Sun-Hyang Park

Harpagoside Inhibits RANKL-Induced Osteoclastogenesis via Syk-Btk-PLCγ2-Ca(2+) Signaling Pathway and Prevents Inflammation-Mediated Bone Loss.

Harpagoside (HAR) is a natural compound isolated from Harpagophytum procumbens (devils claw) that is reported to have anti-inflammatory effects; however, these effects have not been investigated in the context of bone development. The current study describes for the first time that HAR inhibits receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclastogenesis in vitro and suppresses inflammation-induced bone loss in a mouse model. HAR also inhibited the formation of osteoclasts from mouse bone marrow macrophages (BMMs) in a dose-dependent manner as well as the activity of mature osteoclasts, including filamentous actin (F-actin) ring formation and bone matrix breakdown. This involved a HAR-induced decrease in extracellular signal-regulated kinase (ERK) and c-jun N-terminal kinase (JNK) phosphorylation, leading to the inhibition of Syk-Btk-PLCγ2-Ca(2+) in RANKL-dependent early signaling, as well as the activation of c-Fos and nuclear factor of activated T cells cytoplasmic 1 (NFATc1), which resulted in the down-regulation of various target genes. Consistent with these in vitro results, HAR blocked lipopolysaccharide (LPS)-induced bone loss in an inflammatory osteoporosis model. However, HAR did not prevent ovariectomy-mediated bone erosion in a postmenopausal osteoporosis model. These results suggest that HAR is a valuable agent against inflammation-related bone disorders but not osteoporosis induced by hormonal abnormalities.

Esculetin attenuates receptor activator of nuclear factor kappa-B ligand-mediated osteoclast differentiation through c-Fos/nuclear factor of activated T-cells c1 signaling pathway.

Esculetin exerts various biological effects on anti-oxidation, anti-tumors, and anti-inflammation. However, the involvement of esculetin in the bone metabolism process, particularly osteoclast differentiation has not yet been investigated. In the present study, we first confirmed the inhibitory effect of esculetin on receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast formation. We then revealed the relationship between esculetin and the expression of osteoclast-specific molecules to elucidate its underlying mechanisms. Esculetin interfered with the expression of c-Fos and nuclear factor of activated T cell c1 (NFATc1) both at the mRNA and protein level with no involvement in osteoclast-associated early signaling pathways, suppressing the expression of various transcription factors exclusively expressed in osteoclasts such as tartrate-resistant acid phosphatase (Trap), osteoclast-associated receptor (Oscar), dendritic cell-specific transmembrane protein (Dcstamp), osteoclast stimulatory transmembrane protein (Ocstamp), cathepsin K, αvβ3 integrin, and calcitonin receptor (Ctr). Additionally, esculetin inhibited the formation of filamentous actin (F-actin) ring-positive osteoclasts during osteoclast differentiation. However, the development of F-actin structures and subsequent bone resorbing activity of mature osteoclasts, which are observed in osteoclast/osteoblast co-culture systems were not affected by esculetin. Taken together, our results indicate for the first time that esculetin inhibits RANKL-mediated osteoclastogenesis via direct suppression of c-Fos and NFATc1 expression and exerts an inhibitory effect on actin ring formation during osteoclastogenesis.

Lysyl oxidase modulates the osteoblast differentiation of primary mouse calvaria cells.

Lysyl oxidase (LOX) is an extracellular amine oxidase that mediates the formation of collagen fibers. Thus far, five LOX family genes [LOX, lysyl oxidase-like (LOXL)1, LOXL2, LOXL3 and LOXL4] have been identified in humans, each encoding the characteristic C-terminal domains that are required for amine oxidase activity. During osteoblastogenesis, collagen fibers function as a three-dimensional scaffold for organizing mineral deposition. In this study, to assess the functional roles of the LOX family members in osteoblastogenesis, we investigated the temporal expression of these genes as a function of phenotypic development during the osteoblast differentiation of primary cultured mouse calvaria cells. Of the LOX family members, only LOX was prominently expressed during osteoblast differentiation. LOX expression was highest on day 9 of differentiation, as shown by RT-PCR and western blot analysis. The expression pattern of collagen, type I, alpha 2 (COL1A2), which encodes the α2-chain of mouse collagen type I, was similar to that of LOX. The total amine oxidase activity of the differentiating calvaria cells exhibited a temporal pattern that paralleled LOX expression, reaching the highest level on day 9 of differentiation. We also noted that the inhibition of the amine oxidase activity of LOX significantly suppressed both mineral nodule formation and the expression of osteoblast marker genes during the differentiation of primary calvaria cells. Taken together, these findings suggest that the LOX-mediated organization of collagen fibers in the extracellular matrix is an important regulator of osteoblastogenesis.

