Monocrotaline Suppresses RANKL-Induced Osteoclastogenesis In Vitro and Prevents LPS-Induced Bone Loss In Vivo

Background/Aims: Extensive osteoclast formation plays a critical role in bone diseases, including rheumatoid arthritis, periodontitis and the aseptic loosening of orthopedic implants. Thus, identification of agents that can suppress osteoclast formation and bone resorption is important for the treatment of these diseases. Monocrotaline (Mon), the major bioactive component of crotalaria sessiliflora has been investigated for its anti-cancer activities. However, the effect of Mon on osteoclast formation and osteolysis is not known. Methods: The bone marrow macrophages (BMMs) were cultured with M-CSF and RANKL followed by Mon treatment. Then the effects of Mon on osteoclast differentiation were evaluated by counting TRAP (+) multinucleated cells. Moreover, effects of Mon on hydroxyapatite resorption activity of mature osteoclast were studied through resorption areas measurement. The involved potential signaling pathways were analyzed by performed Western blotting and quantitative real-time PCR examination. Further, we established a mouse calvarial osteolysis model to measure the osteolysis suppressing effect of Mon in vivo. Results: In this study, we show that Mon can inhibit RANKL-induced osteoclast formation and function in a dose-dependent manner. Mon inhibits the expression of osteoclast marker genes such as tartrate-resistant acid phosphatase (TRAP) and cathepsin K. Furthermore, Mon inhibits RANKL-induced the activation of p38 and JNK. Consistent with in vitro results, Mon exhibits protective effects in an in vivo mouse model of LPS-induced calvarial osteolysis. Conclusion: Taken together our data demonstrate that Mon may be a potential prophylactic anti-osteoclastic agent for the treatment of osteolytic diseases caused by excessive osteoclast formation and function.

Download Full-text

Inhibitory Effects of N-[2-(4-acetyl-1-piperazinyl) phenyl]-2-(2-chlorophenoxy) acetamide on Osteoclast Differentiation In Vitro via the Downregulation of TRAF6

International Journal of Molecular Sciences ◽

10.3390/ijms20205196 ◽

2019 ◽

Vol 20 (20) ◽

pp. 5196 ◽

Cited By ~ 5

Author(s):

Zhihao Chen ◽

Eunjin Cho ◽

Jinkyung Lee ◽

Sunwoo Lee ◽

Tae-Hoon Lee

Keyword(s):

Bone Resorption ◽

Mononuclear Cells ◽

Osteoclast Differentiation ◽

Bone Diseases ◽

Marker Genes ◽

Tartrate Resistant Acid Phosphatase ◽

Specific Marker ◽

Potential Candidate ◽

Dependent Manner

Osteoclasts are poly-nuclear cells that resorb mineral components from old or damaged bone tissue. Primary mononuclear cells are activated by receptor activator of nuclear factor kappa-Β ligand (RANKL) and differentiate into large multinucleated cells. Dysregulation of osteoclast differentiation can lead to pathological bone loss and destruction. Many studies have focused on the development of new molecules to regulate RANKL-mediated signaling. In this study, N-[2-(4-acetyl-1-piperazinyl)phenyl]-2-(2-chlorophenoxy) acetamide (PPOA-N-Ac-2-Cl) led to a significant decrease in the formation of multinucleated tartrate-resistant acid phosphatase (TRAP)-positive cells in a dose-dependent manner, without inducing significant cytotoxicity. PPOA-N-Ac-2-Cl affected the expression of osteoclast-specific marker genes, such as TRAF6, c-fos, DC-STAMP, NFATc1, MMP9, CtsK, and TRAP (Acp5), during RANKL-mediated osteoclastogenesis. Moreover, PPOA-N-Ac-2-Cl significantly attenuated the protein levels of CtsK, a critical protease involved in bone resorption. Accordingly, bone resorption activity and F-actin ring formation decreased in the presence of PPOA-N-Ac-2-Cl. In conclusion, this study shows that PPOA-N-Ac-2-Cl acts as an inhibitor of osteoclast differentiation and may serve as a potential candidate agent for the treatment of osteoclast-related bone diseases by virtue of attenuating bone resorption.

