Antioxidant α-lipoic acid inhibits osteoclast differentiation by reducing nuclear factor-κB DNA binding and prevents in vivo bone resorption induced by receptor activator of nuclear factor-κB ligand and tumor necrosis factor-α

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Abstract

The relationship between oxidative stress and bone mineral density or osteoporosis has recently been reported. As bone loss occurring in osteoporosis and inflammatory diseases is primarily due to increases in osteoclast number, reactive oxygen species (ROS) may be relevant to osteoclast differentiation, which requires receptor activator of nuclear factor-κB ligand (RANKL). Tumor necrosis factor-α (TNF-α) frequently present in inflammatory conditions has a profound synergy with RANKL in osteoclastogenesis. In this study, we investigated the effects of α-lipoic acid (α-LA), a strong antioxidant clinically used for some time, on osteoclast differentiation and bone resorption. At concentrations showing no growth inhibition, α-LA potently suppressed osteoclastogenesis from bone marrow-derived precursor cells driven either by a high-dose RANKL alone or by a low-dose RANKL plus TNF-α (RANKL/TNF-α). α-LA abolished ROS elevation by RANKL or RANKL/TNF-α and inhibited NF-κB activation in osteoclast precursor cells. Specifically, α-LA reduced DNA binding of NF-κB but did not inhibit IKK activation. Furthermore, α-LA greatly suppressed in vivo bone loss induced by RANKL or TNF-α in a calvarial remodeling model. Therefore, our data provide evidence that ROS plays an important role in osteoclast differentiation through NF-κB regulation and the antioxidant α-lipoic acid has a therapeutic potential for bone erosive diseases.

Introduction

Osteoclasts, cells specialized for resorption (dissolution) of calcified matrix, are continuously generated from discrete subpopulations of hematopoietic cells through differentiation processes. The differentiation events are regulated by various systemic and local factors including hormones, growth factors, and immune mediators in endocrine, paracrine, and autocrine manners. However, the tumor necrosis factor (TNF) family cytokine RANKL (receptor activator of nuclear factor-κB ligand) and its receptor RANK are the fundamental components required for osteoclast differentiation [1], [2], [3], [4]. Many of the factors regulating osteoclast differentiation exert their effects either by changing the expression of RANKL and osteoprotegerin, a decoy receptor for RANKL that interferes with RANKL binding to RANK, or by positively or negatively influencing RANK-dependent signaling [5], [6], [7], [8], [9], [10]. RANKL binding to RANK triggers several intracellular signaling pathways in osteoclast precursor cells, ultimately causing the expression of osteoclast-specific genes. These signaling events include the activation of PI3K/Akt, the cascade of mitogen-activated protein kinase activation, and the stimulation of transcription factors nuclear factor κB (NF-κB) and nuclear factor of activated T cells [11]. The TNF receptor-associated factor family adaptor molecules are recruited to RANK and play a pivotal role in these RANKL-induced signaling responses [11]. RANKL also plays an important role in the activation of differentiated mature osteoclasts.

Reactive oxygen species (ROS), initially recognized as important small molecules for defense against invading organisms and as deleterious metabolic intermediates to kill host cells, have later been increasingly documented for the second messenger or modulator roles in signal transduction [12], [13], [14]. There have been some studies implicating ROS in bone regulation. Bone resorption stimulated by parathyroid hormone and interleukin-1 (IL-1) was inhibited by removal of superoxide anions while addition of hydrogen peroxide increased bone resorption by isolated osteoclasts [15], [16]. Generation of ROS by osteoclasts was shown both in vivo and in vitro, and NADPH oxidase, a ROS generating enzyme, was detected in osteoclasts [15], [17], [18]. Recently, we also demonstrated that RANKL stimulates hydrogen peroxide production in differentiated osteoclasts [19]. These findings indicate that mature osteoclasts produce ROS probably through activated NADPH oxidase and ROS may in turn contribute to their bone resorption function. The possibility of ROS involvement in osteoclast differentiation has been raised based on the correlation between ROS production and the formation of osteoclasts on bone surfaces in vivo, and in in vitro culture of bone marrow cells [15], [20]. However, whether the ROS-mediated contribution was made by osteoblasts/stromal cells or osteoclast precursors and which agonistic factors were directly responsible for stimulating ROS production were not clear in those studies due to the presence of mixed populations of cells. TNF-α is a potential candidate for inducing ROS in osteoclast lineage cells because it exerts strong effects on osteoclast differentiation [7] and has been shown to cause ROS generation in other cell types [13], [21].

