Airborne fine particulate matter induces multiple cell death pathways in human lung epithelial cells

Our group was the first one reporting that autophagy could be triggered by airborne fine particulate matter (PM) with a mean diameter of less than 2.5 μm (PM_2.5) in human lung epithelial A549 cells, which could potentially lead to cell death. In the present study, we further explored the potential interactions between autophagy and apoptosis because it was well documented that PM_2.5 could induce apoptosis in A549 cells. Much to our surprise, we found that PM_2.5-exposure caused oxidative stress, resulting in activation of multiple cell death pathways in A549 cells, that is, the tumor necrosis factor-alpha (TNF-α)-induced pathway as evidenced by TNF-α secretion and activation of caspase-8 and -3, the intrinsic apoptosis pathway as evidenced by increased expression of pro-apoptotic protein Bax, decreased expression of anti-apoptotic protein Bcl-2, disruption of mitochondrial membrane potential, and activation of caspase-9 and -3, and autophagy as evidenced by an increased number of double-membrane vesicles, accompanied by increases of conversion and punctuation of microtubule-associated proteins light chain 3 (LC3) and expression of Beclin 1. It appears that reactive oxygen species (ROS) function as signaling molecules for all the three pathways because pretreatment with N-acetylcysteine, a scavenger of ROS, almost completely abolished TNF-α secretion and significantly reduced the number of apoptotic and autophagic cells. In another aspect, inhibiting autophagy with 3-methyladenine, a specific autophagy inhibitor, enhanced PM_2.5-induced apoptosis and cytotoxicity. Intriguingly, neutralization of TNF-α with an anti-TNF-α special antibody not only abolished activation of caspase-8, but also drastically reduced LC3-II conversion. Thus, the present study has provided novel insights into the mechanism of cytotoxicity and even pathogenesis of diseases associated with PM_2.5 exposure.     

Nap1 regulates Dictyostelium cell motility and adhesion through SCAR-dependent and -independent pathways

SCAR--also known as WAVE--is a key regulator of actin dynamics. Activation of SCAR enhances the nucleation of new actin filaments through the Arp2/3 complex, causing a localized increase in the rate of actin polymerization . In vivo, SCAR is held in a large regulatory complex, which includes PIR121 and Nap1 proteins, whose precise role is unclear. It was initially thought to hold SCAR inactive until needed , but recent data suggest that it is essential for SCAR function . Here, we show that disruption of the gene that encodes Nap1 (napA) causes loss of SCAR function. Cells lacking Nap1 are small and rounded, with diminished actin polymerization and small pseudopods. Furthermore, several aspects of the napA phenotype are more severe than those evoked by the absence of SCAR alone. In particular, napA mutants have defects in cell-substrate adhesion and multicellular development. Despite these defects, napA(-) cells move and chemotax surprisingly effectively. Our results show that the members of the complex have unexpectedly diverse biological roles.     

FGF signals for cell proliferation and migration through different pathways

FGFs are pleiotropic growth factors that control cell proliferation, migration and differentiation. However, FGF transduction studies have so far focused primarily on the mitogenic effect of this growth factor family and it has been difficult to assess if the described intracellular signaling pathways are dedicated solely to cell proliferation, or whether they are equally important for the migratory activity often seen in responsive cells. We review here papers in which the migratory effects of this growth factor family were clearly discriminated from proliferative effects. In toto, these studies suggest that cells use different signaling pathways for migration, such as Src and p38 MAP kinase, from those for proliferation, which tend to upregulate the ERKs. Which signaling pathway a cell uses for proliferation or migration appears to depend on many factors, including the structure and the quantity of available FGF trapped in the basal lamina by heparan sulfate co-factors, the disposition of cognate high affinity receptors and the general environment of the cell. Thus the density of the cell population, the state of the cell cycle, the presence of other factors or receptors will modulate the migratory response of cells to FGF.     

