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

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