RANKL Expression In Osteoblasts Research Articles

Cathepsin K Expression is Upregulated Following Micropulse Vibration Stimulation of Osteoclasts Cultured in Vitro

Aim: To evaluate osteoclast inhibition following micropulse vibration and the effects of micropulse vibration on the expression of IL-1β, TNF-α, and RANKL in osteoblasts and osteoclasts and that of cathepsin K (CatK) in osteoclasts cultivated in vitro. Materials and methods: Primary murine osteoblasts and osteoclasts were cultured in vitro and stimulated with an AcceleDent Aura device for 20 min per day for 0 to 96 hours, with microvibrations of 0.25 N/30 Hz. Subsequently, the levels of IL-1β, TNF-α, and RANKL in the supernatants of the culture medium of both types of cells were measured by enzyme-linked immunosorbent assays (ELISAs). In addition, the level of CatK in osteoclasts was evaluated by flow cytometry. Results: Microvibrations significantly upregulated IL-1β and TNF-α expression and downregulated RANKL expression in osteoblasts compared with the corresponding expression in the control group. Compared to those in the control group, osteoclasts treated with microvibrations showed significant downregulation of IL-1β, TNF-α, and RANKL expression. CatK levels in stimulated osteoclasts were increased compared to those in the control group. Conclusion: The type of microvibration applied in this study inhibits osteoclastogenesis and upregulates the expression of CatK, an enzyme that induces bone matrix degradation in osteoclasts cultured in vitro, which stimulates bone formation by osteoblasts and accelerates bone mineralization.

(D-Ala2)GIP Inhibits Inflammatory Bone Resorption by Suppressing TNF-α and RANKL Expression and Directly Impeding Osteoclast Formation.

Glucose-insulinotropic polypeptide (GIP) is an incretin hormone that induces insulin secretion and decreases blood glucose levels. In addition, it has been reported to suppress osteoclast formation. Native GIP is rapidly degraded by dipeptidyl peptidase-4 (DPP-4). (D-Ala2)GIP is a newly developed GIP analog that demonstrates enhanced resistance to DPP-4. This study aimed to evaluate the influence of (D-Ala2)GIP on osteoclast formation and bone resorption during lipopolysaccharide (LPS)-induced inflammation in vivo and in vitro. In vivo, mice received supracalvarial injections of LPS with or without (D-Ala2)GIP for 5 days. Osteoclast formation and bone resorption were evaluated, and TNF-α and RANKL expression were measured. In vitro, the influence of (D-Ala2)GIP on RANKL- and TNF-α-induced osteoclastogenesis, LPS-triggered TNF-α expression in macrophages, and RANKL expression in osteoblasts were examined. Compared to the LPS-only group, calvariae co-administered LPS and (D-Ala2)GIP led to less osteoclast formation, lower bone resorption, and decreased TNF-α and RANKL expression. (D-Ala2)GIP inhibited osteoclastogenesis induced by RANKL and TNF-α and downregulated TNF-α expression in macrophages and RANKL expression in osteoblasts in vitro. Furthermore, (D-Ala2)GIP suppressed the MAPK signaling pathway. The results suggest that (D-Ala2)GIP dampened LPS-triggered osteoclast formation and bone resorption in vivo by reducing TNF-α and RANKL expression and directly inhibiting osteoclastogenesis.

Generating Bone Marrow Chimeric Mouse Using GPR120 Deficient Mouse for the Study of DHA Inhibitory Effect on Osteoclast Formation and Bone Resorption

Docosahexaenoic acid (DHA) is an omega-3 fatty acid that exerts physiological effects via G protein-coupled receptor 120 (GPR120). In our previous studies, we figured out the inhibitory effects of DHA on TNF-α (Tumor necrosis factor-α)-induced osteoclastogenesis via GPR120 in vivo. Moreover, DHA directly suppressed RANKL expression in osteoblasts via GPR120 in vitro. In this study, we generated bone marrow chimeric mice using GPR120 deficient mice (GPR120-KO) to study the inhibitory effects of DHA on bone resorption and osteoclast formation. Bone marrow cells of wild-type (WT) or GPR120-KO mice were transplanted into irradiated recipient mice, which were WT or GPR120 deficient mice. The resulting chimeric mice contained stromal cells from the recipient and bone marrow cells, including osteoclast precursors, from the donor. These chimeric mice were used to perform a series of histological and microfocus computed tomography (micro-CT) analyses after TNF-α injection for induction of osteoclast formation with or without DHA. Osteoclast number and bone resorption were found to be significantly increased in chimeric mice, which did not express GPR120 in stromal cells, compared to chimeric mice, which expressed GPR120 in stromal cells. DHA was also found to suppress specific signaling pathways. We summarized that DHA suppressed TNF-α-induced stromal-dependent osteoclast formation and bone resorption via GPR120.

