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1.
Gene Ther ; 21(12): 1008-20, 2014 Dec.
Article in English | MEDLINE | ID: mdl-25231175

ABSTRACT

A difficulty in the field of gene therapy is the need to increase the susceptibility of hematopoietic stem cells (HSCs) to ex vivo genetic manipulation. To overcome this obstacle a high-throughput screen was performed to identify compounds that could enhance the transduction of target cells by lentiviral vectors. Of the 1280 compounds initially screened using the myeloid-erythroid-leukemic K562 cell line, 30 were identified as possible enhancers of viral transduction. Among the positive hits were known enhancers of transduction (camptothecin, etoposide and taxol), as well as the previously unidentified phorbol 12-myristate 13-acetate (PMA). The percentage of green fluorescent protein (GFP)-positive-expressing K562 cells was increased more than fourfold in the presence of PMA. In addition, the transduction of K562 cells with a lentiviral vector encoding fVIII was four times greater in the presence of PMA as determined by an increase in the levels of provirus in genetically modified cells. PMA did not enhance viral transduction of all cell types (for example, sca-1(+) mouse hematopoietic cells) but did enhance viral transduction of human bone marrow-derived CD34(+) cells. Notably, the percentage of GFP-positive CD34(+) cells was increased from 7% in the absence of PMA to greater than 22% in the presence of 1 nM PMA. PMA did not affect colony formation of CD34(+) cells or the expression of the hematopoietic markers CD34 and CD45. These data demonstrate that high-throughput screening can be used to identify compounds that increase the transduction efficiency of lentiviral vectors, identifying PMA as a potential enhancer of lentiviral HSC transduction.


Subject(s)
High-Throughput Screening Assays/methods , Lentivirus/genetics , Transduction, Genetic , Animals , Antigens, CD34/metabolism , Camptothecin/pharmacology , Cell Line, Tumor , Colforsin/pharmacology , Genetic Therapy , Genetic Vectors/genetics , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , HEK293 Cells , Hematopoietic Stem Cells/metabolism , Humans , Mice , NIH 3T3 Cells , Sirolimus/pharmacology , Tetradecanoylphorbol Acetate/analogs & derivatives , Tetradecanoylphorbol Acetate/pharmacology , U937 Cells
2.
Gene Ther ; 20(6): 607-15, 2013 Jun.
Article in English | MEDLINE | ID: mdl-22996197

ABSTRACT

We previously compared the expression of several human factor VIII (fVIII) transgene variants and demonstrated the superior expression properties of B domain-deleted porcine fVIII. Subsequently, a hybrid human/porcine fVIII molecule (HP-fVIII) comprising 91% human amino-acid sequence was engineered to maintain the high-expression characteristics of porcine fVIII. The bioengineered construct then was used effectively to treat knockout mice with hemophilia A. In the current study, we focused on optimizing self-inactivating (SIN) lentiviral vector systems by analyzing the efficacy of various lentiviral components in terms of virus production, transduction efficiency and transgene expression. Specifically, three parameters were evaluated: (1) the woodchuck hepatitis post-transcriptional regulatory element (WPRE), (2) HIV versus SIV viral vector systems and (3) various internal promoters. The inclusion of a WPRE sequence had negligible effects on viral production and HP-fVIII expression. HIV and SIV vectors were compared and found to be similar with respect to transduction efficiency in both K562s and HEK-293T cells. However, there was an enhanced expression of HP-fVIII by the SIV system, which was evident in both K562 and BHK-M cell lines. To further compare expression of HP-fVIII from an SIV-based lentiviral system, we constructed expression vectors containing the high expression transgene and a human elongation factor-1 alpha, cytomegalovirus (CMV) or phosphoglycerate kinase promoter. Expression was significantly greater from the CMV promoter, which also yielded therapeutic levels of HP-fVIII in hemophilia A mice. Based on these studies, an optimized vector contains the HP-fVIII transgene driven by a CMV internal promoter within a SIV-based lentiviral backbone lacking a WPRE.


Subject(s)
Factor VIII/genetics , Genetic Therapy , Hemophilia A , Lentivirus/genetics , Recombinant Proteins/genetics , Animals , Factor VIII/administration & dosage , Gene Transfer Techniques , Genetic Vectors , HEK293 Cells , Hemophilia A/genetics , Hemophilia A/therapy , Humans , Mice , Promoter Regions, Genetic , Recombinant Proteins/administration & dosage , Swine , Transduction, Genetic
3.
Oncogene ; 26(27): 3930-40, 2007 Jun 07.
Article in English | MEDLINE | ID: mdl-17213812

ABSTRACT

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is an important negative regulator of cell growth and a tumor suppressor. Its growth-attenuating activity is based on the dephosphorylation of phosphatidylinositol 3,4,5-trisphosphate (PIP3), an essential second messenger for the phosphoinositide 3-kinase/Akt signaling pathway. This activity may require localization of PTEN to cytoplasmic membranes. Yet PTEN can also localize to the cell nucleus where its functions remain unclear. Here we present data that define a short sequence in the N-terminal region of PTEN required for cytoplasmic localization. We will refer to this sequence as cytoplasmic localization signal (CLS). It could function as a non-canonical signal for nuclear export or as a cytoplasmic retention signal of PTEN. Mutations within the CLS induce nuclear localization and impair growth suppressive activities of PTEN while preserving lipid phosphatase activity. We propose that nuclear localization of PTEN is not compatible with plasma membrane-targeted growth suppressive functions of PTEN.


