Randomised trial of genetic testing and targeted intervention to prevent the development and progression of Paget's disease of bone.

INTRODUCTION: Paget's disease of bone (PDB) frequently presents at an advanced stage with irreversible skeletal damage. Clinical outcomes might be improved by earlier diagnosis and prophylactic treatment. METHODS: We randomised 222 individuals at increased risk of PDB because of pathogenic SQSTM1 variants to receive 5 mg zoledronic acid (ZA) or placebo. The primary outcome was new bone lesions assessed by radionuclide bone scan. Secondary outcomes included change in existing lesions, biochemical markers of bone turnover and skeletal events related to PDB. RESULTS: The median duration of follow-up was 84 months (range 0-127) and 180 participants (81%) completed the study. At baseline, 9 (8.1%) of the ZA group had PDB lesions vs 12 (10.8%) of the placebo group. Two of the placebo group developed new lesions versus none in the ZA group (OR 0.41, 95% CI 0.00 to 3.43, p=0.25). Eight of the placebo group had a poor outcome (lesions which were new, unchanged or progressing) compared with none of the ZA group (OR 0.08, 95% CI 0.00 to 0.42, p=0.003). At the study end, 1 participant in the ZA group had lesions compared with 11 in the placebo group. Biochemical markers of bone turnover were significantly reduced in the ZA group. One participant allocated to placebo required rescue therapy with ZA because of symptomatic disease. The number and severity of adverse events did not differ between groups. CONCLUSIONS: Genetic testing for pathogenic SQSTM1 variants coupled with intervention with ZA is well tolerated and has favourable effects on the progression of early PDB. TRIAL REGISTRATION NUMBER: ISRCTN11616770.

The SQSTM1/p62 UBA domain regulates Ajuba localisation, degradation and NF-κB signalling function.

The LIM-domain containing protein Ajuba and the scaffold protein SQSTM1/p62 regulate signalling of NF-κB, a transcription factor involved in osteoclast differentiation and survival. The ubiquitin-associated domain of SQSTM1/p62 is frequently mutated in patients with Paget's disease of bone. Here, we report that Ajuba activates NF-κB activity in HEK293 cells, and that co-expression with SQSTM1/p62 inhibits this activation in an UBA domain-dependent manner. SQSTM1/p62 regulates proteins by targeting them to the ubiquitin-proteasome system or the autophagy-lysosome pathway. We show that Ajuba is degraded by autophagy, however co-expression with SQSTM1/p62 (wild type or UBA-deficient) protects Ajuba levels both in cells undergoing autophagy and those exposed to proteasomal stress. Additionally, in unstressed cells co-expression of SQSTM1/p62 reduces the amount of Ajuba present in the nucleus. SQSTM1/p62 with an intact ubiquitin-associated domain forms holding complexes with Ajuba that are not destined for degradation yet inhibit signalling. Thus, in situations with altered levels and localization of SQSTM1/p62 expression, such as osteoclasts in Paget's disease of bone and various cancers, SQSTM1/p62 may compartmentalize Ajuba and thereby impact its cellular functions and disease pathogenesis. In Paget's, ubiquitin-associated domain mutations may lead to increased or prolonged Ajuba-induced NF-κB signalling leading to increased osteoclastogenesis. In cancer, Ajuba expression promotes cell survival. The increased levels of SQSTM1/p62 observed in cancer may enhance Ajuba-mediated cancer cell survival.

The endoplasmic reticulum-associated protein, OS-9, behaves as a lectin in targeting the immature calcium-sensing receptor.