Mechanisms of gelsolin-dependent and -independent EGF-stimulated cell motility in a human lung epithelial cell line

Acquisition of motility is an important step in malignant progression of tumor cells and involves dynamic changes in actin filament architecture orchestrated by many actin binding proteins. A role for the actin-binding protein gelsolin has been demonstrated in fibroblast motility. In this report, we investigated the role of gelsolin in bronchial epithelial cell motility. The non-tumorigenic bronchial epithelial cell line, NL20 migrated towards EGF in a modified Boyden chamber cell motility assay. However, the tumorigenic NL20-TA cell line derived from the NL20 cells and lacking gelsolin, did not migrate towards EGF. Ectopic expression of gelsolin in NL20-TA cells restored the EGF response, while motility of NL20-TA derived cells towards serum, PDGF, and fibronectin was independent of gelsolin expression. PI3-kinase inhibition failed to block EGF-stimulated motility in gelsolin transfected NL20-TA cells. Furthermore, EGF stimulated a motility response in cells lacking gelsolin in the presence of fibronectin or fibrinogen that was blocked with PI3-kinase inhibition. Thus, EGF-stimulated motility in NL20 cells and its derivatives are gelsolin dependent and PI3-kinase independent, while fibronectin and fibrinogen enhances EGF-stimulated motility through a pathway independent of gelsolin and dependent upon PI3-kinase.     

Autotaxin (ATX): a multi-functional and multi-modular protein possessing enzymatic lysoPLD activity and matricellular properties

Recent studies have established that autotaxin (ATX), also known as phosphodiesterase Ialpha/autotaxin (PD-Ialpha/ATX) or (ecto)nucleotide pyrophosphatase/phosphodiesterase 2 [(E)NPP2], represents a multi-functional and multi-modular protein. ATX was initially thought to function exclusively as a phosphodiesterase/pyrophosphatase. However, it has become apparent that this enzymatically active site, which is ultimately responsible for ATX's originally discovered property of tumor cell motility stimulation, mediates the conversion of lysophosphatidylcholine (LPC) to lysophosphatidic acid (LPA). In addition, a separate functionally active domain, here referred to as the Modulator of Oligodendrocyte Remodeling and Focal adhesion Organization (MORFO) domain, was discovered in studies analyzing the role of ATX during the differentiation of myelinating cells of the central nervous system (CNS), namely oligodendrocytes. This novel domain was found to mediate anti-adhesive, i.e. matricellular, properties and to promote morphological maturation of oligodendrocytes. In this review, we summarize our current understanding of ATX's structure-function domains and discuss their contribution to the presently known main functional roles of ATX.     

Drosophila PTEN regulates cell growth and proliferation through PI3K-dependent and -independent pathways

The control of cell and organ growth is fundamental to the development of multicellular organisms. Here, we show that dPTEN, a Drosophila homolog of the mammalian PTEN tumor suppressor gene, plays an essential role in the control of cell size, cell number, and organ size. In mosaic animals, dPTEN(-) cells proliferate faster than their heterozygous siblings, show an autonomous increase in cell size, and form organs of increased size, whereas overexpression of dPTEN results in opposite phenotypes. The loss-of-function phenotypes of dPTEN are suppressed by mutations in the PI3K target Dakt1 and the translational initiation factor eif4A, suggesting that dPTEN acts through the PI3K signaling pathway to regulate translation. Although activation of PI3K and Akt has been reported to increase rates of cellular growth but not proliferation, loss of dPTEN stimulates both of these processes, suggesting that PTEN regulates overall growth through PI3K/Akt-dependent and -independent pathways. Furthermore, we show that dPTEN does not play a major role in cell survival during Drosophila development. Our results provide a potential explanation for the high frequency of PTEN mutation in human cancer.     

Denatonium inhibits growth and induces apoptosis of airway epithelial cells through mitochondrial signaling pathways

Background

Denatonium, a widely used bitter agonist, activates bitter taste receptors on many cell types and plays important roles in chemical release, ciliary beating and smooth muscle relaxation through intracellular Ca²⁺-dependent pathways. However, the effects of denatonium on the proliferation of airway epithelial cells and on the integrity of cellular components such as mitochondria have not been studied. In this study, we hypothesize that denatonium might induce airway epithelial cell injury by damaging mitochondria.

Methods

Bright-field microscopy, cell counting kit-8 (CCK-8) assay and flow cytometry analysis were used to examine cellular morphology, proliferation and cell cycle, respectively. Transmission electron microscopy (TEM) was used to examine mitochondrial integrity. JC-1 dye and western blotting techniques were used to measure mitochondrial membrane potential and protein expression, respectively.

