The main objective of the project SaMGP was to investigate the role of the matrix Gla protein (MGP) in the mechanisms of extracellular matrix (ECM) mineralization and cell differentiation. MGP exact function is still unclear - possibly calcium binding - and its role as a negative regulator of the ECM mineralization has still to be confirmed. Our strategy, to investigate the role of MGP during mineralization, was based on a multidisciplinary approach which took advantage of recently developed bone-derived cells from the gilthead seabream [Sparus aurata] termed VSa13. The suitability of these cells for our project was demonstrated by experiments showing that (1) they express MGP gene in standard culture conditions, (2) they are able to mineralize their ECM and (3) MGP gene expression is regulated during ECM mineralization. Based on these preliminary results, our objectives were to (1) characterize extracellular calcium effect on the regulation of MGP gene expression by identifying calcium sensing mechanisms, transcription regulatory elements and signal transduction pathways and (2) evaluate the effect of altered MGP levels (increased or decreased production) on ECM mineralization and specific gene expression.
Main achievements of SaMGP project are: (1) the characterization of VSa13 bone-derived cell lines (i.e. culture conditions for ECM mineralization, pattern of bone-related gene expression and fine analysis of MGP gene expression during mineralization), (2) the optimization of DNA delivery into VSa13 cells (i.e. the evaluation of commercially available reagents, the development of an optimized method and the susceptibility of VSa13 cells to antibiotics for stable transfection), (3) the identification of regulators of MGP gene expression (i.e. the characterization of the effect of calcium, ascorbic acid, fibroblast growth factor, retinoic acid and various other agents on MGP gene expression), (4) the identification of the mechanisms mediating regulators effect (i.e. the characterization of regulators effect on MGP transcription, the cloning of SaMGP promoter, the development of a wide set of MGP promoter constructs and the identification of responsive elements and binding factors), (5) the effect of altered MGP levels on ECM mineralization (i.e. characterization of the effect of MGP over-expression and silencing on ECM mineralization), (6) the effect of altered MGP levels on other bone-related gene expression (i.e. through the construction of a DNA macroarray then the utilization of a DNA microarray containing thousands of seabream ESTs and developed within the scope of the Marine Genomics Europe Network of Excellence (MGE NoE), and (7) the effect of increased OC, BMP-2 and IGF levels on MGP gene expression.