Harpagoside Inhibits RANKL-Induced Osteoclastogenesis via Syk-Btk-PLCγ2-Ca²⁺ Signaling Pathway and Prevents Inflammation-Mediated Bone Loss

Harpagoside (HAR) is a natural compound isolated from Harpagophytum procumbens (devil’s 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 <i>in vitro</i> 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-Ca²⁺ 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 <i>in vitro</i> 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

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

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 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

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

Primer sequences used in this study.

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