G. R. Scott Budinger

Mitochondrial metabolism and ROS generation are essential for Kras-mediated tumorigenicity.

Otto Warburgs theory on the origins of cancer postulates that tumor cells have defects in mitochondrial oxidative phosphorylation and therefore rely on high levels of aerobic glycolysis as the major source of ATP to fuel cellular proliferation (the Warburg effect). This is in contrast to normal cells, which primarily utilize oxidative phosphorylation for growth and survival. Here we report that the major function of glucose metabolism for Kras-induced anchorage-independent growth, a hallmark of transformed cells, is to support the pentose phosphate pathway. The major function of glycolytic ATP is to support growth under hypoxic conditions. Glutamine conversion into the tricarboxylic acid cycle intermediate alpha-ketoglutarate through glutaminase and alanine aminotransferase is essential for Kras-induced anchorage-independent growth. Mitochondrial metabolism allows for the generation of reactive oxygen species (ROS) which are required for Kras-induced anchorage-independent growth through regulation of the ERK MAPK signaling pathway. We show that the major source of ROS generation required for anchorage-independent growth is the Qo site of mitochondrial complex III. Furthermore, disruption of mitochondrial function by loss of the mitochondrial transcription factor A (TFAM) gene reduced tumorigenesis in an oncogenic Kras-driven mouse model of lung cancer. These results demonstrate that mitochondrial metabolism and mitochondrial ROS generation are essential for Kras-induced cell proliferation and tumorigenesis.

The Qo site of the mitochondrial complex III is required for the transduction of hypoxic signaling via reactive oxygen species production

Mammalian cells increase transcription of genes for adaptation to hypoxia through the stabilization of hypoxia-inducible factor 1α (HIF-1α) protein. How cells transduce hypoxic signals to stabilize the HIF-1α protein remains unresolved. We demonstrate that cells deficient in the complex III subunit cytochrome b, which are respiratory incompetent, increase ROS levels and stabilize the HIF-1α protein during hypoxia. RNA interference of the complex III subunit Rieske iron sulfur protein in the cytochrome b–null cells and treatment of wild-type cells with stigmatellin abolished reactive oxygen species (ROS) generation at the Qo site of complex III. These interventions maintained hydroxylation of HIF-1α protein and prevented stabilization of HIF-1α protein during hypoxia. Antioxidants maintained hydroxylation of HIF-1α protein and prevented stabilization of HIF-1α protein during hypoxia. Exogenous hydrogen peroxide under normoxia prevented hydroxylation of HIF-1α protein and stabilized HIF-1α protein. These results provide genetic and pharmacologic evidence that the Qo site of complex III is required for the transduction of hypoxic signal by releasing ROS to stabilize the HIF-1α protein.

Minimizing Oxidation and Stable Nanoscale Dispersion Improves the Biocompatibility of Graphene in the Lung

To facilitate the proposed use of graphene and its derivative graphene oxide (GO) in widespread applications, we explored strategies that improve the biocompatibility of graphene nanomaterials in the lung. In particular, solutions of aggregated graphene, Pluronic dispersed graphene, and GO were administered directly into the lungs of mice. The introduction of GO resulted in severe and persistent lung injury. Furthermore, in cells GO increased the rate of mitochondrial respiration and the generation of reactive oxygen species, activating inflammatory and apoptotic pathways. In contrast, this toxicity was significantly reduced in the case of pristine graphene after liquid phase exfoliation and was further minimized when the unoxidized graphene was well-dispersed with the block copolymer Pluronic. Our results demonstrate that the covalent oxidation of graphene is a major contributor to its pulmonary toxicity and suggest that dispersion of pristine graphene in Pluronic provides a pathway for the safe handling and potential biomedical application of two-dimensional carbon nanomaterials.

Flow Cytometric Analysis of Macrophages and Dendritic Cell Subsets in the Mouse Lung

The lung hosts multiple populations of macrophages and dendritic cells, which play a crucial role in lung pathology. The accurate identification and enumeration of these subsets are essential for understanding their role in lung pathology. Flow cytometry is a mainstream tool for studying the immune system. However, a systematic flow cytometric approach to identify subsets of macrophages and dendritic cells (DCs) accurately and consistently in the normal mouse lung has not been described. Here we developed a panel of surface markers and an analysis strategy that accurately identify all known populations of macrophages and DCs, and their precursors in the lung during steady-state conditions and bleomycin-induced injury. Using this panel, we assessed the polarization of lung macrophages during the course of bleomycin-induced lung injury. Alveolar macrophages expressed markers of alternatively activated macrophages during both acute and fibrotic phases of bleomycin-induced lung injury, whereas markers of classically activated macrophages were expressed only during the acute phase. Taken together, these data suggest that this flow cytometric panel is very helpful in identifying macrophage and DC populations and their state of activation in normal, injured, and fibrotic lungs.