Protocatechuic Acid Attenuates Osteoclastogenesis by Downregulating JNK/c-Fos/NFATc1 Signaling and Prevents Inflammatory Bone Loss in Mice

Protocatechuic acid (PCA) plays a critical role in nutritional metabolism; it is a major metabolite of anthocyanins, which are flavonoids with a range of health benefits. PCA has a variety of biological activities including anti‐oxidant, antiinflammatory, anti‐apoptosis, and anti‐microbial activities. However, the pharmacological effect of PCA, especially on osteoclastogenesis, remains unknown. We examined the effect of PCA on receptor activator of NF‐κB ligand (RANKL)‐induced osteoclast differentiation and bone resorption. PCA dose‐dependently inhibited RANKL‐induced osteoclast differentiation in mouse bone marrow macrophages (BMMs) and suppressed the bone‐resorbing activity of mature osteoclasts. At the molecular level, PCA suppressed RANKL‐induced phosphorylation of JNK among MAPKs only, without significantly affecting the early signaling pathway. PCA also suppressed RANKL‐stimulated expression of c‐Fos and nuclear factor of activated T cells c1 (NFATc1) at the mRNA and protein levels, without altering c‐Fos mRNA expression. Additionally, PCA down‐regulated the expression of downstream osteoclastogenesis‐related genes including β3‐integrin, DC‐STAMP, OC‐STAMP, Atp6v0d2, CTR, and CtsK. Mice treated with PCA efficiently recovered from lipopolysaccharide‐induced bone loss in vivo. Thus, PCA inhibits RANKL‐induced osteoclast differentiation and function by suppressing JNK signaling, c‐Fos stability, and expression of osteoclastic marker genes. These results suggest that PCA could be useful in treatment of inflammatory bone disorders. Copyright

Stauntonia hexaphylla (Lardizabalaceae) leaf methanol extract inhibits osteoclastogenesis and bone resorption activity via proteasome-mediated degradation of c-Fos protein and suppression of NFATc1 expression

BackgroundNatural plants, including common vegetables and fruits, have been recognized as essential sources for drug discovery and the development of new, safe, and economical medicaments. Stauntonia hexaphylla (Lardizabalaceae) is widely distributed in Korea, Japan, and China, and is a popular herbal supplement in Korean and Chinese folk medicine owing to its analgesic, sedative, and diuretic properties. However, the exact pharmacological effects of S. hexaphylla extract, particularly its effect on osteoclastogenesis, are not known.MethodsOsteoclast differentiation and function were identified with tartrate-resistant acid phosphatase (TRAP) staining and bone resorption assay, and the underling mechanisms were determined by real-time RT-PCR and western blot analysis.ResultsS. hexaphylla was found to inhibit early-stage receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-mediated osteoclast differentiation in bone marrow macrophages (BMMs) without cytotoxicity and bone-resorbing activity in mature osteoclasts in a dose-dependent manner. This S. hexaphylla-mediated blockade of osteoclastogenesis involved abrogation of the NF-κB, ERK, and c-Src-Btk-PLCγ2 calcium signal pathways. Interestingly, we found that S. hexaphylla down-regulated RANKL-associated c-Fos protein induction by suppressing its translation. Furthermore, ectopic overexpression of c-Fos and NFATc1 rescued the inhibition of osteoclast differentiation by S. hexaphylla. Furthermore, S. hexaphylla inhibited the c-Fos- and NFATc1-regulated expression of genes required for osteoclastogenesis, such as TRAP, OSCAR, β3-integrin, ATP6v0d2, and CtsK.ConclusionsThese findings suggest that S. hexaphylla might be useful for the development of new anti-osteoporosis agents.

