Presence Of RANKL Research Articles

Oestradiol and osteoclast differentiation: Effects on p53 and mitochondrial metabolism.

Oestrogen deficiency increases bone resorption, contributing to osteoporosis development. Yet, the mechanisms mediating the effects of oestrogen on osteoclasts remain unclear. This study aimed to elucidate the early metabolic alteration induced by RANKL, the essential cytokine in osteoclastogenesis and 17-beta-oestradiol (E2) on osteoclast progenitor cells, using RAW 264.7 macrophage cell line and primary bone marrow-derived macrophages as biological models. This research demonstrated that, in osteoclast precursors, RANKL stimulates complex I activity, oxidative phosphorylation (OXPHOS) and mitochondria-derived ATP production as early as 3 h of exposure. This effect on mitochondrial bioenergetics is associated with an increased capacity to oxidize TCA cycle substrates, fatty acids and amino acids. E2 inhibited all effects of RANKL on mitochondria metabolism. In the presence of RANKL, E2 also decreased cell number and stimulated the mitochondrial-mediated apoptotic pathway, detected as early as 3 h. Further, the pro-apoptotic effects of E2 during osteoclast differentiation were associated with an accumulation of p392S-p53 in mitochondria. These findings elucidate the early effects of RANKL on osteoclast progenitor metabolism and suggest novel p53-mediated mechanisms that contribute to postmenopausal osteoporosis.

Natural drug asiaticoside inhibits osteoclast differentiation and promotes repair of steroid-induced osteonecrosis of femoral head via RANKL pathway

Asiaticoside is one of the most classic traditional Chinese medicines. The interaction between Receptor activator of nuclear factor κB (RANK) and Receptor Activator of Nuclear Factor-κ B Ligand (RANKL) activates a series of intracellular signaling pathways. Our research explored the mechanism of asiaticoside inhibiting osteoclast differentiation and repair of steroid-induced osteonecrosis of femoral head (SIONFH) through RANKL pathway. Asiaticoside’s effect on osteoclasts was determined by Microneedle Therapy System (MTS) method, and the number of Tartrate resistant acid phosphatase (TRAcP) multinucleated cells (3 nuclei) was quantitatively analyzed. Under different dose treatments, the changes of bone function were detected by eroded area and TRAcP staining on hydroxyapatite-coated plate, and the expression of different osteocyte formation and osteogenic differentiation were detected. Asiaticoside weakened the osteoclast function induced by RANKL. When the concentration of asiaticoside was 10 μmol/L, osteoclast formation regulatory genes (NFATc1 and Acp5) and osteoclast cell function-related genes (AP-1 and P65) were significantly downregulated in the presence of RANKL. Asiaticoside (10 μmol/L) significantly decreased calcium oscillations induced by RANKL via preventing RANKL-mediated NF-κB activation and Ca2+ oscillation.

Pirfenidone Inhibits Alveolar Bone Loss in Ligature-Induced Periodontitis by Suppressing the NF-κB Signaling Pathway in Mice.

There has been increasing interest in adjunctive use of anti-inflammatory drugs to control periodontitis. This study was performed to examine the effects of pirfenidone (PFD) on alveolar bone loss in ligature-induced periodontitis in mice and identify the relevant mechanisms. Experimental periodontitis was established by ligating the unilateral maxillary second molar for 7 days in mice (n = 8 per group), and PFD was administered daily via intraperitoneal injection. The micro-computed tomography and histology analyses were performed to determine changes in the alveolar bone following the PFD administration. For in vitro analysis, bone marrow macrophages (BMMs) were isolated from mice and cultured with PFD in the presence of RANKL or LPS. The effectiveness of PFD on osteoclastogenesis, inflammatory cytokine expression, and NF-κB activation was determined with RT-PCR, Western blot, and immunofluorescence analyses. PFD treatment significantly inhibited the ligature-induced alveolar bone loss, with decreases in TRAP-positive osteoclasts and expression of inflammatory cytokines in mice. In cultured BMM cells, PFD also inhibited RANKL-induced osteoclast differentiation and LPS-induced proinflammatory cytokine (IL-1β, IL-6, TNF-a) expression via suppressing the NF-κB signal pathway. These results suggest that PFD can suppress periodontitis progression by inhibiting osteoclastogenesis and inflammatory cytokine production via inhibiting the NF-κB signal pathway, and it may be a promising candidate for controlling periodontitis.

