Activated Akt Protects the Lung from Oxidant-Induced Injury and Delays Death of Mice

Oxygen therapy is administered to decrease tissue hypoxia and to relieve arterial hypoxemia in patients with acute and chronic cardiovascular and pulmonary diseases and also to premature babies with respiratory distress syndrome. However, hyperoxia can cause acute lung injury which results in increased lung water with protein-rich edema fluid spilling into the interstitial and alveolar spaces. This is thought to occur after injury to epithelial and capillary endothelial cells resulting in epithelial cell death and increased pulmonary capillary permeability 1,2. Current evidence indicates that the damaging effects of oxygen are caused by reactive oxygen intermediates (ROIs) such as the superoxide anion (O₂⁻) that are formed in the presence of excessive oxygen. Severe oxidant-induced stress can result in respiratory failure and even death 3.

Akt/PKB is homologous to the protein kinases PKA and PKC (and hence the name PKB or related to A and C protein kinase [RAC-PK]; reference 4). Akt is activated by phosphatidylinositol (PI) 3-kinase in response to growth and survival factors 5. It has been shown that after stimulation by growth factors, Akt undergoes phosphorylation at residues Thr308 in the activation loop of the kinase and at Ser473 in the carboxy terminus 6,7. A downstream pathway that is induced by activation of the PI 3-kinase/Akt pathway is the phosphorylation of the Bcl-2 family member Bad, which releases Bcl-XL thereby promoting cell survival 8,9. A second downstream prosurvival mechanism involves the transcription factor forkhead, the phosphorylation of which by Akt prevents its nuclear translocation and activation of genes that promote apoptosis 10. Several studies indicate that Akt activation plays an important role in inhibiting cell apoptosis in fibroblasts, epithelial and lymphoid cell lines, and neuronal cells 11,12,13,14,15,16. However, these studies with Akt have been carried out in vitro and the in vivo significance of this pathway in protection from cell death induced by different stimuli including oxidative stress remains to be investigated. Here we demonstrate that expression of a constitutively active derivative of Akt in the mouse lung by adenovirus gene transfer methods protects lungs and delays death from oxidant-induced injury.

Human primary small airway epithelial (SAE) cells were obtained from Clonetics and grown in supplied medium. The virus packaging cell line 293 was grown in DMEM supplemented with 10% FBS. The generation of adenovirus constructs expressing myr-Akt has been described previously 17.

Akt kinase assay was performed using an “Akt Kinase Assay” kit (Cell Signaling Technology) following the manufacturer's instructions. In brief, SAE cells grown in 100-mm plates were infected by Adeno-myr-Akt virus or control Adeno-green fluorescent protein (GFP) virus at a multiplicity of infection (MOI) of 10. Cells were lysed in immunoprecipitation (IP) buffer and used for immunoprecipitation of Akt with anti-Akt antibody conjugated to agarose beads. The immunoprecipitated Akt was incubated with glycogen synthase kinase (GSK)-3 protein in kinase assay buffer containing ATP. After centrifugation, an aliquot of the supernatant was removed and tested for the presence of phospho-GSK-3 by Western blotting using anti–phospho-GSK-3α/β(Ser 21/9) antibody. The Akt bound to the agarose beads was released by boiling in SDS-sample buffer and subjected to Western blot analysis using anti-Akt antibody.

To address the role of Akt in protection from hyperoxic injury in vivo, a constitutively active form of Akt (myr-Akt; reference 17) was instilled into the trachea of C57BL/6 mice (7–9 wk old). Mice were anesthetized by intraperitoneal injection of a 0.1-ml solution of a ketamine/xylazine mixture (10 mg/ml/1 mg/ml) in pyrogen-free saline. The skin of the mice on the ventral neck was cleaned with a cotton swab soaked in isopropyl alcohol. The mice were next placed on a Deltaphase Isothermal pad (Braintree Scientific Inc.) warmed up to 37°C to reduce hypothermia and quicken recovery time. Trachea were exposed by a small incision in the neck skin. Next, 50 μl of PBS containing adenovirus (Ad-myr-Akt or Ad-EGFP, 10⁹ PFU) was injected into the trachea using a 50-μl Hamilton syringe fitted with a 22 S needle. The incision was closed with wound clips and mice were kept warm and monitored until they recovered from anesthesia (usually 30 min). Then they were transferred to their cages with food and water and the cages were transferred to a hyperoxic chamber and exposed to 100% oxygen in a plexiglass hyperoxia exposure chamber. Oxygen from a liquid source was passed through a bubble humidifier and introduced into the sealed chamber at 10 liters/min. The chamber was fitted with a water manometer to maintain normobaric pressure. CO₂ production by the animals was neutralized in the chamber by absorbing with soda lime filter. The FIO₂ was measured continuously with an oxygen analyzer (VTI O₂ gas analyzer; Vacu Med). The mice were fed food and water ad lib and maintained on a 12-h dark-light cycle at sea level and at room temperature. Mice were followed after exposure to O₂ for different lengths of time and assessed for hyperoxic injury.

Lungs were prepared for histology by perfusing the animal through the right ventricle with PBS to remove all blood as described by us previously 18,19. Lungs were inflated to 20 cm water pressure with Streck Tissue Fixative (STF; Streck Laboratories Inc.) instilled through a tracheostomy tube. 5-μM sections were mounted on slides and stained with hematoxylin and eosin according to established procedures.

DNA fragmentation was assessed using the DeadEnd Colorimetric Apoptosis Detection System (Promega). Tissue sections were deparaffinized by immersing slides in fresh xylene, washed successively in graded ethanol, and subjected to terminal deoxynucleotidyl transferase–mediated dUTP-biotin nick-end labeling (TUNEL) assay following instructions of the manufacturer. Negative controls were incubated in buffer containing all components except TdT enzyme. Nucleotide incorporation was detected by treatment with horseradish peroxidase–conjugated streptavidin and enzyme substrate.

