ECM defects are associated with muscle fibrosis of muscular disorders, and are usually detected at a progressed state of the disease. Muscular dystrophies including OPMD are characterised by the presence of fibrotic muscles. Deregulation of the ECM is found in inherited degenerative muscle diseases such as DMD, FSHD and α-sarcoglycan deficiency [24–26]. ECM thickening in DMD patients are associated with significant dysregulation of collagen and other ECM protein genes . Muscles from symptomatic patients show ECM thickening that is typical of fibrotic tissue. Collagen type I and III are major components of the ECM in skeletal muscle , and in inherited muscular dystrophies an up-regulation of collagen-encoding genes is associated with thickening of the ECM. ECM thickening affects muscle contraction and extracellular signal transduction and increased inflammation . In DMD patients, ECM thickening is associated with up-regulation of collagen encoding genes . Similar to other muscle disorders, in OPMD we found significant molecular dysregulation of ECM-associated genes. Since dysregulation of ECM encoding genes in different cell types are often associated with ECM thickening and profibrotic situations , it is highly likely that the transcriptional changes in the ECM functional group in OPMD are associated with muscle degeneration.
We identified consistent down-regulation of PCOLCE in OPMD patient and cellular and mouse model systems of this condition. In carriers of expPABPN1, down-regulation was found in symptomatic patients but not at the pre-symptomatic stage of the disease. This suggests that the change in PCOLCE expression in muscles is associated with muscle degeneration. PCOLCE is down-regulated in OPMD and is found in PABPN1 nuclear aggregates. Products of dysregulated genes in model systems expressing expPABPN1 often co-localize with PABPN1 within nuclear aggregates [16, 29]. The mechanism as to how this occurs is not fully understood. However, it is possible that a feedback-forward regulatory mechanism between protein aggregation and mRNA accumulation is disturbed when aggregates are formed.
The procollagen I peptide is proteolytically processed to form collagen fibres. Extracellular PCOLCE stimulates the function of procollagen C-proteinase, thereby leading to increased cleavage and processing of procollagen I proteins . Pcolce deficient mice exhibit ECM thickening, aberrant procollagen peptide processing and deformed collagen fibril morphology . Taken together these processes suggest that a reduction in PCOLCE at the extracellular matrix would diminish collagen fibrillogenesis and hence negatively affect ECM maintenance . Of relevance to muscle disorders, Pcolce down-regulation has been found in Myostatin-knockdown mice, and was associated with reduced collagen content and muscle fibrosis . It is therefore likely that in OPMD a reduced expression of PCOLCE causes ECM thickening and consequently contributes to muscle fibrosis.
PCOLCE is a secreted protein. It is synthesized in the lumen of the ER and is transported to the extracellular compartment via the Golgi apparatus and associated secretion pathway . In addition to its known extracellular location at the ECM, we found PCOLCE to also be present within the cell nucleus. We found that PCOLCE sub-cellular localization is dynamic in muscle cells, and changed upon myoblast differentiation to fused myotubes. In degenerated OPMD muscles, a reduction in extracellular PCOLCE is associated with retention of this protein within the nucleus. Since PCOLCE protein biosynthesis is at the secretary Golgi apparatus, this suggests that PCOLCE may shuttle from the extracellular into the cell nuclear compartment. However, the mechanism regulating PCOLCE sub-cellular localization remains to be investigated.
We found that PABPN1 co-immunoprecipitates with PCOLCE (Figure 4B). Since PABPN1 is predominantly localized within the cell nucleus and we found co-localization of PABPN1 and PCOLCE within this sub-cellular compartment (Figures 4A, 5 and 7B), we conclude that PCOLCE binds to nuclear PABPN1 and consequently is entrapped in PABPN1 aggregates.
Co-localization between PCOLCE and aggregated PABPN1 was found to be consistent between cellular and animal model systems of OPMD as well as within OPMD patient muscle samples. Protein entrapment within PABPN1 inclusions is a gradual process where proteins such as ubiquitin or poly(A) polymerase co-localize with small and intermediate sized aggregates, whilst others like heat-shock protein 70 (HSP70) are recruited to large and non-reversible aggregates . Since only limited co-localization of PCOLCE and PABPN1 was found in OPMD nuclei with reduced amounts of aggregated PABPN1 (Figure 7B), it is likely that PCOLCE, as in the case of HSP70, is also recruited to inclusions but not to the intermediate sized PABPN1 aggregated structures.
PABPN1 is a regulator of mRNA processing [32–35]. A functional role for nuclear PCOLCE is, as yet, unknown. PCOLCE contains RNA binding motifs and a role in RNA stabilisation has been suggested . Here we show that PCOLCE binding to PABPN1 is enriched in cells expressing mutant expPABPN1. This suggests that PCOLCE binding to PABPN1 is of relevance to OPMD pathogenesis. It is possible that due to the alanine expansion in expPABPN1 the dynamics of the PCOLCE-PABPN1 complex is reduced and that this in turn could negatively affect PABPN1 function.
In contrast to the paucity of PABPN1 nuclear aggregate formation in affected myonuclei [8, 23], we found a consistent reduction in ECM-localised PCOLCE levels across muscle fibres of OPMD patients, and in a mouse model of this disease. This consistency suggests that this feature could be employed as a marker of affected muscles in OPMD patients. In a skeletal myoblast cell model system expressing expPABPN1, we observed accumulation of nuclear PCOLCE and co-localisation of PCOLCE with aggregated PABPN1. This observation was confirmed in OPMD patient muscle biopsy samples, where nuclear PCOLCE accumulation was more prominent compared with that seen in a healthy control or other muscular dystrophies, namely DMD and FSHD. As both OPMD and MD1 are classified as late onset disease conditions whilst DMD starts within childhood and symptoms in FSHD are typically observed from around 20 years of age, it is possible that the change in PCOLCE sub-cellular localisation may be of greater relevance to late onset muscle disorders. However, the contribution of dysregulation of PCOLCE sub-cellular localisation to disease pathophysiology remains to be demonstrated.