Subject(s)
Cell Proliferation , Cytoplasm/enzymology , PTEN Phosphohydrolase/metabolism , Amino Acid Sequence , Binding Sites/genetics , Cell Line , Cell Line, Tumor , Cell Nucleus/enzymology , Germ-Line Mutation , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Humans , Microscopy, Fluorescence , Molecular Sequence Data , Mutation , PTEN Phosphohydrolase/genetics , Phosphatidylinositol 3-Kinases/metabolism , Phosphatidylinositol 4,5-Diphosphate/metabolism , Phosphatidylinositol Phosphates/metabolism , Protein Sorting Signals/genetics , Proto-Oncogene Proteins c-akt/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sequence Homology, Amino Acid , Signal Transduction , Transfection
4.
J Virol ; 76(20): 10437-43, 2002 Oct.
Article in English | MEDLINE | ID: mdl-12239320

ABSTRACT

Successfully targeting the airway epithelium is essential for gene therapy of some pulmonary diseases. However, the airway epithelium is resistant to virus-mediated gene transfer with commonly used vectors. Vectors that interact with endogenously expressed receptors on the apical surface significantly increase gene transfer efficiency. However, other endogenous components involved in host immunity may hinder virus-mediated gene transfer. We tested the effect of bronchoalveolar lavage liquid (BAL) from patients with cystic fibrosis (CF), BAL from subjects without CF (non-CF BAL), Pseudomonas aeruginosa-derived proteins, and an array of inflammatory proteins on gene transfer mediated by adeno-associated virus type 5 (AAV5) and adenovirus targeted to an apically expressed glycosylphosphatidylinositol-modified coxsackie-adenovirus receptor. We found that neither CF BAL nor its components had a significant effect on gene transfer to human airway epithelium by these vectors. Non-CF BAL significantly impaired adenovirus-mediated gene transfer. Removal of immunoglobulins in non-CF BAL restored gene transfer efficiency. As virus vectors are improved and mechanisms of humoral immunity are elucidated, barriers to successful gene therapy found in the complex environment of the human lung can be circumvented.


Subject(s)
Adenoviruses, Human/immunology , Cystic Fibrosis/immunology , Dependovirus/immunology , Genetic Vectors/immunology , Respiratory Mucosa/immunology , Animals , Bronchoalveolar Lavage Fluid/immunology , COS Cells , Cells, Cultured , Chlorocebus aethiops , Coxsackie and Adenovirus Receptor-Like Membrane Protein , Cystic Fibrosis/therapy , Gene Transfer Techniques , Genetic Therapy , Humans , Lung/immunology , Neutralization Tests , Receptors, Virus/genetics , Receptors, Virus/immunology , Respiratory Mucosa/cytology , Trachea/immunology
5.
Am J Respir Cell Mol Biol ; 25(2): 186-95, 2001 Aug.
Article in English | MEDLINE | ID: mdl-11509328

ABSTRACT

Pseudomonas aeruginosa is an opportunistic human pathogen that causes both an acute lung disease in patients with hospital-acquired pneumonia and a chronic lung disease in individuals with cystic fibrosis. Many of the pathophysiologic effects of P. aeruginosa infection are due to factors secreted by the bacterium. Conditioned media from cultures of P. aeruginosa increased interleukin-8 expression and decreased regulated on activation, normal T cells expressed and secreted (RANTES) expression by human airway epithelial cells. Both of these activities were present in heat-treated, protease-treated, small molecular weight fractions. The activities were not inhibited by polymyxin B and were not extracted into ethyl acetate, suggesting that they were not due to endotoxin or autoinducer. Conversely, results from chloroform extractions and studies with a phenazine-minus mutant suggested that the blue pigment pyocyanin contributes to these activities when present. In addition to the effects of small molecular weight factors on cytokine expression, proteases in bacterial-conditioned media further decreased levels of RANTES. By altering expression, release, and/or activity of inflammatory cytokines, secretory factors from P. aeruginosa could disrupt the delicate balance that constitutes the immune response to bacterial infection and thus could contribute to the lung damage that occurs in P. aeruginosa-infected airways.