The mechanisms responsible for the processing and quality control of the calcium-sensing receptor (CaSR) in the endoplasmic reticulum (ER) are largely unknown. In a yeast two-hybrid screen of the CaSR C-terminal tail (residues 865-1078), we identified osteosarcoma-9 (OS-9) protein as a binding partner. OS-9 is an ER-resident lectin that targets misfolded glycoproteins to the ER-associated degradation (ERAD) pathway through recognition of specific N-glycans by its mannose-6-phosphate receptor homology (MRH) domain. We show by confocal microscopy that the CaSR and OS-9 co-localize in the ER in COS-1 cells. In immunoprecipitation studies with co-expressed OS-9 and CaSR, OS-9 specifically bound the immature form of wild-type CaSR in the ER. OS-9 also bound the immature forms of a CaSR C-terminal deletion mutant and a C677A mutant that remains trapped in the ER, although binding to neither mutant was favored over wild-type receptor. OS-9 binding to immature CaSR required the MRH domain of OS-9 indicating that OS-9 acts as a lectin most likely to target misfolded CaSR to ERAD. Our results also identify two distinct binding interactions between OS-9 and the CaSR, one involving both C-terminal domains of the two proteins and the other involving both N-terminal domains. This suggests the possibility of more than one functional interaction between OS-9 and the CaSR. When we investigated the functional consequences of altered OS-9 expression, neither knockdown nor overexpression of OS-9 was found to have a significant effect on CaSR cell surface expression or CaSR-mediated ERK1/2 phosphorylation.

The Changing Presentation of Paget's Disease of Bone in Australia, A High Prevalence Region.

Studies from several countries suggest that the incidence of Paget's disease of bone (PDB) and the severity of newly diagnosed cases are declining. The aim of this study was to examine secular changes in clinical presentation of PDB in Australia, which historically had the highest prevalence outside the United Kingdom. The participants were 293 patients (61% male) diagnosed between 1956 and 2013 with details recorded in the database of the Paget's Disease Research Group of Western Australia. The mean age at diagnosis was 62 years (range 28-90); 26% of participants had a family history of PDB and 11% had Sequestosome 1 (SQSTM1) mutations. After adjustment for covariates (SQSTM1 mutation status, family history, country of birth, smoking and dog exposure), there was a significant positive relationship between year of diagnosis and age at diagnosis (P < 0.001) and significant negative relationships between year of diagnosis and both pre-treatment total plasma alkaline phosphatase activity (ALP) and number of involved bones (P < 0.001 for each). Patients with SQSTM1 mutations had more extensive disease (P < 0.001) and higher pre-treatment ALP (P = 0.013). In subgroup analyses, relationships between year of diagnosis and each of age at diagnosis, number of involved bones and ALP were similar in patients with sporadic or familial disease, and in patients with and without SQSTM1 mutations. We conclude that the severity of PDB in Western Australia has declined over recent decades. This is likely to reflect altered exposure to one or more environmental agents involved in pathogenesis.

Bafilomycin A1 Attenuates Osteoclast Acidification and Formation, Accompanied by Increased Levels of SQSTM1/p62 Protein.

Vacuolar proton pump H(+)-adenosine triphosphatases (V-ATPases) play an important role in osteoclast function. Further understanding of the cellular and molecular mechanisms of V-ATPase inhibition is vital for the development of anti-resorptive drugs specifically targeting osteoclast V-ATPases. In this study, we observed that bafilomycin A1, a naturally-occurring inhibitor of V-ATPases, increased the protein level of SQSTM1/p62, a known negative regulator of osteoclast formation. Consistently, we found that bafilomycin A1 diminishes the intracellular accumulation of the acidotropic probe lysotracker in osteoclast-like cells; indicative of reduced acidification. Further, bafilomycin A1 inhibits osteoclast formation with attenuation of cell fusion and multi-nucleation of osteoclast-like cells during osteoclast differentiation. Taken together, these data indicate that bafilomycin A1 attenuates osteoclast differentiation in part via increased levels of SQSTM1/p62 protein, providing further mechanistic insight into the effect of V-ATPase inhibition in osteoclasts.

Paget disease of bone-associated UBA domain mutations of SQSTM1 exert distinct effects on protein structure and function.

SQSTM1 mutations are common in patients with Paget disease of bone (PDB), with most affecting the C-terminal ubiquitin-associated (UBA) domain of the SQSTM1 protein. We performed structural and functional analyses of two UBA domain mutations, an I424S mutation relatively common in UK PDB patients, and an A427D mutation associated with a severe phenotype in Southern Italian patients. Both impaired SQSTM1's ubiquitin-binding function in pull-down assays and resulted in activation of basal NF-κB signalling, compared to wild-type, in reporter assays. We found evidence for a relationship between the ability of different UBA domain mutants to activate NF-κB signalling in vitro and number of affected sites in vivo in 1152 PDB patients from the UK and Italy, with A427D-SQSTM1 producing the greatest level of activation (relative to wild-type) of all PDB mutants tested to date. NMR and isothermal titration calorimetry studies were able to demonstrate that I424S is associated with global structural changes in the UBA domain, resulting in 10-fold weaker UBA dimer stability than wild-type and reduced ubiquitin-binding affinity of the UBA monomer. Our observations provide insights into the role of SQSTM1-mediated NF-κB signalling in PDB aetiology, and demonstrate that different mutations in close proximity within loop 2/helix 3 of the SQSTM1 UBA domain exert distinct effects on protein structure and stability, including indirect effects at the UBA/ubiquitin-binding interface.

