Tiotropium bromide, a long acting muscarinic receptor antagonist triggers intracellular calcium signalling in the heart.

BACKGROUND AND PURPOSE: Tiotropium bromide (TB) is a long acting muscarinic receptor antagonist used to manage chronic obstructive pulmonary disease (COPD). Recent meta-analyses suggest an increased risk of cardiovascular events with TB. Ca2+/calmodulin dependent kinase II (CaMKII) and L-type Ca2+ channels regulate Ca2+ concentrations allowing management of Ca2+ across membranes. Pathological increases in Ca2+ are initially slow and progressive, however once the cytosolic concentration rises >1-3 µM from ~100 nM, calcium overload occurs and can lead to cell death. Ipratropium bromide, a short acting muscarinic receptor antagonist has previously been found to induce Ca2+ mediated eryptosis. The aim of this study was to investigate the role of Ca2+ in Tiotropium bromide mediated cardiotoxicity. EXPERIMENTAL APPROACH: Isolated Sprague-Dawley rat hearts were perfused with TB (10-0.1 nM) ±â€¯KN-93 (400 nM) or nifedipine (1 nM). Hearts were stained to determine infarct size (%) using triphenyltetrazolium chloride (TTC), or snap frozen to determine p-CaMKII (Thr286) expression. Cardiomyocytes were isolated using a modified Langendorff perfusion and enzymatic dissociation before preparation for Fluo 3-AM staining and flow cytometric analysis. KEY RESULTS: TB increased infarct size compared to controls by 6.91-8.41%, with no effect on haemodynamic function. KN-93/nifedipine with TB showed a 5.90/7.38% decrease in infarct size compared to TB alone, the combined use of KN-93 with TB also showed a significant increase in left ventricular developed pressure whilst nifedipine with TB showed a significant decrease in coronary flow. TB showed a 42.73% increase in p-CaMKII (Thr286) versus control, and increased Ca2+ fluorescence by 30.63% in cardiomyocytes. CONCLUSIONS AND IMPLICATIONS: To our knowledge, this is the first pre-clinical study to show that Tiotropium bromide induces Ca2+ signalling via CaMKII and L-type Ca2+ channels to result in cell damage. This has significant clinical impact due to long term use of TB in COPD patients, and warrants assessment of cardiac drug safety.

mRNA levels are buffered upon knockdown of RNA decay and translation factors via adjustment of transcription rates in human HepG2 cells.

Evidence from yeast and mammals argues the existence of cross-talk between transcription and mRNA decay. Stabilization of transcripts upon depletion of mRNA decay factors generally leads to no changes in mRNA abundance, attributing this to decreased transcription rates. We show that knockdown of human XRN1, CNOT6 and ETF1 genes in HepG2 cells led to significant alteration in stability of specific mRNAs, alterations in half-life were inversely associated with transcription rates, mostly not resulting in changes in abundance. We demonstrate the existence of the gene expression buffering mechanism in human cells that responds to both transcript stabilization and destabilization to maintain mRNA abundance via altered transcription rates and may involve translation. We propose that this buffering may hold novel cancer therapeutic targets.

A dual role for hypoxia inducible factor-1α in the hepatitis C virus lifecycle and hepatoma migration.

BACKGROUND & AIMS: Hepatitis C virus (HCV) causes progressive liver disease and is a major risk factor for the development of hepatocellular carcinoma (HCC). However, the role of infection in HCC pathogenesis is poorly understood. We investigated the effect(s) of HCV infection and viral glycoprotein expression on hepatoma biology to gain insights into the development of HCV associated HCC. METHODS: We assessed the effect(s) of HCV and viral glycoprotein expression on hepatoma polarity, migration and invasion. RESULTS: HCV glycoproteins perturb tight and adherens junction protein expression, and increase hepatoma migration and expression of epithelial to mesenchymal transition markers Snail and Twist via stabilizing hypoxia inducible factor-1α (HIF-1α). HIF-1α regulates many genes involved in tumor growth and metastasis, including vascular endothelial growth factor (VEGF) and transforming growth factor-beta (TGF-ß). Neutralization of both growth factors shows different roles for VEGF and TGFß in regulating hepatoma polarity and migration, respectively. Importantly, we confirmed these observations in virus infected hepatoma and primary human hepatocytes. Inhibition of HIF-1α reversed the effect(s) of infection and glycoprotein expression on hepatoma permeability and migration and significantly reduced HCV replication, demonstrating a dual role for HIF-1α in the cellular processes that are deregulated in many human cancers and in the viral life cycle. CONCLUSIONS: These data provide new insights into the cancer-promoting effects of HCV infection on HCC migration and offer new approaches for treatment.

