Apoptosis In Lung Epithelial Cells Research Articles

The environmental carcinogen 3-nitrobenzanthrone and its main metabolite 3-aminobenzanthrone enhance formation of reactive oxygen intermediates in human A549 lung epithelial cells

The environmental contaminant 3-nitrobenzanthrone (3-NBA) is highly mutagenic and a suspected human carcinogen. We aimed to evaluate whether 3-NBA is able to deregulate critical steps in cell cycle control and apoptosis in human lung epithelial A549 cells. Increased intracellular Ca 2+ and caspase activities were detected upon 3-NBA exposure. As shown by cell cycle analysis, an increased number of S-phase cells was observed after 24 h of treatment with 3-NBA. Furthermore, 3-NBA was shown to inhibit cell proliferation when added to subconfluent cell cultures. The main metabolite of 3-NBA, 3-ABA, induced statistically significant increases in tail moment as judged by alkaline comet assay. The potential of 3-NBA and 3-ABA to enhance the production of reactive oxygen species (ROS) was demonstrated by flow cytometry using 2′,7′-dichlorofluorescein-diacetate (DCFH-DA). The enzyme inhibitors allopurinol, dicumarol, resveratrol and SKF525A were used to assess the impact of metabolic conversion on 3-NBA-mediated ROS production. Resveratrol decreased dichlorofluorescein (DCF) fluorescence by 50%, suggesting a role for CYP1A1 in 3-NBA-mediated ROS production. Mitochondrial ROS production was significantly attenuated (20% reduction) by addition of rotenone (complex I inhibition) and thenoyltrifluoroacetone (TTFA, complex II inhibition). Taken together, the results of the present study provide evidence for a genotoxic potential of 3-ABA in human epithelial lung cells. Moreover, both compounds lead to increased intracellular ROS and create an environment favorable to DNA damage and the promotion of cancer.

Lung epithelial cell apoptosis during acute lung injury in infancy

Apoptosis of lung epithelial cells is implicated in the pathogenesis of acute lung injury. Most research on this subject has focused on adults. Very little is known about a potential interaction of this process with lung development in children. To summarize the current literature on lung epithelial cell apoptosis and common causes of acute lung injury in infants and young children and to identify new areas of research. A Medline-based literature search. Few studies have focused on lung epithelial cell apoptosis during common causes of acute lung injury in children. Nevertheless, the limited literature suggests that this may be an important mechanism during respiratory distress syndrome of infants and viral respiratory tract infection. Apoptosis is an essential process during lung development and maturation. Insufficient attention has been paid to potential consequences of this for the short- and long-term outcomes of acute lung injury.

Apoptosis induced by crocidolite asbestos in human lung epithelial cells involves inactivation of Akt and MAPK pathways

Exposure of human lung epithelial (A549) cells to asbestos fibers causes apoptosis, which is largely attributed to release of iron and generation of reactive oxygen species (ROS) within the cells. To mimic the highly oxidative environment generated by asbestos exposure in the absence of the actual fibers, we used two chemicals; buthione sulfoximine (BSO), an inhibitor of glutathione (GSH) synthesis and ferric ammonium citrate (FAC), a source of iron. Here, we report that exposure of A549 cells to crocidolite asbestos led to a significant time-dependent inactivation of signaling proteins, i.e. Akt and all mitogen-activated protein kinases (MAPKs) (p38, ERK1/2 and SAPK/JNK), and subsequently to apoptosis. Unlike crocidolite treatment, the use of BSO and FAC, independently or combined, did not change the phosphorylation status of proteins, nor did it induce apoptosis. Taken together, our results presented herein point to the possibility that crocidolite-induced apoptosis of human lung epithelial cells is not a mere consequence of generation of oxidants but also requires inactivation of major cell growth and differentiation pathways.

Gene transfer of soluble transforming growth factor type II receptor by in vivo electroporation attenuates lung injury and fibrosis

Background: Transforming growth factor-β1 (TGF-β1) has the potential to induce acute inflammation and apoptosis in lung epithelial cells and plays a central role in subsequent fibrosis. Aims: To examine a...

