Epithelial A549 Cells Research Articles

The Pseudomonas aeruginosa Exopolysaccharide Psl Facilitates Surface Adherence and NF-κB Activation in A549 Cells

In order for the opportunistic Gram-negative pathogen Pseudomonas aeruginosa to cause an airway infection, the pathogen interacts with epithelial cells and the overlying mucous layer. We examined the contribution of the biofilm polysaccharide Psl to epithelial cell adherence and the impact of Psl on proinflammatory signaling by flagellin. Psl has been implicated in the initial attachment of P. aeruginosa to biotic and abiotic surfaces, but its direct role in pathogenesis has not been evaluated (L. Ma, K. D. Jackson, R. M. Landry, M. R. Parsek, and D. J. Wozniak, J. Bacteriol. 188:8213–8221, 2006). Using an NF-κB luciferase reporter system in the human epithelial cell line A549, we show that both Psl and flagellin are necessary for full activation of NF-κB and production of the interleukin 8 (IL-8) chemokine. We demonstrate that Psl does not directly stimulate NF-κB activity, but indirectly as a result of increasing contact between bacterial cells and epithelial cells, it facilitates flagellin-mediated proinflammatory signaling. We confirm differential adherence of Psl and/or flagellin mutants by scanning electron microscopy and identify Psl-dependent membrane structures that may participate in adherence. Although we hypothesized that Psl would protect P. aeruginosa from recognition by the epithelial cell line A549, we instead observed a positive role for Psl in flagellin-mediated NF-κB activation, likely as a result of increasing contact between bacterial cells and epithelial cells.

Endoplasmic Reticulum Stress Induced by Surfactant Protein C BRICHOS Mutants Promotes Proinflammatory Signaling by Epithelial Cells

Chronic interstitial lung disease in both adults and children is associated with mutations of the surfactant protein C (SP-C) proprotein. Among these, mutations within the distal COOH propeptide, known as the BRICHOS domain, are associated with a severe disease phenotype. We showed that prolonged expression of the BRICHOS mutants, SP-C(Δexon4) and SP-C(L188Q), destabilizes endoplasmic reticulum (ER) quality-control mechanisms (the unfolded protein response, or UPR), resulting in the induction of ER stress signaling, an inhibition of the ubiquitin/proteasome system, and the activation of apoptotic pathways. Based on recent observations that the UPR and ER stress can be linked to the induction of proinflammatory signaling, we hypothesized that the epithelial cell dysfunction mediated by SP-C BRICHOS mutants would activate proinflammatory signaling pathways. In a test of this hypothesis, A549 and human embryonic kidney epithelial (HEK293) cells, transiently transfected with either SP-C(Δexon4) or SP-C(L188Q) mutants, each promoted the upregulation of multiple UPR response genes, including homocysteine-inducible, endoplasmic reticulum stress-inducible, ubiquitin-like domain member 1 (HERPUD1) and GRP78. Commensurate with these results, increases in IL-8 secretion occurred and were accompanied by the activation of c-Jun N-terminal kinase (JNK)/activating protein-1 signaling. The stimulation of IL-8 cytokine release was completely attenuated by treatment with the JNK-specific inhibitor, SP600125. In addition, SP-C(Δexon4), but not SP-C(L188Q), activated NFκB. The treatment of SP-C(Δexon4) transfected cells with 4-phenylbutyric acid, a small molecule chaperone known to improve protein folding, blocked the activation of NFκB, but not the release of IL-8. Taken together, the results support the role of JNK signaling in mediating SP-C BRICHOS-induced cytokine release, and provide a link between SP-C BRICHOS mutants and proinflammatory cytokine signaling.