Subject(s)
Chemokine CCL5/metabolism , Interleukin-8/metabolism , Pseudomonas aeruginosa/immunology , Pseudomonas aeruginosa/pathogenicity , Respiratory System/immunology , Cell Line , Chemokine CCL5/genetics , Culture Media, Conditioned , Cytotoxicity, Immunologic , Epithelial Cells/immunology , Humans , Interleukin-1/pharmacology , Interleukin-8/genetics , Molecular Weight , Mutation , Pneumonia, Bacterial/genetics , Pneumonia, Bacterial/immunology , Pseudomonas Infections/genetics , Pseudomonas Infections/immunology , Pseudomonas aeruginosa/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Respiratory System/cytology , Respiratory System/physiopathology , Solvents , Tumor Necrosis Factor-alpha/pharmacology
6.
Plasmid ; 45(3): 200-8, 2001 May.
Article in English | MEDLINE | ID: mdl-11407915

ABSTRACT

Replication patterns of the miniP1 plasmid pZC176, the miniNR1 plasmid pRR933, and the high-copy miniNR1 derivative pRR942 were examined during the Escherichia coli cell division cycle and compared to the cycle-specific replication pattern of a minichromosome and the cycle nonspecific pattern of pBR322. In E. coli cells growing with doubling times of 40 and 60 min, the miniP1 plasmid was found to replicate with a slight periodicity during the division cycle. The periodicity was not nearly as pronounced as that of the minichromosome, was not affected by the presence of a minichromosome, and was not evident in cells growing more rapidly with a doubling time of 25 min. Both miniNR1 plasmids, pRR933 and pRR942, replicated with patterns indistinguishable from that of pBR322 and clearly different from that of the minichromosome. It is concluded that both P1 and NR1 plasmids can replicate at all stages of the cell cycle but that P1 displays a slight periodicity in replication probability in the cycle of slower growing cells. This periodicity does not appear to be coupled to a specific age in the cycle, but could be associated with the achievement of a specific cell mass per plasmid. During temperature shifts of a dnaC(Ts) mutant, the miniP1 plasmid and pBR322 replicated with similar patterns that differed from that of the minichromosome, but were consistent with a brief eclipse between rounds of replication.


Subject(s)
DNA Replication , Escherichia coli Proteins , Escherichia coli/genetics , Plasmids/genetics , Bacterial Proteins/genetics , Cell Cycle/genetics , Escherichia coli/cytology , Gene Dosage , Mutation , Temperature
7.
J Biol Chem ; 276(25): 22709-14, 2001 Jun 22.
Article in English | MEDLINE | ID: mdl-11331269

ABSTRACT

We have cloned and characterized a new member of the phosphatidylinositol kinase (PIK)-related kinase family. This gene, which we term human SMG-1 (hSMG-1), is orthologous to Caenorhabditis elegans SMG-1, a protein that functions in nonsense-mediated mRNA decay (NMD). cDNA sequencing revealed that hSMG-1 encodes a protein of 3031 amino acids containing a conserved kinase domain, a C-terminal domain unique to the PIK-related kinases and an FKBP12-rapamycin binding-like domain similar to that found in the PIK-related kinase mTOR. Immunopurified FLAG-tagged hSMG-1 exhibits protein kinase activity as measured by autophosphorylation and phosphorylation of the generic PIK-related kinase substrate PHAS-1. hSMG-1 kinase activity is inhibited by high nanomolar concentrations of wortmannin (IC(50) = 105 nm) but is not inhibited by a FKBP12-rapamycin complex. Mutation of conserved residues within the kinase domain of hSMG-1 abolishes both autophosphorylation and substrate phosphorylation, demonstrating that hSMG-1 exhibits intrinsic protein kinase activity. hSMG-1 phosphorylates purified hUpf1 protein, a phosphoprotein that plays a critical role in NMD, at sites that are also phosphorylated in whole cells. Based on these data, we conclude that hSMG-1 is the human orthologue to C. elegans SMG-1. Our data indicate that hSMG-1 may function in NMD by directly phosphorylating hUpf1 protein at physiologically relevant sites.


Subject(s)
Protein Kinases/genetics , Amino Acid Sequence , Animals , Cell Line , Cloning, Molecular , DNA, Complementary , Humans , Metalloendopeptidases , Molecular Sequence Data , Protein Kinases/chemistry , Protein Kinases/metabolism , Sequence Homology, Amino Acid
8.
Biochem J ; 350 Pt 3: 797-804, 2000 Sep 15.
Article in English | MEDLINE | ID: mdl-10970795

ABSTRACT

Because cell-mediated reduction of menadione leads to the generation of reactive oxygen species (ROS), this quinone is widely used to investigate the effects of ROS on cellular functions. We report that A549 human lung epithelial cells exposed to menadione demonstrate a dose-dependent increase in both intracellular calcium ([Ca(2+)](i)) and ROS formation. The concentrations of menadione required to initiate these two events are markedly different, with ROS detection requiring higher levels of menadione. Modulators of antioxidant defences (e.g. buthionine sulphoximine, 3-amino-1,2,4-triazole) have little effect on the [Ca(2+)](i) response to menadione, suggesting that ROS formation does not account for menadione-dependent alterations in [Ca(2+)](i). Additional evidence suggests that menadione photochemistry may be responsible for the observed [Ca(2+)](i) effects. Specifically: (a) EPR studies with the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) show that light exposure (maximum effect at 340 nm) stimulates menadione-dependent formation of the DMPO/(.)OH spin adduct that was not sensitive to antioxidant interventions; (b) DMPO inhibits menadione and light-dependent increases in [Ca(2+)](i); and (c) light (maximum effect at 340 nm) augments the deleterious effects of menadione on cell viability as determined by (51)Cr release. These photo effects do not appear to involve formation of singlet oxygen by menadione, but rather are the result of the oxidizing chemistry initiated by menadione in the triplet state. This work demonstrates that menadione species generated by photo-irradiation can exert biological effects on cellular functions and points to the potential importance of photochemistry in studies of menadione-mediated cell damage.


