The role of Nir2, a lipid-transfer protein, in regulating endothelial cell functions

Lipid transfer proteins regulate the metabolism of phosphoinositides with key roles in cell signaling, membrane and actin dynamics, intracellular trafficking, and diseases. Nir2/PITPNM1 acts as a cellular phosphatidylinositol/phosphatidic acid (PI/PA) exchanger that maintains phosphoinositide signaling at the plasma membrane (PM) and endoplasmic reticulum (ER) membrane contact sites. Here, we assessed the function of Nir2 in human umbilical vein endothelial cells (HUVECs), by analyzing the impacts of Nir2 knockdown (KD) on angiogenesis in vitro, cell viability, proliferation, migration, actin cytoskeletal regulation and vascular endothelial growth factor (VEGF)-mediated downstream cellular signaling pathways. We show that Nir2 KD inhibits angiogenic tube formation in HUVECs, reduces cell viability, proliferation and migration, as well as diminishes actin stress fibers, while Nir2 overexpression increases cell viability and overexpression of an shRNA-resistant Nir2 construct rescues it. Nir2 KD results in decreased activity of AKT and ERK signaling pathways upon VEGF stimulus, plausibly underlying the observed defects in proliferation, migration and angiogenesis. In addition, our interactome analysis confirmed an interaction of Nir2 with the membrane contact site organizer VAPA (vesicle-associated membrane protein-associated protein A), validated by co-immunoprecipitation and co-localization analyses. VAPA KD inhibited angiogenesis similar to that of Nir2, and double KD of the two tended to have even stronger inhibitory effect. A number of other tentative partners of Nir2 were detected; according to STRING analysis, these likely represent indirect interactions driven by a complex with VAPA. The present findings unravel new avenues to understanding the molecular mechanisms by which Nir2 regulates key endothelial functions such as angiogenesis.