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Pearce, L. L., Gandley, R. E., Han, W., Wasserloos, K., Stitt, M., Kanai, A. J., McLaughlin, M. K., Pitt, B. R., and Levitan, E. S. (2000). Role of metallothionein in nitric oxide signaling as revealed by a green fluorescent fusion protein. Proc. Natl. Acad. Sci. USA 97, 477–482. Schwarz, M. A., Lazo, J. S., Yalowich, J. C., Allen, W. P., Whitmore, M., Bergonia, H. A., Tzeng, E., Billiar, T. R., Robbins, P. D., Lancaster, J. R., Jr., and Pitt, B. R. (1995). Cytoplasmic metallothionein overexpression protects NIH 3T3 cells from tert‐butyl hydroperoxide toxicity. Proc. Natl. Acad. Sci. USA 92, 4452–4456. Spahl, D. U., Berendji‐Grun, D., Suschek, C. V., Kolb‐Bachofen, V., and Kroncke, K. D. (2003). Regulation of zinc homeostasis by inducible NO synthase‐derived NO: Nuclear metallothionein translocation and intranuclear Zn2þ release. Proc. Natl. Acad. Sci. USA 100, 13952–13957. St. Croix, C. M., Wasserloos, K. J., Dineley, K. E., Reynolds, I. J., Levitan, E. S., and Pitt, B. R. (2002). Nitric oxide–induced changes in intracellular zinc homeostasis are modulated by metallothionein/thionein. Am. J. Physiol. Lung Cell Mol. Physiol. 282, L185–L192. St. Croix, C. M., Stitt, M. S., Leelavanichkul, K., Wasserloos, K. J., Pitt, B. R., and Watkins, S. C. (2004). Nitric oxide mediated signaling in endothelial cells as determined by spectral fluorescence resonance energy transfer. Free Radic. Biol. Med. 37, 785–792. Stamler, J. S., Singel, D. J., and Loscalzo, J. (1992). Biochemistry of nitric oxide and its redox‐activated forms. Science 258, 1898–1902. Ting, A. Y., Kain, K. H., Klemke, R. L., and Tsien, R. Y. (2001). Genetically encoded fluorescent reporters of protein tyrosine kinase activities in living cells. Proc. Natl. Acad. Sci. USA 98, 15003–15008. Zaccolo, M., De Giorgi, F., Cho, C. Y., Feng, L., Knapp, T., Negulescu, P. A., Taylor, S. S., Tsien, R. Y., Pozzan, T. (2000). A genetically encoded, fluorescent indicator for cyclic AMP in living cells. Nat. Cell Biol. 2, 25–29.
[27] Nitric Oxide Is a Signaling Molecule that Regulates Gene Expression By LORNE J. HOFSETH, ANA I. ROBLES, MICHAEL G. ESPEY , and CURTIS C. HARRIS Abstract
Nitric oxide (NO) is a dynamic and bioreactive molecule that can both participate in and inhibit the genesis of disease. Its ability to have an impact on a wide range of physiological events stems from its capacity to reversibly alter the expression of specific genes and the activities of a wide range of proteins and signaling pathways. Yet, NO remains an enigmatic molecule. Recently developed technologies, including gene‐chips, two‐dimensional electrophoresis, RNA interference, matrix‐assisted laser desorption ionization (MALDI)‐TOF (time-of-flight) mass spectrometry, and protein arrays will allow us to better understand how NO and associated reactive METHODS IN ENZYMOLOGY, VOL. 396 Copyright 2005, Elsevier Inc. All rights reserved.
0076-6879/05 $35.00 DOI: 10.1016/S0076-6879(05)96027-8
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nitrogen species (RNS) regulate both physiology and disease states, toward the development of treatments using NO synthase inhibitors or NO donors. Introduction
In 1998, Drs. Ignarro, Furchgott, and Murad received the Nobel Prize in physiology or medicine for their discoveries concerning NO as a signaling molecule in the cardiovascular system. An explosion of studies followed that have revealed insight into the many roles of NO in normal and disease processes. A characteristic of NO is that it can act as both a ‘‘friend’’ and a ‘‘foe.’’ NO has been considered in the treatment of cardiovascular and respiratory diseases, cancer, and erectile dysfunction. However, nitrosative stress can also play a role in the etiology and progression of many diseases, including cardiovascular disease, cancer, asthma, neurodegeneration, obesity, and diabetes. NO has these many faces because the specific effects of NO exposure change with NO levels, the microenvironment in which it acts (including the presence of other free radicals), and the genetic background of the host/tissue (Espey et al., 2000a; Hofseth et al., 2003a; Thomas et al., 2004; Wink and Mitchell, 1998). Each of these factors will dictate the impact of NO on cell signaling, gene expression, and disease outcome. Nitric Oxide Biochemistry
NO is endogenously formed by a family of enzymes called NO synthases (NOSs), which use L‐arginine as a substrate and molecular oxygen and NADPH as cofactors (Alderton et al., 2001; Marletta, 1994). Because NOS enzymes are responsible for triggering NO signaling cascades, an elaborate network of control mechanisms is available to regulate NOS levels and activity. NOSs are controlled at the transcriptional, posttranscriptional, and translational levels, as well as through posttranslational modifications, protein–protein interactions, and subcellular localization. NOSs are active as homodimers, and there are four isoforms. Two are Ca2þ‐dependent [NOS1 (neuronal NOS) and NOS3 (endothelial NOS)] and constitutively expressed, whereas the Ca2þ‐independent isoform (NOS2 or iNOS) requires induction. There are, however, some exceptions to this general scheme, as NOS1 and 3 have been shown to be inducible (Forstermann et al., 1995), and iNOS is expressed constitutively in some tissues [e.g., bronchus and ileum (Guo et al., 1995; Hoffman et al., 1997)]. Ca2þ‐dependent neuronal (NOS1) and endothelial isoforms (NOS3) produce low levels of NO that range from picomolar to nanomolar concentrations. In contrast, iNOS produces a sustained NO concentration in the
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micromolar range (Beckman et al., 1990; Espey et al., 2000b; Malinski et al., 1993). The fourth isoform, mitochondrial NOS (mtNOS), has only recently been defined (Elfering et al., 2002; Haynes et al., 2003; Lacza, 2003; Nisoli, 2003). It appears that mtNOS could be the alpha form of NOS1, but with posttranslational modifications (Elfering et al., 2002). Its roles in physiology and pathology have been reviewed (Haynes et al., 2003). Nitric Oxide Signaling