Download Full-text

3-Hydroxyolean-12-en-27-oic Acids Inhibit RANKL-Induced Osteoclastogenesis in Vitro and Inflammation-Induced Bone Loss in Vivo

International Journal of Molecular Sciences ◽

10.3390/ijms21155240 ◽

2020 ◽

Vol 21 (15) ◽

pp. 5240

Author(s):

Wonyoung Seo ◽

Suhyun Lee ◽

Phuong Thao Tran ◽

Thi Quynh-Mai Ngo ◽

Okwha Kim ◽

...

Keyword(s):

Bone Destruction ◽

Osteoclast Formation ◽

Bone Diseases ◽

Marker Genes ◽

Tartrate Resistant Acid Phosphatase ◽

Specific Marker ◽

Nuclear Factor ◽

Activated T Cells

Olean-12-en-27-oic acids possess a variety of pharmacological effects. However, their effects and underlying mechanisms on osteoclastogenesis remain unclear. This study aimed to investigate the anti-osteoclastogenic effects of five olean-12-en-27-oic acid derivatives including 3α,23-isopropylidenedioxyolean-12-en-27-oic acid (AR-1), 3-oxoolean-12-en-27-oic acid (AR-2), 3α-hydroxyolean-12-en-27-oic acid (AR-3), 23-hydroxy-3-oxoolean-12-en-27-oic acid (AR-4), and aceriphyllic acid A (AR-5). Among the five olean-12-en-27-oic acid derivatives, 3-hydroxyolean-12-en-27-oic acid derivatives, AR-3 and AR-5, significantly inhibited receptor activator of nuclear factor-κB ligand (RANKL)-induced mature osteoclast formation by reducing the number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts, F–actin ring formation, and mineral resorption activity. AR-3 and AR-5 decreased RANKL-induced expression levels of osteoclast-specific marker genes such as c-Src, TRAP, and cathepsin K (CtsK) as well as c-Fos and nuclear factor of activated T cells cytoplasmic 1 (NFATc1). Mice treated with either AR-3 or AR-5 showed significant protection of the mice from lipopolysaccharide (LPS)-induced bone destruction and osteoclast formation. In particular, AR-5 suppressed RANKL-induced phosphorylation of JNK and ERK mitogen-activated protein kinases (MAPKs). The results suggest that AR-3 and AR-5 attenuate osteoclast formation in vitro and in vivo by suppressing RANKL-mediated MAPKs and NFATc1 signaling pathways and could potentially be lead compounds for the prevention or treatment of osteolytic bone diseases.

Download Full-text

Protective Effect of Ciclopirox against Ovariectomy-Induced Bone Loss in Mice by Suppressing Osteoclast Formation and Function

International Journal of Molecular Sciences ◽

10.3390/ijms22158299 ◽

2021 ◽

Vol 22 (15) ◽

pp. 8299

Author(s):

Hye Jung Ihn ◽

Jiwon Lim ◽

Kiryeong Kim ◽

Sang-Hyeon Nam ◽

Soomin Lim ◽

...

Keyword(s):

Bone Loss ◽

Type I Collagen ◽

Osteoclast Differentiation ◽

Osteoclast Formation ◽

Bone Diseases ◽

Mitogen Activated Protein Kinase ◽

Type I ◽

And Function

Postmenopausal osteoporosis is closely associated with excessive osteoclast formation and function, resulting in the loss of bone mass. Osteoclast-targeting agents have been developed to manage this disease. We examined the effects of ciclopirox on osteoclast differentiation and bone resorption in vitro and in vivo. Ciclopirox significantly inhibited osteoclast formation from primary murine bone marrow macrophages (BMMs) in response to receptor activator of nuclear factor kappa B ligand (RANKL), and the expression of genes associated with osteoclastogenesis and function was decreased. The formation of actin rings and resorption pits was suppressed by ciclopirox. Analysis of RANKL-mediated early signaling events in BMMs revealed that ciclopirox attenuates IκBα phosphorylation without affecting mitogen-activated protein kinase activation. Furthermore, the administration of ciclopirox suppressed osteoclast formation and bone loss in ovariectomy-induced osteoporosis in mice and reduced serum levels of osteocalcin and C-terminal telopeptide fragment of type I collagen C-terminus. These results indicate that ciclopirox exhibits antiosteoclastogenic activity both in vitro and in vivo and represents a new candidate compound for protection against osteoporosis and other osteoclast-related bone diseases.