Reactive Oxygen Species Mediate Caspase Activation and Apoptosis Induced by Lipoic Acid in Human Lung Epithelial Cancer Cells through Bcl-2 Down-Regulation

The antioxidant alpha-lipoic acid (LA) is a naturally occurring compound that has been shown to possess promising anticancer activity because of its ability to preferentially induce apoptosis and inhibit proliferation of cancer cells relative to normal cells. However, the molecular mechanisms underlying the apoptotic effect of LA are not well understood. We report here that LA induced reactive oxygen species (ROS) generation and a concomitant increase in apoptosis of human lung epithelial cancer H460 cells. Inhibition of ROS generation by ROS scavengers or by overexpression of antioxidant enzymes glutathione peroxidase and superoxide dismutase effectively inhibited LA-induced apoptosis, indicating the role of ROS, especially hydroperoxide and superoxide anion, in the apoptotic process. Apoptosis induced by LA was found to be mediated through the mitochondrial death pathway, which requires caspase-9 activation. Inhibition of caspase activity by the pan-caspase inhibitor (z-VAD-FMK) or caspase-9-specific inhibitor (z-LEHD-FMK) completely inhibited the apoptotic effect of LA. Likewise, the mitochondrial respiratory chain inhibitor rotenone potently inhibited the apoptotic and ROS-inducing effects of LA, supporting the role of mitochondrial ROS in LA-induced cell death. LA induced down-regulation of mitochondrial Bcl-2 protein through peroxide-dependent proteasomal degradation, and overexpression of the Bcl-2 protein prevented the apoptotic effect of LA. Together, our findings indicate a novel pro-oxidant role of LA in apoptosis induction and its regulation by Bcl-2, which may be exploited for the treatment of cancer and related apoptosis disorders.

Epithelial Cell Apoptosis by Fas Ligand–Positive Myofibroblasts in Lung Fibrosis

The Fas/Fas ligand (FasL) apoptotic pathway has been shown to be involved in bleomycin-induced lung fibrosis. We examined the hypothesis that myofibroblasts from fibrotic lungs possess a cytotoxic phenotype that causes apoptosis of epithelial cells via the Fas/FasL pathway. We show in vivo epithelial cell apoptosis and associated upregulation of Fas and apoptotic Fas pathway genes in epithelial cells of lungs with bleomycin-induced fibrosis. In addition, we show that FasL surface molecules are overexpressed on alpha-SMA-positive cells in mice with bleomycin-induced fibrosis, and in humans with idiopathic pulmonary fibrosis. This enables the molecules to kill Fas-positive epithelial cells. In contrast, FasL-deficient myofibroblasts lose this myofibroblast cytotoxic phenotype, both in vivo and in vitro. In vivo, there was no bleomycin-induced epithelial cell apoptosis, as assessed by specific M30 staining in chimeric FasL-deficient mice that lacked FasL-positive myofibroblasts. In vitro, FasL-positive, but not FasL-negative myofibroblasts, induce mouse lung epithelial cell apoptosis. Thus myofibroblast cytotoxicity may underlie the absence of re-epithelialization, resulting in persistent lung fibrosis.

The role of Toll-like receptors in non-infectious lung injury

The role of Toll-like receptors (TLRs) in pathogen recognition has been expeditiously advanced in recent years. However, investigations into the function of TLRs in non-infectious tissue injury have just begun. Previously, we and others have demonstrated that fragmented hyaluronan (HA) accumulates during tissue injury. CD44 is required to clear HA during tissue injury, and impaired clearance of HA results in unremitting inflammation. Additionally, fragmented HA stimulates the expression of inflammatory genes by inflammatory cells at the injury site. Recently, we identified that HA fragments require both TLR2 and TLR4 to stimulate mouse macrophages to produce inflammatory chemokines and cytokines. In a non-infectious lung injury model, mice deficient in both TLR2 and TLR4 show an impaired transepithelial migration of inflammatory cells, increased tissue injury, elevated lung epithelial cell apoptosis, and decreased survival. Lung epithelial cell overexpression of high molecular mass HA protected mice against acute lung injury and apoptosis, in part through TLR-dependent basal activation of NF-kappaB. The exaggerated injury in TLR2 and TLR4 deficient mice appears to be due to impaired HA-TLR interactions on epithelial cells. These studies identify that host matrix component HA and TLR interactions provide signals that initiate inflammatory responses, maintain epithelial cell integrity, and promote recovery from acute lung injury.

