Praeruptorin A Inhibits <i>in Vitro</i> Migration of Preosteoclasts and <i>in Vivo</i> Bone Erosion, Possibly Due to Its Potential To Target Calmodulin

Excessive activity and/or increased number of osteoclasts lead to bone resorption-related disorders. Here, we investigated the potential of praeruptorin A to inhibit migration/fusion of preosteoclasts <i>in vitro</i> and bone erosion <i>in vivo</i>. Praeruptorin A inhibited the RANKL-induced migration/fusion of preosteoclasts accompanied by the nuclear translocation of NFATc1, a master regulator of osteoclast differentiation. Antimigration/fusion activity of praeruptorin A was also confirmed by evaluating the mRNA expression of fusion-mediating molecules. <i>In silico</i> binding studies and several biochemical assays further revealed the potential of praeruptorin A to bind with Ca²⁺/calmodulin and inhibit its downstream signaling pathways, including the Ca²⁺/calmodulin-CaMKIV-CREB and Ca²⁺/calmodulin-calcineurin signaling axis responsible for controlling NFATc1. <i>In vivo</i> application of praeruptorin A significantly reduced lipopolysaccharide-induced bone erosion, indicating its possible use to treat bone resorption-related disorders. In conclusion, praeruptorin A has the potential to inhibit migration/fusion of preosteoclasts <i>in vitro</i> and bone erosion <i>in vivo</i> by targeting calmodulin and inhibiting the Ca²⁺/calmodulin-CaMKIV-CREB-NFATc1 and/or Ca²⁺/calmodulin-calcineurin-NFATc1 signaling axis

Acredinone C and the Effect of Acredinones on Osteoclastogenic and Osteoblastogenic Activity

A new inhibitor, acredinone C (<b>1</b>), of receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation was isolated from the culture broth of the fungus <i>Acremonium</i> sp. (F9A015) along with acredinones A (<b>2</b>) and B (<b>3</b>). The structure of acredinone C (<b>1</b>), which incorporates benzophenone and xanthone moieties, was established by the analyses of combined spectroscopic data including 1D and 2D NMR and MS. All of the acredinones studied efficiently inhibited the RANKL-induced formation of TRAP⁺-MNCs in a dose-dependent manner without any cytotoxicity up to 10 μM. Acredinone A showed dual activity in both osteoclast and osteoblast differentiation <i>in vitro</i> and good efficacy in an animal disease model of bone formation

Effect of praeruptorin A on RANKL-induced osteoclast differentiation.

<p>(A) Chemical structure of praeruptorin A. (B) BMMs were pretreated with vehicle (0.1% DMSO) or praeruptorin A for 2 h and then incubated with RANKL (10 ng/ml) and M-CSF (30 ng/ml) for 4 days. Multinucleated cells were fixed, permeabilized, and stained with TRAP solution. Mature TRAP-positive multinucleated osteoclasts (MNCs) were photographed under a light microscope. TRAP-positive MNCs (nuclear number >3) were counted (C), and TRAP activity of osteoclasts was measured (D). (E) The effect of praeruptorin A on the viability of BMMs was evaluated by CCK-8 assay. *, <i>P</i><0.05; **, <i>P</i><0.01; ***<i>P</i><0.001.</p

Anti-Osteoclastogenic Activity of Praeruptorin A via Inhibition of p38/Akt-c-Fos-NFATc1 Signaling and PLCγ-Independent Ca²⁺ Oscillation