Azilsartan inhibits inflammation-triggered bone resorption and osteoclastogenesis in vivo via suppression of TNF-α expression in macrophages.

Hypertension is a major risk factor for cardiovascular disease (CVD) and is associated with increased bone loss due to excessive activity of the local renin-angiotensin system (RAS). Angiotensinogen/Angiotensin (ANG) II/Angiotensin II type 1 receptor (AT1R) axis is considered as the core axis regulating RAS activity. Azilsartan is an FDA-approved selective AT1R antagonist that is used to treat hypertension. This study aimed to determine whether azilsartan affects formation of osteoclast, resorption of bone, and the expression of cytokines linked with osteoclastogenesis during lipopolysaccharide (LPS)-triggered inflammation in vivo. In vivo, following a 5-day supracalvarial injection of LPS or tumor necrosis factor-alpha (TNF-α) with or without azilsartan, the proportion of bone resorption and the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells, which are identified as osteoclasts on mice calvariae were counted. The mRNA expression levels of TRAP, cathepsin K, receptor activator of NF-κB ligand (RANKL), and TNF-α were also evaluated. In vitro, the effect of azilsartan (0, 0.01, 0.1, 1, and 10 μM) on RANKL and TNF-α-triggered osteoclastogenesis were investigated. Also, whether azilsartan restrains LPS-triggered TNF-α mRNA and protein expression in macrophages and RANKL expression in osteoblasts were assessed. Furthermore, western blotting for analysis of mitogen-activated protein kinases (MAPKs) signaling was conducted. Azilsartan-treated calvariae exhibited significantly lower bone resorption and osteoclastogenesis than those treated with LPS alone. In vivo, LPS with azilsartan administration resulted in lower levels of receptor activator of RANKL and TNF-α mRNA expression than LPS administration alone. Nevertheless, azilsartan did not show inhibitory effect on RANKL- and TNF-α-triggered osteoclastogenesis in vitro. Compared to macrophages treated with LPS, TNF-α mRNA and protein levels were lower in macrophages treated by LPS with azilsartan. In contrast, RANKL mRNA and protein expression levels in osteoblasts were the same in cells co-treated with azilsartan and LPS and those exposed to LPS only. Furthermore, azilsartan suppressed LPS-triggered MAPKs signaling pathway in macrophages. After 5-day supracalvarial injection, there is no difference between TNF-α injection group and TNF-α with azilsartan injection group. These findings imply that azilsartan prevents LPS-triggered TNF-α production in macrophages, which in turn prevents LPS-Triggered osteoclast formation and bone resorption in vivo.

IL-17A inhibits the degradation of RANKL in osteoblasts by inhibiting BCL2-Beclin1-autophagy signaling

The inflammatory cytokine IL-17A is known to have the capacity to promote osteoclastogenesis, thereby enhancing bone loss. Moreover, IL-17A can promote the expression of RANKL in osteoblasts, contributing to its pro-osteoclastogenic effect. IL-17A is an autophagy regulator, which is also responsible for its regulation on RANKL expression. However, the specific role of autophagy in IL-17A-regulated RANKL expression and the underlying mechanism of IL-17A-regulated osteoblast autophagy remain unclear. IL-17A is known to inhibit autophagy by preventing BCL2 degradation. This study aimed to explore the significance of BCL2-dependent autophagy in IL-17A-regulated RANKL expression. Our results showed that IL-17A at 50 ng/mL could inhibit autophagic activity and promote RANKL protein expression in MC3T3-E1 osteoblast line. Moreover, the corresponding concentration of IL-17A could enhance BCL2 protein expression and the protein interaction between BCL2 and Beclin1 in MC3T3-E1 cells. However, the protein expression of RANKL and BCL2 promoted by 50 ng/mL of IL-17A was blocked by autophagy activation with Beclin1 pharmacological upregulation. Furthermore, RANKL protein expression promoted by 50 ng/mL of IL-17A was also reversed by autophagy activation with BCL2 knockdown. Importantly, the supernatant from osteoblasts treated with 50 ng/mL of IL-17A made osteoclast precursors (OCPs) form larger osteoclasts, which was reversed by BCL2 knockdown in osteoblasts. In conclusion, high levels of IL-17A prevent the degradation of RANKL by inhibiting BCL2-Beclin1-autophagy activation signal transduction in osteoblasts, thereby indirectly promoting osteoclastogenesis.

