Involvement of tumor necrosis factor alpha in steroid-associated osteonecrosis of the femoral head: friend or foe?

BackgroundThe etiology and pathology osteonecrosis of the femoral head (ONFH) are not completely clarified. As a cytokine participating in systemic inflammation, tumor necrosis factor alpha (TNF) has been shown to be involved in the pathogenesis of ONFH. However, the role of TNF in ONFH is not clearly clarified. In the present study, we investigated the effects of TNF on proliferation, angiogenesis, and osteogenic differentiation of rat bone mesenchymal stem cells (rMSCs) and the underlying mechanisms.MethodsAll femoral bone tissues were separated in surgeries. After extracting total RNA and protein, we evaluated TNF content by ELISA and the relative expression levels of genes by quantitative real-time PCR and western blot. Also, immunohistochemistry staining was performed to observe the expression of Runx2 in the bone samples. Chick embryo chorioallantoic membrane (CAM) assay was performed to observe the effect of TNF on angiogenesis. The genomic DNAs were treated by bisulfite modification, and methylation status of CpG sites in the CpG islands of human and rat Runx2 gene promoter was determined by DNA sequencing. The binding of H3K4me3 and H3K27me3 in Runx2 promoter was checked by ChIP assay. RNA-seq analysis was used to find out the genes and pathways changed by TNF in rMSCs.ResultsThe results demonstrate TNF promotes cell proliferation and angiogenesis whereas inhibits osteogenesis. Epigenetic regulations including DNA methylation and histone modifications play important roles in mediating the effect of TNF on osteogenic differentiation. We find an increased rate of CpG methylation in rat Runx2 promoter in TNF-treated rMSCs, as well as significantly increased occupancy of H3K27me3 in Runx2 gene promoter. The content of TNF in necrotic tissue is much lower than that of normal tissue. And relevantly, human Runx2 promoter is demethylated in necrotic tissue using bone samples from patient with ONFH. In addition, we have observed that Wnt signaling pathway is inhibited by TNF as multiple Wnts are markedly decreased in TNF-treated rMSCs by RNA-seq analysis.ConclusionTaken together, our study shows that TNF plays complicated roles in the pathogenesis of ONFH, including proliferation, angiogenesis, and osteogenesis. Targeting TNF should not be considered as an applicable strategy to inhibit the progression of ONFH.