Tumor necrosis factor-alpha (TNF-alpha) is a multifunctional cytokine that mediates inflammation and induces bone loss caused by excessive bone resorption by osteoclasts. The interaction of TNF-alpha with its receptor activates several signal transduction pathways, including those of mitogen-activated protein (MAP) kinases (p38, JNK, and ERK) and NF-kappa B. Signaling from these molecules has been shown to play an important role in osteoclastogenesis. In the present study, we investigated the mechanism of TNF-alpha-induced osteoclast differentiation in human peripheral blood mononuclear cells (PBMCs). We found that TNF-alpha alone greatly induced differentiation of PBMCs into osteoclasts. The osteoclast differentiation induced by TNF-alpha was independent of RANKL binding to its receptor RANK on PBMCs. Furthermore, TNF-alpha potently activated p38 MAPK, JNK, and NF-kappa B. Western blotting analysis revealed that p21(WAF1/Cip1), a cyclin-dependent kinase (CDK) inhibitor, is significantly induced upon TNF-alpha stimulation. The induction of p21(WAF1/Cip1) during differentiation is responsible for arrest at G(0)/G(1) phase and associated with the JNK pathway. These results suggest that TNF-alpha regulates osteoclast differentiation through p21(WAF1/Cip1) expression and further shows that these events require JNK activity. (c) 2005 Elsevier Inc. All rights reserved.