Protein phosphatase 2C dephosphorylates and inactivates cystic fibrosis transmembrane conductance regulator

cAMP-dependent phosphorylation activates the cystic fibrosis transmembrane conductance regulator (CFTR) in epithelia. However, the protein phosphatase (PP) that dephosphorylates and inactivates CFTR in airway and intestinal epithelia, two major sites of disease, is not certain. We found that in airway and colonic epithelia, neither okadaic acid nor FK506 prevented inactivation of CFTR when cAMP was removed. These results suggested that a phosphatase distinct from PP1, PP2A, and PP2B was responsible. Because PP2C is insensitive to these inhibitors, we tested the hypothesis that it regulates CFTR. We found that PP2Cα is expressed in airway and T84 intestinal epithelia. To test its activity on CFTR, we generated recombinant human PP2Cα and found that it dephosphorylated CFTR and an R domain peptide in vitro. Moreover, in cell-free patches of membrane, addition of PP2Cα inactivated CFTR Cl− channels; reactivation required readdition of kinase. Finally, coexpression of PP2Cα with CFTR in epithelia reduced the Cl− current and increased the rate of channel inactivation. These results suggest that PP2C may be the okadaic acid-insensitive phosphatase that regulates CFTR in human airway and T84 colonic epithelia. It has been suggested that phosphatase inhibitors could be of therapeutic value in cystic fibrosis; our data suggest that PP2C may be an important phosphatase to target.

A membrane lipid imbalance plays a role in the phenotypic expression of cystic fibrosis in cftr−/− mice

A deficiency in essential fatty acid metabolism has been reported in plasma from patients with cystic fibrosis (CF). However, its etiology and role in the expression of disease is unknown. The objective of this study was to determine whether alterations in fatty acid metabolism are specific to CF-regulated organs and whether they play a role in the expression of disease. A membrane lipid imbalance was found in ileum, pancreas, and lung from cftr−/− mice characterized by an increase in phospholipid-bound arachidonic acid and a decrease in phospholipid-bound docosahexaenoic acid (DHA). This lipid imbalance was observed in organs pathologically affected by CF including lung, pancreas, and ileum and was not secondary to impaired intestinal absorption or hepatic biosynthesis of DHA. As proof of concept, oral administration of DHA to cftr−/− mice corrected this lipid imbalance and reversed the observed pathological manifestations. These results strongly suggest that certain phenotypic manifestations of CF may result from remediable alterations in phospholipid-bound arachidonic acid and DHA levels.

Mutations in a NIMA-related kinase gene, Nek1, cause pleiotropic effects including a progressive polycystic kidney disease in mice

We previously have described a mouse model for polycystic kidney disease (PKD) caused by either of two mutations, kat or kat2J, that map to the same locus on chromosome 8. The homozygous mutant animals have a latent onset, slowly progressing form of PKD with renal pathology similar to the human autosomal-dominant PKD. In addition, the mutant animals show pleiotropic effects that include facial dysmorphism, dwarfing, male sterility, anemia, and cystic choroid plexus. We previously fine-mapped the kat2J mutation to a genetic distance of 0.28 ± 0.12 centimorgan between D8Mit128 and D8Mit129. To identify the underlying molecular defect in this locus, we constructed an integrated genetic and physical map of the critical region surrounding the kat2J mutation. Cloning and expression analysis of the transcribed sequences from this region identified Nek1, a NIMA (never in mitosis A)-related kinase as a candidate gene. Further analysis of the Nek1 gene from both kat/kat and kat2J/kat2J mutant animals identified a partial internal deletion and a single-base insertion as the molecular basis for these mutations. The complex pleiotropic phenotypes seen in the homozygous mutant animals suggest that the NEK1 protein participates in different signaling pathways to regulate diverse cellular processes. Our findings identify a previously unsuspected role for Nek1 in the kidney and open a new avenue for studying cystogenesis and identifying possible modes of therapy.

