SLIT3 Promotes Bone Regeneration through Rac1 Inhibition and β-Catenin Activation
Introduction. Bone remodeling is a complex biological process that continues throughout life. It entails two contrary forces of bone formation by osteoblasts and bone resorption by osteoclasts.1-3 A positive balance of bone formation over resorption is needed to repair diseased bone, such as in osteoporosis which is characterized by compromised bone strength and increased risk of fractures.4 Anti-resorptive and anabolic agents are currently used to decrease the bone turnover ratio and are effective but are sometimes limited by intolerance, adverse events, coexisting comorbidities, and inadequate adherence.5,6 SLIT3 is a protein that increases cell mobility and is involved in the homeostasis of multiple tissues including bone. The role of SLIT3 in bone metabolism has been studied recently as a potential novel drug target for regulating the activities of osteoblasts and/or osteoclasts.3 Methods. Both osteoblasts and osteoclasts are used to study the SLIT3 mechanism. In osteoblasts, SHN3 was knocked out in mice; qRT-PCR and Micro-CT were used to measure gene expression and bone density, respectively.7 Osteoblasts were lysed and a binding ELISA was performed to identify the SLIT3 receptor. Western blotting was performed to determine the amount of β-catenin and phosphorylated activated β-catenin after SLIT3 binding. SLIT3 expression was measured in osteoclasts exposed to conditioned media or media spiked with RANKL and p50 NF-κB/CREB, known activators of osteoclasts. ELISA was performed to determine the osteoclast receptors. Finally, western blots were used to determine the effect of knocking out Rac1-specific guanosine triphosphate-activating protein SLIT-Robo GAP2 (Srgap2) on osteoclasts, the Rac1 mechanism, and the activation of osteoclastic activity.8-10 Results. These studies showed that SHN3 knockout in mice increased SLIT3 expression. Micro-CT also showed increased bone mass and bone thickness in SHN3-cKO and SLIT3+ mice. ELISA showed that SLIT3 binds to ROBO1 and ROBO2 on osteoblasts, and ROBO1 and ROBO3 on osteoclasts. After binding, SLIT3 phosphorylates and activates the β-catenin pathway, leading to bone formation in osteoblasts while activating Srgap2 to negatively regulate Rac1 in osteoclasts.7-10 Conclusion. The results showed that SLIT3 promote higher bone mineral density and thickness by stimulating osteoblast migration and suppressing osteoclast formation. There may be a therapeutic potential for SLIT3 as a novel drug target that combines anti-resorptive and anabolic mechanisms.
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