title: Positive feedback regulation of human inducible nitric-oxide synthase expression by Ras protein S-nitrosylation.
abstract: The production of nitric oxide (NO) by inducible NO synthase (iNOS) regulates many aspects of physiology and pathology. The expression of iNOS needs to be tightly regulated to balance the broad ranging properties of NO. We have investigated the feedback regulation of cytokine-induced iNOS expression by NO in human cells. The pharmacological inhibition of iNOS activity reduced iNOS protein levels in response to cytokine stimulation in a human epithelial cell line (A549 cells) as well as in primary human astrocytes and bronchial epithelial cells. The addition of exogenous NO using a NO donor prevented the reduction in iNOS levels caused by blockade of iNOS activity. Examination of signaling pathways affected by iNOS indicated that NO S-nitrosylated Ras. Transfection of cells with a S-nitrosylation-resistant Ras mutant reduced iNOS protein levels, indicating a role for this Ras modification in the amplification of iNOS levels. Further, the induction of iNOS protein levels correlated with the late activation of the phosphatidylinositol 3-kinase/Akt and mammalian target of rapamycin (mTOR) pathways, and inhibition of these signaling molecules reduced iNOS levels. Altogether, our findings reveal a previously unknown regulatory pathway that amplifies iNOS expression in human cells.
authors.affiliation: Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia V5A 1S6, Canada.
authors.forename: Martin
authors.initials: M
authors.lastname: Lee
authors.forename: Jonathan C
authors.initials: JC
authors.lastname: Choy
mesh_terms: D002460:Cell Line; D004790:Enzyme Induction / Q000502:physiology; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D009569:Nitric Oxide / Q000235:genetics / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II / Q000096:biosynthesis* / Q000235:genetics; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D058570:TOR Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; D058570:TOR Serine-Threonine Kinases; D018631:ras Proteins; C496319:NOS2 protein, human; C546842:MTOR protein, human
keywords: Nitric Oxide; Nitric-oxide Synthase; Ras; S-Nitrosylation; Signal Transduction