The Caenorhabditis elegans HNF4α Homolog, NHR-31, Mediates Excretory Tube Growth and Function through Coordinate Regulation of the Vacuolar ATPase

Nuclear receptors of the Hepatocyte Nuclear Factor-4 (HNF4) subtype have been linked to a host of developmental and metabolic functions in animals ranging from worms to humans; however, the full spectrum of physiological activities carried out by this nuclear receptor subfamily is far from established. We have found that the Caenorhabditis elegans nuclear receptor NHR-31, a homolog of mammalian HNF4 receptors, is required for controlling the growth and function of the nematode excretory cell, a multi-branched tubular cell that acts as the C. elegans renal system. Larval specific RNAi knockdown of nhr-31 led to significant structural abnormalities along the length of the excretory cell canal, including numerous regions of uncontrolled growth at sites near to and distant from the cell nucleus. nhr-31 RNAi animals were sensitive to acute challenge with ionic stress, implying that the osmoregulatory function of the excretory cell was also compromised. Gene expression profiling revealed a surprisingly specific role for nhr-31 in the control of multiple genes that encode subunits of the vacuolar ATPase (vATPase). RNAi of these vATPase genes resulted in excretory cell defects similar to those observed in nhr-31 RNAi animals, demonstrating that the influence of nhr-31 on excretory cell growth is mediated, at least in part, through coordinate regulation of the vATPase. Sequence analysis revealed a stunning enrichment of HNF4α type binding sites in the promoters of both C. elegans and mouse vATPase genes, arguing that coordinate regulation of the vATPase by HNF4 receptors is likely to be conserved in mammals. Our study establishes a new pathway for regulation of excretory cell growth and reveals a novel role for HNF4-type nuclear receptors in the development and function of a renal system.     

The Caenorhabditis elegans HNF4α Homolog,NHR-31, Mediates Excretory Tube Growth and Function through Coordinate Regulation of the Vacuolar ATPase

Nuclear receptors of the Hepatocyte Nuclear Factor-4 (HNF4) subtype have been linked to a host of developmental and metabolic functions in animals ranging from worms to humans; however, the full spectrum of physiological activities carried out by this nuclear receptor subfamily is far from established. We have found that the Caenorhabditis elegans nuclear receptor NHR-31, a homolog of mammalian HNF4 receptors, is required for controlling the growth and function of the nematode excretory cell, a multi-branched tubular cell that acts as the C. elegans renal system. Larval specific RNAi knockdown of nhr-31 led to significant structural abnormalities along the length of the excretory cell canal, including numerous regions of uncontrolled growth at sites near to and distant from the cell nucleus. nhr-31 RNAi animals were sensitive to acute challenge with ionic stress, implying that the osmoregulatory function of the excretory cell was also compromised. Gene expression profiling revealed a surprisingly specific role for nhr-31 in the control of multiple genes that encode subunits of the vacuolar ATPase (vATPase). RNAi of these vATPase genes resulted in excretory cell defects similar to those observed in nhr-31 RNAi animals, demonstrating that the influence of nhr-31 on excretory cell growth is mediated, at least in part, through coordinate regulation of the vATPase. Sequence analysis revealed a stunning enrichment of HNF4α type binding sites in the promoters of both C. elegans and mouse vATPase genes, arguing that coordinate regulation of the vATPase by HNF4 receptors is likely to be conserved in mammals. Our study establishes a new pathway for regulation of excretory cell growth and reveals a novel role for HNF4-type nuclear receptors in the development and function of a renal system.     

