Original contributionMAPK pathways mediate hyperoxia-induced oncotic cell death in lung epithelial cells
Introduction
Mechanical ventilation with hyperoxic conditions is often used to treat patients, especially premature newborns, with respiratory distress due to hypoxemia. However, prolonged exposure to hyperoxia causes acute inflammatory lung injury, accompanied by injury and death of both pulmonary endothelial and epithelial cells in animal models, including mice, rats, and rabbits 1, 2, 3, 4, 5. Alveolar epithelial cells are essential in maintaining the integrity of the alveolar-capillary barrier. The compromised integrity of alveolar epithelial cells leads to the leakage of fluid, macromolecules, and lymphatic infiltrates into the air spaces, causing pulmonary edema, lung dysfunction, and even death 6, 7. Therefore, a thorough understanding of the regulation of the pathways leading to alveolar epithelial cell injury and cell death may provide some insights for improving the outcome in these patients.
The terms apoptosis, necrosis, and oncosis are associated with the current thinking about cell death. Whereas necrosis is defined by the loss of plasma membrane integrity, both apoptosis and oncosis are used to describe the cell death processes leading to the loss of plasma membrane integrity 8, 9. Apoptosis is an active process requiring adenosine triphosphate (ATP), characterized by cellular shrinkage, nuclear condensation, and activation of caspase-3 [10]. On the other hand, cell death that is marked by cellular swelling should be called oncosis [11]. Although it has long been confused with necrosis, oncosis describes a process leading to cell death, whereas necrosis refers to the morphological alterations appearing after either apoptosis or oncosis [11]. Much attention has been focused on the signaling pathways that lead to apoptosis induced by various stresses in different cell types. However, aside from its use in classifying dying cells that have swollen organelles, little is known about oncosis 8, 9, 11.
Increasing evidence suggests that bronchial and alveolar epithelia exposed to prolonged hyperoxia suffer from multiple modes of cell injury and cell death, with both oncotic and apoptotic morphologies in hyperoxia-injured animal lungs 3, 12, 13, 14. Morphometric and ultrastructural analyses revealed that these cells exhibit swelling of the intracellular organelles, including the mitochondria and endoplasmic reticulum, typical markers of oncosis. Conversely, characteristics of apoptosis, including chromatin condensation, plasma membrane blebbing, and increased internucleosomal DNA ladders, can be observed concurrently in hyperoxic-injured lungs 5, 14, 15, 16, 17, 18.
Although strategies aimed at attenuating apoptotic cell death may be effective, preventing injury and death of nonapoptotic cells with an oncotic morphology may also be beneficial in preventing tissue injury, especially inflammatory injures. First, because apoptotic cells can be recognized via translocated phosphatidylserine and subsequently cleared, it is reasonable to assume that it is easier to prevent the release of inflammatory-causing contents from apoptotic cells than from nonapoptotic cells. In fact, inflammatory diseases, such as hyperoxic acute lung injury, have been proposed as a result of nonapoptotic cell death [19]. In addition, high mobility group protein-1 (HMG-1) has been shown to be a mediator for acute inflammatory lung injury [20], and the release of HMG-1 from nonapoptotic cells occurs more readily than from apoptotic cells 21, 22. Therefore, interventions focused on pathways leading to oncotic cell injury and cell death may prove to be useful in alleviating inflammatory hyperoxic lung injury.
Mouse lung epithelial (MLE-12) cells have been used as a model system for alveolar epithelial cells by a number of laboratories 23, 24. To investigate the signal transduction pathways to hyperoxia-induced cell injury and cell death in alveolar epithelium, MLE-12 cells were used as a model system in which the detection of signaling events are not complicated by that from other cell types as in the lung. In this report we demonstrate that in hyperoxia, MLE-12 cells died via oncosis, with swollen cell nuclei, cytosolic vacuolation, and disrupted mitochondrial structure and function, in the absence of either increased caspase-3 activity or phosphatidylserine translocation.
Little is known about the pathways leading to oncotic cell death. To begin the dissection of the pathways to hyperoxic oncosis and to examine whether these oncotic cells undergo similar events as oxidative apoptosis, we focused on the upstream pathways that are important in apoptosis. Numerous studies demonstrate that the transcription factor AP-1 and its trans-activators, JNK and p38, are activated by genotoxic stresses [25]. Although AP-1 is involved in modulating both cell proliferation and death in a cell type- and stimulus-dependent manner, sustained activation of AP-1 under persistent genotoxic stress is thought to be more closely associated with apoptosis 26, 27, 28. Under these circumstances, JNK and p38 MAPK play pivotal roles in mediating the induction and sustained activation of AP-1 [25]. Therefore, both AP-1 and its upstream regulators, JNK and p38 MAPK, play essential roles in genotoxic stress-induced apoptosis. However, it is unclear whether such pathways are activated and play causal roles in the less characterized oncotic cell death. In this report, we showed that JNK and p38 MAPK signal transduction pathways mediate not only oxidative apoptosis, but also hyperoxia-induced oncotic cell death.