Download Full-text

Inhibition of RANKL-Induced Osteoclastogenesis by Novel Mutant RANKL

International Journal of Molecular Sciences ◽

10.3390/ijms22010434 ◽

2021 ◽

Vol 22 (1) ◽

pp. 434

Author(s):

Yuria Jang ◽

Hong Moon Sohn ◽

Young Jong Ko ◽

Hoon Hyun ◽

Wonbong Lim

Keyword(s):

Nuclear Translocation ◽

Critical Role ◽

Osteoclast Differentiation ◽

Point Mutations ◽

Tartrate Resistant Acid Phosphatase ◽

Mice Model ◽

Gsk 3Β ◽

Rank Signaling

Background: Recently, it was reported that leucine-rich repeat-containing G-protein-coupled receptor 4 (LGR4, also called GPR48) is another receptor for RANKL and was shown to compete with RANK to bind RANKL and suppress canonical RANK signaling during osteoclast differentiation. The critical role of the protein triad RANK–RANKL in osteoclastogenesis has made their binding an important target for the development of drugs against osteoporosis. In this study, point-mutations were introduced in the RANKL protein based on the crystal structure of the RANKL complex and its counterpart receptor RANK, and we investigated whether LGR4 signaling in the absence of the RANK signal could lead to the inhibition of osteoclastogenesis.; Methods: The effects of point-mutated RANKL (mRANKL-MT) on osteoclastogenesis were assessed by tartrate-resistant acid phosphatase (TRAP), resorption pit formation, quantitative real-time polymerase chain reaction (qPCR), western blot, NFATc1 nuclear translocation, micro-CT and histomorphological assay in wild type RANKL (mRANKL-WT)-induced in vitro and in vivo experimental mice model. Results: As a proof of concept, treatment with the mutant RANKL led to the stimulation of GSK-3β phosphorylation, as well as the inhibition of NFATc1 translocation, mRNA expression of TRAP and OSCAR, TRAP activity, and bone resorption, in RANKL-induced mouse models; and Conclusions: The results of our study demonstrate that the mutant RANKL can be used as a therapeutic agent for osteoporosis by inhibiting RANKL-induced osteoclastogenesis via comparative inhibition of RANKL. Moreover, the mutant RANKL was found to lack the toxic side effects of most osteoporosis treatments.

Download Full-text

Piceatannol attenuates RANKL-induced osteoclast differentiation and bone resorption by suppressing MAPK, NF-κB and AKT signalling pathways and promotes Caspase3-mediated apoptosis of mature osteoclasts

Royal Society Open Science ◽

10.1098/rsos.190360 ◽

2019 ◽

Vol 6 (6) ◽

pp. 190360 ◽

Cited By ~ 5

Author(s):

Liuliu Yan ◽

Lulu Lu ◽

Fangbin Hu ◽

Dattatrya Shetti ◽

Kun Wei

Keyword(s):

