Inhibitory Effects of Rhaponticin on Osteoclast Formation and Resorption by Targeting RANKL-Induced NFATc1 and ROS Activity

Abstract

The extravagant osteoclast formation and resorption is the main cause of osteoporosis. Inhibiting the hyperactive osteoclastic resorption is considered as an efficient treatment for osteoporosis. Rhaponticin (RH) is a small molecule that has been reported to possess anti-inflammatory, anti-allergic, anti-fibrotic, and anti-diabetic activities. However, the influence of RH on osteoclasts differentiation and function is still unclear. To this end, an array of assays including receptor activator of nuclear factor kappa-Β (NF-κB) ligand (RANKL) induced osteoclastogenesis, tartrate-resistant acidic phosphatase (TRAcP) staining, immunofluorescence, and hydroxyapatite resorption were performed in this study. It was found that RH had significant anti-catabolic effects by inhibiting osteoclastogenesis and bone resorption without cytotoxicity. Mechanistically, the expression of NADPH oxidase 1 (Nox1) was found to be suppressed and antioxidant enzymes including catalase, superoxide dismutase 2 (SOD-2), and heme oxygenase-1(HO-1) were enhanced following RH treatment, suggesting RH exhibited antioxidant activity by reducing the generation of reactive oxygen species (ROS) as well as enhancing the depletion of ROS. In addition, MAPKs, NF-κB, and intracellular Ca²⁺ oscillation pathways were significantly inhibited by RH. These changes led to the deactivation of osteoclast master transcriptional factor-nuclear factor of activated T cells 1 (NFATc1), as examined by qPCR and Western blot assay, which led to the decreased expression of downstream integrin β3, c-Fos, cathepsin K, and Atp6v0d2. These results suggested that RH could effectively suppress RANKL-regulated osteoclast formation and bone resorption. Therefore, we propose that RH can represent a novel natural small molecule for the treatment of osteoporosis by inhibiting excessive osteoclast activity.

Keywords: NFATc1; ROS; bone; osteoclast; rhaponticin.

FIGURE 1

Rhaponticin (RH) suppressed RANKL-induced osteoclastogenesis in vitro. (A) Chemical structure and formula of RH. (B) MTS assay of the BMMs cultured with various concentrations of RH. (C, D) Representative images of optical microscope and TRAcP staining of BMMs treated with RH in different concentrations for 6 days (C) and in 50 μM, at different time phase of 1–2 days (D1-2), 3–4 days (D3-4), 5–6 days (D5-6), 1–6 days (D1-6), during differentiation (D) were shown. (E, F) Quantification of TRAcP positive multinucleated osteoclasts (nuclei > 3) with the treatment of RH (n = 3). *p < 0.05, **p < 0.01 relative to RANKL-induced control group. Scale bar = 200 μm.

FIGURE 2

RH inhibited the formation of F-actin belt during the osteoclast formation induced by RANKL. (A) The confocal images of F-actin ring formation were detected with Rhodamine Phalloidin, combined DAPI staining for nuclei respectively. Scale bar, 200 μm. (B) Quantification of the nucleus number per osteoclast under the different concentrations of RH. (C) Quantification of the average of actin ring area per field (n = 3). *p < 0.05, **p < 0.01 relative to RANKL-induced control group. Scale bar = 200 μm.

FIGURE 3

RH attenuated osteoclast hydroxyapatite resorption and osteoclast-specific genes expression. (A) Representative images of the resorption on hydroxyapatite-coated plates and TRAcP staining after treatment of RH for 48 hr. (B) Quantification of TRAcP-positive osteoclasts numbers per well (n = 3). (C) Quantification of resorption area on hydroxyapatite surface per osteoclast (n = 3). (D–G) PCR results of osteoclast-specific genes Nfatc1, c-Fos, Ctsk, and Atp6v0d2. Gene expression levels were standardized to Hprt expression. *p < 0.05, **p < 0.01 relative to RANKL-induced control group. Scale bar = 200 μm.

FIGURE 4

RH suppressed NFATc1 activation and its downstream protein expression. (A) RH inhibited the NFATc1 activity of RAW 264.7 cells transfected with luciferase report construct. The cells were pretreated with RH for 1 hour, followed by RANKL stimulation at 50 ng/ml concentration for 24 hours (n = 4). (B) Representative Western blot images of the effects of RH on the protein expression of NFATc1, integrin β3, CTSK, c-Fos, and V-ATPase-d2 at day 0, 1, 3, and 5 with the stimulation of RANKL (50 ng/mL). (C–G) Quantification of the ratios of band intensity of NFATc1, integrin β3, CTSK, c-Fos, and V-ATPase-d2 relative to β-actin (n = 3). *p < 0.05, **p < 0.01 relative to RANKL-induced control group.

FIGURE 5

RH suppressed RANKL-induced ROS activity and Ca²⁺ oscillation. (A) Representative Western blot images of the Nox1, catalase, HO-1, and SOD-2 expression with the treatment of RH, the Nox1 expression was significantly suppressed by RH, and the antioxidant enzymes of HO-1, catalase and SOD-2 were enhanced. BMMs were stimulated with RANKL (50 ng/mL) with RH at the concentration of 25 and 50 μM or PBS for 2 days before collecting protein. (B–E) Quantification of the ratios of band intensity of Nox1, HO-1, catalase, and SOD-2 relative to β-actin (n = 3 per group). *p < 0.05, **p < 0.01 comparison with the RANKL-induced positive control group. (F–H) Representative images of fluorescence intensity waves of Ca²⁺ oscillation in negative group, RANKL stimulated positive group and RH (50 μM) treated group. There were three colours indicating different cells in each group. (I) Quantification of fluorescence intensity change of Ca²⁺ oscillation in each group. *p < 0.05, **p < 0.01 relative to RANKL-induced positive control group.

FIGURE 6

RH suppressed the activation of NF-κB and the phosphorylation of ERK and P38 induced by RANKL. (A) RH inhibited the NF-κB activity of RAW 264.7 cells with luciferase report construct at the different concentrations of RH as indicated. The cells were pretreated with varying densities of RH and stimulated with RANKL at 50 ng/mL for 6 hours. (B–C) Representative images of the expression level of IĸB-α and β-actin in Western blot assay and the quantification of the ratios of band intensity of IκB-α to β-actin. (B–F) Representative images of the expression phosphorylation level of ERK and P38 with or without the treatment of 50 μM RH, and the quantification of the fold change ratios of band intensity of p-ERK to ERK and p-P38 to P38 (n = 3). *p < 0.05, **p < 0.01 relative to RANKL-induced positive group.

FIGURE 7

A proposed diagram depicts the RH treatment on the inhibition of osteoclast formation and function. RH suppresses NF-κB activity, ERK, and P38 phosphorylation and Ca²⁺ oscillation, eventually leading to the deactivation of NFATc1 and its downstream osteoclast-specific genes. In addition, RH treatment also reduced ROS level by inhibiting ROS production and boosting ROS scavenging activity.