title: The calcineurin/nuclear factor of activated T cells signaling pathway regulates osteoclastogenesis in RAW264.7 cells.
abstract: Although best known for its role in T lymphocyte activation, the calcineurin/nuclear factor of activated T cells (NFAT) signaling pathway is also known to be involved in a wide range of other biological responses in a variety of different cell types. Here we have investigated the role of the calcineurin/NFAT signaling pathway in the regulation of osteoclast differentiation. Osteoclasts are bone-resorbing multinucleated cells that are derived from the monocyte/macrophage cell lineage after stimulation with a member of the tumor necrosis factor family of ligands known as receptor activator of nuclear factor-kappaB ligand (RANKL). We now report that inhibition of calcineurin with either the immunosuppressant drugs cyclosporin A and FK506, or the retrovirally mediated ectopic expression of a specific calcineurin inhibitory peptide, all potently inhibit the RANKL-induced differentiation of the RAW264.7 monocyte/macrophage cell line into mature multinucleated osteoclasts. In addition, we find that NFAT family members are expressed in RAW264.7 cells and that their expression is up-regulated in response to RANKL stimulation. Most importantly, we find that ectopic expression of a constitutively active, calcineurin-independent NFATc1 mutant in RAW264.7 cells is sufficient to induce these cells to express an osteoclast-specific pattern of gene expression and differentiate into morphologically distinct, multinucleated osteoclasts capable of inducing the resorption of a physiological mineralized matrix substrate. Taken together, these data define calcineurin as an essential downstream effector of the RANKL-induced signal transduction pathway leading toward the induction of osteoclast differentiation and furthermore, indicate that the activation of the NFATc1 transcription factor is sufficient to initiate a genetic program that results in the specification of the mature functional osteoclast cell phenotype.
authors.affiliation: Department of Molecular Pharmacology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611, USA.
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Hirotani
authors.forename: Nathaniel A
authors.initials: NA
authors.lastname: Tuohy
authors.forename: Je-Tae
authors.initials: JT
authors.lastname: Woo
authors.forename: Paula H
authors.initials: PH
authors.lastname: Stern
authors.forename: Neil A
authors.initials: NA
authors.lastname: Clipstone
mesh_terms: D000818:Animals; D019703:Calcineurin / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D002460:Cell Line; D016572:Cyclosporine / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007166:Immunosuppressive Agents / Q000494:pharmacology; D008562:Membrane Glycoproteins / Q000378:metabolism; D009154:Mutation; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D010010:Osteoclasts / Q000166:cytology* / Q000378:metabolism*; D010641:Phenotype; D053245:RANK Ligand; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D013601:T-Lymphocytes / Q000378:metabolism*; D016559:Tacrolimus / Q000494:pharmacology; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
chemical_list: D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D007166:Immunosuppressive Agents; D008562:Membrane Glycoproteins; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D053245:RANK Ligand; D014157:Transcription Factors; D016572:Cyclosporine; D019703:Calcineurin; D016559:Tacrolimus