Hexokinase 2-mediated glycolysis promotes receptor activator of NF-κB ligand expression in Porphyromonas gingivalis lipopolysaccharide-treated osteoblasts.

Glucose metabolism plays a pivotal role in sustaining the inflammatory response to microbial stimulation by providing sufficient energy in immune cells. The main purpose of our study was to explore whether hexokinase 2 (HK2)-mediated glycolysis affected the expression of receptor activator of NF-κB Ligand (RANKL) in Porphyromonas gingivalis lipopolysaccharide (P. gingivalis-LPS)-treated osteoblasts and evaluate the potential involvement of the AKT/PI3K pathway activation during HK2-mediated glycolysis. Primary mice osteoblasts were treated with P. gingivalis-LPS, whereas the HK2 inhibitor (Lonidamine, LND) and small interference RNA were used to restrain HK2 expression. Conditioned medium from osteoblasts was utilized for culturing osteoclast precursors. The mRNA and protein levels of genes involved in glycolysis and bone metabolism including RANKL and osteoprotegerin (OPG) were detected by real-time PCR and western blotting. HK2 and lactate levels were detected by ELISA. Tartrate-resistant acid phosphatase (TRAP) staining was utilized to assess osteoclast formation. The involvement of the AKT/PI3K pathway in osteoblasts was explored by Western blotting. P. gingivalis-LPS enhanced HK2 expression along with rising glycolysis in osteoblasts. LND and HK2-knockdown decreased RANKL expression and the RANKL/OPG ratio in osteoblasts, leading to less osteoclast formation from osteoclast precursors as evidenced by TRAP staining, while the osteogenic potential and proliferation of osteoblasts were not affected by HK2-knockdown. Moreover, P. gingivalis-LPS activated the AKT/PI3K pathway, which could regulate HK2 and RANKL expression in osteoblasts. HK2-mediated glycolysis promoted RANKL in osteoblasts and enhanced osteoclast differentiation. Targeting glycolysis may provide novel therapeutic methods for reducing alveolar bone loss.

The HGF/Met/NF-κB Pathway Regulates RANKL Expression in Osteoblasts and Bone Marrow Stromal Cells.

Multiple myeloma (MM)-induced bone disease occurs through hyperactivation of osteoclasts by several factors secreted by MM cells. MM cell-secreted factors induce osteoclast differentiation and activation via direct and indirect actions including enhanced expression of receptor activator of nuclear factor κB ligand (RANKL) in osteoblasts and bone marrow stromal cells (BMSCs). Hepatocyte growth factor (HGF) is elevated in MM patients and is associated with MM-induced bone disease, although the mechanism by which HGF promotes bone disease remains unclear. In the present study, we demonstrated that HGF induces RANKL expression in osteoblasts and BMSCs, and investigated the mechanism of induction. We found that HGF and MM cell supernatants induced RANKL expression in ST2 cells, MC3T3-E1 cells, and mouse BMSCs. In addition, HGF increased phosphorylation of Met and nuclear factor κB (NF-κB) in ST2 cells, MC3T3-E1 cells, or mouse BMSCs. Moreover, Met and NF-κB inhibitors suppressed HGF-induced RANKL expression in ST2 cells, MC3T3-E1 cells, and mouse BMSCs. These results indicated that HGF promotes RANKL expression in osteoblasts and BMSCs via the Met/NF-κB signaling pathway, and Met and NF-κB inhibitors suppressed HGF-induced RANKL expression. Our findings suggest that Met and NF-κB inhibitors are potentially useful in mitigating MM-induced bone disease in patients expressing high levels of HGF.

