Regulation of channel gating by AMP-activated protein kinase modulates cystic fibrosis transmembrane conductance regulator activity in lung submucosal cells

Cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel activity is important for fluid and electrolyte transport in many epithelia including the lung, the site of most cystic fibrosis-associated morbidity. CFTR is unique among ion channels in requiring ATP hydrolysis for its gating, suggesting that its activity is coupled to cellular metabolic status. The metabolic sensor AMP-activated kinase (AMPK) binds to and phosphorylates CFTR, co-localizes with it in various tissues, and inhibits CFTR currents in Xenopus oocytes (Hallows, K. R., Raghuram, V., Kemp, B. E., Witters, L. A. & Foskett, J. K. (2000) J. Clin. Invest. 105, 1711-1721). Here we demonstrate that this AMPK-CFTR interaction has functional implications in human lung epithelial cells. Pharmacologic activation of AMPK inhibited forskolin-stimulated CFTR short circuit currents in polarized Calu-3 cell monolayers. In whole-cell patch clamp experiments, the activation of endogenous AMPK either pharmacologically or by the overexpression of an AMPK-activating non-catalytic subunit mutant (AMPK-gamma1-R70Q) dramatically inhibited forskolin-stimulated CFTR conductance in Calu-3 and CFTR-expressing Chinese hamster ovary cells. Plasma membrane expression of CFTR, assessed by surface biotinylation, was not affected by AMPK activation. In contrast, the single channel open probability of CFTR was strongly reduced in cell-attached patch clamp measurements of Calu-3 cells transfected with the AMPK-activating mutant, an effect due primarily to a substantial prolongation of the mean closed time of the channel. As a metabolic sensor in cells, AMPK may be important in tuning CFTR activity to cellular energy charge, thereby linking transepithelial transport and the maintenance of cellular ion gradients to cellular metabolism.     

cAMP regulation of Cl(-) and HCO(-)(3) secretion across rat fetal distal lung epithelial cells

We isolated and cultured fetal distal lung epithelial (FDLE) cells from 17- to 19-day rat fetuses and assayed for anion secretion in Ussing chambers. With symmetrical Ringer solutions, basal short-circuit currents (I(sc)) and transepithelial resistances were 7.9 +/- 0.5 microA/cm(2) and 1,018 +/- 73 Omega.cm(2), respectively (means +/- SE; n = 12). Apical amiloride (10 microM) inhibited basal I(sc) by approximately 50%. Subsequent addition of forskolin (10 microM) increased I(sc) from 3.9 +/- 0.63 microA/cm(2) to 7.51 +/- 0.2 microA/cm(2) (n = 12). Basolateral bumetanide (100 microM) decreased forskolin-stimulated I(sc) from 7.51 +/- 0.2 microA/cm(2) to 5.62 +/- 0.53, whereas basolateral 4,4'-dinitrostilbene-2,2'-disulfonate (5 mM), an inhibitor of HCO secretion, blocked the remaining I(sc). Forskolin addition evoked currents of similar fractional magnitudes in symmetrical Cl(-)- or HCO(-)(3)-free solutions; however, no response was seen using HCO(-)(3)- and Cl(-)-free solutions. The forskolin-stimulated I(sc) was inhibited by glibenclamide but not apical DIDS. Glibenclamide also blocked forskolin-induced I(sc) across monolayers having nystatin-permeablized basolateral membranes. Immunolocalization studies were consistent with the expression of cystic fibrosis transmembrane conductance regulator (CFTR) protein in FDLE cells. In aggregate, these findings indicate the presence of cAMP-activated Cl(-) and HCO(-)(3) secretion across rat FDLE cells mediated via CFTR.     

