Ii Hisataka, Warraich Sumeeta, Tenn Neil, Quinonez Diana, Holdsworth David W, Hammond James R, Dixon S Jeffrey, Séguin Cheryle A
Department of Physiology and Pharmacology, Schulich School of Medicine & Dentistry, and Bone and Joint Institute, The University of Western Ontario, London, Ontario, Canada; Department of Oral Health School of Life Dentistry at Tokyo, The Nippon Dental University, 1-9-20 Fujimi, Chiyoda-ku, Tokyo 102-8159, Japan.
Department of Physiology and Pharmacology, Schulich School of Medicine & Dentistry, and Bone and Joint Institute, The University of Western Ontario, London, Ontario, Canada.
Bone. 2016 Sep;90:37-49. doi: 10.1016/j.bone.2016.05.008. Epub 2016 May 27.
Equilibrative nucleoside transporter 1 (ENT1) mediates passage of adenosine across the plasma membrane. We reported previously that mice lacking ENT1 (ENT1(-/-)) exhibit progressive ectopic mineralization of spinal tissues resembling diffuse idiopathic skeletal hyperostosis (DISH) in humans. Here, we investigated mechanisms underlying aberrant mineralization in ENT1(-/-) mice. Micro-CT revealed ectopic mineralization of spinal tissues in both male and female ENT1(-/-) mice, involving the annulus fibrosus of the intervertebral discs (IVDs) of older mice. IVDs were isolated from wild-type and ENT1(-/-) mice at 2months of age (prior to disc mineralization), 4, and 6months of age (disc mineralization present) and processed for real-time PCR, cell isolation, or histology. Relative to the expression of ENTs in other tissues, ENT1 was the primary nucleoside transporter expressed in wild-type IVDs and mediated the functional uptake of [(3)H]2-chloroadenosine by annulus fibrosus cells. No differences in candidate gene expression were detected in IVDs from ENT1(-/-) and wild-type mice at 2 or 4months of age. However, at 6months of age, expression of genes that inhibit biomineralization Mgp, Enpp1, Ank, and Spp1 were reduced in IVDs from ENT1(-/-) mice. To assess whether changes detected in ENT1(-/-) mice were cell autonomous, annulus fibrosus cell cultures were established. Compared to wild-type cells, cells isolated from ENT1(-/-) IVDs at 2 or 6months of age demonstrated greater activity of alkaline phosphatase, a promoter of biomineralization. Cells from 2-month-old ENT1(-/-) mice also showed greater mineralization than wild-type. Interestingly, altered localization of alkaline phosphatase activity was detected in the inner annulus fibrosus of ENT1(-/-) mice in vivo. Alkaline phosphatase activity, together with the marked reduction in mineralization inhibitors, is consistent with the mineralization of IVDs seen in ENT1(-/-) mice at older ages. These findings establish that both cell-autonomous and systemic mechanisms contribute to ectopic mineralization in ENT1(-/-) mice.
平衡核苷转运体1（ENT1）介导腺苷穿过质膜。我们之前报道过，缺乏ENT1的小鼠（ENT1(-/-)）表现出脊髓组织的进行性异位矿化，类似于人类的弥漫性特发性骨肥厚（DISH）。在此，我们研究了ENT1(-/-)小鼠异常矿化的潜在机制。显微CT显示，雄性和雌性ENT1(-/-)小鼠的脊髓组织均有异位矿化，涉及老年小鼠椎间盘（IVD）的纤维环。在2月龄（椎间盘矿化之前）、4月龄和6月龄（存在椎间盘矿化）时，从野生型和ENT1(-/-)小鼠中分离出IVD，并进行实时PCR、细胞分离或组织学处理。相对于ENTs在其他组织中的表达，ENT1是野生型IVD中表达的主要核苷转运体，并介导纤维环细胞对[(3)H]2-氯腺苷的功能性摄取。在2或4月龄时，ENT1(-/-)和野生型小鼠的IVD中未检测到候选基因表达的差异。然而，在6月龄时，ENT1(-/-)小鼠IVD中抑制生物矿化的基因Mgp、Enpp1、Ank和Spp1的表达降低。为了评估在ENT1(-/-)小鼠中检测到的变化是否是细胞自主性的，建立了纤维环细胞培养物。与野生型细胞相比，在2或6月龄时从ENT1(-/-) IVD中分离的细胞表现出更高的碱性磷酸酶活性，碱性磷酸酶是生物矿化的促进剂。2月龄ENT1(-/-)小鼠的细胞也比野生型表现出更强的矿化能力。有趣的是，在ENT1(-/-)小鼠体内的纤维环内层检测到碱性磷酸酶活性的定位改变。碱性磷酸酶活性以及矿化抑制剂的显著减少，与老年ENT1(-/-)小鼠中观察到的IVD矿化一致。这些发现表明，细胞自主性和系统性机制都导致了ENT1(-/-)小鼠的异位矿化。
