title: A novel mutation (K378X) in the sequestosome 1 gene associated with increased NF-kappaB signaling and Paget's disease of bone with a severe phenotype.
abstract: Sequestosome 1/p62 (p62) mutations are associated with PDB; however, there are limited data regarding functional consequences. We report a novel mutation in exon 7 (K378X) in a patient with polyostotic Paget's disease of bone. p62 mutants increased NF-kappaB activation and significantly potentiated osteoclast formation and bone resorption in human primary cell cultures.
  
  
  BACKGROUND
  Sequestosome 1/p62 (p62) mutations are associated with Paget's disease of bone (PDB); however, there are limited data regarding functional consequences. One report has linked the common P392L mutation in the p62 ubiquitin binding associated (UBA) domain with increases in NF-kappaB activity, a transcription factor essential for osteoclastogenesis. To further clarify the functional impact of p62 mutations associated with PDB, we assessed the effect of p62 mutation (a novel mutation: K378X, and previously reported mutations: P392L and E396X) on RANK-induced NF-kappaB activation and compared this with the effect of wildtype p62. In addition, we studied the effect of p62 mutation on osteoclast formation and bone resorption.
  
  
  METHODS
  We performed co-transfection experiments with expression plasmids for p62 (wildtype or mutated) and RANK and an NF-kappaB luciferase reporter gene. Luciferase activities were recorded after addition of luciferin to cellular lysates. RAW(264.7) cells stably expressing enhanced green fluorescent protein (EGFP)-tagged p62 (wildtype, K378X, or P392L) or EGFP alone were assessed for changes in cell proliferation. Additionally, these cells were stimulated with RANKL to produce osteoclast-like cells (OLCs). Primary human monocytes collected from the K378X-affected patient and a control subject were stimulated to form OLCs and bone resorption data were obtained.
  
  
  RESULTS
  The novel mutation introduces a premature stop codon in place of Lys-378 and thereby eliminates the entire p62 UBA domain; this and two additional natural mutations (P392L, E396X) increased NF-kappaB activation compared with wildtype p62. Wildtype p62 consistently inhibited NF-kappaB activation compared with empty vector. UBA mutations (K378X and P392L) significantly increased the number of OLCs formed in response to RANKL and also the number of nuclei of the OLCs. K378X-affected human monocytes formed more OLCs with more nuclei and increased bone resorption compared with control monocytes.
  
  
  CONCLUSIONS
  Our data show that mutation of the p62 UBA domain results in increased activation of NF-kappaB and osteoclast formation and function compared with wildtype p62. These results may partially explain the mechanism by which p62 mutation contributes to the pathogenesis of PDB.
authors.affiliation: Laboratory for Molecular Endocrinology, Western Australian Institute for Medical Research, University of Western Australia, Nedlands.
authors.forename: Sarah L
authors.initials: SL
authors.lastname: Rea
authors.forename: John P
authors.initials: JP
authors.lastname: Walsh
authors.forename: Lynley
authors.initials: L
authors.lastname: Ward
authors.forename: Kirk
authors.initials: K
authors.lastname: Yip
authors.forename: Bryan K
authors.initials: BK
authors.lastname: Ward
authors.forename: G Neil
authors.initials: GN
authors.lastname: Kent
authors.forename: James H
authors.initials: JH
authors.lastname: Steer
authors.forename: Jiake
authors.initials: J
authors.lastname: Xu
authors.forename: Thomas
authors.initials: T
authors.lastname: Ratajczak
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000328:Adult; D000818:Animals; D001862:Bone Resorption / Q000235:genetics / Q000378:metabolism; D019556:COS Cells; D002352:Carrier Proteins / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics; D002522:Chlorocebus aethiops; D018388:Codon, Terminator / Q000235:genetics; D006801:Humans; D008297:Male; D008562:Membrane Glycoproteins / Q000494:pharmacology; D016328:NF-kappa B / Q000378:metabolism*; D010001:Osteitis Deformans / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D010010:Osteoclasts / Q000378:metabolism / Q000473:pathology; D017354:Point Mutation; D017434:Protein Structure, Tertiary / Q000235:genetics; D011506:Proteins / Q000235:genetics* / Q000378:metabolism; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B; D000071456:Sequestosome-1 Protein; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics*; D014162:Transfection
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D018388:Codon, Terminator; D008562:Membrane Glycoproteins; D016328:NF-kappa B; D011506:Proteins; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B; C099718:SQSTM1 protein, human; D000071456:Sequestosome-1 Protein; C505686:TNFRSF11A protein, human; C505688:TNFSF11 protein, human