Melatonin Attenuates RANKL-Induced Osteoclastogenesis via Inhibition of Atp6v0d2 and DC-STAMP through MAPK and NFATc1 Signaling Pathways

Melatonin is a hormone secreted by the pineal gland that is involved in the biorhythm of reproductive activities. The present study investigated the inhibitory effects of melatonin on osteoclastogenesis in RAW 264.7 cells according to changes in V-ATPase and the corresponding inhibition of the MAPK and NFATc1 signaling processes. Methods: the cytotoxic effect of melatonin was investigated by MTT assay. Osteoclast differentiation and gene expression of osteoclast-related factors were confirmed via TRAP staining, pit formation assay, immunofluorescence imaging, western blot, and real-time PCR. Results: melatonin was found to inactivate the p38 and JNK of MAP kinase in RAW264.7 cells treated with RANKL and treated with a combination RANKL and melatonin for 1, 3, and 5 days. The melatonin treatment group showed a reduction in osteoclastogenesis transcription factors and ATP6v0d2 gene expression. Conclusions: melatonin inhibits osteoclast differentiation and cell fusion by inhibiting the expression of Atp6v0d2 through the inactivation of MAPK and NFATc1 signaling in RANKL-stimulated RAW264.7 macrophages. The findings of the present study suggest that melatonin could be a suitable therapy for bone loss and imply a potential role of melatonin in bone health.

Download Full-text

MicroRNA-181a-5p regulates inflammatory response of macrophages in sepsis

Open Medicine ◽

10.1515/med-2019-0106 ◽

2019 ◽

Vol 14 (1) ◽

pp. 899-908 ◽

Cited By ~ 4

Author(s):

Zheng Huang ◽

Hang Xu

Keyword(s):

Inflammatory Response ◽

Inflammatory Cytokines ◽

Sirtuin 1 ◽

Raw 264.7 Cells ◽

Inflammatory Factors ◽

Raw 264.7 ◽

Raw264.7 Macrophages ◽

Pathway Activation

AbstractThe aim of this study was to evaluate the role of miR-181a-5p in sepsis, and to further explore the molecular mechanism. RAW 264.7 cells were stimulated with 1 μg/ml LPS for 4 hours. Firstly, qRT-PCR and ELISA was adopted to evaluate the expression of miR-181a-5p and p ro-inflammatory cytokines in RAW 264.7 macrophages a fter LPS stimulation. Results showed that pro-inflammatory cytokines and miR-181a-5p were significantly increased after LPS treatment. Then, we identified that sirtuin-1 (SIRT1) was a direct target of miR-181a-5p and it was down-regulated in LPS treated RAW264.7 macrophages. Furthermore, the data suggested that the miR-181a-5p inhibitor significantly inhibited LPS enhanced inflammatory cytokines expression and NF-κB pathway activation, and these changes were eliminated by SIRT1 silencing. Moreover, the role of the miR-181a-5p inhibitor on sepsis was studied in vivo. We found that the miR-181a-5p inhibitor significantly decreased the secretion of inflammatory factors, and the levels of creatine (Cr), blood urea nitrogen (BUN), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in a serum for mice with sepsis. However, all the effects were reversed by SIRT1-siRNA. In summary, these results indicated that miR-181a-5p was involved in sepsis through regulating the inflammatory response by targeting SIRT1, suggesting that miR-181a-5p may be a potential target for the treatment of sepsis.

Download Full-text

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

International Journal of Molecular Sciences ◽

10.3390/ijms21103486 ◽

2020 ◽

Vol 21 (10) ◽

pp. 3486 ◽

Cited By ~ 3

Author(s):

Wen-Chih Liu ◽

Jia-Fwu Shyu ◽

Paik Seong Lim ◽

Te-Chao Fang ◽

Chien-Lin Lu ◽

...

Keyword(s):

Transcription Factor ◽

Aryl Hydrocarbon Receptor ◽

Critical Role ◽

Osteoclast Differentiation ◽

Indoxyl Sulfate ◽

Raw 264.7 Cells ◽

High Dose ◽

Activated T Cells ◽

Aryl Hydrocarbon

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.

Download Full-text

Thermally induced osteocyte damage initiates pro-osteoclastogenic gene expression in vivo

Journal of The Royal Society Interface ◽

10.1098/rsif.2016.0337 ◽

2016 ◽

Vol 13 (119) ◽

pp. 20160337 ◽

Cited By ~ 4

Author(s):

Eimear B. Dolan ◽

David Tallon ◽

Wing-Yee Cheung ◽

Mitchell B. Schaffler ◽

Oran D. Kennedy ◽

...

