IL-8 inhibits cAMP-stimulated alveolar epithelial fluid transport via a GRK2/PI3K-dependent mechanism.

Patients with acute lung injury (ALI) who retain maximal alveolar fluid clearance (AFC) have better clinical outcomes. Experimental and small clinical studies have shown that ß2-adrenergic receptor (ß2AR) agonists enhance AFC via a cAMP-dependent mechanism. However, two multicenter phase 3 clinical trials failed to show that ß2AR agonists provide a survival advantage in patients with ALI. We hypothesized that IL-8, an important mediator of ALI, directly antagonizes the alveolar epithelial response to ß2AR agonists. Short-circuit current and whole-cell patch-clamping experiments revealed that IL-8 or its rat analog CINC-1 decreases by 50% ß2AR agonist-stimulated vectorial Cl(-) and net fluid transport across rat and human alveolar epithelial type II cells via a reduction in the cystic fibrosis transmembrane conductance regulator activity and biosynthesis. This reduction was mediated by heterologous ß2AR desensitization and down-regulation (50%) via the G-protein-coupled receptor kinase 2 (GRK2)/PI3K signaling pathway. Inhibition of CINC-1 restored ß2AR agonist-stimulated AFC in an experimental model of ALI in rats. Finally, consistent with the experimental results, high pulmonary edema fluid levels of IL-8 (>4000 pg/ml) were associated with impaired AFC in patients with ALI. These results demonstrate a novel role for IL-8 in inhibiting ß2AR agonist-stimulated alveolar epithelial fluid transport via GRK2/PI3K-dependent mechanisms.-Roux, J., McNicholas, C. M., Carles, M., Goolaerts, A., Houseman, B. T., Dickinson, D. A., Iles, K. E., Ware, L. B., Matthay, M. A., Pittet, J.-F. IL-8 inhibits cAMP-stimulated alveolar epithelial fluid transport via a GRK2/PI3K-dependent mechanism.

Transforming growth factor ß suppresses glutamate-cysteine ligase gene expression and induces oxidative stress in a lung fibrosis model.

The concentration of glutathione (GSH), the most abundant intracellular free thiol and an important antioxidant, is decreased in the lung in both fibrotic diseases and experimental fibrosis models. The underlying mechanisms and biological significance of GSH depletion, however, remain unclear. Transforming growth factor ß (TGF-ß) is the most potent and ubiquitous profibrogenic cytokine and its expression is increased in almost all fibrotic diseases. In this study, we show that increasing TGF-ß1 expression in mouse lung to a level comparable to those found in lung fibrotic diseases by intranasal instillation of AdTGF-ß1(223/225), an adenovirus expressing constitutively active TGF-ß1, suppressed the expression of both catalytic and modifier subunits of glutamate-cysteine ligase (GCL), the rate-limiting enzyme in de novo GSH synthesis, decreased GSH concentration, and increased protein and lipid peroxidation in mouse lung. Furthermore, we show that increasing TGF-ß1 expression activated JNK and induced activating transcription factor 3, a transcriptional repressor involved in the regulation of the catalytic subunit of GCL, in mouse lung. Control virus (AdDL70-3) had no significant effect on any of these parameters, compared to saline-treated control. Concurrent with GSH depletion, TGF-ß1 induced lung epithelial apoptosis and robust pulmonary fibrosis. Importantly, lung GSH levels returned to normal, whereas fibrosis persisted at least 21 days after TGF-ß1 instillation. Together, the data suggest that increased TGF-ß1 expression may contribute to the GSH depletion observed in pulmonary fibrosis diseases and that GSH depletion may be an early event in, rather than a consequence of, fibrosis development.

Susceptibility to inhaled flame-generated ultrafine soot in neonatal and adult rat lungs.

Over a quarter of the U.S. population is exposed to harmful levels of airborne particulate matter (PM) pollution, which has been linked to development and exacerbation of respiratory diseases leading to morbidity and mortality, especially in susceptible populations. Young children are especially susceptible to PM and can experience altered anatomic, physiologic, and biological responses. Current studies of ambient PM are confounded by the complex mixture of soot, metals, allergens, and organics present in the complex mixture as well as seasonal and temporal variance. We have developed a laboratory-based PM devoid of metals and allergens that can be replicated to study health effects of specific PM components in animal models. We exposed 7-day-old postnatal and adult rats to a single 6-h exposure of fuel-rich ultrafine premixed flame particles (PFPs) or filtered air. These particles are high in polycyclic aromatic hydrocarbons content. Pulmonary cytotoxicity, gene, and protein expression were evaluated at 2 and 24 h postexposure. Neonates were more susceptible to PFP, exhibiting increased lactate dehydrogenase activity in bronchoalveolar lavage fluid and ethidium homodimer-1 cellular staining in the lung in situ as an index of cytotoxicity. Basal gene expression between neonates and adults differed for a significant number of antioxidant, oxidative stress, and proliferation genes and was further altered by PFP exposure. PFP diminishes proliferation marker PCNA gene and protein expression in neonates but not adults. We conclude that neonates have an impaired ability to respond to environmental exposures that increases lung cytotoxicity and results in enhanced susceptibility to PFP, which may lead to abnormal airway growth.

