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-B 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.Bone is a dynamic tissue that is under a constant state of remodeling or homeostasis. This remodeling process is a delicate balance between the activities of osteoblasts, the cells that deposit bone, and osteoclasts, the cell type that is responsible for bone resorption (1, 2). Interference with this delicate balance can result in very serious human pathologies that affect bone integrity, such as osteoporosis and osteopetrosis. Accordingly, the molecular signaling pathways that regulate osteoblast and osteoclast differentiation and function have come under intense scrutiny.Osteoclasts, the cells that resorb bone, are hematopoietically derived, multinucleated cells that arise from the monocyte/ macrophage lineage (3). It is now clear that osteoclast differentiation is dependent upon the intimate cellular interaction of myeloid preosteoclast precursors with either osteoblasts or stromal cells and is influenced by a wide range of local factors (4, 5). In fact, a wealth of data has indicated that a member of the tumor necrosis factor family of ligands known as receptor activator of nuclear factor-B (NF-B) 1 ligand (RANKL; also known...

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