Inactivation of a Two-Component Signal Transduction System, SaeRS, Eliminates Adherence and Attenuates Virulence of Staphylococcus aureus

Staphylococcus aureus is a major human and animal pathogen. During infection, this organism not only is able to attach to and enter host cells by using its cell surface-associated factors but also exports toxins to induce apoptosis and kill invaded cells. In this study, we identified the regulon of a two-component signal transduction system, SaeRS, and demonstrated that the SaeRS system is required for S. aureus to cause infection both in vitro and in vivo. Using microarray and real-time reverse transcriptase PCR analyses, we found that SaeRS regulates the expression of genes involved in adhesion and invasion (such as those encoding fibronectin-binding proteins and fibrinogen-binding proteins) and genes encoding alpha-, beta-, and gamma-hemolysins. Surprisingly, we found that SaeRS represses the Agr regulatory system since the mutation of saeS up-regulates agrA expression, which was confirmed by using an agr promoter-reporter fusion system. More importantly, we demonstrated that inactivation of the SaeRS system significantly decreases the bacterium-induced apoptosis and/or death of lung epithelial cells (A549) and attenuates virulence in a murine infection model. Moreover, we found that inactivation of the SaeRS system eliminates staphylococcal adhesion and internalization of lung epithelial cells. We also found that both a novel hypothetical protein (the SA1000 protein) and a bifunctional protein (Efb), which binds to extracellular fibrinogen and complement factor C3, might partially contribute to bacterial adhesion to and invasion of epithelial cells. Our results indicate that activation of the SaeRS system may be required for S. aureus to adhere to and invade epithelial cells.

Ultrafine carbon particles induce apoptosis and proliferation in rat lung epithelial cells via specific signaling pathways both using EGF-R

Apoptosis and proliferation are important causes of adverse health effects induced by inhaled ultrafine particles. The molecular mechanisms of particle cell interactions mediating these end points are therefore a major topic of current particle toxicology and molecular preventive medicine. Initial studies revealed that ultrafine particles induce apoptosis and proliferation in parallel in rat lung epithelial cells, dependent on time and dosage. With these end points, two antagonistic reactions seem to be induced by the same extracellular stimulus. It was therefore investigated whether proliferation is induced directly by the particles or as a compensation of particle-caused cell death. Experimental conditions excluding compensatory proliferation demonstrated that both end points are induced independently by specific signaling pathways. Events eliciting signaling cascades leading to apoptosis and proliferation were studied with specific inhibitors of membrane receptors. Epidermal growth factor receptor (EGF-R) kinase activity was identified as essential for apoptosis as well as for proliferation. As ultrafine particle-induced proliferation alone was dependent on the activation of beta1-integrins, these membrane receptors are suggested to mediate the specificity of EGF-R signaling concerning the decision as to whether apoptosis or proliferation is triggered. Accordingly, MAP kinase signaling downstream of EGF-R showed comparable specificity with regard to receptor-dependent induction of apoptosis and proliferation. As key mediators of signaling cascades, the activation of extracellular signal-regulated kinases 1 and 2 proved to be specific for proliferation in a beta1-integrin-dependent manner, whereas phosphorylation of c-Jun NH2-terminal kinases 1 and 2 was correlated with the induction of apoptosis.

TYROSINE PHOSPHORYLATION OF CASPASE-8 ON Y397 PROTECTS A549 LUNG EPITHELIAL CELLS FROM APOPTOSIS

Accelerated apoptosis of epithelial cells in the lung, kidney, and GI tract occurs in animal models of shock, and in human critical illness. Since caspases - the proteolytic enzymes responsible for apoptosis - are constitutively present in epithelial cells, we hypothesized that post-translational modifications might render them quiescent. Methods: A549 lung epithelial cells were cultured in supplemented DMEM. Apoptosis was measured by propidium iodide uptake or by TUNEL staining. A549 cells were transfected using Fugene-6 with a caspase-8/c-myc construct, or with a mutated caspase-8/c-myc in which Y397 was mutated to phenylalanine (Y397F). Protein-protein interactions were evaluated by immunofluorescence microscopy and coimmunoprecipitation studies. Results: Wild-type A549 cells showed minimal evidence of in vitro apoptosis, and minimal activity of caspases-8 and -3. Transfection with wild-type caspase- 8/c-myc resulted in increased apoptosis and rates were further increased when cells were transfected with the Y397F mutant caspase-8:TableTreatment of WT A549 cells with TNF (40 ng/ml) or anti-CD95 antibody resulted in increased apoptosis and decreased tyrosine phosphorylation of caspase-8: Immunofluorescence microscopy revealed increased nuclear localization of caspase-3 in Y397F transfectants. Conclusions: Lung epithelial cell apoptosis is induced by TNFα or engagement of cell surface Fas. Caspase-8 activation is inhibited by its tyrosine phosphorylation on Y397, and accelerated in cells in which this process has been blocked. Post-translational modifications of caspase-8 represent novel targets for epithelial cell cytoprotective strategies in trauma, shock, and sepsis.Figure