Claudin association with CD81 defines hepatitis C virus entry.

Viruses initiate infection by attaching to molecules or receptors at the cell surface. Hepatitis C virus (HCV) enters cells via a multistep process involving tetraspanin CD81, scavenger receptor class B member I, and the tight junction proteins Claudin-1 and Occludin. CD81 and scavenger receptor class B member I interact with HCV-encoded glycoproteins, suggesting an initial role in mediating virus attachment. In contrast, there are minimal data supporting Claudin-1 association with HCV particles, raising questions as to its role in the virus internalization process. In the present study we demonstrate a relationship between receptor active Claudins and their association and organization with CD81 at the plasma membrane by fluorescence resonance energy transfer and stoichiometric imaging methodologies. Mutation of residues 32 and 48 in the Claudin-1 first extracellular loop ablates CD81 association and HCV receptor activity. Furthermore, mutation of the same residues in the receptor-inactive Claudin-7 molecule enabled CD81 complex formation and virus entry, demonstrating an essential role for Claudin-CD81 complexes in HCV infection. Importantly, Claudin-1 associated with CD81 at the basolateral membrane of polarized HepG2 cells, whereas tight junction-associated pools of Claudin-1 demonstrated a minimal association with CD81. In summary, we demonstrate an essential role for Claudin-CD81 complexes in HCV infection and their localization at the basolateral surface of polarized hepatoma cells, consistent with virus entry into the liver via the sinusoidal blood and association with basal expressed forms of the receptors.

Hepatitis C virus infection reduces hepatocellular polarity in a vascular endothelial growth factor-dependent manner.

BACKGROUND & AIMS: Hepatitis C virus (HCV) infection leads to progressive liver disease, frequently culminating in fibrosis and hepatocellular carcinoma. The mechanisms underlying liver injury in chronic hepatitis C are poorly understood. This study evaluated the role of vascular endothelial growth factor (VEGF) in hepatocyte polarity and HCV infection. METHODS: We used polarized hepatoma cell lines and the recently described infectious HCV Japanese fulminant hepatitis (JFH)-1 cell culture system to study the role of VEGF in regulating hepatoma permeability and HCV infection. RESULTS: VEGF negatively regulates hepatocellular tight junction integrity and cell polarity by a novel VEGF receptor 2-dependent pathway. VEGF reduced hepatoma tight junction integrity, induced a re-organization of occludin, and promoted HCV entry. Conversely, inhibition of hepatoma expressed VEGF with the receptor kinase inhibitor sorafenib or with neutralizing anti-VEGF antibodies promoted polarization and inhibited HCV entry, showing an autocrine pathway. HCV infection of primary hepatocytes or hepatoma cell lines promoted VEGF expression and reduced their polarity. Importantly, treatment of HCV-infected cells with VEGF inhibitors restored their ability to polarize, showing a VEGF-dependent pathway. CONCLUSIONS: Hepatic polarity is critical to normal liver physiology. HCV infection promotes VEGF expression that depolarizes hepatoma cells, promoting viral transmission and lymphocyte migration into the parenchyma that may promote hepatocyte injury.

Monoclonal anti-claudin 1 antibodies prevent hepatitis C virus infection of primary human hepatocytes.

