Calcium and protein kinase C dependency of lipoxygenase-derived neutrophil chemotactic activity release from bronchial epithelial cells.

1995 ◽  
Vol 13 (3) ◽  
pp. 366-375 ◽  
Author(s):  
S Koyama ◽  
J G Garcia ◽  
S I Rennard ◽  
R A Robbins
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Epithelial Cells ◽  
Bronchial Epithelial Cells ◽  
Chemotactic Activity ◽  
Bronchial Epithelial ◽  
Kinase C ◽  
Neutrophil Chemotactic Activity

scholarly journals Adducin Promotes Micrometer-Scale Organization of β2-Spectrin in Lateral Membranes of Bronchial Epithelial Cells

2008 ◽  
Vol 19 (2) ◽  
pp. 536-545 ◽  
Author(s):  
Khadar M. Abdi ◽  
Vann Bennett
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Epithelial Cells ◽  
Rho Kinase ◽  
Bronchial Epithelial Cells ◽  
Bronchial Epithelial ◽  
Lateral Membrane ◽  
Kinase C ◽  
Reduced Height ◽  
Interfering Rna

Adducin promotes assembly of spectrin–actin complexes, and is a target for regulation by calmodulin, protein kinase C, and rho kinase. We demonstrate here that adducin is required to stabilize preformed lateral membranes of human bronchial epithelial (HBE) cells through interaction with β2-spectrin. We use a Tet-on regulated inducible small interfering RNA (siRNA) system to deplete α-adducin from confluent HBE cells. Depletion of α-adducin resulted in increased detergent solubility of spectrin after normal membrane biogenesis during mitosis. Conversely, depletion of β2-spectrin resulted in loss of adducin from the lateral membrane. siRNA–resistant α-adducin prevented loss of lateral membrane, but only if α-adducin retained the MARCKS domain that mediates spectrin–actin interactions. Phospho-mimetic versions of adducin with S/D substitutions at protein kinase C phosphorylation sites in the MARCKS domain were not active in rescue. We find that adducin modulates long-range organization of the lateral membrane based on several criteria. First, the lateral membrane of adducin-depleted cells exhibited reduced height, increased curvature, and expansion into the basal surface. Moreover, E-cadherin-GFP, which normally is restricted in lateral mobility, rapidly diffuses over distances up to 10 μm. We conclude that adducin acting through spectrin provides a novel mechanism to regulate global properties of the lateral membrane of bronchial epithelial cells.

scholarly journals Protein Kinase C-α Activity Is Required for Respiratory Syncytial Virus Fusion to Human Bronchial Epithelial Cells

2004 ◽  
Vol 78 (24) ◽  
pp. 13717-13726 ◽  
Author(s):  
Homero San-Juan-Vergara ◽  
Mark E. Peeples ◽  
Richard F. Lockey ◽  
Shyam S. Mohapatra
Keyword(s):  
Protein Kinase C ◽  
Respiratory Syncytial Virus ◽  
Protein Kinase ◽  
Epithelial Cells ◽  
Bronchial Epithelial Cells ◽  
Human Bronchial Epithelial Cells ◽  
Bronchial Epithelial ◽  
Kinase C ◽  
Rsv Infection ◽  
Syncytial Virus

ABSTRACT Respiratory syncytial virus (RSV) infection activates protein kinase C (PKC), but the precise PKC isoform(s) involved and its role(s) remain to be elucidated. On the basis of the activation kinetics of different signaling pathways and the effect of various PKC inhibitors, it was reasoned that PKC activation is important in the early stages of RSV infection, especially RSV fusion and/or replication. Herein, the role of PKC-α during the early stages of RSV infection in normal human bronchial epithelial cells is determined. The results show that the blocking of PKC-α activation by classical inhibitors, pseudosubstrate peptides, or the overexpression of dominant-negative mutants of PKC-α in these cells leads to significantly decreased RSV infection. RSV induces phosphorylation, activation, and cytoplasm-to-membrane translocation of PKC-α. Also, PKC-α colocalizes with virus particles and is required for RSV fusion to the cell membrane. Thus, PKC-α could provide a new pharmacological target for controlling RSV infection.

