Abstract
Familial expansile osteolysis and related disorders are caused by heterozygous tandem duplication mutations in the signal peptide region of the gene encoding receptor activator of NF-κB (RANK), a receptor critical for osteoclast formation and function. Previous studies have shown that overexpression of these mutant proteins causes constitutive activation of NF-κB signaling in vitro, and it has been assumed that this accounts for the focal osteolytic lesions that are seen in vivo. We show here that constitutive activation of NF-κB occurred in HEK293 cells overexpressing wild-type or mutant RANK but not in stably transfected cell lines expressing low levels of each RANK gene. Importantly, only cells expressing wild-type RANK demonstrated ligand-dependent activation of NF-κB. When overexpressed, mutant RANK did not localize to the plasma membrane but localized to extensive areas of organized smooth endoplasmic reticulum, whereas, as expected, wild-type RANK was detected at the plasma membrane and in the Golgi apparatus. This intracellular accumulation of the mutant proteins is probably the result of lack of signal peptide cleavage because, using two in vitro translation systems, we demonstrate that the mutations in RANK prevent cleavage of the signal peptide. In conclusion, signal peptide mutations lead to accumulation of RANK in the endoplasmic reticulum and prevent direct activation by RANK ligand. These results strongly suggest that the increased osteoclast formation/activity caused by these mutations cannot be explained by studying the homozygous phenotype alone but requires further detailed investigation of the heterozygous expression of the mutant RANK proteins. © 2011 American Society for Bone and Mineral Research
Highlights
Osteoclasts are the multinucleated giant cells responsible for bone resorption and are derived from circulating hematopoietic cells of the macrophage-monocyte lineage.[1]. Differentiation of these precursor cells into osteoclasts is critically dependent on the cell surface receptor activator of NF-kB (RANK), a member of the tumor necrosis factor receptor superfamily.[2,3] Interaction of this 66kDa integral membrane protein with RANK ligand (RANKL), expressed on stromal cells, osteoblasts, and activated T cells, causes trimerization of the receptor and initiates a signaling cascade that results in the activation of NF-kB and other transcription factors, leading to osteoclastogenesis.[4,5,6,7]
wild-type RANK (WT-RANK) was identified as a band of approximately 70 kDa (Fig. 1), whereas the bands corresponding to familial expansile osteolysis (FEO), PDB, and expansile skeletal hyperphosphatasia (ESH)-RANK were slightly greater
The disorders are caused by insertion mutations in the signal peptide of RANK that cause amino acid duplications of 6 (FEO, from residue 21), 9, and 5 (ESH, from residue 21), respectively.[10,11] The exact mechanism by which the RANK mutations affect downstream signaling pathways remains unclear
Summary
Osteoclasts are the multinucleated giant cells responsible for bone resorption and are derived from circulating hematopoietic cells of the macrophage-monocyte lineage.[1] Differentiation of these precursor cells into osteoclasts is critically dependent on the cell surface receptor activator of NF-kB (RANK), a member of the tumor necrosis factor receptor superfamily (encoded by TNFRSF11A).(2,3) Interaction of this 66kDa integral membrane protein with RANK ligand (RANKL), expressed on stromal cells, osteoblasts, and activated T cells, causes trimerization of the receptor and initiates a signaling cascade that results in the activation of NF-kB and other transcription factors, leading to osteoclastogenesis.[4,5,6,7]. The aim of this study was to clarify how the signal peptide tandem duplication mutations in RANK affect posttranslational processing and subcellular localization of RANK protein and the downstream activation of NF-kB
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
