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.

Made available in DSpace on 2014-08-20T14:30:15Z (GMT). No. of bitstreams: 1 Dissertacao_ Marcus_Cristian_Muniz_Conde.pdf: 970527 bytes, checksum: a12676103871857eca97235b488f90c1 (MD5) Previous issue date: 2010-04-30 / Isolate high quality RNA form dental tissues is a most critical step to perform gene expression analysis. In some situations it is impossible to achieve the RNA isolation after tooth extraction, which leads to tooth discarding. Since, the aim of this experiment was to verify the effect of different teeth storage methods in the quality of RNA obtained from freshly extracted third molars. The teeth were randomly divided in five groups according to the temperature and storage time conditions. In control group RNA was isolated immediately after tooth extraction in room temperature. Experimental storage conditions evaluated were: liquid nitrogen, -80°C, -20°C (24h) and 4°C (6h). To RNA isolation, teeth were longitudinally sectioned and then pulp and pre-dentin were submerged in TRIzol®. Semi-quantitative RT-PCR was used to analyze the expression of odontoblast makers (DSPP, DMP1, and MEPE), which were normalized against the GAPDH gene. DSPP, DMP1 and MEPE were amplified in all storage conditions evaluated, regardless of storage method or tissue analyzed. Was possible to obtaining high quality RNA from pulp and dentin in all storage conditions appraised, increasing the RNA available to be used as positive control in cell differentiation studies / Extrair RNA de qualidade dos tecidos dentais é um passo crítico para a realização da análise de expressão gênica. Em algumas situações não é possível realizar o isolamento do material genético dos tecidos dentários logo após a exodontia, o que conduz ao descarte do dente. Assim, o objetivo desse estudo foi avaliar o efeito de diferentes formas de armazenamento dos dentes na qualidade do RNA odontoblástico isolado de terceiros molares recém extraídos. Os dentes foram separados de forma aleatória em cinco grupos de acordo com o tempo e a temperatura de armazenamento. No grupo controle o RNA foi isolado imediatamente após o procedimento cirúrgico em temperatura ambiente.As condições experimentais avaliadas foram: armazenamento dos dentes em nitrogênio líquido, -80°C e -20°C durante 24h e armazenamento 4°C durante 6h. Para a extração do RNA os dentes foram seccionados e então o tecido pulpar e a pré-dentina foram imersos, separadamente, em TRIzol. RT-PCR foi utilizado para analisar a efetividade dos métodos de armazenamento através da amplificação dos marcadores da diferenciação odontoblástica (DSPP, DMP1, e MEPE), que foram normalizados contra o gene constitutivo GAPDH. DSPP, DMP1, e MEPE foram amplificados de forma clara em todas as condições avaliadas, independentente do método de armazenamento, ou do tecido avaliado. Foi possível obter RNA de qualidade em polpa e dentina, em todas as condições de armazenamento avaliadas, aumentando assim a disponibilidade de RNA para ser utilizado como controle positivo em estudos de diferenciação celular

Dans le rachitisme hypophosphatémique lié à l’X (XLH), MEPE (Matrix Extracellular PhosphoglycoprotEin), une protéine non collagénique impliquée dans la biominéralisation, subit un clivage pathologique de son extrémité C-terminale. Les peptides ainsi libérés sont porteurs d’un domaine ASARM (acidic serine- and aspartate- rich motif) très conservé dans l’évolution. ASARM inhibe la réabsorption tubulaire du phosphate et la minéralisation de la matrice extracellulaire osseuse. Précédemment, notre équipe a identifié des taux élevés de ce peptide ASARM dérivé de MEPE dans la dentine issue de patients XLH. Ce travail a pour objectif principal d’étudier l’effet d’ASARM sur la minéralisation dentinaire afin de mieux comprendre son implication dans les anomalies dentaires observées chez les malades. Des lattis de collagène ensemencés avec des cellules souches pulpaires SHEDs (Dental pulp stem cells derived from deciduous teeth) englobés dans une tranche de dent humaine ont été cultivés dans des conditions d’induction odontoblastique avec et sans 20 µM de chacune des formes phosphorylé (p-ASARM) ou non phosphorylé (np-ASARM) du peptide recombinant. La minéralisation a été appréciée par microscopie électronique à balayage et colorations de von Kossa. L’expression des marqueurs odontogéniques (DSPP, ostéocalcine, MEPE) a été évaluée par immunohistochimie, qPCR et Western-blot. Parallèlement, des billes d’agarose imprégnées p-ASARM et np-ASARM ont été implantées dans un modèle d’effraction pulpaire chez le rat, dans lequel un pont de dentine de réparation se forme spontanément. La minéralisation dans la chambre pulpaire a été évaluée par micro-CT et immunohistochimie. Dans le modèle in vitro 3D, p-ASARM a inhibé la différenciation des SHEDs, ce qui s’est traduit par 1) l’absence de formation de nodule de minéralisation, 2) la diminution des marqueurs odontogéniques, 3) la surexpression de MEPE, comparativement au contrôle ou au traitement du milieu par np-ASARM. In vivo, p-ASARM a perturbé le processus de réparation dentinaire et a entrainé une surexpression de MEPE. Ces résultats confirment notre hypothèse selon laquelle p-ASARM inhibe la différenciation odonblastique et la minéralisation de la dentine. De plus, l’effet inducteur de p-ASARM sur l’expression de MEPE suggère l’existence d’une boucle de rétrocontrôle positif impliquée dans l’étiopathogénie du XLH. Ainsi, les défauts de minéralisation de la dentine hypophosphatémique sont probablement une conséquence de la libération du peptide ASARM dans la matrice extracellulaire. / In X-linked familial hypophosphatemic rickets (XLH), MEPE (Matrix Extracellular PhosphoglycoprotEin) is cleaved, releasing phosphorylated ASARM (acidic serine- and aspartate- rich motif) peptides that inhibit mineralization of bone extracellular matrix (ECM), and renal tubular phosphate reabsorption. We recently identified high levels of MEPE-derived ASARM peptides in human XLH dentin. The present study was aimed to investigate their effects on dentin mineralization in order to better understand their role in the etiology of tooth abnormalities observed in XLH patients. Dental pulp stem cells derived from deciduous teeth (SHEDs) were seeded in a collagen scaffold, cultured in human tooth slices under mineralizing conditions as a control, and with 20 µM of either phosphorylated (p-ASARM) or non-phosphorylated (np-ASARM) MEPE-derived ASARM peptides. Mineralization was assessed by scanning electron microscopy and von Kossa staining. Odontogenic markers (DSPP, osteocalcin, MEPE) were assessed by immunohistochemistry, RT-PCR and Western blot. In parallel, agarose beads soaked with recombinant ASARM peptides were implanted in a rat pulp injury model where a reparative dentin bridge is spontaneously formed; the repair process was evaluated by micro-CT and IHC. In the tooth slice culture model, p-ASARM inhibited SHED differentiation, with 1) no formation of mineralization nodule, 2) decreased odontogenic marker expression, and 3) up-regulation of MEPE expression, in contrast with np-ASARM and control. In the rat pulp injury model, p-ASARM impaired the formation of the reparative dentin bridge and increased MEPE expression. The present data support our hypothesis that p-ASARM impairs odontogenic differentiation process and the resulting mineralization of dentin. Moreover, the identification of a stimulating effect of p-ASARM on MEPE expression suggests a positive feedback loop in the pathogenicity of XLH disease. Accordingly, the mineralized defects in XLH tooth dentin may be a direct consequence of the release of ASARM peptides in the ECM.