52 research outputs found
Signaling Cascade Involved in Rapid Stimulation of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) by Dexamethasone
Impairment of mucociliary clearance with reduced airway fluid secretion leads to
chronically inflamed airways. Cystic fibrosis transmembrane conductance regulator (CFTR) is
crucially involved in airway fluid secretion and dexamethasone (dexa) has previously been shown to
elevate CFTR activity in airway epithelial cells. However, the pathway by which dexa increases CFTR
activity is largely unknown. We aimed to determine whether the increase of CFTR activity by dexa
is achieved by non-genomic signaling and hypothesized that the phosphoinositide 3-kinase (PI3K)
pathway is involved in CFTR stimulation. Primary rat airway epithelial cells and human bronchial
submucosal gland-derived Calu-3 cells were analyzed in Ussing chambers and kinase activation was
determined byWestern blots. Results demonstrated a critical involvement of PI3K and protein kinase
B (AKT) signaling in the dexa-induced increase of CFTR activity, while serum and glucocorticoid
dependent kinase 1 (SGK1) activity was not essential. We further demonstrated a reduced neural
precursor cell expressed, developmentally downregulated 4-like (NEDD4L) ubiquitin E3 ligase
activity induced by dexa, possibly responsible for the elevated CFTR activity. Finally, increases of
CFTR activity by dexa were demonstrated within 30 min accompanied by rapid activation of AKT.
In conclusion, dexa induces a rapid stimulation of CFTR activity which depends on PI3K/AKT
signaling in airway epithelial cells. Glucocorticoids might thus represent, in addition to their
immunomodulatory actions, a therapeutic strategy to rapidly increase airway fluid secretion
Development and Functional Characterization of Fetal Lung Organoids
Preterminfants frequently suffer frompulmonary complications due to a physiological and
structural lung immaturity resulting in significant morbidity and mortality. Novel in vitro and
in vivo models are required to study the underlying mechanisms of late lung maturation
and to facilitate the development of new therapeutic strategies. Organoids recapitulate
essential aspects of structural organization and possibly organ function, and can be
used to model developmental and disease processes. We aimed at generating fetal
lung organoids (LOs) and to functionally characterize this in vitro model in comparison
to primary lung epithelial cells and lung explants ex vivo. LOs were generated with
alveolar and endothelial cells from fetal rat lung tissue, using a Matrigel-gradient and
air-liquid-interface culture conditions. Immunocytochemical analysis showed that the LOs
consisted of polarized epithelial cell adhesion molecule (EpCAM)-positive cells with the
apical membrane compartment facing the organoid lumen. Expression of the alveolar
type 2 cell marker, RT2-70, and the Club cell marker, CC-10, were observed. Na+
transporter and surfactant protein mRNA expression were detected in the LOs. First
time patch clamp analyses demonstrated the presence of several ion channels with
specific electrophysiological properties, comparable to vital lung slices. Furthermore, the
responsiveness of LOs to glucocorticoids was demonstrated. Finally, maturation of LOs
induced by mesenchymal stem cells confirmed the convenience of the model to test and
establish novel therapeutic strategies. The results showed that fetal LOs replicate key
biological lung functions essential for lung maturation and therefore constitute a suitable
in vitro model system to study lung development and related diseases
Glucocorticoids Equally Stimulate Epithelial Na+ Transport in Male and Female Fetal Alveolar Cells
Preterm infants frequently suffer from respiratory distress syndrome (RDS), possibly due
to lower expression of epithelial Na+ channels (ENaC). RDS incidence is sex-specific, affecting males
almost twice as often. Despite the use of antenatal glucocorticoids (GCs), the sex difference persists.
It is still controversial whether both sexes benefit equally from GCs. We previously showed that
Na+ transport is higher in female compared with male fetal distal lung epithelial (FDLE) cells.
Since GCs increase Na+ transport, we hypothesized that their stimulating effect might be sex-specific.
We analyzed FDLE cells with Ussing chambers and RT-qPCR in the presence or absence of fetal serum.