Ambient particulate matter accelerates coagulation via an IL-6–dependent pathway

The mechanisms by which exposure to particulate matter increases the risk of cardiovascular events are not known. Recent human and animal data suggest that particulate matter may induce alterations in hemostatic factors. In this study we determined the mechanisms by which particulate matter might accelerate thrombosis. We found that mice treated with a dose of well characterized particulate matter of less than 10 microM in diameter exhibited a shortened bleeding time, decreased prothrombin and partial thromboplastin times (decreased plasma clotting times), increased levels of fibrinogen, and increased activity of factor II, VIII, and X. This prothrombotic tendency was associated with increased generation of intravascular thrombin, an acceleration of arterial thrombosis, and an increase in bronchoalveolar fluid concentration of the prothrombotic cytokine IL-6. Knockout mice lacking IL-6 were protected against particulate matter-induced intravascular thrombin formation and the acceleration of arterial thrombosis. Depletion of macrophages by the intratracheal administration of liposomal clodronate attenuated particulate matter-induced IL-6 production and the resultant prothrombotic tendency. Our findings suggest that exposure to particulate matter triggers IL-6 production by alveolar macrophages, resulting in reduced clotting times, intravascular thrombin formation, and accelerated arterial thrombosis. These results provide a potential mechanism linking ambient particulate matter exposure and thrombotic events.

Hypoxic activation of AMPK is dependent on mitochondrial ROS but independent of an increase in AMP/ATP ratio.

AMP-activated protein kinase (AMPK) is a sensor of cellular energy status found in metazoans that is known to be activated by stimuli that increase the cellular AMP/ATP ratio. Full activation of AMPK requires specific phosphorylation within the activation loop of the catalytic domain of the alpha-subunit by upstream kinases such as the serine/threonine protein kinase LKB1. Here we show that hypoxia activates AMPK through LKB1 without an increase in the AMP/ATP ratio. Hypoxia increased reactive oxygen species (ROS) levels and the antioxidant EUK-134 abolished the hypoxic activation of AMPK. Cells deficient in mitochondrial DNA (rho(0) cells) failed to activate AMPK during hypoxia but are able to in the presence of exogenous H(2)O(2). Furthermore, we provide genetic evidence that ROS generated within the mitochondrial electron transport chain and not oxidative phosphorylation is required for hypoxic activation of AMPK. Collectively, these data indicate that oxidative stress and not an increase in the AMP/ATP ratio is required for hypoxic activation of AMPK.

Biocompatible nanoscale dispersion of single-walled carbon nanotubes minimizes in vivo pulmonary toxicity.

Excitement surrounding the attractive physical and chemical characteristics of single walled carbon nanotubes (SWCNTs) has been tempered by concerns regarding their potential health risks. Here we consider the lung toxicity of nanoscale dispersed SWCNTs (mean diameter approximately 1 nm). Because dispersion of the SWCNTs increases their aspect ratio relative to as-produced aggregates, we directly test the prevailing hypothesis that lung toxicity associated with SWCNTs compared with other carbon structures is attributable to the large aspect ratio of the individual particles. Thirty days after their intratracheal administration to mice, the granuloma-like structures with mild fibrosis in the large airways observed in mice treated with aggregated SWCNTs were absent in mice treated with nanoscale dispersed SWCNTs. Examination of lung sections from mice treated with nanoscale dispersed SWCNTs revealed uptake of the SWCNTs by macrophages and gradual clearance over time. We conclude that the toxicity of SWCNTs in vivo is attributable to aggregation of the nanomaterial rather than the large aspect ratio of the individual nanotubes. Biocompatible nanoscale dispersion provides a scalable method to generate purified preparations of SWCNTs with minimal toxicity, thus allowing them to be used safely in commercial and biomedical applications.

