Release of FGF1 and the Pathology of Angiogenesis

FGF1 的释放和血管生成的病理学

• 批准号: 6383803
• 负责人: THOMAS MACIAG
• 金额: $ 29.04万
• 依托单位: MAINEHEALTH
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-01-01 至 2005-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6383803
• 关键词: angiogenesis; biological signal transduction; cell proliferation; exocytosis; fibroblast growth factor; intracellular transport; laboratory mouse; laboratory rabbit; protein structure function; protein transport; secretion; secretory protein; stress; synaptotagmin; vascular endothelium

The long-term goal of this laboratory has been to elucidate the mechanism by which fibroblast growth factor (FGF) 1 regulates the migration and proliferation of human endothelial cells in vitro and to apply this information to augment angiogenic responses during tissue and organ repair in vivo. While the recent application of the FGF prototypes for the generation of human collateral vessel growth in response to ischemic cardiovascular damage argues for the validity of this premise, structure-function studies with the members of the FGF gene family suggest that our understanding of FGF function is still primitive. Indeed, the function of two novel structural features within the FGF prototypes are not well understood and these include the presence of a functional nuclear/nucleolar localization signal(s) and the absence of a classical signal peptide sequence to direct its secretion through the conventional ER-Golgi apparatus-mediated pathway. Interestingly, these features are also found within members of the interleukin (IL)1 gene family which shares structural and crystallographic similarities with members of the FGF gene family. Since FGFs must gain access to the extracellular compartment to signal through their high affinity receptor tyrosine kinases in order to promote a biological response and undergo receptor-dependent nuclear/nucleolar translocation, we have utilized the resources from this award to define the mechanism by which FGF1 is released. Indeed, we have demonstrated during the current funding period that FGF1 but not FGF2 is released in response to biological stresses including heat shock, hypoxia, and serum deprivation in an apoptotic-independent manner. Structure- function analysis of the stress-induced FGF1 release pathway has demonstrated that FGF1 is exported into the extracellular compartment as a latent Cys30 FGF1 homodimer. The FGF1 homodimer is a component of a non-covalent high molecular weight complex which includes the extravesicular domain (p40) of the exocytotic trafficking-protein, p65 synaptotagmin (Syt1) and the annexin (Anx)2-binding protein, S100A13. Interestingly, like FGF1 and IL1alpha, these proteins also lack a classical signal peptide sequence and each is able to associate with acidic phospholipids including phosphatidylserine. In addition, we present preliminary data suggesting that the pathway responsible for the release of the mature but not the precursor form of the signal peptide-less pro-inflammatory cytokine, IL1alpha, is similar to the pathway utilized by FGF1 and that the precursor domain of IL1alpha acts as a dominant negative for the stress-induced release of FGF1. As a result, we request support to confirm and expand these results and propose to further define the mechanism of FGF1 release by (i) defining the structural prerequisites for the release of FGF1 and (ii) characterizing the cellular mechanisms by which these polypeptides facilitate FGF export. We suggest that these studies may not only provide new insight into the regulation of angiogenesis but may also enable access to novel pharmacologic targets for potential therapeutic management in man.

本实验室的长期目标是阐明成纤维细胞生长因子1在体外调节人内皮细胞迁移和增殖的机制，并将这一信息应用于增强体内组织和器官修复过程中的血管生成反应。尽管最近将成纤维细胞生长因子原型应用于人类侧支血管生长以应对缺血性心血管损伤，证明了这一前提的有效性，但对成纤维细胞生长因子基因家族成员的结构-功能研究表明，我们对成纤维细胞生长因子功能的理解仍然是原始的。事实上，成纤维细胞生长因子原型中两个新的结构特征的功能尚不清楚，其中包括存在一个功能性的核/核仁定位信号(S)，以及缺乏一个经典的信号肽序列来引导其通过传统的ER-高尔基体介导的途径分泌。有趣的是，在白介素1基因家族的成员中也发现了这些特征，该家族在结构和晶体结构上与成纤维细胞生长因子基因家族的成员有相似之处。由于FGFs必须进入细胞外室，通过其高亲和力受体酪氨酸激酶发出信号，以促进生物反应和经历受体依赖的核/核仁转位，我们利用该奖项的资源来定义FGF1释放的机制。事实上，在目前的资助期间，我们已经证明了FGF1而不是FGF2是以一种不依赖于细胞凋亡的方式在热休克、低氧和血清剥夺等生物应激反应中被释放的。对应激诱导的FGF1释放途径的结构-功能分析表明，FGF1以潜伏的Cys30 FGF1同源二聚体的形式输出到细胞外腔。FGF1同源二聚体是一个非共价的高分子复合体的组成部分，它包括胞吐运输蛋白的囊外结构域(P40)、突触素(Syt1)和膜联蛋白(ANX)2结合蛋白(S100A13)。有趣的是，像FGF1和IL1α一样，这些蛋白质也缺乏经典的信号肽序列，并且每个蛋白质都能够与包括磷脂酰丝氨酸在内的酸性磷脂结合。此外，我们提供的初步数据表明，负责释放成熟但不是信号肽的促炎细胞因子IL1α的途径类似于FGF1所利用的途径，并且IL1α的前体结构域在应激诱导的FGF1的释放中起主导作用。因此，我们请求支持来确认和扩展这些结果，并建议通过(I)定义FGF1释放的结构先决条件和(Ii)表征这些多肽促进成纤维细胞生长因子输出的细胞机制来进一步定义FGF1释放的机制。我们认为，这些研究不仅可能为血管生成的调控提供新的见解，而且还可能为人类潜在的治疗管理提供新的药理靶点。