Keyword(s):

Gene Expression ◽

Thermal Damage ◽

Bone Cells ◽

Osteoclast Differentiation ◽

Cellular Damage ◽

Sprague Dawley ◽

Related Factors ◽

Thermally Induced ◽

Rat Tibia

Bone is often subject to harsh temperatures during orthopaedic procedures resulting in thermally induced bone damage, which may affect the healing response. Postsurgical healing of bone is essential to the success of surgery, therefore, an understanding of the thermally induced responses of bone cells to clinically relevant temperatures in vivo is required. Osteocytes have been shown to be integrally involved in the bone remodelling cascade, via apoptosis, in micro-damage systems. However, it is unknown whether this relationship is similar following thermal damage. Sprague–Dawley rat tibia were exposed to clinically relevant temperatures (47°C or 60°C) to investigate the role of osteocytes in modulating remodelling related factors. Immunohistochemistry was used to quantify osteocyte thermal damage (activated caspase-3). Thermally induced pro-osteoclastogenic genes ( Rankl , Opg and M-csf ), in addition to genes known to mediate osteoblast and osteoclast differentiation via prostaglandin production ( Cox2 ), vascularization ( Vegf ) and inflammatory ( Il1a ) responses, were investigated using gene expression analysis. The results demonstrate that heat-treatment induced significant bone tissue and cellular damage. Pro-osteoclastogenic genes were upregulated depending on the amount of temperature elevation compared with the control. Taken together, the results of this study demonstrate the in vivo effect of thermally induced osteocyte damage on the gene expression profile.

Download Full-text

A novel mice model of acute flares in osteoarthritis elicited by intra-articular injection of cultured mast cells

Journal of Experimental Orthopaedics ◽

10.1186/s40634-021-00391-6 ◽

2021 ◽

Vol 8 (1) ◽

Author(s):

Junpei Dan ◽

Masashi Izumi ◽

Hiroko Habuchi ◽

Osami Habuchi ◽

Shogo Takaya ◽

...

Keyword(s):

Gene Expression ◽

Mast Cell ◽

Mast Cells ◽

Mice Model ◽

Inhibitory Effects ◽

Histological Changes ◽

New Strategy ◽

Monosodium Iodoacetate ◽

Disease Modifying Treatment

Abstract Purpose Mast cells are multifunctional in osteoarthritis (OA), and infiltration of activated mast cells likely contributes to disease severity and progression. However, the detailed mechanisms of action are unclear. The purpose of this study was to elucidate the role of mast cell infiltration in OA at histological level using a new mice model and to investigate pharmacological inhibitory effects of existing mast cell stabilizers in this model. Methods Mice were injected intra-articularly with monosodium iodoacetate (MIA 0.5 mg) or PBS on day 0, and PBS, with or without mast cells (MC: 1 × 106 cells) on day 14. They were divided into four groups: OA flare (MIA + MC), OA (MIA + PBS), MC non-OA (PBS + MC), and PBS non-OA (PBS + PBS). In OA flare, the MC stabilizer drug (tranilast: 400 mg/kg/day) or PBS was administered intraperitoneally from days 15 to 21. Results Histologically, modified Mankin score of the OA flare was significantly higher than that of OA (7.0 [1.8] vs. 3.3 [1.3], P < 0.05), and a larger number of mast cells was observed in OA flare than in OA (34.5 [6.3]/mm2 vs. 27.2 [2.3]/mm2, P < 0.05) on day 22. OA flare also showed acute exacerbation of pain and increased gene expression of pro-inflammatory cytokines and aggrecanase compared with OA. Administration of tranilast to OA flare-up provoked significant improvements in term of histological changes, pain, and gene expression at day 22. Conclusion Our novel model possibly mimics OA flare conditions, which may open a new strategy of disease-modifying treatment for OA, focused on controlling the multiple functions of mast cells.

Download Full-text

Anti-IFN-γ Antibody Promotes Osteoclastogenesis in Human Bone Marrow Monocyte-Derived Macrophages Co-Cultured with Tuberculosis-Activated Th1 Cells

Cellular Physiology and Biochemistry ◽

10.1159/000493455 ◽

2018 ◽

Vol 49 (4) ◽

pp. 1512-1522

Author(s):

Jiezhong Deng ◽

Dong Sun ◽

Fei Luo ◽

Qiang Zhang ◽

Feifan Chen ◽

...

