Temporal regulation of CFTR expression during ovine lung development: implications for CF gene therapy.

The cystic fibrosis transmembrane conductance regulator (CFTR) protein is a small conductance chloride ion channel that may interact directly with other channels including the epithelial sodium channel (ENaC). CFTR is known to be more abundant in the airway epithelium during the second trimester of human development than after birth. This could be a consequence of the change in function of the respiratory epithelium from chloride secretion to sodium absorption near term. Alternatively it might reflect an additional role for CFTR in the developing airway epithelium. Though the lung epithelia of CF fetuses and infants rarely show gross histological abnormalities, there is often evidence of inflammation. Our aim was to establish whether CFTR expression levels correlated with specific developmental stages or differentiated functions in the ovine fetal lung. We evaluated CFTR expression using a quantitative assay of mRNA at 14 time points through gestation and showed highest levels at the start of the second trimester followed by a gradual decline through to term. In contrast, ENaC expression increased from the start of the third trimester. These results support a role for CFTR in differentiation of the respiratory epithelium and suggest that its expression levels are not merely reflecting major changes in the sodium/chloride bulk flow close to term. These observations may have significant implications for the likely success of CF gene therapy in the postnatal lung.     

Liquid movement across the surface epithelium of large airways

The cystic fibrosis transmembrane conductance regulator CFTR gene is found on chromosome 7 [Kerem, B., Rommens, J.M., Buchanan, J.A., Markiewicz, D., Cox, T.K., Chakravarti, A., Buchwald, M., Tsui, L.C., 1989. Identification of the cystic fibrosis gene: genetic analysis. Science 245, 1073-1080; Riordan, J.R., Rommens, J.M., Kerem, B., Alon, N., Rozmahel, R., Grzelczak, Z., Zielenski, J., Lok, S., Plavsic, N., Chou, J.L., et al., 1989. Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science 245, 1066-1073] and encodes for a 1480 amino acid protein which is present in the plasma membrane of epithelial cells [Anderson, M.P., Sheppard, D.N., Berger, H.A., Welsh, M.J., 1992. Chloride channels in the apical membrane of normal and cystic fibrosis airway and intestinal epithelia. Am. J. Physiol. 263, L1-L14]. This protein appears to have many functions, but a unifying theme is that it acts as a protein kinase C- and cyclic AMP-regulated Cl(-) channel [Winpenny, J.P., McAlroy, H.L., Gray, M.A., Argent, B.E., 1995. Protein kinase C regulates the magnitude and stability of CFTR currents in pancreatic duct cells. Am. J. Physiol. 268, C823-C828; Jia, Y., Mathews, C.J., Hanrahan, J.W., 1997. Phosphorylation by protein kinase C is required for acute activation of cystic fibrosis transmembrane conductance regulator by protein kinase A. J. Biol. Chem. 272, 4978-4984]. In the superficial epithelium of the conducting airways, CFTR is involved in Cl(-) secretion [Boucher, R.C., 2003. Regulation of airway surface liquid volume by human airway epithelia. Pflugers Arch. 445, 495-498] and also acts as a regulator of the epithelial Na(+) channel (ENaC) and hence Na(+) absorption [Boucher, R.C., Stutts, M.J., Knowles, M.R., Cantley, L., Gatzy, J.T., 1986. Na(+) transport in cystic fibrosis respiratory epithelia. Abnormal basal rate and response to adenylate cyclase activation. J. Clin. Invest. 78, 1245-1252; Stutts, M.J., Canessa, C.M., Olsen, J.C., Hamrick, M., Cohn, J.A., Rossier, B.C., Boucher, R.C., 1995. CFTR as a cAMP-dependent regulator of sodium channels. Science 269, 847-850]. In this chapter, we will discuss the regulation of these two ion channels, and how they can influence liquid movement across the superficial airway epithelium.     

