Bone Abstracts

Abstract: Paget’s disease of bone (PDB) is characterised by focal lesions of local bone turnover driven by overactive osteoclasts, which often contain nuclear and cytoplasmic inclusion bodies. Mutations affecting the sequestosome-1 (SQSTM1) ubiquitin-associated (UBA) domain have been identified in individuals with PDB. SQSTM1, also known as p62, is a ubiquitously-expressed scaffold protein of 62 kDa that functions in multiple signalling pathways important for cell survival and osteoclast activity. The mechanisms by which SQSTM1 mutations cause PDB remain unclear. Here, we report our laboratory’s recent advances in understanding the role of SQSTM1 in PDB pathogenesis.

Using molecular and microscopical methods to examine Pagetic bone biopsies, osteoclast cultures and various cell lines, we have identified two isoforms of SQSTM1. In all cell types examined, four SQSTM1 transcripts were detected, differing in their 5′-untranslated region; one transcript encodes p62, while the other three encode 55 kD-SQSTM1. The newly identified isoform also contains the UBA domain mutated in PDB. Using biochemical and microscopical methods, we found that both SQSTM1 isoforms are degraded by autophagy. The isoforms interact with each other and form aggregates upon autophagy inhibition. 55 kD-SQSTM1 is ~45× more abundant in osteoclasts than SQSTM1/p62. Biochemical and microscopical methods showed that mutations in SQSTM1/p62 impair autophagic degradation. Cell lines expressing mutations in SQSTM1/p62 form paracrystalline inclusion bodies, that by immuno-transmission electron microscopy (TEM) were found to contain SQSTM1 and ubiquitin and were ultrastructurally identical to those found in PDB. As observed by TEM, these inclusions can be degraded by autophagy. The effects of mutations in 55 kD-SQSTM1 have yet to be characterised.

Taken together, these data show that mutations in SQSTM1 isoforms impair protein degradation and can lead to inclusion body formation suggesting that PDB results from dysregulated protein degradation in osteoclasts. Further characterisation of the effect of mutations in 55 kD-SQSTM1 in stably transfected cell lines is ongoing.