Mitochondrial reactive oxygen species regulate transforming growth factor-ß signaling.

TGF-ß signaling is required for normal tissue repair; however, excessive TGF-ß signaling can lead to robust profibrotic gene expression in fibroblasts, resulting in tissue fibrosis. TGF-ß binds to cell-surface receptors, resulting in the phosphorylation of the Smad family of transcription factors to initiate gene expression. TGF-ß 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-ß-induced gene expression in primary normal human lung fibroblasts. TGF-ß-induced ROS could be detected in both the mitochondrial matrix and cytosol. Mitochondrially targeted antioxidants markedly attenuated TGF-ß-induced gene expression without affecting Smad phosphorylation or nuclear translocation. Genetically disrupting mitochondrial complex III-generated ROS production attenuated TGF-ß-induced profibrotic gene expression. Furthermore, inhibiting mitochondrial ROS generation attenuated NOX4 (NADPH oxidase 4) expression, which is required for TGF-ß 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-ß-mediated gene expression and that targeting mitochondrial ROS might be beneficial in diseases associated with excessive fibrosis.

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 1alpha (HIF-1alpha) protein. How cells transduce hypoxic signals to stabilize the HIF-1alpha 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-1alpha 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-1alpha protein and prevented stabilization of HIF-1alpha protein during hypoxia. Antioxidants maintained hydroxylation of HIF-1alpha protein and prevented stabilization of HIF-1alpha protein during hypoxia. Exogenous hydrogen peroxide under normoxia prevented hydroxylation of HIF-1alpha protein and stabilized HIF-1alpha 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-1alpha protein.

Epithelial cell death is an important contributor to oxidant-mediated acute lung injury.

RATIONALE: Acute lung injury and the acute respiratory distress syndrome are characterized by increased lung oxidant stress and apoptotic cell death. The contribution of epithelial cell apoptosis to the development of lung injury is unknown. OBJECTIVES: To determine whether oxidant-mediated activation of the intrinsic or extrinsic apoptotic pathway contributes to the development of acute lung injury. METHODS: Exposure of tissue-specific or global knockout mice or cells lacking critical components of the apoptotic pathway to hyperoxia, a well-established mouse model of oxidant-induced lung injury, for measurement of cell death, lung injury, and survival. MEASUREMENTS AND MAIN RESULTS: We found that the overexpression of SOD2 prevents hyperoxia-induced BAX activation and cell death in primary alveolar epithelial cells and prolongs the survival of mice exposed to hyperoxia. The conditional loss of BAX and BAK in the lung epithelium prevented hyperoxia-induced cell death in alveolar epithelial cells, ameliorated hyperoxia-induced lung injury, and prolonged survival in mice. By contrast, Cyclophilin D-deficient mice were not protected from hyperoxia, indicating that opening of the mitochondrial permeability transition pore is dispensable for hyperoxia-induced lung injury. Mice globally deficient in the BH3-only proteins BIM, BID, PUMA, or NOXA, which are proximal upstream regulators of BAX and BAK, were not protected against hyperoxia-induced lung injury suggesting redundancy of these proteins in the activation of BAX or BAK. CONCLUSIONS: Mitochondrial oxidant generation initiates BAX- or BAK-dependent alveolar epithelial cell death, which contributes to hyperoxia-induced lung injury.

Mitochondrial reactive oxygen species trigger hypoxia-inducible factor-dependent extension of the replicative life span during hypoxia.

Physiological hypoxia extends the replicative life span of human cells in culture. Here, we report that hypoxic extension of replicative life span is associated with an increase in mitochondrial reactive oxygen species (ROS) in primary human lung fibroblasts. The generation of mitochondrial ROS is necessary for hypoxic activation of the transcription factor hypoxia-inducible factor (HIF). The hypoxic extension of replicative life span is ablated by a dominant negative HIF. HIF is sufficient to induce telomerase reverse transcriptase mRNA and telomerase activity and to extend replicative life span. Furthermore, the down-regulation of the von Hippel-Lindau tumor suppressor protein by RNA interference increases HIF activity and extends replicative life span under normoxia. These findings provide genetic evidence that hypoxia utilizes mitochondrial ROS as signaling molecules to activate HIF-dependent extension of replicative life span.

Keratinocyte growth factor expression is suppressed in early acute lung injury/acute respiratory distress syndrome by smad and c-Abl pathways.