IL-17A regulates autophagy and promotes osteoclast differentiation through the ERK/mTOR/Beclin1 pathway.

Bone is a frequent target of tumor metastasis, with high incidence rate and poor prognosis. Osteoclasts play a key role in the process of tumor bone metastasis. Interleukin-17A (IL-17A) is an inflammatory cytokine, highly expressed in a variety of tumor cells, that can alter the autophagic activity of other cells, thereby causing corresponding lesions. Previous studies have shown that low concentration IL-17A can promote osteoclastogenesis. The aim of this study was to clarify the mechanism of low concentration IL-17A promoting osteoclastogenesis by regulating autophagic activity. The results of our study showed that IL-17A could promote the differentiation of osteoclast precursors (OCPs) into osteoclasts in the presence of RANKL, and increase the mRNA levels of osteoclast-specific genes. Moreover, IL-17A increased the expression of Beclin1 by inhibiting the phosphorylation of ERK and mTOR, leading to enhanced autophagy of OCPs, accompanied by decreased OCP apoptosis. Furthermore, knockdown of Beclin1 and suppression of autophagy by 3-methyladenine (3-MA) significantly attenuated the enhanced osteoclastogenesis induced by IL-17A. In summary, these results indicate that low concentration IL-17A enhances the autophagic activity of OCPs through the ERK/mTOR/Beclin1 pathway during osteoclastogenesis, and further promotes osteoclast differentiation, suggesting that IL-17A may serve as a potential therapeutic target for cancer-related bone resorption in cancer patients.

CpG Oligodeoxynucleotides Inhibit RANKL-Induced Osteoclast Formation by Upregulating A20 Deubiquitinase in RAW 264.7 Cells.

Objective Activation of toll-like receptor 9 (TLR9) has been proposed to play an inhibitory role in RANKL-induced osteoclastogenesis. A20 deubiquitinase has been found to be related to bone loss. This study investigated the role of CpG oligodeoxynucleotides (CpG-ODNs) through regulation of A20 deubiquitinase in RANKL-induced osteoclast formation. Methods RAW 264.7 cells, a murine monocyte-macrophage cell line, were incubated with or without CpG-ODN in the presence of RANKL. Osteoclast-specific genes and their related signaling molecules were measured by real-time quantitative polymerase chain reaction and Western blot assay. Morphological assessment for osteoclast formation was performed using tartrate-resistant acid phosphatase (TRAP) staining and F-actin ring formation staining. Results RANKL-induced osteoclast-related genes and proteins, c-Fos, NFATc1, TRAP, cathepsin K, and carbonic anhydrase II were significantly inhibited in RAW 264.7 cells stimulated with CpG-ODN. CpG-ODN attenuated TNF receptor-associated factor 6 (TRAF6), p-IκBα, and p-NF-κB expression in RAW 264 cells mediated by RANKL. CpG-ODN increased A20 gene and proteins in time-dependent manner. A20 expression under costimulation with CpG-ODN and RANKL was more decreased than under stimulation with RANKL alone. Cells transfected with A20 siRNA augmented expression of osteoclast-related genes and proteins. Number of TRAP-positive cells transfected with A20 siRNA was higher than those transfected with NC siRNA. A20 expression in cells transfected with IL-1β siRNA in the presence of both RANKL and CpG-ODN was more decreased than those with NC siRNA. Conclusion This study showed that CpG-ODN suppressed RANKL-induced osteoclast formation through regulation of the A20-TRAF6 axis in RAW 264.7 cells.

RANKL Impairs the TLR4 Pathway by Increasing TRAF6 and RANK Interaction in Macrophages.

