Mechanisms of NOS2 regulation by Rho GTPase signaling in airway epithelial cells

2002 ◽  
Vol 283 (3) ◽  
pp. L604-L611 ◽  
Author(s):  
Nathan C. Kraynack ◽  
Deborah A. Corey ◽  
Heather L. Elmer ◽  
Thomas J. Kelley
Keyword(s):  
Promoter Activity ◽  
Rho Gtpase ◽  
Alveolar Epithelial Cell ◽  
Airway Epithelial Cells ◽  
Epithelial Cell Line ◽  
Airway Epithelial ◽  
Protein Levels ◽  
Human Alveolar Epithelial Cell ◽  
Coa Reductase ◽  
Nos2 Expression

The aberrant dysregulation of the inducible form of nitric oxide synthase (NOS2) is thought to play a role in many inflammatory disorders including cystic fibrosis (CF). The complex regulation of NOS2 expression is the subject of intense investigation, and one intriguing regulatory pathway known to influence NOS2 expression is the Rho GTPase cascade. We examined NOS2 regulation in response to inflammatory cytokines in a human alveolar epithelial cell line treated with inhibitors of different upstream and downstream components of the Rho GTPase pathway to better define potential signaling mechanisms. Statin-mediated 3-hydroxy-3-methylglutaryl-CoA reductase inhibition increased cytokine-dependent activation of the NOS2 promoter, reversible by the addition of geranylgeranyl pyrphosphate. However, inhibition of Rho-associated kinase (ROCK) with Y-27632 resulted in a decrease in NOS2 promoter activity, yet an increase in NOS2 mRNA and protein levels. Our results suggest that prenylation events influence NOS2 promoter activity independently of the Rho GTPase pathway and that Rho GTPase signaling mediated through ROCK suppresses NOS2 production downstream of promoter function at the message and protein level.

Zn2+-induced IL-8 expression involves AP-1, JNK, and ERK activities in human airway epithelial cells

2006 ◽  
Vol 290 (5) ◽  
pp. L1028-L1035 ◽  
Author(s):  
Yu-Mee Kim ◽  
William Reed ◽  
Weidong Wu ◽  
Philip A. Bromberg ◽  
Lee M. Graves ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Protein Expression ◽  
Promoter Activity ◽  
Fluorescent Protein ◽  
Airway Epithelial Cells ◽  
Epithelial Cell Line ◽  
Airway Epithelial ◽  
Human Airway ◽  
Mrna And Protein Expression ◽  
Human Airway Epithelial Cells

Exposure to zinc-laden particulate matter in ambient and occupational settings has been associated with proinflammatory responses in the lung. IL-8 is an important proinflammatory cytokine in the human lung and is induced in human airway epithelial cells exposed to zinc. In this study, we examined the cellular mechanisms responsible for Zn2+-induced IL-8 expression. Zn2+ stimulation resulted in pronounced increases in both IL-8 mRNA and protein expression in the human airway epithelial cell line (BEAS-2B). IL-8 promoter activity was significantly increased by Zn2+ exposure in BEAS-2B cells, indicating that Zn2+-induced IL-8 expression is transcriptionally mediated. Mutation of the activating protein (AP)-1 response element in an IL-8 promoter-enhanced green fluorescent protein construct reduced Zn2+-induced IL-8 promoter activity. Moreover, Zn2+ exposure of BEAS-2B cells induced the phosphorylation of the AP-1 proteins c-Fos and c-Jun. We observed that Zn2+ exposure induced the phosphorylation of ERK, JNK, and p38 MAPKs, whereas inhibition of ERK or JNK activity blocked IL-8 mRNA and protein expression in BEAS-2B cells treated with Zn2+. In addition, we investigated the role of protein tyrosine phosphatases in the activation of signaling by Zn2+. Zn2+ treatment inhibited ERK- and JNK-directed phosphatase activities in BEAS-2B cells. These results suggested that Zn2+-induced inhibition of phosphatase activity is an initiating event in MAPK and AP-1 activation that leads to enhanced IL-8 expression by human airway epithelial cells.