Sphingosine-1-Phosphate Is a Novel Regulator of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Activity

The cystic fibrosis transmembrane conductance regulator (CFTR) attenuates sphingosine-1-phosphate (S1P) signaling in resistance arteries and has emerged as a prominent regulator of myogenic vasoconstriction. This investigation demonstrates that S1P inhibits CFTR activity via adenosine monophosphate-activated kinase (AMPK), establishing a potential feedback link. In Baby Hamster Kidney (BHK) cells expressing wild-type human CFTR, S1P (1μmol/L) attenuates forskolin-stimulated, CFTR-dependent iodide efflux. S1P’s inhibitory effect is rapid (within 30 seconds), transient and correlates with CFTR serine residue 737 (S737) phosphorylation. Both S1P receptor antagonism (4μmol/L VPC 23019) and AMPK inhibition (80μmol/L Compound C or AMPK siRNA) attenuate S1P-stimluated (i) AMPK phosphorylation, (ii) CFTR S737 phosphorylation and (iii) CFTR activity inhibition. In BHK cells expressing the ΔF508 CFTR mutant (CFTR^ΔF508), the most common mutation causing cystic fibrosis, both S1P receptor antagonism and AMPK inhibition enhance CFTR activity, without instigating discernable correction. In summary, we demonstrate that S1P/AMPK signaling transiently attenuates CFTR activity. Since our previous work positions CFTR as a negative S1P signaling regulator, this signaling link may positively reinforce S1P signals. This discovery has clinical ramifications for the treatment of disease states associated with enhanced S1P signaling and/or deficient CFTR activity (e.g. cystic fibrosis, heart failure). S1P receptor/AMPK inhibition could synergistically enhance the efficacy of therapeutic strategies aiming to correct aberrant CFTR trafficking.     

Rab11b Regulates the Apical Recycling of the Cystic Fibrosis Transmembrane Conductance Regulator in Polarized Intestinal Epithelial Cells

The cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP/PKA-activated anion channel, undergoes efficient apical recycling in polarized epithelia. The regulatory mechanisms underlying CFTR recycling are understood poorly, yet this process is required for proper channel copy number at the apical membrane, and it is defective in the common CFTR mutant, ΔF508. Herein, we investigated the function of Rab11 isoforms in regulating CFTR trafficking in T84 cells, a colonic epithelial line that expresses CFTR endogenously. Western blotting of immunoisolated Rab11a or Rab11b vesicles revealed localization of endogenous CFTR within both compartments. CFTR function assays performed on T84 cells expressing the Rab11a or Rab11b GDP-locked S25N mutants demonstrated that only the Rab11b mutant inhibited 80% of the cAMP-activated halide efflux and that only the constitutively active Rab11b-Q70L increased the rate constant for stimulated halide efflux. Similarly, RNAi knockdown of Rab11b, but not Rab11a, reduced by 50% the CFTR-mediated anion conductance response. In polarized T84 monolayers, adenoviral expression of Rab11b-S25N resulted in a 70% inhibition of forskolin-stimulated transepithelial anion secretion and a 50% decrease in apical membrane CFTR as assessed by cell surface biotinylation. Biotin protection assays revealed a robust inhibition of CFTR recycling in polarized T84 cells expressing Rab11b-S25N, demonstrating the selective requirement for the Rab11b isoform. This is the first report detailing apical CFTR recycling in a native expression system and to demonstrate that Rab11b regulates apical recycling in polarized epithelial cells.     

Na+/H+ exchanger regulatory factor isoform 1 overexpression modulates cystic fibrosis transmembrane conductance regulator (CFTR) expression and activity in human airway 16HBE14o- cells and rescues DeltaF508 CFTR functional expression in cystic fibrosis cells