High serum levels of osteoprotegerin (OPG) are found in patients with obesity, type 2 diabetes, sepsis, or septic shock and are associated with a high mortality rate in stroke. The primary known function of OPG is to bind to the receptor activator of NF-κB ligand (RANKL), and by doing so, it inhibits the binding between RANKL and its receptor (RANK). TLR4 signaling in macrophages involves TRAF6 recruitment and contributes to low-grade chronic inflammation in adipose tissue. LPS is a classical activator of the TLR4 pathway and induces the expression of inflammatory cytokines in macrophages. We have previously observed that in the presence of RANKL, there is no LPS-induced activation of TLR4 in macrophages. In this study, we investigated the crosstalk between RANK and TLR4 pathways in macrophages stimulated with both RANKL and LPS to unveil the role of OPG in inflammatory processes. We found that RANKL inhibits TLR4 activation by binding to RANK, promoting the binding between TRAF6 and RANK, lowering TLR4 activation and the expression of proinflammatory mediators. Furthermore, high OPG levels aggravate inflammation by inhibiting RANKL. Our findings elect RANKL as a candidate for drug development as a way to mitigate the impact of obesity-induced inflammation in patients.

Rheumatoid arthritis: Development after the emergence of a chemokine for neutrophils in the synovium.

Rheumatoid arthritis (RA) may not be a multifactorial disease; it can be hypothesized that RA is developed through a series of events following a triggering event, which is the emergence of a chemokine for neutrophils in the synovium. IL-17A, secreted by infiltrated neutrophils, stimulates synoviocytes to produce CCL20, which attracts various CCR6-expressing cells, including Th17 cells. Monocytes (macrophages) appear after neutrophil infiltration according to the natural course of inflammation and secrete IL-1β and TNFα. Then, IL-17A, IL-1β, and TNFα stimulate synoviocytes to produce CCL20, amplifying the inflammation. Varieties of chemokines secreted by infiltrating cells accumulate in the synovium and induce synoviocyte proliferation by binding to the corresponding G protein-coupled receptors, thus expanding the synovial tissue. CCL20 in this tissue attracts circulating monocytes that express both CCR6 and receptor activator of NF-κB (RANK), which differentiate into osteoclasts in the presence of RANKL. In this way, pannus is formed, and bone destruction begins.

Effects of fluid flow shear stress to mouse muscle cells on the bone actions of muscle cell-derived extracellular vesicless.

The interactions between skeletal muscle and bone have been recently noted, and muscle-derived humoral factors related to bone metabolism play crucial roles in the muscle/bone relationships. We previously reported that extracellular vesicles from mouse muscle C2C12 cells (Myo-EVs) suppress osteoclast formation in mice. Although mechanical stress is included in extrinsic factors which are important for both muscle and bone, the detailed roles of mechanical stress in the muscle/bone interactions have still remained unknown. In present study, we examined the effects of fluid flow shear stress (FFSS) to C2C12 cells on the physiological actions of muscle cell-derived EV. Applying FFSS to C2C12 cells significantly enhanced muscle cell-derived EV-suppressed osteoclast formation and several osteoclast-related gene levels in mouse bone marrow cells in the presence of receptor activator nuclear factor κB ligand (RANKL). Moreover, FFSS to C2C12 cells significantly enhanced muscle cell-derived EV-suppressed mitochondria biogenesis genes during osteoclast formation with RANKL treatment. In addition, FFSS to C2C12 cells significantly enhanced muscle cell-derived EV-suppressed osteoclast formation and several osteoclast-related gene levels in Raw264.7 cells in the presence of RANKL. Small RNA-seq-analysis showed that FFSS elevated the expression of miR196a-5p and miR155-5p with the suppressive actions of osteoclast formation and low expression in mouse bone cells. On the other hand, muscle cell-derived EVs with or without FFSS to C2C12 cells did not affect the expression of osteogenic genes, alkaline phosphatase activity and mineralization in mouse osteoblasts. In conclusion, we first showed that FFSS to C2C12 cells enhances the suppressive effects of muscle cell-derived EVs on osteoclast formation in mouse cells. Muscle cell-derived EVs might be partly involved in the effects of mechanical stress on the muscle/bone relationships.