Subject(s)
Oxidants/metabolism , Vitamin K/chemistry , Vitamin K/pharmacology , Calcium/metabolism , Cell Line , Cyclic N-Oxides/chemistry , Humans , Hydroxyl Radical/chemistry , Photochemistry , Spectrometry, Fluorescence , Ultraviolet Rays
9.
J Exp Med ; 191(2): 287-302, 2000 Jan 17.
Article in English | MEDLINE | ID: mdl-10637273

ABSTRACT

Complement receptor (CR)-mediated phagocytosis of Mycobacterium tuberculosis by macrophages results in intracellular survival, suggesting that M. tuberculosis interferes with macrophage microbicidal mechanisms. As increases in cytosolic Ca(2+) concentration (¿Ca(2+)(c)) promote phagocyte antimicrobial responses, we hypothesized that CR phagocytosis of M. tuberculosis is accompanied by altered Ca(2+) signaling. Whereas the control complement (C)-opsonized particle zymosan (COZ) induced a 4.6-fold increase in ¿Ca(2+)(c) in human macrophages, no change in ¿Ca(2+)(c) occurred upon addition of live, C-opsonized virulent M. tuberculosis. Viability of M. tuberculosis and ingestion via CRs was required for infection of macrophages in the absence of increased ¿Ca(2+)(c), as killed M. tuberculosis or antibody (Ab)-opsonized, live M. tuberculosis induced elevations in ¿Ca(2+)(c) similar to COZ. Increased ¿Ca(2+)(c) induced by Ab-opsonized bacilli was associated with a 76% reduction in intracellular survival, compared with C-opsonized M. tuberculosis. Similarly, reversible elevation of macrophage ¿Ca(2+)(c) with the ionophore A23187 reduced intracellular viability by 50%. Ionophore-mediated elevation of ¿Ca(2+)(c) promoted the maturation of phagosomes containing live C-opsonized bacilli, as evidenced by acidification and accumulation of lysosomal protein markers. These data demonstrate that M. tuberculosis inhibits CR-mediated Ca(2+) signaling and indicate that this alteration of macrophage activation contributes to inhibition of phagosome-lysosome fusion and promotion of intracellular mycobacterial survival.


Subject(s)
Calcium Signaling , Macrophages/microbiology , Membrane Fusion , Mycobacterium tuberculosis/immunology , Adult , Antibodies, Bacterial/immunology , Calcium/metabolism , Cells, Cultured , Cytosol/metabolism , Humans , Lysosomes , Macrophages/drug effects , Macrophages/immunology , Macrophages/metabolism , Mycobacterium tuberculosis/physiology , Phagocytosis , Phagosomes , Receptors, IgG/immunology , Zymosan/pharmacology
10.
J Virol ; 73(11): 9664-8, 1999 Nov.
Article in English | MEDLINE | ID: mdl-10516081

ABSTRACT

Adenovirus interaction with alphav integrins is important for virus entry. We have examined the effects of adenovirus attachment on intracellular signaling in HeLa cells, with an emphasis on pathways known to be activated following integrin interaction with other ligands. We found no evidence for [Ca(2+)](c)-mediated signaling or for tyrosine phosphorylation of pp125(FAK), p130(CAS), and paxillin. However, adenovirus attachment is known to activate phosphatidylinositol-3 kinase, which in turn may regulate endocytosis via rab5 GTPase. We found that adenovirus uptake was increased by overexpression of wild-type rab5 and decreased by dominant-negative rab5. These results indicate a role for rab5 in adenovirus entry.


Subject(s)
Adenoviruses, Human/physiology , Endocytosis , rab5 GTP-Binding Proteins/metabolism , Adenoviruses, Human/genetics , Gene Expression Regulation, Viral , Gene Transfer Techniques , HeLa Cells , Humans , Phosphorylation , Phosphotyrosine/metabolism , Signal Transduction , Transfection , Tumor Cells, Cultured , rab5 GTP-Binding Proteins/chemistry
11.
J Immunol ; 162(9): 5112-8, 1999 May 01.
Article in English | MEDLINE | ID: mdl-10227981