Regulation of human lung epithelial cell numbers by diesel exhaust particles

Particulate air pollution is associated with respiratory morbidity and has cytotoxic and pro-inflammatory effects. The effects of diesel exhaust particles (DEP) on proliferation and apoptosis of A549 lung epithelial cells were examined. When deprived of serum (serum starvation), epithelial cell numbers fell, but DEP (5-200 microg.mL-1) prevented this. Using flow cytometric analysis of propidium iodide (PI) staining, DEP (10 microg.mL-1) increased cells in the S phase of cell cycle from 12.85 to 18.75% after 48 h, reversing serum starvation-induced G0/1 arrest. DEP also reduced the increase in apoptotic cells, as defined by double expression of annexin V/PI, observed after serum starvation (from 28.35 to 15.46%). The antioxidants, N-acetylcysteine (NAC; 33 mM) and AEOL10113 (10-100 microM), the N-terminal c-jun kinase inhibitor, SP600125 (33 microM), and nuclear factor-kappaB inhibitor, SN50 (33 microM), inhibited DEP-induced cell number increase. NAC inhibited DEP-induced reduction of G0/1 and increase in cells in the S and G2/M phases. Expression of p21CIP1/WAF1 mRNA and protein seen with serum starvation was reduced by DEP. In conclusion, diesel exhaust particles prevented serum starvation-led decreases in A549 epithelial cells by inducing cell cycle progression and preventing apoptosis, processes involving oxidative stress, inhibition of p21CIP1/WAF1 expression and stimulation of N-terminal c-jun kinase and nuclear factor-kappaB. Therefore, low-dose diesel exhaust particle exposure may lead to lung epithelial cell hyperplasia.

Comparison of the effects of erdosteine and N-acetylcysteine on apoptosis regulation in endotoxin-induced acute lung injury

This study was carried out to investigate comparatively the frequency of apoptosis in lung epithelial cells after intratracheal instillation of endotoxin [lipopolysaccharide (LPS)] in rats and the role of tumor necrosis factor alpha (TNF-alpha) on apoptosis, and the effects of erdosteine and N-acetylcysteine on the regulation of apoptosis. Female Wistar rats were given oral erdosteine (10-500 mg kg(-1)) or N-acetylcysteine (10-500 mg kg(-1)) once a day for 3 consecutive days. Then the rats were intratracheally instilled with LPS (5 mg kg(-1)) to induce acute lung injury. The rats were killed at 24 h after LPS administration. Lung tissue samples were stained with hematoxylin-eosin for histopathological assessments. The apoptosis level in the lung bronchial and bronchiolar epithelium was determined using the TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick endlabelling) method. Cytoplasmic TNF-alpha was evaluated by immunohistochemistry. Pretreatment with erdosteine and pretreatment with N-acetylcysteine at a dose of 10 mg kg(-1) had no protective effect on LPS-induced lung injury. When the doses of drugs increased, the severity of the lung damage caused by LPS decreased. It was found that as the pretreatment dose of erdosteine was increased, the rate of apoptosis induced by LPS in lung epithelial cells decreased and this decrease was statistically significant in doses of 300 mg kg(-1) and 500 mg kg(-1). Pretreatment with N-acetylcysteine up to a dose of 500 mg kg(-1) did not show any significant effect on apoptosis regulation. It was noticed that both antioxidants had no significant effect on the local production level of TNF-alpha. These findings suggest that erdosteine could be a possible therapeutic agent for acute lethal lung injury and its mortality.

