Functional Characterization of Thrombospondin-2 in Bone Extracellular Matrix

描述(由申请人提供)：这份KO1申请表中概述的5年职业发展计划的目标是将候选人Alford博士培养成成骨细胞和细胞外基质生物学领域的一名富有成效的独立研究员。在她的导师Steven A Goldstein和Renny T Franceschi博士的指导下，通过与专家顾问Stephen J.Weiss和Kurt D.Hankenson的定期互动，候选人将作为密歇根大学整形外科系整形外科研究实验室的成员开展拟议中的研究。在这里提出的5年研究计划中，我们将利用从TSP2基因缺失的小鼠及其子代获得的原代MSC，在成熟的成骨细胞体外模型中，测试基质结合的TSP2对成骨细胞系进程的特定贡献这一全球假说，这取决于细胞的分化状态，以及与基质组装、成熟和矿化相关的独特的细胞外环境。具体地说，在目标1中，我们将解决工作假说，即在早期成骨细胞分化事件的背景下，TSP2促进富含I型胶原的成骨细胞来源的细胞外基质的组装，从而间接促进对成骨细胞分化至关重要的胶原依赖的信号转导事件。在特定的目标2中，我们将解决的前提是，在基质矿化的背景下，TSP2对成骨细胞的成熟做出了额外的独特贡献。具体地说，我们假设TSP2被蛋白质降解处理以释放影响基质矿化的生物相关片段。总之，这项提案中概述的实验将确定TSP2对成骨细胞谱系进展和基质矿化的依赖于分化阶段的贡献。

DESCRIPTION (provided by applicant): The objective of the 5 year career development plan outlined in this KO1 application is to foster the development of the candidate, Dr. Alford into a productive, independent investigator in the field of osteoblast cellular and extracellular matrix biology. Under the guidance of her mentors, Drs Steven A Goldstein and Renny T Franceschi, and through regular interactions with expert consultants, Stephen J. Weiss and Kurt D. Hankenson, the candidate will carry out the proposed research as a member of the Orthopaedic Research Laboratories in the Department of Orthopaedic Surgery at the University of Michigan. In the 5 year research plan proposed here, we will utilize primary MSC harvested from TSP2-null mice and their littermates in a well established in vitro model of osteoblastogenesis to test the global hypothesis that matrix-bound TSP2 makes specific contributions to osteoblast lineage progression which are dependent on the differentiation state of the cell, as well as on the unique extracellular milieu associated with matrix assembly, maturation and mineralization. Specifically, in aim 1 we will address the working hypothesis that in the context of early osteoblast-differentiation events, TSP2 promotes the assembly of a type I collagen-rich osteoblast-derived extracellular matrix, and as a result, it indirectly promotes collagen-dependent signal transduction events that are critical for osteoblast differentiation. In specific aim 2, we will address the premise that, in the context of matrix mineralization, TSP2 makes an additional unique contribution to osteoblast maturation. Specifically, we hypothesize that TSP2 is proteolytically processed to release biologically relevant fragments that affect matrix mineralization. Together, the experiments outlined in this proposal will define differentiation-stage dependent contributions that TSP2 makes to osteoblast lineage progression and matrix mineralization.