Activin A Suppresses Osteoblast Mineralization Capacity by Altering Extracellular Matrix (ECM) Composition and Impairing Matrix Vesicle (MV) Production

During bone formation, osteoblasts deposit an extracellular matrix (ECM) that is mineralized via a process involving production and secretion of highly specialized matrix vesicles (MVs). Activin A, a transforming growth factor-β (TGF-β) superfamily member, was previously shown to have inhibitory effects in human bone formation models through unclear mechanisms. We investigated these mechanisms elicited by activin A during in vitro osteogenic differentiation of human mesenchymal stem cells (hMSC). Activin A inhibition of ECM mineralization coincided with a strong decline in alkaline phosphatase (ALP 1 ) 1The abbreviations used are:ALPalkaline phosphataseBCAbicinchoninic acidDAVIDDatabase for Annotation, Visualization and Integrated DiscoveryDEXdexamethasoneDMEM/F12-FlexDulbecco's Modified Eagle Medium: nutrient mixture F-12ECMextracellular matrixFBSfetal bovine serumFDRfalse discovery rateGOGene OntologyhMSChuman Mesenchymal Stem CellsIPIinternational protein indexMVmatrix vesiclePBSphosphate buffered salinePiinorganic phosphateSILACstable isotope labeling by amino acids in cell cultureTGF-βtransforming growth factor-β. activity in extracellular compartments, ECM and matrix vesicles. SILAC-based quantitative proteomics disclosed intricate protein composition alterations in the activin A ECM, including changed expression of collagen XII, osteonectin and several cytoskeleton-binding proteins. Moreover, in activin A osteoblasts matrix vesicle production was deficient containing very low expression of annexin proteins. ECM enhanced human mesenchymal stem cell osteogenic development and mineralization. This osteogenic enhancement was significantly decreased when human mesenchymal stem cells were cultured on ECM produced under activin A treatment. These findings demonstrate that activin A targets the ECM maturation phase of osteoblast differentiation resulting ultimately in the inhibition of mineralization. ECM proteins modulated by activin A are not only determinant for bone mineralization but also possess osteoinductive properties that are relevant for bone tissue regeneration.