<div><p>Background</p><p>A decrease of bone mass is a major risk factor for fracture. Several natural products have traditionally been used as herbal medicines to prevent and/or treat bone disorders including osteoporosis. Praeruptorin A is isolated from the dry root extract of <i>Peucedanum praeruptorum</i> Dunn and has several biological activities, but its anti-osteoporotic activity has not been studied yet.</p><p>Materials and Methods</p><p>The effect of praeruptorin A on the differentiation of bone marrow–derived macrophages into osteoclasts was examined by phenotype assay and confirmed by real-time PCR and immunoblotting. The involvement of NFATc1 in the anti-osteoclastogenic action of praeruptorin A was evaluated by its lentiviral ectopic expression. Intracellular Ca²⁺ levels were also measured.</p><p>Results</p><p>Praeruptorin A inhibited the RANKL-stimulated osteoclast differentiation accompanied by inhibition of p38 and Akt signaling, which could be the reason for praeruptorin A-downregulated expression levels of c-Fos and NFATc1, transcription factors that regulate osteoclast-specific genes, as well as osteoclast fusion-related molecules. The anti-osteoclastogenic effect of praeruptorin A was rescued by overexpression of NFATc1. Praeruptorin A strongly prevented the RANKL-induced Ca²⁺ oscillation without any changes in the phosphorylation of PLCγ.</p><p>Conclusion</p><p>Praeruptorin A could exhibit its anti-osteoclastogenic activity by inhibiting p38/Akt-c-Fos-NFATc1 signaling and PLCγ-independent Ca²⁺ oscillation.</p></div

Effect of NFATc1 on anti-osteoclastogenic action of praeruptorin A.

<p>(A) BMMs were infected with retroviruses harboring the control GFP or Ca-NFATc1-GFP vectors. Transduced BMMs were cultured with RANKL (10 ng/ml) and M-CSF (30 ng/ml) in the presence of praeruptorin A (10 µM) or vehicle (DMSO). After incubation for 2 days, GFP expression was visualized under a fluorescence microscope. After 2 additional days, mature TRAP-positive multinucleated osteoclasts were visualized by TRAP staining. (B) TRAP-positive cells (nuclear number >3) were counted as osteoclasts, and TRAP activity was measured at 405 nm. On the differentiation day 2, the mRNA and protein expression levels of osteoclastogenesis-related molecules were analyzed by real-time PCR (C) and Western blot analysis, respectively (D). Densitometric analysis was performed using ImageJ software and the relative, normalized ratios of NFATc1/actin, p-Akt/Akt or p-p38/p38 were presented. *, <i>P</i><0.05; **, <i>P</i><0.01; ***<i>P</i><0.001.</p

Primer sequences used in this study.

<p>Primer sequences used in this study.</p

Effect of praeruptorin A on RANKL-induced activation or expression of osteoclast-specific signaling molecules and transcription factors.

<p>The effects of praeruptorin A on RANKL-induced phosphorylation of MAP kinases and Akt (A) and expression of transcription factors, c-Fos and NFATc1 (B), were evaluated by Western blot analysis. BMMs were pre-treated with praeruptorin A (10 µM) 2 h before treatment with RANKL (10 ng/ml) and M-CSF (30 ng/ml). Actin was used as an internal control. Densitometric analysis was performed using ImageJ software and the relative, normalized ratios of p-p38/p38, p-JNKs/JNKs, p-ERKs/ERK, p-Akt/Akt, c-Fos/actin and NFATc1/actin were presented. (C) The effect of praeruptorin A on the transcriptional activity of NFATc1 was evaluated by luciferase activity assay as described in ‘Materials and Methods’. *, <i>P</i><0.05; **, <i>P</i><0.01.</p

Effect of praeruptorin A on RANKL-induced mRNA expressions of osteoclastic-specific genes.

<p>BMMs were treated with vehicle (DMSO) or praeruptorin A (10 µM) for 2 h and then RANKL (10 ng/ml) was added into cells. The mRNA expression levels of osteoclastic-specific genes were analyzed by real-time PCR. *, <i>P</i><0.05; **, <i>P</i><0.01; ***<i>P</i><0.001.</p

Effect of praeruptorin A on RANKL-induced Ca²⁺ oscillation and PLCγ phosphorylation.

<p>(A) The effect of praeruptorin A on the RANKL-induced Ca²⁺ oscillation was evaluated as described in ‘Materials and Methods’. Each trace presents intracellular Ca²⁺ mobilization in each cell. (B) The effect of praeruptorin A on the RANKL-induced phosphorylation of PLCγ was evaluated by Western blot analysis. BMMs were pre-treated with praeruptorin A for 2 h before treatment with RANKL. Actin was used as an internal control. Densitometric analysis was performed using ImageJ software and the relative, normalized ratio of p-PLCγ2/PLCγ2 was presented.</p