Results

For airway epithelial cells, we observed that denatonium significantly effects cellular morphology, decreases cell proliferation and reduces the number of cells in S phase in a dose-dependent manner. TEM analysis demonstrated that denatonium causes large amplitude swelling of mitochondria, which was confirmed by the loss of mitochondrial membrane potential, the down-regulation of Bcl-2 protein and the subsequent enhancement of the mitochondrial release of cytochrome c and Smac/DIABLO after denatonium treatment.

Conclusions

In this study, we demonstrated for the first time that denatonium damages mitochondria and thus induces apoptosis in airway epithelial cells.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-015-0183-9) contains supplementary material, which is available to authorized users.     

Viral activation of macrophages through TLR-dependent and -independent pathways

Induction of cytokine production is important for activation of an efficient host defense response. Macrophages constitute an important source of cytokines. In this study we have investigated the virus-cell interactions triggering induction of cytokine expression in macrophages during viral infections. We found that viral entry and viral gene products produced inside the cell are responsible for activation of induction pathways leading to IFN-alphabeta expression, indicating that virus-cell interactions on the cell surface are not enough. Moreover, by the use of cell lines expressing dominant negative versions of TLR-associated adaptor proteins we demonstrate that Toll/IL-1 receptor domain-containing adaptor inducing IFN-beta is dispensable for all virus-induced cytokine expression examined. However, a cell line expressing dominant negative MyD88 revealed the existence of distinct induction pathways because virus-induced expression of RANTES and TNF-alpha was totally blocked in this cell line whereas IFN-alphabeta expression was much less affected in the absence of signaling via MyD88. In support of this, we also found that inhibitory CpG motifs, which block TLR9 signaling inhibited early HSV-2-induced TNF-alpha and RANTES expression dramatically whereas IFN-alphabeta induction was only slightly affected. This suggests that virus activates macrophages through distinct pathways, of which some are dependent on TLRs signaling through MyD88, whereas others seem to be independent of TLR signaling. Finally we demonstrate that IFN-alphabeta induction in HSV-2-infected macrophages requires a functional dsRNA-activated protein kinase molecule because cells expressing a dsRNA-dependent protein kinase version unable to bind dsRNA do not express IFN-alphabeta on infection.     

Perfluorocarbon induces alveolar epithelial cell response through structural and mechanical remodeling

During total liquid ventilation, lung cells are exposed to perfluorocarbon (PFC) whose chemophysical properties highly differ from standard aqueous cell feeding medium (DMEM). We herein perform a systematic study of structural and mechanical properties of A549 alveolar epithelial cells in order to characterize their response to PFC exposure, using DMEM as control condition. Changes in F-actin structure, focal adhesion density and glycocalyx distribution are evaluated by confocal fluorescent microscopy. Changes in cell mechanics and adhesion are measured by multiscale magnetic twisting cytometry (MTC). Two different microrheological models (single Voigt and power law) are used to analyze the cell mechanics characterized by cytoskeleton (CSK) stiffness and characteristic relaxation times. Cell–matrix adhesion is analyzed using a stochastic multibond deadhesion model taking into account the non-reversible character of the cell response, allowing us to quantify the adhesion weakness and the number of associated bonds. The roles of F-actin structure and glycocalyx layer are evaluated by depolymerizing F-actin and degrading glycocalyx, respectively. Results show that PFC exposure consistently induces F-actin remodeling, CSK softening and adhesion weakening. These results demonstrate that PFC triggers an alveolar epithelial cell response herein evidenced by a decay in intracellular CSK tension, an adhesion weakening and a glycocalyx layer redistribution. These PFC-induced cell adjustments are consistent with the hypothesis that cells respond to a decrease in adhesion energy at cell surface. This adhesion energy can be even further reduced in the presence of surfactant adsorbed at the cell surface.     

