Growth Factor Signaling and Traffic in Angiogenesis

本实验室的长期目标是阐明FGF调节体内生理和病理事件的机制，特别是参与血管生成的机制。 我们最近克隆和测序锯齿状蛋白1，人类同源的果蝇基因，锯齿状，参与细胞命运的决定通过差异显示的人内皮细胞暴露于纤维蛋白在FGF和VEGF的存在下。 跨膜蛋白，锯齿蛋白1，和它的密切的结构同源物，三角洲，信号通过受体，刻缺，一个事件，其中涉及这些细胞命运的决定因素作为无脊椎动物和脊椎动物细胞生长和分化的关键调节。 事实上，反义实验已经证明了一个特定的增强成纤维细胞生长因子，但不是血管内皮细胞生长因子依赖的反应和锯齿状蛋白1无效小鼠显示正常的血管生成，但异常和致命的胚胎血管生成。 该实验室已经扩展了这些研究，以证明Jagged 1的非跨膜和可溶性形式在体内绒毛尿囊膜测定中刺激血管生成，在体外完全抑制I型胶原合成的表达，并且用可溶性形式的Jagged 1稳定转染的NIH 3 T3细胞表现出I型胶原依赖性索样结构，其类似于内皮细胞在生长中期呈现的索样结构。其体外分化途径的阶段。 此外，FGF诱导可溶性锯齿状1 NIH 3 T3细胞转染子中的显著转化表型，如通过在软琼脂中低细胞种子密度介导的集落形成和Src及其F-肌动蛋白结合蛋白底物corpine的酪氨酸磷酸化的显著增加所评估的。 有趣的是，可溶性锯齿状蛋白1转染子与显性阴性形式的Src的共转染抑制这些细胞表现出I型胶原依赖性腱形成的能力，并恢复I型胶原的生物合成，但不改变FGF 1诱导体外转化表型的能力。 相反，在NIH 3 T3细胞中，Delta 1基因（一种替代Notch配体）的表达在不存在外源性FGF 1的情况下确实导致转化的表型。 因为（i）目前有四种Notch受体基因可作为可溶性Jagged 1依赖性应答的信号传导介质，（ii）尚不清楚其他Jagged同源物是否也能够信号传导类似的生物化学、生理学和病理生理学事件，以及（iii）FGF似乎在体外调节Jagged 1依赖性细胞转化，我们请求支持扩展这些研究，并提出（i）确定负责Jagged 1-、Jagged 2-、Delta 1-和Delta 4-介导的信号传导的Notch受体的身份，评估这些配体作为锯齿蛋白1依赖性反应的潜在激动性和/或拮抗性介质，以剖析负责这些事件的最小锯齿蛋白和δ结构域，以及（ii）探索负责Jagged和FGF介导的信号传导之间的假定协同性的机制。

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.