Musculoskeletal Research Center, Division of Bone and Mineral Diseases, Department of Medicine, Washington University School of Medicine, St. Louis, MO, USA.
Center of Regenerative Medicine, Washington University School of Medicine, St. Louis, MO, USA.
J Bone Miner Res. 2019 Nov;34(11):2087-2100. doi: 10.1002/jbmr.3819. Epub 2019 Jul 31.
Studies from global loss-of-function mutants suggest that alternative NF-κB downstream of NF-κB inducing kinase (NIK) is a cell-intrinsic negative regulator of osteogenesis. However, the interpretation of the osteoblast and/or osteocyte contribution to the bone phenotype is complicated by simultaneous osteoclast defects in these models. Therefore, we turned to a transgenic mouse model to investigate the direct role of NIK in the osteolineage. Osx-Cre;NT3 animals (NT3-Cre +), which bear a constitutively active NIK allele (NT3) driven by Osx-Cre, were compared with their Cre-negative, Control (Ctrl) littermates. NT3-Cre + mice had elevated serum P1NP and CTX levels. Despite this high turnover state, µCT showed that constitutive activation of NIK resulted in a net increase in basal bone mass in both cortical and cancellous compartments. Furthermore, NT3-Cre + mice exhibited a greater anabolic response following mechanical loading compared with controls. We next performed RNA-Seq on nonloaded and loaded tibias to elucidate possible mechanisms underlying the increased bone anabolism seen in NT3-Cre + mice. Hierarchical clustering revealed two main transcriptional programs: one loading-responsive and the other NT3 transgene-driven. Gene ontology (GO) analysis indicated a distinct upregulation of receptor, kinase, and growth factor activities including Wnts, as well as a calcium-response signature in NT3-Cre + limbs. The promoters of these GO-term associated genes, including many known to be bone-anabolic, were highly enriched for multiple κB recognition elements (κB-RE) relative to the background frequency in the genome. The loading response in NT3-Cre + mice substantially overlapped (>90%) with Ctrl. Surprisingly, control animals had 10-fold more DEGs in response to loading. However, most top DEGs shared between genotypes had a high incidence of multiple κB-RE in their promoters. Therefore, both transcriptional programs (loading-responsive and NT3 transgene-driven) are modulated by NF-κB. Our studies uncover a previously unrecognized role for NF-κB in the promotion of both basal and mechanically stimulated bone formation. © 2019 American Society for Bone and Mineral Research.
来自全球功能丧失突变体的研究表明，核因子-κB 诱导激酶（NIK）下游的替代 NF-κB 是成骨细胞内固有负调节因子。然而，由于这些模型中同时存在破骨细胞缺陷，因此对成骨细胞和/或骨细胞对骨表型的贡献的解释变得复杂。因此，我们转向转基因小鼠模型来研究 NIK 在成骨细胞谱系中的直接作用。Osx-Cre;NT3 动物（NT3-Cre+），其带有由 Osx-Cre 驱动的组成型活性 NIK 等位基因（NT3），与它们的 Cre 阴性对照（Ctrl）同窝仔比较。NT3-Cre+小鼠血清 P1NP 和 CTX 水平升高。尽管存在这种高转化率状态，但 µCT 显示，NIK 的组成型激活导致皮质和松质骨腔中基础骨量的净增加。此外，与对照相比，NT3-Cre+小鼠在机械加载后表现出更大的合成代谢反应。接下来，我们对非加载和加载的胫骨进行 RNA-Seq，以阐明 NT3-Cre+小鼠中观察到的增加的骨合成代谢的可能机制。层次聚类显示了两个主要的转录程序：一个是加载反应性的，另一个是 NT3 转基因驱动的。基因本体论（GO）分析表明，Wnt 等受体、激酶和生长因子活性以及钙反应特征明显上调，在 NT3-Cre+肢体中也是如此。这些 GO 术语相关基因的启动子，包括许多已知的骨合成代谢基因，相对于基因组中的背景频率，高度富集多个 κB 识别元件（κB-RE）。NT3-Cre+小鼠的加载反应与 Ctrl 重叠（>90%）。令人惊讶的是，对照动物在响应加载时有 10 倍以上的差异表达基因。然而，在两种基因型之间共享的大多数顶级差异表达基因的启动子中都有大量的 κB-RE。因此，这两个转录程序（加载反应性和 NT3 转基因驱动）都受 NF-κB 调节。我们的研究揭示了 NF-κB 在促进基础和机械刺激骨形成中的先前未被认识的作用。