Cerulein hyperstimulation decreases AMP-activated protein kinase levels at the site of maximal zymogen activation

The premature activation of digestive enzyme zymogens in the pancreatic acinar cell is an important initiating event in acute pancreatitis. We have previously demonstrated that vacuolar ATPase (vATPase) activity is required for zymogen activation. Adenosine monophosphate-activated protein kinase (AMPK) regulates vATPase function in kidney and epididymal clear cells. To determine whether AMPK could affect pancreatitis responses, its effects were first examined in a cellular model of pancreatitis, cerulein-hyperstimulated (100 nM) pancreatic acini. This treatment caused a prominent increase in trypsin and chymotrypsin activities. Pretreatment with AICAR or metformin (AMPK activators) or compound C (an AMPK inhibitor) reduced or increased cerulein-induced zymogen activation, respectively. The association of the vATPase E subunit with membranes, a marker of its activation, tended to be inversely related to AMPK activity (assessed by AICAR and compound C treatments). Cerulein treatment did not change AMPK (α and β) levels but did lead to an increase in its activation (phosphorylation of Thr172) and induced the time-dependent translocation of the enzyme to a Triton-insoluble compartment. Basal in vivo studies showed that AMPK was widely distributed between membrane and soluble fractions generated by differential centrifugation. After cerulein hyperstimulation, AMPK levels selectively decreased in fractions containing the highest levels of active zymogens. These studies suggest that AMPK activity has a protective role in the pancreatic acinar cell that inhibits zymogen activation in the basal state, and this AMPK effect is reduced during pancreatitis. Therapies that prevent the selective reduction of AMPK in compartments that support zymogen activation could reduce injury during pancreatitis.     

Suppressing Dengue-2 Infection by Chemical Inhibition of Aedes aegypti Host Factors

Dengue virus host factors (DENV HFs) that are essential for the completion of the infection cycle in the mosquito vector and vertebrate host represent potent targets for transmission blocking. Here we investigated whether known mammalian DENV HF inhibitors could influence virus infection in the arthropod vector A. aegypti. We evaluated the potency of bafilomycin (BAF; inhibitor of vacuolar H+-ATPase (vATPase)), mycophenolic acid (MPA; inhibitor of inosine-5′-monophosphate dehydrogenase (IMPDH)), castanospermine (CAS; inhibitor of glucosidase), and deoxynojirimycin (DNJ; inhibitor of glucosidase) in blocking DENV infection of the mosquito midgut, using various treatment methods that included direct injection, ingestion by sugar feeding or blood feeding, and silencing of target genes by RNA interference (RNAi). Injection of BAF (5 µM) and MPA (25 µM) prior to feeding on virus-infected blood inhibited DENV titers in the midgut at 7 days post-infection by 56% and 60%, and in the salivary gland at 14 days post-infection by 90% and 83%, respectively, while treatment of mosquitoes with CAS or DNJ did not affect susceptibility to the virus. Ingestion of BAF and MPA through a sugar meal or together with an infectious blood meal also resulted in various degrees of virus inhibition. RNAi-mediated silencing of several vATPase subunit genes and the IMPDH gene resulted in a reduced DENV infection, thereby indicating that BAF- and MPA-mediated virus inhibition in adult mosquitoes most likely occurred through the inhibition of these DENV HFs. The route and timing of BAF and MPA administration was essential, and treatment after exposure to the virus diminished the antiviral effect of these compounds. Here we provide proof-of-principle that chemical inhibition or RNAi-mediated depletion of the DENV HFs vATPase and IMPDH can be used to suppress DENV infection of adult A. aegypti mosquitoes, which may translate to a reduction in DENV transmission.     

Omeprazole inhibits proliferation and modulates autophagy in pancreatic cancer cells

Background

Omeprazole has recently been described as a modulator of tumour chemoresistance, although its underlying molecular mechanisms remain controversial. Since pancreatic tumours are highly chemoresistant, a logical step would be to investigate the pharmacodynamic, morphological and biochemical effects of omeprazole on pancreatic cancer cell lines.