NO is an efficient signaling molecule because (1) it can affect a wide range of cells in its vicinity; (2) it can react with and reversibly generate other free radicals [collectively known as reactive nitrogen species (RNS)] that have their own set of bioreactive properties; (3) it, along with its byproducts (RNS), can reversibly and irreversibly posttranslationally modify proteins and lipids; and as evidence suggests here, (4) it has the ability to alter gene transcription in a discriminatory fashion. Because of their ubiquitous nature, RNS are also unlikely to interact with a single signaling pathway. Although this latter observation means that RNS can influence the expression of many genes (detailed later in this chapter), the pattern of changes is very complicated and often depends on the levels, the microenvironment, and the genetic background of the cell, tissue, or host. The outcome, then, comprises a wide range of physiological and pathological effects observed for RNS that differ, depending on the experimental system (Espey et al., 2001, 2002b; Thomas et al., 2002; Wink and Mitchell, 1998). RNS signaling can broadly be divided into cyclic guanosine monophosphate (cGMP)–dependent and –independent pathways (Fig. 1). In cGMP‐dependent pathways, small fluxes of RNS can bind to the heme iron of soluble guanylate cyclase (sGC) and activate it, which stimulates cGMP formation (Hobbs and Ignarro, 1996; Ignarro, 1992; Stamler, 1994). cGMP then targets a series of proteins (‘‘effector proteins’’), including cGMP‐dependent protein kinases (PKG) I and II (resulting in substrate phosphorylation), cyclic nucleotide–regulated ion channels (resulting in increased ion flux), and phosphodiesterases [resulting in cGMP and/or cyclic adenosine monophosphate (cAMP) hydrolysis]. The regulation of gene expression by cGMP has been detailed elsewhere (Pilz and Casteel, 2003) and, therefore, will not be covered here. cGMP‐independent pathways are generally mediated by larger fluxes of RNS (Wink and Mitchell, 1998), which may damage DNA directly and reversibly or irreversibly damage posttranslationally modified proteins (Beckman, 1996) and lipids (Baker et al., 2004), leading to a dynamic
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FIG. 1. Nitric oxide signaling pathways.
change in the activity of signaling pathways. Depending on the conditions used, different RNSs (e.g., nitrosoperoxycarbonate, peroxynitrite, and nitrogen dioxide) may be formed. For example, peroxynitrite (ONOO) may form via the reaction between NO and superoxide (Beckman and Koppenol, 1996). ONOO can oxidize protein sulfhydryl groups (DeMaster et al., 1995; Kuhn and Geddes, 1999; Radi et al., 1991) and nitrate peptides at tyrosine residues (Beckman, 1996). The presence of nitration is not necessarily indicative of the intermediacy of ONOO. Nitration can also occur
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through the oxidation of nitrite, which forms nitrogen dioxide (NO2). A comparison between ONOO and NO2 showed the latter was a much more efficient effector species (Espey et al., 2002a). ONOO formation requires maintenance of a precise balance between NO and superoxide (O 2 ), whereas nitrite oxidation can occur through various pathways without such spatial and temporal limitations. Because nitration has been shown to influence protein activity (Kuhn and Geddes, 1999; Schopfer et al., 2003), the nitrotyrosine antibodies have been useful as a biomarker of nitrosative stress. Nitrosative stress has been shown to occur in many conditions such as chronic inflammation, cancer, and cardiovascular diseases (Maeda and Akaike, 1998; Turko and Murad, 2002). In addition to nitration, a potential key signaling mechanism is the formation of NO adducts on protein nucleophiles (such as thiols, amines, and alcohols). This may occur via nitrosation by the RNS dinitrogen trioxide (N2O3) formed during the autoxidation of NO (Espey et al., 2001). Alternatively, NO adducts may form through oxidative nitrosylation (Espey et al., 2002b). Improvements in the analysis of nitrosated residues suggest that they may play a more ubiquitous role in physiology than previously thought (Bryan et al., 2004). A key quality of RNS in cell signaling is their ability to both irreversibly and reversibly modify the structure and activity of key signaling proteins (see abbreviated list in Table I). Sometimes this modification results in protein activation, and sometimes it results in protein deactivation. For example, RNS have been shown to both inhibit (Sommer et al., 2002) and activate phosphatases, kinases, and other signaling proteins, including transcription factors and repair proteins (Chen and Wang, 2004; Hofseth et al., 2003b; Klatt et al., 1999; Kibbe et al., 2000; Laval and Wink, 1994; Lin et al., 2003; Mateo et al., 2003; Namkung‐Matthai et al., 2000; Reynaert et al., 2004; Sandau et al., 2001; So et al., 1998; Wink and Laval, 1994; Zhou et al., 2000). Although it is unclear why a particular protein is activated while another is deactivated after posttranslational modification, the types of modifications and the mechanisms of these changes are beginning to be understood. For example, NO can donate electrons and react with iron, copper, and zinc, leading to the formation of metal‐nitrosyl complexes (Ford and Lorkovic, 2002). Because many enzymes, transcription factors, and other proteins (e.g., iron of the heme moiety of sGC and hemoglobin; enzyme sulfide clusters; zinc‐finger proteins) have a transition metal component, the interaction of NO with transition metals is fundamental to NO signaling. Some messenger RNA (mRNA) binding proteins also regulated by transition metals can be functionally modulated by RNS.
