Functional Characterization of Thrombospondin-2 in Bone Extracellular Matrix

描述（由申请人提供）：在这个KO1申请中概述的5年职业发展计划的目标是促进候选人Alford博士在成骨细胞和细胞外基质生物学领域的富有成效的独立研究者的发展。在导师Steven A Goldstein博士和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骨科外科骨科研究实验室的成员开展拟议的研究。在这里提出的5年研究计划中，我们将利用从TSP2缺失的小鼠及其窝代小鼠身上获得的原代间充质干细胞，建立一个成熟的体外成骨细胞形成模型，以验证基质结合的TSP2对成骨细胞谱系进展的特定贡献，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细胞外环境。具体而言，在目标1中，我们将解决在早期成骨细胞分化事件背景下的工作假设，即TSP2促进I型富含胶原的成骨细胞来源的细胞外基质的组装，因此，它间接促进对成骨细胞分化至关重要的胶原依赖性信号转导事件。在具体的目标2中，我们将解决这样一个前提，即在基质矿化的背景下，TSP2对成骨细胞成熟做出了额外的独特贡献。具体来说，我们假设TSP2经过蛋白水解处理以释放影响基质矿化的生物学相关片段。总之，本提案中概述的实验将定义分化阶段依赖于TSP2对成骨细胞谱系进展和基质矿化的贡献。

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.