Physiological consequences of ENOS subcellular targeting

Abstract

Anti-inflammatory effects of NO are thought to involve inhibition of the proinflammatory transcription factor, NF-KB. NO represses the nuclear translocation ofNFKB via the S-nitrosylation of its subunits which decreases the expression of target genes including adhesion molecules. We next investigated the significance of subcellular targeting of eNOS to NF-KB signaling induced by proinflammatory cytokines in human aortic endothelial cells (HAECs). We found that in HAECs stimulated with TNFu, LNAME did not influence the expression of ICAM-1 or VCAM-1. In eNOS "knockdown" HAECs, reconstituted with either PM- or Golgi- restricted forms of eNOS, there was no significant effect of endogenously produced NO on the activation of the NF-KB pathway in response to different concentrations and exposure times of TNFu. Similarly, the endogenous production of NO did not influence the phosphorylation of IkBu and Snitrosylation of IKKP or p65. In contrast, higher concentrations of NO, derived from the use of the exogenous NO donor, DETA NONOate, effectively suppressed the expression of ICAM-1NCAM-1 in response to TNFu and induced more S-nitrosylation of IKKP and p65. These results suggest that neither endogenous eNOS nor eNOS location is an important regulatory influence on inflammatory signaling in HAECs via the NF-KB pathway and that higher NO concentrations are required to suppress NF-KB. A third focus of non-cGMP NO signaling was the NADPH oxidases (Nox), a family of transmembrane oxidoreductases that produce superoxide and other reactive oxygen species (ROS). There are 5 family members (Noxl-5) with Nox5 the last of the conventional Nox isoforms to be identified. Nox5 is a calcium-dependent enzyme that does not depend on accessory subunits for activation. Recently, Nox5 was shown to be expressed in endothelial cells in human blood vessels and therefore we investigated whether endogenous levels of NO can influence Nox5 activity and if so to identify the mechanisms involved. We found that endogenous NO was a potent inhibitor of basal and stimulated Nox5 activity and inhibition was reversible with chronic, but not acute exposure to L-NAME. Nox5 activity was reduced by NO donors, iNOS, eNOS and in endothelial cells and cytokine-stimulated smooth muscle cells in a manner proportional to the NO concentration. ROS production was diminished by NO in an isolated enzyme activity assay replete with surplus calcium and NADPH. There was no evidence for NOdependent changes in tyrosine nitration, glutathiolation or phosphorylation of Nox5 and ROS production was not modified by GAPDH. In contrast, there was evidence for the increased nitrosylation of Nox5 as .determined by the biotin-switch assay and mass spectrometry. Four S-nitrosylation sites were identified and of these, mutation of C694 dramatically lowered Nox5 activity, NO-sensitivity and biotin-labeling. Furthermore, coexpression of the denitrosylation enzymes thioredoxin (Trxl) and GSNO reductase (GSNOR) prevented NO-dependent inhibition ofNox5. The potency of NO against other Nox enzymes was Noxl:;::Nox3>Nox5>Nox2 whereas Nox4 was refractory. These results demonstrate that endogenously produced NO can directly S-nitrosylate and inhibit the activity ofNox5. The overall conclusion of these studies is that the amount of NO is the most important variable influencing protein S-nitrosylation and function.