Oxidants Positively or Negatively Regulate Nuclear Factor κB in a Context-dependent Manner

Redox-based mechanisms play critical roles in the regulation of multiple cellular functions. NF-kappaB, a master regulator of inflammation, is an inducible transcription factor generally considered to be redox-sensitive, but the modes of interactions between oxidant stress and NF-kappaB are incompletely defined. Here, we show that oxidants can either amplify or suppress NF-kappaB activation in vitro by interfering both with positive and negative signals in the NF-kappaB pathway. NF-kappaB activation was evaluated in lung A549 epithelial cells stimulated with tumor necrosis factor alpha (TNFalpha), either alone or in combination with various oxidant species, including hydrogen peroxide or peroxynitrite. Exposure to oxidants after TNFalpha stimulation produced a robust and long lasting hyperactivation of NF-kappaB by preventing resynthesis of the NF-kappaB inhibitor IkappaB, thereby abrogating the major negative feedback loop of NF-kappaB. This effect was related to continuous activation of inhibitor of kappaB kinase (IKK), due to persistent IKK phosphorylation consecutive to oxidant-mediated inactivation of protein phosphatase 2A. In contrast, exposure to oxidants before TNFalpha stimulation impaired IKK phosphorylation and activation, leading to complete prevention of NF-kappaB activation. Comparable effects were obtained when interleukin-1beta was used instead of TNFalpha as the NF-kappaB activator. This study demonstrates that the influence of oxidants on NF-kappaB is entirely context-dependent, and that the final outcome (activation versus inhibition) depends on a balanced inhibition of protein phosphatase 2A and IKK by oxidant species. Our findings provide a new conceptual framework to understand the role of oxidant stress during inflammatory processes.

Interaction between nanoparticles and cytokine proteins: impact on protein and particlefunctionality

There is increased use of nanomaterials in many applications due to their uniqueproperties, such as their high surface area and surface reactivity. However, thepotential health effects to workers, consumers and the environment exposed tonanoparticles (NPs) is unknown. The aim of this study was to investigate whether NPswhich may enter the body could adsorb proteins and whether this interactionaffects both the particle and the protein function. The cytokines IL-8 and TNF-α were adsorbed significantly more by 14 nm carbon black (CB) compared with a similar dose of260 nm CB. Uncoated 14 nm CB particles produced a significant increase in intracellular calcium[Ca2 + ]i which was greater than a similar mass dose of 260 nm CB. The 260 nmCB produced an increase in ICAM-1 expression in A549 epithelialcells at a comparable dose of 14 nm CB, and after coating with TNF-α 260 nm CB produced significantly more ICAM-1 expression compared with control cells. TNF-α bound to 14 nm CB induced a level of ICAM-1 expression thatwas no greater than the control level, suggesting that the TNF-α activity may be inhibited. These results suggest that NP–protein interaction results both ina decrease in protein function and particle activity in the cellular assays tested and this iscurrently being investigated.

M1179 Targeting Epidermal Growth Factor Receptor With Polyethylene Glycol: Applications for Digestive Cancer Prevention

Introduction: H. pylori transactivates the epidermal growth factor (EGFR) and induces extracellular-signal related kinase (ERK) phosphorylation in gastric epithelial cells which may be important in the pathway to gastric carcinogenesis. The green tea catechin epigallocatechin-3-gallate (EGCG) has been reported in some studies to have chemoprotective properties against development of gastric cancer. The aim of this study was to investigate if H. pyloriinduced ERK phosphorylation in A431 epithelial cells was inhibited by EGCG.Methods: H. pylori (NCTC 11637, cag PAI+) were co-cultured with A431 epithelial cells for 2.5 hrs. EGF (100 ng/ml) was used as a positive control. “In-Cell Western” (ICW) assay, which is a rapid quantitative method for determining the inhibitory effects of chemicals of interest on H. pylori epithelial cell signalling responses (REF Du Y et al., 2007), was used to examine the effects of EGCG on ERK1/2 phosphorylation (pERK) in A431 epithelial cells. EGCG (1-200 μM) was pre-incubated with A431 cells for 1 hr prior to the co-culture experiments. Results: EGF significantly (p<0.001, n=4) increased pERK in A431 cells compared to unstimulated controls. EGCG at concentrations greater than 50 μM caused a small but significant (p< 0.01) increase in pERK in A431 cells. EGCG dose-dependently significantly inhibited EGF (100 ng/ml)-induced pERK at a concentration of 10 μM and above (p<0.01, n=4). H. pylori strain NCTC 11637 significantly (p<0.01, n=4) increased pERK in A431 cells compared to unstimulated controls. EGCG dose-dependently inhibited pERK induced by H. pylori. EGCG at 100 μM significantly inhibited (p<0.05, n=4) H. pylori-induced pERK 2.5 hrs postculture. Conclusions: H. pylori-induced ERK phosphorylation in A431 epithelial cells was significantly inhibited by green tea catechin EGCG in a dose-dependent manner. The use of EGCG as dietary phytochemical for H. pylori-induced gastric cancer chemoprevention is plausible, at least in a specific group of patients with higher risk of gastric cancer development but failure for H. pylori eradication.