Use of antioxidants has been suggested to be beneficial in oxidative stress-associated diseases. N-Acetylcysteine (NAC) was shown to decrease the risk of colon cancer in patients with previous adenomatous colonic polyps and to increase immune functions in HIV patients [22], [23]. It was reported that oxidative stress levels were negatively associated with bone mineral density and antioxidant levels were lower in osteoporosis patients [24], [25]. In line with these observations, vitamin C intake showed beneficial effects in increasing bone mineral density in women and, more recently, NAC and vitamin C inhibited ovariectomy-induced bone loss in a rodent osteoporosis model [26], [27]. α-Lipoic acid (α-LA) is a powerful antioxidant that has long been used clinically for treating diabetic neuropathy [28], [29]. This compound also improves glucose uptake in patients with type 2 diabetes mellitus and has antiobesity effects [30], [31]. Recently, interest in the clinical applications of α-LA to many chronic diseases has greatly been elevated, perhaps due to the relative clinical safety and potent antioxidant properties [32]. However, the effect of this antioxidant on bone loss prevention and bone cell activity regulation has not been investigated to date.

In this study, we investigated the effect of α-LA on osteoclast differentiation. We found that α-LA inhibited osteoclastogenesis in bone marrow-derived precursor cell cultures under osteoclastogenic conditions attained with a high-dose RANKL alone or a low-dose RANKL plus TNF-α. The inhibitory effect appeared to be due to suppression of ROS generation and subsequent reduction in the activation of NF-κB induced by those osteoclastogenic stimuli. Furthermore, the potential usefulness of this antioxidant in inhibiting in vivo bone loss was verified by calvarial bone resorption analyses.

Section snippets

Osteoclast culture

Osteoclast generation was achieved using either primary cultures of mouse bone marrow-derived macrophages or the monocyte/macrophage lineage cell line RAW264.7. For generation of bone marrow-derived osteoclasts, monocytes were isolated from tibiae of ICR mice as previously described [33]. Cells were seeded in 6-well plates (2 × 106/well) and cultured in the presence of 30 ng/ml macrophage colony stimulating factor (M-CSF) for 72 h. Cells at this stage were considered M-CSF-dependent bone marrow

α-LA inhibits osteoclast formation

In vitro osteoclastogenesis was investigated by culturing bone marrow-derived macrophages and RAW264.7 cells in the presence of the osteoclastogenic cytokine RANKL and scoring the number of TRAP-positive multinuclear cells (TRAP+ MNC) generated. Before examining the effect of α-lipoic acid on osteoclast differentiation, the potential cytotoxicity of α-LA was tested because cell toxicity would decrease cell numbers during culture and consequently would reduce TRAP+ MNC formation irrelative to

Discussion

Abnormal increase in osteoclast differentiation is the major contributing factor in pathologic bone erosion associated with osteoporosis and inflammatory diseases. Based on well-documented evidence for ROS production in inflammatory conditions and recent reports implicating ROS in osteoporosis [25], [27], we reasoned that osteoclast differentiation might require ROS as a signaling intermediate and that in vivo bone resorption could be controlled by antioxidants. Recently, we found that a

Acknowledgments

We are grateful to Professors Zang Hee Lee and Sakae Tanaka for help with calvarial bone resorption assays. This work was supported by the 21C Frontier Functional Proteomics Project Grant (FPR05C2-280) and the Molecular and Cellular BioDiscovery Research Program (M1-0311-00-0024) from the Ministry of Science and Technology, Korea.

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