The long term goal of this laboratory has been to elucidate the mechanism utilized by FGF to regulate physiologic and pathologic events in vivo especially those involved in angiogenesis. We have recently cloned and sequenced Jagged1, the human homolog of the Drosophila gene, Serrate, involved in the determination of cell fate by a differential display of human endothelial cells exposed to fibrin in the presence of FGF and VEGF. The transmembrane protein, Jagged1, and its close structural homolog, Delta, signal through the receptor, Notch, an event which had implicated these cell fate determinants as key regulators of invertebrate and vertebrate cellular growth and differentiation. Indeed, antisense experiments have demonstrated a specific augmentation of FGF- but not VEGF-dependent responses in endothelial cells and the Jagged1 null mouse displays normal vasculogenesis but abnormal and fatal embryonic angiogenesis. This laboratory has extended these studies to demonstrate that a non-transmembrane and soluble form of Jagged1 stimulates angiogenesis in the chorioallantoic membrane assay in vivo, completely represses the expression of type I collagen synthesis in vitro and NIH 3T3 cells stably transfected with the soluble form of Jagged1 exhibit type I collagen-dependent chord-like structures similar to those presented by endothelial cells during the mid-phase of their differentiation pathway in vitro. In addition, FGF induces a prominent transformed phenotype in the soluble Jagged1 NIH 3T3 cells transfectants as assessed by low cell seed density-mediated colony formation in soft agar and a significant increase in the tyrosine phosphorylation of Src and it F-actin-binding protein substrate, cortactin. Interestingly, cotransfection of the soluble Jagged1 transfectants with a dominant negative form of Src represses the ability of these cells to exhibit a type I collagen-dependent chord formation, and restores type I collagen biosynthesis yet does not alter the ability of FGF1 to induce a transformed phenotype in vitro. In contrast, expression of the Delta1 gene, an alternative Notch ligand, in NIH 3T3 cells does lead to a transformed phenotype in the absence of exogenous FGF1. Because (i) there are presently four Notch receptor genes which may function as signaling mediators of the soluble Jagged1-dependent responses, (ii) it is unclear whether other Jagged homologs are also capable of signaling similar biochemical, physiologic, and pathophysiologic events, and (iii) FGF appears to regulate Jagged1-dependent cell transformation in vitro, we request support to extend these studies and propose (i) to determine the identity of the Notch receptors responsible for Jagged1-, Jagged2-, Delta1- and Delta4-mediated signaling, evaluate these ligands as a potential agonistic and/or antagonistic mediators of Jagged1-dependent responses in order to dissect the minimum Jagged and Delta structural domains responsible for these events and (ii) to explore the mechanism responsible for the putative cooperativity between Jagged- and FGF-mediated signaling.
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