3'4'7-Trihydroxyflavone inhibits RANKL-induced osteoclast formation via NFATc1.

3'4'7-Trihydroxyflavone is a flavonoid from ladino clover, alfalfa, and Albizzia julibrissin. In the present study, we found that 3'4'7-trihydroxyflavone markedly inhibited the receptor activator of nuclear factor kappa B ligand (RANKL) induced osteoclastic differentiation from mouse bone marrow derived macrophages (BMMs). 3'4'7-trihydroxyflavone also reduced the mRNA expression level of osteoclastic marker genes including calcitonin receptor (CTR), Cathepsin K1 v-ATPase V0 subunit d2 (ATP6v0d2), and dendritic cell-specific transmembrane protein (DC-STAMP). In addition, 3'4'7-trihydroxyflavone decreased the bone resorption activity of osteoclasts on dentin slices. We found that 3'4'7-trihydroxyflavone inhibited RANKL-induced expression of nuclear factor of activated T cells c1 (NFATc1), a key transcription factor of osteoclast differentiation. Furthermore, 3'4'7-trihydroxyflavone attenuated RANKL-induced activation of p38 mitogen-activated protein kinase (MAPK) and expression of B lymphocyte-induced maturation protein 1 (Blimp1), a repressor of negative regulators of NFATc1. Taken together, our data suggest that 3'4'7-trihydroxyflavone inhibits osteoclastogenesis via NFATc1.

Iris Koreana NAKAI Inhibits Osteoclast Formation via p38-Mediated Nuclear Factor of Activated T Cells 1 Signaling Pathway.

BACKGROUND: Iris Koreana NAKAI (IKN) is a flowering perennial plant that belongs to the Iridaceae family. In this study, we aimed to demonstrate the effects of IKN on osteoclast differentiation in vitro and in vivo. We also sought to verify the molecular mechanisms underlying its anti-osteoclastogenic effects. METHODS: Osteoclasts were formed by culturing mouse bone marrow macrophage (BMM) cells with macrophage colony-stimulating factor and receptor activator of nuclear factor-κB ligand (RANKL). Bone resorption assays were performed on dentin slices. mRNA expression levels were analyzed by quantitative polymerase chain reaction. Western blotting was performed to detect protein expression or activation. Lipopolysaccharide (LPS)-induced osteoclast formation was performed using a mouse calvarial model. RESULTS: In BMM cultures, an ethanol extract of the root part of IKN suppressed RANKL-induced osteoclast formation and bone resorptive activity. In contrast, an ethanol extract of the aerial parts of IKN had a minor effect on RANKL-induced osteoclast formation. Mechanistically, the root part of IKN suppressed RANKL-induced p38 mitogen-activated protein kinase (MAPK) activation, effectively abrogating the induction of c-Fos and nuclear factor of activated T cells 1 (NFATc1) expression. IKN administration decreased LPS-induced osteoclast formation in a calvarial osteolysis model in vivo. CONCLUSIONS: Our study suggested that the ethanol extract of the root part of IKN suppressed osteoclast differentiation and function partly by downregulating the p38 MAPK/c-Fos/NFATc1 signaling pathways. Thus, the root part.

Fexaramine Inhibits Receptor Activator of Nuclear Factor-κB Ligand-induced Osteoclast Formation via Nuclear Factor of Activated T Cells Signaling Pathways.

BACKGROUND: Osteoclasts are bone resorbing cells and are responsible for bone erosion in diseases as diverse as osteoporosis, periodontitis, and rheumatoid arthritis. Fexaramine has been developed as an agonist for the farnesoid X receptor (FXR). This study investigated the effects of fexaramine on receptor activator of nuclear factor (NF)-κB ligand (RANKL)-induced osteoclast formation and signaling pathways. METHODS: Osteoclasts were formed by culturing mouse bone marrow-derived macrophages (BMMs) with macrophage colony-stimulating factor (M-CSF) and RANKL. Bone resorption assays were performed using dentine slices. The mRNA expression level was analyzed by real-time polymerase chain reaction. Western blotting assays were conducted to detect the expression or activation level of proteins. Lipopolysaccharide-induced osteoclast formation was performed using a mouse calvarial model. RESULTS: Fexaramine inhibited RANKL-induced osteoclast formation, without cytotoxicity. Furthermore, fexaramine diminished the RANKL-stimulated bone resorption. Mechanistically, fexaramine blocked the RANKL-triggered p38, extracellular signal-regulated kinase, and glycogen synthase kinase 3ß phosphorylation, resulting in suppressed expression of c-Fos and NF of activated T cells (NFATc1). Consistent with the in vitro anti-osteoclastogenic effect, fexaramine suppressed lipopolysaccharide-induced osteoclast formation in the calvarial model. CONCLUSIONS: The present data suggest that fexaramine has an inhibitory effect on osteoclast differentiation and function, via downregulation of NFATc1 signaling pathways. Thus, fexaramine could be useful for the treatment of bone diseases associated with excessive bone resorption.