M1176 MicroRNA 132 Regulation in Human Colon Carcinogenesis: Potential Role in NSAID Chemoprevention

Introduction: H. pylori transactivates the epidermal growth factor (EGFR) and induces extracellular-signal related kinase (ERK) phosphorylation in gastric epithelial cells which may be important in the pathway to gastric carcinogenesis. The green tea catechin epigallocatechin-3-gallate (EGCG) has been reported in some studies to have chemoprotective properties against development of gastric cancer. The aim of this study was to investigate if H. pyloriinduced ERK phosphorylation in A431 epithelial cells was inhibited by EGCG.Methods: H. pylori (NCTC 11637, cag PAI+) were co-cultured with A431 epithelial cells for 2.5 hrs. EGF (100 ng/ml) was used as a positive control. “In-Cell Western” (ICW) assay, which is a rapid quantitative method for determining the inhibitory effects of chemicals of interest on H. pylori epithelial cell signalling responses (REF Du Y et al., 2007), was used to examine the effects of EGCG on ERK1/2 phosphorylation (pERK) in A431 epithelial cells. EGCG (1-200 μM) was pre-incubated with A431 cells for 1 hr prior to the co-culture experiments. Results: EGF significantly (p<0.001, n=4) increased pERK in A431 cells compared to unstimulated controls. EGCG at concentrations greater than 50 μM caused a small but significant (p< 0.01) increase in pERK in A431 cells. EGCG dose-dependently significantly inhibited EGF (100 ng/ml)-induced pERK at a concentration of 10 μM and above (p<0.01, n=4). H. pylori strain NCTC 11637 significantly (p<0.01, n=4) increased pERK in A431 cells compared to unstimulated controls. EGCG dose-dependently inhibited pERK induced by H. pylori. EGCG at 100 μM significantly inhibited (p<0.05, n=4) H. pylori-induced pERK 2.5 hrs postculture. Conclusions: H. pylori-induced ERK phosphorylation in A431 epithelial cells was significantly inhibited by green tea catechin EGCG in a dose-dependent manner. The use of EGCG as dietary phytochemical for H. pylori-induced gastric cancer chemoprevention is plausible, at least in a specific group of patients with higher risk of gastric cancer development but failure for H. pylori eradication.

M1175 The Green Tea Catechin Epigallocatechin-3-Gallate Inhibits H. pylori-Induced and EGF-Induced ERK Signaling in A431 Epithelial Cells

M1175 The Green Tea Catechin Epigallocatechin-3-Gallate Inhibits H. pylori-Induced and EGF-Induced ERK Signaling in A431 Epithelial Cells

Selective Targeting of Selenocysteine in Thioredoxin Reductase by the Half Mustard 2-Chloroethyl Ethyl Sulfide in Lung Epithelial Cells

