MAGI1 attenuates osteoarthritis by regulating osteoclast fusion in subchondral bone through the RhoA-ROCK1 signaling pathway

Abstract

Background

Osteoarthritis (OA) is a chronic joint disorder that predominantly affects middle-aged or elderly individuals. Subchondral bone remodeling due to osteoclast hyperactivation is regarded as a major feature of early OA. During osteoclast fusion and multinucleation, the cytoskeleton reorganization leads to the formation of actin belts and ultimately bone resorption. Membrane-associated guanylate kinase with an inverted repeat member 1 (MAGI1) is a scaffolding protein that is crucial for linking the extracellular environment to intracellular signaling pathways and cytoskeleton. However, the role of MAGI1 in subchondral bone osteoclast fusion remains unclear.

Methods

In this study, we collected knee joint samples from OA patients and established the OA mouse model to examine the expression of MAGI1. Furthermore, we established the OA rat model and locally injected rAAV9-mediated shMagi1 into the subchondral bone to knock down MAGI1 expression. Micro-CT, histological staining, and immunofluorescence were employed to assess the effects of MAGI1 knockdown on subchondral bone homeostasis and OA process. We isolated and cultured osteoclasts from femoral and tibial bone marrow. Receptor activator of nuclear factor-κB ligand (RANKL)-stimulated osteoclasts served as an in vitro model for OA and underwent RNA sequencing. We employed gain- and loss-of-function experiments using MAGI1-overexpression plasmids and small interfering RNA to explore the role of MAGI1 in osteoclast differentiation. Further molecular experiments, including RT-qPCR, western blotting, immunofluorescence staining, and LC-MS/MS were performed to investigate underlying mechanisms.

Results

MAGI1 expression was significantly downregulated during RANKL-induced osteoclastogenesis in vitro. Additionally, a progressive decrease in MAGI1 expression was consistently observed in both knee joint samples from OA patients and mouse OA models, correlating with OA progression. Knockdown of MAGI1 in subchondral bone increased osteoclast numbers and worsened subchondral bone microarchitecture and cartilage degeneration; MAGI1 knockdown rats exhibited elevated PDGF-BB, Netrin-1, and CGRP⁺ sensory innervation. Overexpression and knockdown of MAGI1 suppressed and promoted osteoclast differentiation, respectively. Mechanistically, MAGI1 overexpression decreased the levels of RhoA, ROCK1, and p-p65 in RANKL-treated osteoclasts, which was rescued by the addition of RhoA activator narciclasine.

Conclusion

Our results demonstrate that MAGI1 suppresses osteoclast fusion through the RhoA/ROCK1 signaling pathway, targeting MAGI1 in subchondral bone osteoclasts may be a promising therapeutic strategy mitigate the advancement of OA.

The translational potential of this article

This study reveals that the scaffold protein MAGI1 participates in osteoarthritis progression by regulating osteoclast fusion, providing novel theoretical foundations and potential therapeutic targets for osteoarthritis treatment.