Cellular localization and activity of Ad-delivered GFP-CFTR in airway epithelial and tracheal cells

Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, and the cellular trafficking of the CFTR protein is an essential factor that determines its function in cells. The aim of our study was to develop an Ad vector expressing a biologically active green fluorescent protein (GFP)-CFTR chimera that can be tracked by both its localization and chloride channel function. No study thus far has demonstrated a GFP-CFTR construct that displayed both of these functions in the airway epithelia. Tracheal glandular cells, MM39 (CFTRwt) and CF-KM4 (CFTRDeltaF508), as well as human airway epithelial cells from a patient with cystic fibrosis (CF-HAE) and from a healthy donor (HAE) were used for the functional analysis of our Ad vectors, Ad5/GFP-CFTRwt and Ad5/GFP-CFTRDeltaF508. The GFP-CFTRwt protein expressed was efficiently addressed to the plasma membrane of tracheal cells and to the apical surface of polarized CF-HAE cells, while GFP-CFTRDeltaF508 mutant was sequestered intracellularly. The functionality of the GFP-CFTRwt protein was demonstrated by its capacity to correct the chloride channel activity both in CF-KM4 and CF-HAE cells after Ad transduction. A correlation between the proportion of Ad5-transduced CF-KM4 cells and correction of CFTR function showed that 55 to 70% transduction resulted in 70% correction of the Cl- channel function. In reconstituted CF-HAE, GFP-CFTRwt appeared as active as the nontagged CFTRwt protein in correcting the transepithelial Cl- transport. We show for the first time a GFP-CFTR chimera that localized to the apical surface of human airway epithelia and restored epithelial chloride transport to similar levels as nontagged CFTR.     

Opening up of plasmalemma type-1 VDAC to form apoptotic “find me signal” pathways is essential in early apoptosis — Evidence from the pathogenesis of cystic fibrosis resulting from failure of apoptotic cell clearance followed by sterile inflammation

Cell membrane-standing type-1 VDAC is involved in cell volume regulation and thus apoptosis. The channel has been shown to figure as a pathway for osmolytes of varying classes, ATP included. An early event in apoptotic cell death is the release of “find me signals” by cells that enter the apoptotic process. ATP is one of those signals. Apoptotic cells this way attract phagocytes for an immunologically silent cell clearance. Thus, whenever apoptosis fails by a blockade of plasmalemma type-1 VDAC processes of sterile inflammation must be assumed for cell elimination. This is evident from a close look on the pathogenetic process of cystic fibrosis (CF). However, in normal airway epithelia two different anion channels cooperate to guarantee an appropriate volume of airway surface liquid (ASL) necessary for surface clearing: the cystic fibrosis conductance regulator (CFTR) and the outwardly rectifying chloride channel (ORCC) complex also called “alternate chloride channel” and under the control of the CFTR. There are arguments, that type-1 VDAC forms the channel part of the ORCC complex, and it has been shown that CFTR and type-1 VDAC co-localize in the apical membranes of human surface respiratory epithelium. In cystic fibrosis, the central cAMP-dependent regulation of ion and water transport via functional CFTR is lost. Here, CFTR molecules do not reach the apical membranes of airway epithelia anymore or work in an insufficient way, respectively. In addition, type-1 VDAC is no longer available to work as a “find me signal” pathway. In consequence, clearing away of apoptotic cells is blocked. There are experimental data on the channel characteristics of type-1 VDAC under the anion channel blocker DIDS (4,4-diisothiocyanato-stilbenedisulphonic acid) that argue in favor of this hypothesis. Together, type-1 VDAC should be kept as a “find me signal” pathway, which may give way to several classes of such signals.     

Alveolar epithelial CNGA1 channels mediate cGMP-stimulated, amiloride-insensitive, lung liquid absorption