BACKGROUND & AIMS: Hepatitis C virus (HCV) infection is a challenge to prevent and treat because of the rapid development of drug resistance and escape. Viral entry is required for initiation, spread, and maintenance of infection, making it an attractive target for antiviral strategies. The tight junction protein claudin-1 (CLDN1) has been shown to be required for entry of HCV into the cell. METHODS: Using genetic immunization, we produced 6 monoclonal antibodies against the host entry factor CLDN1. The effects of antibodies on HCV infection were analyzed in human cell lines and primary human hepatocytes. RESULTS: Competition and binding studies demonstrated that antibodies interacted with conformational epitopes of the first extracellular loop of CLDN1; binding of these antibodies required the motif W(30)-GLW(51)-C(54)-C(64) and residues in the N-terminal third of CLDN1. The monoclonal antibodies against CLDN1 efficiently inhibited infection by HCV of all major genotypes as well as highly variable HCV quasispecies isolated from individual patients. Furthermore, antibodies efficiently blocked cell entry of highly infectious escape variants of HCV that were resistant to neutralizing antibodies. CONCLUSIONS: Monoclonal antibodies against the HCV entry factor CLDN1 might be used to prevent HCV infection, such as after liver transplantation, and might also restrain virus spread in chronically infected patients.

Hepatoma cell density promotes claudin-1 and scavenger receptor BI expression and hepatitis C virus internalization.

Hepatitis C virus (HCV) entry occurs via a pH- and clathrin-dependent endocytic pathway and requires a number of cellular factors, including CD81, the tight-junction proteins claudin 1 (CLDN1) and occludin, and scavenger receptor class B member I (SR-BI). HCV tropism is restricted to the liver, where hepatocytes are tightly packed. Here, we demonstrate that SR-BI and CLDN1 expression is modulated in confluent human hepatoma cells, with both receptors being enriched at cell-cell junctions. Cellular contact increased HCV pseudoparticle (HCVpp) and HCV particle (HCVcc) infection and accelerated the internalization of cell-bound HCVcc, suggesting that the cell contact modulation of receptor levels may facilitate the assembly of receptor complexes required for virus internalization. CLDN1 overexpression in subconfluent cells was unable to recapitulate this effect, whereas increased SR-BI expression enhanced HCVpp entry and HCVcc internalization, demonstrating a rate-limiting role for SR-BI in HCV internalization.

Polarization restricts hepatitis C virus entry into HepG2 hepatoma cells.

The primary reservoir for hepatitis C virus (HCV) replication is believed to be hepatocytes, which are highly polarized with tight junctions (TJ) separating their basolateral and apical domains. HepG2 cells develop polarity over time, resulting in the formation and remodeling of bile canalicular (BC) structures. HepG2 cells expressing CD81 provide a model system to study the effects of hepatic polarity on HCV infection. We found an inverse association between HepG2-CD81 polarization and HCV pseudoparticle entry. As HepG2 cells polarize, discrete pools of claudin-1 (CLDN1) at the TJ and basal/lateral membranes develop, consistent with the pattern of receptor staining observed in liver tissue. The TJ and nonjunctional pools of CLDN1 show an altered association with CD81 and localization in response to the PKA antagonist Rp-8-Br-cyclic AMPs (cAMPs). Rp-8-Br-cAMPs reduced CLDN1 expression at the basal membrane and inhibited HCV infection, supporting a model where the nonjunctional pools of CLDN1 have a role in HCV entry. Treatment of HepG2 cells with proinflammatory cytokines, tumor necrosis factor alpha and gamma interferon, perturbed TJ integrity but had minimal effect(s) on cellular polarity and HCV infection, suggesting that TJ integrity does not limit HCV entry into polarized HepG2 cells. In contrast, activation of PKC with phorbol ester reduced TJ integrity, ablated HepG2 polarity, and stimulated HCV entry. Overall, these data show that complex hepatocyte-like polarity alters CLDN1 localization and limits HCV entry, suggesting that agents which disrupt hepatocyte polarity may promote HCV infection and transmission within the liver.

Effect of cell polarization on hepatitis C virus entry.

The primary reservoir for hepatitis C virus (HCV) replication in vivo is believed to be hepatocytes within the liver. Three host cell molecules have been reported to be important entry factors for receptors for HCV: the tetraspanin CD81, scavenger receptor BI (SR-BI), and the tight-junction (TJ) protein claudin 1 (CLDN1). The recent discovery of a TJ protein as a critical coreceptor highlighted the importance of studying the effect(s) of TJ formation and cell polarization on HCV entry. The colorectal adenocarcinoma Caco-2 cell line forms polarized monolayers containing functional TJs and was found to express the CD81, SR-BI, and CLDN1 proteins. Viral receptor expression levels increased upon polarization, and CLDN1 relocalized from the apical pole of the lateral cell membrane to the lateral cell-cell junction and basolateral domains. In contrast, expression and localization of the TJ proteins ZO-1 and occludin 1 were unchanged upon polarization. HCV infected polarized and nonpolarized Caco-2 cells to comparable levels, and entry was neutralized by anti-E2 monoclonal antibodies, demonstrating glycoprotein-dependent entry. HCV pseudoparticle infection and recombinant HCV E1E2 glycoprotein interaction with polarized Caco-2 cells occurred predominantly at the apical surface. Disruption of TJs significantly increased HCV entry. These data support a model where TJs provide a physical barrier for viral access to receptors expressed on lateral and basolateral cellular domains.