Interleukin-35 Is Involved in Angiogenesis/Bone Remodeling Coupling Through T Helper 17/Interleukin-17 Axis.

ObjectiveOsteoporosis is a common metabolic bone disease mainly involving bone remodeling and blood vessels. The current study aimed to explore the suppressive role of interleukin (IL)-35 in nuclear factor kappa-B ligand receptor activator (RANKL) and macrophage colony stimulating factor (M-CSF)-induced osteoclastogenesis and angiogenesis in osteoclasts.MethodsOsteoclasts differentiation were induced by incubation of mouse leukemic monocyte/macrophage cell line RAW264.7 cells in the presence of RANKL and M-CSF and was assessed with tartrate-resistant acid phosphatase (TRAP) staining assay. The viability and apoptosis of RAW264.7 was measured using CCK-8 assay and flow cytometry, respectively. The expression of angiogenic genes and proteins were measured using RT-PCR, Western blots and ELISA. The inhibition of Th17/IL-17 axis was examined using plumbagin, which was demonstrated as an IL-17A related signaling pathway inhibitor.ResultsIL-35 inhibited the viability of RAW264.7 cells and promoted the apoptosis of RAW264.7 cells in a dose-dependent manner. Furthermore, IL-35 dose-dependently suppressed the expression of angiogenic markers including VEGF and its receptor. The suppressive effect of IL-35 was confirmed through the activation of Th17/IL-17 axis.ConclusionsWe demonstrated for the first time the immuno-suppressive function of IL-35 on RANKL and M-CSF-induced osteoclastogenesis and angiogenesis through Th17/IL-17 axis. Therapeutic approach involving augmentation of IL-35 regulatory response may serve as a novel treatment option for osteoporosis, especially by suppressing bone resorption and angiogenesis.

Astragalus polysaccharide alleviates alveolar bone destruction by regulating local osteoclastogenesis during periodontitis.

Inflammatory imbalance of bone formation/resorption leads to alveolar bone destruction. Astragalus polysaccharide has been confirmed to have anti-inflammatory effects. We sought to disclose the protective effect and its potential mechanisms of astragalus polysaccharide in the periodontitis model. Experimental periodontitis was induced by cotton ligatures for this study. We measured the alveolar bone damage rate, periodontal osteoclasts, proportion of CD4+Foxp3+, CD4+IL-10+, CD4+TGF-β+ subsets in the gingiva, and RANKL, OPG, TGF-β+, and IL-10+ level in the gingiva. We also cultured osteoclast precursor cells in the presence of RANKL and astragalus polysaccharide. Osteoclasto-like cells were identified by TRAP staining, mRNA of RANK, TRAP, and TRAF6 were evaluated by real time PCR. We found that astragalus polysaccharide caused significant protection of the alveolar bone via reducing local osteoclasts. It also decreased the proportion of CD4+Foxp3+ cells and upregulated the level of CD4+IL-10+ cells, reduced RANKL, and remedied IL-10 levels. In cell culture experiments, astragalus polysaccharide prohibited the RANKL mediated osteoclast differentiation. The findings of this study disclose the functions and possible mechanisms of astragalus polysaccharide engaged in local osteoclastogenesis, and reveal the considerable effect of astragalus polysaccharide in alveolar bone homeostasis and its likely contribution to host immuno-regulation in periodontitis.

4-Hexylresorcinol inhibits osteoclastogenesis by suppressing the NF-κB signaling pathway and reverses bone loss in ovariectomized mice.

