P2Y1 Transient Overexpression Induced Mineralization in Spinal Ligament Cells Derived from Patients with Ossification of the Posterior Longitudinal Ligament of the Cervical Spine

Abstract

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.