Oxytetracycline Inhibits Mucus Secretion and Inflammation in Human Airway Epithelial Cells

Chemotherapy ◽  
2017 ◽  
Vol 62 (5) ◽  
pp. 301-306 ◽  
Author(s):  
Said Ahmad Shah ◽  
Hajime Ishinaga ◽  
Kazuhiko Takeuchi
Keyword(s):  
Gene Expression ◽  
Epithelial Cells ◽  
Airway Epithelial Cells ◽  
Mucus Secretion ◽  
Epithelial Cell Line ◽  
Airway Epithelial ◽  
Human Respiratory Tract ◽  
Mucus Production ◽  
Protein Levels ◽  
Human Airway

Oxytetracycline is a broad-spectrum antibiotic, but its nonantibacterial effects in the human respiratory tract are unknown. In this study, the effects of oxytetracycline on mucus secretion and inflammation were examined by PCR and ELISA in the human airway epithelial cell line NCI-H292. Oxytetracycline (10 μg/mL) significantly inhibited TNF-α-induced MUC5AC gene expression and MUC5AC protein levels in NCI-H292 cells. It also downregulated IL-8 and IL-1β gene expression and IL-1β protein levels. Our findings demonstrated that oxytetracycline suppressed mucus production and inflammation in human respiratory epithelial cells, providing further evidence for the usefulness of oxytetracycline for human airway inflammatory diseases.

Effect of Steroids on the Synthesis of Complement C3 in a Human Alveolar Epithelial Cell Line

1993 ◽  
Vol 19 (5) ◽  
pp. 603-616 ◽  
Author(s):  
Terence L. Zach ◽  
Vicki A. Herrman ◽  
Laura D. Hill ◽  
M. Patricia Leuschen
Keyword(s):  
Epithelial Cell ◽  
Cell Line ◽  
Alveolar Epithelial Cell ◽  
Complement C3 ◽  
Epithelial Cell Line ◽  
Alveolar Epithelial ◽  
Human Alveolar Epithelial Cell

scholarly journals Hepatocyte nuclear factor 4α regulates the expression of intestinal epithelial Na+/H+ exchanger isoform 3

2018 ◽  
Vol 314 (1) ◽  
pp. G14-G21 ◽  
Author(s):  
Saminathan Muthusamy ◽  
Jong Jin Jeong ◽  
Ming Cheng ◽  
Jessica A. Bonzo ◽  
Anoop Kumar ◽  
...  
Keyword(s):  
Promoter Activity ◽  
Core Promoter ◽  
Epithelial Cell Line ◽  
Mrna Levels ◽  
Nuclear Factor ◽  
Intestinal Epithelial ◽  
Hepatocyte Nuclear Factor ◽  
Mobility Shift ◽  
Protein Levels ◽  
Hepatocyte Nuclear Factor 4Α

Na+/H+ exchanger isoform 3 (NHE3) plays a key role in coupled electroneutral NaCl absorption in the mammalian intestine. Reduced NHE3 expression or function has been implicated in the pathogenesis of diarrhea associated with inflammatory bowel disease (IBD) or enteric infections. Our previous studies revealed transcriptional regulation of NHE3 by various agents such as TNF-α, IFN-γ, and butyrate involving transcription factors Sp1 and Sp3. In silico analysis revealed that the NHE3 core promoter also contains a hepatocyte nuclear factor 4α (HNF-4α) binding site that is evolutionarily conserved in several species suggesting that HNF-4α has a role in NHE3 regulation. Nhe3 mRNA levels were reduced in intestine-specific Hnf4α-null mice. However, detailed mechanisms of NHE3 regulation by HNF-4α are not known. We investigated the regulation of NHE3 gene expression by HNF-4α in vitro in the human intestinal epithelial cell line C2BBe1 and in vivo in intestine-specific Hnf4α-null ( Hnf4αΔIEpC) and control ( Hnf4αfl/fl) mice. HNF-4α knockdown by short interfering RNA in C2BBe1 cells significantly decreased NHE3 mRNA and NHE3 protein levels. Gel mobility shift and chromatin immunoprecipitation assays revealed that HNF-4α directly interacts with the HNF-4α motif in the NHE3 core promoter. Site-specific mutagenesis on the HNF-4α motif decreased, whereas ectopic overexpression of HNF-4α increased, NHE3 promoter activity. Furthermore, loss of HNF-4α in Hnf4αΔIEpC mice decreased colonic Nhe3 mRNA and NHE3 protein levels. Our results demonstrate a novel role for HNF-4α in basal regulation of NHE3 expression. These studies represent an important and novel target for therapeutic intervention in IBD-associated diarrhea. NEW & NOTEWORTHY Our studies for the first time show that hepatocyte nuclear factor 4α directly regulates NHE3 promoter activity and its basal expression in the intestine.