Notch and the regulation of osteoclast differentiation and function

Notch 1 through 4 are transmembrane receptors that play a pivotal role in cell differentiation and function; this review addresses the role of Notch signaling in osteoclastogenesis and bone resorption. Notch receptors are activated following interactions with their ligands of the Jagged and Delta-like families. In the skeleton, Notch signaling controls osteoclast differentiation and bone-resorbing activity either directly acting on osteoclast precursors, or indirectly acting on cells of the osteoblast lineage and cells of the immune system. NOTCH1 inhibits osteoclastogenesis, whereas NOTCH2 enhances osteoclast differentiation and function by direct and indirect mechanisms. NOTCH3 induces the expression of RANKL in osteoblasts and osteocytes and as a result induces osteoclast differentiation. There is limited expression of NOTCH4 in skeletal cells. Selected congenital disorders and skeletal malignancies are associated with dysregulated Notch signaling and enhanced bone resorption. In conclusion, Notch signaling is a critical pathway that controls osteoblast and osteoclast differentiation and function and regulates skeletal homeostasis in health and disease.

FRI0376 EFFECT OF CARBAMYLATED LOW-DENSITY LIPOPROTEINS ON BONE CELLS HOMEOSTASIS

Background:Carbamylation is a post-translational modification occurring under several conditions such as uremia, smoking and chronic inflammation as in rheumatoid arthritis (RA). Low-density lipoproteins (LDL) represent a target of carbamylation. Carbamylated-LDL (cLDL) have an increased inflammatory and atherogenic potential. Growing evidence supports an influence of modified lipids on bone cells homeostasis. However, the role of cLDL on bone cells physiology is still unknown.Objectives:Considering the rate of carbamylation and the role of anti-carbamylated proteins antibodies as markers of erosive disease in RA, the purpose of this study is to investigate the effect of cLDL on bone homeostasis.Methods:In-vitrocarbamylation of LDL was performed as previously described by Ok et al. (Kidney Int. 2005). Briefly, native LDL (nLDL) were treated with potassium cyanate (KOCN) for 4 hours, followed by excessive dialysis for 36 hours to remove KOCN. Both osteoclasts (OCs) and osteoblasts (OBLs) were treated at baseline with 20 μg/ml, 100 μg/ml and 200 μg/ml of cLDL or nLDL. To induce osteoclast differentiation, CD14+ monocytes were isolated from peripheral blood of healthy donors by magnetic microbeads separation and then cultured on a 96-wells plate in DMEM media supplemented with RANKL and M-CSF. After 10 days cells were fixed, stained for tartrate-resistant acid phosphatase (TRAP), a marker of OC differentiation, and counted. OBLs were isolated from bone specimens of 3 patients who had undergone to knee or hip arthroplasty for osteoarthritis and treated for 5 days with different concentrations of cLDL and nLDL. OBLs were fixed and stained for alkaline phosphatase positive activity (ALP), a marker of osteogenic differentiation. Total RNA was extracted from cell lysates. Copies of single-stranded complementary DNA (cDNA) were synthesized and analyzed by real-time PCR to evaluate RANKL and Osteoprotegerin (OPG) mRNA expression levels.Results:In OCLs culture, cLDL significantly decreased the number of OC compared to untreated cells (200 μg/ml p=0,0015) and nLDL treated cells (200 μg/ml p= 0,011; 20 μg/ml p= 0,0014) (Fig 1). Moreover, treatment with cLDL induced an increase of not terminally differentiated OCs, reduced dimensions of OCs, less intense TRAP staining and vacuolization (Fig 2). In OBLs culture, cLDL (20, 100 μg/ml) significantly reduced the ALP activity of OBLs compared with untreated cells (p<0.05) (Fig 3). nLDL did not affect the ALP expression. Treatment with cLDL stimulated RANKL mRNA expression in osteoblasts increasing the RANKL/OPG ratio (Fig 4).Fig 1.Fig 2.Fig 3.Fig 4.Conclusion:cLDL induce a significant depression of OC and OBL differentiation. Moreover, cLDL increase RANKL expression in OBL, unbalancing bone tissue turnover towards bone resorption. Accordingly, cLDL could be implicated in the bone loss characterizing several conditions associated to an increased carbamylation, such as RADisclosure of Interests:Bruno Lucchino: None declared, Martina Leopizzi: None declared, Tania Colasanti: None declared, Valeria Di Maio: None declared, cristiano alessandri Grant/research support from: Pfizer, Guido Valesini: None declared, fabrizio conti Speakers bureau: BMS, Lilly, Abbvie, Pfizer, Sanofi, Manuela Di Franco: None declared, Francesca Romana Spinelli Grant/research support from: Pfizer, Consultant of: Novartis, Gilead, Lilly, Sanofi, Celgene, Speakers bureau: Lilly

