Signal peptide mutations in RANK prevent downstream activation of NF-κB

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

Juvenile Paget's disease with heterozygous duplication within TNFRSF11A encoding RANK

“…(5, 36, 37) In 2000, Hughes et al (5) reported that diminished cleavage of the signal peptide from FEO-RANK (84dup18) and PDB2-RANK (75dup27) led to intracellular sequestration of the mutated receptor and enhanced NF-κB signaling. In 2011, Crockett et al (36, 37) confirmed that FEO-RANK (84dup18), PDB2-RANK (75dup27), and ESH-RANK (84dup15) resist signal peptide cleavage, showed that these altered receptors localized differently within the organized smooth endoplasmic reticulum (perhaps somehow explaining the distinctive clinical phenotypes), and were unresponsive to extracellular RANKL. Additionally, they found that overexpression of these mutated RANKs would activate NF-κB, whereas physiologic levels did not enhance down-stream RANK signaling, including NF-κB.…”

“…Additionally, they found that overexpression of these mutated RANKs would activate NF-κB, whereas physiologic levels did not enhance down-stream RANK signaling, including NF-κB. (36, 37) In fact, these altered RANKs, when homozygous, inactivated such signaling. Crockett et al (37) offered that in FEO, PDB2, and ESH perhaps mixed trimers of mutant and wild type RANK interacted somehow to enhance RANK activity, although the mechanism for the differing phenotypes remained unknown.…”

Pathogenesis of Paget Disease of Bone

“…Subsequently, a duplication of 15 bp in the signal peptide region of RANK was found to be the cause of expansile skeletal hyperphosphatasia, another disease with clinical features that overlap to an extent with those of FEO and early-onset familial PDB [85,86]. These mutations prevent normal cleavage of the RANK signal peptide [83], and this causes the mutated RANK molecules to accumulate in the Golgi apparatus [87]. Although the mutated RANK molecules activate NF-jB signaling when overexpressed [83], cells that have been engineered to express a single copy of the mutant RANK show no evidence of a constitutive increase in NF-jB signaling, and they fail to initiate NF-jB or ERK responses to RANKL stimulation, indicating that these are loss-of-function mutations.…”

“…Although the mutated RANK molecules activate NF-jB signaling when overexpressed [83], cells that have been engineered to express a single copy of the mutant RANK show no evidence of a constitutive increase in NF-jB signaling, and they fail to initiate NF-jB or ERK responses to RANKL stimulation, indicating that these are loss-of-function mutations. Paradoxically, however, the mutant RANK molecules stimulate osteoclast formation in vitro when expressed in osteoclast precursors by mechanisms that remain incompletely understood [87].…”

Transfection of Osteoclasts and Osteoclast Precursors

“…Retroviral vectors have been extensively used by some research groups to transfect osteoclast precursors ( 1-3 ) but mature osteoclasts cannot be transfected with this technique since mitosis is required for integration of the retrovirus into the genome of the target cell. In this chapter, we describe methods for transfection of osteoclast precursors using lentiviral vectors (4)(5)(6) and the Amaxa™ Nucleofector ( 7 ) and also describe methods for transduction of mature osteoclasts using adenoviral vectors ( 8 ) , Fig. 1 illustrates the stages of osteoclast differentiation at which each technique is best employed.…”