OBJECTIVE: Keratinocyte growth factor (KGF) is expressed primarily by fibroblasts, is important for alveolar epithelial proliferation/function, and protects against lung injury in multiple animal models. We wished to determine whether acute lung injury/acute respiratory distress syndrome (ALI/ARDS) alveolar fluid induces KGF and fibroblast genes important for alveolar repair. DESIGN: A single-center cohort study enrolling patients between 2004 and 2006. SETTING: A medical intensive care unit of a tertiary care medical center. PATIENTS: Adult patients meeting the American-European Consensus Conference definition of ALI/ARDS. INTERVENTIONS: Patients with ALI/ARDS were enrolled, and lavage fluid was collected within 48 hours of intubation. Lavage fluid was also collected from two control cohorts. The patients with ALI/ARDS were followed for 28 days or until death. MEASUREMENT AND MAIN RESULTS: Fifteen patients with ALI/ARDS, five patients with cardiogenic edema, and five normal lung parenchyma controls were enrolled from 2004 to 2006. Primary normal human lung fibroblasts were incubated with bronchoalveolar lavage fluid and assessed for KGF, connective tissue growth factor, alpha-smooth muscle actin, and collagen 1 expression by real-time reverse transcriptase-polymerase chain reaction. Fibroblasts incubated with ALI/ARDS lavage fluid expressed 50% less KGF messenger RNA than those incubated with lavage fluid from CE patients (p < 0.01) and 33% than normal parenchymal controls (p < 0.03). Lavage fluid from patients with ALI/ARDS induced more connective tissue growth factor (p < 0.05), collagen 1 (p < 0.03), and alpha-smooth muscle actin (p < 0.04) than from CE patients. Preincubation of normal human lung fibroblasts with the transforming growth factor (TGF)-beta1 receptor/smad phosphorylation inhibitor SB431542 increased ALI/ARDS-induced KGF expression by 40% (p < 0.04). In cultured human lung fibroblasts, TGF-beta1 suppressed KGF messenger RNA and protein expression, which were reversed by SB431542 and by the c-Abl inhibitor, imatinib mesylate, but not by the p38 map kinase inhibitor, SB203580. CONCLUSIONS: ALI/ARDS alveolar fluid suppresses KGF expression, in part, due to TGF-beta1. TGF-beta1 suppression of KGF requires both smad phosphorylation and c-Abl activation.

Active transforming growth factor-beta1 activates the procollagen I promoter in patients with acute lung injury.

OBJECTIVE: Fibroproliferation markers like procollagen I predict mortality in patients with acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). We sought to determine whether bronchoalveolar lavage fluid (BALF) from patients with lung injury contained mediators that would activate procollagen I promoter and if this activation predicted important clinical outcomes. DESIGN: Prospective controlled study of ALI/ARDS. SETTING: Intensive care units and laboratory of a university hospital. PATIENTS AND PARTICIPANTS: Acute lung injury/ARDS, cardiogenic edema (negative controls) and pulmonary fibrosis (positive controls) patients. INTERVENTIONS: Bronchoalveolar lavage fluid was collected within 48 h of intubation from ALI/ARDS patients. BALF was also collected from patients with pulmonary fibrosis and cardiogenic pulmonary edema. Human lung fibroblasts were transfected with a procollagen I promoter-luciferase construct and incubated with BALF; procollagen I promoter activity was then measured. BALF active TGF-beta1 levels were measured by ELISA. RESULTS: Twenty-nine ARDS patients, nine negative and six positive controls were enrolled. BALF from ARDS patients induced 41% greater procollagen I promoter activation than that from negative controls (p<0.05) and a TGF-beta1 blocking antibody significantly reduced this activation in ARDS patients. There was a trend toward higher TGF-beta1 levels in the ARDS group compared to negative controls (-1.056 log(10)+/-0.1415 vs -1.505 log(10)+/-0.1425) (p<0.09). Procollagen I promoter activation was not associated with mortality; however, lower TGF-beta1 levels were associated with more ventilator-free and ICU-free days. CONCLUSIONS: Bronchoalveolar lavage fluid from ALI/ARDS patients activates procollagen I promoter, which is due partly to TGF-beta1. Activated TGF-beta1 may impact ARDS outcome independent of its effect on procollagen I activation.

Mitochondrial complex III ROS regulate adipocyte differentiation.

Adipocyte differentiation is characterized by an increase in mitochondrial metabolism. However, it is not known whether the increase in mitochondrial metabolism is essential for differentiation or a byproduct of the differentiation process. Here, we report that primary human mesenchymal stem cells undergoing differentiation into adipocytes display an early increase in mitochondrial metabolism, biogenesis, and reactive oxygen species (ROS) generation. This early increase in mitochondrial metabolism and ROS generation was dependent on mTORC1 signaling. Mitochondrial-targeted antioxidants inhibited adipocyte differentiation, which was rescued by the addition of exogenous hydrogen peroxide. Genetic manipulation of mitochondrial complex III revealed that ROS generated from this complex is required to initiate adipocyte differentiation. These results indicate that mitochondrial metabolism and ROS generation are not simply a consequence of differentiation but are a causal factor in promoting adipocyte differentiation.

Bronchoalveolar lavage fluid from patients with acute lung injury/acute respiratory distress syndrome induces myofibroblast differentiation.