Endogenous parathyroid hormone (PTH) signals through osteoblasts via RANKL during fracture healing to affect osteoclasts

AimTo investigate the effect of endogenous PTH deficiency on osteoclasts during fracture healing and its mechanism. MethodsA femoral fracture model was used to determine the role of endogenous PTH in fracture healing. Immunohistochemistry, qPCR, and Western blot were used to determine the potential functions and mechanisms of endogenous PTH. ResultIn this study, we found that expression of RANKL and CK was lower in PTH knockout (KO) mice than in wild type (WT) mice. In vitro culture of osteoclasts showed that under the same stimulation, there was no statistical difference in the number of osteoclasts and the area of bone resorption areas in PTH WT mice and PTH KO mice. We found that a high concentration of RANKL could promote the number and activity of osteoclasts. Upon induction of osteoblasts in vitro, those from the PTH WT group expressed higher RANKL protein and mRNA than those from the PTH KO group. Lastly, we confirmed that the PI3K/AKT/STAT5 pathway promotes RANKL increase from osteoblasts. ConclusionDuring fracture healing, endogenous PTH deficiency can affect osteoclast activity by reducing RANKL expression in osteoblasts.

IDH2 deficiency increases bone mass with reduced osteoclastogenesis by limiting RANKL expression in osteoblasts.

Mitochondria are not only responsible for cellular energy production but are also involved in signaling, cellular differentiation, cell death, and aging. Mitochondrial NADP+-dependent isocitrate dehydrogenase (IDH2) catalyzes the decarboxylation of isocitrate to α-ketoglutarate, accompanied by NADPH production. IDH2 plays a central role in mitochondrial function in multiple cell types and various organs, including the heart, kidneys, and brain. However, the function of IDH2 in bone tissue is yet to be elucidated. Here, we report that disruption of IDH2 in mice results in high bone mass due to decreased osteoclast number and resorption activity. Although IDH2 played no cell-intrinsic role in osteoclasts, IDH2-deficient animals showed decreased serum markers of osteoclast activity and bone resorption. Bone marrow stromal cells/osteoblasts from Idh2 knockout mice were defective in promoting osteoclastogenesis due to a reduced expression of a key osteoclastogenic factor, receptor activator of nuclear factor-κB ligand (RANKL), in osteoblasts in vivo and in vitro through the attenuation of ATF4-NFATc1 signaling. Our findings suggest that IDH2 is a novel regulator of osteoblast-to-osteoclast communication and bone metabolism, acting via the ATF4-NFATc1-RANKL signaling axis in osteoblasts, and they provide a rationale for further study of IDH2 as a potential therapeutic target for the prevention of bone loss.

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.

Gene Modification of Transforming Growth Factor β (TGF-β) and Interleukin 10 (IL-10) in Suppressing Mt Sonicate Induced Osteoclast Formation and Bone Absorption