Methodology/Principal Findings

Dose-effect curves of omeprazole, pantoprazole, gemcitabine, 5-fluorouracil and the combinations of omeprazole and 5-fluorouracil or gemcitabine were generated for the pancreatic cancer cell lines MiaPaCa-2, ASPC-1, Colo357, PancTu-1, Panc1 and Panc89. They revealed that omeprazole inhibited proliferation at probably non-toxic concentrations and reversed the hormesis phenomena of 5-fluorouracil. Electron microscopy showed that omeprazole led to accumulation of phagophores and early autophagosomes in ASPC-1 and MiaPaCa-2 cells. Signal changes indicating inhibited proliferation and programmed cell death were found by proton NMR spectroscopy of both cell lines when treated with omeprazole which was identified intracellularly. Omeprazole modulates the lysosomal transport pathway as shown by Western blot analysis of the expression of LAMP-1, Cathepsin-D and β-COP in lysosome- and Golgi complex containing cell fractions. Acridine orange staining revealed that the pump function of the vATPase was not specifically inhibited by omeprazole. Gene expression of the autophagy-related LC3 gene as well as of Bad, Mdr-1, Atg12 and the vATPase was analysed after treatment of cells with 5-fluorouracil and omeprazole and confirmed the above mentioned results.

Conclusions

We hypothesise that omeprazole interacts with the regulatory functions of the vATPase without inhibiting its pump function. A modulation of the lysosomal transport pathway and autophagy is caused in pancreatic cancer cells leading to programmed cell death. This may circumvent common resistance mechanisms of pancreatic cancer. Since omeprazole use has already been established in clinical practice these results could lead to new clinical applications.     

Localization of pyrophosphatase in membranes of cauliflower inflorescence cells

Using a polyclonal antiserum specific for the tonoplastic H⁺-pyrophosphatase (tPPase), significant amounts of antigenic polypeptides of the correct molecular mass were detected in Western blots of plasma membrane isolated from cauliflower (Brassica oleracea L.) inflorescence by phase-partitioning and subsequent sucrose density centrifugation. Potassium iodide-stripped plasma membranes continued to give a strong positive signal, indicating that the PPase antigen detected was not a result of contamination through soluble PPase released during homogenisation. The same preparation contained negligible vacuolar (v)H⁺-ATPase activity and the A subunit of the vATPase could not be detected by immunoblotting. Plasma membrane fractions exhibited a proton-pumping activity with ATP as substrate, but such an activity was not measurable with pyrophosphate, although the hydrolysis of this substrate was recorded. By contrast, pyrophosphate supported proton pumping in tonoplast-containing fractions. Immunogold electron microscopy confirmed the presence of PPase at the plasma membrane as well as at the tonoplast, trans Golgi network, and multivesicular bodies. The density of immunogold label was higher at the plasma membrane than at the tonoplast, except for membrane fragments occurring in the lumen of the vacuoles which stained very conspicuously. Received: 29 June 1998 / Accepted: 9 November 1998     

Functional genomic studies of tick cells in response to infection with the cattle pathogen, Anaplasma marginale

The coevolution of ticks and the pathogens that they transmit has ensured their mutual survival. In these studies, we used a functional genomics approach to characterize tick genes regulated in response to Anaplasma marginale infection. Differentially regulated genes/proteins were identified by suppression-subtractive hybridization and differential in-gel electrophoresis analyses of cultured IDE8 tick cells infected with A. marginale. Nine of 17 of these genes were confirmed by real-time RT-PCR to be differentially regulated in ticks and/or IDE8 tick cells in response to A. marginale infection. RNA interference was used for functional studies. Six genes, which encode putative selenoprotein W2a, hematopoietic stem/progenitor cells protein-like, proteasome 26S subunit, ferritin, GST, and subolesin control, were found to affect A. marginale infection in IDE8 tick cells. Four genes, which encode putative GST, salivary selenoprotein M, vATPase, and ubiquitin, affected A. marginale infection in different sites of development in ticks. The results of these studies demonstrated that a molecular mechanism occurs by which tick cell gene expression mediates the A. marginale developmental cycle and trafficking through ticks.     

Silencing of genes involved in Anaplasma marginale-tick interactions affects the pathogen developmental cycle in Dermacentor variabilis

Background  

The cattle pathogen, Anaplasma marginale, undergoes a developmental cycle in ticks that begins in gut cells. Transmission to cattle occurs from salivary glands during a second tick feeding. At each site of development two forms of A. marginale (reticulated and dense) occur within a parasitophorous vacuole in the host cell cytoplasm. However, the role of tick genes in pathogen development is unknown. Four genes, found in previous studies to be differentially expressed in Dermacentor variabilis ticks in response to infection with A. marginale, were silenced by RNA interference (RNAi) to determine the effect of silencing on the A. marginale developmental cycle. These four genes encoded for putative glutathione S-transferase (GST), salivary selenoprotein M (SelM), H+ transporting lysosomal vacuolar proton pump (vATPase) and subolesin.     

