Normoxic Cyclic GMP-independent Oxidative Signaling by Nitrite Enhances Airway Epithelial Cell Proliferation and Wound Healing
The airway epithelium provides important barrier and host defense functions. Recent studies reveal that nitrite is an endocrine reservoir of nitric oxide (NO) bioactivity that is converted to NO by enzymatic reductases along the physiological oxygen gradient. Nitrite signaling has been described as NO dependent activation mediated by reactions with deoxygenated redox active hemoproteins, such as hemoglobin, myoglobin, neuroglobin, xanthine oxidoreductase (XO) and NO synthase at low pH and oxygen tension. However, nitrite can also be readily oxidized to nitrogen dioxide (NO2\xe2\x80\xa2) via heme peroxidase reactions, suggesting the existence of alternative oxidative signaling pathways for nitrite under normoxic conditions. In the present study we examined normoxic signaling effects of sodium nitrite on airway epithelial cell wound healing. In an in vitro scratch injury model under normoxia, we exposed cultured monolayers of human airway epithelial cells to various concentrations of sodium nitrite and compared responses to NO donor. We found sodium nitrite potently enhanced airway epithelium wound healing at physiological concentrations (from 1uM). The effect of nitrite was blocked by the NO and NO2\xe2\x80\xa2 scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (c-PTIO). Interestingly, nitrite treatment did not increase cyclic guanosine monophosphate (cGMP) levels under these normoxic conditions, even in the presence of a phosphodiesterase 5 inhibitor, suggesting cGMP independent signaling. Consistent with an oxidative signaling pathway requiring hydrogen peroxide (H2O2)/heme peroxidase/NO2\xe2\x80\xa2 signaling, the effects of nitrite were potentiated by superoxide dismutase (SOD) and low concentration H2O2, whereas inhibited completely by catalase, followed by downstream extracellular-signal-regulated kinase (ERK) 1/2 activation. Our data represent the first description of normoxic nitrite signaling on lung epithelial cell proliferation and wound healing and suggest novel oxidative signaling pathways involving nitrite-H2O2 reactions, possibly via the intermediary, NO2\xe2\x80\xa2.
1.1 Introduction<!>1.2.1 Cell culture and treatments<!>1.2.2 In vitro wound repair assay<!>1.2.3 Measurement of nitrite levels in conditioned medium and cell lysates<!>1.2.4 Determination of cellular cGMP<!>1.2.5 Cellular ROS detection<!>1.2.6 PicoGreen Cell Proliferation Assay<!>1.2.7 Cell viability assay<!>1.2.7 Statistical analysis<!>1.3.1 Sodium nitrite promotes airway epithelial wound repair<!>1.3.2 NOS independent effects of nitrite<!>1.3.3 Nitrite levels in conditioned medium and cell lysates after sodium nitrite and NO donor treatments<!>1.3.4 c-PTIO blocks sodium nitrite-induced wound repair<!>1.3.5 Non-cGMP signaling induced by nitrite<!>1.3.6 ROS production is involved in sodium nitrite-induced wound repair<!>1.3.7 Sodium nitrite promotes airway epithelial cell proliferation<!>1.3.8 ERK1/2 activation is involved in nitrite-induced wound repair<!>1.4 Discussion
<p>Airway epithelium provides a physical border between host and environment that protects from injurious and infectious stimuli that gain access to the respiratory tract through inspiration or aspiration [1]. Epithelial injury occurs in various lung diseases and repair of the injured epithelium is important to restore its barrier integrity. Immediately after injury, the airway epithelium initiates a repair process in order to restore the barrier integrity. Although wound repair in such in vitro injury models is a complex process involving both cell migration and proliferation, the initial phase of wound repair involves primarily cell migration. Indeed, after migrating cells have covered the denuded and wounded area, the barrier integrity of the bronchial epithelium is restored [1-3]. New therapies that can enhance cell migration and restore barrier integrity hold promise for the treatment of acute lung injury syndromes.