Department of Medicine, University of Oklahoma Health Science Center, Oklahoma City, OK, USA.
Key Laboratory for Zoonosis Research, Jilin University, Changchun, China.
J Bone Miner Res. 2017 Nov;32(11):2207-2218. doi: 10.1002/jbmr.3205. Epub 2017 Aug 2.
Osteoclastogenesis is essential for bone remodeling and normal skeletal maintenance. Receptor activator of NF-κB ligand (RANKL) promotes osteoclast differentiation and function but requires costimulation of immunoreceptor tyrosine-based activation motif (ITAM)-coupled immunoreceptors. Triggering receptor expressed on myeloid cells-2 (TREM2) coupled to ITAM-adaptor protein DNAX activation protein 12kDA (DAP12) provides costimulation of intracellular calcium signaling during osteoclastogenesis. Previously, we found that downstream of kinase-3 (DOK3) physically associates with DAP12 to inhibit toll-like receptor (TLR)-induced inflammatory signaling in macrophages. However, whether and how DOK3 modulates DAP12-dependent osteoclastogenesis is unknown and the focus of this study. Bone microarchitecture and histology of sex- and age-matched wild-type (WT) and DOK3-deficient (DOK3 ) mice were evaluated. Male and female DOK3 mice have significantly reduced trabecular bone mass compared with WT mice with increased TRAP+ osteoclasts in vivo. In vitro, DOK3 bone marrow-derived macrophages (BMMs) have increased macrophage colony-stimulating factor (M-CSF)-induced proliferation and increased sensitivity to RANKL-induced osteoclastogenesis. Compared with WT, DOK3 osteoclasts are significantly larger with more nuclei and have increased resorptive capacity. Mechanistically, DOK3 limits osteoclastogenesis by inhibiting activation of Syk and ERK in response to RANKL and M-CSF. DOK3 is phosphorylated in a DAP12-dependent manner and associates with Grb2 and Cbl. Compared with DAP12 mice with high bone mass, DOK3- and DAP12- doubly deficient mice (DKO) have normalized bone mass, indicating that DOK3 also limits DAP12-independent osteoclastogenesis in vivo. In vitro osteoclasts derived from DKO mice are mononuclear with poor resorptive capacity similar to DAP12 osteoclasts. Histomorphometry reveals that DOK3 mice also have reduced osteoblast parameters. DOK3 osteoblasts have reduced in vitro osteoblastogenesis and increased osteoprotegerin (OPG) to RANKL expression ratio compared with WT osteoblasts. Co-culture of WT and DOK3 osteoblasts with pre-osteoclasts reveals a reduced capacity of DOK3 osteoblasts to support osteoclastogenesis. These data indicate that DOK3 regulates bone remodeling by negatively regulating M-CSF- and RANKL-mediated osteoclastogenesis and positively regulating osteoblastogenesis. © 2017 American Society for Bone and Mineral Research.
破骨细胞的生成对于骨骼重塑和正常骨骼维持至关重要。核因子-κB 受体激活配体（RANKL）可促进破骨细胞分化和功能，但需要免疫受体酪氨酸激活基序（ITAM）偶联免疫受体的共刺激。髓样细胞表达的触发受体-2（TREM2）与 ITAM 衔接蛋白 DNAX 激活蛋白 12kDa（DAP12）偶联，可在破骨细胞生成过程中提供细胞内钙信号的共刺激。此前，我们发现激酶-3（DOK3）下游与 DAP12 物理结合，以抑制巨噬细胞中 Toll 样受体（TLR）诱导的炎症信号。然而，DOK3 是否以及如何调节 DAP12 依赖性破骨细胞生成尚不清楚，这也是本研究的重点。评估了性成熟和年龄匹配的野生型（WT）和 DOK3 缺陷型（DOK3）小鼠的骨微观结构和组织学。雄性和雌性 DOK3 小鼠的骨小梁骨量明显低于 WT 小鼠，体内 TRAP+破骨细胞增多。在体外，DOK3 骨髓来源的巨噬细胞（BMM）的巨噬细胞集落刺激因子（M-CSF）诱导的增殖增加，对 RANKL 诱导的破骨细胞生成的敏感性增加。与 WT 相比，DOK3 破骨细胞的细胞核更多，体积更大，具有更高的吸收能力。从机制上讲，DOK3 通过抑制 RANKL 和 M-CSF 诱导的 Syk 和 ERK 的激活来限制破骨细胞生成。DOK3 以 DAP12 依赖性方式磷酸化，并与 Grb2 和 Cbl 结合。与骨量较高的 DAP12 小鼠相比，DOK3 和 DAP12 双重缺陷型（DKO）小鼠的骨量正常化，表明 DOK3 还限制了体内 DAP12 非依赖性破骨细胞生成。从 DKO 小鼠中分离出的破骨细胞在体外为单核细胞，具有较差的吸收能力，类似于 DAP12 破骨细胞。组织形态计量学显示 DOK3 小鼠的成骨细胞参数也减少。与 WT 成骨细胞相比，DOK3 成骨细胞的体外成骨细胞生成能力降低，骨保护素（OPG）与 RANKL 的表达比值增加。预先与前破骨细胞共培养 WT 和 DOK3 成骨细胞显示 DOK3 成骨细胞支持破骨细胞生成的能力降低。这些数据表明，DOK3 通过负调节 M-CSF 和 RANKL 介导的破骨细胞生成和正调节成骨细胞生成来调节骨重塑。 © 2017 美国骨骼与矿物质研究协会。