Respiratory syncytial virus matrix protein induces lung epithelial cell cycle arrest through a p53 dependent pathway

Respiratory syncytial virus (RSV) is the major cause of viral respiratory infections in children. Our previous study showed that the RSV infection induced lung epithelial cell cycle arrest, which enhanced virus replication. To address the mechanism of RSV-induced cell cycle arrest, we examined the contribution of RSV-matrix (RSV-M) protein. In this report, we show that in both the A549 cell line and primary human bronchial epithelial (PHBE) cells, transfection with RSV-M protein caused the cells to proliferate at a slower rate than in control cells. The cell cycle analysis showed that RSV-M protein induced G1 phase arrest in A549 cells, and G1 and G2/M phase arrest in PHBE cells. Interestingly, RSV-M expression induced p53 and p21 accumulation and decreased phosphorylation of retinoblastoma protein (Rb). Further, induction of cell cycle arrest by RSV-M was not observed in a p53-deficient epithelial cell line (H1299). However, cell cycle arrest was restored after transfection of p53 cDNA into H1299 cells. Taken together, these results indicate that RSV-M protein regulates lung epithelial cell cycle through a p53-dependent pathway, which enhances RSV replication.     

Elastase induces lung epithelial cell autophagy through placental growth factor: A new insight of emphysema pathogenesis

Chronic obstructive pulmonary disease (COPD) is a devastating disease, which is associated with increasing mortality and morbidity. Therefore, there is a need to clearly define the COPD pathogenic mechanism and to explore effective therapies. Previous studies indicated that cigarette smoke (CS) induces autophagy and apoptosis in lung epithelial (LE) cells. Excessive ELANE/HNE (elastase, neutrophil elastase), a factor involved in protease-antiprotease imbalance and the pathogenesis of COPD, causes LE cell apoptosis and upregulates the expression of several stimulus-responsive genes. However, whether or not elastase induces autophagy in LE cell remains unknown. The level of PGF (placental growth factor) is higher in COPD patients than non-COPD controls. We hypothesize that elastase induces PGF expression and causes autophagy in LE cells. In this study, we demonstrated that porcine pancreatic elastase (PPE) induced PGF expression and secretion in LE cells in vitro and in vivo. The activation of MAPK8/JNK1 (mitogen-activated protein kinase 8) and MAPK14/p38alpha MAPK signaling pathways was involved in the PGF mediated regulation of the TSC (tuberous sclerosis complex) pathway and autophagy in LE cells. Notably, PGF-induced MAPK8 and MAPK14 signaling pathways mediated the inactivation of MTOR (mechanistic target of rapamycin), the upregulation of MAP1LC3B/LC3B (microtubule-associated protein 1 light chain 3 β) and the increase of autophagosome formation in mice. Furthermore, the PPE-induced autophagy promotes further apoptosis in vitro and in vivo. In summary, elastase-induced autophagy promotes LE cell apoptosis and pulmonary emphysema through the upregulation of PGF. PGF and its downstream MAPK8 and MAPK14 signaling pathways are potential therapeutic targets for the treatment of emphysema and COPD.     

Autotaxin (lysoPLD/NPP2) protects fibroblasts from apoptosis through its enzymatic product, lysophosphatidic acid, utilizing albumin-bound substrate

Autotaxin (ATX) was originally identified as a potent tumor cell motility-stimulating factor that displays multiple enzymatic activities including ATPase, Type I nucleotide pyrophosphatase/phosphodiesterase, and lysophospholipase D, depending on its substrates. We demonstrate herein that ATX is a key regulator of extracellular lysophosphatidic acid (LPA) that can act as survival factor, in addition to its mitogenic activity in mouse fibroblasts. Introduction of atx gene into NIH3T3 cells resulted in resistance to conditional apoptosis induced by serum-deprivation, and exogenous ATX protein prevented cells from death by starvation. Flow cytometric analysis showed that co-treatment of ATX with lysophosphatidylcholine as substrate rescued NIH3T3 cells from cellular apoptosis, and this survival activity of ATX was also demonstrated by caspase-3 degradation and PARP cleavage resulting from the enzymatic activity of extracellular ATX. Furthermore, the effect of ATX in preventing apoptosis appears to be mediated through the G-protein-coupled receptor pathway followed by the activation of phosphoinositide 3-kinase and Akt pathway leading to enhanced cell survival. These findings provide novel insights into understanding the functions of ATX as a key regulator of bioactive phospholipids and suggest interventions to correct dysfunction in conditions of tumor cell growth and metastasis.     

Polyomavirus middle T antigen induces ribosomal protein S6 phosphorylation through pp60c-src-dependent and -independent pathways.

