CIHR Group in Matrix Dynamics, University of Toronto, Toronto, Ontario, Canada.
J Bone Miner Res. 2010 May;25(5):1077-91. doi: 10.1359/jbmr.091114.
Osteoclastogenesis (OCG) results from the fusion of monocytes after stimulation with macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-kappaB ligand (RANKL). Migration of monocytes into close proximity precedes critical fusion events that are required for osteoclast formation. Cellular migration requires leading-edge actin cytoskeleton assembly that drives cellular locomotion. Filamin A (FLNa) cross-links F-actin filaments in the leading edge of migrating cells and also has been shown to regulate signal transduction during cell migration. However, little is known about the possible role of FLNa in osteoclastogenesis. Our objective in this study was to investigate the role of FLNa in osteoclastogenesis. Bone marrow monocytes isolated from the tibiae and femora of wild type (WT) and Flna-null mice were cultured for 6 days with M-CSF and RANKL, and osteoclasts were identified by tartrate-resistant acid phosphatase (TRACP) staining. The Flna-null mouse skeletal phenotype was characterized using dual-energy X-ray absorptiometry (DXA) to analyze the skeleton, as well as tests on blood chemistry. Osteoclast levels in vivo were quantified by counting of TRACP-stained histologic sections of distal femora. To elucidate the mechanisms by which Flna regulates osteoclastogenesis, migration, actin polymerization, and activation of Rho GTPases, Rac1, Cdc42, and RhoA were assessed in monocytes during in vitro OCG. Deficiencies in migration were rescued using constitutively active Rac1 and Cdc42 TAT fusion proteins. The RANKL signaling pathway was evaluated for activation by monitoring nuclear translocation of NF kappaB and c-jun and expression of key osteoclast genes using quantitative real-time polymerase chain reaction (qRT-PCR). Our results show that Flna-null monocytes formed fewer osteoclasts in vitro, and those that were formed were smaller with fewer nuclei. Decreased OCG was reflected in vivo in TRACP-stained histologic bone sections. Flna-null monocytes experienced impaired migratory ability. When OCG was performed at increasing starting cellular plating densities in order to decrease intercellular distances, there was progressive rescue of Flna-null osteoclast formation comparable with WT levels, confirming that Flna regulates monocyte migration prefusion. Activation of the actin cytoskeleton regulators Rac1, Cdc42, and RhoA and actin free-barbed end generation were partially or completely abrogated in Flna-null monocytes; however, monocyte migration was restored on rescuing with constitutively active Rac1 and Cdc42 TAT fusion proteins. We conclude that filamin A is required for osteoclastogenesis by regulating actin dynamics via Rho GTPases that control monocyte migration.
破骨细胞发生（OCG）是由单核细胞在巨噬细胞集落刺激因子（M-CSF）和核因子 κB 配体受体激活剂（RANKL）刺激后融合而成。单核细胞向紧密融合事件迁移之前，需要进行关键融合事件，这是形成破骨细胞所必需的。细胞迁移需要前缘肌动球蛋白细胞骨架组装，从而驱动细胞运动。细丝蛋白 A（FLNa）交联迁移细胞前缘的 F-肌动蛋白丝，并且已经显示其在细胞迁移过程中调节信号转导。然而，关于 FLNa 在破骨细胞发生中的可能作用知之甚少。我们在这项研究中的目的是研究 FLNa 在破骨细胞发生中的作用。从野生型（WT）和 Flna 基因敲除（Flna-null）小鼠的胫骨和股骨中分离骨髓单核细胞，在 M-CSF 和 RANKL 的作用下培养 6 天，并通过抗酒石酸酸性磷酸酶（TRACP）染色鉴定破骨细胞。使用双能 X 射线吸收法（DXA）分析骨骼来表征 Flna-null 小鼠的骨骼表型，以及血液化学测试。通过对远端股骨的 TRACP 染色组织学切片进行计数来量化体内破骨细胞水平。为了阐明 Flna 调节破骨细胞发生、迁移、肌动蛋白聚合以及 Rho GTPases（Rac1、Cdc42 和 RhoA）激活的机制，在体外 OCG 过程中评估了单核细胞中的迁移、肌动蛋白聚合和 Rho GTPases（Rac1、Cdc42 和 RhoA）激活。使用组成型激活 Rac1 和 Cdc42 TAT 融合蛋白来挽救迁移缺陷。通过监测核易位 NF kappaB 和 c-jun 以及使用定量实时聚合酶链反应（qRT-PCR）表达关键破骨细胞基因来评估 RANKL 信号通路的激活。我们的结果表明，Flna-null 单核细胞在体外形成的破骨细胞较少，并且形成的破骨细胞较小，核较少。在 TRACP 染色的组织学骨切片中，体内 OCG 减少。Flna-null 单核细胞经历迁移能力受损。当在增加起始细胞接种密度时进行 OCG 以减少细胞间距离时，Flna-null 破骨细胞形成的恢复程度与 WT 水平相当，这证实 Flna 通过调节融合前单核细胞迁移来调节肌动球蛋白细胞骨架调节剂 Rac1、Cdc42 和 RhoA 的活性和肌动蛋白游离端生成。我们的结论是，细丝蛋白 A 通过调节 Rho GTPases 控制单核细胞迁移来调节肌动蛋白动力学，从而在破骨细胞发生中是必需的。