</p><p>It is now appreciated that nitrite, previously viewed as a physiologically inert metabolite of nitric oxide (NO), serves as an important source of NO in vasculature and tissues [4]. Nitrite is detected in the airway epithelial lining fluid as an NO metabolite in several studies [5], but the role of this anion in epithelial function is uncertain. Based on a study showing beneficial effects of NO on epithelial growth and wound repair [6], we hypothesized that sodium nitrite may similarly enhance airway epithelial cells wound healing. However, to date, most of the signaling effects of nitrite have been characterized under hypoxic or ischemic conditions which require the reductive formation of NO by deoxygenated redox active hemoproteins [7], while in the present study the signaling effects of nitrite on airway epithelial cells wound healing occur during normoxia. Consistent with alternative normoxic oxidative signaling pathways, the effects of nitrite appear to be independent of cGMP formation and require the intermediary, NO2•.</p><!><p>The immortalized human bronchial epithelial cell line, BEAS-2B (ATCC, Rockville, MD) was grown in bronchial epithelial growth media (BEGM, LONZA, Walkersville, MD). A papilloma virus–immortalized human bronchial epithelial cell line (HBE1), kindly provided by Dr. J. Yankaskas (University of North Carolina, Chapel Hill, NC) and primary normal human bronchial epithelial cells (NHBE) (LONZA, Walkersville, MD) were also grown in BEGM. Experiments were performed in 6- or 12- well collagen I coated plates. After changing the medium or after cell wounding (see next section), cells were treated with sodium nitrite (1-100uM, Sigma, St. Louis, MO) or the NO donor, diethylenetriamine NONOate (DETA-NONOate, Cayman Chemical, Ann Arbor, MI; t1/2 = 20 h at 37°C) for up to 24 h. Cells were pretreated, where indicated, for 15 minutes with the NO or NO2• scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide [8] (c-PTIO, Alexis Biochemicals), nonspecific nitric oxide synthase (NOS) inhibitor, G -nitro-L-Arginine-Methyl Ester (L-NAME, Sigma), polyethylene glycol-superoxide dismutase (PEG-SOD, Sigma) and polyethylene glycol-catalase (PEG-catalase, Sigma). None of the agents used significantly affected cell morphology or viability under these conditions.</p><!><p>To investigate the effects of sodium nitrite on epithelial wound repair, we used a common in vitro wound assay, in which confluent cell monolayers are mechanically wounded by creating a linear scratch. BEAS-2B and HBE1 cells were grown to confluence in 12-well tissue culture plates. After introduction of a linear wound of ~0.5 mm width using a sterile P200 pipette tip, cell monolayers were rinsed with BEGM to remove all cellular debris, fresh media was added to each well, and appropriate reagents were administered. Wound closure was photographed serially for 24 hours, using an inverted microscope (Olympus, Center Valley, PA). Wound closure was expressed as a percentage of the initial wound area, quantitated using NIH ImageJ software ((initial wound area - 24h wound area)/initial wound area × 100%).</p><!><p>The nitrite levels in the conditioned medium and cell lysates were measured by tri-iodide based reductive chemiluminescence using a Model-280 Nitric Oxide Analyzer (NOA) from Sievers Instruments. Confluent BEAS-2B cells grown in 10cm culture dishes were lysed with nitrite preservation solution (0.8 M potassium ferricyanide, 0.1M N-ethylmaleimide, 10% NP-40). 10 μl conditioned medium or 200ul cell lysate samples were injected into the NOA purge vessel containing acidic tri-iodide reagent (prepared freshly: 2.0 g potassium iodide and 1.3 g iodine, 40 ml distilled H2O and 140 ml glacial acetic acid), as previously described [9]. Freshly made sodium nitrite solutions of known concentrations, prepared in phosphate buffered saline, were used as standards. The area under the curve of the samples was divided by the slope from the standard curve (area/pmol) and then divided by the volume of the sample injected (μl). This calculation provides the concentration of nitrite in the samples (μM).</p><!><p>Intracellular cGMP concentration was determined in the cell lysate using a competitive enzyme immunoassay kit (Cayman Chemicals, Ann Arbor, MI). Cells were pretreated with a phosphodiesterase 5 inhibitor, sildenafil (5uM, Sigma) for 15min followed by sodium nitrite (50uM) or DETA-NONOate (25uM) treatment in the presence of sildenafil for 3 hours. After treatments, cells were extracted in cold 0.1 N HCL for 30 min at 4°C, and the protein precipitate was removed by centrifugation at 1,000 × g for 10 min. The cGMP levels were measured in the supernatant, expressed as fmol/mg protein.