Bleomycin initiates apoptosis of lung epithelial cells by ROS but not by Fas/FasL pathway

Epithelial cells are considered to be a main target of bleomycin-induced lung injury, which leads to fibrosis in vivo. We studied the characteristics of in vitro bleomycin-induced apoptosis in a mouse lung epithelial (MLE) cell line. Bleomycin caused an increase of reactive oxygen species (ROS) resulting in oxidative stress, mitochondrial leakage, and apoptosis. These were associated with elevated caspase-8 and resultant caspase-9 activity and with upregulation of Fas expression. Glutathione and inhibitors of caspase-8 or caspase-9, but not of FasL, inhibited these effects, suggesting their dependence on ROS, caspase-8 and -9, in a Fas/FasL-independent pathway. However, postbleomycin-exposed MLE cells were more sensitive to Fas-mediated apoptosis. These results demonstrate that the initial bleomycin-induced oxidative stress causes a direct apoptotic effect in lung epithelial cells involving a regulatory role of caspase-8 on caspase-9. Fas represents an amplification mechanism, and not a direct trigger of bleomycin-induced epithelial cell apoptosis.

Effects of hypoxia and hypercapnia on surfactant protein expression proliferation and apoptosis in A549 alveolar epithelial cells

During lung injury alveolar epithelial cells are directly exposed to changes in PO 2 and PCO 2. Integrity of alveolar epithelial type II cells (AECII) is critical in lung injury but the effect of hypoxia and hypercapnia on AECII function, viability and proliferation has not been clearly investigated. Aim of the present work was to determine the direct effect of hypoxia and hypercapnia on surfactant protein expression, proliferation and apoptosis of lung epithelial cells in vitro. A549 alveolar epithelia cells were subjected to hypoxia (1%O 2–5% CO 2) or hypercapnia (21% O 2– 15% CO 2) and expression of surfactant protein C was measured and compared to normal conditions (21% O 2- 5% CO 2). Cell cycle progression and apoptosis were measured by flow cytometric analysis. Results A549 alveolar epithelial cells produce surfactant proteins, including surfactant protein C, when cultured under normal conditions, which is reduced under hypoxic conditions. Specifically, pro-SpC expression is moderately decreased after 8 h of culture in hypoxia, and is completely attenuated after 48 h. Hypercapnia decreases pro-SpC expression only after 48 h of exposure. Stimulation with TNF-α partly reverses pSPC decrease observed under hypoxic and hypercapnic conditions. Hypoxic culture of A549 cells results in progressive arrest of cells in the G1 phase of the cell cycle and increased apoptosis first observed 4 h following exposure and peaking at 24 h. In contrast hypercapnia has no significant effect on alveolar epithelial cell proliferation or apoptosis. Conclusions Taken together we can conclude that hypoxia rapidly and severely affects AECII function and viability while hypercapnia has an inhibitory effect on pro-SpC production only after prolonged exposure.

Bcl-2 Rescues Ceramide- and Etoposide-induced Mitochondrial Apoptosis through Blockage of Caspase-2 Activation

Recent studies indicate that caspase-2 is involved in the early stage of apoptosis before mitochondrial damage. Although the activation of caspase-2 has been shown to occur in a large protein complex, the mechanisms of caspase-2 activation remain unclear. Here we report a regulatory role of Bcl-2 on caspase-2 upstream of mitochondria. Stress stimuli, including ceramide and etoposide, caused caspase-2 activation, mitochondrial damage followed by downstream caspase-9 and -3 activation, and cell apoptosis in human lung epithelial cell line A549. When A549 cells were pretreated with the caspase-2 inhibitor benzyloxycarbonyl-Val-Asp(-OMe)-Val-Ala-Asp(-OMe)-fluoromethyl ketone or transfected with caspase-2 short interfering RNA, both ceramide- and etoposide-induced mitochondrial damage and apoptosis were blocked. Overexpression of Bcl-2 prevented ceramide- and etoposide-induced caspase-2 activation and mitochondrial apoptosis. Furthermore, caspase-2 was activated when A549 cells were introduced with Bcl-2 short interfering RNA or were treated with Bcl-2 inhibitor, which provided direct evidence of a negative regulatory effect of Bcl-2 on caspase-2. Cell survival was observed when caspase-2 was inhibited in Bcl-2-silencing cells. Blockage of the mitochondrial permeability transition pore and caspase-9 demonstrated that Bcl-2-modulated caspase-2 activity occurred upstream of mitochondria. Further studies showed that Bcl-2 was dephosphorylated at serine 70 after ceramide and etoposide treatment. A protein phosphatase inhibitor, okadaic acid, rescued Bcl-2 dephosphorylation and blocked caspase-2 activation, mitochondrial damage, and cell death. Taken together, ceramide and etoposide induced mitochondria-mediated apoptosis by initiating caspase-2 activation, which was, at least in part, regulated by Bcl-2.

