TNF-α treatment increases DKK1 protein levels in primary osteoblasts via upregulation of DKK1 mRNA levels and downregulation of miR-335-5p

Elucidation of the underlying mechanisms governing osteogenic differentiation is of significant importance to the improvement of therapeutics for bone-related inflammatory diseases. Tumor necrosis factor-α (TNF-α) is regarded as one of the major agents during osteogenic differentiation in an inflammatory environment. miR-335-5p post-transcriptionally downregulates the Dickkopf WNT signaling pathway inhibitor 1 (DKK1) protein level by specifically binding to the DKK1 3′UTR and activating Wnt signaling. The role of miR-335-5p in TNF-α-induced post-transcriptional regulation of DKK1 remains to be elucidated. In the present study, the mRNA and protein levels of DKK1 and the level of miR-335-5p were determined in MC3T3-E1 cells and the primary calvarial osteoblasts treated with or without TNF-α. The role of NF-κB signaling in TNF-α-induced post-transcriptional regulation of DKK1 was also evaluated. The present study determined that although TNF-α treatment exhibited cell-specific effects on DKK1 mRNA expression, the stimulation of TNF-α time- and concentration-dependently upregulated the protein levels of DKK1. In primary calvarial osteoblasts, the decreased miR-335-5p level induced by TNF-α-activated NF-κB signaling served an important role in mediating the post-transcriptional regulation of DKK1 by TNF-α treatment. In MC3T3-E1 cells, the post-transcriptional regulation of DKK1 by TNF-α treatment was more complicated and involved other molecular signaling pathways in addition to the NF-κB signaling. In conclusion, TNF-α treatment served an important role in the post-transcriptional regulation of DKK1 expression, which requires further investigation. The results of the present study not only provided new insights into the regulatory effects of miR-335-5p on osteogenic differentiation in an inflammatory microenvironment, but may also promote the development of potential therapeutic strategies for the treatment of bone-related inflammatory diseases.