SPARC (a Secreted Protein Which is Acidic and Rich in Cysteine) was first described as a stress-related protein released in vitro by both malignant and normal cells derived from all primordial germ layers. It was specifically elevated in endothelial cells after """"""""culture shock"""""""" (characterized by high levels of proliferation and migration) and exposure to endotoxin, and was a major product of mouse embryo parietal yolk sac endoderm and of differentiated F9 teratocarcinoma cells. The cDNA sequence revealed 4 domains including a Ca+2-binding EF hand. Cellular responses to injury and/or developmental signals include the concerted expression of selected genes, one of which is SPARC. Since SPARC is secreted at high levels by proliferating and differentiating cells in vivo and in vitro, we propose that specific Ca+2-binding proteins in the extracellular space influence cellular growth. This application is concerned with the function and regulation of SPARC. Domains that bind Cu+2 will be identified by electroblotting of proteolytic fragments, and the role of the EF hand will be examined after mutation and subsequent expression of the altered sequence. In situ hybridization with SPARC antisense mRNA will be performed on dynamic systems of angiogenesis, wound repair, and somitogenesis. Regulation of SPARC at transcriptional/posttranscriptional levels will be determined by Northern blotting of RNA from endothelial cells after exposure to phorbol esters, transforming growth factor beta, and ionophores. The effect of anti-SPARC antibodies on these processes will be determined. With an expression vector containing an inducible promoter, SPARC will be overexpressed in endothelial cells; inhibition of expression will be achieved by transfection of an antisense RNA into proliferating cells. Genomic clones representative of SPARC regulatory regions will be isolated from a human library, and vectors containing Sparc promoter and a reporter DNA will transfected into endothelial cells and fibroblasts. Our ultimate aim is to understand the regulation of cellular growth by secreted, extracellular proteins that function in both developmental and injurymediated processes, with specific emphasis on vascular endothelium.
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