Thioredoxin reductase (TrxR) is a selenocysteine-containing flavoprotein that catalyzes the NADPH-dependent reduction of oxidized thioredoxin and plays a key role in regulating cellular redox homeostasis. In the present studies, we examined the effects of 2-chloroethyl ethyl sulfide (CEES), a model sulfur mustard vesicant, on TrxR in lung epithelial cells. We speculated that vesicant-induced alterations in TrxR contribute to oxidative stress and toxicity. The treatment of human lung A549 epithelial cells with CEES resulted in a time- and concentration-dependent inhibition of TrxR. Using purified rat liver TrxR, we demonstrated that only the reduced enzyme was inhibited and that this inhibition was irreversible. The reaction of TrxR with iodoacetamide, which selectively modifies free thiol or selenol on proteins, was also markedly reduced by CEES, suggesting that CEES induces covalent modification of the reduced selenocysteine-containing active site in the enzyme. This was supported by our findings that recombinant mutant TrxR, in which selenocysteine was replaced by cysteine, was markedly less sensitive to inhibition by CEES and that the vesicant preferentially alkylated selenocysteine in the C-terminal redox motif of TrxR. TrxR also catalyzes quinone redox cycling, a process that generates reactive oxygen species. In contrast to its inhibitory effects on TrxR activity, CEES was found to stimulate redox cycling. Taken together, these data suggest that sulfur mustard vesicants target TrxR and that this may be an important mechanism mediating oxidative stress and tissue injury.

Spontaneous assembly and active disassembly balance adherens junction homeostasis

The homeostasis of adherens junctions was studied using E-cadherin and its two mutants tagged by the photoconvertible protein Dendra2 in epithelial A-431 cells and in CHO cells lacking endogenous cadherin. The first mutant contained point mutations of two elements, Lys738 and the dileucine motif that suppressed cadherin endocytosis. The second mutant contained, in addition, an extensive truncation that uncoupled the mutant from beta-catenin and p120. Surprisingly, the intact cadherin and its truncated mutant were recruited into the junctions with identical kinetics. The full-size cadherin was actively removed from the junctions by a process that was unaffected by the inactivation of its endocytic elements. The cadherin's apparent half-residence time in the junction was about 2 min. Cadherin clusters made of the truncated mutant exhibited much slower but ATP-independent junctional turnover. Taken together, our experiments showed that adherens junction homeostasis consists of three distinctive steps: cadherin spontaneous recruitment, its lateral catenin-dependent association, and its active release from the resulting clusters. The latter process, whose mechanism is not clear, may play an important role in various kinds of normal and abnormal morphogenesis.

Surfactant lipids regulate LPS-induced interleukin-8 production in A549 lung epithelial cells by inhibiting translocation of TLR4 into lipid raft domains

In addition to providing mechanical stability, growing evidence suggests that surfactant lipid components can modulate inflammatory responses in the lung. However, little is known of the molecular mechanisms involved in the immunomodulatory action of surfactant lipids. This study investigates the effect of the lipid-rich surfactant preparations Survanta, Curosurf, and the major surfactant phospholipid dipalmitoylphosphatidylcholine (DPPC) on interleukin-8 (IL-8) gene and protein expression in human A549 lung epithelial cells using immunoassay and PCR techniques. To examine potential mechanisms of the surfactant lipid effects, Toll-like receptor 4 (TLR4) expression was analyzed by flow cytometry, and membrane lipid raft domains were separated by density gradient ultracentrifugation and analyzed by immunoblotting with anti-TLR4 antibody. The lipid-rich surfactant preparations Survanta, Curosurf, and DPPC, at physiological concentrations, significantly downregulated lipopolysaccharide (LPS)-induced IL-8 expression in A549 cells both at the mRNA and protein levels. The surfactant preparations did not affect the cell surface expression of TLR4 or the binding of LPS to the cells. However, LPS treatment induced translocation of TLR4 into membrane lipid raft microdomains, and this translocation was inhibited by incubation of the cells with the surfactant lipid. This study provides important mechanistic details of the immune-modulating action of pulmonary surfactant lipids.

