Thrombospondin-1 (TSP-1) modulates cell adhesion, migration and growth by directing assembly of macromolecular complexes that integrate the signals produced by growth factors, cytokines and extracellular matrix at the cell surface. The long term goal of this project is to integrate in vitro and in vivo data obtained from protein biochemical, cell biological and genetic approaches. The proposal lists three specific aims.
In Specific Aim 1, functional domains in TSP-1 will be mapped by a combination of approaches including (1) protease fagmentation and isolation of functional domains, (2) generation of fusion proteins with functional activity, (3) generation of polyclonal anti-fusion protein antibody that block function, (4) generation of monoclonal antibodies against fusion proteins that block function, (5) preparation of mutated or domain deleted forms of TSP-1, and (6) preparation of synthetic peptides that inhibit the activity of TSP-1.
Specific Aim 2 will characterize the interaction of TSP-1 with bone and muscle cells with respect to its ability to support attachment, spreading, migration and proliferation. Receptors that interact with the carboxy-terminus of TSP-1 from bone and muscle cell lines will be identified by (1) expression cloning, (2) production of monoclonal antibodies to the cell surface that block binding, and (3) affinity chromatography.
In Specific Aim 3, the applicant will determine the effect of TSP-1 deficiency on mice in two homogeneous genetic backgrounds, C57BL/6 and 129/5V. The phenotypic characterization in these studies will focus on (1) blood cell counts, (2) platelet aggregation, (3) phosphorylation of platelet proteins, (4) cartilage and bone formation, (5) histology of the major organ systems, and (6) angiogenesis. TSP-1-deficient mice will be crossed to mice that are deficient in other members of the TSP family. Finally, the role of TSP-1 in tumor vascularization will be evaluated by crossing the TSP-1-deficient mice to P53-deficient mice and mice carrying the MMTV/c-neu transgene.