ABSTRACT

Delivery of IgA to the mucosal surface occurs via transcytosis of polymeric IgA (pIgA) across the epithelium, a process mediated by the pIgR. Several factors increase pIgR expression in human epithelial cells, including IL-4 and IFN-gamma. Using an RNase protection assay, we found that IL-4 and IFN-gamma increase steady state levels of pIgR mRNA in both human intestinal (HT29) and airway (Calu-3) epithelial cells. Time course studies in HT29 clone 19A cells showed that with each cytokine alone and with both together: 1) there was a significant lag before mRNA levels increased; 2) maximal levels were not reached until 48-72 h after the addition of cytokines; 3) mRNA levels remained elevated in the continued presence of cytokines; and 4) addition of actinomycin D or removal of cytokines led to decreases in mRNA levels with a half-life of approximately 20-28 h. Cytokine-dependent increases in steady state levels of pIgR mRNA were inhibited by cycloheximide and by protein tyrosine kinase inhibitors but not by inhibitors of protein kinase C or cAMP-dependent protein kinase A. Both IFN-gamma and IL-4 increased expression of the inducible transcription factor IFN regulatory factor-1 (IRF-1), but levels of IRF-1 only weakly correlated with levels of pIgR mRNA, suggesting that additional transcription factors are required. These studies provide additional insights into the mechanisms by which cytokines regulate expression of the pIgR, a central player in mucosal immunity.


Subject(s)
Interferon-gamma/physiology , Interleukin-4/physiology , Intestinal Mucosa/metabolism , Lung/metabolism , RNA, Messenger/metabolism , Receptors, Polymeric Immunoglobulin/genetics , Receptors, Polymeric Immunoglobulin/metabolism , Cycloheximide/pharmacology , Cytokines/physiology , DNA-Binding Proteins/biosynthesis , Drug Synergism , Enzyme Inhibitors/pharmacology , Epithelial Cells/immunology , Epithelial Cells/metabolism , HT29 Cells/metabolism , Humans , Interferon Regulatory Factor-1 , Intestinal Mucosa/drug effects , Intestinal Mucosa/immunology , Lung/cytology , Lung/drug effects , Phosphoproteins/biosynthesis , Protein-Tyrosine Kinases/antagonists & inhibitors , Proto-Oncogene Proteins c-jun/biosynthesis , RNA, Messenger/drug effects , Time Factors , Transcription Factors/biosynthesis , Tumor Cells, Cultured
12.
Infect Immun ; 66(12): 5777-84, 1998 Dec.
Article in English | MEDLINE | ID: mdl-9826354

ABSTRACT

Pseudomonas aeruginosa, an opportunistic human pathogen, causes acute pneumonia in patients with hospital-acquired infections and is commonly associated with chronic lung disease in individuals with cystic fibrosis (CF). Evidence suggests that the pathophysiological effects of P. aeruginosa are mediated in part by virulence factors secreted by the bacterium. Among these factors is pyocyanin, a redox active compound that increases intracellular oxidant stress. We find that pyocyanin increases release of interleukin-8 (IL-8) by both normal and CF airway epithelial cell lines and by primary airway epithelial cells. Moreover, pyocyanin synergizes with the inflammatory cytokines tumor necrosis factor alpha and IL-1alpha. RNase protection assays indicate that increased IL-8 release is accompanied by increased levels of IL-8 mRNA. The antioxidant n-acetyl cysteine, general inhibitors of protein tyrosine kinases, and specific inhibitors of mitogen-activated protein kinases diminish pyocyanin-dependent increases in IL-8 release. Conversely, inhibitors of protein kinases C (PKC) and PKA have no effect. In contrast to its effects on IL-8 expression, pyocyanin inhibits cytokine-dependent expression of the monocyte/macrophage/T-cell chemokine RANTES. Increased release of IL-8, a potent neutrophil chemoattractant, in response to pyocyanin could contribute to the marked infiltration of neutrophils and subsequent neutrophil-mediated tissue damage that are observed in Pseudomonas-associated lung disease.


Subject(s)
Interleukin-8/biosynthesis , Lung/immunology , Pseudomonas , Pyocyanine/pharmacology , Acetylcysteine/pharmacology , Antioxidants/pharmacology , Calcium-Calmodulin-Dependent Protein Kinases/antagonists & inhibitors , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Cells, Cultured , Chemokine CCL5/biosynthesis , Drug Synergism , Epithelial Cells/cytology , Epithelial Cells/immunology , Humans , Interleukin-8/genetics , Lung/cytology , Oxidants/metabolism , Protein Kinase C/antagonists & inhibitors , Protein-Tyrosine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/metabolism , RNA, Messenger/analysis , Signal Transduction , Tumor Necrosis Factor-alpha/biosynthesis
13.
Am J Physiol ; 274(6): L893-900, 1998 06.
Article in English | MEDLINE | ID: mdl-9609727

ABSTRACT

Pseudomonas aeruginosa, an opportunistic human pathogen, causes both acute and chronic lung disease. P. aeruginosa exerts many of its pathophysiological effects by secreting virulence factors, including pyocyanine, a redox-active compound that increases intracellular oxidant stress. Because oxidant stress has been shown to affect cytosolic Ca2+ concentration ([Ca2+]c) in other cell types, we studied the effect of pyocyanine on [Ca2+]c in human airway epithelial cells (A549 and HBE). At lower concentrations, pyocyanine inhibits inositol 1,4,5-trisphosphate formation and [Ca2+]c increases in response to G protein-coupled receptor agonists. Conversely, at higher concentrations, pyocyanine itself increases [Ca2+]c. The pyocyanine-dependent [Ca2+]c increase appears to be oxidant dependent and to result from increased inositol trisphosphate and release of Ca2+ from intracellular stores. Ca2+ plays a central role in epithelial cell function, including regulation of ion transport, mucus secretion, and ciliary beat frequency. By disrupting Ca2+ homeostasis, pyocyanine could interfere with these critical functions and contribute to the pathophysiological effects observed in Pseudomonas-associated lung disease.


