Novel Mechanism regulating Aquaporin 1 abundance in Pulmonary Arterial Smooth Muscle cells

Pulmonary arterial hypertension (PAH) is a deadly disease characterized by remodeling of the pulmonary vasculature including vascular medial layer hypertrophy and formation of vaso-occlusive lesions, both of which contain pulmonary arterial smooth muscle cells (PASMCs). We have demonstrated that PASMCs from a well-established rat model of disease have increased expression of the membrane protein aquaporin 1 (AQP1) which is associated with increased cell migration, proliferation, and resistance to apoptosis. These pathologic behaviors are mitigated by decreasing AQP1 expression. Understanding the mechanism(s) driving increased AQP1 levels could lead to novel therapeutic targets that treat and potentially reverse disease. Upregulation of AQP1 is a hypoxia inducible factor-1 – related event, while degradation of the protein occurs via ubiquination and the proteasome. Via in silico analysis, we identified two possible caspase-3 cleavage sites followed by a distal ubiquination site near the 3’ end of the AQP1 cytosolic tail. We hypothesized that caspase-3 cleavage results in loss of the AQP1 ubiquination site, allowing for persistence of the cell membrane protein and its pathologic properties. Methods: A proximity-based BioID assay was performed in human embryonic kidney (HEK 293) cells as they do not express native AQP1, using the AQP1 construct alone or AQP1 fused to biotin ligase. Cells were transfected, incubated with cell-permeable biotin (50 μM), lysed, and biotinylated proteins precipitated and immunoblotted for caspase-3. Next the assay was repeated using adenoviral constructs with caspase-3 fused to biotin ligase at the c-terminal end (CTcas) or n-terminal end (NTcas) to ensure biotin ligase did not interfere with enzyme activation and/or activity. Primary PASMCs isolated from distal pulmonary arteries of rats were obtained via removal of the endothelium and enzymatic digestion of the tissue to obtain a pure smooth muscle population. PASMCs were infected with the BioID caspase constructs or wild-type caspase (WTcas) as a control, and precipitated biotinylated proteins were immunoblotted for AQP1. Lastly, PASMCs were cultured, pre-treated with the irreversible caspase-3 inhibitor DEVD or vehicle (30 min) then treated with PBS or the caspase-3 stimulus hydrogen peroxide (500 μM for 24 h). Cells were collected, lysed and immunoblotted for AQP1. Results: The BioID assays demonstrated an interaction between AQP1 and caspase-3 in live cells. Primary PASMCs treated with hydrogen peroxide demonstrate more AQP1 than controls, an effect which is lost when caspase-3 activity is blocked. Conclusions: These data suggest a novel mechanism to control AQP1 protein abundance in PASMCs. Further studies to elucidate the caspase-3 cleavage site necessary for this interaction are underway. Similar studies using PASMCs from animal models of PAH disease are warranted to further validate this potential novel therapeutic target. T 32 HL 007534; F32 HL165766-01 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.