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TABLE I SELECTED MOLECULES THAT INTERACT WITH NITRIC OXIDE IN CANCER, CARDIOVASCULAR DISEASE, AND NEURONAL PATHOPHYSIOLOGY
Cancer
Cardiovascular disease
Heme oxygenase‐1 HIF‐1 Estrogen receptor VEGF
Heme oxygenase‐1 HIF‐1 Estrogen receptor Beta adrenergic receptors PPAR G‐protein–coupled receptors Kinases Phosphatases Actin Light chain myosin Fibrinogen Kinases Antioxidant enzymes
Kinases Phosphatases Bax p53 Caspases PPAR Microtubules Histones Antioxidant enzymes
Neurotransmission/ neurodegeneration Heme oxygenase‐1 Parkin Estrogen receptor Antioxidant enzymes ‐amyloid Tau protein Synaptophysin GADPH Acetylcholine Dopamine Myelin Presenilin Huntington disease protein ‐synuclein
Other modifications resulting in either loss or gain of protein function include nitration of tryptophan and tyrosine side‐chains (by ONOO or NO2, described earlier). Key proteins shown to be nitrated include albumin, Cu,Zn superoxide dismutase (SOD), cytochrome P450, iNOS, histone, ‐tubulin, and actin (Schopfer et al., 2003). One must take caution, however, in evaluating the significance of RNS adducts formed when proteins are exposed outside the context of the intact cell or tissue. Using two‐dimensional electrophoresis and MALDI‐TOF mass spectrometry, Aulak et al. (2001) showed approximately 31 proteins nitrated in lipopolysaccharide (LPS)‐treated rats, including many proteins not previously recognized as heavily nitrated by RNS, providing evidence for the presence of extensive protein nitration in vivo. S‐nitrosylation (incorporation of a NO group in cysteine thiols), S‐nitrosation, methionine sulfoxidation (oxidation of methionine residues), carbonyl formation at lysine and arginine residues, dityrosine formation, formation of disulfide bonds, and S‐thiolation (S‐glutathionylation; protein mixed disulfide formation between cysteine and GSH) are other posttranslational modifications that play a central role in cell signaling (Klatt and Lamas, 2000). Because of their ability to activate and deactivate both kinases and phosphatases, RNS can influence the phosphorylation
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status of key phosphoproteins such as p53 (Hofseth et al., 2003b; Schneiderhan et al., 2003; Thomas et al., 2004) and pRb (Hofseth et al., unpublished observation; Radisavljevic, 2004). pRb and p53 are cancer suppressor proteins, so this observation is critical to the understanding of RNS in carcinogenesis. Until recently, we have only been able to study the influence of RNS on a protein‐by‐protein basis and test a limited amount of that protein’s function. With the development of high‐throughput technologies such as RNA interference (RNAi), DNA chips, two‐dimensional electrophoresis, MALDI‐ TOF mass spectrometry, protein arrays, and other techniques, we now have the ability to study the expression, modifications, and binding properties of thousands of genes, or the proteins they encode, following RNS exposure. Nitric Oxide and Gene Expression
When released from inflammatory cells or produced endogenously by epithelial, endothelial, or neuronal cells, NO targets a large protein pool. Until the development and use of DNA microarrays, it had been difficult to broadly study the gene expression changes associated with RNS exposure. This technological breakthrough, combined with the more recent development of protein chips, MALDI‐TOF, and RNAi, offers a unique opportunity to uncover pathways altered by RNS that may affect cellular phenotype. At present, relatively few studies in the NO field have reported use of these technologies. A survey of the peer‐reviewed literature yields four studies looking at the effects of RNS on global gene expression in bacteria or plant systems (Firoved et al., 2004; Huang et al., 2002; Ohno et al., 2003; Polverari et al., 2003), three* studies using mammalian cells exposed to RNS (Hemish et al., 2003; Li et al., 2004; Zamora et al., 2002), and two studies that examine the effects of RNS on gene expression in intact rats (Wang et al., 2002) or mice (Okamoto et al., 2004). Although it has long been known that there is an extensive list of individual proteins affected by RNS (abbreviated list in Table I), as will be shown, a key observation arising from microarray studies is that RNS have discriminative properties. For this review, we wanted to tease out trends in gene expression profile arising from the three published data sets using cultured mammalian cells. Although all three studies used different microarray platforms for analysis, we can generalize a few interesting observations. First, there is a consistently * After this paper went to press, two additional references appeared in the literature examining the influence of nitric oxide on gene expression in mammalian cells (Li et al., 2004; Turpaev et al., 2005).
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low percentage of genes that changed significantly in expression after exposure to RNS. Hemish et al. (2003) found 560 of approximately 10,000 genes (5%) to be significantly changed (upregulated or downregulated) after exposure of mouse NIH‐3T3 cells to NO (250 M of the NO donor, SNAP). Zamora et al. (2002) found 200 of approximately 6500 genes (3%) significantly changed (upregulated or downregulated) after exposure of mouse hepatocytes to NO (infection with adenovirus expressing human iNOS). We have characterized p53‐dependent apoptotic signaling associated with NO exposure (Li et al., 2004). Overall, we found 358 (3.9%) of 9180 genes significantly changed (upregulated or downregulated) in TK6 (p53 wild‐type) human lymphoblastoid cells exposed to NO (390 mol pure NO gas). Second, even though at first glance there is very limited overlap in individual gene expression changes, a more detailed analysis reveals that RNS exposure modulates similar signal transduction pathways in all three experimental systems. Some of the conserved changes include alteration of expression of kinase and phosphatases, heat shock proteins, cyclins, zinc‐finger proteins, transcription factors, cell energy (ATP)–related proteins, apoptosis, serine proteinase inhibitors, and members of the ubiquitin family (data not shown). Third, more extensive overlap is found within the same species and type of exposure. Direct comparison of genes significantly changed by RNS exposure in the two studies examining murine cells (Hemish et al., 2003; Zamora et al., 2002) yields 12 genes in common (Table II). Upon methodically searching for mouse genes with homology to human genes significantly changed in our study (Li et al., 2004) using HomoloGene (http://www. ncbi.nlm.nih.gov/entrez/query.fcgi), only nine genes were found to overlap with those of Hemish et al. (2003), even though both used exogenous NO as the exposure route (Table III). Again, some of the conserved changes include alteration of kinase and phosphatase expression, heat shock proteins, cyclins, zinc‐finger proteins, transcription factors, cell energy (ATP)–related proteins, apoptosis, serine proteinase inhibitors, and members of the ubiquitin family. Additionally, there was commonality in extracellular matrix proteins (integrins) that were affected by RNS exposure in both mouse and human cells. Perhaps not surprisingly, the least amount of overlap was found between our study (Li et al., 2004) and the published data set from Zamora et al. (2002), which differ in both species and route of exposure. There were only three genes in common: ribonucleotide reductase M1 polypeptide, proliferating cell nuclear antigen (PCNA), and heme oxygenase‐1 (HO‐1). As shown in Tables II and III, the only two proteins with significantly changed expression in all three studies using mammalian cells exposed to
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signaling and gene expression TABLE II GENES SIGNIFICANTLY CHANGED IN BOTH PUBLISHED STUDIES EXAMINING THE EFFECTS OF RNS ON MURINE CELL GENE EXPRESSIONa Description
Proliferating cell nuclear antigen Heme oxygenase‐1 Macrophage migration inhibitory factor Thymidylate synthase
CD81 antigen Adrenomedullin Cystein and glycine‐rich protein 1 Eukaryotic translation initiation factor 3 Glutamate‐cysteine ligase, catalytic subunit
Keratin 19 Ferritin light chain 1 Adaptor protein complex AP‐1, 1‐subunit
Hemish et al.
