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

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 (KD) 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.