4-Hexylresorcinol (4HR) is a small organic compound that is widely used as an antiseptic and antioxidant. In the present study, its role in osteoclastogenesis was investigated. Bone marrow-derived macrophages from mice were used to examine the role of 4HR in osteogenesis. An ovariectomy (OVX) mouse model was constructed to examine the effect of 4HR in vivo, followed by hematoxylin and eosin and tartrate resistant acid phosphatase staining. In the present study, 4HR effectively suppressed receptor activator of NF-κB ligand-induced osteoclastogenesis in a dose-dependent manner. 4HR was also found to significantly suppress the expression of osteoclast (OC)-specific markers, including tartrate-resistant acid phosphatase, cathepsin K, nuclear factor of activated T-cell cytoplasmic 1 and c-Fos in the presence of RANKL in BMMs. Furthermore, 4HR inhibited osteoclastogenesis by inhibiting the activation of the NF-κB signaling pathway in BMMs. Consistent with the in vitro results, 4HR effectively ameliorated OVX-induced bone loss and markedly reduced OC number in the proximal tibia in vivo. In conclusion, the present results suggested that 4HR inhibited osteoclastogenesis in vitro and rescued bone loss in vivo, suggesting that 4HR may serve as a novel therapeutic agent for osteoporosis treatment.

Bone-protective and anti-tumor effect of baicalin in osteotropic breast cancer via induction of apoptosis.

Research suggested that bone is the specific target organ for breast cancer metastasis. The related tumor causes significant morbidity due to a reduction in quality of life and physical function. Increased osteoclast function is implicated in the bone microenvironment during the outgrowth of breast cancer. In the present experimental study, we examined the potential bone-protective effect of baicalin osteotropic breast Cancer and explored the possible mechanism of action. In vitro cell viability effect of baicalin was assessed on the breast cancer cell lines (MDA-MB-231 and MCF-7). We also estimated the in vitro osteoclast and bone resorption. Further, baicalin-regulated osteoblastogenesis and osteoclastogenesis were also estimated in vitro. Finally, the role of the baicalin in the expansion of osteolytic bone disease was scrutinized in a breast cancer bone metastases model. Baicalin significantly (p < 0.001) downregulated the viability of murine and human cancer cell lines and diminished the osteoclastogenesis of osteoclast progenitors via estimation with the help of qRT-PCR. Baicalin showed the downregulation in the mRNA expression of OCN and ALP. Baicalin reduced the TRAP-positive cells in the presence of RANKL. Baicalin considerably upregulated the cytochrome c secretion into the cytoplasm. Baicalin markedly increased the DNA fragmentation, caspase-3, caspase-8, and caspase-9. Baicalin significantly (p < 0.001) reduced the metastatic growth of MDA-MB-231 cells,preserving the bone mass in a bone metastasis model. Collectively, we can conclude that these results highlight the bone-protective effect of baicalin, which also highlighted the anti-tumor effect; further research is needed into the likely effects on bone health in the bone metastases and osteoporosis populations, such as post-menopausal women with breast cancer.

Spi-C positively regulates RANKL-mediated osteoclast differentiation and function

Spi-C is an SPI-group erythroblast transformation-specific domain transcription factor expressed during B-cell development. Here, we report that Spi-C is a novel receptor activator of nuclear factor-κB ligand (RANKL)-inducible protein that positively regulates RANKL-mediated osteoclast differentiation and function. Knockdown of Spi-C decreased the expression of RANKL-induced nuclear factor of activated T-cells, cytoplasmic 1, receptor activator of nuclear factor-κB (RANK), and tartrate-resistant acid phosphatase (TRAP), resulting in a marked decrease in the number of TRAP-positive multinucleated cells. Spi-C-transduced bone marrow-derived monocytes/macrophages (BMMs) displayed a significant increase in osteoclast formation in the presence of RANKL. In addition, Spi-C-depleted cells failed to show actin ring formation or bone resorption owing to a marked reduction in the expression of RANKL-mediated dendritic cell-specific transmembrane protein and the d2 isoform of vacuolar (H+) ATPase V0 domain, which are known osteoclast fusion-related genes. Interestingly, RANKL stimulation induced the translocation of Spi-C from the cytoplasm into the nucleus during osteoclastogenesis, which was specifically blocked by inhibitors of p38 mitogen-activated protein kinase (MAPK) or PI3 kinase. Moreover, Spi-C depletion prevented RANKL-induced MAPK activation and the degradation of inhibitor of κB-α (IκBα) in BMMs. Collectively, these results suggest that Spi-C is a novel positive regulator that promotes both osteoclast differentiation and function.

