Role of endosomal RANKL-LGR4 signaling during osteoclast differentiation

Area of Science:

• Cell Biology
• Molecular Biology
• Endocrinology

Background:

• Osteoclastogenesis, crucial for bone homeostasis, is regulated by complex signaling pathways.
• Leucine-rich repeat-containing G-protein-coupled receptor 4 (LGR4) negatively regulates the RANK signaling cascade.
• The interplay between cell surface receptor signaling and endocytic trafficking is increasingly recognized as bidirectional.

Purpose of the Study:

• To investigate the distinct signaling roles of membrane-bound versus endosomal LGR4.
• To determine if LGR4 signaling influences RANK-RANKL signaling during osteoclastogenesis.
• To elucidate the role of membrane-to-endosomal trafficking in regulating LGR4 signaling.

Main Methods:

• CRISPR-Cas9 technology to create LGR4 conditional knock-out (CKO) RAW 264.7 cells.
• Generation of Drg2 knockout (KO) mice.
• Analysis of LGR4 endocytosis, localization within endosomes (RAB5-positive), and downstream signaling events (GSK-3β phosphorylation, NFATC1 translocation).

Main Results:

• LGR4 is endocytosed into early endosomes upon binding RANKL in osteoclast precursor cells.
• Internalized LGR4 activates LGR4-RANKL signaling within early endosomes.
• Lgr4 CKO cells exhibited increased early endosome signaling and decreased GSK-3β inhibition.
• RANKL treatment enhanced NFATC1 nuclear translocation in Lgr4 CKO cells and Drg2 KO mice.

Conclusions:

• RANKL-LGR4 signaling is dynamically regulated by membrane-to-endosomal trafficking.
• Endosomal localization of LGR4 is critical for its inhibitory role in osteoclastogenesis.
• These findings reveal a novel mechanism linking receptor trafficking to bone remodeling regulation.