BackgroundOsteoarticular tuberculosis is an osteolytic lesion caused by Mycobacterium tuberculosis (MTB). Inflammatory factors such as TNF-α play a critical role in anti-tuberculosis immunity by regulating osteoblast and osteoclast functions. Both TGF-β and IL-10 have immune suppression effects to downregulate secretion and release of inflammatory factors, such as TNF-α, that play roles in regulating osteoblast and osteoclast functions. This study thus investigated the effects of osteoclast with modified TGF-β and IL-10 gene expression on MTB-induced osteoclast formation and bone absorption.Material/MethodsBone marrow mononuclear cells were induced to differentiate into osteoblasts and osteoclasts in vitro to generate a co-culture system. MTB powder lysed by ultrasound (Mt sonicate) were added in gradients to observe osteoblast formation and osteoclast absorption. Cell apoptosis was measured by flow cytometry, while ELISA was used assess TNF-α, TGF-β, and IL-10. Viral vectors carrying TGF-β or IL-10 gene were used to transfect osteoclasts, followed by ELISA assay. Bone absorption and osteoblast apoptosis were compared among groups.ResultsMt sonicate significantly facilitated osteoclast formation and bone formation. It upregulated contents of TNF-α, TGF-β, and IL-10, induced osteoblast apoptosis, enhanced RANKL expression in osteoblasts, and decreased OPG expression. Overexpression of TGF-β and/or IL-10 significantly decreased its upregulation effect on TNF-α by Mt sonicate, and hindered Mt sonicate-induced osteoblast apoptosis, osteoclast formation, and bone absorption.ConclusionsOverexpression of TGF-β and IL-10 significantly inhibits TMB-induced TNF-α synthesis and release, suppresses osteoblast apoptosis, and hinders osteoclast formation and bone absorption.

Effect of Runx2 silencing on autophagy and RANKL expression in osteoblasts

ObjectiveThis study aimed to investigate the effect of Runx2 silencing on autophagy and RANKL expression in mouse osteoblasts, and provide an experimental basis to assess obstacles in dental eruption. MethodsIn accordance with previously reported methods, LVpFU-GW-016PSC60109-1 virus was used to transfect mouse osteoblasts (MOI = 40). Target gene expression was assessed via cytometer, and the effect of silencing Runx2 was assessed via a two-step quantitative real-time polymerase chain reaction (qRT-PCR)-based method. Western blotting was performed to assess LC3, Beclin-1 and RANKL expression. ResultsAs confirmed via qRT-PCR analysis, Runx2 was efficiently silenced in the experimental group (>90% efficiency). Western blotting revealed that LC3 and RANKL proteins were significantly down -regulated in the experimental group (group KD), their expression levels being particularly lower than those in the control group (group NC). However, Beclin-1 protein expression was not significantly different from that of the control. ConclusionUpon Runx2 silencing, autophagy-related proteins and RANKL were repressed in osteoblasts, thereby potentially causing the tooth eruption disorder.

The Natural Product 6-Gingerol Inhibits Inflammation-Associated Osteoclast Differentiation via Reduction of Prostaglandin E₂ Levels.

The natural product 6-gingerol, a major bioactive component of the rhizome of ginger (Zingiber officinale), is known to have several beneficial effects on health, including anti-inflammatory activity. The present study aimed to investigate the effects of 6-gingerol on osteoclast differentiation associated with inflammation. 6-Gingerol inhibited osteoclast differentiation in co-cultures of osteoblasts and osteoclast precursor cells in response to the pro-inflammatory cytokine, interleukin (IL)-1. However, it did not affect osteoclast precursor differentiation into osteoclasts induced by the receptor activator of nuclear factor-κB ligand (RANKL), a key cytokine causing osteoclast differentiation. 6-Gingerol inhibited IL-1-induced RANKL expression in osteoblasts, and the addition of RANKL to the co-cultures overcame 6-gingerol-mediated inhibition of osteoclast differentiation. It also suppressed IL-1-induced prostaglandin E2 (PGE2) production in osteoblasts, and the addition of exogenous PGE2 reversed 6-gingerol-mediated inhibition of IL-induced RANKL expression in osteoblasts and osteoclast differentiation in the co-cultures. We found that 6-gingerol reduced PGE2 levels by suppressing enzymatic activities of cyclooxygenase and PGE synthase, which cooperatively catalyze the conversion of arachidonic acid to PGE2. Our findings demonstrate that 6-gingerol inhibits IL-1-induced osteoclast differentiation via suppression of RANKL expression in osteoblasts though reduction of PGE2 levels, suggesting its potential use in treating inflammatory bone destruction associated with excessive PGE2 production.