Subject(s)
Calcium/metabolism , Pseudomonas aeruginosa/physiology , Pulmonary Alveoli/drug effects , Pulmonary Alveoli/metabolism , Pyocyanine/pharmacology , Signal Transduction , Adenosine Triphosphate/pharmacology , Cell Line , Epithelial Cells/drug effects , Epithelial Cells/metabolism , GTP-Binding Proteins/metabolism , Homeostasis , Humans , Inositol 1,4,5-Trisphosphate/metabolism , Oxidation-Reduction , Receptors, Cell Surface/physiology
14.
Blood ; 90(1): 372-81, 1997 Jul 01.
Article in English | MEDLINE | ID: mdl-9207473

ABSTRACT

Calreticulin is a soluble endoplasmic reticulum protein comprising the major storage reservoir for inositol trisphosphate-releasable calcium. Although its highly conserved primary structure and a wide range of functions have been well described, less attention has been paid to its biosynthesis, particularly in human tissues. We report analyses of synthesis, proteolytic processing and glycosylation of human calreticulin. In both HL-60 and PLB-985 myeloid cell lines calreticulin was immunoprecipitated as a single 60-kD species without evidence of precursor forms. However, in vitro cell-free synthesis produced a 62-kD primary translation product, which in the presence of microsomal membranes, was processed by cotranslational signal peptide cleavage to a 60-kD species that comigrated with mature calreticulin produced in myeloid cells. Neither tunicamycin treatment of the cells nor endoglycosidase digestion of calreticulin resulted in any forms other than the 60-kD protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, suggesting that the potential site for N-glycosylation at asparagine-327 was unmodified. However, oxidative derivatization of carbohydrate components with digoxigenin showed that human calreticulin produced in either HL-60 cells or Sf9 insect cells is glycosylated, indicating that glycosylated and nonglycosylated human calreticulin have indistinguishable electrophoretic mobilities. Direct measurement by phenol-H2SO4 confirmed the presence of carbohydrate on recombinant human calreticulin. These data show that human myeloid calreticulin undergoes cotranslational signal peptide cleavage and posttranslational N-linked glycosylation. Although glycosylation of calreticulin has been shown in rat liver and bovine liver and brain, it has been reported to be lacking in other tissues including human lymphocytes.


Subject(s)
Calcium-Binding Proteins/biosynthesis , Ribonucleoproteins/biosynthesis , Animals , Calreticulin , Cattle , Glycosylation , HL-60 Cells , Humans , Peptide Biosynthesis , Protein Processing, Post-Translational , Rats
15.
J Immunol ; 156(12): 4807-14, 1996 Jun 15.
Article in English | MEDLINE | ID: mdl-8648128

ABSTRACT

IL-4 and IFN-gamma increase release of secretory component (SC), the polymeric IgA (plgA)-binding segment of the plgA receptor (plgAR), by the human intestinal epithelial cell line HT29. Moreover, these two cytokines synergistically increase plgA binding and cell surface staining for the receptor. To understand better the mechanism by which these cytokines regulate plgAR, we did quantitative immunoblotting using Abs against secretory component. We found that synergy occurs at the level of total cellular plgAR. Additionally, time course studies indicated that maximal receptor levels required >24-h incubation, that reaching maximal levels required at least 18 h of cytokine treatment, and that receptor levels remained elevated as long as cytokines were present. Conversely, if cytokines were removed, then cellular plgAR levels decreased with an approximate t1/2 of 20 h. Finally, synergy required the simultaneous presence of both cytokines throughout the treatment period. Direct measurement of second messengers and inhibitor studies suggest that Ca2+, cAMP, protein kinase A, and protein kinase C do not play major roles in regulating cellular plgAR levels by either cytokine, and do not contribute to the mechanism of synergy. In contrast, protein tyrosine kinase inhibitors potently inhibited all cytokine-dependent increases in total cellular plgAR. These results suggest that IL-4 and IFN-gamma increase cellular plgAR levels in HT29 cells predominantly by activating protein tyrosine kinase-dependent signaling pathways.