Section snippets
Cell culture
Murine lung epithelium MLE-12 cells were obtained from American Type Culture Collection (ATCC; Rockville, MD, USA), and cultured in Hite's medium (as recommended by ATCC) supplemented with 2% FBS, 1000 U/ml penicillin, and 500 μg/ml streptomycin in 5% CO2 at 37°C as described [29]. MLE-12 cells expressing dominant negative constructs of JNK1 were cultured with an additional 200 μg/ml G418 (Gibco, Grand Island, NY, USA). MCF-7 cells, a human breast carcinoma cell line from ATCC, were maintained
Oncotic cell death in hyperoxia
It has been previously shown that human and mink alveolar epithelial cells undergo hyperoxic cell death without apoptotic morphology and DNA fragmentation 40, 41, 42, whereas apoptotic phenotypes were observed in cultured murine macrophages [43] and mouse lung epithelial cells (Dr. Patty Lee, personal communication). To further clarify whether exposure of mouse alveolar epithelial cells to hyperoxia induces apoptosis, we exposed MLE-12 cells to 95% O2 and performed both biochemical and
Discussion
In this report we show that hyperoxia induces an oncotic cell death in MLE-12 cells with swollen organelles, marked cytoplasmic vacuolation, and disrupted mitochondrial structures and function in the absence of either increased caspase-3 activity or phosphatidylserine translocation. Characterization of the signal transduction pathways associated with hyperoxic oncotic cell death indicates that AP-1, JNK, or p38 MAPK play causal roles in such oxidative oncosis. Interestingly, oxidative apoptosis
Acknowledgements
We thank Drs. Jeff Kazzaz, Yuchi Li, and Hank Simms for their suggestions, criticisms, and scientific insights; Drs. Roger J. Davis (a Howard Hughs Medical Institute Investigator), Yvonne Janssen-Heininger, and Punya Ranjan for providing the pcDNA3-Flag-JNK1(APF) and vector control plasmids; Dr. Daniel Grande and Pasquale Razzano for their kind help in performing the tritium-labeled thymidine incorporation experiments; Drs. Marc Symons, Ping Wang, and Maria Ruggieri for their invaluable
References (67)
- et al.
Oxygen toxicity
Clin. Chest Med.
(1988) - et al.
Effect of ICRF-187 on the pulmonary damage induced by hyperoxia in the rat
Toxicology
(1992) - et al.
A c-Jun dominant negative mutant protects sympathetic neurons against programmed cell death
Neuron
(1995) - et al.
Nuclear factor-κB is activated by hyperoxia but does not protect from cell death
J. Biol. Chem.
(1997) - et al.
Characterization of the structure and function of a new mitogen-activated protein kinase (p38beta)
J. Biol. Chem.
(1996) - et al.
IB1, a JIP-1-related nuclear protein present in insulin-secreting cells
J. Biol. Chem.
(1998) - et al.
Hyperoxia inhibits oxidant-induced apoptosis in lung epithelial cells
J. Biol. Chem.
(2001) - et al.
JNK1a protein kinase stimulated by UV light and Ha-Ras that binds and phosphorylates the c-Jun activation domain
Cell
(1994) - et al.
Identification of c-Jun NH2-terminal protein kinase (JNK)-activating kinase 2 as an activator of JNK but not p38
J. Biol. Chem.
(1997) - et al.
Suppression of nuclear factor-KAPPA B activity by nitric oxide and hyperoxia in oxygen resistant cells
J. Biol. Chem.
(2002)
Cellular oxygen toxicity. Oxidant injury without apoptosis
J. Biol. Chem.
Suppression of apoptosis by all-trans-retinoic acid. Dual intervention in the c-Jun n-terminal kinase-AP-1 pathway
J. Biol. Chem.
Signal transduction by the c-Jun N-terminal kinase (JNK)—from inflammation to development
Curr. Opin. Cell Biol.
Regulation and function of the JNK subgroup of MAP kinases
Biochim. Biophys. Acta
Role of glutathione S-transferases in protection against lipid peroxidation. Overexpression of hGSTA2-2 in K562 cells protects against hydrogen peroxide-induced apoptosis and inhibits JNK and caspase 3 activation
J. Biol. Chem.
Characterization of oxygen-resistant Chinese hamster ovary cells. III. Relative resistance of succinate and alpha-ketoglutarate dehydrogenases to hyperoxic inactivation
Free Radic. Biol. Med.
Regulation of cellular oncosis by uncoupling protein 2
J. Biol. Chem.
Necrotic cell death in response to oxidant stress involves the activation of the apoptogenic caspase-8/bid pathway
J. Biol. Chem.
Structural and biochemical changes in rat lungs occurring during exposures to lethal and adaptive doses of oxygen
Am. Rev. Respir. Dis.
Morphologic changes in pulmonary oxygen toxicity
Annu. Rev. Physiol.
Glutathione ethyl ester supplementation prevents mortality in newborn rats exposed to hyperoxia
Biol. Neonate
Progressive alveolar septal injury in primates exposed to 60% oxygen for 14 days
Am. J. Physiol.
Oxygen toxicity
New Horiz
The nomenclature of cell deathrecommendations of an ad hoc Committee of the Society of Toxicologic Pathologists
Toxicol. Pathol.
Morphological and biochemical aspects of apoptosis, oncosis and necrosis
Anat. Histol. Embryol.
Apoptosiscell death defined by caspase activation
Cell Death Differ
Apoptosis, oncosis, and necrosis. An overview of cell death
Am. J. Pathol.
Oxygen toxicity in the infant rhesus monkey lung. Light microscopic and ultrastructural studies
Histol. Histopathol.
Oxygen toxicity in mouse lungpathways to cell death
Am. J. Respir. Cell Mol. Biol.
Unscheduled apoptosis during acute inflammatory lung injury
Cell Death Differ
Targeted lung expression of interleukin-11 enhances murine tolerance of 100% oxygen and diminishes hyperoxia-induced DNA fragmentation
J. Clin. Invest.
DNA damage and cell cycle checkpoints in hyperoxic lung injurybraking to facilitate repair
Am. J. Physiol. Lung Cell. Mol. Physiol.
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