Aconitum pseudo-laeve var. erectum Inhibits Receptor Activator of Nuclear Factor Kappa-B Ligand-Induced Osteoclastogenesis via the c-Fos/nuclear Factor of Activated T-Cells, Cytoplasmic 1 Signaling Pathway and Prevents Lipopolysaccharide-Induced Bone Loss in Mice

Aconitum pseudo-laeve var. erectum (APE) has been widely shown in herbal medicine to have a therapeutic effect on inflammatory conditions. However, there has been no evidence on whether the extract of APE is involved in the biological bone metabolism process, particularly osteoclast-mediated bone resorption. In this study, we confirmed that the administration of APE could restore normal skeletal conditions in a murine model of lipopolysaccharide (LPS)-induced bone loss via a decrease in the receptor activator of nuclear factor kappa-B ligand (RANKL)/osteoprotegerin (OPG) ratio and osteoclast number. We then investigated the effect of APE on the RANKL-induced formation and function of osteoclasts to elucidate its underlying molecular mechanisms. APE suppressed the formation of tartrate-resistant acid phosphatase (TRAP)-positive cells, as well as the bone-resorbing activity of mature osteoclasts. Furthermore, APE attenuated nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) and c-Fos without affecting any early signal pathway of osteoclastogenesis. Subsequently, APE significantly downregulated the expression of various genes exclusively expressed in osteoclasts. These results demonstrate that APE restores LPS-induced bone loss through a decrease of the serum RANKL/OPG ratio, and inhibits osteoclast differentiation and function, suggesting the promise of APE as a potential cure for various osteoclast-associated bone diseases.

Dual Effect of Chrysanthemum indicum Extract to Stimulate Osteoblast Differentiation and Inhibit Osteoclast Formation and Resorption In Vitro

The risk of bone-related diseases increases due to the imbalance between bone resorption and bone formation by osteoclasts and osteoblasts, respectively. The goal in the development of antiosteoporotic treatments is an agent that will improve bone through simultaneous osteoblast stimulation and osteoclast inhibition without undesirable side effects. To achieve this goal, numerous studies have been performed to identify novel approaches using natural oriental herbs to treat bone metabolic diseases. In the present study, we investigated the effect of Chrysanthemum indicum extract (CIE) on the differentiation of osteoclastic and osteoblastic cells. CIE inhibited the formation of TRAP-positive mature osteoclasts and of filamentous-actin rings and disrupted the bone-resorbing activity of mature osteoclasts in a dose-dependent manner. CIE strongly inhibited Akt, GSK3β, and IκB phosphorylation in RANKL-stimulated bone marrow macrophages and did not show any effects on MAP kinases, including p38, ERK, and JNK. Interestingly, CIE also enhanced primary osteoblast differentiation via upregulation of the expression of alkaline phosphatase and the level of extracellular calcium concentrations during the early and terminal stages of differentiation, respectively. Our results revealed that CIE could have a potential therapeutic role in bone-related disorders through its dual effects on osteoclast and osteoblast differentiation.

The Inhibitory Effect of Angelica tenuissima Water Extract on Receptor Activator of Nuclear Factor-Kappa-B Ligand-Induced Osteoclast Differentiation and Bone Resorbing Activity of Mature Osteoclasts

Angelica tenuissima has been traditionally used in oriental medicine for its therapeutic effects in headache, toothache, and flu symptoms. It also exerts anti-inflammatory activity via the inhibition of the expression of cyclooxygenase-2 (COX-2). However, the effect of Angelica tenuissima on osteoclast differentiation has not been identified until recently. In this study, we first confirmed that Angelica tenuissima water extract (ATWE) significantly interrupted the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNCs) in a dose-dependent manner without any cytotoxicity. Next, we clarified the underlying mechanisms linking the suppression effects of ATWE on the receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis. At the molecular level, ATWE induced the dephosphorylation of c-Jun N-terminal kinase (JNK) and Akt and decreased the degradation of IκB in RANKL-dependent early signaling pathways. Subsequently, ATWE caused impaired activation of the protein and mRNA levels of c-Fos and nuclear factor of activated T cell c1 (NFATc1). Moreover, the disassembly of filamentous actin (F-actin) ring and anti-resorptive activity of mature osteoclasts were triggered by ATWE treatment. Although ATWE did not enhance osteogenesis in primary osteoblasts, our results showed that ATWE is a potential candidate for anti-resorptive agent in osteoporosis, a common metabolic bone disorder.