Impairment of lung liquid absorption can lead to severe respiratory symptoms, such as those observed in pulmonary oedema. In the adult lung, liquid absorption is driven by cation transport through two pathways: a well-established amiloride-sensitive Na(+) channel (ENaC) and, more controversially, an amiloride-insensitive channel that may belong to the cyclic nucleotide-gated (CNG) channel family. Here, we show robust CNGA1 (but not CNGA2 or CNGA3) channel expression principally in rat alveolar type I cells; CNGA3 was expressed in ciliated airway epithelial cells. Using a rat in situ lung liquid clearance assay, CNG channel activation with 1?mM 8Br-cGMP resulted in an approximate 1.8-fold stimulation of lung liquid absorption. There was no stimulation by 8Br-cGMP when applied in the presence of either 100?μM L: -cis-diltiazem or 100?nM pseudechetoxin (PsTx), a specific inhibitor of CNGA1 channels. Channel specificity of PsTx and amiloride was confirmed by patch clamp experiments showing that CNGA1 channels in HEK 293 cells were not inhibited by 100?μM amiloride and that recombinant αβγ-ENaC were not inhibited by 100?nM PsTx. Importantly, 8Br-cGMP stimulated lung liquid absorption in situ, even in the presence of 50?μM amiloride. Furthermore, neither L: -cis-diltiazem nor PsTx affected the β(2)-adrenoceptor agonist-stimulated lung liquid absorption, but, as expected, amiloride completely ablated it. Thus, transport through alveolar CNGA1 channels, located in type I cells, underlies the amiloride-insensitive component of lung liquid reabsorption. Furthermore, our in situ data highlight the potential of CNGA1 as a novel therapeutic target for the treatment of diseases characterised by lung liquid overload.     

Ontogeny of CLCN3 chloride channel gene expression in human pulmonary epithelium

Human fetal bronchopulmonary epithelia secrete liquid, and this chloride (Cl)-dependent process is important for normal lung growth. At the time of birth there is a maturational transition from a secretory to an absorptive phenotype. The pathways for Cl exit from the apical membrane which are required for fetal lung liquid secretion are unknown but are thought to be independent of the cystic fibrosis transmembrane conductance regulator. We determined the ontogeny of expression of the CLCN family of voltage-dependent Cl channel genes (CLCN2 through 6, K(a) and K(b)) in the human lung to identify potential pathways for pulmonary liquid secretion. Only CLCN3 and CLCN6 messenger RNA were detected by Northern analysis of fetal whole lung tissue. Ribonuclease protection assays confirmed the expression of CLCN3 and also revealed expression of CLCN2. The ontogeny of expression of these two channels was similar, peaking in midgestation and declining postnatally. In situ hybridization localized the CLCN2 and CLCN3 messages to airway and distal pulmonary epithelia and to pulmonary blood vessels. We conclude that CLCN3 is expressed in human airway epithelia and expression is developmentally regulated. The contribution of these channels to pulmonary epithelial liquid transport and lung development remains to be determined.     

ATP regulation of ciliary beat frequency in rat tracheal and distal airway epithelium

Ciliary beat frequency (CBF) was measured by video-optical microscopy in rat tracheal and distal airway ciliary cells using a slice preparation. In tracheal ciliary cells (tracheal slice), ATP or 2-methylthio ATP (MeSATP) increased CBF, which was inhibited by suramin (100 microm, an inhibitor of purinergic receptor). Ionomycin (5 microm) or thapsigargin (2 microm) increased CBF similarly. Ca2+-free solution or addition of Ni2+ (1 mm) decreased CBF gradually by approximately 25% and subsequent stimulation with ATP (10 microm) increased CBF transiently. The purinergic agonist experiments demonstrated that ATP increases CBF in tracheal ciliary cells via both P2X and P2Y receptors. ATP increased the intracellular calcium concentration ([Ca2+]i) in tracheal ciliary cells. However, in distal airway ciliary cells (lung slice), ATP did not increase CBF and [Ca2+]i, although a Ca2+-free solution decreased CBF, and ionomycin (5 microm) or thapsigargin (2 microm) increased it. Moreover, acetylcholine (100 microm) did not increase CBF in distal airway ciliary cells, although it increased CBF in tracheal ciliary cells. Terbutaline (10 microm), a selective beta2-adrenergic agonist, increased CBF in both tracheal and distal airway ciliary cells. These observations suggest that the Ca2+-mobilization mechanisms via purinergic or muscarinic receptors of the distal airway ciliary cell may be different from those of the tracheal ciliary cell. In conclusion, the CBF increase is differently regulated in the tracheal and distal airway epithelia of the rat.     

