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

信号因子 BMP2、TGF-β3、Smad8 L MH2 的生物学和功能

• 批准号: 269986331
• 负责人: Professorin Dr. Andrea Hoffmann
• 金额: --
• 依托单位: Arbeitsgruppe Biologische Grundlagen für Biohybridimplantate
• 依托单位国家: 德国
• 项目类别: Research Units
• 财政年份: 2015
• 资助国家: 德国
• 起止时间: 2014-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/269986331?language=en
• 关键词: Biology; function; signaling; factors; BMP2

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.

子项目1涉及与研究单位相关的三种信号传导因子：骨形态发生蛋白-2（BMP-2），转化生长因子（TGF）-β和Smad 8 L+ MH 2（一种基因工程形式的转录因子，代表生物活性因子领域的创新成分）。第二个资助期的主要目标是关注非常规因子Smad 8 L+ MH 2的生物学作用机制。此外，子项目1将与子项目2一起产生和表征用于修饰植入物的重组蛋白。总之，子项目1致力于生物因子分级释放的时间和空间方面，设计其量化策略，并通过与其他子项目，特别是P3至P5的反馈和相互作用来优化时间和空间释放。除了计划的体外研究之外，还有来自骨髓的原代人MSC，此外，细胞系C3 H10 T1/2将用于特定应用。为了增强我们对Smad 8的激活和Smad 8与BMP 2组合的肌腱细胞诱导能力的理解，将对L+ MH 2细胞进行慢病毒修饰，并在体外以及通过体内异位增殖研究它们的分化。此外，可溶性和分泌性蛋白质e。例如，在一个实施例中，将用于表征该转录因子的活化。将通过激活腱调节蛋白启动子-荧光素酶构建体来评估所选转录因子的腱细胞诱导能力。随后将结合具有肌腱形成能力的转录因子进行优化。此外，在第一个资助期内对miRNAs的筛选揭示了肌腱细胞形成的有趣候选者，这将进一步表征。一个新增加的工作包用于表征蛋白质与Smad 1内特定短结构域（仅39个氨基酸）的相互作用，然而，Smad 8版本与我们的研究单位相关。此外，还将阐明Smad 8和Smad 1之间的蛋白质-蛋白质相互作用（所有工作包AP 1 -1）。在工作包AP 1 -2中，将与子项目2密切合作，进一步克隆和表征细胞可渗透的可溶性Smad 8 L+ MH 2（具有蛋白转导结构域，PTD）。此外，将产生BMP-2变体以分别允许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功能活性将完成蛋白及其变体的表征。在AP 1 -3中，将重点研究人骨髓源性MSC的细胞相容性和诱导分化。