Bone Resorption ◽

Osteoclast Differentiation ◽

Giant Cells ◽

Inhibitory Effect ◽

Osteoclast Formation ◽

Bone Diseases ◽

Signalling Pathways ◽

Dependent Manner ◽

Underlying Mechanisms ◽

And Function

Osteoclasts are multinuclear giant cells that have unique ability to degrade bone. The search for new medicines that modulate the formation and function of osteoclasts is a potential approach for treating osteoclast-related bone diseases. Piceatannol (PIC) is a natural organic polyphenolic stilbene compound found in diverse plants with a strong antioxidant and anti-inflammatory effect. However, the effect of PIC on bone health has not been scrutinized systematically. In this study, we used RAW264.7, an osteoclast lineage of cells of murine macrophages, to investigate the effects and the underlying mechanisms of PIC on osteoclasts. Here, we demonstrated that PIC treatment ranging from 0 to 40 µM strongly inhibited osteoclast formation and bone resorption in a dose-dependent manner. Furthermore, the inhibitory effect of PIC was accompanied by the decrease of osteoclast-specific genes. At the molecular level, PIC suppressed the phosphorylation of c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK1/2), NF-κB p65, IκBα and AKT. Besides, PIC promoted the apoptosis of mature osteoclasts by inducing caspase-3 expression. In conclusion, our results suggested that PIC inhibited RANKL-induced osteoclastogenesis and bone resorption by suppressing MAPK, NF-κB and AKT signalling pathways and promoted caspase3-mediated apoptosis of mature osteoclasts, which might contribute to the treatment of bone diseases characterized by excessive bone resorption.

Download Full-text

Anti-Osteoporosis Effects of the Eleutherococcus senticosus, Achyranthes japonica, and Atractylodes japonica Mixed Extract Fermented with Nuruk

Nutrients ◽

10.3390/nu13113904 ◽

2021 ◽

Vol 13 (11) ◽

pp. 3904

Author(s):

So Young Eun ◽

Yoon-Hee Cheon ◽

Gyeong Do Park ◽

Chong Hyuk Chung ◽

Chang Hoon Lee ◽

...

Keyword(s):

Bone Loss ◽

Bone Diseases ◽

Marker Genes ◽

Tartrate Resistant Acid Phosphatase ◽

Specific Marker ◽

Eleutherococcus Senticosus ◽

Achyranthes Japonica ◽

Atractylodes Japonica

Vigeo is a mixture of fermented extracts of Eleutherococcus senticosus Maxim (ESM), Achyranthes japonica (Miq.) Nakai (AJN), and Atractylodes japonica Koidzumi (AJK) manufactured using the traditional Korean nuruk fermentation method. Although the bioactive effects of ESM, AJN, and AJK have already been reported, the pharmacological effects of Vigeo have not been proven. Therefore, in this study, we investigated whether Vigeo had inhivitory effects on lipopolysaccharide (LPS)-induced inflammatory bone loss in vivo and receptor activator of nuclear factor-B ligand (RANKL)-induced osteoclastogenesis and the related mechanism in vitro. Vigeo administration conferred effective protection against bone loss induced by excessive inflammatory response and activity of osteoclasts in LPS-induced inflammatory osteoporosis mouse model. In addition, Vigeo significantly suppressed the formation of tartrate-resistant acid phosphatase-positive osteoclasts induced by RANKL and inhibited F-actin formation and bone resorbing activity without any cytotoxicity. Moreover, Vigeo significantly inhibited RANKL-induced phosphorylation of p38, ERK, JNK, IκB, and AKT and degradation of IkB. Additionally, Vigeo strongly inhibited the mRNA and protein expression of c-FOS and NFATc1 and subsequently attenuated the expression of osteoclast specific marker genes induced by RANKL. We demonstrated for the first time the anti-osteoporosis effect of Vigeo, suggesting that it could be a potential therapeutic candidate for the treatment of osteoclast-mediated inflammatory bone diseases.

Download Full-text

Butyrate Inhibits Osteoclast Activity In Vitro and Regulates Systemic Inflammation and Bone Healing in a Murine Osteotomy Model Compared to Antibiotic-Treated Mice

Mediators of Inflammation ◽

10.1155/2021/8817421 ◽

2021 ◽

Vol 2021 ◽

pp. 1-17

Author(s):

Alexandra Wallimann ◽

Walker Magrath ◽

Brenna Pugliese ◽

Nino Stocker ◽

Patrick Westermann ◽

...

