Receptor activator of NF-B ligand (RANKL) is a transmembrane glycoprotein that has an essential role in the development of osteoclasts. The extracellular portion of RANKL is cleaved proteolytically to produce soluble RANKL, but definite RANKL sheddase(s) and the physiologic function of RANKL shedding have not yet been determined. In the present study, we found that matrix metalloproteinase (MMP) 14 and a disintegrin and metalloproteinase (ADAM) 10 have strong RANKL shedding activity. In Western blot analysis, soluble RANKL was detected as two different molecular weight products, and RNA interference of MMP14 and ADAM10 resulted in a reduction of both the lower and higher molecular weight products. Suppression of MMP14 in primary osteoblasts increased membrane-bound RANKL and promoted osteoclastogenesis in cocultures with macrophages. Soluble RANKL produced by osteoblasts from MMP14-deficient mice was markedly reduced, and their osteoclastogenic activity was promoted, consistent with the findings of increased osteoclastogenesis in vivo. RANKL shedding is an important process that down-regulates local osteoclastogenesis.Receptor activator of NF-B ligand (RANKL), 2 also known as TNF-related activation-induced cytokine, osteoprotegerin ligand, and osteoclast differentiation factor, is a type II transmembrane glycoprotein of the TNF ligand family. RANKL is expressed on the plasma membrane of osteoblasts, bone marrow stromal cells, and T-lymphocytes (1-5) and exerts its activity through binding to its TNF family receptor RANK, which is expressed on monocyte-macrophage lineage osteoclast precursors (3, 4, 6). Consequently, RANK activates downstream signaling pathways such as NF-B, p38 mitogen-activated protein kinase, c-Jun N-terminal kinase, and nuclear factor of activated T cells c1 and leads to the differentiation, activation, and survival of osteoclasts (6 -9). Both RANKL-and RANK-deficient mice develop severe osteopetrosis due to a lack of osteoclasts (10, 11). Conversely, a deficiency of osteoprotegerin (OPG), a natural inhibitor of RANKL, causes severe osteoporosis due to enhanced osteoclastogenesis (12). These findings suggest a crucial role for the RANKL/RANK/OPG axis in osteoclast development.A number of transmembrane proteins undergo proteolysis and are released from the plasma membrane, a process called ectodomain shedding. The biologic and pathologic significance of ectodomain shedding varies between substrate proteins. For example, cytokines such as TNF-␣ and epidermal growth factor are released from local environments and exert their activity in paracrine and endocrine signaling by ectodomain shedding (13-15). In some cases, ectodomain shedding is necessary even for the local effects of growth factors such as epidermal growth factor (16). Although membrane-bound RANKL is converted to a soluble form through ectodomain shedding (17, 18) similar to other TNF family members, such as TNF-␣ (13, 14) and Fas ligand (19), the RANKL sheddases involved in physiologic and pathologic bone resorption, and the role of...

The role of Rac1 in osteoclast survival and bone-resorbing activity was examined using adenovirus vector expression systems. Rac1 is critically involved in M-CSF receptor signaling and mediates survival signaling primarily through PI3K/Akt pathways. Rac1 also plays a significant role in bone resorptive activity, probably by regulating the motility of osteoclasts.Introduction: Rac1 is a member of Rho family small G-proteins, and recent studies have revealed that it mediates anti-apoptotic signals in some types of cells. Rac1 is reported to be required for the cytoskeletal organization and bone-resorbing activity of osteoclasts, but their roles in osteoclast survival and function are not fully elucidated. Materials and Methods:We constructed the adenovirus vector carrying cDNA of either the dominant negative Rac1 (Rac1

The life span of osteoclasts is critically regulated by various cytokines, and therapeutics such as bisphosphonates act directly on osteoclasts and induce apoptosis of the cells. This article will focus on the molecular mechanism of osteoclast apoptosis and summarize the recent advances in this field with an emphasis on the role of intracellular signaling pathways.

Proapoptotic Bcl-2 family member Bim plays an essential role in the osteoclast apoptosis and is degraded through ubiquitin/proteasome pathways in a caspase-3-dependent manner. This negative feedback loop in the Bim-caspase-3 axis is important for regulating the survival and activity of osteoclasts.Introduction: Bim is a member of the proapoptotic Bcl-2 family and regulates the mitochondrial apoptosis pathway. Bim expression is post-translationally regulated in osteoclasts (OCs) through ubiquitin/proteasome pathways, and Bim is critical for their survival and activity. Materials and Methods: Time-course of change in the expression of Bim in the course of OC apoptosis was examined, and the effect of various proteinase inhibitors on the degradation of Bim was analyzed. The role of caspase-3 and caspase-7 on Bim degradation was studied using RNA interference technique and caspase-3

During endochondral ossification, chondrocytes undergo hypertrophic differentiation and die by apoptosis. The level of inorganic phosphate (P i ) elevates at the site of cartilage mineralization, and when chondrocytes were treated with P i , they underwent rapid apoptosis. Gene silencing of the proapoptotic Bcl-2 homology 3-only molecule bnip3 significantly suppressed P i -induced apoptosis. Conversely, overexpression of Bcl-xL suppressed, and its knockdown promoted, the apoptosis of chondrocytes. Bnip3 was associated with Bcl-xL in chondrocytes stimulated with P i . Bcl-xL was expressed uniformly in the growth plate chondrocytes, whereas Bnip3 expression was exclusively localized in the hypertrophic chondrocytes. Finally, we generated chondrocyte-specific bcl-x knock-out mice using the Cre-loxP recombination system, and we provided evidence that the hypertrophic chondrocyte layer was shortened in those mice because of an increased apoptosis of prehypertrophic and hypertrophic chondrocytes, with the mice afflicted with dwarfism as a result. These results suggest the pivotal role of Bcl-2 family members in the regulation of chondrocyte apoptosis.Endochondral ossification is an essential process for skeletal development, fracture healing, and pathologic conditions such as osteoarthritis and ectopic ossification (1). In this process, chondrocytes first proliferate and then differentiate into mature hypertrophic chondrocytes, which mineralize the surrounding matrix that is finally replaced by bone (1). There is controversy as to the cell fate of hypertrophic chondrocytes, and several studies have shown that they undergo apoptosis after terminal differentiation (2-4). Apoptosis is a form of programmed cell death that is characterized by specific morphological and biochemical features, and is tightly regulated by extracellular stimuli and intracellular signaling pathways (5). Morphologically, apoptosis is characterized by a series of structural changes in dying cells as follows: blebbing of the plasma membrane, condensation of the cytoplasm and the nucleus, and cellular fragmentation into membrane apoptotic bodies. Biochemically, apoptosis is characterized by the degradation of chromatin, initially into large fragments of 50 -300 kb and subsequently into smaller fragments that are monomers and multimers of 200 bases. Not only does apoptosis regulate various aspects of the biological activity, but it also can trigger cancer, autoimmune diseases, and degenerative disorders (6). Several molecules such as Sox5, -6, and -9 and Runx2 have been reported to regulate proliferation and hypertrophic differentiation of chondrocytes (7-9). However, the physiologic and pathologic significance of chondrocyte apoptosis and key molecules that regulate this process remain to be elucidated. Previous studies have shown the possible involvement of cytokines, such as tumor necrosis factor-␣ and Fas ligand, and hormones, such as glucocorticoids and parathyroid hormonerelated peptide (PTHrP), 2 in the localized hypoxia and increased generati...