Receptor Activator of NF-κB (RANK) Ligand Induces Ectodomain Shedding of RANK in Murine RAW264.7 Macrophages

Abstract

Osteoclastogenesis is a highly sophisticated process that involves a variety of membrane-bound proteins expressed in osteoblasts and osteoclast precursors. Over the past several years, proteolytic cleavage and release of the ectodomain of membrane-bound proteins, also referred to as ectodomain shedding, has emerged as an important posttranslational regulatory mechanism for modifying the function of cell surface proteins. In line with this notion, several membrane-bound molecules involved in osteoclastogenesis, including CSF-1R and receptor activator of NF-κB ligand (RANKL), are proteolytically cleaved and released from the cell surface. In this study, we investigated whether receptor activator of NF-κB (RANK), one of the most essential molecules in osteoclastogenesis, undergoes ectodomain shedding. The results showed that RANK is released in the form of a soluble monomeric protein and that TNF-α–converting enzyme is involved in this activity. We also identified potential cleavage sites in the juxtamembrane domain of RANK and found that rRANKL induces RANK shedding in a macrophage-like cell line RAW264.7 via TNFR-associated factor 6 and MAPK pathways. Furthermore, we found that RANKL-induced osteoclastogenesis is accelerated in TNF-α–converting enzyme-deficient osteoclast precursors. These observations suggest the potential involvement of ectodomain shedding in the regulation of RANK functions and may provide novel insights into the mechanisms of osteoclastogenesis.

Conditional Inactivation of TNFα-Converting Enzyme in Chondrocytes Results in an Elongated Growth Plate and Shorter Long Bones

“…On the contrary, we found no skeletal defects in Egfr flox/flox /LysM-cre mice (Fig. 4F) or in Tace flox/flox /LysM-cre mice [17], [30] (data not shown), suggesting that the EGFR ligands released from cartilage cells in the growth plate act in an autocrine or paracrine manner and not as a chemoattractant for osteoclasts and their precursors. Furthermore, X-ray analysis revealed that the long bones of mutant mice that specifically lack EGFR in the chondrocytes are shorter than those of their wild-type littermates at 8 weeks of age (Fig.…”

New knowledge on critical osteoclast formation and activation pathways from study of rare genetic diseases of osteoclasts: focus on the RANK/RANKL axis

“…TNFα converting enzyme (TACE; ADAM-17) is one such enzyme and generates the soluble forms of TNFα as well as RANKL [59,80]. Inhibition of TACE activity upregulates surface RANK expression on monocytes [81], and, most recently, RANKL was shown to upregulate TACE-mediated shedding of the RANK ectodomain in a TRAF6-dependent manner indicating a negative feedback mechanism to regulate RANK surface expression [82]. Therefore, these observations strongly suggest that activation or inhibition of TACE could indeed play a significant role in the regulation of RANK signalling.…”

“…Therefore, the patient phenotype seen as a result of harbouring such truncating mutations is likely the combined functional effect of the expression of the truncated protein together with that of reduced levels of RANK expression. The effect of the mutation A244S in the cytoplasmic domain of RANK is not clear as deletion of this region does not affect osteoclast formation [82], and the patient carrying this mutation was a compound heterozygote carrying C175R on the other allele complicating the functional analysis.…”

ADAM17 Deletion in Thymic Epithelial Cells Alters Aire Expression without Affecting T Cell Developmental Progression

“…ADAM17 has recently been shown to cleave the RANK receptor in macrophages [51]. RANK signaling is a key signaling pathway in the development of mature medullary Aire-expressing TECs at the fetal stage [52], [53], [54].…”