Growth Factor Signaling and Traffic in Angiogenesis

这个实验室的长期目标是阐明成纤维细胞生长因子在体内调节生理和病理事件的机制，特别是那些涉及血管生成的事件。我们最近克隆了果蝇基因Serrate的人类同源物Jagged1，并对其进行了测序。Jagged1参与了细胞命运的决定，方法是通过差异显示人内皮细胞在成纤维细胞生长因子和血管内皮生长因子的存在下暴露于纤维蛋白。跨膜蛋白Jagged1和它的结构相近的同系物Delta通过受体Notch发出信号，这一事件表明这些细胞命运决定因素是无脊椎动物和脊椎动物细胞生长和分化的关键调节因素。事实上，反义实验已经证明了内皮细胞中成纤维细胞生长因子-但不是血管内皮生长因子依赖的反应的特异性增强，并且Jagged1基因缺失的小鼠表现出正常的血管生成但异常的和致命的胚胎血管生成。本实验室扩大了这些研究，以证明非跨膜和可溶性形式的Jagged1在体内的绒毛尿囊膜实验中刺激血管生成，在体外完全抑制I型胶原合成的表达，并且稳定转染Jagged1可溶性形式的NIH 3T3细胞在体外分化途径的中期阶段表现出类似于内皮细胞的I型胶原依赖的弦样结构。此外，通过低细胞种子密度介导的软琼脂集落形成和显著增加Src及其F-肌动蛋白结合蛋白底物Cortactin的酪氨酸磷酸化水平，Fgf可诱导可溶性Jagged1 NIH 3T3细胞发生显著的转化表型。有趣的是，将可溶性Jagged1转染体与主要阴性形式的Src共转染可抑制这些细胞展示I型胶原依赖的弦形成的能力，并恢复I型胶原的生物合成，但不改变FGF1在体外诱导转化表型的能力。相反，在没有外源FGF1的情况下，Delta1基因(一种替代的Notch配体)在NIH 3T3细胞中的表达确实会导致表型转化。由于(I)目前有四个Notch受体基因可以作为可溶性Jagged1依赖反应的信号媒介，(Ii)尚不清楚其他Jagged同源物是否也能够发出类似的生化、生理和病理生理事件的信号，以及(Iii)成纤维细胞生长因子似乎在体外调节Jagged1依赖的细胞转化，我们请求支持延长这些研究，并建议(I)确定负责Jagged1、Jagged2-、Delta1和Delta4介导的信号传递的Notch受体的身份。评估这些配体作为Jagged1依赖反应的潜在激动剂和/或拮抗剂的作用，以剖析导致这些事件的最小锯齿和Delta结构域，以及(Ii)探索锯齿和成纤维细胞生长因子介导的信号之间可能的协同作用的机制。

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.