Toru Nyunoya

Constitutive ERK MAPK Activity Regulates Macrophage ATP Production and Mitochondrial Integrity

A unique feature of human alveolar macrophages is their prolonged survival in the face of a stressful environment. We have shown previously that the ERK MAPK is constitutively active in these cells and is important in prolonging cell survival. This study examines the role of the ERK pathway in maintaining mitochondrial energy production. The data demonstrate that ATP levels in alveolar macrophages depend on intact mitochondria and optimal functioning of the electron transport chain. Significant levels of MEK and ERK localize to the mitochondria and inhibition of ERK activity induces an early and profound depletion in cellular ATP coincident with a loss of mitochondrial transmembrane potential. The effect of ERK suppression on ATP levels was specific, since it did not occur with PI3K/Akt, p38, or JNK suppression. ERK inhibition led to cytosolic release of mitochondrial proteins and caspase activation. Both ERK inhibition and mitochondrial blockers induced loss of plasma membrane permeability and cell death. The cell death induced by ERK inhibition had hallmarks of both apoptotic (caspase activation) and necrotic (ATP loss) cell death. By blocking ERK inhibition-induced reactive oxygen species, caspase activation was prevented, although necrotic pathways continued to induce cell death. This suggests that mitochondrial dysfunction caused by ERK inhibition generates both apoptotic and necrotic cell death-inducing pathways. As a composite, these data demonstrate a novel mitochondrial role for ERK in maintaining mitochondrial membrane potential and ATP production in human alveolar macrophages.

Cigarette Smoke Induces Cellular Senescence via Werner's Syndrome Protein Down-regulation

RATIONALE Werners syndrome is a genetic disorder that causes premature aging due to loss-of-function mutations in a gene encoding a member of the RecQ helicase family. Both Werners syndrome and cigarette smoking accelerate aging. No studies have examined the effect of cigarette smoke on Werners syndrome protein. OBJECTIVES To investigate the role of Werners syndrome protein in cigarette smoke-induced cellular senescence. METHODS Cellular senescence and amounts of Werners syndrome protein were measured in fibroblasts isolated from patients with emphysema and compared with age-matched nonsmokers. The in vitro effects of cigarette smoke on amounts of Werners syndrome protein, function, and senescence were also evaluated in primary human lung fibroblasts and epithelial cells. MEASUREMENTS AND MAIN RESULTS Cultured lung fibroblasts isolated from patients with emphysema exhibited a senescent phenotype accompanied by a decrease in Werners syndrome protein. Cigarette smoke extract decreased Werners syndrome protein in cultured fibroblasts and epithelial cells. Werners syndrome protein-deficient fibroblasts were more susceptible to cigarette smoke-induced cellular senescence and cell migration impairment. In contrast, exogenous overexpression of Werners syndrome protein attenuated the cigarette smoke effects. CONCLUSIONS Cigarette smoke induces cellular senescence and cell migration impairment via Werners syndrome protein down-regulation. Rescue of Werners syndrome protein down-regulation may represent a potential therapeutic target for smoking-related diseases.

Molecular Processes that Drive Cigarette Smoke–Induced Epithelial Cell Fate of the Lung

Cigarette smoke contains numerous chemical compounds, including abundant reactive oxygen/nitrogen species and aldehydes, and many other carcinogens. Long-term cigarette smoking significantly increases the risk of various lung diseases, including chronic obstructive pulmonary disease and lung cancer, and contributes to premature death. Many in vitro and in vivo studies have elucidated mechanisms involved in cigarette smoke-induced inflammation, DNA damage, and autophagy, and the subsequent cell fates, including cell death, cellular senescence, and transformation. In this Translational Review, we summarize the known pathways underlying these processes in airway epithelial cells to help reveal future challenges and describe possible directions of research that could lead to better management and treatment of these diseases.

Softwares and methods for estimating genetic ancestry in human populations

The estimation of genetic ancestry in human populations has important applications in medical genetic studies. Genetic ancestry is used to control for population stratification in genetic association studies, and is used to understand the genetic basis for ethnic differences in disease susceptibility. In this review, we present an overview of genetic ancestry estimation in human disease studies, followed by a review of popular softwares and methods used for this estimation.