Increasing epithelial junction permeability enhances gene transfer to airway epithelia In vivo

Gene transfer to airway epithelia is the most direct approach for treating the progressive lung disease associated with cystic fibrosis. However, the transduction efficiency is poor when viral vectors are applied to the mucosal surface. We reported previously that gene transfer via the apical surface of human airway epithelia in vitro was improved by formulating vectors with ethyleneglycol-bis-(2-aminoethyl ether)- N,N,N',N'-tetraacetic acid (EGTA) in a hypotonic buffer. First, we investigated the mechanism for this enhancement. When 100-nm fluorescent beads were applied to the apical surface in the presence of EGTA, paracellular deposition of the particles was noted. Transmission electron microscopy verified that the epithelial junction complex was disrupted under these conditions. The Ca(2+) chelators EGTA, 1,2-bis (2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA), and ethylenediaminetetraacetic acid all caused a rapid, reversible drop in transepithelial resistance and facilitated gene transfer with retrovirus or adenovirus in vitro. When Ca(2+) chelators were applied to rabbit tracheal epithelia or human nasal epithelia in vivo, the transepithelial voltage decreased, and amiloride sensitivity was lost, suggesting that epithelial junctions opened. Importantly, this novel formulation enhanced both retroviral- and adenoviral-mediated gene transfer to rabbit tracheal epithelia in vivo. This technique may have applications for vector or drug delivery to airway epithelia and other polarized cells.     

Adenosine regulation of cystic fibrosis transmembrane conductance regulator through prostenoids in airway epithelia

Cystic fibrosis is caused by dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) protein, leading to altered ion transport, chronic infection, and excessive inflammation. Here we investigated regulation of CFTR in airway cell monolayers by adenosine, adenosine receptors, and arachidonic acid. Our studies demonstrate that the A2B adenosine receptor is expressed at high levels relative to the other adenosine receptor subtypes, with a characteristic low-affinity profile for adenosine-stimulated CFTR Cl- currents in both Calu-3 cells and CFBE41o- airway cell monolayers stably transduced with wild-type CFTR. The levels of adenosine found in sputum from patients with cystic fibrosis with moderate to severe lung disease stimulated apical prostaglandin release in Calu-3 and CFBE41o- cells, implicating adenosine regulation of phospholipase A2 (PLA2) activity. A2B adenosine receptor and arachidonic acid stimulation produced CFTR-dependent currents in airway monolayers and increased cAMP levels that were sensitive to cyclooxygenase inhibition. Arachidonic acid demonstrated dual regulation of CFTR, stimulating CFTR and Cl- currents in intact airway monolayers, and potently inhibiting PKA-activated Cl- currents in excised membrane patches. Cl- currents produced by arachidonic acid were sensitive to inhibition of PKA, cyclooxygenase, and 5-lipoxygenase. Together, the results provide a converging mechanism to link regulation of CFTR and airway cell inflammation through adenosine and adenosine receptors.     

Species-specific differences in mouse and human airway epithelial biology of recombinant adeno-associated virus transduction

Differences in airway epithelial biology between mice and humans have presented challenges to evaluating gene therapies for cystic fibrosis (CF) using murine models. In this context, recombinant adeno-associated virus (rAAV) type 2 and rAAV5 vectors have very different transduction efficiencies in human air-liquid interface (ALI) airway epithelia (rAAV2 approximately = rAAV5) as compared with mouse lung (rAAV5 > rAAV2). It is unclear if these differences are due to species-specific airway biology or limitations of ALI cultures to reproduce in vivo airway biology. To this end, we compared rAAV2 and rAAV5 transduction biology in mouse and human ALI cultures, and investigated the utility of murine deltaF508 cystic fibrosis transmembrane conductance regulator (CFTR) ALI epithelia to study CFTR complementation. Our results demonstrate that mouse ALI epithelia retain in vivo preferences for rAAV serotype transduction from the apical membrane (rAAV5 > rAAV2) not seen in human epithelia (rAAV2 approximately = rAAV5). Viral binding of rAAV2 and rAAV5 to the apical surface of mouse ALI airway epithelia was not significantly different, and proteasome-modulating agents significantly enhanced rAAV2 transduction to a level equivalent to that of rAAV5 in the presence of these agents, suggesting that the ubiquitin/proteasome pathway represents a more significant intracellular block for rAAV2 transduction of mouse airway epithelia. Interestingly, cAMP-inducible chloride currents were enhanced in deltaF508CFTR mouse ALI cultures, making this model incompatible with CFTR complementation studies. These studies emphasize species-specific differences in airway biology between mice and humans that significantly influence the use of mice as surrogate models for rAAV transduction and gene therapy for CF.     

