Uniaxial Cyclic Stretch Promotes Osteogenic Differentiation and Synthesis of BMP2 in the C3H10T1/2 Cells with BMP2 Gene Variant of rs2273073 (T/G)

Abstract

Ossification of the posterior longitudinal ligament of the cervical spine (OPLL) is characterized by the replacement of ligament tissues with ectopic bone formation, and this result is strongly affected by genetic and local factors. Two single nucleotide polymorphisms (SNPs) of rs2273073 (T/G) and rs235768 (A/T) of bone morphogenetic protein 2 (BMP2) gene which are associated with OPLL have been reported in our previous report. In this study, we confirmed the connection in 18 case samples analysis of BMP2 gene in OPLL patients; additionally, it was also shown from the OPLL patients with ligament tissues that enchondral ossification and expression of BMP2 were significantly higher compared with the non-OPLL patients by histological examination, immunohistochemistry and Western blotting analysis. To investigate the underlying mechanism, we studied the effect of SNPs in cell model. The C3H10T1/2 cells with different BMP2 gene variants were constructed and then subjected to uniaxial cyclic stretch (0.5 Hz, 10% stretch). In the presence of mechanical stress, the expression of BMP2 protein in C3H10T1/2 cells transfected by BMP2 (rs2273073 (T/G)) and BMP2 (rs2273073 (T/G), rs235768 (A/T)) were significantly higher than the corresponding static groups (P<0.05). In conclusion, these results suggested that BMP2 gene variant of rs2273073 (T/G) could not only increase cell susceptibility to bone transformation similar to pre-OPLL change, but also increase the sensibility to mechanical stress which might play an important role during the progression of OPLL.

Targeted next-generation sequencing reveals multiple deleterious variants in OPLL-associated genes

“…Likewise, variants in BMP-2 has been found to be involved in different stages of development of ectopic ossification 27 , such as Ser37Ala (T/G) (i.e., rs2273073, a relatively common SNP with MAF 0.03786 in ExAc) and Ser87Ser (A/G) (i.e., rs1049007, a common SNP with MAF 0.6723 in ExAc) 8 . As shown in a recent study, a forced expression of BMP2 containing the rs2273073 in C3H10T1/2 cells (mouse embryonic fibroblasts) was found to increase cell susceptibility to bone transformation similar to pre-OPLL change as well as the sensibility to mechanical stress which might play an important role during the progression of OPLL 28 . Therefore, what we observed an increased level of BMP2 with p.Q89E in the patient’s cells revealed its potential role in the pathogenicity of OPLL.…”

“…As shown in a recent study, a forced expression of BMP2 containing the rs2273073 in C3H10T1/2 cells (mouse embryonic fibroblasts) was found to increase cell susceptibility to bone transformation similar to pre-OPLL change as well as the sensibility to mechanical stress which might play an important role during the progression of OPLL28. Therefore, what we observed an increased level of BMP2 with p.Q89E in the patient’s cells revealed its potential role in the pathogenicity of OPLL.…”

Role of Bone Morphogenetic Protein 2 Polymorphisms in Bone Mineral Density after the Start of Treatment with Atorvastatin

“…Besides, both intronic polymorphisms involve changes in the recognition sites for transcription factors. In addition, these polymorphisms were previously reported in association studies of alleles related to bone disease . The BMP‐2 gene localization is 20p12.3.…”

The Pathogenesis of Ossification of the Posterior Longitudinal Ligament

“…Several reports have showed that frequencies for the rs2273073 (T/G) and rs235768 (A/T) polymorphisms of BMP2 are significant higher in the OPLL than those in control groups. Both SNPs are associated with the occurrence of OPLL in Chinese Han population [ 52 - 55 ]. The functional analysis of rs2273073 (T/G) SNP has showed this mutant aberrantly activates BMP-Smad signaling.…”

“…For example, transfection of this BMP2 mutant promotes the activation of p-Smad1/5/8, expression of Smad4 and activity of alkaline phosphatase (ALP) [ 55 ]. Uniaxial cyclic stretch promotes osteogenic differentiation and synthesis of BMP2 in the C3H10T1/2 Cells with BMP2 gene variant of rs2273073 (T/G) [ 52 ]. However, Kim et al reported that both BMP2 SNPs showed no significant difference between OPLL and non-OPLL groups in Korean populations [ 35 ].…”