Cigarette Smoke Alters Respiratory Syncytial Virus–Induced Apoptosis and Replication

Individuals exposed to cigarette smoke have a greater number and severity of viral infections, including respiratory syncytial virus (RSV) infections, than do nonsmokers, but the cellular mechanism is unknown. Our objective was to determine the mechanism by which cigarette smoke augments viral infection. We hypothesize that cigarette smoke causes necrosis and prevents virus-induced cellular apoptosis, and that this is associated with increased inflammation and viral replication. Primary airway epithelial cells were exposed to cigarette smoke extract for 2 days, followed by 1 day of RSV exposure. Western blot detection of cleaved caspases 3 and 7 showed less apoptosis when cells were treated with cigarette smoke before viral infection. This finding was confirmed with ELISA and TUNEL detection of apoptosis. Measures of cell viability, including propidium iodide staining, ATP assay, and cell counts, indicated that cigarette smoke causes necrosis rather than virus-induced apoptosis. Using plaque assay and fluorescently-labeled RSV, we showed that although there were less live cells in the cigarette smoke-pretreated group, viral load was increased. The effect was inhibited by pretreatment of cells with N-acetylcysteine and aldehyde dehydrogenase, suggesting that the effect was primarily mediated by reactive aldehydes. Cigarette smoke causes necrosis rather than apoptosis in viral infection, resulting in increased inflammation and enhanced viral replication.

RIP1 potentiates BPDE-induced transformation in human bronchial epithelial cells through catalase-mediated suppression of excessive reactive oxygen species.

Cell survival signaling is important for the malignant phenotypes of cancer cells. Although the role of receptor-interacting protein 1 (RIP1) in cell survival signaling is well documented, whether RIP1 is directly involved in cancer development has never been studied. In this report, we found that RIP1 expression is substantially increased in human non-small cell lung cancer and mouse lung tumor tissues. RIP1 expression was remarkably increased in cigarette smoke-exposed mouse lung. In human bronchial epithelial cells (HBECs), RIP1 was significantly induced by cigarette smoke extract or benzo[a]pyrene diol epoxide (BPDE), the active form of the tobacco-specific carcinogen benzo(a)pyrene. In RIP1 knockdown HBECs, BPDE-induced cytotoxicity was significantly increased, which was associated with induction of cellular reactive oxygen species (ROS) and activation of mitogen-activated protein kinases (MAPKs), including c-jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) and p38. Scavenging ROS suppressed BPDE-induced MAPK activation and inhibiting ROS or MAPKs substantially blocked BPDE-induced cytotoxicity, suggesting ROS-mediated MAPK activation is involved in BPDE-induced cell death. The ROS-reducing enzyme catalase is destabilized in an ERK- and JNK-dependent manner in RIP1 knockdown HBECs and application of catalase effectively blocked BPDE-induced ROS accumulation and cytotoxicity. Importantly, BPDE-induced transformation of HBECs was significantly reduced when RIP1 expression was suppressed. Altogether, these results strongly suggest an oncogenic role for RIP1, which promotes malignant transformation through protecting DNA-damaged cells against carcinogen-induced cytotoxicity associated with excessive ROS production.

RIP1 maintains DNA integrity and cell proliferation by regulating PGC-1α-mediated mitochondrial oxidative phosphorylation and glycolysis.

Aerobic glycolysis or the Warburg effect contributes to cancer cell proliferation; however, how this glucose metabolism pathway is precisely regulated remains elusive. Here we show that receptor-interacting protein 1 (RIP1), a cell death and survival signaling factor, regulates mitochondrial oxidative phosphorylation and aerobic glycolysis. Loss of RIP1 in lung cancer cells suppressed peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) expression, impairing mitochondrial oxidative phosphorylation and accelerating glycolysis, resulting in spontaneous DNA damage and p53-mediated cell proliferation inhibition. Thus, although aerobic glycolysis within a certain range favors cancer cell proliferation, excessive glycolysis causes cytostasis. Our data suggest that maintenance of glycolysis by RIP1 is pivotal to cancer cell energy homeostasis and DNA integrity and may be exploited for use in anticancer therapy.

