The principal objective of this revised SERCA application is to investigate the function of the extra cellular matrix (ECM) protein TSP2 in regulating murine bone formation. TSP2 is a protein with multiple functional domains and mice with a targeted disruption of the TSP2 gene (TSP2-null) have a number of non-lethal abnormalities, including a long-bone phenotype that is characterized by increased endosteal bone production. In association, TSP2-null osteoprogenitors [marrow stromal cells (MSCs)] are increased in number and show increased basic fibroblast growth factor (bFGF)-mediated proliferation. Local mechanisms that control mammalian bone formation are poorly understood; thus the TSP2 null mouse represents a unique model in which to study the function of an ECM protein in the regulation of osteoblast development and subsequent bone formation.
In Specific Aim #1, the candidate proposes studying the in vivo proliferation dynamics and osteoblast synthetic activity of MSCs from TSP2-null mice.
In Specific Aim #2 the role of the TSP2 type I domain in bFGF binding and growth inhibition will be studied. The significance of the multiple domain structure in the function of TSP2 is unknown.
This aim will test whether the type I repeats of TSP2 are necessary to impart negative regulation of bFGF-mediated proliferation. The mechanism by which TSP2 inhibits bFGF-mediated MSC proliferation will be examined in Specific Aim #3, specifically, whether TSP2 binding to bFGF inhibits signaling. In phase two of this proposal (Specific Aim #4), the applicant will generate and evaluate the phenotype of a new murine model. Mice lacking the type I repeats will be generated by replacing the endogenous Thbs2 allele with a Thbs2 gene lacking the coding region for the type I repeats. By studying the phenotype of the """"""""knockin,"""""""" in comparison to the TSP2-null mice, the importance of the type I repeats in the in vivo function of TSP2 will be determined. UW is one of the premier biomedical research institutions in the country and its faculty includes a large group of ECM investigators, a highly-regarded orthopedic research group, and a very active transgenic program headed by Dr. Ladiges (the secondary mentor). The primary mentor, Dr. Bornstein, has been one of the pre-eminent ECM investigators since the early 1970s and his outstanding contributors to the field have been recognized with an NIH MERIT award. The laboratory is currently involved in the generation and analysis of a number of ECM knockout and transgenic mice. The applicant earned a MS from Purdue University, following two years of equine practice, and is in his third year of a PhD program at UW. By completing an analysis of the role of TSP2 in endosteal bone formation, he will master techniques in molecular and cell biology, gene targeting, and mouse pathobiology, directed at the study of ECM biology and orthopedic science. The applicant's career goal is to obtain an academic position as an instructor of comparative morphology and to develop a research program that generates new murine models to study the function of ECM proteins in orthopedic healing and remodeling.
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