Functional Characterization of Thrombospondin-2 in Bone Extracellular Matrix

描述（由申请人提供）：本KO1申请中概述的5年职业发展计划的目标是促进候选人阿尔福德博士发展成为成骨细胞和细胞外基质生物学领域的高效独立研究者。在导师Steven A Goldstein博士和Renny T Franceschi博士的指导下，并通过与专家顾问Stephen J.韦斯和Kurt D.候选人Hankenson将作为密歇根大学矫形外科系矫形研究实验室的一员进行拟议的研究。在这里提出的5年研究计划中，我们将在成骨细胞生成的良好体外模型中利用从TSP2缺失小鼠及其同窝小鼠收获的原代MSC来测试以下总体假设：基质结合的TSP2对成骨细胞谱系进展做出特定贡献，这取决于细胞的分化状态，以及与基质组装相关的独特细胞外环境，成熟和矿化。具体而言，在目标1中，我们将解决工作假设，即在早期成骨细胞分化事件的背景下，TSP 2促进I型胶原丰富的成骨细胞衍生的细胞外基质的组装，因此，它间接促进胶原依赖性信号转导事件，这对成骨细胞分化至关重要。在具体目标2中，我们将解决的前提是，在基质矿化的背景下，TSP 2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是蛋白水解处理释放生物相关的片段，影响基质矿化。总之，本提案中概述的实验将定义TSP2对成骨细胞谱系进展和基质矿化的分化阶段依赖性贡献。

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.