Resveratrol prevents RANKL-induced osteoclast differentiation of murine osteoclast progenitor RAW 264.7 cells through inhibition of ROS production

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Abstract

The bone protective effects of resveratrol have been demonstrated in several osteoporosis models while the underlying mechanism is largely unclear. In the present study, we evaluated the effects of resveratrol on differentiation and apoptosis of murine osteoclast progenitor RAW 264.7 cells. We found that resveratrol at non-toxic concentrations dose-dependently inhibited RANKL-induced osteoclast differentiation and induced apoptosis. Resveratrol has been shown to be an activator of Sirt1, a NAD+ dependent protein deacetylase, and has been demonstrated to mimic estrogen. However, we found that although Sirt1 protein was abundantly expressed in RAW264.7 cells, the specific Sirt1 inhibitor EX-527 could not attenuate the inhibition of osteoclastogenesis mediated by resveratrol. Also, the effects of resveratrol could not be attenuated by ICI-182780, a high affinity estrogen receptor antagonist. The central role of reactive oxygen species (ROS) in RANKL-induced osteoclast differentiation has recently been clarified. We found that resveratrol suppressed RANKL-induced ROS generation in a concentration dependent manner. We postulate that the direct inhibitory effects of resveratrol on osteoclastogenesis are mediated via inhibition of ROS generation.

Research highlights

Resveratrol inhibits RANKL-induced osteoclastogenesis. ► Resveratrol induces apoptosis of differentiated osteoclasts. ► Sirt1 and ER(s) pathways are not involved in the inhibition of osteoclastogenesis by resveratrol. ► The inhibition of osteoclastogenesis is correlated with the inhibitory effects of resveratrol on ROS production.

Introduction

Resveratrol (RSV) is a polyphenol produced by certain plant species, including nuts, berries, and grapes. It is known to exert a variety of health benefits in mammals, among which the cancer chemopreventive and cardio- and neuro-protective properties have attracted most attention [1]. Its protective effects in bone have also been demonstrated. In ovariectomized rats, RSV feeding significantly inhibited the bone loss induced by estrogen deficiency [2], [3]; In rats under tail suspension, prior RSV treatment preserved trabecular bone in the hind-limb from disuse osteopenia [4]; In aged-mice, the onset of senile osteoporosis was markedly delayed after long-term feeding with RSV-containing foods [5]. These findings indicate that this natural substance might be useful in the treatment or prevention of osteoporosis but the underlying mechanism remains to be clarified.

Bone remodeling is dependent on a delicate balance between the activities of osteoblasts and osteoclasts. It has been shown that, through activation of estrogen receptors (ERs), RSV increased osteoblast proliferation, enhanced alkaline phosphatase (ALP) activity [6], and stimulated BMP-2 production [7]. In addition, through activation of Sirt1, a NAD-dependent protein deacetylase, RSV promoted mesenchymal stem cell differentiation toward osteoblastic lineage [8]. The direct effects of RSV on osteoclasts are relatively unclear. Studies of Naruse et al. [9] and Voronov et al. [10] showed that RSV did not affect osteoclastogenesis but could reverse the inhibition of osteoclastic resorption mediated by arylhydrocarbon receptor (AhR) agonists. In contrast, Boissy et al. [11] and Gabbay et al. [12] showed that RSV directly inhibited RANKL-induced osteoclastogenesis and attenuated the bone loss in castrated male mice. However, neither of these studies has investigated the intracellular mechanism(s) by which RSV exerts the effects.

Osteoclasts are formed by fusion of monocyte/macrophage precursors [13]. The receptor activator of NF-κB (nuclear factor-κB) ligand (RANKL) produced by osteoblasts plays a key role in osteoclast differentiation and activation [14]. In physiological milieu, the binding of RANKL to its receptor RANK on the surface of osteoclast precursors leads to the activation of TNF receptor-associated factor 6 (TRAF6), which subsequently stimulates the transcription of osteoclast associated genes (such as cathepsin K) and leads to the formation of multinucleated tartrate-resistant acid phosphatase (TRACP) positive giant cells [13]. As a widely used pre-osteoclast model, the murine macrophage/osteoclast precursor cell line RAW 264.7 cells differentiate into osteoclasts when stimulated with RANKL [15]. In the present study, RAW 264.7 cells were used to evaluate the effects of RSV on RANKL-induced osteoclastogenesis. We found that RSV dose-dependently inhibited RANKL-induced osteoclast differentiation and increased apoptosis of differentiated cells. We also found that the inhibition of RSV on RANKL-induced osteoclast differentiation was independent on both the Sirt1 and ERs pathways but was correlated with the inhibition of intracellular reactive oxygen species (ROS) production.

Section snippets

Reagents

Modified Eagle’s medium alpha (α-MEM), fetal bovine serum (FBS), L-Glutamine and Gentamicin were purchased from Gibco (Invitrogen). Recombinant mouse RANKL (462-TEC) was purchased from R&D Systems; RSV was purchased from Sigma–Aldrich, and EX-527 and ICI-182780 were from Tocris.

Cell culture

The murine RAW264.7 macrophage cell line was obtained from the American Type Culture Collection (ATCC). Cells were cultured in α-MEM medium supplemented with 10% FBS, 2 mM L-Glutamine and 50 μg/ml Gentamicin at 37 °C in a

RSV inhibited RANKL-induced osteoclast formation of RAW 264.7 cells

Different concentrations of RSV (0, 0.3, 1, 3, and 10 μM) were added to RAW 264.7 cells after stimulation with RANKL. As shown in Fig. 1, the number of TRACP-positive multinucleated cells reduced with increasing RSV concentration. To substantiate this finding, we further measured the expression of the specific osteoclast marker, cathepsin K. We found that the expression of cathepsin K was downregulated at both mRNA (Fig. 3E) and protein level (Fig. 3F) with the treatment of RSV. Taken together,

Discussion

At present the anti-resorptives remain the mainstay of osteoporosis management, however, the simultaneous inhibitory effects on bone remodeling can be a problem [16]. Bone anabolic compounds that stimulate osteoblast activity would be preferred for the treatment of established osteoporosis, but the application of agents such as parathyroid hormone (PTH), is limited by cost and concerns regarding its long-term safety [21]. From a botanical point of view, RSV belongs to a class of defence

Acknowledgments

This work is supported by the Ulla and Gustaf af Ugglas Foundation and Loo and Hans Ostermans Foundation for geriatric research.

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