Wang Hao, Jin Weitao, Li Haibin
Department of Orthopaedics, Beijing Tiantan Hospital, Capital Medical University, 6 TiantanXili, Dongcheng District, Beijing, 100050, China.
Department of Epidemiology and Biostatistics, School of Public Health, Capital Medical University, Beijing, 100069, China.
BMC Musculoskelet Disord. 2018 Feb 20;19(1):61. doi: 10.1186/s12891-018-1966-1.
The present study investigated the molecular mechanisms underlying the 4A > C and -349C > T single nucleotide polymorphisms (SNPs) in bone morphogenetic protein receptor type IA (BMPR-IA) gene, which significantly associated with the occurrence and the extent of ossification of the posterior longitudinal ligament (OPLL) in the cervical spine.
The SNPs in BMPR-IA gene were genotyped, and the association with the occurrence and severity of OPLL were evaluated in 356 OPLL patients and 617 non-OPLL controls. In stably transfected mouse embryonic mesenchymal stem cells (C3H10T1/2), the expression levels of the BMPR-IA gene and Smad4 protein as well as phosphorylated Smad1/5/8 were detected by Western blotting. In addition, the alkaline phosphatase (ALP) and osteocalcin (OC) activity of osteogenesis specificity protein was assessed using the ALP quantitation and osteocalcin radioimmunoassay kit, respectively.
The 4A > C and the -349C > T polymorphisms of BMPR-IA gene were significantly associated with the development of OPLL in the cervical spine. The C allele type in 4A > C polymorphism significantly increases the occurrence and the extent of OPLL. The T allele type in -349C > T polymorphism significantly increases the susceptibility to OPLL, but not the extent of OPLL. The current results further validate our previous observations. The expression levels of BMPR-IA gene were significantly increased in pcDNA3.1/BMPR-IA (mutation type, MT -349C > T; MT 4A > C; MT -349C > T and 4A > C) vector-transfected C3H10T1/2 cells compared to the wild type (WT) vector-transfected cells. The levels of phosphorylated Smad1/5/8 and ALP activity were significantly increased in pcDNA3.1/BMPR-IA (MT -349C > T) vector-transfected C3H10T1/2 cells compared to the WT vector-transfected cells. However, no significant differences were observed in the protein levels of phosphorylated Smad1/5/8 and the ALP activity between MT A/C and WT vector-transfected cells. In addition, no significant differences were observed in the Smad4 protein levels among the experimental groups, as well as in the OC activity between WT vector-transfected and MT C/T, MT A/C, MT C/T and MT A/C vector-transfected cells.
Our results suggest that Smad signaling pathway may play important roles in the pathological process of OPLL induced by SNPs in BMPR-IA gene. These results will help to clarify the molecular mechanisms underlying the SNP and gene susceptibility to OPLL.
本研究调查了骨形态发生蛋白受体IA型（BMPR-IA）基因中4A＞C和-349C＞T单核苷酸多态性（SNP）的分子机制，这些多态性与颈椎后纵韧带骨化（OPLL）的发生及程度显著相关。
对BMPR-IA基因的SNP进行基因分型，并在356例OPLL患者和617例非OPLL对照中评估其与OPLL发生及严重程度的相关性。在稳定转染的小鼠胚胎间充质干细胞（C3H10T1/2）中，通过蛋白质免疫印迹法检测BMPR-IA基因、Smad4蛋白以及磷酸化Smad1/5/8的表达水平。此外，分别使用碱性磷酸酶定量试剂盒和骨钙素放射免疫分析试剂盒评估成骨特异性蛋白的碱性磷酸酶（ALP）和骨钙素（OC）活性。
BMPR-IA基因的4A＞C和-349C＞T多态性与颈椎OPLL的发生显著相关。4A＞C多态性中的C等位基因类型显著增加了OPLL的发生及程度。-349C＞T多态性中的T等位基因类型显著增加了OPLL的易感性，但未增加OPLL的程度。目前的结果进一步验证了我们之前的观察结果。与野生型（WT）载体转染的细胞相比，pcDNA3.1/BMPR-IA（突变型，MT -349C＞T；MT 4A＞C；MT -349C＞T和4A＞C）载体转染的C3H10T1/2细胞中BMPR-IA基因的表达水平显著升高。与WT载体转染的细胞相比，pcDNA3.1/BMPR-IA（MT -349C＞T）载体转染的C3H10T1/2细胞中磷酸化Smad1/5/8的水平和ALP活性显著增加。然而，MT A/C和WT载体转染的细胞之间，磷酸化Smad1/5/8的蛋白水平和ALP活性未观察到显著差异。此外，各实验组之间Smad4蛋白水平以及WT载体转染与MT C/T、MT A/C、MT C/T和MT A/C载体转染的细胞之间OC活性均未观察到显著差异。
我们的结果表明，Smad信号通路可能在BMPR-IA基因SNP诱导的OPLL病理过程中发挥重要作用。这些结果将有助于阐明SNP和基因对OPLL易感性的分子机制。