Analysis of the liver mitochondrial proteome in response to ethanol and S-adenosylmethionine treatments: novel molecular targets of disease and hepatoprotection.

S-adenosylmethionine (SAM) minimizes alcohol hepatotoxicity; however, the molecular mechanisms responsible for SAM hepatoprotection remain unknown. Herein, we use proteomics to determine whether the hepatoprotective action of SAM against early-stage alcoholic liver disease is linked to alterations in the mitochondrial proteome. For this, male rats were fed control or ethanol-containing liquid diets +/- SAM and liver mitochondria were prepared for proteomic analysis. Two-dimensional isoelectric focusing (2D IEF/SDS-PAGE) and blue native gel electrophoresis (BN-PAGE) were used to determine changes in matrix and oxidative phosphorylation (OxPhos) proteins, respectively. SAM coadministration minimized alcohol-dependent inflammation and preserved mitochondrial respiration. SAM supplementation preserved liver SAM levels in ethanol-fed rats; however, mitochondrial SAM levels were increased by ethanol and SAM treatments. With use of 2D IEF/SDS-PAGE, 30 proteins showed significant changes in abundance in response to ethanol, SAM, or both. Classes of proteins affected by ethanol and SAM treatments were chaperones, beta oxidation proteins, sulfur metabolism proteins, and dehydrogenase enzymes involved in methionine, glycine, and choline metabolism. BN-PAGE revealed novel changes in the levels of 19 OxPhos proteins in response to ethanol, SAM, or both. Ethanol- and SAM-dependent alterations in the proteome were not linked to corresponding changes in gene expression. In conclusion, ethanol and SAM treatment led to multiple changes in the liver mitochondrial proteome. The protective effects of SAM against alcohol toxicity are mediated, in part, through maintenance of proteins involved in key mitochondrial energy conserving and biosynthetic pathways. This study demonstrates that SAM may be a promising candidate for treatment of alcoholic liver disease.

gamma-Glutamyl transpeptidase is induced by 4-hydroxynonenal via EpRE/Nrf2 signaling in rat epithelial type II cells.

gamma-Glutamyl transpeptidase (GGT) plays key roles in glutathione homeostasis and metabolism of glutathione S-conjugates. Rat GGT is transcribed via five tandemly arranged promoters into seven transcripts. The transcription of mRNA V is controlled by promoter 5. Previously we found that GGT mRNA V-2 was responsible for the induction of GGT in rat alveolar epithelial cells by 4-hydroxynonenal (HNE). In the current study, the underlying mechanism was investigated. Reporter deletion and mutation analysis demonstrated that an electrophile-response element (EpRE) in the proximal region of GGT promoter 5 (GP5) was responsible for the basal- and HNE-induced promoter activity. Gel-shift assays showed an increased binding activity of GP5 EpRE after HNE exposure. The nuclear content of NF-E2-related factor 2 (Nrf2) was significantly increased by HNE. The recruitment of Nrf2 to GP5 EpRE after HNE treatment was demonstrated by supershift and chromatin immunoprecipitation assays. The tissue expression pattern of GGT mRNA V was previously unknown. Using polymerase chain reaction, we found that GGT mRNA V-2 was expressed in many tissues in rat. Taken together, GGT mRNA V-2 is widely expressed in rat tissues and its basal and HNE-induced expression is mediated through EpRE/Nrf2 signaling.

Protective effect of L-trans-pyrrolidine-2,4-dicarboxilic acid preload against cell death induced by oxygen/glucose deprivation in differentiated PC12 cells.

