Nitric oxide (NO) generated by the endothelial NO synthase (eNOS) regulates vascular tone and endothelial homeostasis to counteract vascular inflammation. The effects of NO are attributable to its interaction with heme-containing proteins e.g., soluble guanylyl cyclase, or to the post-translational modification of proteins i.e., S-nitrosation. It is well known that eNOS is localized to the cell membrane and the Golgi apparatus. However, the protein has also been detected in the endothelial cell nucleus. In this study we assessed the potential role of nuclear eNOS in endothelial cells.

Confocal microscopy studies confirmed the presence of eNOS in the nucleus of unstimulated human and murine endothelial cells and stimulation with vascular endothelial growth factor (VEGF – 50 ng/ml, 10 minutes) elicited eNOS nuclear translocation. Co-precipitation studies coupled with proteomics revealed the association of nuclear eNOS with 81 proteins, most of which are known to be S-nitrosated and involved in RNA binding and processing. One of the S-nitrosated eNOS-interacting proteins was double-stranded RNA-specific adenosine deaminase 1 (ADAR1), an enzyme involved in RNA editing via the deamination of adenosine to inosine in double-stranded RNA (dsRNA). The shRNA-mediated knockdown of eNOS in endothelial cells was associated with an increase in the amount of dsRNA (immunofluorescence). In HEK cells dsRNA content was decreased by introducing either wild-type eNOS or a gain-of-function mutant (eNOS-Y657F), while a loss-of-function mutant (eNOS-Y657D) had no effect. ADAR1-mediated A-I editing (RNA sequencing) was significantly altered in the absence of eNOS, underscoring the importance of the eNOS-ADAR1 interaction and ADAR1 S-nitrosation for ADAR1 function.

In addition to editing, nuclear eNOS affected transcription and the shRNA-mediated knockdown of eNOS in human endothelial cells altered the expression of >4000 genes. Indeed, nuclear eNOS also physically interacted with core components of paraspeckles, specialized nuclear membrane-less organelles involved in the regulation of gene expression. Studies unravelling the exact role of eNOS/S-nitrosation in paraspeckle dynamics are ongoing and will help to elucidate the impact of NO signalling on endothelial cell gene expression.

These results demonstrate that eNOS and NO signalling modulate nuclear processes that are essential for the regulation of endothelial cell gene expression.

Keywords: eNOS, S-nitrosation, ADAR, paraspeckles