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

Der Einfluss von Glukokortikoiden auf die tracheale Chloridsekretion

Ionenbewegungen über das Atemwegsepithel bilden die Grundlage einer definierten Zusammensetzung intraluminaler pulmonaler Flüssigkeit. Ein perinataler Anstieg maternaler und damit einhergehend fetaler Glukokortikoid (GC)-Serumlevel ist vergesellschaftet mit einer Veränderung im epithelialen Ionentransport der Lunge. So erfährt das respiratorische Epithel in einer perinatalen Anpassungsreaktion eine Transition fetaler Fruchtwasserproduktion hin zu überwiegend flüssigkeitsabsorbierenden Vorgängen. In den distalen Abschnitten spiegelt sich dies in einer Aktivierung flüssigkeitsabsorptiv wirkender epithelialer Natriumkanäle (ENaC) wider. Vor Beginn der Transition überwiegt ein sekretorisch aktiver, apikaler Chloridkanal, der cystic fibrosis transmembrane conductance regulator (CFTR). Als Gegenspieler zum ENaC gewährleistet der CFTR einen transepithelialen Flüssigkeitsstrom in das Lumen des respiratorischen Systems. Diese Prozesse ermöglichen den pulmonalen Gastaustausch sowie die Bereitstellung eines periziliären Flüssigkeitsfilms, welcher die postnatale mukoziliäre Clearance (MCC) gewährleistet. Während bereits gezeigt werden konnte, dass GC den ENaC vor allem in distalen Atemwegsepithelien in Expression und Aktivität steigern (Thome et al. 2003; Venkatesh und Katzberg 1997), gibt es bisher nur wenige Daten zum Einfluss von GC auf den CFTR. Zusammenfassend kann durch die vorliegende Arbeit gezeigt werden, dass es durch GC zu einer Zunahme des CFTR-abhängigen Ionentransportes kommt und dass dieser aktivitätssteigernde Einfluss von der PI3K sowie der AKT abhängig ist, während die SGK1 nicht wesentlich in diesen Wirkmechanismus involviert zu sein scheint. Es ließ sich nachweisen, dass es durch GC zu einer gesteigerten Phosphorylierung von AKT, SGK1 sowie NEDD4L kommt, woraus eine Aktivitätssteigerung der AKT und der SGK1 sowie ein Rückgang in der NEDD4L-Aktivität resultieren. Eine AKT-Inhibition verhindert die zunehmende NEDD4L Phosphorylierung, während eine Inhibition der SGK1 keinen Einfluss auf den NEDD4L-Phosphorylierungsstatus zeigt. Des Weiteren ergibt sich ein stimulierender Einfluss von GC auf die CFTR-Aktivität innerhalb von 30 Minuten. Im Gegensatz zum Ionentransport zeigten GC keinen Einfluss auf den Epithelwiderstand und damit die Barrierefunktion der Zellkultur.:1. Abkürzungsverzeichnis S. 3 2. Abbildungsverzeichnis S. 5 3. Bibliographische Beschreibung S. 6 4. Einleitung S. 7 4.1. Der CFTR und seine Rolle im epithelialen Chloridtransport S. 7 4.2.Die Struktur und Regulation des CFTR S. 8 4.3.Die (Patho-)Physiologie des CFTR S. 10 4.4.Alternative Chloridkanäle S. 11 4.5.Methodik – Etablierung der Methode S. 12 5. Originalpublikation 5.1. “Glucocorticoids Distinctively Modulate the CFTR Channel with Possible Implications in Lung Development and Transition into Extrauterine Life” S. 14 5.2. “Signaling Cascade involved in Rapid Stimulation of Cystic Fibrosis Conductance Regulator (CFTR) by Dexamethasone” S. 36 6. Zusammenfassung der Ergebnisse S. 52 6.1 Hintergrund und Ziel S. 52 6.2 Ergebnisse S. 53 7. Diskussion der Ergebnisse S. 54 8. Literaturverzeichnis S. 57 9. Anmerkungen zur Originalpublikation – Darstellung des eigenen Beitrags S. 64 10. Erklärung über die eigenständige Abfassung der Arbeit S. 65 11. Curriculum vitae S. 66 12. Danksagung S. 6

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 by Western 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

Dexamethasone reduces Cftr mRNA expression in FDLE cells.

<p>Graphs represent the mean + SEM for normalized Cftr mRNA expression acquired by RT-qPCR. <b>A:</b> Dose-response curve of dexamethasone effect (1 nM–1 μM for 24 h, n = 4, ** p<0.01; *** p<0.001, ANOVA with Dunnett's <i>post hoc</i> test compared to control monolayers without dexamethasone addition). <b>B:</b> Time course of Cftr mRNA expression in response to 100 nM dexamethasone for 6, 12 and 24 h (n = 4, * p<0.05; ** p<0.01; *** p<0.001, T-test).</p

Dexamethasone reduces CFTR/Cftr mRNA expression and increases channel activity in airway epithelial cells.

<p><b>A/B:</b> Graphs represent the mean + SEM for normalized CFTR/Cftr mRNA expression in response to 100 nM dexamethasone for 24 h acquired by RT-qPCR. <b>A:</b> 16HBE14o- cells (n = 8, *** p<0.001, T-test compared to control cells without dexamethasone addition). <b>B:</b> Primary rat airway epithelial cells (n = 7–8, *** p<0.001, T-test compared to control cells without dexamethasone addition). <b>C:</b> Typical current tracing of primary airway epithelial cells. D/E: Graphs represent the mean + SEM of I_SC in response to 100 nM dexamethasone for 24 h measured in Ussing chambers. <b>D:</b> Forskolin-induced I_SC (n = 20–23, ** p<0.01, T-test compared to control cells without dexamethasone addition). E: CFTR₁₇₂inh-sensitive I_SC (n = 19–20, ** p<0.01, T-test compared to control cells without dexamethasone addition).</p

Dexamethasone reduces CFTR/Cftr mRNA expression and channel activity in adult alveolar cells.

<p><b>A/B:</b> primary adult ATII cells. <b>A:</b> Graph represents the mean + SEM for normalized Cftr mRNA expression in response to 100 nM dexamethasone for 24 h acquired by RT-qPCR (n = 6, *** p<0.001 by T-test compared to control monolayers without dexamethasone addition). <b>B:</b> Graph represents the mean + SEM of I_SC in response to 100 nM dexamethasone for 24 h measured in Ussing chambers. Forskolin-induced I_SC (n = 29–31, * p<0.05 by T-test compared to control monolayers without dexamethasone addition) and glibenclamide-sensitive I_SC (n = 27–28). <b>C:</b> A549 cells. Graph represents the mean + SEM for normalized CFTR mRNA expression in response to 100 nM dexamethasone for 24 h acquired by RT-qPCR (n = 8, *** p<0.001, T-test compared to control cells without dexamethasone addition).</p