Biology and function of the signaling factors BMP2, TGF-beta3, Smad8 L+MH2

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Subproject 1 deals with three signaling factors with central relevance for the research unit: bone morphogenetic protein-2 (BMP-2), transforming growth factor (TGF)-beta and Smad8 L+MH2 (a genetically engineered version of a transcription factor representing an innovative component in the field of bioactive factors). Major aim in the second funding period is to focus on the biological mechanisms of action of the non-conventional factor Smad8 L+MH2. In addition, subproject 1 will – together with subproject 2 – generate and characterize recombinant proteins for modification of implants. In summary, subproject 1 is dedicated to the temporal and spatial aspects of graded release of biological factors, devises strategies for their quantification, and contributes to optimization of the temporal and spatial release via feedback and interaction with the other subprojects, in particular P3 to P5.Pivotal to the planned in vitro-investigations are primary human MSCs from bone marrow, in addition the cell line C3H10T½ will be used in specific applications. In order to enhance our understanding of the activation of Smad8 and the tendon cell-inducing capacity of Smad8 L+MH2 in combination with BMP2 cells will be lentivirally modified and their differentiation investigated in vitro as well as by ectopic implantations in vivo. Additionally, soluble and secreted proteins e. g., from the extracellular matrix, will be used for characterization of the activation of this transcription factor. The tendon cell-inducing capacity of selected transcription factors will be evaluated by activation of a tenomodulin promoter-luciferase construct. Transcription factors with tendon-forming capacity will subsequently be combined for optimization. Additionally, a screening of miRNAs during the first funding period revealed interesting candidates for tendon cell formation which will be further characterized. A newly added work package serves to characterize protein interactions with a particular short domain (only 39 amino acids) within Smad1 which is, however, missing in the Smad8 version relevant for our research unit. In addition protein-protein interactions between Smad8 and Smad1 will be elucidated (all working packages AP1-1). Within working package AP1-2, in close collabo¬ration with subproject 2, further cloning and characterization of a cell-permeable soluble Smad8 L+MH2 (with protein transduction domain, PTD) will be performed. Also, BMP-2 variants will be generated to allow targeted fluorescence modification and optimized interaction with the extracellular matrix, respectively. The determination of biological-functional activity of all recombinant proteins via reporter gene analyses will coomplete the characterization of proteins and variants thereof. Within AP1-3 focussed investigations of cytocompatibility and induction of differentiation of human bone marrow-derived MSCs will be performed.