</p><!><p>Intracellular superoxide production was detected using the superoxide-sensitive fluorophore dihydroethidine (DHE, Sigma). DHE is oxidized to oxoethidium by superoxide. Oxoethidium binds to DNA, becoming highly fluorescent. After treatment, cells were loaded with 5 μM DHE in fresh media at 37°C in the incubator for 30 min. The cells were then rinsed with fresh media and were imaged immediately. Images were taken on an inverted fluorescent microscope.</p><!><p>Cell proliferation was determined by measuring the DNA content of cell lysates using a picoGreen® dsDNA Quantification Kit (Invitrogen). BEAS-2B or NHBE Cells were grown in 24 well plates with or without nitrite treatment. At 24 hour the culture medium was replaced with 0.1%v/v Triton X-100 in PBS and frozen at −80°C. Cells were then lysed by three freeze/thaw cycles and the assay performed as per manufacturer's instructions. Samples were analysed at wavelengths of 480nm (excitation) and 520nm (emission).</p><!><p>BEAS-2B or HBE1 cells (2 × 104 per well) were loaded in 96-well plates and maintained in BEGM. Cell viability was determined with the CellTiter 96 AQueous One Solution cell proliferation assay (Promega Corp., Madison, WI, USA) according to the manufacturer's instructions. Absorbances at 490 nm (test wavelength) and at 650 nm (reference wavelength) were measured using an enzyme-linked immunosorbent assay (ELISA) microplate reader (Bio-TEK Instruments Inc., Winooski, VT, USA).</p><!><p>All data are expressed as the mean ± SD. Student's t-test or one-way analysis of variance (ANOVA) were used as appropriate and indicated. A p value of ≤ 0.05 was considered significant.</p><!><p>To evaluate the effect of sodium nitrite on airway epithelial wound repair in vitro, confluent monolayers of BEAS-2B cells were mechanically damaged and then exposed to various concentrations (1-100uM) of sodium nitrite. The cells treated with epithelial growth factor (EGF), known to potently increase wound healing, served as positive control. As illustrated in Figure 1, sodium nitrite significantly and potently (from 1 uM) enhanced wound repair in a linear wound assay in BEAS-2B cells at 24h compared to untreated control cells. The effect of sodium nitrite on wound closure was dose-dependent in the concentration range lower than 50uM, but inhibited at higher nitrite concentrations (100μM, Figure 1A). In addition, the effect on wound repair induced by nitrite was similar to the effect with an equivalent concentration of authentic NO, delivered using the NO donor DETA-NONOate, which produces an expected 2 moles of NO per mole (Figure 1B). To confirm the results of studies with BEAS-2B cells, the experiments were also performed with another human bronchial epithelial cell line, HBE1. Similar effects were observed with nitrite in HBE1 cell wound healing (Figure 1D). Treatment with sodium nitrite for 24 hours did not affect BEAS-2B cell viability (see Supplemental Figure 1).</p><!><p>Since respiratory epithelium constitutively expresses nitric oxide synthase (NOS), which may account for the endogenous NO production [10], we treated cells with nitrite (50uM) in the presence of the nonspecific NOS inhibitor L-NAME (1mM) to rule out this possibility. We found exposure to L-NAME did not attenuate the wound repair induced by nitrite, suggesting a NOS-independent effect (Figure 2).</p><!