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

Distribution of patients^* into GOLD categories according to symptom assessment (mMRC versus CAT scores).

<p>*N = 474</p><p>^†Meets FEV1 criteria (<50% FEV1 predicted).</p><p>^‡Meets exacerbation criteria (≥2 COPD exacerbations or ≥1 hospitalization or visit to the emergency department because of COPD exacerbation in past 12 months).</p><p>^§Meets exacerbation and FEV1 criteria (<50% FEV1 predicted and ≥2 COPD exacerbations or ≥1 hospitalization or visit to the emergency department because of COPD exacerbation in the past 12 months).</p><p>Distribution of patients^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115787#t004fn001" target="_blank">*</a>} into GOLD categories according to symptom assessment (mMRC versus CAT scores).</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

Medical histories reported in the self-administered questionnaire.

<p>Data are presented as numbers (%).</p><p>COPD: chronic obstructive pulmonary disease.</p><p>*Fisher’s exact test.</p><p>Medical histories reported in the self-administered questionnaire.</p

Selection of subjects for the analysis of COPD prevalence among surgical candidates.

<p>BD: bronchodilator, FEV1/FVC: forced expiratory volume in 1 s/forced vital capacity, COPD: chronic obstructive pulmonary disease.</p

Distribution of airflow limitation, level of symptoms, and exacerbation of COPD among COPD patients.

<p>Data are presented as numbers (%).</p><p>Post-BD: postbronchodilator, FEV1: forced expiratory volume in 1 s, mMRC: modified Medical Research Council, CAT: COPD assessment test.</p><p>Distribution of airflow limitation, level of symptoms, and exacerbation of COPD among COPD patients.</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 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