Candida albicans actively modulates intracellular membrane trafficking in mouse macrophage phagosomes

The intracellular trafficking/survival strategies of the opportunistic human pathogen Candida albicans are poorly understood. Here we investigated the infection of RAW264.7 macrophages with a virulent wild-type (WT) filamentous C. albicans strain and a hyphal signalling-defective mutant ( efg1 Δ /cph1 Δ). A comparative analysis of the acquisition by phagosomes of actin, and of early/late endocytic organelles markers of the different fungal strains was performed and related to Candida's survival inside macrophages. Our results show that both fungal strains have evolved a similar mechanism to subvert the 'lysosomal' system, as seen by the inhibition of the phagosome fusion with compartments enriched in the lysobisphosphatidic acid and the vATPase, and thereby the acquisition of a low pH from the outset of infection. Besides, the virulent WT strain displayed additional specific survival strategies to prevent its targeting to compartmentsdisplaying late endosomal/lysosomal features, such as induction of active recycling out of phagosomes of the lysosomal membrane protein LAMP-1, the lysosomal protease cathepsin D and preinternalized colloidal gold. Finally, both virulent and efg1 Δ /cph1 Δ mutant fungal strains actively suppressed the production of macrophage nitric oxide (NO), although their cell wall extracts were potent inducers of NO.     

Biogenesis of Salmonella typhimurium-containing vacuoles in epithelial cells involves interactions with the early endocytic pathway

In epithelial cells, the intracellular pathogen Salmonella typhimurium resides and replicates within a unique cytoplasmic organelle, the Salmonella -containing vacuole (SCV). In vitro studies have shown that the SCV is a dynamic organelle that selectively acquires lysosomal glycoproteins (lgps) without fusing directly with lyosomes. Here, we have investigated early events in SCV biogenesis using immunofluorescence microscopy and epitope-specific flow cytometry. We show that proteins specific to the early endocytic pathway, EEA1 and transferrin receptor (TR), are present on early SCVs. The association of these proteins with SCVs is transient, and both proteins are undetectable at later time points when lgp and vATPase are acquired. Analysis of the fraction of SCVs containing both TR and lamp-1 showed that TR is lost from SCVs as the lgp is acquired, and that these processes occur progressively and not as the result of a single fusion/fission event. These experiments reveal a novel mechanism of SCV biogenesis, involving previously undetected initial interactions with the early endocytic pathway followed by the sequential delivery of lgp. The pathway does not involve interactions with the late endosome/prelysosome and is distinct from traditional phagocytic and endocytic pathways. Our study indicates that intracellular S. typhimurium occupies a unique niche, branching away from the traditional endocytic pathway between the early and late endosomal compartments.     

Regulation of the V-ATPase along the endocytic pathway occurs through reversible subunit association and membrane localization

The lumen of endosomal organelles becomes increasingly acidic when going from the cell surface to lysosomes. Luminal pH thereby regulates important processes such as the release of internalized ligands from their receptor or the activation of lysosomal enzymes. The main player in endosomal acidification is the vacuolar ATPase (V-ATPase), a multi-subunit transmembrane complex that pumps protons from the cytoplasm to the lumen of organelles, or to the outside of the cell. The active V-ATPase is composed of two multi-subunit domains, the transmembrane V(0) and the cytoplasmic V(1). Here we found that the ratio of membrane associated V(1)/Vo varies along the endocytic pathway, the relative abundance of V(1) being higher on late endosomes than on early endosomes, providing an explanation for the higher acidity of late endosomes. We also found that all membrane-bound V-ATPase subunits were associated with detergent resistant membranes (DRM) isolated from late endosomes, raising the possibility that association with lipid-raft like domains also plays a role in regulating the activity of the proton pump. In support of this, we found that treatment of cells with U18666A, a drug that leads to the accumulation of cholesterol in late endosomes, affected acidification of late endosome. Altogether our findings indicate that the activity of the vATPase in the endocytic pathway is regulated both by reversible association/dissociation and the interaction with specific lipid environments.     