Subject(s)
Immunity, Mucosal , Interferon-gamma/administration & dosage , Interleukin-4/administration & dosage , Protein-Tyrosine Kinases/physiology , Receptors, Fc/metabolism , Receptors, Polymeric Immunoglobulin/metabolism , Blotting, Western , Cells, Cultured , Drug Synergism , Humans , Protein-Tyrosine Kinases/antagonists & inhibitors , Signal Transduction , Time Factors
16.
In Vitro Cell Dev Biol Anim ; 30A(9): 622-35, 1994 Sep.
Article in English | MEDLINE | ID: mdl-7529626

ABSTRACT

Most of the pancreatic exocrine epithelium consists of acinar and intralobular duct (ductular) cells, with the balance consisting of interlobular and main duct cells. Fragments of mouse acinar/ductular epithelium can be isolated by partial digestion with collagenase and purified by Ficoll density gradient centrifugation. We investigated whether previously developed culture conditions used for duct epithelium would result in the selective survival and proliferation of ductular cells from the acinar/ductular fragments. The fragments were cultured on nitrocellulose filters coated with extracellular matrix. After 2 to 4 wk the filters were covered with proliferating cells resembling parallel cultures of duct epithelium by the following criteria: protein/DNA ratio, light and electron microscopic appearance, the presence of duct markers (carbonic anhydrase [CA] activity, CA II mRNA, the cystic fibrosis transmembrane conductance regulator), the near absence of acinar cell markers (amylase and chymotrypsin), a similar polypeptide profile after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the presence of spontaneous and secretin-stimulated electrogenic ion transport. Both duct and ductular epithelia formed fluid-filled cysts in collagen gels and both could be subcultured. We conclude that acinar/ductular tissue gives rise to ductular cells in culture by some combination of acinar cell death and/or transdifferentiation to a ductular phenotype, accompanied by proliferation of these cells and preexisting ductular cells. These cultures may be used to investigate the properties of this part of the pancreatic duct system, from which most of the pancreatic juice water and electrolytes probably originates.


Subject(s)
Pancreas/cytology , Pancreatic Ducts/cytology , Amylases/analysis , Animals , Carbonic Anhydrases/analysis , Cell Division , Cell Survival , Cells, Cultured , Chymotrypsin/analysis , Culture Media , DNA/analysis , Electric Conductivity , Epithelial Cells , Male , Mice , Microscopy, Electron , Microscopy, Electron, Scanning , Proteins/analysis
17.
Am J Physiol ; 267(3 Pt 1): C776-83, 1994 Sep.
Article in English | MEDLINE | ID: mdl-7524335

ABSTRACT

Ca2+ plays a central role in regulating transepithelial fluid and electrolyte transport in intestinal epithelial cells. To investigate the mechanisms regulating the cytosolic free Ca2+ concentration ([Ca2+]c), we examined the effect of secretory agonists on [Ca2+]c in the intestinal epithelial cell line HT-29 clone 19A cells. We found that [Ca2+]c increased after addition of either adenosine 3',5'-cyclic monophosphate (cAMP)-dependent agonists or a D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]dependent agonist carbachol. Several lines of evidence suggest that cAMP- and Ins(1,4,5)P3-dependent agonists act through separate pathways. First, isoproterenol and forskolin increased cellular levels of cAMP but not Ins(1,4,5)P3, whereas carbachol increased cellular levels of Ins(1,4,5)P3 and stimulated inositol phosphate turnover without increasing cAMP. Second, carbachol increased [Ca2+]c by stimulating the release of Ca2+ from intracellular stores and influx of extracellular Ca2+. In contrast, cAMP agonists increased [Ca2+]c by stimulating Ca2+ influx alone. Third, the responses to maximal concentrations of cAMP agonists and carbachol were approximately additive. Finally, Ins(1,4,5)P3- but not cAMP agonist-dependent Ca2+ influx was inhibited by inorganic Ca2+ channel blockers. Thus, in intestinal epithelial cells, [Ca2+]c is regulated by at least two different second-messenger pathways, involving Ins(1,4,5)P3 or cAMP. In addition, cAMP stimulates influx of extracellular Ca2+ through a pathway distinct from that mediated by Ins(1,4,5)P3.


Subject(s)
Calcium/metabolism , Cyclic AMP/physiology , Inositol 1,4,5-Trisphosphate/physiology , Intestinal Mucosa/metabolism , 1-Methyl-3-isobutylxanthine/pharmacology , Calcium Channel Blockers/pharmacology , Carbachol/pharmacology , Cell Line , Intestinal Mucosa/cytology
18.
Am J Physiol ; 266(4 Pt 1): L405-13, 1994 Apr.
Article in English | MEDLINE | ID: mdl-7513963

ABSTRACT

Cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl- channel regulated by adenosine 3',5'-cyclic monophosphate (cAMP)-dependent phosphorylation and by intracellular nucleotides. The function of CFTR, like other recombinant ion channels, has generally been studied in single cells using voltage-clamp techniques. However, because CFTR is normally located in the apical membrane of epithelia we wanted to develop a system to study the function of recombinant CFTR expressed in an epithelium. We chose Fischer rat thyroid (FRT) epithelia for two reasons. First, when grown on permeable filter supports, FRT cells form polarized epithelia with a high transepithelial resistance. Second, they have no endogenous cAMP-regulated Cl- channels in their apical membrane. We expressed CFTR in FRT epithelia either transiently, using recombinant vaccinia virus, or stably, using a retrovirus. To measure apical membrane Cl- currents, we permeabilized the basolateral membrane to monovalent ions with nystatin and imposed a large transepithelial Cl- concentration gradient. cAMP agonists stimulated apical membrane Cl- currents in FRT epithelia infected with wild-type CFTR (vTF-CFTR) but not in FRT epithelia infected with either control virus (vTF7-3) or CFTR containing the delta F508 mutation (vTF-delta F508). These Cl- currents had properties similar to those of cAMP-activated Cl- currents in cells expressing endogenous or recombinant CFTR.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Genetic Techniques , Membrane Proteins/metabolism , Thyroid Gland/metabolism , Animals , Cell Membrane/metabolism , Cells, Cultured , Chloride Channels/physiology , Cystic Fibrosis Transmembrane Conductance Regulator , Electric Conductivity , Epithelial Cells , Epithelium/metabolism , Epithelium/physiology , Rats , Rats, Inbred F344 , Thyroid Gland/cytology , Thyroid Gland/physiology
19.
J Biol Chem ; 269(6): 4254-9, 1994 Feb 11.
Article in English | MEDLINE | ID: mdl-8307989