Keyword(s):

Bone Marrow ◽

Bone Resorption ◽

Osteoclast Differentiation ◽

Osteoclast Formation ◽

Th1 Cells ◽

Actin Ring ◽

Tnf Α ◽

Ifn Γ ◽

Trap Staining

Background/Aims: Tuberculosis induces bone loss and activates Th1 cells that play an important role in the host defense of Bacille Calmette-Guérin tuberculosis vaccine. However, the role of tuberculosis-activated Th1 cells in differentiation of osteoclast precursors to osteoclasts is unclear. As secretion of IFN-γ in Th1 cells is induced by tuberculosis, we aimed to investigate the role of anti-IFN-γ antibody on the differentiation and activation of osteoclasts in bone marrow monocyte-derived macrophages (BMMs). Methods: BMMs were isolated and co-cultured with CD4+T helper 1 cells (Th1 cells), pretreated with anti-IFN-γ antibody. Then, cell proliferation, expression and release of cytokines, formation of actin ring, differentiation of osteoclasts and bone resorption function were measured by CCK8 assay, qRT-PCR/Western blot/flow cytometry, ELISA, immunofluorescence, tartrate-resistant acidic phosphatase (TRAP) staining and bone absorbance assay, respectively. Results: Anti-IFN-γ antibody inhibited the cell viability of BMMs, and induced the expressions of RANKL, TNF-α, NF-κB and TRAF6 in BMMs. In addition, it led to increased expression levels of RANK on cell surfaces, and increased production of RANKL, TNF-α, MCP-1 and SDF-1. Anti-IFN-γ antibody also induced the expression of osteoclast differentiation factor and actin ring formation, but inhibited the expression of osteoprotegerin. TRAP staining and bone resorption assays showed that anti-IFN-γ antibody induced an increase in osteoclast formation and bone resorption. Conclusion: The anti-IFN-γ antibody induced osteoclast formation, and is probably mediated by RANKL-induced activation of NF-κB, that induces TRAF6 in the RANKL-RANK signaling pathway. Our data suggest an inhibitory role for IFN-γ in osteoclast formation induced by tuberculosis.

Download Full-text

MiRNAs Expression Profiling in Raw264.7 Macrophages after Nfatc1-Knockdown Elucidates Potential Pathways Involved in Osteoclasts Differentiation

Biology ◽

10.3390/biology10111080 ◽

2021 ◽

Vol 10 (11) ◽

pp. 1080

Author(s):

Roberta Russo ◽

Francesca Zito ◽

Nadia Lampiasi

Keyword(s):

Potential Role ◽

Osteoclast Differentiation ◽

Negative Control ◽

Differentially Expressed ◽

Bone Homeostasis ◽

Disease States ◽

Raw264.7 Macrophages ◽

Mirnas Expression ◽

Rankl Stimulation

Differentiation of macrophages toward osteoclasts is crucial for bone homeostasis but can be detrimental in disease states, including osteoporosis and cancer. Therefore, understanding the osteoclast differentiation process and the underlying regulatory mechanisms may facilitate the identification of new therapeutic targets. Hereby, we tried to reveal new miRNAs potentially involved in the regulation of early steps of osteoclastogenesis, with a particular focus on those possibly correlated with NFATc1 expression, by studying miRNAs profiling. During the first 24 h of osteoclastogenesis, 38 miRNAs were differentially expressed between undifferentiated and RANKL-stimulated RAW264.7 cells, while 10 miRNAs were differentially expressed between RANKL-stimulated cells transfected with negative control or NFATc1-siRNAs. Among others, the expression levels of miR-411, miR-144 and members of miR-29, miR-30, and miR-23 families changed after RANKL stimulation. Moreover, the potential role of miR-124 during osteoclastogenesis was explored by transient cell transfection with anti-miR-124 or miR-124-mimic. Two relatively unknown miRNAs, miR-880-3p and miR-295-3p, were differentially expressed between RANKL-stimulated/wild-type and RANKL-stimulated/NFATc1-silenced cells, suggesting their possible correlation with NFATc1. KEGG enrichment analyses showed that kinase and phosphatase enzymes were among the predicted targets for many of the studied miRNAs. In conclusion, our study provides new data on the potential role and possible targets of new miRNAs during osteoclastogenesis.