Where appropriate, data are expressed as means ± SEM. The significance of difference between two groups was analyzed using the Student's unpaired t test. Differences in means were considered significant if P < 0.05. Differences in survival between mice infected with control virus and Akt-expressing virus were assessed by χ² analysis.

In cell culture systems, activation of Akt has been shown to result in phosphorylation of protein substrates that are involved in cytoprotection. To study the in vivo function of activated Akt, we generated replication-defective adenovirus containing a constitutively active form of Akt (myristoylated Akt; Ad-myr-Akt). The functional activity of myr-Akt was assessed by infecting primary SAE cells with either Ad-myr-Akt or control replication-defective adenovirus (Ad-con) and by performing Akt kinase assay by immunoprecipitating Akt from cell extracts and using GSK-3 as substrate. Fig. 1 A demonstrates that myr-Akt is an active kinase as revealed by phosphorylation of GSK-3. One mechanism by which Akt promotes cell survival involves phosphorylation of the Bcl-family member Bad 8,9. Phosphorylation of Bad at Ser-136 by Akt results in its dissociation from Bcl-X_L and association with the adapter protein 14-3-3. The free Bcl-X_L released from sequestration by Bad promotes cell survival. Another downstream substrate of activated Akt involved in cell survival is the transcription factor forkhead 10. Phosphorylation of forkhead by Akt inhibits its apoptotic function by preventing its nuclear translocation. Fig. 1b and Fig. C, show the ability of myr-Akt to cause phosphorylation of Bad and forkhead. Therefore, these data show that myr-Akt is able to phosphorylate its signaling substrates that are involved in cytoprotection.

Having confirmed the ability of myr-Akt to induce activation of downstream signaling pathways in lung epithelial cells, we investigated whether expression of Ad-myr-Akt could prevent oxidant-induced lung injury and death in animals. In these experiments, control adenovirus or adenovirus expressing myr-Akt 17 was introduced intratracheally into mice and the mice were immediately exposed to 100% O₂. Although all of the control mice died within 72 h of initiation of exposure to 100% O₂, all of the mice infected with Ad-myr-Akt lived longer (Fig. 2). The mice were killed at 120 h at which point they had begun to display respiratory distress. Thus, mice expressing activated Akt showed a significant increase in survival time (P < 0.0001). Histological examination of lung sections showed hyaline membrane formation, hemorrhage, inflammation, and gross pulmonary edema in the lungs of the control mice at 72 h (Fig. 3 A). In contrast, the lungs of mice infected with Ad-myr-Akt appeared normal and free of any hyaline membrane, hemorrhage, inflammation, or edema at 72 h after hyperoxia. At 120 h of exposure to 100% O₂, the lungs of the myr-Akt–expressing mice displayed hyaline membrane formation, hemorrhage, inflammation, and edema. To confirm expression and activation of myr-Akt in the lungs of the infected mice, lung extracts were immunoprecipitated with anti-hemagglutinin (HA) antibody and the immunoprecipitates were analyzed by immunoblotting with antibody to phosphorylated Akt. As shown in Fig. 3 B, adenovirus-derived transgenic expression of activated Akt was evident within 24 h of infection in mice infected with Ad-myr-Akt but not in those infected with the control virus, and the expression was detectable at 96 h after infection, the last time point tested in this assay. It is well established that hyperoxic injury involves an initiation phase after exposure to hyperoxia which is followed by an inflammatory phase and a destructive phase. Collectively, our data suggest that activated Akt can significantly delay the initiation phase of acute lung injury.

We next examined whether expression of constitutively active Akt is sufficient to suppress cell death induced by hyperoxia. In these experiments, lung sections from mice infected with control adenovirus or adenovirus expressing constitutively active Akt were subjected to TUNEL assay for nuclear DNA fragmentation, an indicator of cell death. As shown in Fig. 4 A, mice infected with control virus and subjected to hyperoxia displayed extensive TUNEL⁺ nuclei in the airway epithelium as well as in alveolar epithelial cells at 72 h after hyperoxia. In contrast, at this time point, cell death in the airway epithelium was almost completely blocked and alveolar epithelial cell death was partially blocked when mice were infected with Ad-myr-Akt. The better protection of the airway epithelium was probably due to the fact that the airway epithelium is more accessible to adenovirus mediated gene transfer than the alveolar epithelium. At 120 h after exposure to hyperoxia, when hemorrhage, inflammation, and edema were evident in the lungs of the Ad-myr-Akt–infected mice (Fig. 4 A), correspondingly, cell death was apparent in both airway and alveolar epithelial cells. Thus, the increased survival of Ad-myr-Akt–expressing animals at 72 h after hyperoxia correlated with decreased hemorrhage, inflammation, and edema in the lung and a dramatic suppression of airway cell death. These results suggest that a better means of activating Akt in alveolar epithelial cells will provide a better survival advantage to the animals.

In summary, our data demonstrate that Akt activation in vivo promotes resistance to oxidant-induced cell death and significantly delays the onset of acute lung injury resulting in increased survival of animals. Recently, activation of Akt by the 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitor simvastatin was shown to promote angiogenesis in the ischemic limbs of normocholesterolemic animals 20. Similarly, Akt activation may be a useful adjunct to oxygen therapy in hypoxic stress in cardiovascular and pulmonary diseases.

The authors thank R. Homer for advice on lung pathologies and members of the Yale Pulmonary Section for helpful discussions.

This work was supported by grants HL60207 and HL60995 from the National Institutes of Health to P. Ray.