There is evidence that cystic fibrosis transmembrane conductance regulator (CFTR) interacting proteins play critical roles in the proper expression and function of CFTR. The Na(+)/H(+) exchanger regulatory factor isoform 1 (NHERF1) was the first identified CFTR-binding protein. Here we further clarify the role of NHERF1 in the regulation of CFTR activity in two human bronchial epithelial cell lines: the normal, 16HBE14o-, and the homozygous DeltaF508 CFTR, CFBE41o-. Confocal analysis in polarized cell monolayers demonstrated that NHERF1 distribution was associated with the apical membrane in 16HBE14o- cells while being primarily cytoplasmic in CFBE41o- cells. Transfection of 16HBE14o- monolayers with vectors encoding for wild-type (wt) NHERF1 increased both apical CFTR expression and apical protein kinase A (PKA)-dependent CFTR-mediated chloride efflux, whereas transfection with NHERF1 mutated in the binding groove of the PDZ domains or truncated for the ERM domain inhibited both the apical CFTR expression and the CFTR-dependent chloride efflux. These data led us to hypothesize an important role for NHERF1 in regulating CFTR localization and stability on the apical membrane of 16HBE14o- cell monolayers. Importantly, wt NHERF1 overexpression in confluent DeltaF508 CFBE41o- and DeltaF508 CFT1-C2 cell monolayers induced both a significant redistribution of CFTR from the cytoplasm to the apical membrane and a PKA-dependent activation of CFTR-dependent chloride secretion.     

Potentiation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl- currents by the chemical solvent tetrahydrofuran

The chemical solvent tetrahydrofuran (THF) increases short-circuit current (I(sc)) in renal epithelia endogenously expressing the cystic fibrosis transmembrane conductance regulator (CFTR). To understand how THF increases I(sc), we employed the Ussing chamber and patch-clamp techniques to study cells expressing recombinant human CFTR. THF increased I(sc) in Fischer rat thyroid (FRT) epithelia expressing wild-type CFTR with half-maximal effective concentration (K(D)) of 134 mM. This THF-induced increase in I(sc) was enhanced by forskolin (10 microM), inhibited by the PKA inhibitor H-89 (10 microM) and the thiazolidinone CFTR(inh)-172 (10 microM) and attenuated greatly in FRT epithelia expressing the cystic fibrosis mutants F508del- and G551D-CFTR. By contrast, THF (100 mM) was without effect on untransfected FRT epithelia, while other solvents failed to increase I(sc) in FRT epithelia expressing wild-type CFTR. In excised inside-out membrane patches, THF (100 mM) potentiated CFTR Cl(-) channels open in the presence of ATP (1 mM) alone by increasing the frequency of channel openings without altering their duration. However, following the phosphorylation of CFTR by PKA (75 nM), THF (100 mM) did not potentiate channel activity. Similar results were obtained with the triangle upR-S660A-CFTR Cl(-) channel that is not regulated by PKA-dependent phosphorylation and using 2'deoxy-ATP, which gates wild-type CFTR more effectively than ATP. Our data suggest that THF acts directly on CFTR to potentiate channel gating, but that its efficacy is weak and dependent on the phosphorylation status of CFTR.     

Role of Binding and Nucleoside Diphosphate Kinase A in the Regulation of the Cystic Fibrosis Transmembrane Conductance Regulator by AMP-activated Protein Kinase

Cystic fibrosis transmembrane conductance regulator (CFTR) Cl⁻ channel mutations cause cystic fibrosis lung disease. A better understanding of CFTR regulatory mechanisms could suggest new therapeutic strategies. AMP-activated protein kinase (AMPK) binds to and phosphorylates CFTR, attenuating PKA-activated CFTR gating. However, the requirement for AMPK binding to CFTR and the potential role of other proteins in this regulation are unclear. We report that nucleoside diphosphate kinase A (NDPK-A) interacts with both AMPK and CFTR in overlay blots of airway epithelial cell lysates. Binding studies in Xenopus oocytes and transfected HEK-293 cells revealed that a CFTR peptide fragment that binds AMPK (CFTR-1420-57) disrupted the AMPK-CFTR interaction. Introduction of CFTR-1420-57 into human bronchial Calu-3 cells enhanced forskolin-stimulated whole cell conductance in patch clamp measurements. Similarly, injection of CFTR-1420-57 into Xenopus oocytes blocked the inhibition of cAMP-stimulated CFTR conductance by AMPK in two-electrode voltage clamp studies. AMPK also inhibited CFTR conductance with co-expression of WT NDPK-A in two-electrode voltage clamp studies, but co-expression of a catalytically inactive H118F mutant or various Ser-120 NDPK-A mutants prevented this inhibition. In vitro phosphorylation of WT NDPK-A was enhanced by purified active AMPK, but phosphorylation was prevented in H118F and phosphomimic Ser-120 NDPK-A mutants. AMPK does not appear to phosphorylate NDPK-A directly but rather promotes an NDPK-A autophosphorylation event that involves His-118 and Ser-120. Taken together, these results suggest that NDPK-A exists in a functional cellular complex with AMPK and CFTR in airway epithelia, and NDPK-A catalytic function is required for the AMPK-dependent regulation of CFTR.     