In serum-free medium, GCs increased the ENaC activity and mRNA expression, independent of sex.
In contrast, GCs did not increase the Na+ transport in serum-supplemented media and abolished the
otherwise observed sex difference. Inhibition of the GC receptor in the presence of serum did not
equalize Na+ transport between male and female cells. The GC-induced surfactant protein mRNA
expression was concentration and sex-specific. In conclusion, female and male FDLE cells exhibit no
sex difference in response to GCs with regard to Na+ transport, and GR activity does not contribute
to the higher Na+ transport in females
Albumin Stimulates Epithelial Na+ Transport and Barrier Integrity by Activating the PI3K/AKT/SGK1 Pathway
Albumin is a major serum protein and is frequently used as a cell culture supplement. It is
crucially involved in the regulation of osmotic pressure and distribution of fluid between different
compartments. Alveolar epithelial Na+
transport drives alveolar fluid clearance (AFC), enabling air
breathing. Whether or not albumin affects AFC and Na+
transport is yet unknown. We therefore
determined the acute and chronic effects of albumin on Na+
transport in fetal distal lung epithelial
(FDLE) cells and the involved kinase pathways. Chronic BSA treatment strongly increased epithelial
Na+
transport and barrier integrity in Ussing chambers. BSA did not elevate mRNA expression of
Na+
transporters in FDLE cells after 24 h. Moreover, acute BSA treatment for 45 min mimicked the
chronic effects. The elevated Na+
transport was caused by an increased maximal ENaC activity, while
Na,K-ATPase activity remained unchanged. Acute and chronic BSA treatment lowered membrane
permeability, confirming the increased barrier integrity observed in Ussing chambers. Western blots
demonstrated an increased phosphorylation of AKT and SGK1, and PI3K inhibition abolished the
stimulating effect of BSA. BSA therefore enhanced epithelial Na+
transport and barrier integrity by
activating the PI3K/AKT/SGK1 pathwa
Y It Matters—Sex Differences in Fetal Lung Development
Within this review, sex-specific differences in alveolar epithelial functions are discussed with special focus on preterm infants and the respiratory disorders associated with premature birth. First, a short overview about fetal lung development, the challenges the lung faces during perinatal lung transition to air breathing and respiratory distress in preterm infants is given. Next, clinical observations concerning sex-specific differences in pulmonary morbidity of human preterm infants are noted. The second part discusses potential sex-specific causes of pulmonary complications, including pulmonary steroid receptors and local lung steroid metabolism. With regard to pulmonary steroid metabolism, it is important to highlight which steroidogenic enzymes are expressed at which stage during fetal lung development. Thereafter, we review the knowledge concerning sex-specific aspects of lung growth and maturation. Special focus is given to alveolar epithelial Na+ transport as a driver of perinatal lung transition and the sex differences that were noted in this process
Rapid elevation of sodium transport through insulin is mediated by AKT in alveolar cells