Protein kinase A-dependent step(s) in hepatitis C virus entry and infectivity.

Viruses exploit signaling pathways to their advantage during multiple stages of their life cycle. We demonstrate a role for protein kinase A (PKA) in the hepatitis C virus (HCV) life cycle. The inhibition of PKA with H89, cyclic AMP (cAMP) antagonists, or the protein kinase inhibitor peptide reduced HCV entry into Huh-7.5 hepatoma cells. Bioluminescence resonance energy transfer methodology allowed us to investigate the PKA isoform specificity of the cAMP antagonists in Huh-7.5 cells, suggesting a role for PKA type II in HCV internalization. Since viral entry is dependent on the host cell expression of CD81, scavenger receptor BI, and claudin-1 (CLDN1), we studied the role of PKA in regulating viral receptor localization by confocal imaging and fluorescence resonance energy transfer (FRET) analysis. Inhibiting PKA activity in Huh-7.5 cells induced a reorganization of CLDN1 from the plasma membrane to an intracellular vesicular location(s) and disrupted FRET between CLDN1 and CD81, demonstrating the importance of CLDN1 expression at the plasma membrane for viral receptor activity. Inhibiting PKA activity in Huh-7.5 cells reduced the infectivity of extracellular virus without modulating the level of cell-free HCV RNA, suggesting that particle secretion was not affected but that specific infectivity was reduced. Viral particles released from H89-treated cells displayed the same range of buoyant densities as did those from control cells, suggesting that viral protein association with lipoproteins is not regulated by PKA. HCV infection of Huh-7.5 cells increased cAMP levels and phosphorylated PKA substrates, supporting a model where infection activates PKA in a cAMP-dependent manner to promote virus release and transmission.

CD81 and claudin 1 coreceptor association: role in hepatitis C virus entry.

Hepatitis C virus (HCV) is an enveloped positive-stranded RNA hepatotropic virus. HCV pseudoparticles infect liver-derived cells, supporting a model in which liver-specific molecules define HCV internalization. Three host cell molecules have been reported to be important entry factors or receptors for HCV internalization: scavenger receptor BI, the tetraspanin CD81, and the tight junction protein claudin-1 (CLDN1). None of the receptors are uniquely expressed within the liver, leading us to hypothesize that their organization within hepatocytes may explain receptor activity. Since CD81 and CLDN1 act as coreceptors during late stages in the entry process, we investigated their association in a variety of cell lines and human liver tissue. Imaging techniques that take advantage of fluorescence resonance energy transfer (FRET) to study protein-protein interactions have been developed. Aequorea coerulescens green fluorescent protein- and Discosoma sp. red-monomer fluorescent protein-tagged forms of CD81 and CLDN1 colocalized, and FRET occurred between the tagged coreceptors at comparable frequencies in permissive and nonpermissive cells, consistent with the formation of coreceptor complexes. FRET occurred between antibodies specific for CD81 and CLDN1 bound to human liver tissue, suggesting the presence of coreceptor complexes in liver tissue. HCV infection and treatment of Huh-7.5 cells with recombinant HCV E1-E2 glycoproteins and anti-CD81 monoclonal antibody modulated homotypic (CD81-CD81) and heterotypic (CD81-CLDN1) coreceptor protein association(s) at specific cellular locations, suggesting distinct roles in the viral entry process.

Regulation of neuronal excitability through pumilio-dependent control of a sodium channel gene.

