Investigating the role of optineurin in bone biology and Paget's disease of bone
Abstract
Paget’s disease of bone (PDB) is a common disease with a strong genetic
component. Approaches such as linkage analysis and candidate gene studies have
shown that mutations in Sequestosome 1 (SQSTM1) explain up to 40% of familial
cases and 10% of sporadic cases, however the majority of PDB patients have no
mutations in this gene. Genome-wide association studies (GWAS) have recently
identified new susceptibility loci for PDB including variants at CSF1, TNFRSF11A,
OPTN, TM7SF4, PML, NUP205 and RIN3 loci. These loci were confirmed to be
associated with PDB in various European populations. OPTN encodes optineurin, a
widely expressed protein involved in many cellular processes but its role in bone
metabolism is yet unknown. The aim of this PhD thesis was to investigate the role of
OPTN in bone metabolism and PDB using in vitro and in vivo studies. In chapter 3,
the OPTN rs1561570 identified by previous GWAS was examined for its association
with the severity and clinical outcome of PDB in patients without SQSTM1 mutations.
The results showed that rs1561570 was significantly associated with total disease
severity score so that carriers of the risk allele “T” had higher severity score compared
to non-carriers (P < 0.05). A trend for reduced quality of life physical scores (SF36)
was also associated with the rs1561570 risk allele, but the relationship was not
statistically significant. In order to identify functional variants within OPTN, the
coding regions as well as the exon-intron boundaries were sequenced in 24 familial
PDB cases and 19 controls. No mutation was found that could be predicted as
pathogenic suggesting that disease susceptibility could be mediated by regulatory
polymorphisms that influence gene expression. In chapter 4, the role of OPTN was
investigated in osteoclast development using in vitro knockdown experiments. Optn
was expressed in mouse bone marrow derived macrophages (BMDMs) as well as all
stages of osteoclast development and it was significantly increased three days post
RANKL treatment. Optn expression was knocked down in BMDMs and cells were
induced to form osteoclast in the presence of RANKL and M-CSF. Compared to non-targeted
cells, Optn depleted cells formed significantly more and larger osteoclasts (P<
0.05). Optn knockdown was also found to enhance osteoclast survival as well as
RANKL-induced NFκB activation. In chapter 5, the role of OPTN was investigated in
vitro from cells obtained from knock in mice with a loss-of-function mutation in Optn
(OptnD477N/D477N). In agreement with the in vitro knockdown experiments, osteoclasts
were significantly higher and larger in mutant mice compared to WT and the NF-B
activity measured by luciferase reporter assay was significantly higher in cells from
OptnD477N/D477N compared to WT during most stages of osteoclast development. OPTN
from mutant and WT mice was co-precipitated with its CYLD binding-partner, which
acts as a negative regulator to RANK signalling by inhibiting the TRAF6 downstream
signalling. The data from this immunoprecipitation (IP) experiment revealed that
defective OPTN interacted less with CYLD from mutant mice compared to WT. This
study also showed that OPTN was expressed in osteoblasts and the expression rate did
not change during osteoblast development. The data obtained from the mineralization
assay revealed no significant difference between OptnD477N/D477N and WT. In chapter
6, I investigated the effect of the D477N loss of function mutation in Optn on bone
metabolism. Bone Histomorphometrical analysis of OptnD477N/D477N mice showed
higher bone resorption parameters (Oc.N/BS and Oc.S/BS) compared to wild type
(WT). Osteoid analysis showed evidence of increased bone formation parameters
(OS/BS and OV/BV) in mutant mice compared to WT. Calcein labelling showed a
significant difference in mineral apposition rate (MAR) from mutant mice compared
to WT. Analysis of serum biomarkers of bone turnover showed evidence of enhanced
bone turnover in mutant mice compared to WT. Micro computed tomography (μCT)
analysis of 4 and 14 months old mice showed no significant differences in bone
morphology between WT and OptnD477N/D477N mice of both sexes.
In conclusion, this study has shown for the first time that OPTN plays a role in
regulating bone turnover by acting as a negative regulator of osteoclast differentiation.
The data obtained from this study strongly suggest the crucial role of OPTN in RANK
signalling. The effect of OPTN on osteoblast activity may be direct or indirect
compensation for increased osteoclast activity. Further detailed studies will be
required to explore the underlying mechanism of OPTN including downstream RANK
signalling and a complete knockout model to corroborate these findings.