Mechanisms Driving Apoptosis Resistance in Pulmonary Hypertension

点击翻译按钮获取中文摘要

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 Exciting new data from an unpublished proteomics study using lung lysates demonstrates that AQP1 immunoprecipitates with total caspase-3, a enzymatic protein activated in apoptosis which is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.