Download Full-text

IL-4 suppresses osteoclast development and mature osteoclast function by a STAT6-dependent mechanism: irreversible inhibition of the differentiation program activated by RANKL

Blood ◽

10.1182/blood-2002-11-3437 ◽

2003 ◽

Vol 102 (3) ◽

pp. 1078-1086 ◽

Cited By ~ 95

Author(s):

Jose L. Moreno ◽

Michele Kaczmarek ◽

Achsah D. Keegan ◽

Mehrdad Tondravi

Keyword(s):

Gene Expression ◽

Alternative Pathway ◽

Osteoclast Differentiation ◽

Precursor Cells ◽

Interleukin 4 ◽

Tartrate Resistant Acid Phosphatase ◽

Mature Osteoclast ◽

Raw264.7 Macrophages ◽

Osteoclast Function ◽

Dependent Mechanism

Abstract Numerous reports have described the effects of interleukin-4 (IL-4) on bone biology. Previous studies, performed using complex coculture systems, demonstrated the effects of IL-4 on osteoblasts and osteoclasts. To directly test the effect of IL-4 on osteoclasts, we took advantage of a simplified system using recombinant receptor activator of nuclear factor κB ligand (RANKL) as the osteoclast differentiation factor. We analyzed the ability of IL-4 to directly regulate osteoclast differentiation and mature osteoclast function. We found that IL-4 inhibited the differentiation of osteoclasts from bone marrow precursors in an irreversible manner and also inhibited the resorptive capacity of mature osteoclasts. In the presence of IL-4, we detected the appearance of tartrate-resistant acid phosphatase (TRAP)–negative multinucleated giant (MNG) cells. Both IL-4 effects were dependent on signal transducer and activator of transcription 6 (STAT6). We found that IL-4 suppresses RANK mRNA expression in the developing precursor cells. When RANK was ectopically expressed under the cytomegalovirus (CMV) promoter in RAW264.7 macrophages, IL-4 treatment did not inhibit osteoclast development. Furthermore, when osteoclastogenesis was induced independently of RANKL by using tumor necrosis factor-α (TNF-α), IL-4 inhibited osteoclast differentiation through a STAT6-dependent mechanism. These results suggest that IL-4 regulates osteoclast development by regulating gene expression, including RANK. We propose that IL-4 irreversibly regulates the lineage commitment of precursor cells by regulating gene expression, resulting in the suppression of osteoclast development and the generation of MNG cells as an alternative pathway of differentiation.

Download Full-text

Protocatechualdehyde Inhibits the Osteoclast Differentiation of RAW264.7 and BMM Cells by Regulating NF-κB and MAPK Activity

BioMed Research International ◽

10.1155/2021/6108999 ◽

2021 ◽

Vol 2021 ◽

pp. 1-11

Author(s):

Yunyun Qu ◽

Xin Liu ◽

Shuai Zong ◽

Huanxin Sun ◽

Shuang Liu ◽

...

Keyword(s):

Salvia Miltiorrhiza ◽

Osteoclast Differentiation ◽

Cell Activity ◽

Cell Types ◽

Underlying Mechanism ◽

Rt Pcr ◽

Macrophage Cell ◽

Mapk Activity ◽

Trap Staining

Protocatechualdehyde (PCA), an important component of Salvia miltiorrhiza, has many activities, such as anti-inflammatory and antisepsis activities. However, the role of PCA in osteoclasts is not clear. We used RAW264.7 cells (a mouse leukemic monocyte/macrophage cell line) and bone marrow macrophages (BMMs) to probe the role of PCA in osteoclasts and the underlying mechanism. The effects of PCA on cell activity were evaluated with CCK-8 assays. TRAP staining detected mature osteoclasts. Corning Osteo Assay Surface plates were used to examine absorption. The levels of RNA and protein were analyzed, respectively, using RT-PCR and Western blotting. PCA (5 μg/ml) was not toxic to the two cell types but reduced the formation of osteoclasts and bone absorption. Furthermore, PCA restrained the expression of mRNAs encoding proteins associated with osteoclasts and reduced the phosphorylation of proteins in important signaling pathways. The results indicate that PCA inhibits osteoclast differentiation by suppressing NF-κB and MAPK activity.

Download Full-text

FNDC4 Inhibits RANKL-Induced Osteoclast Formation by Suppressing NF-κB Activation and CXCL10 Expression

BioMed Research International ◽

10.1155/2018/3936257 ◽

2018 ◽

Vol 2018 ◽

pp. 1-9 ◽

Cited By ~ 2

Author(s):

Zheng-tao Lv ◽

Shuang Liang ◽

Kun Chen ◽

Jia-ming Zhang ◽

Peng Cheng ◽

...

