Mesenchymal Stem Cell-Conditioned Medium Rescues LPS-Impaired ENaC Activity in Mouse Trachea via WNK4 Pathway

2020 ◽  
Vol 26 (29) ◽  
pp. 3601-3607 ◽  
Author(s):  
Yapeng Hou ◽  
Zhiyu Zhou ◽  
Hongfei Liu ◽  
Honglei Zhang ◽  
Yan Ding ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Conditioned Medium ◽  
Airway Epithelium ◽  
Short Circuit ◽  
Ussing Chamber ◽  
Great Promise ◽  
Mrna Levels ◽  
Tracheal Epithelial Cells ◽  
Airway Injury ◽  
Tracheal Epithelial

Background: Airway epithelium plays an essential role in maintaining the homeostasis and function of respiratory system as the first line of host defense. Of note, epithelial sodium channel (ENaC) is one of the victims of LPS-induced airway injury. Regarding the great promise held by mesenchymal stem cells (MSCs) for regenerative medicine in the field of airway injury and the limitations of cell-based MSCs therapy, we focused on the therapeutic effect of MSCs conditioned medium (MSCs-CM) on the ENaC activity in mouse tracheal epithelial cells. Methods: Ussing chamber apparatus was applied to record the short-circuit currents in primary cultured mouse tracheal epithelial cells, which reflects the ENaC activity. Expressions of α and γ ENaC were measured at the protein and mRNA levels by western blot and real-time PCR, respectively. The expression of with-no-lysinekinase- 4 (WNK4) and ERK1/2 were measured at protein levels, and the relationship between WNK4 and ERK1/2 was determined by WNK4 knockdown. Results: MSCs-CM restored the LPS-impaired ENaC activity, as well as enhanced the mRNA and protein expressions of ENaC in primary cultured mouse tracheal epithelial cells. Meanwhile, WNK4 and ERK1/2, both negative-regulators of ENaC, were suppressed accordingly after the administration of MSCs-CM in LPS-induced airway injury. After WNK4 gene was knocked down by siRNA, the level of ERK1/2 phosphorylation decreased. Conclusion: In light of the key role of ENaC in fluid reabsorption and the beneficial effects of MSCs-CM in the injury of airway epithelium, our results suggest that MSCs-CM is effective in alleviating LPS-induced ENaC dysfunction through WNK4-ERK1/2 pathway, which will provide a potent direction for the therapy of airway injury.

Endothelin stimulates chloride secretion across canine tracheal epithelium

1991 ◽  
Vol 261 (2) ◽  
pp. L188-L194 ◽  
Author(s):  
P. I. Plews ◽  
Z. A. Abdel-Malek ◽  
C. A. Doupnik ◽  
G. D. Leikauf
Keyword(s):  
Epithelial Cells ◽  
Short Circuit ◽  
Second Messengers ◽  
Short Circuit Current ◽  
Chloride Secretion ◽  
Tracheal Epithelium ◽  
Membrane Phospholipids ◽  
Cl Secretion ◽  
Tracheal Epithelial Cells ◽  
Tracheal Epithelial

The endothelins (ET) are a group of isopeptides produced by a number of cells, including canine tracheal epithelial cells. Because these compounds are endogenous peptides that may activate eicosanoid metabolism, we investigated the effects of ET on Cl secretion in canine tracheal epithelium. Endothelin 1 (ET-1) was found to produce a dose-dependent change in short-circuit current (Isc) that increased slowly and reached a maximal value within 10-15 min. When isopeptides of ET were compared, 300 nM ET-1 and ET-2 produced comparable maximal increases in Isc, whereas ET-3 produced smaller changes in Isc (half-maximal concentrations of 2.2, 7.2, and 10.4 nM, respectively). Ionic substitution of Cl with nontransported anions, iodide and gluconate, reduced ET-1-induced changes in Isc. Furthermore, the response was inhibited by the NaCl cotransport inhibitor, furosemide. In paired tissues, ET-1 significantly increased mucosal net 36Cl flux without significant effect on 22Na flux. The increase in Isc induced by ET was diminished by pretreatment with indomethacin. The second messengers mediating the increase in Isc were investigated in cultured canine tracheal epithelial cells. ET-1 stimulated the release of [3H]arachidonate from membrane phospholipids, increased intracellular Ca2+ (occasionally producing oscillations), and increased adenosine 3',5'-cyclic monophosphate accumulation. The latter was diminished by indomethacin. Thus ET is a potent agonist of Cl secretion (with the isopeptides having the following potency: ET-1 greater than or equal to ET-2 greater than ET-3) and acts, in part, through a cyclooxygenase-dependent mechanism.