Activation of AMP-activated protein kinase reduces cAMP-mediated epithelial chloride secretion

AMP-activated protein kinase (AMPK) is activated in response to fluctuations in cellular energy status caused by oxidative stress. One of its targets is the cystic fibrosis transmembrane conductance regulator (CFTR), which is the predominant Cl- secretory channel in colonic tissue. The aim of this study was to determine the role of AMPK in the modulation of colonic chloride secretion under conditions of oxidative stress and chronic inflammation. Chloride secretion and AMPK activity were examined in colonic tissue from adult IL-10-deficient and wild-type 129 Sv/Ev mice in the presence and absence of pharmacological AMPK inhibitors and activators, respectively. Apical levels of CFTR were measured in brush-border membrane vesicles. Cell culture studies in human colonic T84 monolayers examined the effect of hydrogen peroxide and pharmacological activation of AMPK on forskolin-stimulated chloride secretion. Inflamed colons from IL-10-deficient mice exhibited hyporesponsiveness to forskolin stimulation in association with reductions in surface CFTR expression and increased AMPK activity. Inhibition of AMPK restored tissue responsiveness to forskolin, whereas stimulation of AMPK with 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR) induced tissue hyporesponsivness in wild-type mice. T84 cells exposed to hydrogen peroxide demonstrated a time-dependent increase in AMPK activity and reduction of forskolin-stimulated chloride secretion. Inhibition of AMPK prevented the reduction in chloride secretion. Treatment of cells with the AMPK activator, AICAR, resulted in a decreased chloride secretion. In conclusion, AMPK activation is linked with reductions in cAMP-mediated epithelial chloride flux and may be a contributing factor to the hyporesponsiveness seen under conditions of chronic inflammation.     

AICAR decreases the activity of two distinct amiloride-sensitive Na+-permeable channels in H441 human lung epithelial cell monolayers

Transepithelial transport of Na(+) across the lung epithelium via amiloride-sensitive Na(+) channels (ENaC) regulates fluid volume in the lung lumen. Activators of AMP-activated protein kinase (AMPK), the adenosine monophosphate mimetic AICAR, and the biguanide metformin decreased amiloride-sensitive apical Na(+) conductance (G(Na+)) in human H441 airway epithelial cell monolayers. Cell-attached patch-clamp recordings identified two distinct constitutively active cation channels in the apical membrane that were likely to contribute to G(Na+): a 5-pS highly Na(+) selective ENaC-like channel (HSC) and an 18-pS nonselective cation channel (NSC). Substituting NaCl with NMDG-Cl in the patch pipette solution shifted the reversal potentials of HSC and NSC, respectively, from +23 mV to -38 mV and 0 mV to -35 mV. Amiloride at 1 microM inhibited HSC activity and 56% of short-circuit current (I(sc)), whereas 10 microM amiloride partially reduced NSC activity and inhibited a further 30% of I(sc). Neither conductance was associated with CNG channels as there was no effect of 10 microM pimoside on I(sc), HSC, or NSC activity, and 8-bromo-cGMP (0.3-0.1 mM) did not induce or increase HSC or NSC activity. Pretreatment of H441 monolayers with 2 mM AICAR inhibited HSC/NSC activity by 90%, and this effect was reversed by the AMPK inhibitor Compound C. All three ENaC proteins were identified in the apical membrane of H441 monolayers, but no change in their abundance was detected after treatment with AICAR. In conclusion, activation of AMPK with AICAR in H441 cell monolayers is associated with inhibition of two distinct amiloride-sensitive Na(+)-permeable channels by a mechanism that likely reduces channel open probability.     

