Heat-shock response increases lung injury caused by Pseudomonas aeruginosa via an interleukin-10-dependent mechanism in mice.

Background: The heat-shock response (HSR) protects from insults, such as ischemia-reperfusion injury, by inhibiting signaling pathways activated by sterile inflammation. However, the mechanisms by which the HSR activation would modulate lung damage and host response to a bacterial lung infection remain unknown.

Methods: HSR was activated with whole-body hyperthermia or by intraperitoneal geldanamycin in mice that had their lungs instilled with Pseudomonas aeruginosa 24 h later (at least six mice per experimental group).

HMGB1 accelerates alveolar epithelial repair via an IL-1β- and αvβ6 integrin-dependent activation of TGF-β1.

High mobility group box 1 (HMGB1) protein is a danger-signaling molecule, known to activate an inflammatory response via TLR4 and RAGE. HMGB1 can be either actively secreted or passively released from damaged alveolar epithelial cells. Previous studies have shown that IL-1β, a critical mediator acute lung injury in humans that is activated by HMGB1, enhances alveolar epithelial repair, although the mechanisms are not fully understood.

Activation of the heat shock response attenuates the interleukin 1β-mediated inhibition of the amiloride-sensitive alveolar epithelial ion transport.

Acute lung injury (ALI) is a clinical syndrome characterized by hypoxia, which is caused by the breakdown of the alveolar capillary barrier. Interleukin 1β (IL-1β), a cytokine released within the airspace in ALI, downregulates the α subunit of the epithelial sodium channel (αENaC) transcription and protein expression via p38 MAP kinase-dependent signaling. Although induction of the heat shock response can restore alveolar fluid clearance compromised by IL-1β following the onset of severe hemorrhagic shock in rats, the mechanisms are not fully understood.

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 β2-adrenergic receptor (β2AR) agonists enhance AFC via a cAMP-dependent mechanism. However, two multicenter phase 3 clinical trials failed to show that β2AR agonists provide a survival advantage in patients with ALI.

Stroke-induced activation of the α7 nicotinic receptor increases Pseudomonas aeruginosa lung injury.

Infectious complications, predominantly pneumonia, are the most common cause of death in the postacute phase of stroke, although the mechanisms underlying the corresponding immunosuppression are not fully understood. We tested the hypothesis that activation of the α7 nicotinic acetylcholine receptor (α7nAChR) pathway is important in the stroke-induced increase in lung injury caused by Pseudomonas aeruginosa pneumonia in mice. Prior stroke increased lung vascular permeability caused by P.

PAI-1 is an essential component of the pulmonary host response during Pseudomonas aeruginosa pneumonia in mice.

Rationale: Elevated plasma and bronchoalveolar lavage fluid plasminogen activator inhibitor 1 (PAI-1) levels are associated with adverse clinical outcome in patients with pneumonia caused by Pseudomonas aeruginosa. However, whether PAI-1 plays a pathogenic role in the breakdown of the alveolar-capillary barrier caused by P aeruginosa is unknown.

Objectives: The role of PAI-1 in pulmonary host defence and survival during P aeruginosa pneumonia in mice was tested.

Cytoprotective-selective activated protein C attenuates Pseudomonas aeruginosa-induced lung injury in mice.

Inhibition of the small GTPase RhoA attenuates the development of pulmonary edema and restores positive alveolar fluid clearance in a murine model of Pseudomonas aeruginosa pneumonia. Activated protein C (aPC) blocks the development of an unfavorably low ratio of small GTPase Rac1/RhoA activity in lung endothelium through endothelial protein C receptor (EPCR)/protease-activated receptor-1 (PAR-1)-dependent signaling mechanisms that include transactivating the sphingosine-1-phosphate (S1P) pathway. However, whether aPC's cytoprotective effects can attenuate the development of pulmonary edema and death associated with P.

Reactive species and pulmonary edema.

Pulmonary edema occurs when fluid flux into the lung interstitium exceeds its removal, resulting in hypoxemia and even death. Noncardiogenic pulmonary edema (NPE) generally results when microvascular and alveolar permeability to plasma proteins increase, one possible etiology being oxidant injury. Reactive oxygen and nitrogen species (RONS) can modify or damage ion channels, such as epithelial sodium channels, which alters fluid balance.

Influenza virus M2 protein inhibits epithelial sodium channels by increasing reactive oxygen species.

The mechanisms by which replicating influenza viruses decrease the expression and function of amiloride-sensitive epithelial sodium channels (ENaCs) have not been elucidated. We show that expression of M2, a transmembrane influenza protein, decreases ENaC membrane levels and amiloride-sensitive currents in both Xenopus oocytes, injected with human alpha-, beta-, and gamma-ENaCs, and human airway cells (H441 and A549), which express native ENaCs. Deletion of a 10-aa region within the M2 C terminus prevented 70% of this effect.

Fatty acid transduction of nitric oxide signaling: nitrolinoleic acid mediates protective effects through regulation of the ERK pathway.

In vivo and in vitro studies revealed that nitroalkenes serve as protective mediators in the lung by inducing the cytoprotective enzyme heme oxygenase-1 (HO-1). Nitrolinoleic acid (LNO2) increased HO-1 mRNA, protein, and activity in cultured pulmonary epithelial cells treated with 5 to 50 microM LNO2 and in lungs of rats injected intraperitoneally with 2.6 mg/kg LNO2 twice daily for 20 days.

