OPTN negatively regulates osteoclast formation in vitro

OPTN encodes a ubiquitously expressed cytoplasmic protein called optineurin which contains an ubiquitin binding domain, similar to that present in NEMO. Mutations in OPTN have been linked to glaucoma and motor neuron disease but the role of optineurin in bone metabolism is yet unknown. We recently identified variants in the OPTN gene that predispose to Paget's disease of bone using genome wide association studies (Albagha et al., Nat Genet, 2011) suggesting a role for this gene in bone metabolism. The aim of this study was to investigate the role of optineurin in osteoclast development using in vitro knock-down experiments. First we investigated the expression of OPTN during osteoclast formation. Bone marrow cells from wild type CD1 mice were isolated and cultured for 48 h in the presence of M-CSF (50 ng/ml) followed by stimulation of adherent cells with RANKL (100 ng/ml) and M-CSF (25 ng/ml) for 5 days until osteoclast were formed. OPTN expression was determined using western blot analysis. Expression of OPTN was detected in bone marrow derived macrophages (BMDMs) but not in total bone marrow cells. No significant difference in expression levels was found after the first day of RANKL treatment but optineurin expression significantly increased from day 2 to day 5. We then investigated the effect of OPTN depletion on osteoclast formation in mouse bone marrow cultures. The expression of OPTN in mouse BMDMs was knocked down using lentiviral particles expressing shRNA targeted against the OPTN gene. Non-targeting shRNA lentiviral particles were used as a negative control and OPTN knock down was confirmed (> 85%) using western blot analysis. OPTN-depleted BMDMs were stimulated with M-CSF and RANKL until osteoclasts were formed. TRAP positive multinucleated osteoclasts (> 3 nuclei) were counted and numbers were compared to the non-targeted negative control. We found that the number of osteoclasts formed from OPTN-depleted BMDMs was significantly higher than those formed from nontargeted cells (number ± SD; 151 ± 21 vs. 69 ± 18; P = 0.002). In conclusion we have shown for the first time that optineurin negatively regulates osteoclast formation in vitro. Our data are consistent with the previously reported negative effect of OPTN on TNF-alpha induced NFκB activation. These data also provide a possible mechanism by which variants in OPTN increase susceptibility to Paget's disease of bone but further in vivo studies will be required to confirm these findings.