Basolateral chloride current in human airway epithelia

Electrolyte transport by airway epithelia regulates the quantity and composition of liquid covering the airways. Previous data indicate that airway epithelia can absorb NaCl. At the apical membrane, cystic fibrosis transmembrane conductance regulator (CFTR) provides a pathway for Cl(-) absorption. However, the pathways for basolateral Cl(-) exit are not well understood. Earlier studies, predominantly in cell lines, have reported that the basolateral membrane contains a Cl(-) conductance. However, the properties have varied substantially in different epithelia. To better understand the basolateral Cl(-) conductance in airway epithelia, we studied primary cultures of well-differentiated human airway epithelia. The basolateral membrane contained a Cl(-) current that was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). The current-voltage relationship was nearly linear, and the halide selectivity was Cl(-) > Br(-) > I(-). Several signaling pathways increased the current, including elevation of cellular levels of cAMP, activation of protein kinase C (PKC), and reduction of pH. In contrast, increasing cell Ca(2+) and inducing cell swelling had no effect. The basolateral Cl(-) current was present in both cystic fibrosis (CF) and non-CF airway epithelia. Likewise, airway epithelia from wild-type mice and mice with disrupted genes for ClC-2 or ClC-3 all showed similar Cl(-) currents. These data suggest that the basolateral membrane of airway epithelia possesses a Cl(-) conductance that is not due to CFTR, ClC-2, or ClC-3. Its regulation by cAMP and PKC signaling pathways suggests that coordinated regulation of Cl(-) conductance in both apical and basolateral membranes may be important in controlling transepithelial Cl(-) movement.     

Flagellin-stimulated Cl- secretion and innate immune responses in airway epithelia: role for p38

Activation of an innate immune response in airway epithelia by the human pathogen Pseudomonas aeruginosa requires bacterial expression of flagellin. Addition of flagellin (10(-7) M) to airway epithelial cell monolayers (Calu-3, airway serous cell-like) increased Cl(-) secretion (I(Cl)) beginning after 3-10 min, reaching a plateau after 20-45 min at DeltaI(Cl) = 15-50 microA/cm(2). Similar, although 10-fold smaller, responses were observed in well-differentiated bronchial epithelial cultures. Flagellin stimulated I(Cl) in the presence of maximally stimulating doses of the purinergic agonist ATP, but had no effects following forskolin. IL-1beta (produced by both epithelia and neutrophils during infections) stimulated I(Cl) similar to flagellin. Flagellin-, IL-1beta-, ATP-, and forskolin-stimulated I(Cl) were inhibited by cystic fibrosis transmembrane conductance regulator (CFTR) blockers GlyH101, CFTRinh172, and glibenclamide. Neither flagellin nor IL-1beta altered transepithelial fluxes of membrane-impermeant dextran (10 kDa) or lucifer yellow (mol wt = 457), but both activated p38, NF-kappaB, and IL-8 secretion. Blockers of p38 (SB-202190 and SB-203580) reduced flagellin- and IL-1beta-stimulated I(Cl) by 33-50% but had smaller effects on IL-8 and NF-kappaB. It is concluded that: 1) flagellin and IL-1beta activated p38, NF-kappaB, IL-8, and CFTR-dependent anion secretion without altering tight junction permeability; 2) p38 played a role in regulating I(Cl) and IL-8 but not NF-kappaB; and 3) p38 was more important in flagellin- than IL-1beta-stimulated responses. During P. aeruginosa infections, flagellin and IL-1beta are expected to increase CFTR-dependent ion and fluid flow into and bacterial clearance from the airways. In cystic fibrosis, the secretory response would be absent, but activation of p38, NF-kappaB, and IL-8 would persist.     