The aging lysosome: An essential catalyst for late-onset neurodegenerative diseases

Lysosomes figure prominently in theories of aging as the proteolytic system most responsible for eliminating growing burdens of damaged proteins and organelles in aging neurons and other long lived cells. Newer evidence shows that diverse experimental measures known to extend lifespan in invertebrate aging models share the property of boosting lysosomal clearance of substrates through the autophagy pathway. Maintaining an optimal level of lysosome acidification is particularly crucial for these anti-aging effects. The exceptional dependence of neurons on fully functional lysosomes is reflected by the neurological phenotypes that develop in congenital lysosomal storage disorders, which commonly present as severe neurodevelopmental or neurodegenerative conditions even though the lysosomal deficit maybe systemic. Similar connections are now being appreciated between primary lysosomal deficit and the risk for late age-onset neurodegenerative disorders. In diseases such as Alzheimer's and Parkinson's, as in aging alone, primary lysosome dysfunction due to acidification impairment is emerging as a frequent theme, supported by the growing list of familial neurodegenerative disorders that involve primary vATPase dysfunction. The additional cellular roles played by intraluminal pH in sensing nutrient and stress and modulating cellular signaling have further expanded the possible ways that lysosomal pH dysregulation in aging and disease can disrupt neuronal function. Here, we consider the impact of cellular aging on lysosomes and how the changes during aging may create the tipping point for disease emergence in major late-age onset neurodegenerative disorders.     

Vacuolar membrane localization of the Arabidopsis 'two-pore' K+ channel KCO1

Potassium (K+) channels play multiple roles in higher plants, and have been characterized electrophysiologically in various subcellular membranes. The K+ channel AtKCO1 from Arabidopsis thaliana is the prototype of a new family of plant K+ channels. In a previous study the protein has been functionally characterized after heterologous expression in Baculovirus-infected insect cells. In order to obtain further information on the physiological function of AtKCO1, the gene expression pattern and subcellular localization of the protein in plants were investigated. The regulatory function of the 5' region of the AtKCO1 gene was examined in transgenic A. thaliana plants carrying beta-glucuronidase (GUS) fusion constructs. Our analysis demonstrates that the AtKCO1 promoter is active in various tissues and cell types, and the highest GUS activity could be detected in mitotically active tissues of the plant. Promoter activity was strongly dependent on the presence of a 5' leader intron. The same overall structure was identified in two genes encoding AtKCO1-like K+ channels from Solanum tuberosum (StKCO1alpha and StKCO1beta). To investigate the subcellular localization of AtKCO1, the channel protein, as well as a fusion protein of AtKCO1 with green fluorescence protein (GFP), were expressed in transgenic tobacco BY2 cells. In sucrose density gradients, both proteins co-fractionate with tonoplast markers (Nt-TIPa, vATPase). In fluorescence images from transgenic AtKCO1-GFP BY2 cells fluorescence was exclusively detected in the tonoplast. Thus AtKCO1 is the first cloned K+ channel demonstrated to be a vacuolar K+ channel.     

Massive excretion of calcium oxalate from late prepupal salivary glands of Drosophila melanogaster demonstrates active nephridial‐like anion transport

