Regulation of inducible nitric oxide synthase expression in human cells

Nitric oxide (NO) is a bioactive gas that has multiple roles in innate and adaptive immune responses and dysregulated expression of its production by inducible nitric oxide synthase (iNOS) is implicated in the pathogenesis of various inflammatory diseases and cancer. The mechanisms by which the expression of iNOS is regulated in human cells is incompletely understood. I show that NO positively regulates iNOS expression through a positive feedback mechanism that involves S-nitrosylation and activation of the small Ras GTPase, which in turn activates downstream PI3K/Akt and mTOR pathways. This feedback mechanism acts in a post-translational manner to increase iNOS protein levels by reducing its ubiquitination and proteasomal degradation. In addition to examining the feedback regulation of iNOS protein expression, I also studied how gene expression of this enzyme is controlled. I focused on the hypoxia inducible factor (HIF) because of the central role of this transcription factor in controlling many aspects of cell biology. The human iNOS gene promoter has three predicted HIF binding sites and mutation of one of these sites at -4.9 kb reduced the induction of iNOS gene promoter activity by cytokines. Further, HIF-1a gene editing by CRISPR/Cas9 eliminated HIF activity and reduced the induction of iNOS mRNA and protein expression by cytokines. Altogether, I discovered two mechanisms whereby iNOS expression is positively regulated at the protein and gene expression levels in an inflammatory setting. The role of NO and HIF in augmenting iNOS expression may provide insight into new therapeutic strategies for inflammatory diseases and cancer.