Magnolol Inhibits Osteoclast Differentiation via Suppression of RANKL Expression

Magnolol, a compound from the traditional Korean herb Magnolia sp., has been exhaustively investigated as a therapeutic agent against several diseases including systemic and local inflammation. We examined the effects of magnolol on osteoclastic differentiation associated with inflammation. Magnolol markedly reduced interleukin (IL)-1-induced osteoclast formation in co-cultures of murine osteoblasts and bone marrow cells, whereas it had no effect on receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast formation in bone marrow macrophage cultures. In osteoblasts, magnolol markedly inhibited both the up-regulation of RANKL expression and the production of prostaglandin E2 (PGE2) in response to IL-1 treatment. Addition of exogenous PGE2 reversed the inhibitory effects of magnolol on IL-1-induced RANKL expression in osteoblasts and osteoclast formation in co-cultures. Magnolol inhibited IL-1-induced PGE2 production, at least in part by suppressing cyclooxygenase-2 (COX-2) expression. Taken together, these results demonstrate that magnolol inhibits IL-1-induced RANKL expression in osteoblasts through suppression of COX-2 expression and PGE2 production, resulting in inhibition of osteoclast differentiation in co-cultures.

α-Tocopheryl Succinate Inhibits Osteolytic Bone Metastasis of Breast Cancer by Suppressing Migration of Cancer Cells and Receptor Activator of Nuclear Factor-κB Ligand Expression of Osteoblasts.

BackgroundBreast cancer is one of the most common cancers affecting women and has a high incidence of bone metastasis, causing osteolytic lesions. The elevated expression of receptor activator of nuclear factor-κB ligand (RANKL) in cancer activates osteoclasts, leading to bone destruction. We previously reported that α-tocopheryl succinate (αTP-suc) inhibited interleukin-1-induced RANKL expression in osteoblasts. Here, we examined the effect of αTP-suc on osteolytic bone metastasis in breast cancer.MethodsTo examine the effect of αTP-suc on the metastatic capacity of breast cancer, MDA-MB-231-FL cells were injected into the left cardiac ventricle of BALB/c nude mice along with intraperitoneal injection of αTP-suc. The mice were then analyzed by bioluminescence imaging. To investigate the effect of αTP-suc on osteolysis, 4T1 cells were directly injected into the femur of BALB/c mice along with intraperitoneal injection of αTP-suc. Microcomputed tomography analysis and histomorphometric analysis of the femora were performed.ResultsαTP-suc inhibited cell migration and cell growth of 4T1 cells. In line with these results, bone metastasis of MDA-MB-231-FL cells was reduced in mice injected with αTP-suc. In addition, αTP-suc decreased osteoclastogenesis by inhibiting 4T1-induced RANKL expression in osteoblasts. Consistent with these results, 4T1-induced bone destruction was ameliorated by αTP-suc, with in vivo analysis showing reduced tumor burden and osteoclast numbers.ConclusionsOur findings suggest that αTP-suc may be efficiently utilized to prevent and treat osteolytic bone metastasis of breast cancer with dual effects.

Resolvin E1 Inhibits Osteoclastogenesis and Bone Resorption by Suppressing IL-17-induced RANKL Expression in Osteoblasts and RANKL-induced Osteoclast Differentiation

Resolvin E1 (RvE1) derived from the ω-3 polyunsaturated fatty acid eicosapentaenoic acid is known to be a potent pro-resolving lipid mediator that prevents chronic inflammation and osteoclastogenesis. We investigated the inhibitory effects of RvE1 on osteoclastogenesis and bone resorption to clarify its therapeutic potential for rheumatoid arthritis (RA). Receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation was assessed with tartrate-resistant acid phosphatase staining. RANKL-induced bone resorption was assessed by the measurement of pit formation using calcium phosphate-labeled fluorescent polyanionic molecules in RAW264.7 cells as osteoclast precursors. The effects of RvE1 on the RANKL-induced mRNA expression of osteoclast-specific genes and transcriptional factors such as c-fos and nuclear factor of activated T cells c1 (NFATc1) in RAW264.7 cells were measured by quantitative real-time PCR. The distribution of NFATc1 induced by RANKL was evaluated by immunofluorescence staining in RAW264.7 cells. To analyze the mechanism of the inhibitory effect of RvE1 on osteoclastogenesis, we measured IL-17-induced RANKL mRNA expression in MC3T3-E1 osteoblast cells treated with RvE1 using quantitative real-time PCR and determined the level of prostaglandin E2 (PGE2) production by enzyme-linked immunosorbent assay. RvE1 significantly suppressed RANKL-induced osteoclast differentiation and bone resorption. RvE1 inhibited the RANKL-induced mRNA expression of osteoclast-specific genes along with the transcription factors NFATc1 and c-fos. Moreover, NFATc1 translocation from the cytoplasm to the nucleus of RAW264.7 cells was suppressed following RvE1 treatment. RvE1 also inhibited IL-17-induced RANKL mRNA expression and PGE2 production in MC3T3-E1 cells. RvE1 inhibited osteoclastogenesis and bone resorption by suppressing RANKL-induced NFATc1 and c-fos expression in osteoclasts and IL-17-induced RANKL expression through the autocrine action of PGE2 in osteoblasts. Our data suggest RvE1 as a new therapeutic target of RA.

