National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS), National Institutes of Health (NIH), Bethesda, MD, USA.
Biosciences Division, College of Dentistry, The Ohio State University, Columbus, OH, USA.
Bone. 2017 Dec;105:134-147. doi: 10.1016/j.bone.2017.08.027. Epub 2017 Sep 1.
Although acellular cementum is essential for tooth attachment, factors directing its development and regeneration remain poorly understood. Inorganic pyrophosphate (PP), a mineralization inhibitor, is a key regulator of cementum formation: tissue-nonspecific alkaline phosphatase (Alpl/TNAP) null mice (increased PP) feature deficient cementum, while progressive ankylosis protein (Ank/ANK) null mice (decreased PP) feature increased cementum. Bone sialoprotein (Bsp/BSP) and osteopontin (Spp1/OPN) are multifunctional extracellular matrix components of cementum proposed to have direct and indirect effects on cell activities and mineralization. Studies on dentoalveolar development of Bsp knockout (Bsp) mice revealed severely reduced acellular cementum, however underlying mechanisms remain unclear. The similarity in defective cementum phenotypes between Bsp mice and Alpl mice (the latter featuring elevated PP and OPN), prompted us to examine whether BSP is operating by modulating PP-associated genes. Genetic ablation of Bsp caused a 2-fold increase in circulating PP, altered mRNA expression of Alpl, Spp1, and Ank, and increased OPN protein in the periodontia. Generation of a Bsp knock-out (KO) cementoblast cell line revealed significantly decreased mineralization capacity, 50% increased PP in culture media, and increased Spp1 and Ank mRNA expression. While addition of 2μg/ml recombinant BSP altered Spp1, Ank, and Enpp1 expression in cementoblasts, changes resulting from this dose were not dependent on the integrin-binding RGD motif or MAPK/ERK signaling pathway. Decreasing PP by genetic ablation of Ank on the Bsp mouse background reestablished cementum formation, allowing >3-fold increased acellular cementum volume compared to wild-type (WT). However, deleting Ank did not fully compensate for the absence of BSP. Bsp; Ank double-deficient mice exhibited mean 20-27% reduced cementum thickness and volume compared to Ank mice. From these data, we conclude that the perturbations in PP metabolism are not solely driving the cementum pathology in Bsp mice, and that PP is more potent than BSP as a cementum regulator, as shown by the ability to override loss of BSP by lowering PP. We propose that BSP and PP work in concert to direct mineralization in cementum and likely other mineralized tissues.
虽然无细胞牙骨质对于牙齿附着是必不可少的，但指导其发育和再生的因素仍知之甚少。无机焦磷酸酯（PP），一种矿化抑制剂，是牙骨质形成的关键调节剂：组织非特异性碱性磷酸酶（Alpl/TNAP）缺失小鼠（PP 增加）表现出牙骨质缺陷，而进行性粘连蛋白（Ank/ANK）缺失小鼠（PP 减少）表现出牙骨质增加。骨唾液蛋白（Bsp/BSP）和骨桥蛋白（Spp1/OPN）是牙骨质的多功能细胞外基质成分，据推测对细胞活性和矿化具有直接和间接的影响。对 Bsp 敲除（Bsp）小鼠牙牙槽发育的研究表明，无细胞牙骨质严重减少，但其潜在机制尚不清楚。Bsp 小鼠和 Alpl 小鼠（后者表现出升高的 PP 和 OPN）的牙骨质缺陷表型之间的相似性促使我们研究 BSP 是否通过调节 PP 相关基因起作用。Bsp 的遗传缺失导致循环 PP 增加 2 倍，改变了 Alpl、Spp1 和 Ank 的 mRNA 表达，并增加了牙周组织中的 OPN 蛋白。生成 Bsp 敲除（KO）牙骨质细胞系显示出矿化能力显著降低，培养基中 PP 增加 50%，Spp1 和 Ank mRNA 表达增加。虽然添加 2μg/ml 重组 BSP 改变了牙骨质细胞中的 Spp1、Ank 和 Enpp1 表达，但该剂量引起的变化不依赖于整合素结合 RGD 基序或 MAPK/ERK 信号通路。在 Bsp 小鼠背景下通过遗传缺失 Ank 降低 PP 重建了牙骨质形成，与野生型（WT）相比，无细胞牙骨质体积增加了 3 倍以上。然而，删除 Ank 并不能完全弥补 BSP 的缺失。Bsp;ANK 双缺失小鼠的牙骨质厚度和体积比 Ank 小鼠分别减少 20-27%。从这些数据中，我们得出结论，PP 代谢的改变并不是导致 Bsp 小鼠牙骨质病变的唯一因素，而且 PP 作为牙骨质调节剂比 BSP 更有效，如通过降低 PP 来克服 BSP 缺失的能力所示。我们提出 BSP 和 PP 协同作用以指导牙骨质和可能其他矿化组织的矿化。