Dynamic changes in synaptic connectivity and strength, which occur during both embryonic development and learning, have the tendency to destabilize neural circuits. To overcome this, neurons have developed a diversity of homeostatic mechanisms to maintain firing within physiologically defined limits. In this study, we show that activity-dependent control of mRNA for a specific voltage-gated Na+ channel [encoded by paralytic (para)] contributes to the regulation of membrane excitability in Drosophila motoneurons. Quantification of para mRNA, by real-time reverse-transcription PCR, shows that levels are significantly decreased in CNSs in which synaptic excitation is elevated, whereas, conversely, they are significantly increased when synaptic vesicle release is blocked. Quantification of mRNA encoding the translational repressor pumilio (pum) reveals a reciprocal regulation to that seen for para. Pumilio is sufficient to influence para mRNA. Thus, para mRNA is significantly elevated in a loss-of-function allele of pum (pum(bemused)), whereas expression of a full-length pum transgene is sufficient to reduce para mRNA. In the absence of pum, increased synaptic excitation fails to reduce para mRNA, showing that Pum is also necessary for activity-dependent regulation of para mRNA. Analysis of voltage-gated Na+ current (I(Na)) mediated by para in two identified motoneurons (termed aCC and RP2) reveals that removal of pum is sufficient to increase one of two separable I(Na) components (persistent I(Na)), whereas overexpression of a pum transgene is sufficient to suppress both components (transient and persistent). We show, through use of anemone toxin (ATX II), that alteration in persistent I(Na) is sufficient to regulate membrane excitability in these two motoneurons.

Latrophilin is required for toxicity of black widow spider venom in Caenorhabditis elegans.

Black widow spider venom (BWSV) kills Caenorhabditis elegans after injection owing to the presence of heat- and detergent-sensitive components, which are high-molecular-mass latrotoxins. A C. elegans homologue of latrophilin/CIRL (calcium-independent receptor for latrotoxin), B0457.1, was identified and shown to have five conserved domains. RNAi (RNA interference) of this gene rendered C. elegans resistant to BWSV, whereas RNAi for CYP37A1 or a neurexin I homologue, and a deletion mutant of the related B0286.2 gene, had no effect on BWSV toxicity. The latrophilin RNAi mutants exhibit changes in defaecation cycle and alterations in drug sensitivity. These results demonstrate that latrophilin mediates the toxicity of BWSV and provide evidence for a physiological function of this receptor.

Microarray methods in Drosophila neurobiology.

In the relatively short period since their development, DNA microarrays have been used increasingly in the study of genetic and cellular processes, thereby offering a genome-wide approach to gene expression studies. With the advent of genome sequencing programs for organisms from yeast to man, the number of organisms which now have ready-made commercial arrays continues to increase. Here, the principle of DNA microarrays is introduced, with particular attention being given to the role of this technology in studies of the nervous system of the fruitfly Drosophila melanogaster. The importance of experimental design and sample preparation, in line with minimum information about microarray experiment (MIAME) compliance, is emphasised. The technical platforms available to the Drosophila neurobiologist have been illustrated and a brief number of data analysis tools that are readily available reviewed.

Clearance of persistent hepatitis C virus infection in humanized mice using a claudin-1-targeting monoclonal antibody.

Hepatitis C virus (HCV) infection is a leading cause of liver cirrhosis and cancer. Cell entry of HCV and other pathogens is mediated by tight junction (TJ) proteins, but successful therapeutic targeting of TJ proteins has not been reported yet. Using a human liver-chimeric mouse model, we show that a monoclonal antibody specific for the TJ protein claudin-1 (ref. 7) eliminates chronic HCV infection without detectable toxicity. This antibody inhibits HCV entry, cell-cell transmission and virus-induced signaling events. Antibody treatment reduces the number of HCV-infected hepatocytes in vivo, highlighting the need for de novo infection by means of host entry factors to maintain chronic infection. In summary, we demonstrate that an antibody targeting a virus receptor can cure chronic viral infection and uncover TJ proteins as targets for antiviral therapy.

EGFR and EphA2 are host factors for hepatitis C virus entry and possible targets for antiviral therapy.