Abstract Alveolar fluid clearance is driven by vectorial Na + transport and promotes postnatal lung adaptation. The effect of insulin on alveolar epithelial Na + transport was studied in isolated alveolar cells from 18-19-day gestational age rat fetuses. Equivalent short-circuit currents (I SC ) were measured in Ussing chambers and different kinase inhibitors were used to determine the pathway of insulin stimulation. In Western Blot measurements the activation of mediators stimulated by insulin was analyzed. The I SC showed a fast dose-dependent increase by insulin, which could be attributed to an increased ENaC (epithelial Na + channel) activity in experiments with permeabilized apical or basolateral membrane. 5-(N-Ethyl-N-isopropyl)amiloride inhibition of I SC was not affected, however, benzamil-sensitive I SC was increased in insulin-stimulated monolayers. The application of LY-294002 and Akti1/2 both completely blocked the stimulating effect of insulin on I SC . PP242 partly blocked the effect of insulin, whereas Rapamycin evoked no inhibition. Western Blot measurements revealed an increased phosphorylation of AKT after insulin stimulation. SGK1 activity was also increased by insulin as shown by Western Blot of pNDRG1. However, in Ussing chamber measurements, GSK650394, an inhibitor of SGK1 did not prevent the increase in I SC induced by insulin. The application of IGF-1 mimicked the effect of insulin and increased the ENaC activity. In addition, an increased autophosphorylation of the IGF-1R/IR was observed after insulin stimulation. We conclude that insulin rapidly increases epithelial Na + transport by enhancing the activity of endogenous ENaC through activation of PI3K/AKT in alveolar cells
Glucocorticoids distinctively modulate the CFTR channel with possible implications in lung development and transition into extrauterine life: Glucocorticoids distinctively modulate the CFTR channel with possible implications in lung development and transition intoextrauterine life
During fetal development, the lung is filled with fluid that is secreted by an active Cltransport promoting lung growth. The basolateral Na+,K+,2Cl- cotransporter (NKCC1) participates in Cl- secretion. The apical Cl- channels responsible for secretion are unknown but studies suggest an involvement of the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR is developmentally regulated with a high expression in early fetal development and a decline in late gestation. Perinatal lung transition is triggered by hormones that
stimulate alveolar Na+ channels resulting in fluid absorption. Little is known on how hormones affect pulmonary Cl- channels. Since the rise of fetal cortisol levels correlates with the decrease in fetal CFTR expression, a causal relation may be assumed. The aim of this
study was to analyze the influence of glucocorticoids on pulmonary Cl- channels. Alveolar cells from fetal and adult rats, A549 cells, bronchial Calu-3 and 16HBE14o- cells, and primary rat airway cells were studied with real-time quantitative PCR and Ussing chambers. In fetal and adult alveolar cells, glucocorticoids strongly reduced Cftr expression and channel activity, which was prevented by mifepristone. In bronchial and primary airway cells CFTR mRNA expression was also reduced, whereas channel activity was increased which was prevented by LY-294002 in Calu-3 cells. Therefore, glucocorticoids strongly reduce CFTR expression while their effect on CFTR activity depends on the physiological function of the cells. Another apical Cl- channel, anoctamin 1 showed a glucocorticoid-induced reduction of mRNA expression in alveolar cells and an increase in bronchial cells. Furthermore, voltage-gated chloride channel 5 and anoctamine 6 mRNA expression were increased in alveolar cells. NKCC1 expression was reduced by glucocorticoids in alveolar and bronchial cells alike. The results demonstrate that glucocorticoids differentially modulate pulmonary Clchannels and are likely causing the decline of CFTR during late gestation in preparation for
perinatal lung transition
Glucocorticoids Distinctively Modulate the CFTR Channel with Possible Implications in Lung Development and Transition into Extrauterine Life.
