) of co-culture system were less than OPG(0.84±0.06) and NF-κB(1.13±0.03) of single culture. While RANKL protein expression (0.54±0.03)of co-culture system was more than single culture RANKL(0.31±0.03), and had statistically differences, which was in agreement of the trend of gene expression change. Co-culture system of mouse MC3T3-E1 cells and RAW264.7 cell was viable in Transwell chamber, and the activity of osteoblasts is higher than osteoclasts in co-culture system.

Psoralen inhibits RAW264.7 differentiation into osteoclasts and bone resorption by regulating CD4+T cell differentiation

This paper aimed to investigate whether psoralen inhibits the differentiation and bone resorption by regulating CD4+T cell differentiation in RANKL-induced osteoclastogenesis in RAW264.7 cells, and elucidate its mechanism for osteoporosis. CD4+T cells were isolated from spleen cells of Balb/c mice by immunomagnetic separation method. The cells were divided into blank control group and psoralen group. The cells were cultured in 24-well plates and cultured for 3 days, and then they were collected for co-culture experiments after 4 days. Co-culture experiments were divided into RAW264.7 cell group, psoralen+RAW264.7 cell group, without psoralen treatment of CD4+T cells+RAW264.7 cell group, psoralen treatment of CD4+T cells+RAW264.7 cell group. After 5 days of co-culture, TRAP staining was used to detect the number of osteoclasts, and after 8 days of co-culture, bone resorption was evaluated by toluidine blue staining. The expressions of RORγt, Foxp3, IL-17, TNF-α, TGF-β and IL-10 in CD4+T cells and osteoclast differentiation-related genes MMP-9, TRAP and Cat-K were detected by Real-time polymerase chain reaction (RT-PCR); ELISA kit was used to detect IL-17, TNF-α, TGF-β and IL-10 and other cytokines levels. Our data confirmed that the psoralen significantly promoted the expression of Foxp3, TGF-β and IL-10 in CD4+T, and inhibited the expression of RORγt, IL-17 and TNF-α in CD4+T, the CD4+T cells without treatment by psoralen can significantly promote RANKL-induced differentiation of RAW264.7 to osteoclasts, and psoralen treatment of CD4+T can significantly inhibit RANKL-induced RAW264.7 osteoclast differentiation and bone resorption. Taken together, psoralen inhibits the differentiation and bone resorption of RAW264.7 into osteoclasts by promoting the development of CD4+ CD25+ Treg/Th17 balance in CD4+T cells to CD4+CD25+T.

The histone methyltransferase DOT1L inhibits osteoclastogenesis and protects against osteoporosis

Osteoclasts are absorptive cells that play a critical role in homeostatic bone remodeling and pathological bone resorption. Emerging evidence suggests an important role of epigenetic regulation in osteoclastogenesis. In this study, we investigated the role of DOT1L, which regulates gene expression epigenetically by histone H3K79 methylation (H3K79me), during osteoclast formation. Using RANKL-induced RAW264.7 macrophage cells as an osteoclast differentiation model, we found that DOT1L and H3K79me2 levels were upregulated during osteoclast differentiation. Small molecule inhibitor- (EPZ5676 or EPZ004777) or short hairpin RNA-mediated reduction in DOT1L expression promoted osteoclast differentiation and resorption. In addition, DOT1L inhibition increased osteoclast surface area and accelerated bone-mass reduction in a mouse ovariectomy (OVX) model of osteoporosis without alter osteoblast differentiation. DOT1L inhibition increase reactive oxygen species (ROS) generation and autophagy activity, and cell migration in pre-osteoclasts. Moreover, it strengthened expression of osteoclast fusion and resorption-related protein CD9 and MMP9 in osteoclasts derived from RAW264.7. Our findings support a new mechanism of DOT1L-regulated, H3K79me2-mediated, epigenetic regulation of osteoclast differentiation, implicating DOT1L as a new therapeutic target for osteoclast dysregulation-induced disease.

The Simultaneous Inhibitory Effect of Niclosamide on RANKL-Induced Osteoclast Formation and Osteoblast Differentiation.