SQSTM1 mutations--bridging Paget disease of bone and ALS/FTLD.

Paget disease of bone (PDB) is a skeletal disorder common in Western Europe but extremely rare in the Indian subcontinent and Far East. The condition has a strong genetic element with mutations affecting the SQSTM1 gene, encoding the p62 protein, frequently identified. Recently SQSTM1 mutations have also been reported in a small number of patients with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), neurodegenerative disorders in which significant coexistence with PDB has not been previously recognized. Although several SQSTM1 mutations are common to both ALS/FTLD and PDB, many are ALS/FTLD-specific. The p62 protein regulates various cellular processes including NF-κB signaling and autophagy pathways. Here we consider how knowledge of the impact of PDB-associated SQSTM1 mutations (several of which are now known to be relevant for ALS/FTLD) on these pathways, as well as the locations of the mutations within the p62 primary sequence, may provide new insights into ALS/FTLD disease mechanisms.

Common susceptibility alleles and SQSTM1 mutations predict disease extent and severity in a multinational study of patients with Paget's disease.

Paget's disease of bone (PDB) has a strong genetic component. Here, we investigated possible associations between genetic variants that predispose to PDB and disease severity. Allelic variants identified as predictors of PDB from genome-wide association studies were analyzed in 1940 PDB patients from the United Kingdom, Italy, Western Australia, and Spain. A cumulative risk allele score was constructed by adding the variants together and relating this to markers of disease severity, alone and in combination with SQSTM1 mutations. In SQSTM1-negative patients, risk allele scores in the highest tertile were associated with a 27% increase in disease extent compared with the lowest tertile (p < 0.00001) with intermediate values in the middle tertile (20% increase; p = 0.0007). The effects were similar for disease severity score, which was 15% (p = 0.01) and 25% (p < 0.00001) higher in the middle and upper tertiles, respectively. Risk allele score remained a significant predictor of extent and severity when SQSTM-positive individuals were included, with an effect size approximately one-third of that observed with SQSTM1 mutations. A genetic risk score was developed by combining information from both markers, which identified subgroups of individuals with low, medium, and high levels of severity with a specificity of 70% and sensitivity of 55%. Risk allele scores and SQSTM1 mutations both predict extent and severity of PDB. It is possible that with further refinement, genetic profiling may be of clinical value in identifying individuals at high risk of severe disease who might benefit from enhanced surveillance and early intervention.

The helix 1-3 loop in the glucocorticoid receptor LBD is a regulatory element for FKBP cochaperones.

The heat-shock protein 90 (Hsp90) cochaperone FK506-binding protein 52 (FKBP52) upregulates, whereas FKBP51 inhibits, hormone binding and nuclear targeting of the glucocorticoid receptor (GR). Decreased cortisol sensitivity in the guinea pig is attributed to changes within the helix 1 to helix 3 (H1-H3) loop of the guinea pig GR (gpGR) ligand-binding domain. It has been proposed that this loop serves as a contact point for FKBP52 and/or FKBP51 with receptor. We examined the role of the H1-H3 loop in GR activation by FKBP52 using a Saccharomyces cerevisiae model. The activity of rat GR (rGR) containing the gpGR H1-H3 loop substitutions was still potentiated by FKBP52, confirming the loop is not involved in primary FKBP52 interactions. Additional assays also excluded a role for other intervening loops between ligand-binding domain helices in direct interactions with FKBP52 associated with enhanced receptor activity. Complementary studies in FKBP51-deficient mouse embryo fibroblasts and HEK293 cells demonstrated that substitution of the gpGR H1-H3 loop residues into rGR dramatically increased receptor repression by FKBP51 without enhancing receptor-FKBP51 interaction and did not alter recruitment of endogenous Hsp90 and the p23 cochaperone to receptor complexes. FKBP51 suppression of the mutated rGR did not require FKBP51 peptidylprolyl cis-trans isomerase activity and was not disrupted by mutation of the FK1 proline-rich loop thought to mediate reciprocal FKBP influences on receptor activity. We conclude that the gpGR-specific mutations within the H1-H3 loop confer global changes within the GR-Hsp90 complex that favor FKBP51 repression over FKBP52 potentiation, thus identifying the loop as an important target for GR regulation by the FKBP cochaperones.