Ampelopsis brevipedunculata Extract Prevents Bone Loss by Inhibiting Osteoclastogenesis in Vitro and in Vivo

Osteoclasts play a critical role in bone resorbing disorders such as osteoporosis, periodontitis, and rheumatoid arthritis. Therefore, discovery of agents capable of suppressing osteoclast differentiation may aid the development of a therapeutic access for the treatment of pathological bone loss. Ampelopsis brevipedunculata has been used as herbal folk medicine to treat liver diseases and inflammation in Asia. However, its effects on osteoclast differentiation are unknown. We were aimed to investigate the anti-osteoclastogenic activity in vitro and in vivo and to elucidate the underlying mechanism of Ampelopsis brevipedunculata extract (ABE). In this study, ABE inhibited receptor activator of NF-κB ligand (RANKL)-induced osteoclast differentiation, the formation of filamentous actin rings and the bone resorbing activity of mature osteoclasts. ABE inhibited RANKL-induced p38 and IκB phosphorylation and IκB degradation. Also, ABE suppressed the mRNA and protein expression of nuclear factor of activated T cells c1 (NFATc1) and c-Fos, and the mRNA expression of genes required for cell fusion and bone resorption, such as osteoclast-associated receptor (OSCAR), tartrate resistant acid phosphatase (TRAP), cathepsin K, dendritic cell-specific transmembrane protein (DC-STAMP), β3-integrin and osteoclast stimulatory transmembrane protein (OC-STAMP). Furthermore, results of micro-CT and histologic analysis indicated that ABE remarkably prevented lipopolysaccharide (LPS)-induced bone erosion. These results demonstrate that ABE prevents LPS-induced bone erosion through inhibition of osteoclast differentiation and function, suggesting the promise of ABE as a potential cure for various osteoclast-associated bone diseases.

Ethanolic extract of Schizonepeta tenuifolia attenuates osteoclast formation and activation in vitro and protects against lipopolysaccharide-induced bone loss in vivo

BackgroundExcessive osteoclast activity is a major cause of metabolic bone disorders, such as osteopenia, rheumatoid arthritis, and osteoporosis. Thus, discovery of agents targeting osteoclast differentiation and bone resorption is important for development of novel treatments for bone diseases. It has been demonstrated that ethanolic extract of schizonepeta tenuifolia (EEST) has potent anti-oxidant and anti-inflammatory activities. However, the beneficial effects of EEST on bone metabolism have not been studied. Therefore, we intend to investigate the effects of EEST on osteoclast differentiation.MethodsWe examined the effects and mechanisms of action of the EEST on osteoclastogenesis in vitro in bone marrow macrophages (BMMs) stimulated with receptor activator of nuclear factor kappa-B ligand (RANKL) and in vivo using a mouse model of lipopolysaccharide (LPS)-induced bone destruction.ResultsWe found that EEST inhibited phosphorylation of Akt and IkB at early stages of RANKL-induced osteoclastogenesis. Furthermore, EEST negatively controlled the transcription and translation levels of nuclear factor of activated T cells c1 (NFATc1) and the translation level of c-Fos at the final stage of osteoclast differentiation. Reflecting these effects, EEST blocked both filamentous actin (F-actin) ring formation and bone resorbing activity of mature osteoclasts in vitro. The inhibitory effects of EEST on osteoclast formation and activity were observed in an LPS-mediated bone erosion mouse model using micro-CT and histological analysis.ConclusionsEEST is a potential agent that is able to treat osteoclast-related bone diseases, such as osteoporosis.