Defective regulatory volume decrease in human cystic fibrosis tracheal cells because of altered regulation of intermediate conductance Ca2+-dependent potassium channels

The cystic fibrosis transmembrane conductance regulator (CFTR) protein has the ability to function as both a chloride channel and a channel regulator. The loss of these functions explains many of the manifestations of the cystic fibrosis disease (CF), including lung and pancreatic failure, meconium ileus, and male infertility. CFTR has previously been implicated in the cell regulatory volume decrease (RVD) response after hypotonic shocks in murine small intestine crypts, an effect associated to the dysfunction of an unknown swelling-activated potassium conductance. In the present study, we investigated the RVD response in human tracheal CF epithelium and the nature of the volume-sensitive potassium channel affected. Neither the human tracheal cell line CFT1, expressing the mutant CFTR-ΔF508 gene, nor the isogenic vector control line CFT1-LC3, engineered to express the βgal gene, showed RVD. On the other hand, the cell line CFT1-LCFSN, engineered to express the wild-type CFTR gene, presented a full RVD. Patch-clamp studies of swelling-activated potassium currents in the three cell lines revealed that all of them possess a potassium current with the biophysical and pharmacological fingerprints of the intermediate conductance Ca2+-dependent potassium channel (IK, also known as KCNN4). However, only CFT1-LCFSN cells showed an increase in IK currents in response to hypotonic challenges. Although the identification of the molecular mechanism relating CFTR to the hIK channel remains to be solved, these data offer new evidence on the complex integration of CFTR in the cells where it is expressed.

Role of the cystic fibrosis transmembrane conductance regulator in innate immunity to Pseudomonas aeruginosa infections

Chronic Pseudomonas aeruginosa infection occurs in 75–90% of patients with cystic fibrosis (CF). It is the foremost factor in pulmonary function decline and early mortality. A connection has been made between mutant or missing CF transmembrane conductance regulator (CFTR) in lung epithelial cell membranes and a failure in innate immunity leading to initiation of P. aeruginosa infection. Epithelial cells use CFTR as a receptor for internalization of P. aeruginosa via endocytosis and subsequent removal of bacteria from the airway. In the absence of functional CFTR, this interaction does not occur, allowing for increased bacterial loads in the lungs. Binding occurs between the outer core of the bacterial lipopolysaccharide and amino acids 108–117 in the first predicted extracellular domain of CFTR. In experimentally infected mice, inhibiting CFTR-mediated endocytosis of P. aeruginosa by inclusion in the bacterial inoculum of either free bacterial lipopolysaccharide or CFTR peptide 108–117 resulted in increased bacterial counts in the lungs. CFTR is also a receptor on gastrointestinal epithelial cells for Salmonella enterica serovar Typhi, the etiologic agent of typhoid fever. There was a significant decrease in translocation of this organism to the gastrointestinal submucosa in transgenic mice that are heterozygous carriers of a mutant ΔF508 CFTR allele, suggesting heterozygous CFTR carriers may have increased resistance to typhoid fever. The identification of CFTR as a receptor for bacterial pathogens could underlie the biology of CF lung disease and be the basis for the heterozygote advantage for carriers of mutant alleles of CFTR.

Submucosal gland secretions in airways from cystic fibrosis patients have normal [Na+] and pH but elevated viscosity

Fluid and macromolecule secretion by submucosal glands in mammalian airways is believed to be important in normal airway physiology and in the pathophysiology of cystic fibrosis (CF). An in situ fluorescence method was applied to measure the ionic composition and viscosity of freshly secreted fluid from airway glands. Fragments of human large airways obtained at the time of lung transplantation were mounted in a humidified perfusion chamber and the mucosal surface was covered by a thin layer of oil. Individual droplets of secreted fluid were microinjected with fluorescent indicators for measurement of [Na+], [Cl−], and pH by ratio imaging fluorescence microscopy and viscosity by fluorescence recovery after photobleaching. After carbachol stimulation, 0.1–0.5 μl of fluid accumulated in spherical droplets at gland orifices in ≈3–5 min. In gland fluid from normal human airways, [Na+] was 94 ± 8 mM, [Cl−] was 92 ± 12 mM, and pH was 6.97 ± 0.06 (SE, n = 7 humans, more than five glands studied per sample). Apparent fluid viscosity was 2.7 ± 0.3-fold greater than that of saline. Neither [Na+] nor pH differed in gland fluid from CF airways, but viscosity was significantly elevated by ≈2-fold compared to normal airways. These results represent the first direct measurements of ionic composition and viscosity in uncontaminated human gland secretions and indicate similar [Na+], [Cl−], and pH to that in the airway surface liquid. The elevated gland fluid viscosity in CF may be an important factor promoting bacterial colonization and airway disease.

