Effects of bone morphogenetic protein 2 on ossification of posterior longitudinal ligament and possible mechanism

Abstract

Objective To investigate the effect of bone morphogenetic protein 2(BMP2)on ossification of the posterior longitudinal ligament (OPLL) and its relationship with transforming growth factor-β (TGF-β)/Smad signaling pathway. Methods The expression vectors of wild type pcDNA3.1-BMP2 (WT), mutant pcDNA3.1-BMP2 (37G), mutant pcDNA3.1-BMP2 (190T) and mutant pcDNA3.1-BMP2 (37G/190T) were constructed and identified by agarose gel electrophoresis. The constructed vector was transfected into mouse embryonic fibroblasts C3H10T1/2 mediated by liposome to detect the expression of BMP2. Six groups were divided according to the transfection situation: (1) the non-transfection group; (2) empty vector pcDNA3.1 transfection group; (3) pcDNA3.1-bmp2 (WT) transfection group; (4) pcDNA3.1-bmp2 (37G) transfection group; (5) pcDNA3.1-bmp2 (190T) transfection group; (6) pcDNA3.1-bmp2 (37G/190T) transfection group. The experimental and control group were defined according to whether BMP2 polymorphism was included. Therefore, the non-transfection group and empty vector pcDNA3.1 transfection group were control groups, and the other groups were experimental groups. The expression of phosphorylated Smad1/5/8 and Smad4 in positive cell clones were detected by western blotting, and the alkaline phosphatase (ALP) was detected by quantitative detection kits. The protein expressions were compared among the experimental groups. Results Two fragments digested from pcDNA3.1-BMP2 represented 1.2 kb and 5.4 kb by agarose electrophoresis. The direct sequencing results were in accordance with target gene sequence. BMP2 gene was successfully transfected and stably expressed in C3H10T1/2 cells. Western blotting showed that the expression of phosphorylated Smad1/5/8 protein in the experimental groups was increased significantly after transfection, with significant difference between the experimental groups and the control groups (P 0.05). The expressions of Smad4 protein transfected by wild or mutation type pcDNA3.1-BMP2 were significantly higher than those in the control groups (P 0.05). However, there were significant differences between the two groups and other experimental groups (P<0.05). The Ser37Ala (T/G) polymorphism in exon 2 of BMP2 gene was positively correlated with ALP activity in stably transfected C3H10T1/2 cells. Conclusion The Ser37Ala (T/G) polymorphism in exon 2 of BMP2 gene promotes OPLL ossification through TGF-β/Smad signaling pathway, the possible mechanism for which is to up-regulate the protein expressions of Smad4 and ALP. Key words: Ossification, posterior longitudinal ligament; Bone morphogenetic protein 2; Signal transduction; Single nucleotide polymorphisms