Specific RANK Cytoplasmic Motifs Drive Osteoclastogenesis

Area of Science:

• Cell Biology
• Immunology
• Bone Biology

Background:

• Receptor activator of NF-κB ligand (RANKL) binding to RANK initiates osteoclast formation via TRAF recruitment.
• Two intracellular RANK motifs (Motif 2: PVQEET560-565, Motif 3: PVQEQG604-609) are identified as critical for TRAF binding and osteoclastogenesis in vitro.

Purpose of the Study:

• To validate the in vitro findings on RANK motifs' role in osteoclast formation using knock-in mouse models.
• To investigate the necessity of RANK Motifs 2 and 3 in Porphyromonas gingivalis (Pg)-mediated osteoclastogenesis.
• To explore the potential of RANK-derived peptides as therapeutic agents for blocking osteoclast formation.

Main Methods:

• Generation of knock-in (KI) mice with inactivating mutations in RANK Motifs 2 and 3 (RANKKI/KI).
• Assessment of bone mass, tooth eruption, and osteoclast formation in wild-type, heterozygous, and homozygous KI mice.
• In vitro assays using bone marrow macrophages (BMMs) stimulated with M-CSF, RANKL, and Pg, analyzing signaling pathways (NF-κB, ERK, p38, JNK, c-Fos, NFATc1).
• Administration of cell-penetrating peptide (CPP) conjugates targeting RANK motifs.

Main Results:

• Homozygous RANKKI/KI mice exhibit significantly increased trabecular bone mass compared to wild-type and heterozygous littermates.
• RANKKI/KI BMMs fail to form osteoclasts in vitro upon RANKL stimulation and are unresponsive to Pg-induced osteoclastogenesis.
• RANKL activates canonical NF-κB and MAPK pathways in KI cells but fails to induce c-Fos and NFATc1, which are crucial for osteoclast differentiation.
• CPP conjugates targeting RANK Motifs 2 or 3 effectively block RANKL- and Pg-mediated osteoclastogenesis by inhibiting c-Fos and NFATc1 expression.

Conclusions:

• RANK Motifs 2 and 3 are essential for osteoclast formation in vivo and Porphyromonas gingivalis-induced osteoclastogenesis.
• These motifs mediate osteoclastogenesis by regulating c-Fos and NFATc1 expression during RANKL priming and subsequent Pg stimulation.
• Targeting RANK motifs with CPP conjugates presents a promising therapeutic strategy for controlling osteoclast-related bone diseases.