Proteinase-Activated Receptor-1 Mediates Elastase-Induced Apoptosis of Human Lung Epithelial Cells

Apoptosis of distal lung epithelial cells plays a pivotal role in the pathogenesis of acute lung injury. In this context, proteinases, either circulating or leukocyte-derived, may contribute to epithelial apoptosis and lung injury. We hypothesized that apoptosis of lung epithelial cells induced by leukocyte elastase is mediated via the proteinase activated receptor (PAR)-1. Leukocyte elastase, thrombin, and PAR-1-activating peptide, but not the control peptide, induced apoptosis in human airway and alveolar epithelial cells as assessed by increases in cytoplasmic histone-associated DNA fragments and TUNEL staining. These effects were largely prevented by a specific PAR-1 antagonist and by short interfering RNA directed against PAR-1. To ascertain the mechanism of epithelial apoptosis, we determined that PAR-1AP, thrombin, and leukocyte elastase dissipated mitochondrial membrane potential, induced translocation of cytochrome c to the cytosol, enhanced cleavage of caspase-9 and caspase-3, and led to JNK activation and Akt inhibition. In concert, these observations provide strong evidence that leukocyte elastase mediates apoptosis of human lung epithelial cells through PAR-1-dependent modulation of the intrinsic apoptotic pathway via alterations in mitochondrial permeability and by modulation of JNK and Akt.

Apoptosis of mink lung epithelial cells by co-treatment of low-dose staurosporine and transforming growth factor-β1 depends on the enhanced TGF-β signaling and requires the decreased phosphorylation of PKB/Akt

We demonstrate how co-treatment of low-dose staurosporine (STS) and TGF-β1, which alone have little effect on cell death, markedly induces apoptosis in Mv1Lu mink lung epithelial cells, but not in its clonal variant R1B cells lacking functional TGF-β signaling. This process was associated with mitochondria-dependent apoptosis and the enhanced TGF-β/Smad signaling in Mv1Lu cells. When R1B cells were infected with adenovirus carrying wild-type ALK5, a functional TGF-β type I receptor gene, the apoptotic cell death was significantly restored in these cells following co-treatment of low-dose STS and TGF-β1. Treatment of Mv1Lu cells with both low-dose STS and TGF-β1 decreased the activity of phospho-Akt, which is involved in cell survival signal. In addition, pre-treatments of PI3 kinase inhibitors, LY294002 and wortmannin, further increased the apoptosis of MvlLu cells induced by co-treatment of low-dose STS and TGF-β1. And overexpression of constitutively active Akt (myr-Akt) using adenoviral expression system inhibited the apoptotic cell death of Mv1Lu cells by about 50% upon co-treatment of low-dose STS and TGF-β1. These results suggest that co-treatment of low-dose STS and TGF-β1 induces apoptosis of mink lung epithelial cells by enhancing TGF-β signaling and in part suppressing cytoprotective signaling.

Keratinocyte growth factor induces Akt kinase activity and inhibits Fas-mediated apoptosis in A549 lung epithelial cells.

Acute respiratory distress syndrome (ARDS) is a syndrome characterized by the rapid influx of protein-rich edema fluid into the air spaces. The magnitude of alveolar epithelial cell injury is a key determinant of disease severity and an important predictor of patient outcome. The alveolar epithelium is positioned at the interface of the host response in the initiation, progression, and recovery phase of the disease. Keratinocyte growth factor (KGF) is a potent survival factor unique to the epithelium that promotes lung epithelial cell survival, accelerates wound closure, and reduces fibrosis. We therefore hypothesized that KGF preserves lung function by inhibiting apoptosis through activation of a signal transduction pathway responsible for cell survival. To test this hypothesis we determined that KGF inhibits death following Fas activation, a relevant apoptosis pathway, and then determined that cell survival is mediated through activation of the phosphatidylinositol 3'-kinase (PI3K)/Akt kinase signal transduction pathway. We found that KGF induces a dose- and time-dependent increase in Akt kinase activity and that, as expected, activation of Akt via KGF is PI3K dependent. KGF inhibited Fas-induced apoptosis as measured by a reduction in apoptotic cells and caspase-3 activity. This investigation supports our original hypothesis that KGF protects the lung epithelium by inhibiting apoptosis and that protection occurs through activation of PI3K/Akt-mediated cell survival pathway. Our results are in agreement with other reports that identify the PI3K/Akt axis as a key intracellular pathway in the lung epithelium that may serve as a therapeutic target to preserve epithelial integrity during inflammation.