Inhibition of MEK/ERK upregulates GSH production and increases RANKL-induced osteoclast differentiation in RAW 264.7 cells

Osteoclasts (OCs) are multinucleated cells that are phylogenetically evolved from monocyte–macrophage lineage and are essential for skeletal coupling processes. During bone development, bone formation by osteoblasts and bone resorption by OCs are tightly coupled and are involved in bone homeostasis. Therefore, it is essential to understand the mechanisms that regulate OC differentiation in order to develop effective therapeutics for the treatment of OC-associated diseases. This study aimed to determine the molecular mechanisms regulating OC differentiation. The mitogen-activated protein kinases and extracellular signal-regulated kinase (ERK) are recognised to be crucial factors regulating OC differentiation and activation. RAW 264.7 cells were differentiated into OCs in the presence of RANKL and were treated with inhibitors of several signal pathways. Although PD98059 is an ERK inhibitor, it inhibited the phosphorylation of ERK, JNK, Akt, and Src kinase. PD98059 increased OC differentiation and expression of OC markers, such as TRAP, calcitonin receptor, and cathepsin K, and increased the expression of NFATc1. Moreover, it also increased the expression of glutamate-cysteine ligase and production of glutathione (GSH). Thus, we examined the involvement of GSH in OC differentiation and observed that GSH treatment alone increased the OC numbers and cotreatment with PD98059 further enhanced OC differentiation. Our results suggested that inhibition of the ERK pathway may promote OC differentiation via upregulation of GSH. These findings reveal that ERK and GSH modulate the signal pathway necessary for OC differentiation, and this may form the basis of a new therapeutic strategy for treating OC-related diseases.

Negative Regulation of Osteoclast Commitment by Intracellular Protein Phosphatase Magnesium-Dependent 1A.

Increased protein phosphatase magnesium-dependent 1A (PPM1A) levels in patients with ankylosing spondylitis regulate osteoblast differentiation in bony ankylosis; however, the potential mechanisms that regulate osteoclast differentiation in relation to abnormal bone formation remain unclear. This study was undertaken to investigate the relationship of PPM1A to osteoclast differentiation by generating conditional gene-knockout (PPM1Afl/fl ;LysM-Cre) mice and evaluating their bone phenotype. The bone phenotypes of LysM-Cre mice (n = 6) and PPM1Afl/fl ;LysM-Cre mice (n = 6) were assessed by micro-computed tomography. Osteoclast differentiation was induced by culturing bone marrow-derived macrophages in the presence of RANKL and macrophage colony-stimulating factor (M-CSF), and was evaluated by counting tartrate-resistant acid phosphatase-positive multinucleated cells. Levels of messenger RNA for PPM1A, RANK, and osteoclast-specific genes were examined by real-time quantitative polymerase chain reaction, and protein levels were determined by Western blotting. Surface RANK expression was analyzed by fluorescence flow cytometry. The PPM1Afl/fl ;LysM-Cre mice displayed reduced bone mass (P < 0.001) and increased osteoclast differentiation (P < 0.001) and osteoclast-specific gene expression (P < 0.05) compared with their LysM-Cre littermates. Mechanistically, reduced PPM1A function in osteoclast precursors in PPM1Afl/fl ;LysM-Cre mice induced osteoclast lineage commitment by up-regulating RANK expression (P < 0.01) via p38 MAPK activation in response to M-CSF. PPM1A expression in macrophages was decreased by Toll-like receptor 4 activation (P < 0.05). The Ankylosing Spondylitis Disease Activity Score was negatively correlated with the expression of PPM1A in peripheral blood mononuclear cells from patients with axial spondyloarthritis (SpA) (γ = -0.7072, P < 0.0001). The loss of PPM1A function in osteoclast precursors driven by inflammatory signals contributes to osteoclast lineage commitment and differentiation by elevating RANK expression, reflecting a potential role of PPM1A in dynamic bone metabolism in axial SpA.

