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 a6?1 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 a6?1 integrin. a6?1 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. a4?1 integrin supports adhesion of venous endothelial cells but not of microvascular endothelial cells on immobilized thrombospondin-1 and thrombospondin-2. Chemotactic activities of this region of thrombospondin-1 and thrombospondin-2 are also mediated by a4?1, whereas antagonism of fibroblast growth factor-2-stimulated chemotaxis is not mediated by this region. Immobilized N-terminal regions of thrombospondin-1 and thrombospondin-2 promote endothelial cell survival and proliferation in an a4?1-dependent manner. Soluble a4?1 antagonists inhibit angiogenesis in the chick chorioallantoic membrane and neovascularization of mouse muscle explants. The latter inhibition is thrombospondin-1-dependent and not observed in explants from thrombospondin-1 -/- mice. Antagonizing a4?1 may in part block pro-angiogenic activities of thrombospondin-1 and thrombospondin-2, because N-terminal regions of thrombospondin-1 and thrombospondin-2 containing the a4?1 binding sequence stimulate angiogenesis in vivo. Therefore, a4?1 is an important endothelial cell receptor for mediating motility and proliferative responses to thrombospondins and for modulation of angiogenesis. In addition to the three known ?1 integrin recognition sites in the N-module of TSP1, we found that ?1 integrins mediate cell adhesion to the type 1 and type 2 repeats. The type 1 repeats of TSP1 differ from typical integrin ligands in that recognition is pan-?1 specific. ?1 integrins recognize both the second and third type 1 repeats, and each type 1 repeat shows pan-?1 specificity and divalent cation-dependence for promoting cell adhesion. ?1 integrin expression is necessary for cell adhesion to the type 1 or type 2 repeats, and ?1 integrins bind in a divalent cation-dependent manner to a type 1 repeat affinity column. These results provide a new mechanism for previously reported biological activities of this domain of TSP1. 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. These studies identified the actin cytoskeleton modulators cofilin and hsp27 which show increased phosphorylation and several genes that have altered mRNA expression. Cofilin and hsp27 phosphorylation may mediate the inhibition of endothelial cell motility, which is characteristic of angiogenesis inhibitors, defining a convergent signaling node for modulating angiogenesis.
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