TNF‐α expression is transcriptionally regulated by RANK ligand

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Abstract
Tumor necrosis factor (TNF)-α is known for its osteoclastogenic and resorptive activities. Induction of osteoclastogenesis by receptor activator of NF-κB ligand (RANKL) is accompanied by increased TNF-α expression. In the present study we investigated the mechanism by which RANKL induces expression of TNF-α in osteoclast precursors. The macrophage-like cell-line, RAW 264.7 was used as a model for osteoclast precursors. To examine if RANKL-mediated increase in TNF-α expression involves increased stability of its transcript, RAW264.7 cells were treated with or without RANKL, and then a transcription inhibitor was added. At different time points, TNF-α and L32 mRNA levels were examined. TNF-α mRNA stability was not altered by RANKL. We next measured directly the transcription rate of TNF-α by a run-on assay and found that RANKL increases TNF-α transcription rate by 2.9-fold in RAW264.7 cells. We further characterized this transcriptional induction of TNF-α by RANKL. Gel shift assays using nuclear extracts derived from RANKL-treated RAW264.7 cells show increased specific NF-κB binding activity on the murine TNF-α promoter. Gliotoxin, known for its ability to inhibit NF-κB activation blocked RANKL-induced TNF-α expression. We finally used 1,260 bp of the murine TNF-α promoter fused to luciferase, as well as four mutants of this promoter carrying mutations in each of the four NF-κB sites to stably transfect RAW 264.7 cells. Reporter activity was increased in response to RANKL in wild type promoter transfected cells, whereas treatment of the mutants' transfected cells did not elicit reporter activity. In conclusion, RANKL induces TNF-α expression via a transcriptional mechanism, depending on the NF-κB sites in the TNF promoter.