Thrombospondin (TS) is a regulator of the adhesion, migration and proliferation of a number of cell types. Exogenous TS inhibits the migration and proliferation of endothelial cells in response to angiogenic agents such as basic FGF. TS also inhibits angiogenesis in three in vivo assays. Until now, the only known product of the TS gene (on chromosome 15) has been platelet TS, a trimer of 180 KDa subunits. TSp180 has multiple domains for binding matrix proteins, glycans and at least 4 cellular receptors. Another product of the TS gene, generated by alternative splicing, has been characterized using cDNA cloning and PCR of RNA (cDNA). PCR of genomic DNA established the intron-exon structure of the TS gene in the region of splicing, and sequencing of the amplified DNA confirmed the presence of consensus splice sites and a polyadenylation/cleavage signal for the new mRNA. The protein product is designated TSp70 (Mr=70 KDa) and contains the first 356 amino acids of 1152 residue TSpl80 grafted onto a unique 97 residue C terminal tail. Antipeptide antibodies against the unique region identify TSp70 in a number of human cell lines and tissues. Further experiments have identified a group of RNA species, one of which appears to encode the human homolog of gpl40 (TSpl40). This hamster TS isoform, which lacks the N terminal 293 residues of TSpl80, has been identified as an endogenous inhibitor of angiogenesis. This proposal is aimed at (1) characterization of the cellular localization, tissue distribution and function of TSp70; (2) identification and characterization of the TS isoforms encoded by the gpl40-like RNA species, and (3) determination of the factors that regulate TS isoform expression during development, neoplasia and angiogenesis. These include soluble factors such as bFGF and TGFbeta, matrix components and products of tumor suppressor genes. These studies will result in a better understanding of the distribution and function of these newly described TS isoforms, and in particular their regulation during angiogenesis, both in the cells eliciting neovascularization (tumors, new tissue) and in the responding endothelial cells. These data will also help to elucidate the suspected role of TS as an autocrine agent in endothelial growth and differentiation.
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