It has been postulated that cellular glutamate is released into the extracellular fluid when the energy supply of the brain is compromised (i.e., anoxia or oxygen/glucose deprivation), and there the amino acid triggers the so-called excitotoxic cascade, causing neuronal death. Several mechanisms for this release have been postulated, and, by using glutamate transporter inhibitors, several authors have established that reversed uptake is the major mechanism through which glutamate is released in acute oxygen/glucose deprivation. We have studied the effect of the slowly transported glutamate analogue L-trans-pyrrolidine-2,4-dicarboxilic acid (PDC) preload on glutamate release and cell death in an in vitro model of oxygen plus glucose deprivation with differentiated PC12 cells. As expected, we found that PDC preload inhibits glutamate release induced by oxygen/glucose deprivation, supporting the conclusion that it occurs via reverse transport. In addition, we show that PDC preload but not the nontransportable glutamate uptake inhibitor DL-threo-beta-benzyloxyaspartate (TBOA) protects cells against the death induced by oxygen/glucose deprivation, indicating that PDC entry into the cell is necessary for this protective effect. This protection does not correlate with the extracellular glutamate concentration or changes in proteins synthesis rate and eukaryotic initiation 2 phosphorylation. Oxygen/glucose deprivation induces a significant increase in glutathione levels in both unloaded and PDC-preloaded cells, but this increase is not due to up-regulation of glutamate cysteine ligase levels. Intracellular glutathione disulfide (GSSG) significantly increased after oxygen/glucose deprivation. It was also interesting that intracellular GSSG levels in PDC-preloaded cells under oxygen/glucose deprivation strongly correlate with the protection exerted by this compound against cell death.

HNE increases HO-1 through activation of the ERK pathway in pulmonary epithelial cells.

Heme oxygenase-1 (HO-1) is a key cytoprotective enzyme and an established marker of oxidative stress. Increased HO-1 expression has been found in the resident macrophages in the alveolar spaces of smokers. The lipid peroxidation product 4-hydroxynonenal (HNE) is also increased in the bronchial and alveolar epithelium in response to cigarette smoke. This suggests a link between a chronic environmental stress, HNE formation, and HO-1 induction. HNE is both an agent of oxidative stress in vivo and a potent cell signaling molecule. We hypothesize that HNE acts as an endogenously produced pulmonary signaling molecule that elicits an adaptive response culminating in the induction of HO-1. Here we demonstrate that HNE increases HO-1 mRNA, protein, and activity in pulmonary epithelial cells and identify ERK as a key pathway involved. Treatment with HNE increased ERK phosphorylation, c-Fos protein, JNK phosphorylation, c-Jun phosphorylation, and AP-1 binding. Whereas inhibiting the ERK pathway with the MEK inhibitor PD98059 significantly decreased HNE-mediated ERK phosphorylation, c-Fos protein induction, AP-1 binding, and HO-1 protein induction, inhibition of the ERK pathway had no effect on HNE-induced HO-1 mRNA. This suggests that ERK is involved in the increase in HO-1 through regulation of translation rather than transcription.

Nitric oxide-induced resistance to hydrogen peroxide stress is a glutamate cysteine ligase activity-dependent process.

Nitric oxide (*NO) is a reactive nitrogen species known to be involved in cytotoxic processes. Cells respond to cytotoxic injury by stress response induction leading to the development of cellular resistance. This report describes an *NO-induced stress response in Chinese hamster fibroblasts (HA1), which leads to glutathione synthesis-dependent resistance to H2O2-mediated oxidative stress. The development of resistance to H2O2 was completely abolished by the inhibition of glutamate cysteine ligase (GCL) during the first 8 h of recovery after *NO exposure. Altered thiol metabolism was observed immediately after *NO exposure as demonstrated by up to 75% decrease in intracellular thiol pools (glutathione, gamma-glutamylcysteine, and cysteine), which then reaccumulated during the *NO-mediated development of resistance. Immunoreactive protein and activity associated with GCL decreased immediately after exposure to *NO and then reaccumulated during the development of resistance to H2O2 challenge. Moreover, compared to N2 controls the activity levels of GCL in *NO-exposed cells increased approximately twofold 24 h after H2O2 challenge. These results demonstrate that *NO exposure is capable of inducing an adaptive response to H2O2-mediated oxidative stress in mammalian cells, which involves alterations in thiol metabolism and is dependent upon glutathione synthesis and increased GCL activity.

Human glutamate cysteine ligase gene regulation through the electrophile response element.

Glutathione (GSH) is the primary nonprotein thiol in the cell. It has many important roles in cell function, including regulating redox-dependent signal transduction pathways. The content of GSH within the cell varies with stress. In many cases, a process involving GSH synthesis results in adaptation to subsequent stressors. Sustained increases in GSH content are controlled primarily through induction of two genes, Gclc and Gclm, leading to the synthesis of the rate-limiting enzyme for GSH synthesis, glutamate cysteine ligase. Each of these genes in humans has a number of putative enhancer elements in their promoters. Overall, the most important element in both Gclc and Gclm expression is the electrophile response element. We review the evidence that has led to this conclusion and the implications for the redox-dependent regulation of this critical intracellular antioxidant.