Phosphorylation of ribosomal protein S6 is elevated in polyomavirus-infected cells. This elevation results only in part from activation of S6 kinase activity. These effects appear to reflect independent activities of wild-type middle T antigen. Hr-t mutant NG59, encoding a defective middle T protein, and mutant Py808A, encoding no middle T protein, were unable to induce S6 kinase activity or elevate S6 phosphorylation. Two other site-directed mutants encoding altered middle T proteins did elevate S6 phosphorylation while only weakly stimulating S6 kinase activity. These results suggest at least two independent pathways leading to elevation of S6 phosphorylation. One pathway leads to induction of S6 kinase activity following activation of pp60c-src by transformation-competent middle T antigen. Another pathway operates independently of S6 kinase induction and can be regulated by transformation-defective middle T mutants such as Py1387T. This mutant, encoding a truncated middle T protein that failed to associate with the plasma membrane and to activate pp60c-src, caused increased levels of S6 phosphorylation without detectably increasing S6 kinase activity. The ability of mutants such as Py1387T to induce S6 phosphorylation correlated with their ability to increase phosphorylation of VP1, an event linked to maturation of infectious virions.     

CXCL12 induces lung cancer cell migration by polarized mtDNA redistribution

Instability of mitochondrial DNA (mtDNA) has been associated with the initiation and development of cancer, but the specific role of mtDNA in the invasiveness and migration of cancer cells remains unclear. In this study, we investigated whether the chemokine CXCL12 causes intact mitochondria to redistribute in cancer cells and, in this way, to increase cell invasiveness and migration. A549 lung cancer cells with intact mtDNA (mtDNA+) and ρ⁰A549 cells depleted of mtDNA (mtDNA?) by long-term ethidium bromide incubation were examined for their responses to CXCL12 in a transwell migration assay and for mitochondrial distribution by fluorescence microscopy. Intact A549 cells showed significantly increased migration and increased polar distribution of mitochondria (asymmetry) in response to CXCL12. However, ρ⁰A549 cells showed no changes in mitochondrial distribution in response to CXCL12, and only a few ρ⁰A549 cells migrated across the transwell membrane after CXCL12 treatment. These results demonstrate that, in A549 lung cancer cells, intact mitochondrial DNA is necessary for mitochondrial redistribution and a chemotactic response to CXCL12.     

Attenuated Leishmanial sphingolipid induces apoptosis in A375 human melanoma cell via both caspase-dependent and -independent pathways

A fraction of attenuated Leishmanial lipid (ALL) rich in sphingolipids, previously shown to have apoptosis inducing activity in mouse melanoma (B16F10) and human melanoma (A375) cells, was resolved to isolate the bioactive sphingolipid. The mechanism of apoptosis induction by this bioactive attenuated Leishmanial sphingolipid (ALSL) was studied in A375 cells. Apoptosis induced by ALSL in A375 cells was found to be dose and time-dependent. Exposure of cells to ALSL resulted in a rapid increase in reactive oxygen species generation. Pretreatment of cells with the antioxidant N-acetyl-cystein reduced ROS generation and attenuated apoptosis induced by ALSL. Again, ALSL sensitization resulted in the activation of caspase-3 and -9 but not caspase-8. However, inhibitors of these caspases could not protect the cells completely from ALSL-induced apoptosis. N-acetyl-cystein pretreatment was again found to attenuate the activation of caspase-3 and -9. ALSL treatment also resulted in the alteration of mitochondrial membrane potential, and release of pro-apoptotic factors such as cytochrome c and apoptosis inducing factor (AIF) from mitochondria. Furthermore, c-Jun N-terminal kinase was activated that resulted in apoptosis of A375 cells, whereas p38 MAPK was activated to counteract the stress generated in cells in response to ALSL treatment. Taken together, our results indicate that ALSL-induced apoptosis of A375 cells is mediated by both mitochondrial caspase-dependent and -independent pathways and it involves ROS and JNK activation in the mitogen-activated protein kinase cascade.     

