Mechanisms Driving Apoptosis Resistance in Pulmonary Hypertension

项目总结 尽管有可用的治疗方法，肺动脉高压(PAH)仍然是一个致命的诊断。多环芳烃是 以广泛的肺血管重塑为特征的，涉及血管闭塞的形成 病变和血管壁增厚的内侧层，两者都含有肺动脉平滑肌 细胞(PASMC)。已证实，从已建立的PAH大鼠模型中分离出的PASMC是 在基础和刺激条件下都能抵抗细胞凋亡。细胞膜蛋白水通道蛋白1 (AQP1)最初被描述为一条水路运输通道，但最近被牵连到其他 包括迁移和增殖在内的细胞功能，以及在几种不同的癌症类型中，都与 具有抗细胞凋亡作用。从大鼠PAH模型分离的PASMC中，AQP1表达上调，表明AQP1是一种 恶性疾病模型，尽管水通道蛋白1‘S在细胞凋亡抵抗中的确切作用尚不清楚。令人振奋的新数据来自 一项未发表的使用肺裂解物的蛋白质组学研究表明，AQP1免疫沉淀与总 Caspase-3，一种激活细胞凋亡的酶蛋白，它被转移到启动细胞的细胞核 死亡。在利用生物素连接酶技术进行的邻近研究中，我已经证明了AQP1和总caspase- 3在活细胞中非常接近。此外，在电子计算机分析中，AQP1蛋白揭示了3个潜在的 Caspase-3裂解位点，这为这种蛋白质-蛋白质相互作用提供了机制。总而言之，这些数据 提示AQP1与caspase-3相互作用，在Caspase-3和AQP1之间提供了一种新的关系 级联反应，以及对细胞凋亡的抵抗。这个应用程序提供了一种培训工具，帮助我探索 AQP1在PAH中调节细胞凋亡的机制。AIM 1旨在确定 AQP1上胞浆caspase-3裂解位点(S)是AQP1/caspase-3相互作用所必需的，AIM 2的作用是 评估AQP1对Capase-3核定位的影响，最终目标3将确定是否增加 AQP1是抵抗细胞凋亡所必需的和/或充分的。用来实现这些目标的技术 包括但不限于蛋白质表达和定点突变、生物素连接酶邻近分析 免疫共沉淀、PAH动物模型及原代细胞分离、免疫荧光和共聚焦 显微镜、细胞核/胞质分裂、caspase-3/7活性测定、Hoechst染色和 TUNEL染色。该项目的完成将为AQP1和AQP1之间的相互作用提供新的见解 Caspase-3和AQP1在细胞凋亡抵抗中的作用以及为新的治疗提供新途径 目标。在本研究的设计和实施过程中获得的技能以及实验结果将提供 获得K奖的必要基础，以及作为独立职业者开始职业生涯的良好平台 资助的临床医生科学家专注于PAH。从这项工作中获得的见解也可能产生更多的影响 在任何诊断中，细胞对凋亡的抵抗对疾病的发展是必不可少的。

PROJECT SUMMARY Pulmonary arterial hypertension (PAH) remains a fatal diagnosis despite available therapies. PAH is characterized by extensive remodeling of the pulmonary vasculature involving the formation of vaso-occlusive lesions and a thickened medial layer of the vascular wall, both of which contain pulmonary arterial smooth muscle cells (PASMCs). It has been demonstrated that PASMCs isolated from a well-established rat model of PAH are resistant to apoptosis under both basal and stimulated conditions. The cell membrane protein aquaporin 1 (AQP1) was initially described as a water transport channel, but more recently has been implicated in other cellular functions including migration and proliferation, and in several distinct cancer types, has been associated with apoptosis resistance. AQP1 is upregulated in PASMCs isolated from rat models of PAH suggesting a ‘quasi- malignant’ disease model, although AQP1’s exact role in apoptosis resistance is unclear. Exciting new data from an unpublished proteomics study using lung lysates demonstrates that AQP1 immunoprecipitates with total caspase-3, a enzymatic protein activated in apoptosis which is translocated to the nucleus where it initiates cell death. In proximity studies utilizing biotin ligase techniques, I have demonstrated that AQP1 and total caspase- 3 come within close proximity in live cells. Furthermore, in silico analysis of the AQP1 protein reveals 3 potential caspase-3 cleavage sites, which provide a mechanism for this protein-protein interaction. Together, these data suggest that AQP1 interacts with caspase-3, providing a novel relationship between AQP1, the caspase cascade, and resistance to apoptosis. This application serves to provide a training vehicle as I explore a potential mechanism by which AQP1 regulates apoptosis during PAH. Aim 1 is designed to determine whether the cytosolic caspase-3 cleavage site(s) on AQP1 are necessary for AQP1/caspase-3 interaction, Aim 2 serves to evaluate the impact of AQP1 on nuclear localization of capsase-3, and finally Aim 3 will establish if increased AQP1 is necessary and/or sufficient to confer apoptosis resistance. Techniques utilized to address these aims include but are not limited to protein expression and site directed mutagenesis, biotin ligase proximity assays, co-immunoprecipitation, animal models of PAH and primary cell isolation, immunofluorescence and confocal microscopy, nuclear/cytosolic fractionation, luminescent caspase-3/7 activity assay, Hoechst staining and TUNEL staining. Completion of this project will provide novel insight into the interaction between AQP1 and caspase-3 and the role for AQP1 in apoptosis resistance as well as provide a novel pathway for new therapeutic targets. The skills obtained in the design and execution of this study and the experimental results will provide the necessary foundation for a K award and an excellent platform on which to start a career as an independently funded clinician scientist focused on PAH. Insights gained from this work also could have implications beyond PAH to any diagnosis in which cellular resistance to apoptosis is essential to the development of disease.