骨吸收破骨细胞(OCL)是通过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。破骨细胞生成过程中紧密调节的细胞融合导致可吸收的OCL的形成,其大小落在可预测的生理范围内。调节OCL融合的发生及其随后的停滞的分子机制是,然而,很大程度上未知。我们以前已经表明,从小鼠中培养的OCLs在囊泡运输相关蛋白分选nexin10中的R51Q突变纯合,该突变在人类和小鼠中诱导常染色体隐性遗传性骨硬化症,显示失调和连续融合,产生巨大的,非活动OCL。因此,成熟OCL的融合被一个活跃的,基因编码,细胞自主,和SNX10依赖机制。为了直接检查SNX10是否在体内发挥类似的作用,我们产生了SNX10缺陷(SKO)小鼠,并证明它们表现出大量的骨硬化,并且它们的OCLs在培养中不可控制地融合,纯合R51QSNX10(RQ/RQ)小鼠也是如此。缺乏SNX10的OCL在其外周表现出DC-STAMP蛋白的持续存在,这可能有助于它们不受控制的融合。为了在其天然骨环境中可视化内源性SNX10突变OCLs,我们对野生型OCLs进行了遗传标记,使用EGFP的SKO和RQ/RQ小鼠,然后通过双光子可视化常驻OCL和细胞周围骨基质的三维组织,共焦,和二次谐波生成显微镜。我们展示了卷,表面积和,特别是,两种突变株的OCLs中的细胞核数量平均比野生型小鼠的OCLs大2-6倍,表明放松管制,在突变小鼠中发生过度融合。我们得出结论,OCL的融合,因此它们的大小,在体内受到成熟OCL融合的SNX10依赖性阻滞的调节。
破骨细胞(OCL)是降解骨骼的细胞。这些细胞通过单核细胞前体细胞的融合产生,但是调节这一过程并最终逮捕它的机制是未知的。我们先前已经表明,从蛋白质分选nexin10(SNX10)中携带R51Q突变的小鼠培养的OCL失去了吸收能力,并且由于不受控制的融合而变得巨大。为了检查是否需要SNX10在体内进行OCL融合阻滞,我们灭活了小鼠的Snx10基因,并荧光标记了它们的OCLs和R51QSNX10小鼠的OCLs,隔离他们的股骨,并使用先进的3D显微镜方法来可视化骨基质内的OCL。不出所料,缺乏SNX10的小鼠表现出过多的骨量,表示它们的OCL处于非活动状态。两种突变小鼠品系的骨骼中的OCL平均比对照小鼠大2-6倍,并按比例包含更多的原子核。我们得出结论,OCL融合被控制,但不是SNX10突变体,老鼠,表明成熟OCL的大小在体内受到活性物质的限制,抑制细胞融合的SNX10依赖性机制。
Bone-resorbing osteoclasts (OCLs) are formed by differentiation and fusion of monocyte precursor cells, generating large multi-nucleated 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. In order 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. In order to visualize endogenous SNX10-mutant OCLs in their native bone environment we genetically labelled the OCLs of wild-type, SKO and RQ/RQ mice with EGFP, and then visualized the three-dimensional organization of resident OCLs and the pericellular bone matrix by two-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 wild-type mice, indicating that deregulated, excessive fusion occurs in the mutant mice. We conclude that the fusion of OCLs, and consequently their size, are regulated in vivo by SNX10-dependent arrest of fusion of mature OCLs.
Osteoclasts (OCLs) are cells that degrade bone. These cells are generated by fusion of monocyte precursor cells, but the mechanisms that regulate this process and eventually arrest it are unknown. We had previously shown that OCLs cultured from mice carrying the R51Q mutation in the protein sorting nexin 10 (SNX10) lose their resorptive capacity and become gigantic due to uncontrolled fusion. To examine whether
SNX10 is required for OCL fusion arrest also in vivo, we inactivated the
Snx10 gene in mice and fluorescently labelled their OCLs and OCLs of R51Q
SNX10 mice, isolated their femurs, and used advanced 3D microscopy methods to visualize OCLs within the bone matrix. As expected, mice lacking
SNX10 exhibited excessive bone mass, indicating that their OCLs are inactive. OCLs within bones of both mutant mouse strains were on average 2-6-fold larger than in control mice, and contained proportionally more nuclei. We conclude that OCL fusion is arrested in control, but not SNX10 mutant, mice, indicating that the sizes of mature OCLs are limited in vivo by an active, SNX10-dependent mechanism that suppresses cell fusion.