Variable regulation of glutamate cysteine ligase subunit proteins affects glutathione biosynthesis in response to oxidative stress.

Glutamate cysteine ligase (GCL), composed of a catalytic (GCLC) and modulatory (GCLM) subunit, catalyzes the first step of glutathione (GSH) biosynthesis. Using 4-hydroxy-2-nonenal (4HNE), 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), and tertiary-butylhydroquinone (tBHQ) as models of oxidative stress which are known to work through different mechanisms, we measured changes in cellular GSH, GCL mRNA, and GCL protein. 4HNE and tBHQ treatments increased cellular GSH levels, while DMNQ exposure depleted GSH. Furthermore, changes in the two GCL mRNAs largely paralleled changes in the GCL proteins; however, the magnitudes differed, suggesting some form of translational control. The molar ratio of GCLC:GCLM ranged from 3:1 to 17:1 in control human bronchial epithelial (HBE1) cells and all treatments further increased this ratio. Data from several mouse tissues show molar ratios of GCLC:GCLM that range from 1:1 to 10:1 in support of these findings. These data demonstrate that alterations in cellular GSH are clearly correlated with GCLC to a greater extent than GCLM. Surprisingly, both control HBE1 cells and some mouse tissues have more GCLC than GCLM and GCLM increases to a much lesser extent than GCLC, suggesting that the regulatory role of GCLM is minimal under physiologically relevant conditions of oxidative stress.

Cellular mechanisms of redox cell signalling: role of cysteine modification in controlling antioxidant defences in response to electrophilic lipid oxidation products.

The molecular mechanisms through which oxidized lipids and their electrophilic decomposition products mediate redox cell signalling is not well understood and may involve direct modification of signal-transduction proteins or the secondary production of reactive oxygen or nitrogen species in the cell. Critical in the adaptation of cells to oxidative stress, including exposure to subtoxic concentrations of oxidized lipids, is the transcriptional regulation of antioxidant enzymes, many of which are controlled by antioxidant-responsive elements (AREs), also known as electrophile-responsive elements. The central regulator of the ARE response is the transcription factor Nrf2 (NF-E2-related factor 2), which on stimulation dissociates from its cytoplasmic inhibitor Keap1, translocates to the nucleus and transactivates ARE-dependent genes. We hypothesized that electrophilic lipids are capable of activating ARE through thiol modification of Keap1 and we have tested this concept in an intact cell system using induction of glutathione synthesis by the cyclopentenone prostaglandin, 15-deoxy-Delta12,14-prostaglandin J2. On exposure to 15-deoxy-Delta12,14-prostaglandin J2, the dissociation of Nrf2 from Keap1 occurred and this was dependent on the modification of thiols in Keap1. This mechanism appears to encompass other electrophilic lipids, since 15-A(2t)-isoprostane and the lipid aldehyde 4-hydroxynonenal were also shown to modify Keap1 and activate ARE. We propose that activation of ARE through this mechanism will have a major impact on inflammatory situations such as atherosclerosis, in which both enzymic as well as non-enzymic formation of electrophilic lipid oxidation products are increased.

Decreased synthetic capacity underlies the age-associated decline in glutathione content in Fisher 344 rats.

Although it is well documented that the concentration of glutathione (GSH), the most abundant intracellular free thiol and an important antioxidant, declines with age in many tissues of different animal species, the underlying mechanism is not well understood. In a previous study, we showed that the expression of the glutamate cysteine ligase genes was down-regulated with age, accompanied by a decline in GSH content in the liver, kidney, and lung of Fisher 344 rats. The aim of this study was to examine the age-associated changes in the activities of three other enzymes, which also play important roles in GSH biosynthesis, to further explore the mechanism underlying the age-associated decline in GSH content in Fisher 344 rats. The results showed for the first time that the activity and gene expression of glutathione synthase, which catalyzes the second reaction in de novo GSH synthesis, were also decreased with increased age in the lung and kidney, but not in the liver or heart. No age-associated change in the activity of either gamma-glutamyltranspeptidase or glutathione reductase was observed in any of the organs examined. The results further indicate that decreased GSH synthetic capacity is responsible for the age-associated decline in GSH content in Fisher 344 rats.

HNE--signaling pathways leading to its elimination.