><p>In order to compare nitrite levels with both treatments, we measured nitrite concentration in BEAS-2B cell media at 24h after treatment (Figure 3A). The nitrite levels measured in conditioned medium were lower than the added concentration, at approximately 70% of the added nitrite, indicating consumption of extracellular nitrite by the cells. However, only approximately 30% of the added NO was measured as nitrite after DETA-NONOate treatment. These data suggest that despite similar effects on epithelial wound healing, the actual nitrite exposure levels were different in sodium nitrite and equivalent NO treated cells. These studies indicate to us that nitrite might be mediating NO-independent signaling effects on airway epithelial wound healing. With increased extracellular nitrite treatment, the nitrite levels in cell lysates went up (Figure 3B). However, the intracellular nitrite levels were substantially lower than the extracellular levels, suggesting nitrite serves as a signaling molecule at very low concentrations, and the movement of nitrite across cell membrane is a highly controlled process, which to date has not been characterized.</p><!><p>To further characterize mechanisms of nitrite-induced wound closure, cells were pretreated with c-PTIO. The protective effects of sodium nitrite and NO donor on wound repair were markedly diminished in the presence of 100uM c-PTIO, whereas c-PTIO alone did not affect wound repair in control cells (Figure 4). It should be noted that the NO scavenger c-PTIO also scavenges NO2• rapidly. In fact, the rate constants for the reaction of NO2• with c-PTIO to form the corresponding oxoammonium cations (PTIO+s) and nitrite have been measured at 1.5 - 2 × 107 M−1 sec−1 [8].</p><!><p>In order to distinguish NO signaling via soluble guanylyl cyclase (sGC) versus alternative signaling pathways via NO2•, we directly measured intracellular cGMP levels since one cellular target involved in NO-mediated signaling is the heme center of sGC, activation of which induces the production of cGMP [11]. As illustrated in Figure 5, we found that cellular cGMP production was not increased by nitrite treatment under normoxic condition (21% oxygen) while the NO donor produced robust cGMP formation, which suggested that nitrite enhanced wound healing via a non-cGMP signaling pathway.</p><!><p>To investigate whether the stimulatory effect of sodium nitrite on airway epithelial wound closure involves reactive oxidative species (ROS) signaling, we detected superoxide formation during wounding using DHE staining. As shown in Figure 6A, mechanical wounding induced significant superoxide generation at the wound edges. We next exposed wounded BEAS-2B monolayers to cell permeable antioxidants PEG-SOD and PEG-catalase. PEG-SOD (400U/ml) pretreatment significantly increased BEAS-2B wound closure, while PEG-catalase (1kU/ml) inhibited the effects of nitrite on wound closure (Figure 6B), suggesting that H2O2 formation may be needed in the nitrite mediated signaling pathway. To further confirm this we evaluated the effect of low concentrations (5-25uM) of H2O2 on nitrite mediated wound healing, revealing enhanced basal wound repair and trends towards enhanced nitrite-induced wound repair by H2O2 (Figure 6C). We performed a number of inhibitor studies showing that the ROS is not produced by XO, mitochondria, or uncoupled eNOS (data not shown). This supports a possible role for dual oxidase (Duox), which has been previously described [12]. We did Western Blot analysis for 3-nitrotyrosine formation in the cells but did not see any difference with or without nitrite treatment (Supplemental Figure 2). However this is not unexpected as the formation of 3-nitrotyrosine requires 2 nitrogen dioxide equivalents and our nitrite concentrations are very low. It is more likely that NO2• radical formed in these reactions would oxidize glutathione and protein thiol targets.</p><!><p>To identify the effects of nitrite on airway epithelial cell proliferation, we used Picogreen assay to measure DNA content after nitrite treatment. As shown in Figure 7, sodium nitrite (10-50uM) promoted BEAS-2B and primary NHBE cell proliferation in a dose dependent manner, while catalase inhibited the effect. This result demonstrates the proliferative signaling effects of nitrite on airway epithelial cells and the role of H2O2 in bronchial epithelial cell proliferation as well.</p><!><p>To gain insight into the possible downstream intracellular signaling which regulates cell migration and proliferation, a specific inhibitor of the ERK1/2 activation, PD098059 was used. Prior studies of in vitro epithelial cell migration and wound repair suggest that the ERK1/2 pathway plays a critical role in cell migration and proliferation [13, 14], which can be activated by ROS and reactive nitrogen species (RNS) [15]. We found pretreatment with PD098059 (10uM) attenuated the stimulatory effect of nitrite after wounding while PD098059 by itself had no significant effect on the wound closure, suggesting downstream ERK1/2 activation is involved in nitrite-induced wound healing (Figure 8).