Stimulation of protein kinase C activity by tumor necrosis factor-α in bovine bronchial epithelial cells

1997 ◽  
Vol 273 (5) ◽  
pp. L1007-L1012 ◽  
Author(s):  
Todd A. Wyatt ◽  
Harumasa Ito ◽  
Thomas J. Veys ◽  
John R. Spurzem
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Tumor Necrosis Factor ◽  
Epithelial Cells ◽  
Tumor Necrosis ◽  
Bronchial Epithelial ◽  
Calphostin C ◽  
Kinase C ◽  
Tnf Α ◽  
Necrosis Factor

Bronchial epithelial cell migration, attachment, and proliferation are important processes in response to airway injury. We have shown that tumor necrosis factor (TNF)-α stimulates the migration of bovine bronchial epithelial cells (BBEC) in vitro. We hypothesized that protein kinase C (PKC) may be one of the intracellular signaling mediators of TNF-α in BBEC. In this study, we have identified multiple PKC isoforms in BBEC and measured total cellular PKC activity. Polyclonal antibodies to the PKC-α, -β2, -δ, and -ε isoforms recognized protein bands around 80–90 kDa. BBEC primary cultures treated with either 500 U/ml TNF-α for 2–4 h or 100 ng/ml 12- O-tetradecanoylphorbol 13-acetate for 15 min resulted in three- to fivefold increases in PKC activity in the particulate fractions of crude cell lysates. This activity was inhibited by 1 μM calphostin C or 10 μM H-7. Similarly, TNF-α-stimulated BBEC migration was reduced at least twofold in the presence of H-7 or calphostin C. These studies suggest that the activation of PKC is necessary for TNF-α-stimulated BBEC migration.

Bronchial epithelial cells release neutrophil chemotactic activity in response to tachykinins

1992 ◽  
Vol 263 (2) ◽  
pp. L226-L231 ◽  
Author(s):  
S. G. Von Essen ◽  
S. I. Rennard ◽  
D. O'Neill ◽  
R. F. Ertl ◽  
R. A. Robbins ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Proteinase Inhibitors ◽  
Bronchial Epithelial Cells ◽  
Chemotactic Activity ◽  
Induced Response ◽  
Neurokinin A ◽  
Maximal Effect ◽  
Neutrophil Chemotaxis ◽  
Bronchial Epithelial ◽  
Neutrophil Chemotactic Activity

The purpose of this study was to determine whether substance P (SP), neurokinin A (NKA), and neurokinin B (NKB) induce the release of neutrophil chemotactic activity (NCA) from bovine bronchial epithelial cells (BBEC) and whether neutral endopeptidase (NEP), a membrane-bound metalloenzyme that hydrolyzes tachykinins, modulates these effects. BBEC monolayers were exposed to SP, NKA, and NKB in the absence or presence of phosphoramidon (10(-6) M), a selective NEP inhibitor, for 72 h. Using a modified blind-well in vitro neutrophil chemotaxis assay, we found that tachykinin-exposed BBEC culture supernatant fluids induced significant neutrophil chemotaxis compared with supernatants obtained from unstimulated BBEC. Maximal effect was observed after 48 h of incubation and at SP concentration of 10(-13) M [92 +/- 3 (SP) vs. 64 +/- 2 (media) cells/high-power field (HPF), mean +/- SE, n = 7, P less than 0.05]. Release of NCA was mediated by the COOH-terminal of the SP molecule. The rank order of potency of tachykinins in inducing release of NCA was SP greater than NKA = NKB. SP-induced response was significantly potentiated by phosphoramidon (109 +/- 3 vs. 92 +/- 3 cells/HPF, n = 7, P less than 0.05), whereas other proteinase inhibitors had no effect. The released NCA was composed of protein and lipid-soluble components. These data indicate that mammalian tachykinins induce the release of NCA from BBEC and that NEP modulates these effects. We suggest that tachykinins regulate neutrophil recruitment into the lower respiratory tract, in part, by inducing the release of NCA from airway epithelial cells.

Cigarette Smoke Stimulates Release of Neutrophil Chemotactic Activity from Cultured Bovine Bronchial Epithelial Cells

1995 ◽  
Vol 88 (3) ◽  
pp. 337-344 ◽  
Author(s):  
Shunsuke Shoji ◽  
Ronald F. Ertl ◽  
Sekiya Koyama ◽  
Richard Robbins ◽  
George Leikauf ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Cyclic Amp ◽  
Cigarette Smoke ◽  
Fetal Calf Serum ◽  
Calf Serum ◽  
Bronchial Epithelial Cells ◽  
Chemotactic Activity ◽  
Bronchial Epithelial ◽  
Lipoxygenase Inhibitors ◽  
Neutrophil Chemotactic Activity