Zamora et al.
Increase (1.3‐fold at 12 h) Increase (2.4‐fold at 12 h) Increase (1.9‐fold at 16 h) Increase (max 1.3‐fold at 0.5 h) and decrease (max 2.6‐fold at 0.75 h) Increase (1.5‐fold at 16 h)b Increase (1.4‐fold at 8 h) Increase (1.5‐fold at 4 h) Decrease (1.5‐fold at 48 h) Increase (1.8‐fold at 12 h) and decrease (1.2‐fold at 48 h) Increase (1.4‐fold at 4 h) Increase (1.5‐fold at 16 h) Decrease (1.14‐fold at 12 h)
Increase (5.9‐fold) Increase (6.2‐fold) Increase (2‐fold) Increase (4.2‐fold)
Decrease (40‐fold) Increase (3.1‐fold) Increase (5.2‐fold) Decrease (6.5‐fold) Decrease (6‐fold)
Increase (4.2‐fold) Decrease (2.8‐fold) Increase (2‐fold)
a
Italicized text indicates that the gene was significantly changed in human TK6 cells exposed to NO gas (Li et al., 2004). b Initial slight decrease (1.2‐fold) at 0.75 h.
NO were PCNA and HO‐1. Both of these genes were increased after exposure in every case. PCNA was first recognized in 1981 as a nuclear antigen associated with cell proliferation and blast transformation in the sera of patients with systemic lupus erythematosus (Takasaki et al., 1981). First used as a proliferation marker, it has now been shown that PCNA functions as a sliding clamp protein of the DNA polymerase complex, is intimately involved in DNA repair, and is an executive molecule controlling critical cellular decision pathways (Matunis, 2002; Paunesku et al., 2001).
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TABLE III GENES SIGNIFICANTLY CHANGED IN BOTH PUBLISHED STUDIES EXAMINING THE EFFECTS OF EXOGENOUS RNS ON MAMMALIAN CELL GENE EXPRESSIONa Description
Hemish et al.
Li et al.
Heme oxygenase‐1 Proliferating cell nuclear antigen Hypoxia inducible factor 1‐alpha Cyclin E2 Solute carrier family 3 (activators of dibasic and neutral amino acid transport) member 2 b GADD45‐ c p21Cip1/Waf1 d MDM2 Tumor necrosis factor receptor superfamily, member 6
Increase (2.4‐fold at 12 h) Increase (1.3‐fold at 12 h)
Increase (2.1‐fold at 24 h) Increase (2.3‐fold at 24 h)
Increase (1.3‐fold at 8 h)
Increase (1.9‐fold at 12 h)
Increase (1.6‐fold at 12 h) Increase (1.6‐fold at 12 h)
Increase (2.2‐fold at 24 h) Increase (1.8‐fold at 24 h)
Increase (8‐fold at 16 h) Increase (11‐fold at 16 h) Increase (8‐fold at 16 h) Decrease (2.2‐fold at 8 h)
Increase Increase Increase Increase
(3.2‐fold at 12 h) (6‐fold at 24 h) (3.6‐fold at 24 h) (3.3‐fold at 24 h)
a
Italicized text indicates that the gene was significantly changed in mouse hepatocytes overexpressing human inducible nitric oxide synthase (Zamora et al., 2002). b Induced also in a study by Li et al. (2004). c Induced also in studies by Li et al. (2004) and Turpaev et al. (2005). d Induded also in a study by Li et al. (2004).
HO‐1 has previously been found to be induced by oxidative or nitrosative stress, cytokines, and other mediators produced during inflammatory processes (Bouton and Demple, 2000; Terry et al., 1999; Vile et al., 1994). HO‐1 is involved in the regulatory defense system that leads to the production of mediators that modulate the inflammatory response. For example, it controls intracellular levels of ‘‘free’’ heme (a prooxidant), produces biliverdin (an antioxidant), improves nutritive perfusion via CO release, and fosters the synthesis of the Fe‐binding protein ferritin (Bauer and Bauer, 2002). HO‐1 activity results in the inhibition of oxidative damage and apoptosis, with significant reductions in inflammatory events (Alcaraz et al., 2003). An obvious reason for the low overlap in individual gene expression affected by RNS from study to study is the use of different microarray platforms, which can lead to gene representation bias. However, more relevant physiological reasons for the lack of consistency can reflect previous observations that the response to RNS depends on the microenvironment, the genetic background, and the type and level of RNS exposure (Hofseth et al., 2003a).
