Nitric Oxide Signaling in Endothelial Cells
Nitric oxide signaling and S-nitrosylation
Nitric oxide (NO) produced by endothelial NO synthase (eNOS) mediates various cellular processes important for endothelial cell functions and regulates vascular response. In addition to conventional NO signaling via the activation of soluble guanylyl cyclase (sGC) to produce cGMP as a second messenger, protein modification by NO via S-nitrosylation has also received considerable attention. S-nitrosylation is the coupling of an NO moiety to a reactive thiol side chain of cysteine to form an S-nitroso-thiol and is considered an important mechanism for dynamic, post-translational regulation of many classes of proteins (Figure 1). A growing body of evidence suggests that eNOS-derived NO can S-nitrosylate specific proteins, influence cellular processes in endothelial cells, and modulate a variety of endothelial cell functions, such as inflammation, apoptosis, permeability, migration, and cell growth (Iwakiri (2011), Nitric Oxide). Our lab investigates protein S-nitrosylation by eNOS-derived NO, cellular processes regulated by S-nitrosylated proteins and the subsequent effects of these cellular processes on endothelial and liver cell functions.
Unique cellular localization of eNOS
Endothelial NOS (eNOS) is unique among the NOS family members by being localized mainly near specific intracellular membrane domains including the cytoplasmic face of the Golgi apparatus and plasma membrane caveolae (Garcia-Cardena et al. PNAS. 1996). The functional significance of this unique localization of eNOS in endothelial cells remains largely unknown (Iwakiri (2011), Nitric Oxide).
Nitric oxide is confined to the region where eNOS is localized
In comparison to the actions of NO-regulating metal-centered processes (activation of sGC and inhibition of cytochrome oxidase) or its interaction with other radicals, the primary biological reactions involving S-nitrosylation of a thiol by NO are thought to require higher concentrations of NO to sustain them in spite of its diffusible nature (Gow et al. (2002), JBC).
To demonstrate the association of localized sites of active NOS with the presence of a concentrated NO pool, we generated red fluorescent protein (RFP)-tagged eNOS constructs and monitored the simultaneous presence of both red (for eNOS localization) and green (DAF-2, for areas of NO generated) fluorescence in transfected COS-7 cells using confocal microscopy (Iwakiri et al. (2006), PNAS). Upon stimulation of the cells with ATP, the highest amount of DAF-2 fluorescence was detected in a perinuclear region overlapping with or adjacent to RFP-tagged eNOS WT. To further confirm that eNOS-derived NO acts locally, we performed identical experiments in cells transfected with an RFP-tagged eNOS-NLS mutant which localizes in the nucleus. As expected, the presence of concentrated NO (imaged by DAF-2) also co-localized in the nucleus. Thus, these data using RFP-tagged eNOS constructs clearly suggest that NO is preferentially channeled to sites in proximity to those showing eNOS activity (Figure 2).
Additionally, we visualized cellular NO distribution generated by NO donors such as S-nitroso-glutathione (GSNO) and S-nitroso-N-acetylpenicillamine (SNAP) (Iwakiri et al. (2004), PNAS). The dye-loaded COS-7 cells showed an increase in fluorescent intensity immediately after adding the NO donor. The fluorescent signal was diffusely distributed throughout the cells, suggesting that DAF is not concentrated on specific organelles in COS-7 cells. Collectively, these observations suggest that eNOS and NO donors provide different spatial NO concentration (Figure 3).
S-nitrosylated Golgi proteins by eNOS (Sangwung, Iwakiri et al. (2012), PLoS ONE)
We identified 9 Golgi proteins target for S-nitrosylation. Focusing on CD147/EMMPRIN/basigin, we investigate how S-nitrosylation regulates CD147 function and how S-nitrosylation of CD147 is involved in arterial remodeling (i.e., thinning) in the splanchnic circulation in cirrhosis with portal hypertension. Furthermore, we are interested in studying how S-nitrosylation of CD147 regulates hepatocyte functions, since hepatocytes are the major cell type that expresses CD147 in the liver.
References
Iwakiri, Y. S-nitrosylation of proteins: A new insight into endothelial cell function regulated by eNOS-derived NO. Nitric Oxide. 2011;25(2):95-101.
Garcia-Cardena,G., Oh,P., Liu, J., Schnitzer, J.E., and Sessa, W.C. Targeting of nitric oxide synthase to endothelial cell caveolae via palmitoylation: implications for nitric oxide signaling. Proc. Natl. Acad. Sci. USA, 1996;93: 6448–6453.
Iwakiri, Y., Satoh, A., Chatterjee, S., Toomre, D.K., Chalouni, C.M., Fulton, D., Groszmann, R.J., Shah, V.H., and Sessa, W.C. Nitric oxide synthase generates nitric oxide locally to regulate compartmentalized protein S-nitrosylation and protein trafficking. Proc. Natl. Acad. Sci. USA. 2006;103:19777–19782.
Gow, A.J., Chen, Q., Hess, D.H., Day, B.J., Ischiropoulos, H., and Stamler, J.S. Basal and stimulated protein S-nitrosylation in multiple cell types and tissues. J. Biol. Chem. 2002; 277: 9637–9640.
Sangwung, P., Greco, T.M., Wang, Y., Ischiropoulos, H., Sessa, W.C., and Iwakiri, Y. Proteomic Identification of S-nitrosylated Golgi Proteins: New Insights into Endothelial Cell Regulation by eNOS-derived NO. PLoS ONE. 2012; 7(2):e31564. PMC3283662