Abstract
Thoracic ossification of the ligamentum flavum (TOLF) is characterized by ectopic bone formation in the ligamentum flavum and is considered to be a leading cause of thoracic spinal canal stenosis and myelopathy. However, the underlying etiology is not well understood. An iTRAQ proteomics was used to reveal the involvement of inflammation factors in TOLF. TNF-α is a pro-inflammatory cytokine implicated in the pathogenesis of many human diseases. Protein profiling analysis showed that the protein level of TNF-α increased in the ossified ligamentum flavum of TOLF, which was confirmed by western blot. The effects of TNF-α on primary ligamentum flavum cells was examined. Cell proliferation assay demonstrated that primary cells from the ossified ligamentum flavum of TOLF grew faster than the control. Flow cytometry assay indicated that the proportions of cells in S phase of cell cycle of primary cells increased after TNF-α stimulation. To address the effect of TNF-α on gene expression, primary cells were derived from ligamentum flavum of TOLF patients. Culture cells were stimulated by TNF-α. RNA was isolated and analyzed by quantitative RT-PCR. G1/S-specific proteins cyclin D1 and c-Myc were upregulated after TNF-α stimulation. On the other hand, osteoblast differentiation related genes such as Bmp2 and Osterix (Osx) were upregulated in the presence of TNF-α. TNF-α activated Osx expression in a dose-dependent manner. Interestingly, a specific mitogen-activated protein kinase ERK inhibitor U0126, but not JNK kinase inhibitor SP600125, abrogated TNF-α activation of Osx expression. This suggests that TNF-α activates Osx expression through the mitogen-activated protein kinase ERK pathway. Taken together, we provide the evidence to support that TNF-α involves in TOLF probably through regulating cell proliferation via cyclin D1 and c-Myc, and promoting osteoblast differentiation via Osx.
Highlights
Ossification of ligamentum flavum (OLF) of the spine is characterized by ectopic bone formation in the ligamentum flavum, and it is more likely to affect Asian population, including the Chinese, Japanese and Korean [1,2,3,4,5]
Our preliminary data showed that the serum Tumor necrosis factor (TNF)-α of patients with Thoracic ossification of the ligamentum flavum (TOLF) was significantly higher compared with the control; Secondly, our preliminary results of gene Microarray indicated that the gene expression level of TNF-α increased in the ossified ligamentum flavum in TOLF, supporting our proteomic observation in this study; Thirdly, previously reports have suggested an important role of TNF-α in fracture healing in transgenic animal model [25] or a promoting effect of TNF-α on osteogenic differentiation from mesenchymal stem cells [31]
Protein was purified from the cell lysates of primary cells of the ossified ligamentum flavum, and TNF-α protein expression was detected by western blot
Summary
Ossification of ligamentum flavum (OLF) of the spine is characterized by ectopic bone formation in the ligamentum flavum, and it is more likely to affect Asian population, including the Chinese, Japanese and Korean [1,2,3,4,5]. It has been reported that OLF mostly occurs in the lower thoracic spine [4]. Thoracic ossification of the ligamentum flavum (TOLF) has been considered to be a leading cause of thoracic spinal stenosis and myelopathy [6]. One paper indicates that over 70% of patients suffering from thoracic spinal stenosis have been diagnosed with TOLF [7]. It has been suggested that some related factors are associated with TOLF, such as genetic factors, basic metabolic elements, and mechanical effects [8,9]. The underlying etiology of TOLF is not well understood
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