The receptor activator of NF-B ligand (RANKL) induces osteoclast differentiation from bone marrow cells in the presence of macrophage colony-stimulating factor. We found that treatment of bone marrow cells with SB203580 inhibited osteoclast differentiation via inhibition of the RANKL-mediated signaling pathway. To elucidate the role of p38 mitogen-activated protein (MAP) kinase pathway in osteoclastogenesis, we employed RAW264 cells which could differentiate into osteoclastlike cells following treatment with RANKL. In a dose-dependent manner, SB203580 but not PD98059, inhibited RANKL-induced differentiation. Among three MAP kinase families tested, this inhibition profile coincided only with the activation of p38 MAP kinase. Expression in RAW264 cells of the dominant negative form of either p38␣ MAP kinase or MAP kinase kinase (MKK) 6 significantly inhibited RANKL-induced differentiation of the cells. These results indicate that activation of the p38 MAP kinase pathway plays an important role in RANKLinduced osteoclast differentiation of precursor bone marrow cells.Bone morphogenesis, remodeling, and resorption are controlled in part by osteoclasts. These cells differentiate from hematopoietic myeloid precursors of monocyte/macrophage lineage under control of osteotropic hormones and local factors produced by supporting cells such as osteoblasts and stromal cells (1-9).The receptor activator of NF-B ligand (RANKL) 1 (10), also refereed to as osteoclast differentiation factor (11), tumor necrosis factor-related activation-induced cytokine (12), or osteoprotegerin ligand (13), was shown to be highly expressed in supporting cells and to directly induce the differentiation and maturation of osteoclasts (7,(13)(14)(15). To describe RANKL-induced osteoclastogenesis, the sequential phenotype progression model was proposed. The model includes the appearance of mononuclear osteoclasts, the fusion process prior to multinucleated osteoclast formation, and the osteoclast maturation process (6). Moreover, it has been shown that mutant mice disrupted with either RANKL or its receptor RANK revealed severe osteopetrosis and osteoclast defects (16,17), indicating that the RANKL-RANK signaling system plays an essential role in osteoclast differentiation.It has been shown recently that RANK is associated with tumor necrosis factor receptor-associated factors (TRAFs) (18 -21). The intracellular domain of RANK contains two distinct TRAF-binding domains, each of which recognizes different TRAF proteins specifically (18,19). While the C-terminal region of RANK interacts with TRAF2 and TRAF5, the TRAF6-binding domain resides in the middle of the RANK intracellular region. Overexpression of RANK C-terminal deletion mutants has revealed that activation of the RANK-mediated signaling pathway results in the activation of NF-B and c-Jun N-terminal kinase (JNK) which correlate with the TRAF6 interaction activity of mutants (18,19). In addition, mice with disrupted TRAF6 gene exhibit an osteopetrotic phenotype due to a defect in bone resorptio...

The receptor activator of NF-B ligand (RANKL) induces various osteoclast-specific marker genes during osteoclast differentiation mediated by mitogen-activated protein (MAP) kinase cascades. However, the results of transcriptional programming of an osteoclastspecific cathepsin K gene are inconclusive. Here we report the regulatory mechanisms of RANKL-induced cathepsin K gene expression during osteoclastogenesis in a p38 MAP kinase-dependent manner. The reporter gene analysis with sequential 5-deletion constructs of the cathepsin K gene promoter indicates that limited sets of the transcription factors such as NFATc1, PU.1, and microphthalmia transcription factor indeed enhance synergistically the gene expression when overexpressed in RAW264 cells. In addition, the activation of p38 MAP kinase is required for the maximum enhancement of the gene expression. RANKL-induced NFATc1 forms a complex with PU.1 in nuclei of osteoclasts following the nuclear accumulation of NFATc1 phosphorylated by the activated p38 MAP kinase. These results suggest that the RANKL-induced cathepsin K gene expression is cooperatively regulated by the combination of the transcription factors and p38 MAP kinase in a gradual manner.

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Interferon regulatory factor-1 (IRF-1) acts as a transcriptional activator in the interferon system and as a tumor suppressor. The loss of functional IRF-1 has been observed in a signi®cant number of patients with myelodysplastic syndrome (MDS) and leukemia, suggesting a potentially critical role of IRF-1 in human oncostasis. Here we report an alternative mechanism by which IRF-1 may be inactivated. We puri®ed an IRF-1 association molecule which was revealed to be identical to a nuclear factor nucleophosmin (NPM)/B23/numatrin. Functional analysis showed that NPM inhibited the DNA-binding and transcriptional activity of IRF-1. Moreover, NPM was overexpressed in several clinical leukemia samples and human-derived leukemia cell lines. Finally, overexpression of NPM in NIH3T3 cells resulted in malignant transformation. These results suggest the possible involvement of NPM in inactivating IRF-1-dependent anti-oncogenic surveillance in human cancer development.