OBJECTIVE: Myofibroblasts express alpha-smooth muscle actin (alphaSMA), are important in tissue repair, and are present in the early phase of acute lung injury/acute respiratory distress syndrome (ALI/ARDS). We wished to determine whether bronchoalveolar lavage fluid (BALF) from ALI/ARDS patients can induce myofibroblast differentiation and if this induction is associated with outcome. DESIGN: A single-center cohort study enrolling patients between 2002 and 2005. SETTING: Medical intensive care unit of a tertiary care medical center. PATIENTS: Adult patients meeting the American-European Consensus Conference definition of ALI/ARDS. INTERVENTIONS: BALF was collected from ALI/ARDS patients within 48 hrs of intubation and incubated with normal human lung fibroblasts in vitro, and alphaSMA expression was assessed by reverse transcription polymerase chain reaction. BALF was also collected and tested from negative control patients. ALI/ARDS patients were followed for 28 days or death. MEASUREMENTS AND MAIN RESULTS: Thirty-one lung injury and 11 negative control patients were enrolled from 2002 to 2005. ALI/ ARDS BALF demonstrated potent alphaSMA induction with a mean value 92% greater than negative controls (34.5% +/- 7.6% vs. 18% +/- 2.4% of maximal transforming growth factor [TGF]-beta1 [5 ng/mL], p < .02). The specific TGF-beta1 receptor inhibitor SB431542 reduced ALI/ARDS BALF-stimulated alphaSMA induction by 52% (p < .005). There was no correlation between ALI/ARDS BALF-induced alphaSMA and procollagen 3 induction (r = -.08, p = .66). The odds ratio for survival was 6.75 (1.15-39.80) times higher for ALI/ARDS patients with alphaSMA induction between 15% and 75% of maximal TGF-beta1 induction (5 ng/mL) than outside this range. CONCLUSIONS: ALI/ARDS BALF-induced myofibroblast differentiation is partially attributable to TGF-beta1. Procollagen 3 and alphaSMA are regulated by distinct mechanisms in ALI/ARDS and there may be an optimal level of myofibroblast induction that is associated with better outcome.

Proapoptotic Bid is required for pulmonary fibrosis.

The molecular mechanisms of pulmonary fibrosis are poorly understood. Previous reports indicate that activation of TGF-beta1 is essential for the development of pulmonary fibrosis. Here, we report that the proapoptotic Bcl-2 family member Bid is required for the development of pulmonary fibrosis after the intratracheal instillation of bleomycin. Mice lacking Bid exhibited significantly less pulmonary fibrosis in response to bleomycin compared with WT mice. The attenuation in pulmonary fibrosis was observed despite similar levels of inflammation, lung injury, and active TGF-beta1 in bronchoalveolar lavage fluid 5 days after the administration of bleomycin in mice lacking Bid and in WT controls. Bleomycin induced similar levels cell death in vitro in alveolar epithelial cells isolated from WT and bid(-/-) mice. By contrast, alveolar epithelial cells from bid(-/-) mice were resistant to TGF-beta1-induced cell death. These results indicate that Bcl-2 family members are critical regulators for the development of pulmonary fibrosis downstream of TGF-beta1 activation.

Bleomycin induces alveolar epithelial cell death through JNK-dependent activation of the mitochondrial death pathway.

Exposure to bleomycin in rodents induces lung injury and fibrosis. Alveolar epithelial cell death has been hypothesized as an initiating mechanism underlying bleomycin-induced lung injury and fibrosis. In the present study we evaluated the contribution of mitochondrial and receptor-meditated death pathways in bleomycin-induced death of mouse alveolar epithelial cells (MLE-12 cells) and primary rat alveolar type II cells. Control MLE-12 cells and primary rat alveolar type II cells died after 48 h of exposure to bleomycin. Both MLE-12 cells and rat alveolar type II cells overexpressing Bcl-X(L) did not undergo cell death in response to bleomycin. Dominant negative Fas-associating protein with a death domain failed to prevent bleomycin-induced cell death in MLE-12 cells. Caspase-8 inhibitor CrmA did not prevent bleomycin-induced cell death in primary rat alveolar type II cells. Furthermore, fibroblast cells deficient in Bax and Bak, but not Bid, were resistant to bleomycin-induced cell death. To determine whether the stress kinase JNK was an upstream regulator of Bax activation, MLE-12 cells were exposed to bleomycin in the presence of an adenovirus encoding a dominant negative JNK. Bleomycin-induced Bax activation was prevented by the expression of a dominant negative JNK in MLE-12 cells. Dominant negative JNK prevented cell death in MLE-12 cells and in primary rat alveolar type II cells exposed to bleomycin. These data indicate that bleomycin induces cell death through a JNK-dependent mitochondrial death pathway in alveolar epithelial cells.