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Such conditions were also different in the three published studies we examined here. It would be interesting to compare the response of the same cell type to different types of RNS and exposure methods or different mammalian cell types to the same RNS and exposure methods in carefully controlled experiments. Such studies have yet to be published, but we are continuing our efforts to delineate the response to RNS in human cells. Two studies have examined the influence of NO on global gene expression in animals. Wang et al. (2002) examined cells from the thoracic aorta in rats receiving RNS through nitroglycerin infusion. They found that 447 of approximately (11.6%) 3531 genes were significantly changed (upregulated or downregulated). Okamoto et al. (2004) observed that 106 of (0.9%) 12,451 genes were significantly changed in lung tissues from iNOSþ/þ versus iNOS/ mice exposed to LPS. Only one gene (aldehyde dehydrogenase) was consistently changed in association with RNS in both studies, which again underscores the observation that RNS affects physiology differently, based on the levels used, the surrounding microenvironment, and the genetic background of the tissue/host. Conclusion
RNS are a ubiquitous and complicated group of free radicals that reversibly and irreversibly posttranslationally modify proteins and dynamically alter the activity of cell signaling pathways. With advances in genomics, and more recently in proteomics, we have the tools to better understand key genes expressed and pathways changed that lead to the plethora of phenotypes associated with RNS exposure. Choosing the appropriate system is critical to proper interpretation of study results. Careful attention has to be paid to the RNS used (e.g., an NO donor versus pure NO gas), the environment in which it is used (e.g., media properties or degree of hypoxia in the tissue examined), the levels at which it is used (e.g., low levels can inhibit apoptosis, whereas high levels can stimulate apoptosis), and the genetic background of the host (e.g., cell type and p53 status). Only when these systems are controlled can we compare genomic and proteomic results and perhaps better evaluate the usefulness of RNS or their inhibition in the treatment of cancer, cardiovascular and respiratory diseases, neurodegeneration, and diabetes, which are all among the major causes of death in humans. References Alderton, W. K., Cooper, C. E., and Knowles, R. G. (2001). Nitric oxide synthases: Structure, function and inhibition. Biochem. J. 357, 593–615. Alcaraz, M. J., Fernandez, P., and Guillen, M. I. (2003). Anti‐inflammatory actions of the heme oxygenase‐1 pathway. Curr. Pharm. Des. 9, 2541–2551.
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[27] Nitric Oxide Is a Signaling Molecule that Regulates Gene Expression By LORNE J. HOFSETH, ANA I. ROBLES, MICHAEL G. ESPEY , and CURTIS C. HARRIS Abstract
Nitric oxide (NO) is a dynamic and bioreactive molecule that can both participate in and inhibit the genesis of disease. Its ability to have an impact on a wide range of physiological events stems from its capacity to reversibly alter the expression of specific genes and the activities of a wide range of proteins and signaling pathways. Yet, NO remains an enigmatic molecule. Recently developed technologies, including gene‐chips, two‐dimensional electrophoresis, RNA interference, matrix‐assisted laser desorption ionization (MALDI)‐TOF (time-of-flight) mass spectrometry, and protein arrays will allow us to better understand how NO and associated reactive METHODS IN ENZYMOLOGY, VOL. 396 Copyright 2005, Elsevier Inc. All rights reserved.
0076-6879/05 $35.00 DOI: 10.1016/S0076-6879(05)96027-8
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nitrogen species (RNS) regulate both physiology and disease states, toward the development of treatments using NO synthase inhibitors or NO donors. Introduction
In 1998, Drs. Ignarro, Furchgott, and Murad received the Nobel Prize in physiology or medicine for their discoveries concerning NO as a signaling molecule in the cardiovascular system. An explosion of studies followed that have revealed insight into the many roles of NO in normal and disease processes. A characteristic of NO is that it can act as both a ‘‘friend’’ and a ‘‘foe.’’ NO has been considered in the treatment of cardiovascular and respiratory diseases, cancer, and erectile dysfunction. However, nitrosative stress can also play a role in the etiology and progression of many diseases, including cardiovascular disease, cancer, asthma, neurodegeneration, obesity, and diabetes. NO has these many faces because the specific effects of NO exposure change with NO levels, the microenvironment in which it acts (including the presence of other free radicals), and the genetic background of the host/tissue (Espey et al., 2000a; Hofseth et al., 2003a; Thomas et al., 2004; Wink and Mitchell, 1998). Each of these factors will dictate the impact of NO on cell signaling, gene expression, and disease outcome. Nitric Oxide Biochemistry
NO is endogenously formed by a family of enzymes called NO synthases (NOSs), which use L‐arginine as a substrate and molecular oxygen and NADPH as cofactors (Alderton et al., 2001; Marletta, 1994). Because NOS enzymes are responsible for triggering NO signaling cascades, an elaborate network of control mechanisms is available to regulate NOS levels and activity. NOSs are controlled at the transcriptional, posttranscriptional, and translational levels, as well as through posttranslational modifications, protein–protein interactions, and subcellular localization. NOSs are active as homodimers, and there are four isoforms. Two are Ca2þ‐dependent [NOS1 (neuronal NOS) and NOS3 (endothelial NOS)] and constitutively expressed, whereas the Ca2þ‐independent isoform (NOS2 or iNOS) requires induction. There are, however, some exceptions to this general scheme, as NOS1 and 3 have been shown to be inducible (Forstermann et al., 1995), and iNOS is expressed constitutively in some tissues [e.g., bronchus and ileum (Guo et al., 1995; Hoffman et al., 1997)]. Ca2þ‐dependent neuronal (NOS1) and endothelial isoforms (NOS3) produce low levels of NO that range from picomolar to nanomolar concentrations. In contrast, iNOS produces a sustained NO concentration in the
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micromolar range (Beckman et al., 1990; Espey et al., 2000b; Malinski et al., 1993). The fourth isoform, mitochondrial NOS (mtNOS), has only recently been defined (Elfering et al., 2002; Haynes et al., 2003; Lacza, 2003; Nisoli, 2003). It appears that mtNOS could be the alpha form of NOS1, but with posttranslational modifications (Elfering et al., 2002). Its roles in physiology and pathology have been reviewed (Haynes et al., 2003). Nitric Oxide Signaling
NO is an efficient signaling molecule because (1) it can affect a wide range of cells in its vicinity; (2) it can react with and reversibly generate other free radicals [collectively known as reactive nitrogen species (RNS)] that have their own set of bioreactive properties; (3) it, along with its byproducts (RNS), can reversibly and irreversibly posttranslationally modify proteins and lipids; and as evidence suggests here, (4) it has the ability to alter gene transcription in a discriminatory fashion. Because of their ubiquitous nature, RNS are also unlikely to interact with a single signaling pathway. Although this latter observation means that RNS can influence the expression of many genes (detailed later in this chapter), the pattern of changes is very complicated and often depends on the levels, the microenvironment, and the genetic background of the cell, tissue, or host. The outcome, then, comprises a wide range of physiological and pathological effects observed for RNS that differ, depending on the experimental system (Espey et al., 2001, 2002b; Thomas et al., 2002; Wink and Mitchell, 1998). RNS signaling can broadly be divided into cyclic guanosine monophosphate (cGMP)–dependent and –independent pathways (Fig. 1). In cGMP‐dependent pathways, small fluxes of RNS can bind to the heme iron of soluble guanylate cyclase (sGC) and activate it, which stimulates cGMP formation (Hobbs and Ignarro, 1996; Ignarro, 1992; Stamler, 1994). cGMP then targets a series of proteins (‘‘effector proteins’’), including cGMP‐dependent protein kinases (PKG) I and II (resulting in substrate phosphorylation), cyclic nucleotide–regulated ion channels (resulting in increased ion flux), and phosphodiesterases [resulting in cGMP and/or cyclic adenosine monophosphate (cAMP) hydrolysis]. The regulation of gene expression by cGMP has been detailed elsewhere (Pilz and Casteel, 2003) and, therefore, will not be covered here. cGMP‐independent pathways are generally mediated by larger fluxes of RNS (Wink and Mitchell, 1998), which may damage DNA directly and reversibly or irreversibly damage posttranslationally modified proteins (Beckman, 1996) and lipids (Baker et al., 2004), leading to a dynamic
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FIG. 1. Nitric oxide signaling pathways.