1. Recruitment of neutrophils into the airway is a prominent feature of chronic bronchitis, a syndrome often associated with exposure to cigarette smoke. Since bronchial epithelial cells are the ‘first’ lung cells to come into contact with smoke, these cells may be responsible for neutrophil recruitment into the airway by release of neutrophil chemotactic activity in response to cigarette smoke. 2. To evaluate this hypothesis, we prepared bovine bronchial epithelial cells and measured their ability to release neutrophil chemotactic activity following exposure to cigarette smoke. Bronchial epithelial cells were prepared by overnight digestion with protease, filtered through 100-μm Nitex mesh and resuspended in Dulbecco's modified Eagle medium supplemented with 10% fetal calf serum and antibiotics and cultured at 2×106 cells in 2 ml of medium in 35-mm culture dishes. After 4 days, dishes were rinsed and refed with medium without fetal calf serum and incubated with and without 1:10 diluted smoke extract for 6 h at 37°C. The neutrophil chemotactic activity of the supernatant fluids was measured by the blindwell chamber technique. 3. Cigarette smoke itself added to medium did not stimulate chemotaxis. In contrast, cigarette smoke did stimulate the release of neutrophil chemotactic activity from bovine bronchial epithelial cells [15 ± 3 (control) versus 74 ± 5 (smoke), P < 0.01]. 4. This neutrophil chemotactic activity was dialysable, pepsin and acid stable, heat sensitive and lipid extractable. Sephadex G-75 column chromatography demonstrated two peaks of neutrophil chemotactic activity. 5. The lipoxygenase inhibitors diethylcarbamazine and nordihydroguaratic acid both diminished the release of chemotactic activity, suggesting that the activity may be a lipoxygenase product(s). 6. Dibutyryl cyclic AMP also blocked the smoke-stimulated release of neutrophil chemotactic activity, suggesting that its release may be regulated by intracellular cyclic AMP. 7. Thus, bovine bronchial epithelial cells release neutrophil chemotactic activity in response to cigarette smoke, suggesting that bronchial epithelial cells may be modulators of the airway inflammatory response caused by cigarette smoke.

Antiproteases Attenuate the release of Neutrophil Chemotactic Activity from Bronchial Epithelial Cells Induced by Smoke

1996 ◽  
Vol 22 (1) ◽  
pp. 1-19 ◽  
Author(s):  
Sekiya Koyama ◽  
Stephen I. Rennard ◽  
George D. Leikauf ◽  
Ronald F. Ertl ◽  
Richard A. Robbins
Keyword(s):  
Epithelial Cells ◽  
Bronchial Epithelial Cells ◽  
Chemotactic Activity ◽  
Bronchial Epithelial ◽  
Neutrophil Chemotactic Activity

Viral Infection of Bovine Bronchial Epithelial Cells Induces Increased Neutrophil Chemotactic Activity and Neutrophil Adhesion

1993 ◽  
Vol 85 (6) ◽  
pp. 753-760 ◽  
Author(s):  
Meir Raz ◽  
Richard A. Robbins ◽  
Clayton L. Kelling ◽  
Lisa C. Stine ◽  
George D. Leikauf ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Viral Infection ◽  
Herpes Virus ◽  
Bronchial Epithelial Cells ◽  
Chemotactic Activity ◽  
Bronchial Epithelial ◽  
Lipoxygenase Inhibitors ◽  
Bovine Herpes Virus 1 ◽  
Herpès Virus ◽  
Neutrophil Chemotactic Activity

1. Acute bronchitis secondary to viral infection is associated with an influx of neutrophils. We hypothesized that bronchial epithelial cells are capable of releasing neutrophil chemotactic activity in response to viral infection. 2. To test this hypothesis, primary cultures of bovine bronchial epithelial cells were inoculated with a bovine respiratory pathogen, bovine herpes virus-1. 3. Supernatants collected from inoculated cells, before signs of toxicity, demonstrated significant neutrophil chemotactic activity using a blind well chamber neutrophil chemotaxis assay. Lipoxygenase inhibitors markedly reduced the amount of neutrophil chemotactic activity released after bovine herpes virus-1 inoculation. Analysis of arachidonic acid metabolites in cell supernatants by reverse-phase h.p.l.c. confirmed that leukotriene B4, a potent neutrophil chemoattractant, was released. 4. We also confirmed that adhesion of neutrophils to bovine herpes virus-1-inoculated bronchial epithelial cells was increased and mediated in part by the neutrophil integrin, LFA-1. 5. Thus, virally infected airway epithelial cells release leucocyte chemoattractants and hence adhesive interactions, functions that are likely to be important in the inflammatory acute response to viral infection.

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