title: TNF-alpha expression is transcriptionally regulated by RANK ligand.
abstract: Tumor necrosis factor (TNF)-alpha is known for its osteoclastogenic and resorptive activities. Induction of osteoclastogenesis by receptor activator of NF-kappaB ligand (RANKL) is accompanied by increased TNF-alpha expression. In the present study we investigated the mechanism by which RANKL induces expression of TNF-alpha 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-alpha 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-alpha and L32 mRNA levels were examined. TNF-alpha mRNA stability was not altered by RANKL. We next measured directly the transcription rate of TNF-alpha by a run-on assay and found that RANKL increases TNF-alpha transcription rate by 2.9-fold in RAW264.7 cells. We further characterized this transcriptional induction of TNF-alpha by RANKL. Gel shift assays using nuclear extracts derived from RANKL-treated RAW264.7 cells show increased specific NF-kappaB binding activity on the murine TNF-alpha promoter. Gliotoxin, known for its ability to inhibit NF-kappaB activation blocked RANKL-induced TNF-alpha expression. We finally used 1,260 bp of the murine TNF-alpha promoter fused to luciferase, as well as four mutants of this promoter carrying mutations in each of the four NF-kappaB 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-alpha expression via a transcriptional mechanism, depending on the NF-kappaB sites in the TNF promoter.
authors.affiliation: The H Hubert Humphrey Center for Experimental Medicine and Cancer Research, The Hebrew University Faculty of Medicine, Jerusalem, Israel.
authors.forename: W
authors.initials: W
authors.lastname: Zou
authors.forename: A
authors.initials: A
authors.lastname: Amcheslavsky
authors.forename: S
authors.initials: S
authors.lastname: Takeshita
authors.forename: H
authors.initials: H
authors.lastname: Drissi
authors.forename: Z
authors.initials: Z
authors.lastname: Bar-Shavit
mesh_terms: D000818:Animals; D001853:Bone Marrow; D002352:Carrier Proteins / Q000494:pharmacology / Q000502:physiology*; D002460:Cell Line; D005786:Gene Expression Regulation / Q000502:physiology*; D008264:Macrophages / Q000378:metabolism; D008297:Male; D008562:Membrane Glycoproteins / Q000494:pharmacology / Q000502:physiology*; D051379:Mice; D008807:Mice, Inbred BALB C; D016328:NF-kappa B / Q000378:metabolism; D010010:Osteoclasts / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000187:drug effects; D053245:RANK Ligand; D012333:RNA, Messenger / Q000378:metabolism; D053246:Receptor Activator of Nuclear Factor-kappa B; D013234:Stem Cells / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects / Q000502:physiology*; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics* / Q000378:metabolism*
chemical_list: D002352:Carrier Proteins; D008562:Membrane Glycoproteins; D016328:NF-kappa B; D053245:RANK Ligand; D012333:RNA, Messenger; D053246:Receptor Activator of Nuclear Factor-kappa B; C505687:Tnfrsf11a protein, mouse; C505689:Tnfsf11 protein, mouse; D014409:Tumor Necrosis Factor-alpha