change in the activity of signaling pathways. Depending on the conditions used, different RNSs (e.g., nitrosoperoxycarbonate, peroxynitrite, and nitrogen dioxide) may be formed. For example, peroxynitrite (ONOO) may form via the reaction between NO and superoxide (Beckman and Koppenol, 1996). ONOO can oxidize protein sulfhydryl groups (DeMaster et al., 1995; Kuhn and Geddes, 1999; Radi et al., 1991) and nitrate peptides at tyrosine residues (Beckman, 1996). The presence of nitration is not necessarily indicative of the intermediacy of ONOO. Nitration can also occur
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through the oxidation of nitrite, which forms nitrogen dioxide (NO2). A comparison between ONOO and NO2 showed the latter was a much more efficient effector species (Espey et al., 2002a). ONOO formation requires maintenance of a precise balance between NO and superoxide (O 2 ), whereas nitrite oxidation can occur through various pathways without such spatial and temporal limitations. Because nitration has been shown to influence protein activity (Kuhn and Geddes, 1999; Schopfer et al., 2003), the nitrotyrosine antibodies have been useful as a biomarker of nitrosative stress. Nitrosative stress has been shown to occur in many conditions such as chronic inflammation, cancer, and cardiovascular diseases (Maeda and Akaike, 1998; Turko and Murad, 2002). In addition to nitration, a potential key signaling mechanism is the formation of NO adducts on protein nucleophiles (such as thiols, amines, and alcohols). This may occur via nitrosation by the RNS dinitrogen trioxide (N2O3) formed during the autoxidation of NO (Espey et al., 2001). Alternatively, NO adducts may form through oxidative nitrosylation (Espey et al., 2002b). Improvements in the analysis of nitrosated residues suggest that they may play a more ubiquitous role in physiology than previously thought (Bryan et al., 2004). A key quality of RNS in cell signaling is their ability to both irreversibly and reversibly modify the structure and activity of key signaling proteins (see abbreviated list in Table I). Sometimes this modification results in protein activation, and sometimes it results in protein deactivation. For example, RNS have been shown to both inhibit (Sommer et al., 2002) and activate phosphatases, kinases, and other signaling proteins, including transcription factors and repair proteins (Chen and Wang, 2004; Hofseth et al., 2003b; Klatt et al., 1999; Kibbe et al., 2000; Laval and Wink, 1994; Lin et al., 2003; Mateo et al., 2003; Namkung‐Matthai et al., 2000; Reynaert et al., 2004; Sandau et al., 2001; So et al., 1998; Wink and Laval, 1994; Zhou et al., 2000). Although it is unclear why a particular protein is activated while another is deactivated after posttranslational modification, the types of modifications and the mechanisms of these changes are beginning to be understood. For example, NO can donate electrons and react with iron, copper, and zinc, leading to the formation of metal‐nitrosyl complexes (Ford and Lorkovic, 2002). Because many enzymes, transcription factors, and other proteins (e.g., iron of the heme moiety of sGC and hemoglobin; enzyme sulfide clusters; zinc‐finger proteins) have a transition metal component, the interaction of NO with transition metals is fundamental to NO signaling. Some messenger RNA (mRNA) binding proteins also regulated by transition metals can be functionally modulated by RNS.
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TABLE I SELECTED MOLECULES THAT INTERACT WITH NITRIC OXIDE IN CANCER, CARDIOVASCULAR DISEASE, AND NEURONAL PATHOPHYSIOLOGY
Cancer
Cardiovascular disease
Heme oxygenase‐1 HIF‐1 Estrogen receptor VEGF
Heme oxygenase‐1 HIF‐1 Estrogen receptor Beta adrenergic receptors PPAR G‐protein–coupled receptors Kinases Phosphatases Actin Light chain myosin Fibrinogen Kinases Antioxidant enzymes
Kinases Phosphatases Bax p53 Caspases PPAR Microtubules Histones Antioxidant enzymes
Neurotransmission/ neurodegeneration Heme oxygenase‐1 Parkin Estrogen receptor Antioxidant enzymes ‐amyloid Tau protein Synaptophysin GADPH Acetylcholine Dopamine Myelin Presenilin Huntington disease protein ‐synuclein
Other modifications resulting in either loss or gain of protein function include nitration of tryptophan and tyrosine side‐chains (by ONOO or NO2, described earlier). Key proteins shown to be nitrated include albumin, Cu,Zn superoxide dismutase (SOD), cytochrome P450, iNOS, histone, ‐tubulin, and actin (Schopfer et al., 2003). One must take caution, however, in evaluating the significance of RNS adducts formed when proteins are exposed outside the context of the intact cell or tissue. Using two‐dimensional electrophoresis and MALDI‐TOF mass spectrometry, Aulak et al. (2001) showed approximately 31 proteins nitrated in lipopolysaccharide (LPS)‐treated rats, including many proteins not previously recognized as heavily nitrated by RNS, providing evidence for the presence of extensive protein nitration in vivo. S‐nitrosylation (incorporation of a NO group in cysteine thiols), S‐nitrosation, methionine sulfoxidation (oxidation of methionine residues), carbonyl formation at lysine and arginine residues, dityrosine formation, formation of disulfide bonds, and S‐thiolation (S‐glutathionylation; protein mixed disulfide formation between cysteine and GSH) are other posttranslational modifications that play a central role in cell signaling (Klatt and Lamas, 2000). Because of their ability to activate and deactivate both kinases and phosphatases, RNS can influence the phosphorylation