Activation by extracellular nucleotides of chloride secretion in the airway epithelia of patients with cystic fibrosis.

BACKGROUND. Cystic fibrosis is characterized by abnormal electrolyte transport across the epithelia of the airways. In particular, there is excessive sodium absorption and deficient chloride secretion. Drugs that block excessive sodium absorption may provide clinical benefit in cystic fibrosis, but there are no available therapeutic agents to improve chloride secretion. In vitro studies in cultured human-airway epithelia indicate that triphosphate nucleotides (ATP and UTP) induce chloride secretion through apical-membrane purinergic receptors. METHODS. We tested the ability of nucleotides to induce chloride secretion in vivo in 9 normal subjects and 12 patients with cystic fibrosis by measuring responses of nasal transepithelial potential difference (PD) to superfusion of nucleotides. Changes in transepithelial bioelectric properties and the permeability of the apical membrane to chloride in response to extracellular (apical) UTP were determined with ion-selective microelectrodes in cultured nasal epithelia. RESULTS. ATP and UTP induced chloride secretion in vivo in both groups. At their maximal effective concentrations of 10(-4) M, ATP and UTP were more effective chloride secretagogues in the patients with cystic fibrosis (mean [+/- SE] change in PD, -19.8 +/- 1.4 mV and -15.0 +/- 1.7 mV, respectively) than in the normal subjects (-6.9 +/- 0.6 mV and -8.1 +/- 0.9 mV, respectively). Microelectrode studies established that extracellular UTP stimulated a larger increase in PD and chloride secretory current in epithelial cells from patients with cystic fibrosis than in cells from normal subjects, by actions localized to the apical membrane. CONCLUSIONS. Extracellular nucleotides are effective in vivo chloride secretagogues in the nasal epithelia of patients with cystic fibrosis. The equipotency of ATP and UTP suggests that the effect is mediated by P2 nucleotide receptors. Selected nucleotides, such as UTP or nucleotide analogues, should be investigated as therapeutic agents for lung disease in cystic fibrosis.     

Retinal bipolar neurons express the cyclic nucleotide-gated channel of cone photoreceptors

Cyclic nucleotide-gated (CNG) channels link intracellular cyclic nucleotides to changes in membrane ionic conductance in a variety of physiological contexts. In the retina, in addition to their central role in phototransduction, CNG channels may be involved in nitric oxide signaling in bipolar neurons or in the hyperpolarizing synaptic response to glutamate in ON-type (depolarizing) bipolar cells. Despite their potential physiological significance, however, expression of CNG channels has not yet been demonstrated in bipolar cells. To identify CNG channel subtypes in retinal bipolar neurons, we used single-cell molecular biological techniques in morphologically distinctive ON bipolar cells from goldfish retina. Both single-cell in situ hybridization and single-cell RT-PCR demonstrated in ON bipolar cells the presence of mRNA for the CNG channel subtype that is also found in cone photoreceptors. Other bipolar cells, which likely represent OFF cells, did not express the cone CNG channel. Thus the CNG channel of cone photoreceptors is expressed in ON bipolar cells, where it may be involved in physiological responses to nitric oxide, or in the sign-inverting glutamatergic synapse that gives rise to the ON visual pathway.     

Lung epithelial fluid transport and the resolution of pulmonary edema

The discovery of mechanisms that regulate salt and water transport by the alveolar and distal airway epithelium of the lung has generated new insights into the regulation of lung fluid balance under both normal and pathological conditions. There is convincing evidence that active sodium and chloride transporters are expressed in the distal lung epithelium and are responsible for the ability of the lung to remove alveolar fluid at the time of birth as well as in the mature lung when pathological conditions lead to the development of pulmonary edema. Currently, the best described molecular transporters are the epithelial sodium channel, the cystic fibrosis transmembrane conductance regulator, Na+-K+-ATPase, and several aquaporin water channels. Both catecholamine-dependent and -independent mechanisms can upregulate isosmolar fluid transport across the distal lung epithelium. Experimental and clinical studies have made it possible to examine the role of these transporters in the resolution of pulmonary edema.     