The role of apoptosis in the pathophysiology of Acute Respiratory Distress Syndrome (ARDS): An up-to-date cell-specific review

ARDS pathophysiology is characterized by complex mechanisms that involve cells of inflammation, lung tissue cells, cytokines, chemokines, as well as apoptosis activators and inhibitors. There are two important theories that link apoptosis with ARDS and suggest that epithelial cell apoptosis, as well as the accumulation of neutrophils in the lung, may contribute to a cascade of events and, finally, ARDS. The activation of the Fas/FasL pathway is an important mechanism of alveolar epithelial injury in the lungs of patients with ALI. In addition, neutrophilic inflammation in the alveolar spaces is characteristic of ALI in humans and in most animal models of ALI. The enhanced phagocytosis of apoptotic neutrophils could lead to resolution of inflammation and repair during ARDS. In this review, we will focus on elucidating the role of apoptosis in the pathophysiology of ARDS and the contribution of Fas-mediated inflammation in ARDS. Furthermore, we will give evidence that TNF-alpha, IL-1beta and IL-13 attenuate the pro-cell death effects of Fas/CD95 on A549 epithelial cells, at least partially, by the NF-kB and PI3-K pathways, suggesting that induction of the expression of antiapoptotic genes protects the epithelial cells from cell death.

Annexin A2 on lung epithelial cell surface is recognized by severe acute respiratory syndrome-associated coronavirus spike domain 2 antibodies

Severe acute respiratory syndrome-associated coronavirus (SARS-CoV) infection causes lung failure characterized by atypical pneumonia. We previously showed that antibodies against SARS-CoV spike domain 2 (S2) in the patient sera can cross-react with human lung epithelial cells; however, the autoantigen is not yet identified. In this study, we performed proteomic studies and identified several candidate autoantigens recognized by SARS patient sera in human lung type II epithelial cell A549. Among the candidate proteins, annexin A2, which was identified by mass spectrometry analysis and had the highest score by Mascot data search, was further characterized and investigated for its role as an autoantigen. By confocal microscopic observation, SARS patient sera and anti-S2 antibodies were co-localized on A549 cells and both of them were co-localized with anti-annexin A2 antibodies. Anti-annexin A2 antibodies bound to purified S2 proteins, and anti-S2 bound to immunoprecipitated annexin A2 from A549 cell lysate in a dose-dependent manner. Furthermore, an increased surface expression and raft-structure distribution of annexin A2 was present in A549 cells after stimulation with SARS-induced cytokines interleukin-6 and interferon-γ. Cytokine stimulation increased the binding capability of anti-S2 antibodies to human lung epithelial cells. Together, the upregulated expression of annexin A2 by SARS-associated cytokines and the cross-reactivity of anti-SARS-CoV S2 antibodies to annexin A2 may have implications in SARS disease pathogenesis.

Kinome-Wide RNAi Studies in Human Multiple Myeloma Identify a Lymphoid Restricted Kinase GRK6 as a Selectively Vulnerable Target That Regulates STAT3/MCL1.