Attenuation of RANKL-induced Osteoclast Formation via p38-mediated NFATc1 Signaling Pathways by Extract of Euphorbia Lathyris L.

BACKGROUND: Osteoclasts are the only cell type capable of breaking down bone matrix, and its excessive activation is responsible for the development of bone-destructive diseases. Euphorbia lathyris L. (ELL) is an herbal plant that belongs to the Euphorbiaceae family. This study investigated the effects of the methanol extract of the aerial part of ELL on receptor activator of nuclear factor-kappa B ligand (RANKL)-induced osteoclast formation and signaling pathways. METHODS: Osteoclasts were formed by co-culturing mouse bone marrow with osteoblasts or by culturing mouse bone marrow-derived macrophages (BMMs) with macrophage colony-stimulating factor (M-CSF) and RANKL. Bone resorption assays were performed using dentine slices. The expression level of mRNA was analyzed by real-time polymerase chain reaction (PCR) or reverse transcription (RT)-PCR. Western blotting assays were performed to detect the expression or activation level of proteins. RESULTS: ELL inhibited RANKL-induced osteoclast formation without cytotoxicity. Furthermore, the RANKL-stimulated bone resorption was diminished by ELL. Mechanistically, ELL blocked the RANKL-triggered p38 mitogen-activated protein kinase (MAPK) phosphorylation, which resulted in the suppression of the expression of c-Fos and nuclear factor of activated T cells (NFATc1). In osteoblasts, ELL had little effect on the mRNA expression of RANKL and osteoprotegerin (OPG). CONCLUSIONS: The present data suggest that ELL has an inhibitory effect on osteoclast differentiation and function via downregulation of the p38/c-Fos/NFATc1 signaling pathways. Thus, ELL could be useful for the treatment of bone diseases associated with excessive bone resorption.

L-type Ca(2+) channel agonist inhibits RANKL-induced osteoclast formation via NFATc1 down-regulation.

AIMS: BayK 8644 is an L-type Ca(2+) channel agonist that enhances Ca(2+) influx and elevates cytosolic Ca(2+). As intracellular calcium plays a key role in osteoclast formation, we investigated the effects of BayK 8644 in cultures of bone marrow-derived precursor cells with macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappaB ligand (RANKL). MAIN METHODS: We performed an osteoclast formation assay, a pit formation assay, real-time PCR, and Western blot analysis. KEY FINDINGS: BayK 8644 concentration-dependently suppressed osteoclastogenesis, as well as the expression of osteoclastic marker genes. It also decreased osteoclastic bone resorption on a dentine slice. While the RANKL-stimulated induction of IL-1ß and IL-6 was not affected, TNF-α induction was reduced by BayK 8644 treatment. In addition, BayK 8644 blocked IκB degradation and the induction of nuclear factor of activated T cells c1 (NFATc1), the master regulator of osteoclast differentiation, following RANKL stimulation. Finally, forced expression of NFATc1 reversed the inhibitory effect of BayK 8644 on osteoclastogenesis, suggesting that NFATc1 is a downstream target for the anti-osteoclastogenic action of BayK 8644. Taken together, our data suggest that BayK 8644 has an anti-osteoclastogenic effect by inhibiting RANKL-induced activation of NF-κB pathways, thereby suppressing the gene expression of NFATc1 in osteoclast precursors. SIGNIFICANCE: Our results provide a molecular understanding of the inhibitory effect of the L-type Ca(2+) channel agonist, BayK 8644, on osteoclastogenesis.