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.

Osteoporosis is the most common metabolic disease of skeleton with reduced bone density and weaker bone. Qianggu Capsule as a traditional chinese medicine has been widely used to treat osteoporosis. The potential pharmacological mechanism of its active ingredient Gusuibu is not well understood. The purpose of this work is to analyze the anti-osteoporosis function of Gusuibu based on network pharmacology, and further explore the potential mechanism of Qianggu Capsule. The active compounds and their corresponding targets of Gusuibu were obtained from TCMSP, TCMID, and BATMAN-TCM databases. Potential therapeutic targets for osteoporosis were obtained through DisGeNET, TTD, GeneCards, MalaCards, CTD, and OMIM databases. The overlapping targets of Gusuibu and osteoporosis were obtained. GO and KEGG pathway enrichment analysis were performed. The “Gusuibu-active compounds-target genes-osteoporosis” network and protein-protein interaction (PPI) network were constructed, and the top hub genes were screened by using the plug-in CytoHubba. Molecular docking was used to verify the binding activity of hub genes and key compounds. We identified 21 active compounds and 140 potential therapeutic targets that may be related to Gusuibu and 10 hub genes (AKT1, IL6, JUN, TNF, MAPK3, VEGFA, EGFR, MAPK1, CASP3, PTGS2). Molecular docking analysis demonstrated that four key active small molecules in Gusuibu (including Luteolin, Naringenin, Kaempferol, and Beta-sitosterol) have excellent binding affinity to the target proteins encoded by the top 10 hub genes. Our new findings indicated that one key active compound kaempferol activated the expression of osteoblast specific transcription factor OSX through JNK kinase pathway.

Chemical modification of RNAs is important for posttranscriptional gene regulation. The METTL3-METTL14 complex generates most N ⁶ -methyladenosine (m ⁶ A) modifications in messenger RNAs (mRNAs), and dysregulated methyltransferase expression has been linked to cancers. Here we show that a changed sequence context for m ⁶ A can promote oncogenesis. A gain-of-function missense mutation from patients with cancer, METTL14 ^R298P , increases malignant cell growth in culture and transgenic mice without increasing global m ⁶ A levels in mRNAs. The mutant methyltransferase preferentially modifies noncanonical sites containing a GGAU motif, in vitro and in vivo. The m ⁶ A in GGAU context is detected by the YTH family of readers similarly to the canonical sites but is demethylated less efficiently by an eraser, ALKBH5. Combining the biochemical and structural data, we provide a model for how the cognate RNA sequences are selected for methylation by METTL3-METTL14. Our work highlights that sequence-specific m ⁶ A deposition is important and that increased GGAU methylation can promote oncogenesis.

Thoracic ossification of the ligamentum flavum (TOLF) is a heterotopic ossification of spinal ligaments. TOLF is the major cause of thoracic spinal canal stenosis and myelopathy, and its underlying mechanisms are not clear. Bone formation is a complex developmental process involving the differentiation of mesenchymal stem cells to osteoblasts, and regulated by BMP2, RUNX2, Osterix (OSX), etc. In this study, we continue to further characterize properties of TOLF. Our immunohistochemistry experiments showed that expressions of osteoblastic factors such as BMP2 and RUNX2 increased in TOLF. According to flow cytometry analysis the proportion of S phase of cell cycle in primary TOLF cells was 9% higher than the control. Alizarin red staining and ALP staining observations were consistent with immunohistochemistry results. It was also observed that inflammatory cytokine IL-6 level dramatically increased in the culture supernatant of primary TOLF cells. We propose the hypothesis that IL-6 is involved in TOLF. To testify the hypothesis, we examined the effect of IL-6. Our results showed that IL-6 was able to activate expressions of osteoblastic factors such as BMP2, RUNX2, OSX, OCN and ALP, and that expressions of cell proliferation factors cyclin D1 and cyclin C increased in the presence of IL-6. Moreover, IL-6-induced BMP2 expression was inhibited by p38 inhibitor SB203580, indicating that IL-6 regulated the osteogenic BMP2 activation through p38 MAPK pathway. These data suggest that IL-6 is involved in TOLF.