New insights into the role of sequestosome 1/p62 mutant proteins in the pathogenesis of Paget's disease of bone.

Paget's disease of bone (PDB) is characterized by focal areas of aberrant and excessive bone turnover, specifically increased bone resorption and disorganized bone formation. Germline mutations in the sequestosome 1/p62 (SQSTM1/p62) gene are common in PDB patients, with most mutations affecting the ubiquitin-associated domain of the protein. In vitro, osteoclast precursor cells expressing PDB-mutant SQSTM1/p62 protein are associated with increases in nuclear factor κB activation, osteoclast differentiation, and bone resorption. Although the precise mechanisms by which SQSTM1/p62 mutations contribute to disease pathogenesis and progression are not well defined, it is apparent that as well as affecting nuclear factor κB signaling, SQSTM1/p62 is a master regulator of ubiquitinated protein turnover via autophagy and the ubiquitin-proteasome system. Additional roles for SQSTM1/p62 in the oxidative stress-induced Keap1/Nrf2 pathway and in caspase-mediated apoptosis that were recently reported are potentially relevant to the pathogenesis of PDB. Thus, SQSTM1/p62 may serve as a molecular link or switch between autophagy, apoptosis, and cell survival signaling. The purpose of this review is to outline recent advances in understanding of the multiple pathophysiological roles of SQSTM1/p62 protein, with particular emphasis on their relationship to PDB, including challenges associated with translating SQSTM1/p62 research into clinical diagnosis and treatment.

The S349T mutation of SQSTM1 links Keap1/Nrf2 signalling to Paget's disease of bone.

Mutations affecting the Sequestosome 1 (SQSTM1) gene commonly occur in patients with the skeletal disorder Paget's disease of bone (PDB), a condition characterised by defective osteoclast differentiation and function. Whilst most mutations cluster within the ubiquitin-associated (UBA) domain of the SQSTM1 protein, and are associated with dysregulated NFκB signalling, several non-UBA domain mutations have also been identified. Keap1 is a SQSTM1-interacting protein that regulates the levels and activity of the Nrf2 transcription factor. This in turn controls the expression of numerous cytoprotective genes that contribute to the cell's capacity to defend itself against chemical and oxidative stress, through binding to the antioxidant response element (ARE). The PDB-associated S349T mutation maps to the Keap1-interacting region (KIR) of SQSTM1, however the effects of PDB mutant SQSTM1 on Keap1 function have not been investigated. Here we show that unlike other SQSTM1 mutations, the S349T mutation results in neither impaired ubiquitin-binding function in pull-down assays, nor dysregulated NFκB signalling in luciferase reporter assays. Keap1 is expressed in differentiating osteoclast-like cells and the S349T mutation selectively impairs the SQSTM1-Keap1 interaction in co-immunoprecipitations, which molecular modelling indicates results from effects on critical hydrogen bonds required to stabilise the KIR-Keap1 complex. Further, S349T mutant SQSTM1, but not other PDB-associated mutants, showed reduced ability to activate Nrf2 signalling as assessed by ARE-luciferase reporter assays. Thus, SQSTM1-mediated dysregulation of the Keap1-Nrf2 axis, which could potentially lead to aberrant production of oxidative response genes, may contribute to disease aetiology in a subset of PDB patients.

Genome-wide association identifies three new susceptibility loci for Paget's disease of bone.