RANKL-independent osteoclastogenesis in the SH3BP2 cherubism mice

Even though the receptor activator of the nuclear factor-κB ligand (RANKL) and its receptor RANK have an exclusive role in osteoclastogenesis, the possibility of RANK/RANKL-independent osteoclastogenesis has been the subject of a long-standing debate in bone biology. In contrast, it has been reported that calvarial injection of TNF-ɑ elicits significant osteoclastogenesis in the absence of RANK/RANKL in NF-κB2- and RBP-J-deficient mice, suggesting that inflammatory challenges and secondary gene manipulation are the prerequisites for RANK/RANKL-deficient mice to develop osteoclasts in vivo. Here we report that, even in the absence of RANKL (Rankl−/−), cherubism mice (Sh3bp2KI/KI) harboring the homozygous gain-of-function mutation in SH3-domain binding protein 2 (SH3BP2) develop tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclasts spontaneously. The Sh3bp2KI/KIRankl−/− mice exhibit an increase in tooth exposure and a decrease in bone volume/total volume compared to Sh3bp2+/+Rankl−/− mice. The multinucleated cells were stained positively for cathepsin K. Osteoclastic marker gene expression in bone and serum TRAP5b levels were elevated in Sh3bp2KI/KIRankl−/− mice. Elevation of the serum TNF-ɑ levels suggested that TNF-ɑ is a driver for the RANKL-independent osteoclast formation in Sh3bp2KI/KI mice. Our results provide a novel mutant model that develops osteoclasts independent of RANKL and establish that the gain-of-function of SH3BP2 promotes osteoclastogenesis not only in the presence of RANKL but also in the absence of RANKL.

Puerarin inhibits the osteoclastogenesis by inhibiting RANKL-dependent and \u2013independent autophagic responses

BackgroundPuerarin exerts therapeutic effect on osteoporosis due to its inhibitory effect on the formation of osteoclasts. Puerarin is also widely established as an autophagy inhibitor. The study aimed to investigate the significance of autophagy in Puerarin-treated osteoclast formation.MethodsOsteoclast precursors (OCPs) derived from bone marrow-derived macrophages (BMMs) were treated with Puerarin along with RANKL or without RANKL, and then the autophagic parameters of OCPs (including autophagic proteins, LC3 transformation, autophagosome or LC3-puncta) were observed through Western Blotting, Transmission Electron Microscopy and Immunofluorescence assays. Next, after using overexpression vectors of autophagic genes (Atg7, Atg5 and BECN1) to alter autophagy activity, OCP proliferation was measured by Ethynyl deoxyuridine (EdU) assays and Cell Counting Kit-8 (CCK-8) kit, and osteoclast differentiation was assessed by Tartrate-resistant acid phosphatase (TRAP) staining.ResultsThe results showed that Puerarin could directly inhibit the autophagy and proliferation of OCPs. Importantly, overexpression of autophagic genes Atg5, Atg7 and BECN1 reversed Puerarin-inhibited OCP autophagy and proliferation. What’s more, RANKL could promote the autography of OCPs, which was recovered by Puerarin treatment. Interestingly, different from single-Puerarin treatment, we found that in the presence of RANKL, only BECN1 overexpression significantly reversed Puerarin-inhibited osteoclast differentiation and OCP autophagy.ConclusionIn conclusion, Puerarin could inhibit the OCP autophagy in the presence or absence of RANKL, which blocked the OCP proliferation and osteoclast differentiation respectively. Moreover, BECN1 plays an essential role in Puerarin-inhibited osteoclastogenesis. Our study provides potential clue to further complete the intrinsic mechanism of Puerarin in treating osteoporosis.

