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

Julie C Crockett, David J Mellis, Kathleen I J Shennan, Angela Duthie, John Greenhorn, Debbie I Wilkinson, Stuart H Ralston, Miep H Helfrich, Michael J Rogers

Research output: Contribution to journalArticle

17 Citations (Scopus)

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-kappaB (RANK), a receptor critical for osteoclast formation and function. Previous studies have shown that overexpression of these mutant proteins causes constitutive activation of NF-kappaB 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-kappaB 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-kappaB. 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.
Original languageEnglish
Pages (from-to)1926-1938
Number of pages13
JournalJournal of Bone and Mineral Research
Volume26
Issue number8
Early online date6 Apr 2011
DOIs
Publication statusPublished - Aug 2011

Fingerprint

Receptor Activator of Nuclear Factor-kappa B
Protein Sorting Signals
Mutation
NF-kappa B
Osteoclasts
Mutant Proteins
Cell Membrane
Ligands
Smooth Endoplasmic Reticulum
HEK293 Cells
Golgi Apparatus
Endoplasmic Reticulum
Genes

Keywords

  • rank
  • familial expansile osteolysis
  • early-onset Paget's disease
  • expansile skeletal hyperphosphatasia
  • osteoclast
  • NFKB

Cite this

Crockett, J. C., Mellis, D. J., Shennan, K. I. J., Duthie, A., Greenhorn, J., Wilkinson, D. I., ... Rogers, M. J. (2011). Signal peptide mutations in RANK prevent downstream activation of NF-κB. Journal of Bone and Mineral Research, 26(8), 1926-1938. https://doi.org/10.1002/jbmr.399

Signal peptide mutations in RANK prevent downstream activation of NF-κB. / Crockett, Julie C; Mellis, David J; Shennan, Kathleen I J; Duthie, Angela; Greenhorn, John; Wilkinson, Debbie I; Ralston, Stuart H; Helfrich, Miep H; Rogers, Michael J.

In: Journal of Bone and Mineral Research, Vol. 26, No. 8, 08.2011, p. 1926-1938.

Research output: Contribution to journalArticle

Crockett, JC, Mellis, DJ, Shennan, KIJ, Duthie, A, Greenhorn, J, Wilkinson, DI, Ralston, SH, Helfrich, MH & Rogers, MJ 2011, 'Signal peptide mutations in RANK prevent downstream activation of NF-κB', Journal of Bone and Mineral Research, vol. 26, no. 8, pp. 1926-1938. https://doi.org/10.1002/jbmr.399
Crockett, Julie C ; Mellis, David J ; Shennan, Kathleen I J ; Duthie, Angela ; Greenhorn, John ; Wilkinson, Debbie I ; Ralston, Stuart H ; Helfrich, Miep H ; Rogers, Michael J. / Signal peptide mutations in RANK prevent downstream activation of NF-κB. In: Journal of Bone and Mineral Research. 2011 ; Vol. 26, No. 8. pp. 1926-1938.
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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-kappaB (RANK), a receptor critical for osteoclast formation and function. Previous studies have shown that overexpression of these mutant proteins causes constitutive activation of NF-kappaB 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-kappaB 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-kappaB. 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. {\circledC} 2011 American Society for Bone and Mineral Research.",
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AU - Wilkinson, Debbie I

AU - Ralston, Stuart H

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AU - Rogers, Michael J

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AB - 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-kappaB (RANK), a receptor critical for osteoclast formation and function. Previous studies have shown that overexpression of these mutant proteins causes constitutive activation of NF-kappaB 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-kappaB 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-kappaB. 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.

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