DOK3 Modulates Bone Remodeling by Negatively Regulating Osteoclastogenesis and Positively Regulating Osteoblastogenesis
DOK3 Modulates Bone Remodeling by Negatively Regulating Osteoclastogenesis and Positively Regulating Osteoblastogenesis
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DOI：
10.1002/jbmr.3205
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发表时间：
2017-11-01
影响因子：
6.2
通讯作者：
Humphrey, Mary Beth
中科院分区：
文献类型：
--
作者：
Cai, Xiaofeng;Xing, Junjie;Humphrey, Mary Beth
破骨细胞生成对于骨重建和正常骨骼的维持是必不可少的。NF-κ B配体受体激活因子（RANKL）促进破骨细胞分化和功能，但需要基于免疫受体酪氨酸激活基序（ITAM）偶联免疫受体的共刺激。髓样细胞上表达的触发受体2（TREM 2）偶联至ITAM-衔接蛋白DNAX活化蛋白12 kDA（DAP 12），在破骨细胞生成期间提供细胞内钙信号传导的共刺激。以前，我们发现激酶3（DOK 3）的下游与DAP 12物理结合，以抑制巨噬细胞中Toll样受体（TLR）诱导的炎症信号传导。然而，DOK 3是否以及如何调节DAP 12依赖性破骨细胞生成尚不清楚，也是本研究的重点。评估了性别和年龄匹配的野生型（WT）和DOK 3缺陷（DOK 3（-/-））小鼠的骨微结构和组织学。与体内TRAP+破骨细胞增加的WT小鼠相比，雄性和雌性DOK 3（-/-）小鼠的骨小梁质量显著降低。在体外，DOK 3（-/-）骨髓源性巨噬细胞（BK 3）增加了巨噬细胞集落刺激因子（M-CSF）诱导的增殖，并增加了对RANKL诱导的破骨细胞生成的敏感性。与WT相比，DOK 3（-/-）破骨细胞显著更大，具有更多的细胞核，并且具有增加的再吸收能力。从机制上讲，DOK 3通过抑制Syk和ERK响应RANKL和M-CSF的活化来限制破骨细胞生成。DOK 3以DAP 12依赖性方式磷酸化，并与Grb 2和Cbl缔合。与具有高骨量的DAP 12（-/-）小鼠相比，DOK 3-和DAP 12-双缺陷小鼠（DKO）具有正常化的骨量，表明DOK 3也限制了体内不依赖于DAP 12的破骨细胞生成。DKO小鼠的体外破骨细胞是单核细胞，与DAP 12（-/-）破骨细胞相似，具有较差的再吸收能力。组织形态学显示DOK 3（-/-）小鼠也具有降低的成骨细胞参数。与WT成骨细胞相比，DOK 3（-/-）成骨细胞的体外成骨细胞生成减少，骨保护素（OPG）与RANKL的表达比率增加。WT和DOK 3（-/-）成骨细胞与前破骨细胞的共培养揭示了DOK 3（-/-）成骨细胞支持破骨细胞生成的能力降低。这些数据表明DOK 3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。(C)2017年美国骨与矿物质研究学会。
Osteoclastogenesis is essential for bone remodeling and normal skeletal maintenance. Receptor activator of NF-kappa B 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. (C) 2017 American Society for Bone and Mineral Research.