Introduction: Phosphatase 1 Nuclear Targeting Subunit (PNUTS, official gene name PPP1R10) is a nuclear protein described to act as a binding platform for the PP1 phosphatase complex. PNUTS has recently found to be implicated in cell cycle, cell proliferation in cancer cells and protection against DNA damage response and apoptosis. PNUTS expression is markedly repressed in the heart during aging.
Hypothesis: Our objective is to reveal the potential role of PNUTS in the endothelium and determine the mechanism by which PNUTS regulates endothelial cell function.
Methods: We used siRNAs to knock-down PNUTS in human umbilical vein endothelial cells (HUVECs) and studied the effect of PNUTS repression in vitro. As an in vivo model, we found the constitutive deletion of PNUTS to be embryonically lethal, therefore we generated an endothelial-specific inducible PNUTS-deficient mouse line (Cdh5-CreERT2;PNUTSfl/fl).
Results: We confirmed that PNUTS is repressed by cell culture-induced senescence in HUVECs. PNUTS silencing in HUVECs increases apoptosis and senescence and decreases proliferation and angiogenic sprouting. We also studied the state of endothelial barrier function and found that endothelial cell-cell interaction is severely impaired in PNUTS-silenced cells. Using RNA Immunoprecipitation Sequencing (RIP-Seq) in HUVECs, we have found that PNUTS binds lncRNAs and mRNAs related with angiogenesis and endothelial cell junctions. In vivo, Cdh5-CreERT2;PNUTSfl/fl mice were treated with tamoxifen to induce endothelial-specific KO of PNUTS. Two weeks later, KO mice presented distress, peritoneal ascites and premature death. Aortic-ring sprouting assays showed a dramatic decrease of angiogenic capability of endothelial cells in KO mice. Histology revealed pulmonary edema and a renal uremic phenotype, indicative of vascular leakage in multiple organs. We performed RNA-Seq analysis of lung tissue, revealing significant changes in the expression of genes related with cell-cell junctions in KO animals.
Conclusions: This work suggests that PNUTS is essential for correct endothelial cell junction function. Ongoing work is aimed at determining the mechanism by which PNUTS regulates cell-cell interaction in endothelial cells.