A Jak1/2 inhibitor, baricitinib, inhibits osteoclastogenesis by suppressing RANKL expression in osteoblasts in vitro.

The Janus kinases (Jaks) are hubs in the signaling process of more than 50 cytokine or hormone receptors. However, the function of Jak in bone metabolism remains to be elucidated. Here, we showed that the inhibition of Jak1 and/or Jak2 in osteoblast-lineage cells led to impaired osteoclastogenesis due to the reduced expression of receptor activator of nuclear factor-κB ligand (RANKL). Murine calvaria-derived osteoblasts induced differentiation of bone marrow cells into osteoclasts in the presence of 1,25-dihydroxyvitamin D3 (1,25D3) and prostaglandin E2 (PGE2) in vitro. However, treatment with the Jak1/2 inhibitor, baricitinib, markedly inhibited osteoclastogenesis in the co-culture. On the other hand, baricitinib did not inhibit RANKL-induced osteoclast differentiation of bone marrow macrophages. These results indicated that baricitinib acted on osteoblasts, but not on bone marrow macrophages. Baricitinib suppressed 1,25D3 and PGE2-induced up-regulation of RANKL in osteoblasts, but not macrophage colony-stimulating factor expression. Moreover, the addition of recombinant RANKL to co-cultures completely rescued baricitinib-induced impairment of osteoclastogenesis. shRNA-mediated knockdown of Jak1 or Jak2 also suppressed RANKL expression in osteoblasts and inhibited osteoclastogenesis. Finally, cytokine array revealed that 1,25D3 and PGE2 stimulated secretion of interleukin-6 (IL-6), IL-11, and leukemia inhibitory factor in the co-culture. Hence, Jak1 and Jak2 represent novel therapeutic targets for osteoporosis as well as inflammatory bone diseases including rheumatoid arthritis.

Use of ethanol extracts of Terminalia chebula to prevent periodontal disease induced by dental plaque bacteria

BackgroundThe fruit of the Terminalia chebula tree has been widely used for the treatment of various disorders. Its anti-diabetic, anti-mutagenic, anti-oxidant, anti-bacterial, anti-fungal, and anti-viral effects have been studied. Dental plaque bacteria (DPB) are intimately associated with gingivitis and periodontitis. In the quest for materials that will prove useful in the treatment and prevention of periodontal disease, we investigated the preventive effects of an ethanol extract of Terminalia chebula (EETC) on DPB-induced inflammation and bone resorption.MethodsThe anti-bacterial effect of EETC was analyzed using the disc diffusion method. The anti-inflammatory effect of EETC was determined by molecular biological analysis of the DPB-mediated culture cells. Prevention of osteoclastic bone resorption by EETC was explored using osteoclast formation and pit formation assays.ResultsEETC suppressed the growth of oral bacteria and reduced the induction of inflammatory cytokines and proteases, abolishing the expression of PGE2 and COX-2 and inhibiting matrix damage. By stimulating the DPB-derived lipopolysaccharides, EETC inhibited both osteoclast formation in osteoclast precursors and RANKL expression in osteoblasts, thereby contributing to the prevention of bone resorption.ConclusionsEETC may be a beneficial supplement to help prevent DPB-mediated periodontal disease.