The cystic fibrosis transmembrane conductance regulator activates aquaporin 3 in airway epithelial cells

Enhanced osmotic water permeability has been observed in Xenopus oocytes expressing cystic fibrosis transmembrane conductance regulator (CFTR) protein. Subsequent studies have shown that CFTR activates an endogenous water permeability in oocytes, but that CFTR itself is not the water channel. Here, we show CFTR-dependent activation of endogenous water permeability in normal but not in cystic fibrosis human airway epithelial cells. Cell volume was measured by novel confocal x-z laser scanning microscopy. Glycerol uptake and antisense studies suggest CFTR-dependent regulation of aquaporin 3 (AQP3) water channels in airway epithelial cells. Regulatory interaction was confirmed by coexpression of CFTR and AQP3 cloned from human airways in Xenopus oocytes and of CFTR and rat AQP3 in Chinese hamster ovary cells. These findings indicate that CFTR is a regulator of AQP3 in airway epithelial cells.     

Improved oxygenation promotes CFTR maturation and trafficking in MDCK monolayers

Culturing airway epithelial cells with most of the apical media removed (air-liquid interface) has been shown to enhance cystic fibrosis transmembrane conductance regulator (CFTR)-mediated Cl(-) secretory current. Thus we hypothesized that cellular oxygenation may modulate CFTR expression. We tested this notion using type I Madin-Darby canine kidney cells that endogenously express low levels of CFTR. Growing monolayers of these cells for 4 to 5 days with an air-liquid interface caused a 50-fold increase in forskolin-stimulated Cl(-) current, compared with conventional (submerged) controls. Assaying for possible changes in CFTR by immunoprecipitation and immunocytochemical localization revealed that CFTR appeared as an immature 140-kDa form intracellularly in conventional cultures. In contrast, monolayers grown with an air-liquid interface possessed more CFTR protein, accompanied by increases toward the mature 170-kDa form and apical membrane staining. Culturing submerged monolayers with 95% O(2) produced similar improvements in Cl(-) current and CFTR protein as air-liquid interface culture, while increasing PO(2) from 2.5% to 20% in air-liquid interface cultures yielded graded enhancements. Together, our data indicate that improved cellular oxygenation can increase endogenous CFTR maturation and/or trafficking.     

Elevated Paracellular Glucose Flux across Cystic Fibrosis Airway Epithelial Monolayers Is an Important Factor for Pseudomonas aeruginosa Growth

People with cystic fibrosis (CF) who develop related diabetes (CFRD) have accelerated pulmonary decline, increased infection with antibiotic-resistant Pseudomonas aeruginosa and increased pulmonary exacerbations. We have previously shown that glucose concentrations are elevated in airway surface liquid (ASL) of people with CF, particularly in those with CFRD. We therefore explored the hypotheses that glucose homeostasis is altered in CF airway epithelia and that elevation of glucose flux into ASL drives increased bacterial growth, with an effect over and above other cystic fibrosis transmembrane conductance regulator (CFTR)-related ASL abnormalities. The aim of this study was to compare the mechanisms governing airway glucose homeostasis in CF and non-CF primary human bronchial epithelial (HBE) monolayers, under normal conditions and in the presence of Ps. aeruginosa filtrate. HBE-bacterial co-cultures were performed in the presence of 5 mM or 15 mM basolateral glucose to investigate how changes in blood glucose, such as those seen in CFRD, affects luminal Ps. aeruginosa growth. Calu-3 cell monolayers were used to evaluate the potential importance of glucose on Ps. aeruginosa growth, in comparison to other hallmarks of the CF ASL, namely mucus hyperviscosity and impaired CFTR-dependent fluid secretions. We show that elevation of basolateral glucose promotes the apical growth of Ps. aeruginosa on CF airway epithelial monolayers more than non-CF monolayers. Ps. aeruginosa secretions elicited more glucose flux across CF airway epithelial monolayers compared to non-CF monolayers which we propose increases glucose availability in ASL for bacterial growth. In addition, elevating basolateral glucose increased Ps. aeruginosa growth over and above any CFTR-dependent effects and the presence or absence of mucus in Calu-3 airway epithelia-bacteria co-cultures. Together these studies highlight the importance of glucose as an additional factor in promoting Ps. aeruginosa growth and respiratory infection in CF disease.     