Abstract 601A paucity of validated kinase targets in human multiple myeloma (MM) has delayed clinical deployment of kinase inhibitors in treatment strategies. We therefore conducted a kinome-wide small interfering RNA (siRNA) lethality study in MM tumor lines bearing common t(4;14), t(14;16) and t(11;14) translocations to identify critically vulnerable kinases in MM tumor cells without regard to preconceived mechanistic notions. Primary screening was performed in duplicate using an 1800-oligo siRNA library in a single-siRNA-per-well format. siRNA were transfected at low concentration (13nM) to minimize off-target effects using conditions that resulted in transfection of >95% cells and <5% background cytotoxicity. After 96 hours, viability was measured by ATP-dependent luminescence. Fifteen kinases were consistently vulnerable in MM cells, including AKT1, AK3L1, AURKA, AURKB, CDC2L1, CDK5R2, FES, FLT4, GAK, GRK6, HK1, PKN1, PLK1, SMG1, and TNK2. While several kinases (PLK1, HK1) were equally vulnerable in epithelial cells, others and particularly the G-protein coupled receptor kinase, GRK6, appeared selectively vulnerable in MM. GRK6 inhibition is selectively lethal to KMS11, OPM1, H929, KMS18 and OCI-MY5 myeloma cells and has minimal effect on 293, MCF7, SF767, A375 or A549 epithelial cells. Persistent expression of FLAG-GRK6 via cDNA rescued KMS11 cells from the lethal effect of a 3'UTR-targeted GRK6 siRNA, but not from control siRNA, validating identification of GRK6 as an essential myeloma survival kinase. Furthermore, concordant results were obtained using four different exon-based GRK6 siRNA, all of which induced GRK6 silencing and similar inhibition of KMS11 proliferation and viability. Significantly, GRK6 is ubiquitously expressed in lymphoid tissues and myeloma, but by comparison appears absent or only weakly expressed in most primary human somatic tissues. From co-immunoprecipitation experiments we demonstrate that GRK6 is highly expressed in myeloma cells via direct association with the HSP90 chaperone. Inhibition of HSP90 with geldanamycin blocks GRK6 protein expression. Importantly, direct GRK6 silencing causes rapid and selective suppression of STAT3 phosphorylation that is associated with sustained reductions in total MCL1 protein levels and MCL1 phosphorylation (within 24 hours), providing a potent mechanism for the cytotoxicity of GRK6 inhibition in MM tumor cells. GF109203X is an inhibitor of both protein kinase C and of GRK6 that causes near total inhibition of these kinases in vitro at distinct concentrations of 0.1μM and 1-10μM respectively. Notably, GF109203X was substantially cytotoxic to 10/14 myeloma tumor lines at concentrations most consistent with GRK6 inhibition (5-20μM), and was selectively more cytotoxic to myeloma tumor cells than to non-myeloma cell lines (P=0.01), highlighting the potential of GRK6 as a pharmaceutical target for selective therapeutic intervention in myeloma. As mice that lack GRK6 are healthy, inhibition of GRK6 represents a uniquely targeted novel therapeutic strategy in human multiple myeloma. Disclosures:Perkins:MMRC: Employment. Reeder:Celgene: Research Funding; Millennium: Research Funding. Fonseca:Otsuka: Consultancy; BMS: Consultancy; Amgen: Consultancy; Medtronic: Consultancy; Genzyme: Consultancy.

Direct effects of protein S in ameliorating acute lung injury.

Protein S may exert an anticoagulant activity by enhancing the anticoagulant activity of activated protein C and/or by directly inhibiting the prothrombinase complex. Protein S itself may also directly regulate inflammatory responses and apoptosis. The role of protein S in acute lung injury (ALI) was unknown. This study evaluated the effect of protein S on ALI in the mouse. Animal ALI was induced in C57/BL6 mice by intratracheal instillation of lipopolysaccharide (LPS). Mice were treated with protein S or saline by intraperitoneal injection 1 h before LPS instillation. Activated protein or protein S alone and combined activated protein C + protein S therapy decreased inflammatory markers and cytokines in mice with acute lung injury. In LPS-treated mice compared with controls ALI was induced as shown by significantly increased levels of total protein, tumor necrosis factor-alpha, interleukin-6 and monocyte chemoattractant protein-1 in the bronchoalveolar lavage fluid. Mice with ALI treated with protein S had significantly decreased concentrations of tumor necrosis factor-alpha and interleukin-6 in the lung compared with untreated animals. Thrombin-antithrombin III, a marker of the activity of the coagulation cascade, was unchanged. Protein S inhibited the expression of cytokines in vitro and increased activation of the Axl tyrosine kinase pathway in A549 epithelial cells. Protein S protects against LPS-induced ALI, possibly by directly inhibiting the local expression of inflammatory cytokines without affecting coagulation.

Divergent intracellular pathways regulate interleukin-1β-induced miR-146a and miR-146b expression and chemokine release in human alveolar epithelial cells

We have previously reported that IL-β-induced miR-146a and miR-146b expression negatively regulates IL-8 and RANTES release in human alveolar A549 epithelial cells. To determine the intracellular pathways that regulate this response, we demonstrate IL-1β-induced activation of the nuclear factor (NF)-κB, extracellular regulated kinase (ERK)-1/2, c-jun N-terminal kinase (JNK)-1/2 and p38 mitogen activated kinase (MAP) kinase pathways. Subsequent pharmacological studies show that IL-1β-induced miR-146a, IL-8 and RANTES production was regulated via NF-κB and JNK-1/2 whilst miR-146b expression was mediated via MEK-1/2 and JNK-1/2. These divergent intracellular pathways likely explain the differential expression and biological action of the miR-146 isoforms.

