Vascular cell migration and VHL gene function in flies

果蝇血管细胞迁移和 VHL 基因功能

• 批准号: 6478299
• 负责人: TIEN HSU
• 金额: $ 14.3万
• 依托单位: MEDICAL UNIVERSITY OF SOUTH CAROLINA
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-01 至 2004-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6478299
• 关键词: Drosophilidae; arthropod genetics; cell migration; chimeric proteins; developmental genetics; gene induction /repression; gene interaction; gene targeting; genetically modified animals; green fluorescent proteins; hypoxia; hypoxia inducible factor 1; invertebrate embryology; larva; protein protein interaction; recombinant proteins; tumor suppressor genes; tumor suppressor proteins; yeast two hybrid system

Angiogenesis is an integral part of solid tumor growth and holds promise as a target for effective therapeutics with limited deleterious side effects. Various anti-angiogenic therapies have focused on the growth factors, their cognate receptors or metalloproteinase inhibitors. These therapies, however, have not been overwhelmingly successful. Additional studies on angiogenic mechanisms can help identify new therapeutic targets. One interesting unconventional angiogenic regulator is the von Hippel- Lindau tumor suppressor gene (VHL). Mutations in the VHL tumor suppressor gene predispose individuals to highly vascularized tumors. We have recently isolated the Drosophila homolog of VHL (D-VHL) and showed that it preserved the anti-angiogenic function in the Drosophila tracheal system, which provides an accessible and relevant in vivo model for studying vascular tubule formation and cell migration. The prevailing model suggests that VHL may function as a subunit of a ubiquitin ligase, which facilitates degradation of its targets. Among its proposed targets is the alpha-subunit of hypoxia-inducible factor 1alpha (Hif-1alpha) that is a transcriptional activator of the vascular-endothelial growth factor (VEGF) and is highly unstable in normal oxygen condition (normoxia). Thus, the hyper-vascularized phenotype of VHL mutant can be explained by an up-regulation of Hif-1alpha in normoxia. Thus, the hyper-vascularized phenotype of VHL mutant can be explained by an up-regulation of Hif-1alpha in normoxia, resulting in over-expression of the potent angiogenic factor VEGF. This interesting model has not been fully validated in vivo, however. Our recent work on D-VHL further suggests that it can modulate cell migration, and perhaps cell division, by directly regulating the components of the actin filaments and microtubules. These cytoskeletal components may constitute a new battery of anti-angiogenic targets. We have also devised a promising new assay system and a provocative conjecture for analyzing the relationship between VHL and Hif-1alpha in vivo. Finally, our recent results suggest that VHL is a multi-functional and may regulate cell division. These results also indicate that VHL may be more than a ubiquitination enzyme as suggested by the current model. In order to formulate a thorough and focused research project, we need to first accomplish three short-term goals, summarized in three Specific Aims in this exploratory study (R21); (1) To determine if the Drosophila Hif-1alpha activity is regulated by hypoxia in vivo. (2) To assess the relevance of the potential D-VHL interacting proteins and complete the ongoing yeast 2-hybrid screen for D-VHL interacting proteins. (4) Generating genomic D-VHL mutant.

血管生成是实体瘤生长的一个组成部分，并且有望成为具有有限有害副作用的有效治疗的靶点。各种抗血管生成疗法集中在生长因子、其同源受体或金属蛋白酶抑制剂上。然而，这些疗法并没有取得压倒性的成功。对血管生成机制的进一步研究可以帮助确定新的治疗靶点。一个有趣的非传统的血管生成调节因子是von Hippel-Lindau肿瘤抑制基因（VHL）。VHL肿瘤抑制基因的突变使个体易患高度血管化的肿瘤。我们最近分离了果蝇VHL同源物（D-VHL），并表明它保留了果蝇气管系统中的抗血管生成功能，这为研究血管小管形成和细胞迁移提供了一个可访问的和相关的体内模型。流行的模型表明，VHL可能作为一个亚基的泛素连接酶，这有利于降解其目标。在其提出的目标是缺氧诱导因子1 α（Hif-1 α）的α-亚基，它是血管内皮生长因子（VEGF）的转录激活因子，在正常氧条件下（常氧）高度不稳定。因此，VHL突变体的过度血管化表型可以解释为在常氧条件下HIF-1 α的上调。因此，VHL突变体的过度血管化表型可以通过常氧条件下Hif-1 alpha的上调来解释，从而导致强效血管生成因子VEGF的过度表达。然而，这个有趣的模型尚未在体内得到充分验证。我们最近对D-VHL的研究进一步表明，它可以通过直接调节肌动蛋白丝和微管的组分来调节细胞迁移，也许还可以调节细胞分裂。这些细胞骨架成分可能构成一个新的电池的抗血管生成的目标。我们还设计了一个有前途的新的检测系统和一个挑衅性的猜想，用于分析VHL和HIF-1 α在体内的关系。最后，我们最近的研究结果表明，VHL是一个多功能的，并可能调节细胞分裂。这些结果也表明，VHL可能是一个泛素化酶比目前的模型所建议的。为了制定一个全面和集中的研究项目，我们需要首先完成三个短期目标，总结在本探索性研究的三个具体目标（R21）：（1）确定果蝇Hif-1 α活性是否受体内缺氧的调节。(2)评估潜在的D-VHL相互作用蛋白的相关性，并完成正在进行的酵母双杂交筛选D-VHL相互作用蛋白。(4)产生基因组D-VHL突变体。