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status of key phosphoproteins such as p53 (Hofseth et al., 2003b; Schneiderhan et al., 2003; Thomas et al., 2004) and pRb (Hofseth et al., unpublished observation; Radisavljevic, 2004). pRb and p53 are cancer suppressor proteins, so this observation is critical to the understanding of RNS in carcinogenesis. Until recently, we have only been able to study the influence of RNS on a protein‐by‐protein basis and test a limited amount of that protein’s function. With the development of high‐throughput technologies such as RNA interference (RNAi), DNA chips, two‐dimensional electrophoresis, MALDI‐ TOF mass spectrometry, protein arrays, and other techniques, we now have the ability to study the expression, modifications, and binding properties of thousands of genes, or the proteins they encode, following RNS exposure. Nitric Oxide and Gene Expression
When released from inflammatory cells or produced endogenously by epithelial, endothelial, or neuronal cells, NO targets a large protein pool. Until the development and use of DNA microarrays, it had been difficult to broadly study the gene expression changes associated with RNS exposure. This technological breakthrough, combined with the more recent development of protein chips, MALDI‐TOF, and RNAi, offers a unique opportunity to uncover pathways altered by RNS that may affect cellular phenotype. At present, relatively few studies in the NO field have reported use of these technologies. A survey of the peer‐reviewed literature yields four studies looking at the effects of RNS on global gene expression in bacteria or plant systems (Firoved et al., 2004; Huang et al., 2002; Ohno et al., 2003; Polverari et al., 2003), three* studies using mammalian cells exposed to RNS (Hemish et al., 2003; Li et al., 2004; Zamora et al., 2002), and two studies that examine the effects of RNS on gene expression in intact rats (Wang et al., 2002) or mice (Okamoto et al., 2004). Although it has long been known that there is an extensive list of individual proteins affected by RNS (abbreviated list in Table I), as will be shown, a key observation arising from microarray studies is that RNS have discriminative properties. For this review, we wanted to tease out trends in gene expression profile arising from the three published data sets using cultured mammalian cells. Although all three studies used different microarray platforms for analysis, we can generalize a few interesting observations. First, there is a consistently * After this paper went to press, two additional references appeared in the literature examining the influence of nitric oxide on gene expression in mammalian cells (Li et al., 2004; Turpaev et al., 2005).
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low percentage of genes that changed significantly in expression after exposure to RNS. Hemish et al. (2003) found 560 of approximately 10,000 genes (5%) to be significantly changed (upregulated or downregulated) after exposure of mouse NIH‐3T3 cells to NO (250 M of the NO donor, SNAP). Zamora et al. (2002) found 200 of approximately 6500 genes (3%) significantly changed (upregulated or downregulated) after exposure of mouse hepatocytes to NO (infection with adenovirus expressing human iNOS). We have characterized p53‐dependent apoptotic signaling associated with NO exposure (Li et al., 2004). Overall, we found 358 (3.9%) of 9180 genes significantly changed (upregulated or downregulated) in TK6 (p53 wild‐type) human lymphoblastoid cells exposed to NO (390 mol pure NO gas). Second, even though at first glance there is very limited overlap in individual gene expression changes, a more detailed analysis reveals that RNS exposure modulates similar signal transduction pathways in all three experimental systems. Some of the conserved changes include alteration of expression of kinase and phosphatases, heat shock proteins, cyclins, zinc‐finger proteins, transcription factors, cell energy (ATP)–related proteins, apoptosis, serine proteinase inhibitors, and members of the ubiquitin family (data not shown). Third, more extensive overlap is found within the same species and type of exposure. Direct comparison of genes significantly changed by RNS exposure in the two studies examining murine cells (Hemish et al., 2003; Zamora et al., 2002) yields 12 genes in common (Table II). Upon methodically searching for mouse genes with homology to human genes significantly changed in our study (Li et al., 2004) using HomoloGene (http://www. ncbi.nlm.nih.gov/entrez/query.fcgi), only nine genes were found to overlap with those of Hemish et al. (2003), even though both used exogenous NO as the exposure route (Table III). Again, some of the conserved changes include alteration of kinase and phosphatase expression, heat shock proteins, cyclins, zinc‐finger proteins, transcription factors, cell energy (ATP)–related proteins, apoptosis, serine proteinase inhibitors, and members of the ubiquitin family. Additionally, there was commonality in extracellular matrix proteins (integrins) that were affected by RNS exposure in both mouse and human cells. Perhaps not surprisingly, the least amount of overlap was found between our study (Li et al., 2004) and the published data set from Zamora et al. (2002), which differ in both species and route of exposure. There were only three genes in common: ribonucleotide reductase M1 polypeptide, proliferating cell nuclear antigen (PCNA), and heme oxygenase‐1 (HO‐1). As shown in Tables II and III, the only two proteins with significantly changed expression in all three studies using mammalian cells exposed to
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signaling and gene expression TABLE II GENES SIGNIFICANTLY CHANGED IN BOTH PUBLISHED STUDIES EXAMINING THE EFFECTS OF RNS ON MURINE CELL GENE EXPRESSIONa Description
Proliferating cell nuclear antigen Heme oxygenase‐1 Macrophage migration inhibitory factor Thymidylate synthase
CD81 antigen Adrenomedullin Cystein and glycine‐rich protein 1 Eukaryotic translation initiation factor 3 Glutamate‐cysteine ligase, catalytic subunit
Keratin 19 Ferritin light chain 1 Adaptor protein complex AP‐1, 1‐subunit
Hemish et al.
Zamora et al.
