Ellagic acid blocks RANKL–RANK interaction and suppresses RANKL-induced osteoclastogenesis by inhibiting RANK signaling pathways

Highlights

• Ellagic acid (EA) inhibited RANKL-induced osteoclast differentiation and F-actin ring formation.

• EA directly bound to RANK and RANKL with strong affinities and blocked RANKL–RANK interaction.

• EA suppressed canonical RANK signaling pathways in osteoclast precursors.

• EA downregulated master transcriptional factors and osteoclast-specific genes and proteins expression.

Abstract

Ellagic acid (EA) is a naturally occurring polyphenolic compound that has been shown to exhibit diverse beneficial pharmacological activities including anti-osteoclastogenesis effect. However, the molecular mechanism by which EA inhibits osteoclastogenesis remains to be elucidated. The protein-protein interaction between receptor activator of nuclear factor (NF)-κB ligand (RANKL) and its receptor RANK contributes to osteoclast differentiation and activation in bone remodeling, and is regarded as an important therapeutic target for the treatment of osteoporosis. The current study is focused on investigating whether EA can directly bind to RANKL and/or RANK and block the interaction between RANKL and RANK, thereby inhibiting downstream signaling pathways. Interestingly, we found that EA had strong affinities to RANK and RANKL, with the estimated equilibrium dissociation constants (K_D) of 2.485 × 10⁻¹¹ and 1.688 × 10⁻⁹ M, respectively, and could disrupt the interaction between RANKL and RANK, thereby inhibiting RANKL-induced canonical RANK signaling pathways (p65, JNK, ERK, and p38) and expression of downstream master transcriptional factors (NFATc1 and c-Fos) and osteoclast-specific genes and proteins (TRAP, c-Src, and cathepsin K), which could ultimately suppress RANKL-induced osteoclast differentiation and F-actin ring formation. Taken together, our results revealed that EA could block RANKL–RANK interaction and suppress RANKL-induced osteoclastogenesis by inhibiting RANK signaling pathways in RAW 264.7 murine macrophages.

Introduction

Bone is continuously being remodelled and bone homeostasis relies on the balance between bone formation by osteoblasts and bone resorption by osteoclasts [1,2]. The imbalance of bone homeostasis in favour of osteoclasts can result in pathologic bone diseases, such as osteoporosis, osteoarthritis, periodontitis, and Paget's disease of bone [3,4], which affect millions of people. Therefore, inhibiting excessive osteoclast differentiation and activation is a potential therapeutic strategy for the prevention and treatment of bone metabolic diseases.

Osteoclasts, giant multinucleated cells, are differentiated from hematopoietic stem cells by the cooperative action of receptor activator of nuclear factor (NF)-κB ligand (RANKL) and macrophage-colony stimulating factor (M-CSF) [5]. RANKL, a critical cytokine of the tumor necrosis factor (TNF) family, can directly bind to its receptor RANK and subsequently activate downstream signaling pathways, including NF-κB and mitogen-activated protein kinase (MAPK), which induce osteoclast differentiation via activation of nuclear factor of activated T cell c1 (NFATc1) and c-Fos [6]. During osteoclastogenesis, these transcription factors regulate the expression of osteoclast-related marker genes and proteins, including tartrate-resistant acid phosphatase (TRAP), c-Src, matrix metallopeptidase-9 (MMP-9), and cathepsin K [7]. The RANKL–RANK interaction has become a therapeutic target for osteoclastogenesis with the successful launch of RANKL antagonists [8]. However, these biologic therapies have exhibited inevitable adverse effects [9]. As such, the identification of natural compounds that can disrupt RANKL–RANK interaction is a promising and current focus area.

Ellagic acid (EA) is a naturally occurring polyphenolic compound that is widely found in numerous fruits and other foods, including pomegranates, grapes, strawberries, blackberries, raspberries, nuts as well as green tea [10]. Accumulating evidence has demonstrated that EA possesses strong anti-inflammatory [[11], [12], [13]], antioxidant [10], anti-diabetic [14], and antitumor properties [15]. Moreover, EA has been shown to exhibit anti-osteoclastogenesis activity [16,17]. However, the detailed molecular mechanism by which EA suppresses osteoclast differentiation and activation remains to be elucidated.

Given that the protein-protein interaction between RANKL and RANK contributes to osteoclastogenesis in bone remodeling, and is regarded as an important therapeutic target for the treatment of osteoporosis, we hypothesized that EA might directly bind to RANKL and/or RANK and block the interaction between RANKL and RANK, thereby inhibiting downstream signaling pathways during osteoclastogenesis. In this study, we showed that EA can directly bind to RANK and RANKL with strong affinities and block RANKL–RANK interaction, thereby inhibiting RANKL-induced canonical RANK signaling pathways and downstream key regulatory factors in osteoclast precursors, which could ultimately suppress osteoclastogenesis.