Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada.
Bone. 2012 Jul;51(1):145-52. doi: 10.1016/j.bone.2012.04.018. Epub 2012 May 4.
Modulation of Rho GTPases Rac1 and Rac2 impacts bone development, remodeling, and disease. In addition, GTPases are considered treatment targets for dysplastic and erosive bone diseases including Neurofibromatosis type 1. While it is important to understand the effects of Rac modulation on osteoclast function, two-dimensional resorption pit area measurements fall short in elucidating the volume aspect of bone resorption activity. Bone marrow from wild-type, Rac1 and Rac2 null mice was isolated from femora. Osteoclastogenesis was induced by adding M-CSF and RANKL in culture plates containing dentin slices and later stained with Picro Sirius Red to image resorption lacunae. Osteoclasts were also plated on glass cover slips and stained with phalloidin and DAPI to measure their surface area and the number of nuclei. Volumetric images were collected on a laser-scanning confocal system. Sirius Red confocal imaging provided an unambiguous, continuous definition of the pit boundary compared to reflected and transmitted light imaging. Rac1- and Rac2-deficient osteoclasts had fewer nuclei in comparison to wild-type counterparts. Rac1-deficient osteoclasts showed reduced resorption pit volume and surface area. Lacunae made by single Rac2 null osteoclasts had reduced volume but surprisingly surface area was unaffected. Surface area measures are deceiving since volume changed independently in resorption pits made by individual Rac2 null osteoclasts. Our innovative confocal imaging technique allows us to derive novel conclusions about Rac1 and Rac2 in osteoclast function. The data and method can be applied to study effects of genes and drugs including Rho GTPase modulators on osteoclast function and to develop pharmacotherapeutics to treat bone lytic disorders.
Rho GTPases Rac1 和 Rac2 的调节作用影响骨骼的发育、重塑和疾病。此外，GTPases 被认为是治疗包括神经纤维瘤病 1 型在内的发育不良和侵蚀性骨疾病的靶点。虽然了解 Rac 调节对破骨细胞功能的影响很重要，但二维吸收陷窝面积测量无法阐明骨吸收活性的体积方面。从野生型、Rac1 和 Rac2 基因敲除小鼠的股骨中分离骨髓。在含有牙本质切片的培养板中加入 M-CSF 和 RANKL 诱导破骨细胞生成，然后用 Picrosirius Red 染色来观察吸收陷窝。还将破骨细胞铺在玻璃载玻片上，用鬼笔环肽和 DAPI 染色来测量它们的表面积和细胞核数量。在激光共聚焦系统上采集体积图像。与反射和透射光成像相比，Sirius Red 共聚焦成像提供了明确的、连续的陷窝边界定义。与野生型相比，Rac1 和 Rac2 基因敲除的破骨细胞的细胞核数量较少。Rac1 基因敲除的破骨细胞的吸收陷窝体积和表面积减少。单个 Rac2 基因敲除的破骨细胞形成的陷窝体积减小，但令人惊讶的是，表面积不受影响。表面积测量是具有欺骗性的，因为在单个 Rac2 基因敲除的破骨细胞形成的吸收陷窝中，体积的变化是独立的。我们的创新共聚焦成像技术使我们能够对 Rac1 和 Rac2 在破骨细胞功能中的作用得出新的结论。该数据和方法可用于研究基因和药物（包括 Rho GTPase 调节剂）对破骨细胞功能的影响，并开发治疗溶骨性骨疾病的药物疗法。