Paget's disease of bone (PDB) is a common disorder characterized by focal abnormalities of bone remodeling. We previously identified variants at the CSF1, OPTN and TNFRSF11A loci as risk factors for PDB by genome-wide association study. Here we extended this study, identified three new loci and confirmed their association with PDB in 2,215 affected individuals (cases) and 4,370 controls from seven independent populations. The new associations were with rs5742915 within PML on 15q24 (odds ratio (OR) = 1.34, P = 1.6 × 10(-14)), rs10498635 within RIN3 on 14q32 (OR = 1.44, P = 2.55 × 10(-11)) and rs4294134 within NUP205 on 7q33 (OR = 1.45, P = 8.45 × 10(-10)). Our data also confirmed the association of TM7SF4 (rs2458413, OR = 1.40, P = 7.38 × 10(-17)) with PDB. These seven loci explained ∼13% of the familial risk of PDB. These studies provide new insights into the genetic architecture and pathophysiology of PDB.

Proteasome inhibitors impair RANKL-induced NF-kappaB activity in osteoclast-like cells via disruption of p62, TRAF6, CYLD, and IkappaBalpha signaling cascades.

Proteasome inhibitors represent a promising therapy for the treatment of relapsed and/or refractory multiple myeloma, a disease that is concomitant with osteolysis and enhanced osteoclast formation. While blockade of the proteosome pathway has been recently shown to influence osteoclast formation and function, the precise molecular cascade underlying these effects is presently unclear. Here, we provide evidence that proteasome inhibitors directly impair osteoclast formation and function via the disruption of key RANK-mediated signaling cascades. Disruption of the proteosome pathway using selective inhibitors (MG-132, MG-115, and epoxomicin) resulted in the accumulation of p62 and CYLD, and altered the subcellular targeting and distribution of p62 and TRAF6 in osteoclast-like cells. Proteosome inhibition also blocked RANKL-induced NF-kappaB activation, IkappaBalpha degradation and nuclear translocation of p65. The disruption in RANK-signaling correlated dose-dependently with an impairment in osteoclastogenesis, with relative potency epoxomicin > MG-132 > MG-115 based on equimolar concentrations. In addition, these inhibitors were found to impact osteoclastic microtubule organization and attenuate bone resorption. Based on these data we propose that deregulation of key RANK-mediated signaling cascades (p62, TRAF6, CYLD, and IkappaBalpha) underscores proteasome-mediated inhibition of osteolytic bone conditions.

Sequestosome 1 mutations in Paget's disease of bone in Australia: prevalence, genotype/phenotype correlation, and a novel non-UBA domain mutation (P364S) associated with increased NF-kappaB signaling without loss of ubiquitin binding.

Previously reported Sequestosome 1(SQSTM1)/p62 gene mutations associated with Paget's disease of bone (PDB) cluster in, or cause deletion of, the ubiquitin-associated (UBA) domain. The aims of this study were to examine the prevalence of SQSTM1 mutations in Australian patients, genotype/phenotype correlations and the functional consequences of a novel point mutation (P364S) located upstream of the UBA. Mutation screening of the SQSTM1 gene was conducted on 49 kindreds with PDB. In addition, 194 subjects with apparently sporadic PDB were screened for the common P392L mutation by restriction enzyme digestion. HEK293 cells stably expressing RANK were co-transfected with expression plasmids for SQSTM1 (wildtype or mutant) or empty vector and a NF-kappaB luciferase reporter gene. GST-SQSTM1 (wildtype and mutant) proteins were used in pull-down assays to compare monoubiquitin-binding ability. We identified SQSTM1 mutations in 12 of 49 families screened (24.5%), comprising 9 families with the P392L mutation and 1 family each with the following mutations: K378X, 390X, and a novel P364S mutation in exon 7, upstream of the UBA. The P392L mutation was found in 9 of 194 (4.6%) patients with sporadic disease. Subjects with SQSTM1 mutations had more extensive disease, but not earlier onset, compared with subjects without mutations. In functional studies, the P364S mutation increased NF-kappaB activation compared with wildtype SQSTM1 but did not reduce ubiquitin binding. This suggests that increased NF-kappaB signaling, but not the impairment of ubiquitin binding, may be essential in the pathogenesis of PDB associated with SQSTM1 mutations.

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.

UNLABELLED: 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. INTRODUCTION: 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. MATERIALS AND 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.