Nickel induces intracellular calcium mobilization and pathophysiological responses in human cultured airway epithelial cells.

Environmental exposure to nickel is associated to respiratory disorders and potential toxicity in the lung but molecular mechanisms remain incompletely explored. The extracellular Ca(2+)-sensing receptor (CaSR) is widely distributed and may be activated by divalent cations. In this study, we investigated the presence of CaSR in human cultured airway epithelial cells and its activation by nickel. Nickel transiently increased intracellular calcium (-logEC(50)=4.67+/-0.06) in A549 and human bronchial epithelial cells as measured by epifluorescence microscopy. Nickel (20muM)-induced calcium responses were reduced after thapsigargin or ryanodine exposure but not by Ca(2+)-free medium. Inhibition of phospholipase-C or inositol trisphosphate release reduced intracellular calcium responses to nickel indicating activation of G(q)-signaling. CaSR mRNA and protein expression in epithelial cells was demonstrated by RT-PCR, western blot and immunofluorescence. Transfection of specific siRNA inhibited CaSR expression and suppressed nickel-induced intracellular calcium responses in A549 cells thus confirming nickel-CaSR activation. NPS2390, a CaSR antagonist, abolished the calcium response to nickel. Nickel-induced contraction, proliferation, alpha(1)(I)collagen production and inflammatory cytokines mRNA expression by epithelial cells as measured by traction microscopy, BrdU assay and RT-PCR, respectively. These responses were blocked by NPS2390. In conclusion, micromolar nickel concentrations, relevant to nickel found in the lung tissue of humans exposed to high environmental nickel, trigger intracellular Ca(2+) mobilization in human airway epithelial cells through the activation of CaSR which translates into pathophysiological outputs potentially related to pulmonary disease.

Enolase from Paracoccidioides brasiliensis: isolation and identification as a fibronectin-binding protein

Paracoccidioides brasiliensis yeast cells can enter mammalian cells and may manipulate the host cell environment to favour their own growth and survival. Moreover, fibronectin and several other host extracellular matrix proteins are recognized by various components of the yeast cell extracts. The present study was designed to isolate and characterize a fibronectin-binding protein from P. brasiliensis. We also compared P. brasiliensis strain 18, tested before (Pb18a) and after (Pb18b) animal passage, in relation to its adhesion and invasion processes. Extracts from both samples, when cultured on blood agar solid medium, showed higher levels of protein expression than when the same samples were cultured on Fava-Netto solid medium, as demonstrated by two-dimensional electrophoresis and SDS-PAGE. Also, both Pb18a and Pb18b exhibited stronger adhesion to A549 epithelial cells when cultured on blood agar medium than when cultured on Fava-Netto medium. Ligand affinity binding assays revealed a protein of 54 kDa and pI 5.6 in P. brasiliensis cell-free extracts with the properties of a fibronectin-binding adhesin, which was characterized by tryptic digestion and mass spectroscopy as a homologue of enolase from P. brasiliensis. Antibody raised against this 54 kDa protein abolished 80 % of P. brasiliensis adhesion to A549 epithelial cells. Our results demonstrate that P. brasiliensis produces a fibronectin-binding adhesin, irrespective of the culture medium, and that this activity can be inhibited by a specific antibody and is involved in the adhesion of the fungus to pulmonary epithelial cells.