CFTR, chloride concentration and cell volume: could mammalian protein histidine phosphorylation play a latent role?

A considerable body of evidence indicates that the intracellular chloride concentration ([Cl-]i) is an important regulatory signal in epithelial ion transport. [Cl-]i regulates the open channel probability of sodium and chloride channels, the rate of chloride channel recycling to the apical membrane, cell volume homeostasis, the activity of sodium-coupled chloride entry pathways and G-protein activity. Cell volume goes awry in epithelial cells bearing mutant forms of the cystic fibrosis (CF) transmembrane conductance regulator protein (CFTR); however, the pathways that mediate this [Cl-]i effect at the apical membrane of polarized epithelia are unknown. Recently, we proposed a mechanism for the transduction of in vitro chloride concentration into a phosphorylation signal to proteins within the apical membrane of respiratory epithelia. Our studies show that an apically enriched plasma membrane fraction from a variety of species, including sheep, human and mouse airway, contains at least two membrane-bound protein kinases which exhibit a number of novel properties. Firstly, the phosphate is located on histidine residues within different families of proteins; one kinase(s) utilizes GTP rather than ATP as a phosphate donor and each kinase has its own unique profile of membrane protein phosphorylation (which itself varies with anion species). Secondly, both kinases mediate Cl- -dependent phosphorylation of an apical membrane protein around the established physiological values for [Cl-]i in airway epithelial cells ( approximately 40 mM); associated phosphatases also alter the net phosphoprotein profile of the apical membrane. These findings are reviewed and their potential roles explored in relation to the pathogenesis of CF using the control of cell volume as a model for disrupted cellular function in CF-affected epithelia.     

Morphological analysis of the trachea and pattern of breathing in βENaC-Tg mice

Cystic fibrosis (CF) is caused by mutations in the CFTR (cystic fibrosis transmembrane conductance regulator) gene which is a Cl- channel and a regulator of the epithelial Na+ channel (ENaC). We have recently shown that newborn CFTR-deficient mice exhibit abnormalities of the tracheal cartilage leading to altered ventilation (Bonvin et al., 2008). However, the mechanism by which a lack of CFTR causes tracheal cartilage defects remains unknown. The main goal of the present study was to determine whether the development of airway cartilage defects is related to ENac channel dysfunction. We thus performed macroscopic analysis of the trachea and explored ventilatory function in adult βENaC-overexpressing (βENaC-Tg) mice with airway Na+ hyperabsorption and "CF-lung" lung disease, at 2 and 5 month of age. Only minor cartilaginous abnormalities were observed in 8 out of 16 βENaC-Tg mice and in 2 out of 20 littermate controls. Breathing pattern was progressively altered in βENaC-Tg mice as evidenced by a significant decrease in respiratory frequency. Our results suggest that Na+ hyperabsorption alone is not a major contributor to the development of tracheal malformation observed in CF mice and that breathing pattern changes in βENaC-Tg mice likely reflect airflow limitation due to airway mucus obstruction.     

Quantitative evaluation of the development of tracheal submucosal glands in infants with cystic fibrosis and control infants.

The development of the tracheal submucosal glands has been determined quantitatively in 22 infants with cystic fibrosis and in 25 control infants, all under 4 months of age. In cross-sections of normal trachea significant relationships were found between postconceptional age (PCA) and gland area (P less than 0.001), submucosal area (P less than 0.02), tracheal airway diameter (P less than 0.05), and acinar diameter (P less than 0.001). In infants with cystic fibrosis the pattern of development was similar to that of the control infants. No statistically significant differences were found between three subgroups of infants with cystic fibrosis, which included those with meconium ileus with no lung infection, those with meconium ileus with lung infection, and those with lung infection and no history of meconium ileus. The normal pattern of development of tracheal submucosal glands in infants with cystic fibrosis was in contrast to the deficiency of normal maturation seen in the exocrine pancreas of these infants. The lumen fraction, an index of dilatation of acinar lumina, showed no significant relationship with PCA in either the control group or the group with cystic fibrosis. However, statistically significant dilatation of acini was observed in the tracheal submucosal glands of infants with cystic fibrosis (0.14, P less than 0.005).