Modification of biophysical properties of lung epithelial Na+ channels by dexamethasone

2000 ◽  
Vol 279 (3) ◽  
pp. C762-C770 ◽  
Author(s):  
A. Lazrak ◽  
A. Samanta ◽  
K. Venetsanou ◽  
P. Barbry ◽  
S. Matalon
Keyword(s):  
Cell Line ◽  
Single Channel ◽  
Alveolar Epithelial Cell ◽  
A549 Cells ◽  
Alveolar Epithelium ◽  
Housekeeping Gene ◽  
Epithelial Cell Line ◽  
Mrna Levels ◽  
Open Probability ◽  
Protein Levels

There is considerable interest in identifying the basic mechanisms by which dexamethasone alters ion transport across the adult alveolar epithelium. Herein, we incubated synchronized A549 cells, a human alveolar epithelial cell line, with dexamethasone (1 μM) for 24–48 h. When normalized to HPRT (a housekeeping gene), A549 β- and γ-subunit mRNA levels for the human amiloride-sensitive epithelial sodium channel (hENaC), assessed by RT-PCR, increased by 1.6- and 17-fold respectively, compared with control values ( P < 0.05). These changes were abolished by actinomycin D, indicating transcriptional regulation. Western blotting studies revealed that dexamethasone also increased expression of β- and γ-hENaC protein levels. In contrast, α-hENaC mRNA increased by onefold ( P > 0.05) and α-hENaC protein level was unchanged. Incubation of A549 cells with dexamethasone increased their whole cell amiloride-sensitive sodium currents twofold and decreased the K 0.5 for amiloride from 833 ± 69 to 22 ± 5.4 nM (mean ± SE; P < 0.01). Single channel recordings in the cell-attached mode showed that dexamethasone treatment increased single channel open time and open probability threefold and decreased channel conductance from 8.63 ± 0.036 to 4.4 ± 0.027 pS (mean ± SE; P < 0.01). We concluded that dexamethasone modulates the amiloride-sensitive Na+ channels by differentially regulating the expression of β- and γ-subunits at the mRNA and protein levels in the human A549 cell line, with little effect on α-hENaC subunit.

Effect of defensins on interleukin-8 synthesis in airway epithelial cells

1997 ◽  
Vol 272 (5) ◽  
pp. L888-L896 ◽  
Author(s):  
S. Van Wetering ◽  
S. P. Mannesse-Lazeroms ◽  
M. A. Van Sterkenburg ◽  
M. R. Daha ◽  
J. H. Dijkman ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Neutrophil Elastase ◽  
Proteinase Inhibitor ◽  
De Novo ◽  
Tissue Injury ◽  
A549 Cells ◽  
Airway Epithelial Cells ◽  
Epithelial Cell Line ◽  
Mrna Levels ◽  
Airway Epithelial

Neutrophils play an important role in inflammatory processes in the lung and may cause tissue injury through, for example, release of proteinases such as neutrophil elastase. In addition to neutrophil elastase, stimulated neutrophils also release small nonenzymatic and cationic polypeptides termed defensins. The aim of the present study was to investigate whether defensins induce interleukin (IL)-8 expression in cells of the A549 lung epithelial cell line and in human primary bronchial epithelial cells (PBEC). Supernatants of defensin-treated A549 cells contained increased neutrophil chemotactic activity (16-fold) that was inhibited by antibodies against IL-8. Concurrently, within 3 and 6 h, defensins significantly increased the IL-8 levels in supernatants of both A549 cells (n = 6, P < 0.05 and P < 0.01, respectively) and PBEC (n = 4, P < 0.001 and P < 0.001, respectively). This defensin-induced increase was fully inhibited by the serine proteinase inhibitor alpha 1-proteinase inhibitor. In addition, defensins also increased IL-8 mRNA levels (12-fold); this increase was dependent on de novo mRNA synthesis and did not require protein synthesis. Furthermore, defensins did not affect IL-8 mRNA stability, indicating that the enhanced IL-8 expression was due to increased transcription. Our findings suggest that defensins, released by stimulated neutrophils, stimulate IL-8 synthesis by airway epithelial cells and thus may mediate the recruitment of additional neutrophils into the airways.

scholarly journals TGF-β2 reduces nitric oxide synthase mRNA through a ROCK-dependent pathway in airway epithelial cells