Glutathione suppresses TGF-beta-induced PAI-1 expression by inhibiting p38 and JNK MAPK and the binding of AP-1, SP-1, and Smad to the PAI-1 promoter.

Transforming growth factor (TGF)-beta upregulates plasminogen activator inhibitor type 1 (PAI-1) in a variety of cell types, and PAI-1 is considered to be an essential factor for the development of fibrosis. Our previous studies demonstrated that TGF-beta decreased intracellular glutathione (GSH) content in murine embryonic fibroblasts (NIH/3T3 cells), whereas treatment of the cells with GSH, which restored intracellular GSH concentration, inhibited TGF-beta-induced collagen accumulation by blocking PAI-1 expression and enhancing collagen degradation. In the present study, we demonstrate that GSH blocks TGF-beta-induced PAI-1 promoter activity in NIH/3T3 cells, which is associated with an inhibition of TGF-beta-induced JNK and p38 phosphorylation.

The ADP-stimulated NADPH oxidase activates the ASK-1/MKK4/JNK pathway in alveolar macrophages.

The role of H2O2 as a second messenger in signal transduction pathways is well established. We show here that the NADPH oxidase-dependent production of O2*(-) and H2O2 or respiratory burst in alveolar macrophages (AM) (NR8383 cells) is required for ADP-stimulated c-Jun phosphorylation and the activation of JNK1/2, MKK4 (but not MKK7) and apoptosis signal-regulating kinase-1 (ASK1). ASK1 binds only to the reduced form of thioredoxin (Trx).

Fatty acid transduction of nitric oxide signaling: nitrolinoleic acid potently activates endothelial heme oxygenase 1 expression.

Nitroalkenes are a class of cell signaling mediators generated by NO and fatty acid-dependent redox reactions. Nitrated fatty acids such as 10- and 12-nitro-9,12-octadecadienoic acid (nitrolinoleic acid, LNO(2)) exhibit pluripotent antiinflammatory cell signaling properties. Heme oxygenase 1 (HO-1) is up-regulated as an adaptive response to inflammatory mediators and oxidative stress.

Fatty acid transduction of nitric oxide signaling: multiple nitrated unsaturated fatty acid derivatives exist in human blood and urine and serve as endogenous peroxisome proliferator-activated receptor ligands.

Mass spectrometric analysis of human plasma and urine revealed abundant nitrated derivatives of all principal unsaturated fatty acids. Nitrated palmitoleic, oleic, linoleic, linolenic, arachidonic and eicosapentaenoic acids were detected in concert with their nitrohydroxy derivatives. Two nitroalkene derivatives of the most prevalent fatty acid, oleic acid, were synthesized (9- and 10-nitro-9-cis-octadecenoic acid; OA-NO2), structurally characterized and determined to be identical to OA-NO2 found in plasma, red cells, and urine of healthy humans.

4-Hydroxynonenal induces rat gamma-glutamyl transpeptidase through mitogen-activated protein kinase-mediated electrophile response element/nuclear factor erythroid 2-related factor 2 signaling.

Gamma-glutamyl transpeptidase (GGT) plays critical roles in glutathione homeostasis and metabolism. Rat GGT is a single-copy gene from which seven types of GGT mRNA with a common protein encoding sequence, but different 5'-untranslated regions, may be transcribed. We previously showed that type V-2 was the predominant form of GGT mRNA in rat L2 epithelial cells, and that it could be induced by 4-hydroxynonenal (HNE) through the electrophile response element (EpRE) located in GGT promoter 5 (GP5).

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.

Mechanisms of glutamate cysteine ligase (GCL) induction by 4-hydroxynonenal.

4-Hydroxynonenal (HNE) is one of the major end-products of lipid peroxidation and is increased in response to cellular stress and in many chronic and/or inflammatory diseases. HNE can in turn function as a potent signaling molecule to induce the expression of many genes including glutamate cysteine ligase (GCL), the rate-limiting enzyme in de novo glutathione (GSH) biosynthesis. GSH, the most abundant nonprotein thiol in the cell, plays a key role in antioxidant defense.

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.

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.

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.

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.

Macrophage signaling and respiratory burst.

Macrophages are key defenders of the lung and play an essential role in mediating the inflammatory response. Critical to this is the activation of the NADPH oxidase. Through receptor-mediated interactions, extracellular stimuli activate pathways that signal for the phosphorylation and assembly of the NADPH oxidase.

4-hydroxynonenal induces glutamate cysteine ligase through JNK in HBE1 cells.

Glutathione is the most abundant non-protein thiol in the cell, with roles in cell cycle regulation, detoxification of xenobiotics, and maintaining the redox tone of the cell. The glutathione content is controlled at several levels, the most important being the rate of de novo synthesis, which is mediated by two enzymes, glutamate cysteine ligase (GCL), and glutathione synthetase (GS), with GCL being rate-limiting generally. The GCL holoenzyme consists of a catalytic (GCLC) and a modulatory (GCLM) subunit, which are encoded by separate genes.

A549 subclones demonstrate heterogeneity in toxicological sensitivity and antioxidant profile.

In A549 cell culture, significant variability was found in sensitivity to actinomycin D. Using limiting dilution, actinomycin D-susceptible (G4S) and -resistant (D3R) subclones were isolated. G4S cells were also susceptible to protein synthesis inhibitors, a redox cycling quinone, and an electrophile with concomitant activation of caspases 3 and 9.