Keyword(s):

Gene Expression ◽

Bone Resorption ◽

Molecular Mechanisms ◽

Osteoclast Differentiation ◽

Differentially Expressed Gene ◽

Osteoclast Formation ◽

Luciferase Assay ◽

Raw 264.7 Cells ◽

Inflammatory Factor ◽

Dependent Manner

FNDC4 acts as an anti-inflammatory factor on macrophages and improves mouse model of induced colitis. Considering osteoclast formation is characterized by the activation of inflammation-related pathways, we thus speculated that FNDC4 may play a pivotal role in this process. RT-qPCR analysis was performed to confirm the expression of osteoclast formation related genes in primary murine bone marrow macrophages (BMMs). RANKL-treated BMMs were cultured with FNDC4 to evaluate the effect of FNDC4 on osteoclast differentiation. TRAP staining and bone resorption pits assay were used to assess osteoclast formation and bone resorption, respectively. Luciferase assay and western blotting analysis were conducted to determine whether FNDC4 inhibited osteoclast formation via NF-κB signaling in RAW 264.7 cells. Furthermore, to identify gene signatures in FNDC4-treated BMMs and to use these to elucidate the underlying molecular mechanisms during osteoclast formation, we adopted a bioinformatics approach by downloading the GSE76172 gene expression profiling dataset from the Gene Expression Omnibus (GEO) database. FNDC4 inhibited RANKL-induced osteoclastogenesis and mature osteoclast resorptive function in a dose-dependent manner. Results of NF-κB luciferase assay suggested that FNDC4 could significantly suppress the RANKL-induced NF-κB transcriptional activity. Based on the protein-protein interaction network, CXCL10 was identified as the differentially expressed gene with the highest connectivity degree (degree = 23); it was drastically downregulated in the presence of FNDC4, but supplementation of CXCL10 (10 ng/mL) partially ameliorated the FNDC4-induced inhibition of osteoclast formation. Taken together, we speculated that FNDC4 could suppress osteoclast formation via NF-κB pathway and downregulation of CXCL10.

Download Full-text

RNA sequencing analysis reveals differential gene expression of CXCL2 in ACPA-positive and ACPA-negative rheumatoid arthritis

10.21203/rs.3.rs-21873/v1 ◽

2020 ◽

Author(s):

Lin Sun ◽

Xinyu Wang ◽

Ning He ◽

Zhuo An ◽

Ruohan Yu ◽

...

Keyword(s):

Rheumatoid Arthritis ◽

Gene Expression ◽

Rna Sequencing ◽

Differentially Expressed Genes ◽

Differentially Expressed ◽

Enzyme Linked Immunosorbent Assay ◽

Sequencing Analysis ◽

Healthy Controls ◽

Raw264.7 Macrophages

Abstract Background. Anti-citrullinated protein/peptide antibodies (ACPA) play important roles in the pathogenesis of rheumatoid arthritis (RA), and are associated with RA severity. It has been suggested that ACPA-positive (ACPA+) and ACPA-negative (ACPA-) RA are different disease subsets with distinct differences in genetic variation and clinical outcomes. The aims of the present study were to compare gene expression profiles in ACPA + and ACPA- RA and identify novel candidate gene signatures that might serve as therapeutic targets. Methods. Comprehensive transcriptome analysis of peripheral blood mononuclear cells (PBMCs) from ACPA + and ACPA- RA patients, and healthy controls was performed via RNA sequencing. Genes with significantly different expressions were analyzed by cluster analysis, Gene Ontology analysis and Ingenuity Pathway analysis. A validation cohort was used to further investigate differentially expressed genes via real-time PCR and enzyme-linked immunosorbent assay. Spearman's correlation test was used to evaluate the correlation of differentially expressed genes and the clinical and laboratory data of the patients. The role of differentially expressed genes in osteoclastogenesis was further investigated. Results. There were significant differences in the expression levels of both genes and gene isoforms between ACPA + and ACPA- RA samples. Expression of C-X-C motif chemokine ligand 2 (CXCL2) was significantly increased in ACPA + RA patients than in ACPA- RA patients and healthy controls. Validation of candidate genes expression showed that CXCL2 levels in PBMCs and serum were higher in ACPA + RA patients than in ACPA- RA patients and healthy controls. CXCL2 promoted the migration of CD14 + monocytes and increased osteoclast differentiation in RA patients. RAW264.7 macrophages were used to investigate specific mechanisms, and the results suggested that CXCL2 stimulated osteoclastogenesis via ERK MAPK and NFκB pathways. Conclusion. Novel pathways associated with ACPA + RA were identified via RNA sequencing, and CXCL2 was highly expressed in ACPA + RA than in ACPA- RA. These results reveal a previously unreported role of CXCL2 during osteoclastogenesis in RA, and suggest that the blockade of CXCL2 might be a novel strategy for the treatment of RA.

Download Full-text