Phorbol ester and okadaic acid regulation of Na-2Cl-K cotransport in rabbit tracheal epithelial cells

1996 ◽  
Vol 271 (1) ◽  
pp. C338-C346 ◽  
Author(s):  
C. M. Liedtke ◽  
L. Thomas
Keyword(s):  
Epithelial Cells ◽  
Okadaic Acid ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Term Treatment ◽  
Secretory Cells ◽  
Adrenergic Stimulation ◽  
Short Term Treatment ◽  
Tracheal Epithelial Cells ◽  
Tracheal Epithelial

We evaluated a role for protein kinase C (PKC) in the regulation of rabbit tracheal epithelial Na-Cl(K) cotransport. Short-term treatment with phorbol 12-myristate 13-acetate (PMA) dose dependently increased bumetanide-sensitive Na and Cl efflux and elevated staurosporine- and bumetanide-sensitive Na, Cl, and K uptake. PMA and the alpha 2A-adrenergic agonist guanabenz both induced contransport with a stoichiometry of 2 Cl:1 Na and 2 Cl:1 Rb and elevated staurosporine-sensitive PKC activity in cytosolic and particulate fractions. Prolonged PMA treatment did not sustain bumetanide-sensitive 2 Cl:1 Na and 2 Cl:1 Rb transport but did block stimulation of bumetanide-sensitive transport by PMA or guanabenz and elevation of PKC activity by PMA and guanabenz in a particulate fraction. Cells treated with okadaic acid exhibited a staurosporine- and bumetanide-sensitive 2 Cl:1 Na and 2 Cl:1 Rb uptake. In cultured monolayers, basolateral perfusion with epinephrine, isoproterenol, or PMA increased short-circuit current (Isc). Basolateral application of bumetanide reduced elevated Isc to baseline levels, indicating a role for Cl secretory cells in a reconstituted tracheal epithelium. Pretreatment of transmonolayer cultures with PMA diminished the stimulatory response to epinephrine. These results indicate that, in rabbit tracheal epithelial cells, alpha-adrenergic stimulation activated Na-2Cl-K cotransport and that PKC is a critical effector in this process.

Regulation of type I (epidermal) transglutaminase mRNA levels during squamous differentiation: down regulation by retinoids

1989 ◽  
Vol 9 (11) ◽  
pp. 4846-4851
Author(s):  
E E Floyd ◽  
A M Jetten
Keyword(s):  
Retinoic Acid ◽  
Epithelial Cells ◽  
Cdna Clones ◽  
Type I ◽  
Mrna Levels ◽  
Type Ii ◽  
Squamous Differentiation ◽  
Tracheal Epithelial Cells ◽  
Tracheal Epithelial ◽  
Epidermal Transglutaminase

Squamous differentiation of rabbit tracheal epithelial cells is accompanied by an approximately 50-fold increase in the activity of type I (epidermal) transglutaminase, while the levels of type II (tissue) transglutaminase remain almost undetectable. To identify a cDNA encoding type I transglutaminase, we screened a library of cDNA clones prepared from poly(A)+ RNA isolated from squamous-differentiated rabbit tracheal epithelial cells. Four overlapping clones (represented by clone pTG-7) which span a range of 2.8 kilobases were identified; partial sequencing of pTG-7 indicated that it encodes a transglutaminaselike protein. pTG-7 hybridized to a 3.6-kilobase mRNA which is distinct from that for type II transglutaminase. pTG-7 mRNA levels were low in proliferative cells, increased dramatically in squamous-differentiated cells, and could be further enhanced by growth of the cells in high concentrations (2 mM) of calcium ions. Retinoic acid, which blocks the expression of the squamous phenotype, prevented this increase in pTG-7 mRNA levels. These changes in levels of pTG-7 mRNA parallel the changes in type I transglutaminase activity observed under similar culture conditions. These data indicate that pTG-7 encodes the mRNA for transglutaminase type I and that expression of this mRNA is negatively regulated by retinoic acid.

MODELING THE AIRWAY EPITHELIUM IN ALLERGIC ASTHMA: INTERLEUKIN-13– INDUCED EFFECTS IN DIFFERENTIATED MURINE TRACHEAL EPITHELIAL CELLS

2005 ◽  
Vol 41 (7) ◽  
pp. 217 ◽  
Author(s):  
SUSAN M. LANKFORD ◽  
MARIANGELA MACCHIONE ◽  
ANNE L. CREWS ◽  
SHAUN A. MCKANE ◽  
NANCY J. AKLEY ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Allergic Asthma ◽  
Airway Epithelium ◽  
Interleukin 13 ◽  
Tracheal Epithelial Cells ◽  
Tracheal Epithelial

scholarly journals Regulation of type I (epidermal) transglutaminase mRNA levels during squamous differentiation: down regulation by retinoids.