ABSTRACT

Platelet-activating factor (PAF) is a potent mediator which produces a wide range of biological responses by binding to specific, high affinity receptors on the target cell surface. In addition, we and others have observed cellular responses to PAF which are not receptor-mediated. We report here that in HBE-16 human bronchial epithelial cells, PAF produces a biphasic increase in [Ca2+]i consisting of a rapid initial increase due to release from intracellular stores followed by a gradual, sustained phase caused by influx of extracellular Ca2+. Under certain conditions, the PAF receptor antagonist L-659,989 completely blocks the release of Ca2+ from intracellular stores, suggesting a complete block of the receptor-mediated response. Under these same conditions, a residual influx of extracellular Ca2+ is observed, suggesting a possible receptor-independent response. HBE-16 cells partially metabolize PAF to 1-O-alkyl-2-acetyl-sn-glycerol (AAG), a bioactive diacylglycerol analog. Moreover, AAG stimulates Ca2+ influx in these cells; the response to AAG is at least 100-fold more potent than that to PAF. Taken together, these results suggest that PAF may stimulate Ca2+ influx in HBE-16 cells through a receptor-independent pathway mediated by AAG. Thus these studies suggest a previously unrecognized dual-pathway regulatory mechanism for PAF in the airway.


Subject(s)
Bronchi/metabolism , Calcium/metabolism , Glyceryl Ethers/metabolism , Platelet Activating Factor/pharmacology , Platelet Membrane Glycoproteins/physiology , Receptors, Cell Surface , Receptors, G-Protein-Coupled , Bronchi/cytology , Cells, Cultured , Epithelium/metabolism , Furans/pharmacology , Humans , In Vitro Techniques , Platelet Activating Factor/metabolism , Platelet Membrane Glycoproteins/antagonists & inhibitors
20.
Am J Respir Cell Mol Biol ; 9(6): 578-85, 1993 Dec.
Article in English | MEDLINE | ID: mdl-7504926

ABSTRACT

During development the fetal lung secretes fluid that is osmotically linked to chloride (Cl-) transport. One possible pathway for Cl- secretion across the fetal pulmonary epithelium is through the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR is expressed in epithelia and functions as a Cl- channel regulated by cyclic adenosine monophosphate (cAMP)-dependent protein kinase and intracellular ATP. Previous studies have shown that CFTR mRNA is expressed throughout the human fetal pulmonary epithelium and CFTR protein can be immunoprecipitated from human fetal lung homogenates. In cultured fetal lung tissue explants, CFTR mRNA was localized to alveolar epithelial cells. To test the hypothesis that fetal alveolar epithelial cells express functional CFTR, we immunolocalized CFTR in human fetal lung and looked for evidence of Cl- secretion in cultured alveolar epithelial cell monolayers. Monoclonal anti-CFTR antibodies localized CFTR in cultured lung explants to the epithelial cells, predominantly at the apical surface. Bioelectric properties of cultured monolayers of midgestation fetal alveolar epithelial cells were measured in modified Ussing chambers. In unstimulated monolayers, transepithelial electrical potential difference (psi t) = -1.1 +/- 0.1 mV, transepithelial resistance (Rt) = 768 +/- 58 omega.cm2, and short-circuit current (Isc) = 1.9 +/- 0.2 microA/cm2 (mean +/- SE, n = 17). Addition of amiloride to the apical surface significantly decreased basal Isc. Apical diphenylamine-2-carboxylate (DPC), a Cl- channel inhibitor, caused no significant change in basal Isc. In the presence of apical amiloride, isoproterenol significantly increased Isc, a response that was inhibited by apical DPC and submucosal bumetanide. The cAMP agonists forskolin and IBMX also stimulated Isc.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Chloride Channels/metabolism , Cyclic AMP/physiology , Membrane Proteins/metabolism , Pulmonary Alveoli/embryology , Pulmonary Alveoli/metabolism , Antibodies, Monoclonal , Biological Transport/physiology , Cells, Cultured , Cystic Fibrosis Transmembrane Conductance Regulator , Epithelial Cells , Epithelium/embryology , Epithelium/metabolism , Fetus/cytology , Fetus/metabolism , Humans , Immunohistochemistry , Membrane Proteins/analysis , Pulmonary Alveoli/cytology
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