Characterising the role of Valosin Containing Protein (VCP) in autophagy and cell differentiation.

Valosin containing protein (VCP)/p97 is a hexameric ATPase of the AAA family, which regulates a wide array of essential cellular processes. Dominant mutations in the N-domain of the VCP give rise to the complex disease syndrome known as Inclusion body myopathy with Paget disease of the bone and frontotemporal dementia (IBMPFD). VCP plays a key role in the ubiquitin-proteasome dependent protein degradation although mutations in VCP seem to result in a late stage autophagy defect. Osteoclast precursors containing VCP mutations are hyper-responsive to RANKL and M-CSF treatment. This suggests that under normal homeostasis VCP plays an important role in regulating the response of osteoclasts to bone microenvironment. However, the mechanisms by which VCP mutations stimulate osteoclast differentiation in Paget disease of the bone (PDB) are not completely understood. To gain insight into disease phenotype associated with VCP mutations I examined the role of VCP in autophagy and the role of autophagy on osteoclastogenesis. I have shown that VCP co-localises with p62 and LC3 at the subcellular level in cells undergoing autophagy and that VCP co-immunoprecipitates with p62 in the autophagy-dependant manner. I have also examined the stability of VCP in the cell and shown that p62 has a role in stabilising the VCP protein and that the mutant protomers seem to be less stable than the normal VCP protomers. Initiation of autophagy in RAW264.7 cells in the presence of RANKL resulted in marked reduction in osteoclast formation, regardless of the time point at which the treatment begun. I also found that RANKL and TNFα induced NFκB activation is increased (in an autophagy dependent manner) in macrophages from the heterozygous VCP mouse compared to normal macrophages. These data together with the already existing knowledge on VCP, and the link with PDB, suggest that modulation of the autophagy pathway by VCP may represent a major regulator of bone remodelling and maintenance. Autophagy has direct effect on the fate of osteoclast progenitor cells thus regulation of osteoclastogenesis is a key process underlying the pathogenesis of PDB. This work acts to further our understanding of the pathogenic mechanism of VCP-related disease and will facilitate the search for modifiers of the disease phenotype

Assessment of C3-Epi-25-Hydroxyvitamin D concentration in adult serum: LC-MS/MS determination using [2H3] 3-epi-25OHD3 internal standard and NIST traceable commercial 3-epi-25OHD calibrators.

Background: The C-3 Epimer of 25 Hydroxyvitamin D3 (3-Epi-25OHD3) is produced in the liver by the epimerisation pathway of 25-hydroxy vitamin D3. It differs from 25OHD3 in configuration of the hydroxyl group at the third carbon (C-3) position. Despite the fact that little is known regarding its clinical significance, concerns have been raised that isobaric interference may result in over-estimation of total 25OHD when measured by liquid chromatography tandem mass spectrometry (LC-MS/MS). Objective: The aim of the study was to assess the occurrence of 3-Epi-25OHD3 in adult serum samples. A LC-MS/MS technique was developed to resolve and quantify 3-Epi-25OHD3 from 25OHD3. The newly available NIST (SRM972a) traceable 3-Epi-25OHD commercial standards were used to ensure assay accuracy. Method: Serum was precipitated with zinc sulphate and acetonitrile containing [2H3]-3-epi-25OHD3 as internal standard. The extract was chromatographed using a 2.6µm 100 x 2.1mm I.D. solid core particle column. Mass detection and quantification were performed by positive electrospray ionization with MS/MS in multiple reaction monitoring mode. Results: The method was able to fully resolved 3-Epi-25OHD3 from 25OHD3. The intraassay CVs for the epimer were 6.3% and 4.1% at 25.4 and 62.1 nmol/L respectively; and interassay CVs were 8.3% and 6.5% at 27.6 and 63.2 nmol/L, respectively. In our sample cohort with 25OHD3 ranged between 3.4 – 165 nmol/L, 3-Epi-25OHD3 was detected in 91.9% of samples (mean = 3.8 nmol/L). No detectable 3-Epi-25OHD2 was found in our sample study. One patient sample had total 25OHD3 of 187 nmol/L that was shown to contain 141 nmol/L of 25OHD3 and 44 nmol/L of 3-Epi-25OHD3. This patient was receiving a high dose of vitamin D supplementation. Conclusion: Using [2H3]-3-epi-25OHD3 as internal standard and NIST aligned calibrators enabled us to obtain an accurate assessment of 3-epi-25OHD concentration in adult serum. Although the concentration of serum 3-epi-25OHD3 was found to be low the presence was observed in the majority of our samples. The findings in this study showed that 3-epi-25OHD3 contributed to the overestimation of 25OHD3 that could potentially resulted in misinterpretation of total vitamin D status