Production of CCL20 from lung cancer cells induces the cell migration and proliferation through PI3K pathway

Tumour inflammatory microenvironment is considered to play a role in the sensitivity of tumour cells to therapies and prognosis of patients with lung cancer. The expression of CCL20, one of the critical chemoattractants responsible for inflammation cells recruitment, has been shown overexpressed in variety of tumours. This study aimed at investigating potential mechanisms of CCL20 function and production in human non‐small cell lung cancer (NSCLC). Expression of CCL20 gene and protein in lung tissues of patients with NSCLC and NSCLC cells (A549) were determined. The interleukin (IL)‐1β‐induced signal pathways in A549 and the effect of CCL20‐induced A549 cell migration and proliferation were determined using migration assays and cell‐alive monitoring system. Mechanisms of signal pathways involved in the migration of CCL20 were also studied. We initially found that NSCLC tumour tissues markedly overexpressed CCL20 in comparison with normal lung samples. In addition, IL‐1β could directly promote CCL20 production in lung cancer cells, which was inhibited by extracellular signal‐regulated kinase (ERK)1/2 inhibitor, p38 mitogen‐activated protein kinase (p38 MARP) inhibitor or PI3K inhibitors. CCL20 promoted lung cancer cells migration and proliferation in an autocrine manner via activation of ERK1/2‐MAPK and PI3K pathways. Our data indicated that IL‐1β could stimulate CCL20 production from lung cancer cells through the activation of MAPKs and PI3K signal pathways, and the auto‐secretion of CCL20 could promote lung cancer cell migration and proliferation through the activation of ERK and PI3K signal pathways. Our results may provide a novel evidence that CCL20 could be a new therapeutic target for lung cancer.     

Tumor necrosis factor-alpha inhibits myogenesis through redox-dependent and -independent pathways

Muscle wasting accompanies diseases that are associated with chronic elevated levels of circulating inflammatory cytokines and oxidative stress. We previously demonstrated that tumor necrosis factor-alpha (TNF-alpha) inhibits myogenic differentiation via the activation of nuclear factor-kappaB (NF-kappaB). The goal of the present study was to determine whether this process depends on the induction of oxidative stress. We demonstrate here that TNF-alpha causes a decrease in reduced glutathione (GSH) during myogenic differentiation of C(2)C(12) cells, which coincides with an elevated generation of reactive oxygen species. Supplementation of cellular GSH with N-acetyl-l-cysteine (NAC) did not reverse the inhibitory effects of TNF-alpha on troponin I promoter activation and only partially restored creatine kinase activity in TNF-alpha-treated cells. In contrast, the administration of NAC before treatment with TNF-alpha almost completely restored the formation of multinucleated myotubes. NAC decreased TNF-alpha-induced activation of NF-kappaB only marginally, indicating that the redox-sensitive component of the inhibition of myogenic differentiation by TNF-alpha occurred independently, or downstream of NF-kappaB. Our observations suggest that the inhibitory effects of TNF-alpha on myogenesis can be uncoupled in a redox-sensitive component affecting myotube formation and a redox independent component affecting myogenic protein expression.     

Mismatch in mechanical and adhesive properties induces pulsating cancer cell migration in epithelial monolayer

The mechanical and adhesive properties of cancer cells significantly change during tumor progression. Here we assess the functional consequences of mismatched stiffness and adhesive properties between neighboring normal cells on cancer cell migration in an epithelial-like cell monolayer. Using an in vitro coculture system and live-cell imaging, we find that the speed of single, mechanically soft breast carcinoma cells is dramatically enhanced by surrounding stiff nontransformed cells compared with single cells or a monolayer of carcinoma cells. Soft tumor cells undergo a mode of pulsating migration that is distinct from conventional mesenchymal and amoeboid migration, whereby long-lived episodes of slow, random migration are interlaced with short-lived episodes of extremely fast, directed migration, whereas the surrounding stiff cells show little net migration. This bursty migration is induced by the intermittent, myosin II-mediated deformation of the soft nucleus of the cancer cell, which is induced by the transient crowding of the stiff nuclei of the surrounding nontransformed cells, whose movements depend directly on the cadherin-mediated mismatched adhesion between normal and cancer cells as well as α-catenin-based intercellular adhesion of the normal cells. These results suggest that a mechanical and adhesive mismatch between transformed and nontransformed cells in a cell monolayer can trigger enhanced pulsating migration. These results shed light on the role of stiff epithelial cells that neighbor individual cancer cells in early steps of cancer dissemination.