We are investigating the mechanisms by which the adhesive glycoproteins TSP1 and TSP2 regulate tumor growth, metastasis, and angiogenesis. Data from tumor xenograft and transgenic mouse models demonstrate that TSP1 and TSP2 are suppressors of tumor progression. Although the suppressive activity of TSP1 was initially ascribed to inhibition of angiogenesis, we have obtained evidence for significant direct effects of TSP1 on both tumor cells and the host immune response. Using synthetic peptides, recombinant TSP1 fragments, and mutagenesis, we are defining receptors and recognition sequences that mediate activities of TSP1 toward each of these cell types. We are defining the cell-specific signal transduction pathways for multiple TSP receptors using TSP1-null transgenic mice and by identifying genes that are regulated by these TSP1-initiated signals in specific cell types. We have identified peptide sequences in TSP1 that mimic the anti-angiogenic activities of the whole molecule. Stable analogs of these peptides inhibited angiogenesis in several animal models and are being developing for therapeutic applications. We have now identified two additional pro-angiogenic sequences in TSP1 and their endothelial cell receptors. We discovered that a6b1 recognizes both TSP1 and TSP2. A conserved amino acid sequence in the N-terminal domains of TSP1 and TSP2 was identified that is recognized by a6b1 integrin. a6b1 is an adhesion receptor for TSP1 and TSP2 in HT-1080 fibrosarcoma cells and microvascular endothelial cells. This integrin was also found to play an important role in mediating the chemotactic activity of TSP1 and TSP2 for microvascular endothelial cells. To identify downstream targets of angiogenesis inhibitors, we have examined changes in endothelial protein and mRNA expression induced by thrombospondins and three other angiogenesis inhibitors. Several of the proteins identified by this approach are modulators of the actin cytoskeleton. Among these, we demonstrated that alterations in cofilin and hsp27 are due to increased phosphorylation of the proteins. This is associated with changes in their subcellular localization, increased actin stress fibers, increased focal adhesions, and loss of lamellipodia. Thus, cofilin and hsp27 phosphorylation may mediate the inhibition of cell motility which is characteristic of angiogenesis inhibitors, and these actin regulators define a convergent signaling node for modulating angiogenesis. The specific responses of tumor cells, T lymphocytes, and endothelial cells to TSP1 arise from the utilization of distinct combinations of cell surface TSP1 receptors and result in different intracellular signals in each cell type. T lymphocytes interact with TSP1 via CD47, heparan sulfate proteoglycan, and a4b1 integrin. We previously reported that regulation of T cell chemotaxis and matrix metalloproteinase gene expression required a4b1 integrin and indirectly required CD47. We now find that CD47 positively regulates its function. In addition to T cells, we demonstrate that a4b1 integrin in melanoma cells is regulated by CD47 and by CD47-binding peptides from TSP1. This activity involves interaction of CD47 with a4b1 integrin, and the two receptors form a detergent stable complex in the membrane. Unexpectedly, we also discovered that CD47-binding peptides from TSP1 have CD47-independent activities to modulate a4b1 integrin function that depends on an unknown G protein-dependent receptor.
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