</p><!><p>The airway epithelium serves a primary role as a protective barrier against inhaled environmental toxins and microorganisms. In response to airway epithelial injury, repair processes are initiated and early events in the repair process include cell spreading, migration and proliferation [3, 16]. The main finding of our present study is that sodium nitrite enhances cell proliferation and wound healing of human airway epithelial cells in normoxic physiological concentrations. Moreover, our studies reveal that intracellular signaling after stimulation with nitrite under normoxia is independent of NO and cGMP generation, but involves oxidative reactions with hydrogen peroxide and downstream ERK1/2 activation.</p><p>In our study, the initiation of wound closure by sodium nitrite occurs at relatively low levels that are well within the range of the physiological conditions found in the human body. It has been reported that the nitrite levels in human plasma range from 100 nM to 1 uM [17-20]. The salivary nitrite levels can approach 1-2 mM after a dietary nitrate load [7, 21]. Nitrite is also detected in exhaled breath condensate [22], suggesting higher levels in airway lining fluid. However, the exact concentrations of nitrite to which pulmonary epithelial cells are physiologically exposed are not known. Our studies suggest that extracellular physiological concentrations of nitrite potently promote airway epithelial cell proliferation and repair in response to mechanical injury in vitro. However, very high non-physiologic levels of extracellular nitrite slowed the wound repair.</p><p>Prior studies suggest nitrite protects against tissue injury in a variety of ischemic diseases (liver and heart ischemia-reperfusion injury, hypoxic pulmonary vasoconstriction, and hemorrhagic stroke) via NO-dependent mechanisms [23, 24]. Nitrite is converted to bioactive NO at low pH and oxygen tension by reduction by deoxyhemoglobin, deoxymyoglobin, xanthine oxidase and other hemoproteins [25-29]. Virtually all studies showing authentic NO generation from nitrite are conducted under anaerobic conditions. However, it is not clear how cells could convert nitrite to NO under normoxic conditions. In the present study a number of findings suggest that nitrite is signaling independent of NO. The first is that all the experiments were performed under normoxic conditions. Providing more direct evidence, we found no formation of the NO-downstream second messenger cGMP after exposure to nitrite in these studies. While c-PTIO inhibited the wound healing effect of both NO and nitrite, c-PTIO can also rapidly scavenge NO2• [8]. Based on lack of cGMP stimulation but potent inhibition by c-PTIO, we considered the possibility that nitrite oxidation to NO2• could mediate signaling. Nitrite can be oxidized to NO2• via heme catalzyzed peroxidase chemistry. In this reaction H2O2 is required which reacts with a peroxidase or redox active heme (hemoglobin, myoglobin, cytochrome C, etc.) to form an oxoiron intermediate analogous to peroxidase compound I and II (FeIV= O) which can oxidize nitrite to NO2• [30, 31]. For example, myoglobin reacts according to a peroxidase-like cycle forming two active intermediates, which can induce one-electron oxidation of the substrates. The MbFe(IV)==O intermediate oxidizes nitrite to NO2•. In the second mechanism, H2O2 reacts with iron-bound nitrite to produce an active nitrating species, which has been assumed to be a protein-bound peroxynitrite species, MbFe(III)--N(O)OO.