Keyword(s):

Fracture Healing ◽

Systemic Inflammation ◽

Bone Healing ◽

Bone Turnover Marker ◽

Osteoclast Differentiation ◽

Short Chain Fatty Acids ◽

Osteoclast Formation ◽

Dependent Manner

Short-chain fatty acids (SCFAs) produced by the gut microbiota have previously been demonstrated to play a role in numerous chronic inflammatory diseases and to be key mediators in the gut-bone signaling axis. However, the role of SCFAs in bone fracture healing and its impact on systemic inflammation during the regeneration process has not been extensively investigated yet. The aim of this study was to first determine the effects of the SCFA butyrate on key cells involved in fracture healing in vitro, namely, osteoclasts and mesenchymal stromal cells (MSCs), and second, to assess if butyrate supplementation or antibiotic therapy impacts bone healing, systemic immune status, and inflammation levels in a murine osteotomy model. Butyrate significantly reduced osteoclast formation and resorption activity in a dose-dependent manner and displayed a trend for increased calcium deposits in MSC cultures. Numerous genes associated with osteoclast differentiation were differentially expressed in osteoclast precursor cells upon butyrate exposure. In vivo, antibiotic-treated mice showed reduced SCFA levels in the cecum, as well as a distinct gut microbiome composition. Furthermore, circulating proinflammatory TNFα, IL-17a, and IL-17f levels, and bone preserving osteoprotegerin (OPG), were increased in antibiotic-treated mice compared to controls. Antibiotic-treated mice also displayed a trend towards delayed bone healing as revealed by reduced mineral apposition at the defect site and higher circulating levels of the bone turnover marker PINP. Butyrate supplementation resulted in a lower abundance of monocyte/macrophages in the bone marrow, as well as reduced circulating proinflammatory IL-6 levels compared to antibiotic- and control-treated mice. In conclusion, this study supports our hypothesis that SCFAs, in particular butyrate, are important contributors to successful bone healing by modulating key cells involved in fracture healing as well as systemic inflammation and immune responses.

Download Full-text

BCPA {N,N′-1,4-Butanediylbis[3-(2-chlorophenyl)acrylamide]} Inhibits Osteoclast Differentiation through Increased Retention of Peptidyl-Prolyl cis-trans Isomerase Never in Mitosis A-Interacting 1

International Journal of Molecular Sciences ◽

10.3390/ijms19113436 ◽

2018 ◽

Vol 19 (11) ◽

pp. 3436 ◽

Cited By ~ 6

Author(s):

Eugene Cho ◽

Jin-Kyung Lee ◽

Jee-Young Lee ◽

Zhihao Chen ◽

Sun-Hee Ahn ◽

...

Keyword(s):

Mononuclear Cells ◽

Transmembrane Protein ◽

Osteoclast Differentiation ◽

Osteoclast Formation ◽

Tartrate Resistant Acid Phosphatase ◽

Mrna Levels ◽

Dependent Manner ◽

Nuclear Factor ◽

Activated T Cells ◽

And Function

Osteoporosis is caused by an imbalance of osteoclast and osteoblast activities and it is characterized by enhanced osteoclast formation and function. Peptidyl-prolyl cis-trans isomerase never in mitosis A (NIMA)-interacting 1 (Pin1) is a key mediator of osteoclast cell-cell fusion via suppression of the dendritic cell-specific transmembrane protein (DC-STAMP). We found that N,N′-1,4-butanediylbis[3-(2-chlorophenyl)acrylamide] (BCPA) inhibited receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclastogenesis in a dose-dependent manner without cytotoxicity. In addition, BCPA attenuated the reduction of Pin1 protein during osteoclast differentiation without changing Pin1 mRNA levels. BCPA repressed the expression of osteoclast-related genes, such as DC-STAMP and osteoclast-associated receptor (OSCAR), without altering the mRNA expression of nuclear factor of activated T cells (NFATc1) and cellular oncogene fos (c-Fos). Furthermore, Tartrate-resistant acid phosphatase (TRAP)-positive mononuclear cells were significantly decreased by BCPA treatment compared to treatment with the Pin1 inhibitor juglone. These data suggest that BCPA can inhibit osteoclastogenesis by regulating the expression of the DC-STAMP osteoclast fusion protein by attenuating Pin1 reduction. Therefore, BCPA may be used to treat osteoporosis.