Increase (1.3‐fold at 12 h) Increase (2.4‐fold at 12 h) Increase (1.9‐fold at 16 h) Increase (max 1.3‐fold at 0.5 h) and decrease (max 2.6‐fold at 0.75 h) Increase (1.5‐fold at 16 h)b Increase (1.4‐fold at 8 h) Increase (1.5‐fold at 4 h) Decrease (1.5‐fold at 48 h) Increase (1.8‐fold at 12 h) and decrease (1.2‐fold at 48 h) Increase (1.4‐fold at 4 h) Increase (1.5‐fold at 16 h) Decrease (1.14‐fold at 12 h)
Increase (5.9‐fold) Increase (6.2‐fold) Increase (2‐fold) Increase (4.2‐fold)
Decrease (40‐fold) Increase (3.1‐fold) Increase (5.2‐fold) Decrease (6.5‐fold) Decrease (6‐fold)
Increase (4.2‐fold) Decrease (2.8‐fold) Increase (2‐fold)
a
Italicized text indicates that the gene was significantly changed in human TK6 cells exposed to NO gas (Li et al., 2004). b Initial slight decrease (1.2‐fold) at 0.75 h.
NO were PCNA and HO‐1. Both of these genes were increased after exposure in every case. PCNA was first recognized in 1981 as a nuclear antigen associated with cell proliferation and blast transformation in the sera of patients with systemic lupus erythematosus (Takasaki et al., 1981). First used as a proliferation marker, it has now been shown that PCNA functions as a sliding clamp protein of the DNA polymerase complex, is intimately involved in DNA repair, and is an executive molecule controlling critical cellular decision pathways (Matunis, 2002; Paunesku et al., 2001).
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TABLE III GENES SIGNIFICANTLY CHANGED IN BOTH PUBLISHED STUDIES EXAMINING THE EFFECTS OF EXOGENOUS RNS ON MAMMALIAN CELL GENE EXPRESSIONa Description
Hemish et al.
Li et al.
Heme oxygenase‐1 Proliferating cell nuclear antigen Hypoxia inducible factor 1‐alpha Cyclin E2 Solute carrier family 3 (activators of dibasic and neutral amino acid transport) member 2 b GADD45‐ c p21Cip1/Waf1 d MDM2 Tumor necrosis factor receptor superfamily, member 6
Increase (2.4‐fold at 12 h) Increase (1.3‐fold at 12 h)
Increase (2.1‐fold at 24 h) Increase (2.3‐fold at 24 h)
Increase (1.3‐fold at 8 h)
Increase (1.9‐fold at 12 h)
Increase (1.6‐fold at 12 h) Increase (1.6‐fold at 12 h)
Increase (2.2‐fold at 24 h) Increase (1.8‐fold at 24 h)
Increase (8‐fold at 16 h) Increase (11‐fold at 16 h) Increase (8‐fold at 16 h) Decrease (2.2‐fold at 8 h)
Increase Increase Increase Increase
(3.2‐fold at 12 h) (6‐fold at 24 h) (3.6‐fold at 24 h) (3.3‐fold at 24 h)
a
Italicized text indicates that the gene was significantly changed in mouse hepatocytes overexpressing human inducible nitric oxide synthase (Zamora et al., 2002). b Induced also in a study by Li et al. (2004). c Induced also in studies by Li et al. (2004) and Turpaev et al. (2005). d Induded also in a study by Li et al. (2004).
HO‐1 has previously been found to be induced by oxidative or nitrosative stress, cytokines, and other mediators produced during inflammatory processes (Bouton and Demple, 2000; Terry et al., 1999; Vile et al., 1994). HO‐1 is involved in the regulatory defense system that leads to the production of mediators that modulate the inflammatory response. For example, it controls intracellular levels of ‘‘free’’ heme (a prooxidant), produces biliverdin (an antioxidant), improves nutritive perfusion via CO release, and fosters the synthesis of the Fe‐binding protein ferritin (Bauer and Bauer, 2002). HO‐1 activity results in the inhibition of oxidative damage and apoptosis, with significant reductions in inflammatory events (Alcaraz et al., 2003). An obvious reason for the low overlap in individual gene expression affected by RNS from study to study is the use of different microarray platforms, which can lead to gene representation bias. However, more relevant physiological reasons for the lack of consistency can reflect previous observations that the response to RNS depends on the microenvironment, the genetic background, and the type and level of RNS exposure (Hofseth et al., 2003a).
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Such conditions were also different in the three published studies we examined here. It would be interesting to compare the response of the same cell type to different types of RNS and exposure methods or different mammalian cell types to the same RNS and exposure methods in carefully controlled experiments. Such studies have yet to be published, but we are continuing our efforts to delineate the response to RNS in human cells. Two studies have examined the influence of NO on global gene expression in animals. Wang et al. (2002) examined cells from the thoracic aorta in rats receiving RNS through nitroglycerin infusion. They found that 447 of approximately (11.6%) 3531 genes were significantly changed (upregulated or downregulated). Okamoto et al. (2004) observed that 106 of (0.9%) 12,451 genes were significantly changed in lung tissues from iNOSþ/þ versus iNOS/ mice exposed to LPS. Only one gene (aldehyde dehydrogenase) was consistently changed in association with RNS in both studies, which again underscores the observation that RNS affects physiology differently, based on the levels used, the surrounding microenvironment, and the genetic background of the tissue/host. Conclusion
RNS are a ubiquitous and complicated group of free radicals that reversibly and irreversibly posttranslationally modify proteins and dynamically alter the activity of cell signaling pathways. With advances in genomics, and more recently in proteomics, we have the tools to better understand key genes expressed and pathways changed that lead to the plethora of phenotypes associated with RNS exposure. Choosing the appropriate system is critical to proper interpretation of study results. Careful attention has to be paid to the RNS used (e.g., an NO donor versus pure NO gas), the environment in which it is used (e.g., media properties or degree of hypoxia in the tissue examined), the levels at which it is used (e.g., low levels can inhibit apoptosis, whereas high levels can stimulate apoptosis), and the genetic background of the host (e.g., cell type and p53 status). Only when these systems are controlled can we compare genomic and proteomic results and perhaps better evaluate the usefulness of RNS or their inhibition in the treatment of cancer, cardiovascular and respiratory diseases, neurodegeneration, and diabetes, which are all among the major causes of death in humans. References Alderton, W. K., Cooper, C. E., and Knowles, R. G. (2001). Nitric oxide synthases: Structure, function and inhibition. Biochem. J. 357, 593–615. Alcaraz, M. J., Fernandez, P., and Guillen, M. I. (2003). Anti‐inflammatory actions of the heme oxygenase‐1 pathway. Curr. Pharm. Des. 9, 2541–2551.
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