Department of Pharmacology, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki, Aomori 036-8562, Japan.
Calcif Tissue Int. 2011 Apr;88(4):263-71. doi: 10.1007/s00223-010-9456-y. Epub 2011 Jan 6.
Ossification of the posterior longitudinal ligament of the spine (OPLL) is characterized by ectopic bone formation in the spinal ligaments. We previously reported that P2 purinoceptor Y1 (P2Y1) expression is elevated in the spinal ligament cells of OPLL patients, but the role of P2Y1 in the spinal ligament calcification process is unknown. To verify the hypothesis that P2Y1 expression causes ossification of the spinal ligaments, we forced expression of P2Y1 in spinal ligament cells obtained from OPLL and non-OPLL patients using a cytomegaloviral vector. The expression of mRNA and protein was investigated by quantitative real-time polymerase chain reaction and immunofluorescence staining, respectively. After transfection, bone morphogenetic protein-2 (BMP-2) and Sox9 mRNA expression was significantly increased in spinal ligament cells derived from OPLL patients (4.36- and 6.44-fold, respectively) compared with cells from non-OPLL patients (0.57- and 3.64-fold, respectively) 2 days after P2Y1 transient transfection. Furthermore, a statistically significant correlation was observed between BMP-2 and P2Y1 mRNA expression levels in cells obtained from OPLL patients but not from non-OPLL patients. Immunofluorescence analysis showed that BMP-2 and P2Y1 expression was increased in OPLL patients only, while Sox9 expression was increased in OPLL and non-OPLL patients. MRS2279, a selective P2Y1 antagonist, blocked the upregulation of Sox9 and BMP-2 after forced expression of P2Y1. Furthermore, 4 days after transient transfection of P2Y1, mineralization was observed only in spinal ligament cells from OPLL patients. These results suggest that P2Y1 expression plays an important role in ectopic bone formation in the spinal ligaments of OPLL patients.
脊柱后纵韧带骨化症（OPLL）的特征是脊柱韧带中异位骨形成。我们之前报道过，P2 嘌呤受体 Y1（P2Y1）在 OPLL 患者的脊柱韧带细胞中表达升高，但 P2Y1 在脊柱韧带钙化过程中的作用尚不清楚。为了验证 P2Y1 表达导致脊柱韧带骨化的假说，我们使用巨细胞病毒载体在来自 OPLL 和非 OPLL 患者的脊柱韧带细胞中强制表达 P2Y1。通过定量实时聚合酶链反应和免疫荧光染色分别研究 mRNA 和蛋白质的表达。转染后，来自 OPLL 患者的脊柱韧带细胞中骨形态发生蛋白 2（BMP-2）和 Sox9 mRNA 的表达分别显著增加（分别为 4.36 倍和 6.44 倍）与非 OPLL 患者的细胞（分别为 0.57 倍和 3.64 倍）相比，在 P2Y1 瞬时转染后 2 天。此外，在来自 OPLL 患者的细胞中观察到 BMP-2 和 P2Y1 mRNA 表达水平之间存在统计学显著相关性，但在来自非 OPLL 患者的细胞中没有观察到这种相关性。免疫荧光分析显示，仅在 OPLL 患者中观察到 BMP-2 和 P2Y1 表达增加，而 Sox9 表达在 OPLL 和非 OPLL 患者中均增加。MRS2279 是一种选择性 P2Y1 拮抗剂，可阻断 P2Y1 强制表达后 Sox9 和 BMP-2 的上调。此外，在 P2Y1 瞬时转染 4 天后，仅在来自 OPLL 患者的脊柱韧带细胞中观察到矿化。这些结果表明，P2Y1 表达在 OPLL 患者脊柱韧带中的异位骨形成中起重要作用。