1989 ◽  
Vol 9 (11) ◽  
pp. 4846-4851 ◽  
Author(s):  
E E Floyd ◽  
A M Jetten
Keyword(s):  
Retinoic Acid ◽  
Epithelial Cells ◽  
Cdna Clones ◽  
Type I ◽  
Mrna Levels ◽  
Type Ii ◽  
Squamous Differentiation ◽  
Tracheal Epithelial Cells ◽  
Tracheal Epithelial ◽  
Epidermal Transglutaminase

Squamous differentiation of rabbit tracheal epithelial cells is accompanied by an approximately 50-fold increase in the activity of type I (epidermal) transglutaminase, while the levels of type II (tissue) transglutaminase remain almost undetectable. To identify a cDNA encoding type I transglutaminase, we screened a library of cDNA clones prepared from poly(A)+ RNA isolated from squamous-differentiated rabbit tracheal epithelial cells. Four overlapping clones (represented by clone pTG-7) which span a range of 2.8 kilobases were identified; partial sequencing of pTG-7 indicated that it encodes a transglutaminaselike protein. pTG-7 hybridized to a 3.6-kilobase mRNA which is distinct from that for type II transglutaminase. pTG-7 mRNA levels were low in proliferative cells, increased dramatically in squamous-differentiated cells, and could be further enhanced by growth of the cells in high concentrations (2 mM) of calcium ions. Retinoic acid, which blocks the expression of the squamous phenotype, prevented this increase in pTG-7 mRNA levels. These changes in levels of pTG-7 mRNA parallel the changes in type I transglutaminase activity observed under similar culture conditions. These data indicate that pTG-7 encodes the mRNA for transglutaminase type I and that expression of this mRNA is negatively regulated by retinoic acid.

scholarly journals Transcriptional Regulation of β-Defensin Gene Expression in Tracheal Epithelial Cells

2000 ◽  
Vol 68 (1) ◽  
pp. 113-119 ◽  
Author(s):  
Gill Diamond ◽  
Vicki Kaiser ◽  
Janice Rhodes ◽  
John P. Russell ◽  
Charles L. Bevins
Keyword(s):  
Innate Immunity ◽  
Transcription Factors ◽  
Epithelial Cells ◽  
Antimicrobial Peptides ◽  
Binding Sites ◽  
Binding Activity ◽  
Mrna Levels ◽  
Tracheal Epithelial Cells ◽  
Tracheal Epithelial ◽  
Flanking Region

ABSTRACT Innate immunity provides an ever-present or rapidly inducible initial defense against microbial infection. Among the effector molecules of this defense in many species are broad-spectrum antimicrobial peptides. Tracheal antimicrobial peptide (TAP) was the first discovered member of the β-defensin family of mammalian antimicrobial peptides. TAP is expressed in the ciliated epithelium of the bovine trachea, and its mRNA levels are dramatically increased upon stimulation with bacteria or bacterial lipopolysaccharide (LPS). We report here that this induction by LPS is regulated at the level of transcription. Furthermore, the transfection of reporter gene constructs into tracheal epithelial cells indicates that DNA sequences in the 5′ flanking region of the TAP gene, within 324 nucleotides of the transcription start site, are responsible in part for mediating gene induction. This region includes consensus binding sites for NF-κB and nuclear factor interleukin-6 (NF IL-6) transcription factors. Gel mobility shift assays indicate that LPS induces NF-κB binding activity in the nuclei of these cells, while NF IL-6 binding activity is constitutively present. The gene encoding human β-defensin 2, a human homologue of TAP with similar inducible expression patterns in the airway, was cloned and found to have conserved NF-κB and NF IL-6 consensus binding sites in its 5′ flanking region. Previous studies of antimicrobial peptides from insects indicated that their induction by infectious microbes and microbial products also occurs via activation of NF-κB-like and NF IL-6-like transcription factors. Together, these observations indicate that a strategy for the induction of peptide-based antimicrobial innate immunity is conserved among evolutionarily diverse organisms.

Electrical properties of dog tracheal epithelial cells grown in monolayer culture

1984 ◽  
Vol 246 (3) ◽  
pp. C355-C359 ◽  
Author(s):  
D. L. Coleman ◽  
I. K. Tuet ◽  
J. H. Widdicombe
Keyword(s):  
Electrical Properties ◽  
Epithelial Cells ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Airway Epithelia ◽  
Tracheal Epithelial Cells ◽  
Monolayer Cultures ◽  
Tracheal Epithelial ◽  
Pg E2 ◽  
Water And Ion Transport

Epithelial cells from dog trachea, when grown in tissue culture, formed confluent monolayers 5-6 days after plating. After 8-10 days, domes [mean diam 356 +/- (SE) 53 micron] appeared in monolayers grown in collagen-coated flasks. When grown on polycarbonate filters coated with collagen, a detectable resistance (greater than 5 omega X cm2) and transepithelial potential difference (PD) (greater than 0.1 mV) developed 6 days after plating and increased to approximately 15 omega X cm2 and 15 mV at 10 days. Serosal ouabain (10(-4) M) abolished PD and short-circuit current (Isc). Luminal ouabain had no effect. Luminal amiloride (10(-4) M) and serosal bumetanide (10(-4) M) each decreased PD and Isc. However, a combination of both of these drugs did not abolish Isc. Isoproterenol (10(-5) M), dibutyryl adenosine 3',5'-cyclic monophosphate (10(-3) M), vasoactive intestinal peptide (10(-7) M), prostaglandin (PG) E2 (10(-5) M), PGF2 alpha (10(-5) M), and bradykinin (10(-5) M) each increased PD and Isc. Thus these monolayer cultures maintain electrical properties resembling those of the original tissue. This preparation may prove useful for the study of water and ion transport by airway epithelia.

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