The Acinetobacter baumannii 19606 OmpA Protein Plays a Role in Biofilm Formation on Abiotic Surfaces and in the Interaction of This Pathogen with Eukaryotic Cells

The ability of Acinetobacter baumannii to adhere to and persist on surfaces as biofilms could be central to its pathogenicity. The production of pili and a biofilm-associated protein and the expression of antibiotic resistance are needed for robust biofilm formation on abiotic and biotic surfaces. This multistep process also depends on the expression of transcriptional regulatory functions, some of which could sense nutrients available to cells. This report extends previous observations by showing that although outer membrane protein A (OmpA) of A. baumannii 19606 plays a partial role in the development of robust biofilms on plastic, it is essential for bacterial attachment to Candida albicans filaments and A549 human alveolar epithelial cells. In contrast to abiotic surfaces, the interaction with biotic surfaces is independent of the CsuA/BABCDE-mediated pili. The interaction of A. baumannii 19606 with fungal and epithelial cells also results in their apoptotic death, a response that depends on the direct contact of bacteria with these two types of eukaryotic cells. Furthermore, the bacterial adhesion phenotype correlates with the ability of bacteria to invade A549 epithelial cells. Interestingly, the killing activity of cell-free culture supernatants proved to be protease and temperature sensitive, suggesting that its cytotoxic activity is due to secreted proteins, some of which are different from OmpA.

The Role of Recombinant Human Elafin in the Resistance of A549 Cells against Pseudomonas aeruginosa Biofilm

Background:Pseudomonas aeruginosa, an opportunistic respiratory pathogen, often causes pulmonary infection in patients with chronic inflammatory airway diseases and forms drug-resistant biofilm. Elafin, a natural antibiotic, has antimicrobial properties against Gram-negative bacteria. Objectives: This study investigated the effects of elafin on the resistance of epithelial cells against P.aeruginosa biofilm. Methods: A549 epithelial cells transfected with pEGFP-N1-elafin or pEGFP-N1 were stimulated with tumor necrosis factor (TNF), P.aeruginosa supernatant and Escherichia coli supernatant followed by co-incubation for 24 h to form P.aeruginosa biofilms. Results: We found structural changes in the biofilms of all groups, especially the P.aeruginosa group. Bacterial counts in the TNF (3.238 ± 0.356 × 10⁶ CFU/cm²) and P.aeruginosa (3.317 ± 0.247 × 10⁶ CFU/cm²) groups were lower than in the control group (5.946 ± 0.453 × 10⁶ CFU/cm²; p < 0.01). In the P.aeruginosa group, the bacterial count was lower than that of the E. coli group (5.138 ± 0.391× 10⁶ CFU/cm²; p < 0.05). Bacterial clearance rates were higher in P.aeruginosa (44.2%) and TNF groups (45.6%) than in the E. coli group (13.6%). Cell intactness rates in pEGFP-N1-elafin-transfected groups were consistently higher than those of the control group (p < 0.01); the P.aeruginosa group rate at 24 h was the highest. Compared to the control group, the TNF, P.aeruginosa and E. coli groups showed increased elafin mRNA and protein in cells (p < 0.05), and contents in the TNF and P.aeruginosa group were higher than in the E. coli group (p< 0.05). Conclusion: Overexpression of elafin in epithelial cells attenuated the damage of P.aeruginosa biofilm on epithelia. P.aeruginosa has the potential to induce elafin expression.

Nitration of distinct tyrosine residues causes inactivation of histone deacetylase 2

Histone deacetylases (HDACs) are key molecules involved in epigenetic regulation of gene expression. We have previously demonstrated that oxidative stress caused a reduction in HDAC2, resulting in amplified inflammation and reduced corticosteroid responsiveness. Here we showed nitrative/oxidative stress reduced HDAC2 expression via nitration of distinct tyrosine residues. Peroxynitrite, hydrogen peroxide and cigarette smoke-conditioned medium reduced HDAC2 expression in A549 epithelial cells in vitro. This reduction was due to increased proteasomal degradation following ubiquitination rather than reduction of mRNA expression or stability. HDAC2 was nitrated under nitrative/oxidative stress and in the peripheral lung tissues of smokers and patients with chronic obstructive pulmonary disease. Mutagenesis studies replacing tyrosine (Y) residues with alanine revealed that Y253 is at least partly responsible for the proteasomal degradation of HDAC2 under nitrative stress. Thus, nitration of distinct tyrosine residues modifies both the expression and activity of HDAC2, having an impact on epigenetic regulation.