The Drosophila salivary glands (SGs) were well known for the puffing patterns of their polytene chromosomes and so became a tissue of choice to study sequential gene activation by the steroid hormone ecdysone. One well‐documented function of these glands is to produce a secretory glue, which is released during pupariation to fix the freshly formed puparia to the substrate. Over the past two decades SGs have been used to address specific aspects of developmentally‐regulated programmed cell death (PCD) as it was thought that they are doomed for histolysis and after pupariation are just awaiting their fate. More recently, however, we have shown that for the first 3–4 h after pupariation SGs undergo tremendous endocytosis and vacuolation followed by vacuole neutralization and membrane consolidation. Furthermore, from 8 to 10 h after puparium formation (APF) SGs display massive apocrine secretion of a diverse set of cellular proteins. Here, we show that during the period from 11 to 12 h APF, the prepupal glands are very active in calcium oxalate (CaOx) extrusion that resembles renal or nephridial excretory activity. We provide genetic evidence that Prestin, a Drosophila homologue of the mammalian electrogenic anion exchange carrier SLC26A5, is responsible for the instantaneous production of CaOx by the late prepupal SGs. Its positive regulation by the protein kinases encoded by fray and wnk lead to increased production of CaOx. The formation of CaOx appears to be dependent on the cooperation between Prestin and the vATPase complex as treatment with bafilomycin A₁ or concanamycin A abolishes the production of detectable CaOx. These data demonstrate that prepupal SGs remain fully viable, physiologically active and engaged in various cellular activities at least until early pupal period, that is, until moments prior to the execution of PCD.     

Early trafficking and intracellular replication of Legionella longbeachaea within an ER-derived late endosome-like phagosome

Legionella pneumophila is the predominant cause of Legionnaires' disease in the USA and Europe in contrast to Legionella longbeachaea, which is the leading cause of the disease in Western Australia. The ability of L. pneumophila to replicate intracellularly is triggered at the post-exponential phase along with expression of other virulence traits, such as motility. We show that while motility of L. longbeachaea is triggered upon growth transition into post-exponential phase, its ability to proliferate intracellularly is totally independent of the bacterial growth phase. Within macrophages, L. pneumophila replicates in a phagosome that excludes early and late endocytic markers and is surrounded by the rough endoplasmic reticulum (RER). In contrast, the L. longbeachaea phagosome colocalizes with the early endosomal marker early endosomal antigen 1 (EEA1) and the late endosomal markers lysosomal associated membrane glycoprotein 2 (LAMP-2) and mannose 6-phosphate receptor (M6PR), and is surrounded by the RER. The L. longbeachaea phagosome does not colocalize with the vacuolar ATPase (vATPase) proton pump, and the lysosomal luminal protease Cathepsin D, or the lysosomal tracer Texas red Ovalbumin (TROV). Intracellular proliferation of L. longbeachaea occurs in LAMP-2-positive phagosomes that are remodelled by the RER. Despite their distinct trafficking, both L. longbeachaea and L. pneumophila can replicate in communal phagosomes whose biogenesis is predominantly modulated by L. longbeachaea into LAMP-2-positive phagosomes. In addition, the L. pneumophila dotA mutant is rescued for intracellular replication if it co-inhabits the phagosome with L. longbeachaea. During late stages of infection, L. longbeachaea escape into the cytoplasm, prior to lysis of the macrophage, similar to L. pneumophila. We conclude that the L. longbeachaea phagosome matures to a non-acidified late endosome-like stage that is remodelled by the RER, indicating an idiosyncratic trafficking of L. longbeachaea compared with other intracellular pathogens, and a divergence in its intracellular lifestyle from L. pneumophila. In addition, re-routing biogenesis of the L. pneumophila phagosome into a late endosome controlled by L. longbeachaea has no effect on intracellular replication.     

Divalent cation mobility throughout exponential growth and sporulation of Bacillus megaterium.

Each of the five elements considered was taken up by Bacillus megaterium during exponential growth. Initial Mg and Mn uptake was rapid and ended by mid-log. For Ca, Fe, and Zn, uptake continued throughout exponential growth. Elements were released from the cells immediately following initial uptake. For Mn, egression continued to t2, with release of 36% of total accumulated. Secondary uptake followed immediately and continued through stage V. Magnesium egression continued to t1 with release of 33% accumulated. Secondary uptake began by t5 (stage IV) and continued slowly through sporulation. Calcium egression ceased by t4 with release of 25% total accumulated. Secondary uptake began by t6 (stage V) and continued until depleted. Zinc egression stopped by t5 with release of 34% accumulated with some secondary uptake by stage V. Iron egression terminated at t4 with release of 59% of total accumulated. This was followed by secondary uptake after t12 (stage VI).