2011 ◽  
Vol 301 (3) ◽  
pp. L361-L367 ◽  
Author(s):  
Jingjing Jiang ◽  
Steven C. George
Keyword(s):  
Epithelial Cells ◽  
Gas Phase ◽  
Airway Epithelial Cells ◽  
No Synthase ◽  
Arginase Activity ◽  
Inos Mrna ◽  
Airway Epithelial ◽  
Protein Levels ◽  
No Release ◽  
Dependent Pathway

Exhaled NO (eNO) is a potential noninvasive biomarker of inflammation in asthma. The significant intersubject variability of eNO within clinically similar patients has contributed to its limited clinical application. Arginase and NO synthase (NOS) utilize the same substrate (l-arginine) and contribute to the fibrotic and inflammatory features of asthma, respectively. Interestingly, TGF-β2 can increase the expression of arginase, stimulates fibrosis, and is overexpressed in asthma. We hypothesized that TGF-β2-enhanced arginase activity would decrease gas phase NO release from lung epithelial cells by limiting l-arginine availability for NOS. Our results show that TGF-β2 (5 ng/ml) significantly enhances total arginase activity up to two- to threefold in both primary small airway epithelial cells (SAECs) and the A549 cell line. Preincubation with TGF-β2 prior to cytokine (IL-1β, TNF-α, and IFN-γ, 10 ng/ml each) stimulation decreases gas phase NO release to baseline levels (from 1.66 ± 0.52 to 0.30 ± 0.12 pl·s−1·cm−2 and from 0.27 ± 0.03 pl·s−1·cm−2 to near zero in SAEC and A549 cells, respectively). Addition of arginase inhibitor ( Nω-hydroxy-nor-l-arginine) or small interfering RNA only partly reverses the reduction. In contrast, Rho-kinase (ROCK) pathway inhibitor (Y-27632) completely recovers the cytokine-induced NO flux in the present of TGF-β2. Inducible NO synthase (iNOS) mRNA and protein levels change in a similar trend as NO release from the cells. We conclude that TGF-β2 impacts cytokine-induced NO production in airway epithelial cells by reducing iNOS mRNA and protein levels through a ROCK-dependent pathway.

scholarly journals Transport of AOPP-Albumin into Human Alveolar Epithelial A549 Cell

2018 ◽  
Vol 21 ◽  
pp. 247-255 ◽  
Author(s):  
Masashi Kawami ◽  
Tadashi Shimonakamura ◽  
Ryoko Yumoto ◽  
Mikihisa Takano
Keyword(s):  
Alveolar Epithelial Cell ◽  
A549 Cells ◽  
Alveolar Epithelial Cells ◽  
Epithelial Cell Line ◽  
Ros Generation ◽  
Alveolar Epithelial ◽  
Lung Injuries ◽  
Km Value ◽  
Human Alveolar Epithelial Cell ◽  
Fluorescence Spectrometer

Purpose. Alveolar clearance of proteins, such as albumin, plays an essential role in recovery from lung injuries. Albumin is known to be oxidized by reactive oxygen species (ROS), leading to generation of advanced oxidation protein products (AOPP)-albumin in the alveolar lining fluid. In this study, we aimed to characterize the uptake of FITC-labeled AOPP-albumin (FITC-AOPP-albumin) into human alveolar epithelial cell line, A549. Methods. FITC-AOPP-albumin uptake into A549 cells and its effect of ROS generation was evaluated using fluorescence spectrometer and flow cytometry, respectively. Results. FITC-AOPP-albumin was taken up by A549 cells in a time- and temperature-dependent fashion, and showed saturation kinetics with a Km value of 0.37 mg/mL. The uptake of FITC-AOPP-albumin was suppressed by phenylarsine oxide, a clathrin-mediated endocytosis inhibitor, but not by indomethacin and nystatin, caveolae-mediated endocytosis inhibitors, or 5-(N-ethyl-N-isopropyl) amiloride, a macropinocytosis inhibitor. AOPP-albumin induced ROS generation in A549 cells, suggesting that alveolar clearance of AOPP-albumin should be important to prevent further ROS generation. Conclusion. AOPP-albumin is transported into alveolar epithelial cells through clathrin-mediated endocytosis, which may be important to prevent further ROS generation. This article is open to POST-PUBLICATION REVIEW. Registered readers (see “For Readers”) may comment by clicking on ABSTRACT on the issue’s contents page.

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