During fetal development, the lung is filled with fluid that is secreted by an active Cl- transport promoting lung growth. The basolateral Na+,K+,2Cl- cotransporter (NKCC1) participates in Cl- secretion. The apical Cl- channels responsible for secretion are unknown but studies suggest an involvement of the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR is developmentally regulated with a high expression in early fetal development and a decline in late gestation. Perinatal lung transition is triggered by hormones that stimulate alveolar Na+ channels resulting in fluid absorption. Little is known on how hormones affect pulmonary Cl- channels. Since the rise of fetal cortisol levels correlates with the decrease in fetal CFTR expression, a causal relation may be assumed. The aim of this study was to analyze the influence of glucocorticoids on pulmonary Cl- channels. Alveolar cells from fetal and adult rats, A549 cells, bronchial Calu-3 and 16HBE14o- cells, and primary rat airway cells were studied with real-time quantitative PCR and Ussing chambers. In fetal and adult alveolar cells, glucocorticoids strongly reduced Cftr expression and channel activity, which was prevented by mifepristone. In bronchial and primary airway cells CFTR mRNA expression was also reduced, whereas channel activity was increased which was prevented by LY-294002 in Calu-3 cells. Therefore, glucocorticoids strongly reduce CFTR expression while their effect on CFTR activity depends on the physiological function of the cells. Another apical Cl- channel, anoctamin 1 showed a glucocorticoid-induced reduction of mRNA expression in alveolar cells and an increase in bronchial cells. Furthermore, voltage-gated chloride channel 5 and anoctamine 6 mRNA expression were increased in alveolar cells. NKCC1 expression was reduced by glucocorticoids in alveolar and bronchial cells alike. The results demonstrate that glucocorticoids differentially modulate pulmonary Cl- channels and are likely causing the decline of CFTR during late gestation in preparation for perinatal lung transition
Signaling Cascade Involved in Rapid Stimulation of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) by Dexamethasone
Impairment of mucociliary clearance with reduced airway fluid secretion leads to
chronically inflamed airways. Cystic fibrosis transmembrane conductance regulator (CFTR) is
crucially involved in airway fluid secretion and dexamethasone (dexa) has previously been shown to
elevate CFTR activity in airway epithelial cells. However, the pathway by which dexa increases CFTR
activity is largely unknown. We aimed to determine whether the increase of CFTR activity by dexa
is achieved by non-genomic signaling and hypothesized that the phosphoinositide 3-kinase (PI3K)
pathway is involved in CFTR stimulation. Primary rat airway epithelial cells and human bronchial
submucosal gland-derived Calu-3 cells were analyzed in Ussing chambers and kinase activation was
determined byWestern blots. Results demonstrated a critical involvement of PI3K and protein kinase
B (AKT) signaling in the dexa-induced increase of CFTR activity, while serum and glucocorticoid
dependent kinase 1 (SGK1) activity was not essential. We further demonstrated a reduced neural
precursor cell expressed, developmentally downregulated 4-like (NEDD4L) ubiquitin E3 ligase
activity induced by dexa, possibly responsible for the elevated CFTR activity. Finally, increases of
CFTR activity by dexa were demonstrated within 30 min accompanied by rapid activation of AKT.
In conclusion, dexa induces a rapid stimulation of CFTR activity which depends on PI3K/AKT
signaling in airway epithelial cells. Glucocorticoids might thus represent, in addition to their
immunomodulatory actions, a therapeutic strategy to rapidly increase airway fluid secretion
Signaling Cascade Involved in Rapid Stimulation of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) by Dexamethasone
Impairment of mucociliary clearance with reduced airway fluid secretion leads to
chronically inflamed airways. Cystic fibrosis transmembrane conductance regulator (CFTR) is
crucially involved in airway fluid secretion and dexamethasone (dexa) has previously been shown to
elevate CFTR activity in airway epithelial cells. However, the pathway by which dexa increases CFTR
activity is largely unknown. We aimed to determine whether the increase of CFTR activity by dexa
is achieved by non-genomic signaling and hypothesized that the phosphoinositide 3-kinase (PI3K)
pathway is involved in CFTR stimulation. Primary rat airway epithelial cells and human bronchial
submucosal gland-derived Calu-3 cells were analyzed in Ussing chambers and kinase activation was
determined byWestern blots. Results demonstrated a critical involvement of PI3K and protein kinase
B (AKT) signaling in the dexa-induced increase of CFTR activity, while serum and glucocorticoid
dependent kinase 1 (SGK1) activity was not essential. We further demonstrated a reduced neural
precursor cell expressed, developmentally downregulated 4-like (NEDD4L) ubiquitin E3 ligase
activity induced by dexa, possibly responsible for the elevated CFTR activity. Finally, increases of
CFTR activity by dexa were demonstrated within 30 min accompanied by rapid activation of AKT.
In conclusion, dexa induces a rapid stimulation of CFTR activity which depends on PI3K/AKT
signaling in airway epithelial cells. Glucocorticoids might thus represent, in addition to their
immunomodulatory actions, a therapeutic strategy to rapidly increase airway fluid secretion
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