Depletion of mmu-miR-185 enhances osteocyte connectivity and suppresses bone fragility through the interaction between ARL8B and RAB5A.

backgroundOsteocytes, the most abundant cells embedded in bone, possess extensive cytoplasmic extensions that form a complex network facilitating direct communication among osteocytes and with other effector cells in the bone and bone marrow. The mechanisms underlying osteocyte dendrite formation during “osteocytogenesis” remain poorly understood. MicroRNAs play a crucial role in regulating bone homeostasis.

methodsTo induce osteoporosis, bilateral ovariectomy was performed on 8-week-old female C57BL/6J mice of both wild-type (WT) and miR-185 knockout (KO) genotypes. Ploton silver staining was used to visualize the effects of miR-185 KO on the osteocyte lacunar-canalicular network (LCN), while micro-computed tomography assessed bone microstructure. Bone biomechanical properties were evaluated via three-point bending tests. To investigate the mechanisms by which miR-185 regulates osteocyte LCN, miR-185 KO MLO-Y4 cells were generated using CRISPR/Cas9 technology. Bioinformatics analysis, Western blotting and dual-luciferase reporter assays were used to identify the target gene of miR-185. Co-immunoprecipitation and GST pull-down experiments were utilized to validate the interacting partner of target gene. The levels of pathway-related molecules and the activation of downstream signaling were examined using cell surface protein biotinylation, Western blotting and Lysosome immunoprecipitation. The role of miR-185 KO in osteocyte-mediated regulation of osteoblast and osteoclast differentiation was evaluated using ALP staining, alizarin red staining and TRAP staining.

resultsIn this study, we demonstrated that dendrite formation and osteocyte connectivity were significantly enhanced in miR-185 KO ovariectomized (OVX) mice. Furthermore, both bone quality and mass were improved in miR-185 KO OVX mice. In miR-185 KO MLO-Y4 cells, we observed similar osteocyte morphology change. We identified Arl8b, a small GTP-binding protein that promotes lysosomal anterograde trafficking, as a target gene of miR-185. Knockdown of Arl8b significantly inhibited the enhanced dendritic process formation phenotype observed in miR-185 KO cells. Additionally, we discovered that RAB5A is a novel interacting protein of ARL8B. Downregulation of RAB5A similarly impaired osteocyte connectivity, suggesting a functional interaction between ARL8B and RAB5A. Further studies revealed that knockdown Arl8b suppressed RAB5A-mediated ITGB1 endosomal recycling, promoted its entry into lysosomes, reduced ITGB1 expression, inhibited FAK activation, and consequently diminished osteocyte dendritic process formation. Moreover, it was observed that the culture supernatant from miR-185 KO MLO-Y4 cells enhanced osteoblast mineralization and inhibited osteoclast differentiation.

conclusionsThis study elucidates the mechanism of miR-185 in regulating osteocyte LCN and its involvement in estrogen deficiency-induced osteoporosis progression, identifying novel therapeutic targets and informing potential intervention strategies for skeletal disorders.