Autophagy exerts pivotal roles in regulatory effects of 1α,25-(OH)2D3 on the osteoclastogenesis

As an active form of vitamin D3, 1α,25-(OH)2D3 has a positive therapeutical effect on osteoporosis. However, 1α,25-(OH)2D3 can not only promote the osteoclastogenesis, but also inhibit the proliferation of osteoclast precursors (OCPs). Autophagy is regulated by 1α,25-(OH)2D3 and is considered to promote the osteoclastogenesis. Nevertheless, the role of 1α,25-(OH)2D3 in OCPs' autophagy remains unknown. Our study aims to explore whether the effect of 1α,25-(OH)2D3 on osteoclastogenesis is related to its regulation in autophagy. The results showed that 1α,25-(OH)2D3 exhibited a direct inhibitory effect on the autophagy activity and the proliferation of OCPs derived from bone marrow-derived macrophages (BMMs), which was reversed by the overexpression of autophagy-related gene. In presence of RANKL, the autophagy capacity of OCPs and the differentiation from OCPs into mature osteoclasts were significantly enhanced by 1α,25-(OH)2D3, while the suppression of autophagy with spautin-1 or 3-MA downregulated the osteoclastogenesis capacity. In summary, 1α,25-(OH)2D3 can directly suppress OCPs autophagy, which negatively regulates the proliferation of OCPs without RANKL. 1α,25-(OH)2D3 can indirectly upregulate the autophagy response of OCPs, thereby enhancing the osteoclasts formation in presence of RANKL. Therefore, our study found that 1α,25-(OH)2D3 had a dual effect on osteoclastogenesis by regulating autophagy, suggesting that some drugs targeting autophagy may act as an effective supplement of 1α,25-(OH)2D3 in treating osteoporosis.

Suppression of hematopoietic cell kinase ameliorates the bone destruction associated with inflammation

Objectives: To investigate the role of non-receptor tyrosine kinases (NRTKs) in inflammation-induced osteoclastogenesis.Methods: Microarray analyses of global mRNA expression during receptor activator of NF-κB ligand (RANKL) and RANKL plus tumor necrosis factor (TNF)-α-induced osteoclast differentiation were performed. The inhibitory effect on TNF-α-induced osteoclast differentiation of A-419259, a potent inhibitor of hematopoietic cell kinase (Hck), was examined. The in vivo therapeutic effect of A-419259 treatment on lipopolysaccharide (LPS)-induced inflammatory bone destruction was evaluated.Results: We confirmed that Hck expression was selectively increased among the NRTKs during the osteoclast differentiation induced by RANKL and TNF-α, but not by RANKL alone. RANKL and TNF-α-induced osteoclast differentiation and they were dose-dependently inhibited by A-419259 treatment through inhibition of the expression of key regulators of osteoclastogenesis, including Prdm1 and Nfatc1. Notably, LPS-induced inflammatory bone loss in murine calvarial bones was ameliorated by the administration of A-419259.Conclusions: Our results demonstrate that the administration of A-419259 is effective for the inhibition of osteoclast differentiation induced by TNF-α in the presence of RANKL. Therefore, an inhibitor of Hck may be useful as a potent anti-osteoclastogenic agent for the treatment of inflammatory bone destruction.

S100A12 facilitates osteoclast differentiation from human monocytes.

Osteoclasts play a critical role not only in bone homeostasis but also in inflammatory osteolysis, such as that occurring in inflammatory arthritis and systemic inflammation. In both inflammation conditions, inflammatory cytokines like Interleukin (IL)-1, IL-6 and tumor necrosis factor (TNF)-α induce RANKL expression in osteoblasts, but the roles of these cytokines in osteoclast activation remain unclear. S100A12, an S100 family member, is a low-molecular-weight calcium-binding protein. Although it has a pro-inflammatory role, its effects on osteoclast differentiation have been unclear. Here we examined the direct effects of S100A12 on human osteoclasts in vitro. S100A12 facilitated osteoclast formation in the presence of RANKL, as judged by the cells’ morphology and elevated expression of osteoclast-related molecules, including NFATc1, ACP5, CALCR, and ITGβ3. In addition, S100A12 administration markedly enhanced the osteoclasts’ bone resorption ability, consistent with their increased expression levels of CTSK and CA2. Blocking RAGE and TLR4 cancelled the effects of S100A12. Our results indicate that S100A12 is a potential therapeutic target for inflammatory osteolysis.