Department of Molecular Genetics, The Weizmann Institute of Science, Rehovot 76100, Israel.
Institute of Comparative Molecular Endocrinology, University of Ulm, Ulm 89081, Germany.
J Bone Miner Res. 2024 Sep 26;39(10):1503-1517. doi: 10.1093/jbmr/zjae125.
Bone-resorbing osteoclasts (OCLs) are formed by differentiation and fusion of monocyte precursor cells, generating large multinucleated cells. Tightly regulated cell fusion during osteoclastogenesis leads to formation of resorption-competent OCLs, whose sizes fall within a predictable physiological range. The molecular mechanisms that regulate the onset of OCL fusion and its subsequent arrest are, however, largely unknown. We have previously shown that OCLs cultured from mice homozygous for the R51Q mutation in the vesicle trafficking-associated protein sorting nexin 10, a mutation that induces autosomal recessive osteopetrosis in humans and in mice, display deregulated and continuous fusion that generates gigantic, inactive OCLs. Fusion of mature OCLs is therefore arrested by an active, genetically encoded, cell-autonomous, and SNX10-dependent mechanism. To directly examine whether SNX10 performs a similar role in vivo, we generated SNX10-deficient (SKO) mice and demonstrated that they display massive osteopetrosis and that their OCLs fuse uncontrollably in culture, as do homozygous R51Q SNX10 (RQ/RQ) mice. OCLs that lack SNX10 exhibit persistent presence of DC-STAMP protein at their periphery, which may contribute to their uncontrolled fusion. To visualize endogenous SNX10-mutant OCLs in their native bone environment, we genetically labeled the OCLs of WT, SKO, and RQ/RQ mice with enhanced Green Fluorescent Protein (EGFP), and then visualized the 3D organization of resident OCLs and the pericellular bone matrix by 2-photon, confocal, and second harmonics generation microscopy. We show that the volumes, surface areas and, in particular, the numbers of nuclei in the OCLs of both mutant strains were on average 2-6-fold larger than those of OCLs from WT mice, indicating that deregulated, excessive fusion occurs in the mutant mice. We conclude that the fusion of OCLs, and consequently their size, is regulated in vivo by SNX10-dependent arrest of fusion of mature OCLs.
破骨细胞（OCL）是由单核细胞前体细胞的分化和融合形成的，产生大的多核细胞。在破骨细胞发生过程中，细胞融合受到严格调控，导致形成具有吸收能力的 OCL，其大小落在可预测的生理范围内。然而，调节 OCL 融合及其随后的停止的分子机制在很大程度上是未知的。我们之前已经表明，从小鼠中分离出来的 OCL 中，囊泡运输相关蛋白分选连接蛋白 10（SNX10）的 R51Q 突变纯合子，在人类和小鼠中诱导常染色体隐性骨硬化症，其显示出失调和连续融合，产生巨大的、无活性的 OCL。成熟的 OCL 融合因此被一种活跃的、遗传编码的、细胞自主的和 SNX10 依赖的机制所阻止。为了直接检查 SNX10 是否在体内发挥类似的作用，我们生成了 SNX10 缺陷（SKO）小鼠，并证明它们表现出严重的骨质硬化症，并且它们的 OCL 在培养中不受控制地融合，正如纯合 R51Q SNX10（RQ/RQ）小鼠一样。缺乏 SNX10 的 OCL 在外周持续存在 DC-STAMP 蛋白，这可能有助于它们的不受控制的融合。为了在其天然骨环境中可视化内源性 SNX10 突变的 OCL，我们用增强型绿色荧光蛋白（EGFP）对 WT、SKO 和 RQ/RQ 小鼠的 OCL 进行基因标记，然后通过双光子、共聚焦和二次谐波产生显微镜观察驻留 OCL 的 3D 组织和细胞周围骨基质。我们表明，两种突变株的 OCL 的体积、表面积，特别是核的数量，平均比 WT 小鼠的 OCL 大 2-6 倍，表明在突变小鼠中发生了失调的、过度的融合。我们得出结论，SNX10 依赖性阻止成熟 OCL 融合调节体内 OCL 的融合，从而调节其大小。
