Bone Abstracts

Paget’s disease of Bone (PDB) is caused by mutations in the gene encoding Sequestosome-1 (Q17STM1 or p62) that affect the C-terminal Ubiquitin-Associated (UBA) domain. A second isoform of Q17STM1 exists (referred to hereafter as 55kDa-Q17STM1), which lacks the N-terminal Phox and Bem1 (PB1) domain and has previously been reported to be ~45x more abundant than Q17STM1/p62 in osteoclasts. Mutations in the UBA domain will also occur in this isoform. Several of the UBA mutations in 62kDa-Q17STM1 have been reported to increase the activation of NF-κB in overexpression systems. No study has yet examined the effect of mutations in 55kD-Q17STM1 on NF-κB signalling.

Human Embryonic Kidney (HEK293)-derived cell lines were stably transfected with wild-type (WT) or mutated (392L, 396X or 425R) 55kDa- or 62kDa-Q17STM1. Expression of endogenous Q17STM1 renders these cell lines heterozygous for each mutation. NF-κB activation for all cell lines was assessed using dual luciferase reporter assays.

55kDa-WT and 62kDa-WT cell lines similarly activated NF-κB. Marked differences were observed in NF-κB activation between mutations in the two isoforms: 62kDa-P392L exhibited 80.1% NF-κB activity of 62kDa-WT whereas 55kDa-P392L increased activation by 17.3% compared to 55kDa-WT. For the E396X mutation the results were opposite (62kDa-E396X increased activation by 57.5% and 55kDa-E396X decreased activation by 26.1%). No differences between isoforms were seen for the G425R substitution, where NF-κB activation was reduced by 35% compared to 62kDa-WT for both isoforms.

In our physiologically-relevant cell model, we found that the effect of UBA domain mutations on NF-κB signalling is context-dependent, with opposing effects occurring depending on which isoform the mutation is present within. In vivo, this may regulate NF-κB signalling in a manner related to isoform abundance, and this warrants further investigation. The 55kDa isoform could, therefore, play an important role in the development and/or limitation of PDB exclusively in osteoclasts.