Bcl-2 Family Members and Functional Electron Transport Chain Regulate Oxygen Deprivation-Induced Cell Death

ABSTRACT The mechanisms underlying cell death during oxygen deprivation are unknown. We report here a model for oxygen deprivation-induced apoptosis. The death observed during oxygen deprivation involves a decrease in the mitochondrial membrane potential, followed by the release of cytochrome c and the activation of caspase-9. Bcl-XL prevented oxygen deprivation-induced cell death by inhibiting the release of cytochrome c and caspase-9 activation. The ability of Bcl-XL to prevent cell death was dependent on allowing the import of glycolytic ATP into the mitochondria to generate an inner mitochondrial membrane potential through the F1F0-ATP synthase. In contrast, although activated Akt has been shown to inhibit apoptosis induced by a variety of apoptotic stimuli, it did not prevent cell death during oxygen deprivation. In addition to Bcl-XL, cells devoid of mitochondrial DNA (ρ° cells) that lack a functional electron transport chain were resistant to oxygen deprivation. Further, murine embryonic fibroblasts from bax −/− bak −/− mice did not die in response to oxygen deprivation. These data suggest that when subjected to oxygen deprivation, cells die as a result of an inability to maintain a mitochondrial membrane potential through the import of glycolytic ATP. Proapoptotic Bcl-2 family members and a functional electron transport chain are required to initiate cell death in response to oxygen deprivation.

Mitochondrial Reactive Oxygen Species Regulate Transforming Growth Factor-β Signaling

Background: Although reactive oxygen species (ROS) are integral for TGF-β signaling, the source of ROS is not clear. Results: Inhibition of TGF-β-induced mitochondrial ROS generation attenuates profibrotic gene expression. Conclusion: ROS generated by complex III of the electron transport chain are required for TGF-β-mediated transcription in normal human lung fibroblasts. Significance: Mitochondrial ROS might be a novel target to prevent TGF-β-mediated induced fibrosis. TGF-β signaling is required for normal tissue repair; however, excessive TGF-β signaling can lead to robust profibrotic gene expression in fibroblasts, resulting in tissue fibrosis. TGF-β binds to cell-surface receptors, resulting in the phosphorylation of the Smad family of transcription factors to initiate gene expression. TGF-β also initiates Smad-independent pathways, which augment gene expression. Here, we report that mitochondrial reactive oxygen species (ROS) generated at complex III are required for TGF-β-induced gene expression in primary normal human lung fibroblasts. TGF-β-induced ROS could be detected in both the mitochondrial matrix and cytosol. Mitochondrially targeted antioxidants markedly attenuated TGF-β-induced gene expression without affecting Smad phosphorylation or nuclear translocation. Genetically disrupting mitochondrial complex III-generated ROS production attenuated TGF-β-induced profibrotic gene expression. Furthermore, inhibiting mitochondrial ROS generation attenuated NOX4 (NADPH oxidase 4) expression, which is required for TGF-β induced myofibroblast differentiation. Lung fibroblasts from patients with pulmonary fibrosis generated more mitochondrial ROS than normal human lung fibroblasts, and mitochondrially targeted antioxidants attenuated profibrotic gene expression in both normal and fibrotic lung fibroblasts. Collectively, our results indicate that mitochondrial ROS are essential for normal TGF-β-mediated gene expression and that targeting mitochondrial ROS might be beneficial in diseases associated with excessive fibrosis.

Nuclear β-Catenin Is Increased in Systemic Sclerosis Pulmonary Fibrosis and Promotes Lung Fibroblast Migration and Proliferation

Pulmonary fibrosis is a disease that results in loss of normal lung architecture, but the signaling events that drive tissue destruction are incompletely understood. Wnt/β-catenin signaling is important in normal lung development, but whether abnormal signaling occurs in lung fibrosis due to systemic sclerosis and the consequences of β-catenin signaling toward the fibrogenic phenotype remain poorly defined. In this study, we show nuclear β-catenin accumulation in fibroblastic foci from lungs of patients with systemic sclerosis-associated advanced pulmonary fibrosis. Forced activation of β-catenin signaling in three independently derived sources of normal human lung fibroblasts promotes proliferation and migratory activities but is not sufficient to activate classic markers of fibroblast activation, such as TGF-β, type 1 collagen, α-smooth muscle actin, and connective tissue growth factor. These findings indicate that activation of β-catenin signaling in pulmonary fibroblasts may be a common feature of lung fibrosis, contributing to fibroproliferative and migratory activities associated with the disease.