Up-regulation of AMP-activated kinase by dysfunctional cystic fibrosis transmembrane conductance regulator in cystic fibrosis airway epithelial cells mitigates excessive inflammation

AMP-activated kinase (AMPK) is a ubiquitous metabolic sensor that inhibits the cystic fibrosis (CF) transmembrane conductance regulator (CFTR). To determine whether CFTR reciprocally regulates AMPK function in airway epithelia and whether such regulation is involved in lung inflammation, AMPK localization, expression, and activity and cellular metabolic profiles were compared as a function of CFTR status in CF and non-CF primary human bronchial epithelial (HBE) cells. As compared with non-CF HBE cells, CF cells had greater and more diffuse AMPK staining and had greater AMPK activity than their morphologically matched non-CF counterparts. The cellular [AMP]/[ATP] ratio was higher in undifferentiated than in differentiated non-CF cells, which correlated with AMPK activity under these conditions. However, this nucleotide ratio did not predict AMPK activity in differentiating CF cells. Inhibiting channel activity in non-CF cells did not affect AMPK activity or metabolic status, but expressing functional CFTR in CF cells reduced AMPK activity without affecting cellular [AMP]/[ATP]. Therefore, lack of functional CFTR expression and not loss of channel activity in CF cells appears to up-regulate AMPK activity in CF HBE cells, presumably through non-metabolic effects on upstream regulatory pathways. Compared with wild-type CFTR-expressing immortalized CF bronchial epithelial (CFBE) cells, DeltaF508-CFTR-expressing CFBE cells had greater AMPK activity and greater secretion of tumor necrosis factor-alpha and the interleukins IL-6 and IL-8. Further pharmacologic AMPK activation inhibited inflammatory mediator secretion in both wild type- and DeltaF508-expressing cells, suggesting that AMPK activation in CF airway cells is an adaptive response that reduces inflammation. We propose that therapies to activate AMPK in the CF airway may be beneficial in reducing excessive airway inflammation, a major cause of CF morbidity.     

Na+/H+ Exchanger Regulatory Factor 1 Overexpression-dependent Increase of Cytoskeleton Organization Is Fundamental in the Rescue of F508del Cystic Fibrosis Transmembrane Conductance Regulator in Human Airway CFBE41o- Cells

We have demonstrated that Na⁺/H⁺ exchanger regulatory factor 1 (NHERF1) overexpression in CFBE41o- cells induces a significant redistribution of F508del cystic fibrosis transmembrane conductance regulator (CFTR) from the cytoplasm to the apical membrane and rescues CFTR-dependent chloride secretion. Here, we observe that CFBE41o- monolayers displayed substantial disassembly of actin filaments and that overexpression of wild-type (wt) NHERF1 but not NHERF1-Δ Ezrin-Radixin-Moesin (ERM) increased F-actin assembly and organization. Furthermore, the dominant-negative band Four-point one, Ezrin, Radixin, Moesin homology (FERM) domain of ezrin reversed the wt NHERF1 overexpression-induced increase in both F-actin and CFTR-dependent chloride secretion. wt NHERF1 overexpression enhanced the interaction between NHERF1 and both CFTR and ezrin and between ezrin and actin and the overexpression of wt NHERF1, but not NHERF1-ΔERM, also increased the phosphorylation of ezrin in the apical region of the cell monolayers. Furthermore, wt NHERF1 increased RhoA activity and transfection of constitutively active RhoA in CFBE41o- cells was sufficient to redistribute phospho-ezrin to the membrane fraction and rescue both the F-actin content and the CFTR-dependent chloride efflux. Rho kinase (ROCK) inhibition, in contrast, reversed the wt NHERF1 overexpression-induced increase of membrane phospho-ezrin, F-actin content, and CFTR-dependent secretion. We conclude that NHERF1 overexpression in CFBE41o- rescues CFTR-dependent chloride secretion by forming the multiprotein complex RhoA-ROCK-ezrin-actin that, via actin cytoskeleton reorganization, tethers F508del CFTR to the cytoskeleton stabilizing it on the apical membrane.     