Download Full-text

Inhibitory Effect ofChrysanthemum zawadskiiHerbich var.latilobumKitamura Extract on RANKL-Induced Osteoclast Differentiation

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2013/509482 ◽

2013 ◽

Vol 2013 ◽

pp. 1-10 ◽

Cited By ~ 5

Author(s):

Dong Ryun Gu ◽

Jin-Ki Hwang ◽

Munkhsoyol Erkhembaatar ◽

Kang-Beom Kwon ◽

Min Seuk Kim ◽

...

Keyword(s):

Rheumatoid Arthritis ◽

Inflammatory Diseases ◽

Osteoclast Differentiation ◽

Ethanol Extract ◽

Inhibitory Effect ◽

Bone Diseases ◽

Dependent Manner ◽

Erk Activation

Chrysanthemum zawadskii Herbichvar.latilobum Kitamura, known as “Gujulcho” in Korea, has been used in traditional medicine to treat various inflammatory diseases, including rheumatoid arthritis. However, these effects have not been tested on osteoclasts, the bone resorbing cells that regulate bone metabolism. Here, we investigated the effects ofC. zawadskiiHerbich var.latilobumKitamura ethanol extract (CZE) on osteoclast differentiation induced by treatment with the receptor activator of NF-κB ligand (RANKL). CZE inhibited osteoclast differentiation and formation in a dose-dependent manner. The inhibitory effect of CZE on osteoclastogenesis was due to the suppression of ERK activation and the ablation of RANKL-stimulated Ca2+-oscillation via the inactivation of PLCγ2, followed by the inhibition of CREB activation. These inhibitory effects of CZE resulted in a significant repression of c-Fos expression and a subsequent reduction of NFATc1, a key transcription factor for osteoclast differentiation, fusion, and activationin vitroandin vivo. These results indicate that CZE negatively regulates osteoclast differentiation and may be a therapeutic candidate for the treatment of various bone diseases, such as postmenopausal osteoporosis, rheumatoid arthritis, and periodontitis.

Download Full-text

Berberine Suppresses RANKL-Induced Osteoclast Differentiation by Inhibiting c-Fos and NFATc1 Expression

The American Journal of Chinese Medicine ◽

10.1142/s0192415x19500228 ◽

2019 ◽

Vol 47 (02) ◽

pp. 439-455 ◽

Cited By ~ 3

Author(s):

Sang-Yong Han ◽

Yun-Kyung Kim

Keyword(s):

Bone Loss ◽

Osteoclast Differentiation ◽

Osteoclast Formation ◽

Bone Diseases ◽

Mrna Levels ◽

Nuclear Factor ◽

Activated T Cells ◽

Murine Bone Marrow

Osteoporosis is a common disorder of bone remodeling, marked by excessive osteoclast formation. Recent studies indicated that berberine (BBR) is a potential natural drug for the treatment of various bone diseases. However, it still needs to be further studied for the treatment of osteoporosis. The current study investigated the inhibitory effects of BBR on receptor activator of nuclear factor-[Formula: see text]B ligand (RANKL)-induced osteoclast differentiation in vitro and in vivo. Cell-based assays were performed using osteoclasts generated in cultures of murine bone marrow-derived macrophages (BMMs) treated with RANKL and M-CSF. The effects of BBR on in vivo lipopolysaccharide (LPS)-mediated bone loss were evaluated using ICR mice. BBR significantly inhibited TRAP-positive osteoclast formation induced by RANKL. BBR also inhibited RANKL-induced Akt, p38 and ERK phosphorylation and I[Formula: see text]B degradation, and suppressed RANKL-induced expression of c-Fos and nuclear factor of activated T cells c1 (NFATc1), which is a key transcription factors for osteoclast formation. BBR reduced the mRNA levels of osteoclast markers, including TRAP, osteoclast-associated receptor (OSCAR), cathepsin K, and ATPase H[Formula: see text] transporting V0 subunit d2 (ATP6v0d2). Moreover, BBR prevented LPS-mediated bone loss in vivo. We suggest BBR as a natural compound that can be a potential therapeutic agent for osteoclast-related bone diseases.

Download Full-text