Negative Regulation of Osteoclastogenesis by Ectodomain Shedding of Receptor Activator of NF-κB Ligand

Abstract

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...

Variations along the 24-hour cycle of circulating osteoprotegerin and soluble RANKL: a rhythmometric analysis

“…RANKL is produced as a 45 kDa membrane-associated protein, as a 31 kDa soluble protein by proteolytic cleavage, or as a 39.5 kDa soluble protein following expression of the hRANKL3 isoform [24]. In murine models, the membrane form is more active than the soluble ones [24][25][26]. sRANKL is present in blood circulation both in the free form and as a complex with OPG, but the sensitivity of available assays is still insufficient to detect the free form of sRANKL in a significant proportion of healthy individuals [3,4].…”

Global Characterization of Protein Secretion from Human Macrophages Following Non-canonical Caspase-4/5 Inflammasome Activation

“…metalloproteinases, matrix metalloproteinase 14 (MMP14) and a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10), which we identified from enriched EV fraction in response to LPS transfection (Table I). MMP14 and ADAM10 are known to convert transmembrane molecules into a soluble form through a proteolytic process called ectodomain shedding, and they also share common substrates (35,36). In addition, it has also been shown that a close relative and functionally similar protease to ADAM10, ADAM17 (37), can induce proteolytic processing and shedding of macrophage colony-stimulating factor (M-CSF) (38).…”

The effect of mechanical vibration on orthodontically induced root resorption

“…7,11,12 RANKL is expressed as a transmembrane protein in osteoblasts, whereas OPG is a protein secreted by the osteoblasts acting as a decoy receptor blocking the RANKL-RANK ligand interaction, thus antagonizing the formation of mature osteoclasts. 7,13,14 Our null hypothesis is that there is no difference between the root volumes resulting from the effect of different LFMVs. We sought to study the effects of different LFMVs on orthodontically induced RR and to understand the underlying cellular mechanism of LFMV.…”