Aldehyde dehydrogenase 3A1 protects airway epithelial cells from cigarette smoke-induced DNA damage and cytotoxicity

Aldehyde dehydrogenase 3A1 (ALDH3A1), an ALDH superfamily member, catalyzes the oxidation of reactive aldehydes, highly toxic components of cigarette smoke (CS). Even so, the role of ALDH3A1 in CS-induced cytotoxicity and DNA damage has not been examined. Among all of the ALDH superfamily members, ALDH3A1 mRNA levels showed the greatest induction in response to CS extract (CSE) exposure of primary human bronchial epithelial cells (HBECs). ALDH3A1 protein accumulation was accompanied by increased ALDH enzymatic activity in CSE-exposed immortalized HBECs. The effects of overexpression or suppression of ALDH3A1 on CSE-induced cytotoxicity and DNA damage (γH2AX) were evaluated in cultured immortalized HBECs. Enforced expression of ALDH3A1 attenuated cytotoxicity and downregulated γH2AX. SiRNA-mediated suppression of ALDH3A1 blocked ALDH enzymatic activity and augmented cytotoxicity in CSE-exposed cells. Our results suggest that the availability of ALDH3A1 is important for cell survival against CSE in HBECs.

Antioxidant Diet Protects Against Emphysema, but Increases Mortality in Cigarette Smoke-Exposed Mice

Oxidative stress plays an important role in cigarette smoke-induced lung inflammation and emphysema. We produced an enriched diet by adding freeze-dried fruits and vegetables and additional supplements to the 8604 Teklad Rodent Diet, a standard rodent diet. In this study, we examined the effects of the antioxidant-enriched diet on cigarette smoke-induced lung inflammation and emphysema. CH3/HeN mice were fed either a regular diet or the supplemented diet. These mice were exposed to filtered air, a low concentration of cigarette smoke (total particulate matter: 100 mg/m3) or a high concentration of cigarette smoke (total particulate matter: 250 mg/m3) for 6 h/day, 5 days/week for total 16 weeks. Surprisingly, increased mortality (53%) was observed in the high concentration of cigarette smoke-exposed mice fed the antioxidant diet compared to the high concentration of cigarette smoke-exposed mice that were fed a regular diet (13%). The necropsy analysis revealed nasal passage obstruction due to mucous plugging in cigarette smoke-exposed mice on the antioxidant diet. However, the antioxidant diet significantly reduced neutrophilic inflammation and emphysema in the high concentration of cigarette smoke-exposed mice as compared to the regular diet /high concentration of cigarette smoke controls. The antioxidant capacity in the bronchoalveolar fluid or oxidative damage to the lung tissue was not affected by the antioxidant diet. Pro-MMP-2, MMP-2, and MMP-9 activity did not correlate with the protective effects of AOD on cigarette smoke-induced emphysema. These data suggest that the antioxidant diet reduced cigarette smoke-induced inflammation and emphysema, but increased mortality in the obligate nose-breathing mice.

Autophagy‐Mediated Degradation of IAPs and c‐FLIPL Potentiates Apoptosis Induced by Combination of TRAIL and Chal‐24

Combination chemotherapy is an effective strategy for increasing anticancer efficacy, reducing side effects and alleviating drug resistance. Here we report that combination of the recently identified novel chalcone derivative, chalcone‐24 (Chal‐24), and TNF‐related apoptosis‐inducing ligand (TRAIL) significantly increases cytotoxicity in lung cancer cells. Chal‐24 treatment significantly enhanced TRAIL‐induced activation of caspase‐8 and caspase‐3, and the cytotoxicity induced by combination of these agents was effectively suppressed by the pan‐caspase inhibitor z‐VAD‐fmk. Chal‐24 and TRAIL combination suppressed expression of cellular FLICE (FADD‐like IL‐1β‐converting enzyme)‐inhibitory protein large (c‐FLIPL) and cellular inhibitor of apoptosis proteins (c‐IAPs), and ectopic expression of c‐FLIPL and c‐IAPs inhibited the potentiated cytotoxicity. In addition, TRAIL and Chal‐24 cooperatively activated autophagy. Suppression of autophagy effectively attenuated cytotoxicity induced by Chal‐24 and TRAIL combination, which was associated with attenuation of c‐FLIPL and c‐IAPs degradation. Altogether, these results suggest that Chal‐24 potentiates the anticancer activity of TRAIL through autophagy‐mediated degradation of c‐FLIPL and c‐IAPs, and that combination of Chal‐24 and TRAIL could be an effective approach in improving chemotherapy efficacy. J. Cell. Biochem. 117: 1136–1144, 2016.