Hepatitis C virus (HCV) is a major cause of liver disease, but therapeutic options are limited and there are no prevention strategies. Viral entry is the first step of infection and requires the cooperative interaction of several host cell factors. Using a functional RNAi kinase screen, we identified epidermal growth factor receptor and ephrin receptor A2 as host cofactors for HCV entry. Blocking receptor kinase activity by approved inhibitors broadly impaired infection by all major HCV genotypes and viral escape variants in cell culture and in a human liver chimeric mouse model in vivo. The identified receptor tyrosine kinases (RTKs) mediate HCV entry by regulating CD81-claudin-1 co-receptor associations and viral glycoprotein-dependent membrane fusion. These results identify RTKs as previously unknown HCV entry cofactors and show that tyrosine kinase inhibitors have substantial antiviral activity. Inhibition of RTK function may constitute a new approach for prevention and treatment of HCV infection.

Neuronal nitric oxide synthase gene transfer decreases [Ca2+]i in cardiac sympathetic neurons.

Gene transfer of neuronal nitric oxide synthase (nNOS) can decrease cardiac sympathetic outflow and facilitate parasympathetic neurotransmission. The precise pathway responsible for nitric oxide (NO) mediated inhibition of sympathetic neurotransmission is not known, but may be related to NO-cGMP activation of cGMP-stimulated phosphodiesterase (PDE2) that enhances the breakdown of cAMP to deactivate protein kinase A (PKA), resulting in a decrease in Ca(2+) influx mediated exocytosis of the neurotransmitter. We investigated depolarization evoked Ca(2+) influx in nNOS gene transduced sympathetic neurons from stellate ganglia with a noradrenergic cell specific vector (Ad.PRS-nNOS or empty vector), and examined how nNOS gene transfer affected cAMP and cGMP levels in these neurons. We found that targeting nNOS into these sympathetic neurons reduced amplitudes of voltage activated Ca(2+) transients by 44%. nNOS specific inhibition by N-[(4S)-4-Amino-5-[(2-aminoetyl](amino] pentyl]-N'-nitroguanidine (AAAN) reversed this response. nNOS gene transfer also increased intracellular cGMP (47%) and decreased cAMP (29%). A PDE2 specific inhibitor Bay60-7557 reversed the reduction in cAMP caused by Ad.PRS-nNOS. These results suggest that neuronal NO modulates cGMP and PDE2 to regulate voltage gated intracellular Ca(2+) transients in sympathetic neurons. Therefore, we propose this as a possible key step involved in NO decreasing cardiac sympathetic neurotransmission.

The homeobox transcription factor Even-skipped regulates acquisition of electrical properties in Drosophila neurons.

BACKGROUND: While developmental processes such as axon pathfinding and synapse formation have been characterized in detail, comparatively less is known of the intrinsic developmental mechanisms that regulate transcription of ion channel genes in embryonic neurons. Early decisions, including motoneuron axon targeting, are orchestrated by a cohort of transcription factors that act together in a combinatorial manner. These transcription factors include Even-skipped (Eve), islet and Lim3. The perdurance of these factors in late embryonic neurons is, however, indicative that they might also regulate additional aspects of neuron development, including the acquisition of electrical properties. RESULTS: To test the hypothesis that a combinatorial code transcription factor is also able to influence the acquisition of electrical properties in embryonic neurons we utilized the molecular genetics of Drosophila to manipulate the expression of Eve in identified motoneurons. We show that increasing expression of this transcription factor, in two Eve-positive motoneurons (aCC and RP2), is indeed sufficient to affect the electrical properties of these neurons in early first instar larvae. Specifically, we observed a decrease in both the fast K+ conductance (IKfast) and amplitude of quantal cholinergic synaptic input. We used charybdotoxin to pharmacologically separate the individual components of IKfast to show that increased Eve specifically down regulates the Slowpoke (a BK Ca2+-gated potassium channel), but not Shal, component of this current. Identification of target genes for Eve, using DNA adenine methyltransferase identification, revealed strong binding sites in slowpoke and nAcRalpha-96Aa (a nicotinic acetylcholine receptor subunit). Verification using real-time PCR shows that pan-neuronal expression of eve is sufficient to repress transcripts for both slo and nAcRalpha-96Aa. CONCLUSION: Taken together, our findings demonstrate, for the first time, that Eve is sufficient to regulate both voltage- and ligand-gated currents in motoneurons, extending its known repertoire of action beyond its already characterized role in axon guidance. Our data are also consistent with a common developmental program that utilizes a defined set of transcription factors to determine both morphological and functional neuronal properties.