The oxidation of polyunsaturated fatty acids results in the production of HNE, which can react through both non-enzymatic and enzyme catalyzed reactions to modify a number of cellular components, including proteins and DNA. Multiple pathways for its enzyme catalyzed elimination include oxidation of the aldehyde to a carboxylic acid, reduction of the aldehyde to an alcohol, and conjugation of the carbon-carbon double bond to glutathione (GSH). Interestingly, the enzymes that result in HNE elimination are induced by HNE itself although the chemical mechanism for signaling is not well understood. One of the striking effects of HNE is that after a transient decrease in GSH, synthesis of GSH is elevated through induction of glutamate cysteine ligase (GCL), which catalyzes the first step in de novo synthesis of GSH. GCL has two subunits, which are transcriptionally regulated by a wide variety of agents, including oxidants and electrophiles, such as HNE, which elevates both. The transcriptional regulation of GCL has been the subject of many investigations yielding a complex picture in which the pathways for up-regulation of the subunits appear to be independent and vary with inducing agent and cell type. We have found that in human bronchial epithelial cells, HNE acts through AP-1 activation with signaling through the JNK pathway, and that neither the ERK nor p38(MAPK) pathways is involved. With these results we review what is currently known about the signaling mechanisms for removal of HNE, focusing principally on conjugation mechanisms involving GSH.

Cytoprotection against oxidative stress and the regulation of glutathione synthesis.

Adaptation to oxidative and nitrosative stress occurs in cells first exposed to a nontoxic stress, resulting in the ability to tolerate a toxic challenge of the same or a related oxidant. Adaptation is observed in a wide variety of cells including endothelial cells on exposure to nitric oxide or oxidized lipids, and lung epithelial cells exposed to air-borne pollutants and toxicants. This acquired characteristic has been related to the regulation of a family of stress responding proteins including those that control the synthesis of the intracellular antioxidant glutathione. The focus of this article, which includes a review of recent results along with new data, is the regulation and signaling of glutathione biosynthesis, especially those relating to adaptive mechanisms. These concepts are illustrated with examples using nitric oxide and oxidized low density lipoprotein mediated adaptation to oxidative stress. These data are discussed in the context of other adaptive mechanisms relating to glutathione synthesis including those from dietary constituents such as curcumin.

Bio-effectiveness of Tat-catalase conjugate: a potential tool for the identification of H2O2-dependent cellular signal transduction pathways.

Reactive oxygen species such as hydrogen peroxide (H(2)O(2)) have taken center stage as bona fide second messengers in various signaling pathways. Here, we report the synthesis, metabolic fate, and effectiveness in modulating such pathways of a Tat-catalase conjugate. Incubation of L2 cells with Tat-catalase greatly increased cell-associated enzymatic activity, reaching close to a plateau by 30 min. The cell-associated catalase activity and antibody-detectable Tat-derivatives declined over time after changing medium, although still remaining at significantly higher levels than baseline even at 4h. While most cell-associated Tat-catalase was apparently tightly attached to the cell surface, a small fraction entered the cells as the proteasome inhibitor MG-132 slightly prevented the disappearance of the enzyme. Tat-catalase, either membrane-bound or intracellular, but not native catalase, inhibited serum-induced Elk phosphorylation and anisomycin- and/or MG-132-induced ERK phosphorylation, suggesting the involvement of H(2)O(2). Thus, Tat-catalase should be a useful tool to dissect H(2)O(2)-dependent events in signaling pathways.

Curcumin alters EpRE and AP-1 binding complexes and elevates glutamate-cysteine ligase gene expression.

Dietary use of curcumin, the active component of tumeric, one of the most widely used spices, is linked to several beneficial health effects, although the underlying molecular mechanisms remain largely unknown. Correlations have been established between curcumin exposure and increases in enzymes for glutathione synthesis, particularly glutamate-cysteine ligase (GCL), and metabolism as well as glutathione content, suggesting the eliciting of an adaptive response to stress. In this study, using HBE1 cells, we found that the mechanism of curcumin-induced GCL elevation occurred via transcription of the two Gcl genes. Gcl transcription has been shown in several systems to be mediated through binding of transcription factor complexes to TRE and EpRE elements. Studies herein showed that curcumin caused modest but sustained increases in binding of proteins to DNA sequences for both cis elements but, more importantly, altered the compositions and nuclear content of proteins in these complexes. Curcumin exposure increased JunD and c-Jun content in AP-1 complexes and increased JunD while decreasing MafG/MafK in EpRE complexes. Thus, the beneficial effects elicited by curcumin appear to be due to changes in the pool of transcription factors that compose EpRE and AP-1 complexes, affecting gene expression of GCL and other phase II enzymes.