</p><p>We therefore explored the role of H2O2, based on the known chemistry of heme catalyzed peroxidase oxidation of nitrite to NO2•. The inhibition of nitrite effects on wound healing by catalase, and augmentation with SOD and H2O2, suggest an important role of H2O2 in this signaling process, strongly indicating that nitrite oxidation to NO2• may mediate these effects. H2O2 is now known to serve as a cell signaling messenger in numerous contexts, such as cell survival, wound healing etc. The signaling role of H2O2 is effective at very low levels and excessive H2O2 poses the clear risk of oxidative stress [32]. In zebra fish wound margin tissue, the H2O2 signal peaked 20 minutes after wounding [33] and low levels of H2O2 generated by NADPH oxidases (NOX) enable leukocyte recruitment and wound healing [34]. In airway epithelial cells it is reported that ROS is generated by a core component homolog dual oxidase 1 (Duox1) [12, 35, 36] and the wound edge is a major source of reactive oxygen species generated via Duox [12]. With increased extracellular nitrite and H2O2 levels, nitrogen dioxide (NO2•), the one-electron oxidation product of nitrite may be generated, which could drive a number of post-translational protein modifications, including nitration of critical tyrosine residues, oxidation of thiols to form sulfenic and sulfinic groups, and S-nitrosation via thiyl radical formation or N2O3 formation. All of these modifications could alter downstream protein activities. In the present study, our results are consistent with previous reports, and demonstrate the importance of downstream ERK1/2 activation in epithelial cell proliferation and wound healing, supported by the result that pretreatment with PD098059 attenuated the stimulatory effect of nitrite. ERK1/2 can be activated by ROS and RNS via phosphorylation [37-41] or tyrosine nitration [42]. In addition, a variety of potential targets of ROS and RNS could contribute to ERK1/2 activation, including cell surface receptors, G proteins, upstream kinases, protein phosphatases and proteasome components, each of which could be direct or indirect targets of reactive oxygen or nitrogen species, thereby modulating the duration and magnitude of ERK1/2 activation [15].</p><p>Here we propose a new hypothesis for nitrite signaling under normoxic and hypoxic conditions based on the unique redox properties of nitrite. Nitrite is very stable in vivo with a half-life in humans of approximately 30-minutes [43, 44]. Its unique redox properties provide an opportunity for a one-electron oxidation to NO2• and a one electron reduction to NO under different oxygen conditions. The one proton adduct, nitrous acid, is readily reduced to NO and also can directly nitrosate thiol targets. Additional reactions with NO and NO2•, or with nitrite, NO and ferric hemoglobin can generate N2O3 [45, 46]. Finally, the peroxidase reactions with hemoproteins and the autocatalytic reaction of nitrite with oxyhemoglobin can produce NO2• [47]. In Figure 9 we propose a model for nitrite signaling via oxidative and reductive pathways in biology.</p><p>There are some potential limitations to this study. First, the simulative effect of nitrite on wound healing was detected mostly in immortalized cell lines. We did not see nitrite enhanced the wound closure of primary NHBEs (data not shown). However, we found nitrite increased NHBE cell proliferation. Since the wound repair process includes cell spreading, migration and cell proliferation, the effects of nitrite on wound healing may vary in different cell types. Second, in the present study, airway epithelial cells were grown in isolated monolayers, rather than in contact with extracellular matrix and endothelial/mesenchymal cells. It is possible that the wound repair is different from the conditions in vivo. Finally, we have not directly measured NO2• levels, as these are very low in cells. Further biochemical and in vivo studies are needed to confirm the validity of these findings and our hypothesis that nitrite signals via oxidative, and specifically NO2• dependent pathways.</p><p>In conclusion, our findings demonstrate that low, physiologically relevant concentrations of nitrite promote airway epithelial cell proliferation and wound repair via cGMP independent pathway. Interaction with H2O2 and downstream activation of the ERK1/2 pathway plays a central role in this process. This study provides new information about the mechanisms involved in nitrite mediated signaling under normoxia. The role of nitrite in promoting wound healing raises the possibility of therapeutic application of this previously considered inert anion.</p>