Role of MEPE in chondrocyte matrix mineralisation

Abstract

Matrix Extracellular Phosphoglycoprotein (MEPE) is a member of a family of proteins called small integrin-binding ligand, N-linked glycoproteins (SIBLINGs) which play key roles in biomineralisation. Altered MEPE expression is associated with several phosphate and bone-mineral metabolic disorders such as oncogenic osteomalacia and hypophosphatemic rickets. Despite this, it remains undetermined what impact MEPE has on the growth plate; the cartilage anlagen from which endochondral ossification, the process responsible for linear bone growth, occurs. The work of this thesis has characterised the ATDC5 cell line and the metatarsal organ culture as useful in vitro models of endochondral ossification. These will prove vital in the pursuit of underpinning the molecular mechanisms involved in endochondral bone growth. These models form the basis of the further studies in this thesis examining the role of MEPE within this highly orchestrated process. Before such role can be defined, this thesis details the spatial and temporal localisation patterns of MEPE in 10-day- and 4-week-old murine growth plates. More specifically, MEPE protein and mRNA were preferentially expressed by the hypertrophic chondrocytes as shown by immunohistochemistry and in situ hybridisation respectively. Microdissection of the murine growth plate confirmed this. Localisation of the cleavage product of MEPE, a 2.2kDa acidic serine- and aspirate-rich motif (ASARM) peptide, followed a similar pattern of expression. The localisation of MEPE to sites of mineralisation serves to strengthen its potential role in chondrocyte matrix mineralisation. This thesis identified this role in both mineralising ATDC5 cells and the metatarsal organ culture. The ASARM peptide was found to be the functional component of MEPE and this function was dependent upon its post-translational phosphorylation. Phosphorylated (p)ASARM peptides significantly inhibited chondrocyte matrix mineralisation without altering the proliferation or differentiation of the chondrocyte cells, or their ability to produce an extracellular matrix. mRNA analysis by qPCR indicted a feedback system by which the pASARM peptide functions to allow the release of further ASARM peptides. Moreover, the pASARM peptide inhibited mRNA expression of markers of vascular angiogenesis highlighting a novel mechanism by which they may inhibit chondrocyte matrix mineralisation. This thesis also determines the regulatory cross-talk between the chondrocytes of the murine growth plate, with the most abundant bone cell type, the osteocyte. This cross-talk inhibits chondrocyte matrix mineralisation and is attributed to sclerostin, an osteocyte-specific secretory protein. Furthermore, it is shown that sclerostin acts through the MEPE-ASARM axis to regulate chondrocyte matrix mineralisation and thus endochondral ossification. The work described herein has characterised and validated in vitro models of growth plate chondrocyte matrix mineralisation and has used these to identify the role of MEPE within chondrocyte matrix mineralisation.