Apoptosis and lung injury

Apoptosis is important in developmental biology and in remodeling of tissues during repair. Apoptosis also plays important roles in the progression of many diseases. The cellular and molecular mechanisms of apoptosis, in general, have been extensively demonstrated. However, the causes and the roles of apoptosis of various cell types in the lung are not well understood. We have determined that adenosine/homocysteine causes lung vascular endothelial cell apoptosis by inhibition of carboxyl methylation of the small GTPase, Ras, through inhibition of isoprenylcysteine carboxyl methyltransferase(ICMT) activity, leading to inactivation of Ras and the subsequent disruption of focal adhesion complexes, resulting in cell-extracellular matrix detachment and anoikis. Apoptosis can either ameliorate or exacerbate lung injury, depending upon the cell type. Although apoptosis of polymorphonuclear leukocytes in the lung prevents inflammation and the development of acute respiratory distress syndrome during acute lung injury, Fas/FasL-mediated alveolar epithelial cell apoptosis promotes acute lung injury and pulmonary fibrosis. Lung epithelial and endothelial cell apoptosis also contributes to the development of emphysema. This article focuses on elucidating the mechanisms of adenosine/homocysteine-induced endothelial cell apoptosis. We also review the current understanding of the role of lung cell apoptosis in acute lung injury, pulmonary fibrosis and emphysema.

Cytochrome c release is required for phosphatidylserine peroxidation during fas‐triggered apoptosis in lung epithelial A549 cells

Oxidation of phosphatidylserine (PtdSer) has been shown to play a pivotal role in signaling during cell apoptosis and subsequent recognition of apoptotic cells by phagocytes. However, the redox catalytic mechanisms involved in selective PtdSer oxidation during apoptosis remain poorly understood. Here we employed anti-Fas antibody CH-11-treated A549 cells as a physiologically relevant model to investigate the involvement of PtdSer oxidation and its potential mechanism during apoptosis. We demonstrated that ligation of CH-11 with its cognate receptor initiated execution of apoptotic program in interferon gamma-pretreated A549 cells as evidenced by activation of caspase and DNA fragmentation. A significant increase of cytochrome c (cyt c) content in the cytosol as early as 2 h after CH-11 exposure was detected indicating that Fas-induced apoptosis in A549 cells proceeds via extrinsic type II pathway and includes mitochondrial signaling. PtdSer was selectively oxidized 3 h after anti-Fas triggering while two more abundant phospholipids--phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn)--and the major intracellular antioxidant, glutathione, remained nonoxidized. A pan-caspase inhibitor, z-VAD, fully blocked cyt c release and oxidation of PtdSer in Fas-treated A549 cells. On the other hand, z-DQMD, a caspase-3 inhibitor, completely inhibited caspase-3 activity but did not fully block caspase-8 activation and release of cyt c. Importantly, z-DQMD failed to protect PtdSer from oxidation. In addition, in a model system, we demonstrated that peroxidase activity of cyt c was greatly enhanced in the presence of dioleoylphosphatidylserine containing liposomes by monitoring oxidation of 2',7'-dichlorodihydrofluorescein to 2',7'-dichlorofluorescein. We further showed that peroxidase activity of cyt c catalyzed oxidation of 1-palmitoyl-2-arachidonoyl-3-glycero-phosphoserine using a newly developed HPLC assay. MS analysis of 1-palmitoyl-2-arachidonoyl-3-glycero-phosphoserine revealed that in addition to its mono- and dihydroperoxides, several different PtdSer oxidation products can be formed. Overall, we concluded that cyt c acts as a catalyst of PtdSer oxidation during Fas-triggered A549 cell apoptosis.