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

分项目1涉及对研究单位具有核心意义的三个信号因子：骨形态发生蛋白-2、转化生长因子-β和Smad8 L+MH2(代表生物活性因子领域的创新成分的转录因子的基因工程版本)。第二个资助期的主要目标是重点研究非常规因子Smad8、L+MH2的生物学作用机制。此外，子项目1将与子项目2一起产生和鉴定用于修饰植入物的重组蛋白质。总而言之，子项目1致力于生物因子分级释放的时间和空间方面，设计了它们的量化策略，并通过与其他子项目，特别是P3至P5的反馈和相互作用，促进了时间和空间释放的优化。计划的体外研究的重点是来自骨髓的原代人MSCs，此外，细胞系C3H10T 1将用于特定的应用。为了加深我们对Smad8的激活和肌腱细胞诱导分化能力的了解，我们将对Smad8+MH2与BMP2细胞联合进行慢病毒修饰，并在体外和体内通过异位植入的方法研究其分化情况。此外，可溶的和分泌的蛋白质，例如来自细胞外基质的蛋白质，将被用于表征该转录因子的激活。所选转录因子的肌腱细胞诱导能力将通过肌腱调节蛋白启动子-荧光素酶结构的激活来评估。随后将结合具有肌腱形成能力的转录因子进行优化。此外，在第一个资助期对miRNAs的筛选发现了肌腱细胞形成的有趣的候选者，这将被进一步表征。一个新增加的工作包用于表征蛋白质与Smad1中特定短域(只有39个氨基酸)的相互作用，然而，这在与我们研究单位相关的Smad8版本中是缺失的。此外，还将阐明Smad8和Smad1之间的蛋白质-蛋白质相互作用(所有工作包AP1-1)。在AP1-2工作包中，与子项目2密切合作，将进一步克隆和鉴定一个细胞透性的可溶性Smad8 L+MH2(带蛋白转导结构域，Ptd)。此外，还将产生BMP-2变体，以分别实现靶向荧光修饰和优化与细胞外基质的相互作用。通过报告基因分析确定所有重组蛋白的生物学功能活性，将完善蛋白质及其变体的特征。在AP1-3中，将进行人骨髓来源的MSCs的细胞相容性和诱导分化的重点研究。

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.