The bone destruction disease including osteoporosis and rheumatoid arthritis are caused by the imbalance between osteoblastogenesis and osteoclastogenesis. Inhibition of the NF-κB pathway was responsible for decreased osteoclastogenesis. Recently many studies indicated that niclosamide, the FDA approved an antihelminth drug, inhibits prostate and breast cancer cells growth by targeting NF-κB signaling pathways. This study evaluated the effects of niclosamide on osteoclast and osteoblast differentiation and function in vitro.In RANKL-induced murine osteoclast precursor cell RAW264.7 and M-CSF/RANKL-stimulated primary murine bone marrow-derived macrophages (BMM), niclosamide dose-dependently inhibited the formation of TRAP-positive multinucleated osteoclasts and resorption pits formation between 0.5uM and 1uM. In addition, niclosamide suppressed the expression of nuclear factor of activated T cells c1 (NFATc1) and osteoclast differentiated-related genes in M-CSF/ RANKL-stimulated BMM by interference with TRAF-6, Erk1/2, JNK and NF-κB activation pathways. However, the cytotoxic effects of niclosamide obviously appeared at the effective concentrations for inhibiting osteoclastogenesis (0.5-1uM) with increase of apoptosis through caspase-3 activation in osteoblast precursor cell line, MC3T3-E1. Niclosamide also inhibited ALP activity, bone mineralization and osteoblast differentiation-related genes expression in MC3T3-E1. Therefore, our findings suggest the new standpoint that niclosamide's effects on bones must be considered before applying it in any therapeutic treatment.

Wedelolactone enhances osteoblastogenesis by regulating Wnt/β-catenin signaling pathway but suppresses osteoclastogenesis by NF-κB/c-fos/NFATc1 pathway.

Bone homeostasis is maintained by formation and destruction of bone, which are two processes tightly coupled and controlled. Targeting both stimulation on bone formation and suppression on bone resorption becomes a promising strategy for treating osteoporosis. In this study, we examined the effect of wedelolactone, a natural product from Ecliptae herba, on osteoblastogenesis as well as osteoclastogenesis. In mouse bone marrow mesenchymal stem cells (BMSC), wedelolactone stimulated osteoblast differentiation and bone mineralization. At the molecular level, wedelolactone directly inhibited GSK3β activity and enhanced the phosphorylation of GSK3β, thereafter stimulated the nuclear translocation of β-catenin and runx2. The expression of osteoblastogenesis-related marker gene including osteorix, osteocalcin and runx2 increased. At the same concentration range, wedelolactone inhibited RANKL-induced preosteoclastic RAW264.7 actin-ring formation and bone resorption pits. Further, wedelolactone blocked NF-kB/p65 phosphorylation and abrogated the NFATc1 nuclear translocation. As a result, osteoclastogenesis-related marker gene expression decreased, including c-src, c-fos, and cathepsin K. In ovariectomized mice, administration of wedelolactone prevented ovariectomy-induced bone loss by enhancing osteoblast activity and inhibiting osteoclast activity. Together, these data demonstrated that wedelolactone facilitated osteoblastogenesis through Wnt/GSK3β/β-catenin signaling pathway and suppressed RANKL-induced osteoclastogenesis through NF-κB/c-fos/NFATc1 pathway. These results suggested that wedelolacone could be a novel dual functional therapeutic agent for osteoporosis.

Aspirin inhibits osteoclastogenesis by suppressing the activation of NF-κB and MAPKs in RANKL-induced RAW264.7 cells.

Aspirin is a commonly used medicine as an effective antipyretic, analgesic and anti-inflammatory drug. Previous studies have demonstrated its potential effects of anti-postmenopausal osteoporosis, while the molecular mechanisms remain unclear. The effects of aspirin on receptor-activator of nuclear factor κB (NF-κB) ligand (RANKL)-induced osteoclasts were investigated in RAW264.7 cells in the current study. Using tartrate-resistant acid phosphatase (TRAP) staining, it was observed that aspirin inhibited the differentiation of RANKL-induced RAW264.7 cells. The mRNA expression of osteoclastic marker genes, including cathepsin K, TRAP, matrix metalloproteinase 9 and calcitonin receptor, were suppressed by aspirin as identified using reverse transcription-quantitative polymerase chain reaction analysis. The immunofluorescence assay indicated that aspirin markedly inhibited NF-κB p65 translocation to the nucleus in RANKL-induced RAW264.7 cells. In addition, aspirin also suppressed the phosphorylation of mitogen-activated protein kinases (MAPKs), observed by western blot analysis. Taken together, these data identified that aspirin inhibits osteoclastogenesis by suppressing the activation of NF-κB and MAPKs in RANKL-induced RAW264.7 cells, implying that aspirin may possess therapeutic potential for use in the prevention and treatment of osteoporosis.