Beta-oestradiol rescues DeltaF508CFTR functional expression in human cystic fibrosis airway CFBE41o- cells through the up-regulation of NHERF1.

BACKGROUND INFORMATION: CF (cystic fibrosis) is a disease caused by mutations within the CFTR (CF transmembrane conductance regulator) gene. The most common mutation, DeltaF508 (deletion of Phe-508), results in a protein that is defective in folding and trafficking to the cell surface but is functional if properly localized in the plasma membrane. We have recently demonstrated that overexpression of the PDZ protein NHERF1 (Na(+)/H(+)-exchanger regulatory factor 1) in CF airway cells induced both a redistribution of DeltaF508CFTR from the cytoplasm to the apical membrane and the PKA (protein kinase A)-dependent activation of DeltaF508CFTR-dependent chloride secretion. In view of the potential importance of the targeted up-regulation of NHERF1 in a therapeutic context, and since it has been demonstrated that oestrogen treatment increases endogenous NHERF1 expression, we tested the hypothesis that oestrogen treatment can increase NHERF1 expression in a human bronchiolar epithelial CF cell line, CFBE41o(-), with subsequent rescue of apical DeltaF508CFTR chloride transport activity. RESULTS: We found that CFBE41o(-) cells do express ERs (oestrogen receptors) in the nuclear fraction and that beta-oestradiol treatment was able to significantly rescue DeltaF508CFTR-dependent chloride secretion in CFBE41o(-) cell monolayers with a peak between 6 and 12 h of treatment, demonstrating that the DeltaF508CFTR translocated to the apical membrane can function as a cAMP-responsive channel, with a significant increase in chloride secretion noted at 1 nM beta-oestradiol and a maximal effect observed at 10 nM. Importantly, knock-down of NHERF1 expression by transfection with siRNA (small interfering RNA) for NHERF1 inhibited the beta-oestradiol-dependent increase in DeltaF508CFTR protein expression levels and completely prevented the beta-oestradiol-dependent rescue of DeltaF508CFTR transport activity. CONCLUSIONS: These results demonstrate that beta-oestradiol-dependent up-regulation of NHERF1 significantly increases DeltaF508CFTR functional expression in CFBE41o(-) cells.     

Differentiation of adult rat bone marrow stem cells into epithelial progenitor cells in culture

We have previously obtained monoclonal bone marrow stem cells from adult rats (rMSCs) and induced them into phenotypic neurons. In the present study, we aimed to induce rMSCs into epithelial cells by culturing them onto compartmentalized permeable supports, which have been used for growing a variety of polarized epithelia in culture. Hematoxylin staining showed that after 4 days grown on permeable supports, rMSCs formed an epithelial-like monolayer. Immunofluorescence of the permeably-supported monolayers, but not the rMSCs grown in culture flasks, showed positive signals for epithelial markers, cytokeratin 5 & 8. RT-PCR results also showed the mRNA expression of epithelial sodium channel (ENaC) and cystic fibrosis transmembrane conductance regulator (CFTR) as well as tight junction protein ZO-1 in the rMSC-derived monolayers grown on permeable supports but absent from those grown in culture flasks. However, western blot only detected protein expression of ZO-1 but not ENaC nor CFTR. The short-circuit current measurements showed that the rMSC-derived monolayers grown on permeable supports exhibited a trans-monolayer resistance of 30-50 Omega cm(2); however, the monolayers did not respond to activators or blockers of CFTR or ENaC. The results suggest that compartmentalized or polarized culture conditions provide a suitable environment for rMSCs to differentiate into epithelial progenitor cells with tight junction formation; however, this condition is not sufficient for functional expression of epithelial ion channels associated with well-differentiated epithelia.