Polycystin, the polycystic kidney disease 1 protein, is expressed by epithelial cells in fetal, adult, and polycystic kidney.

Polycystic kidney disease 1 (PKD1) is the major locus of the common genetic disorder autosomal dominant polycystic kidney disease. We have studied PKD1 mRNA, with an RNase protection assay, and found widespread expression in adult tissue, with high levels in brain and moderate signal in kidney. Expression of the PKD1 protein, polycystin, was assessed in kidney using monoclonal antibodies to a recombinant protein containing the C terminus of the molecule. In fetal and adult kidney, staining is restricted to epithelial cells. Expression in the developing nephron is most prominent in mature tubules, with lesser staining in Bowman's capsule and the proximal ureteric bud. In the nephrogenic zone, detectable signal was observed in comma- and S-shaped bodies as well as the distal branches of the ureteric bud. By contrast, uninduced mesenchyme and glomerular tufts showed no staining. In later fetal (>20 weeks) and adult kidney, strong staining persists in cortical tubules with moderate staining detected in the loops of Henle and collecting ducts. These results suggest that polycystin's major role is in the maintenance of renal epithelial differentiation and organization from early fetal life. Interestingly, polycystin expression, monitored at the mRNA level and by immunohistochemistry, appears higher in cystic epithelia, indicating that the disease does not result from complete loss of the protein.

Uptake of fluorescent dyes associated with the functional expression of the cystic fibrosis transmembrane conductance regulator in epithelial cells.

Specific mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), the most common autosomal recessive fatal genetic disease of Caucasians, result in the loss of epithelial cell adenosine 3',5'-cyclic-monophosphate (cAMP)-stimulated Cl- conductance. We show that the influx of a fluorescent dye, dihydrorhodamine 6G (dR6G), is increased in cells expressing human CFTR after retrovirus- and adenovirus-mediated gene transfer. dR6G influx is stimulated by cAMP and is inhibited by antagonists of cAMP action. Dye uptake is ATP-dependent and inhibited by Cl- removal or the addition of 10 mM SCN-. Increased staining is associated with functional activation of CFTR Cl- permeability. dR6G staining enables both the fluorescent assessment of CFTR function and the identification of successfully corrected cells after gene therapy.

Impaired cell volume regulation in intestinal crypt epithelia of cystic fibrosis mice.

Cystic fibrosis is a disease characterized by abnormalities in the epithelia of the lungs, intestine, salivary and sweat glands, liver, and reproductive systems, often as a result of inadequate hydration of their secretions. The primary defect in cystic fibrosis is the altered activity of a cAMP-activated Cl- channel, the cystic fibrosis transmembrane conductance regulator (CFTR) channel. However, it is not clear how a defect in the CFTR Cl- channel function leads to the observed pathological changes. Although much is known about the structural properties and regulation of the CFTR, little is known of its relationship to cellular functions other than the cAMP-dependent Cl- secretion. Here we report that cell volume regulation after hypotonic challenge is also defective in intestinal crypt epithelial cells isolated from CFTR -/- mutant mice. Moreover, the impairment of the regulatory volume decrease in CFTR -/- crypts appears to be related to the inability of a K+ conductance to provide a pathway for the exit of this cation during the volume adjustments. This provides evidence that the lack of CFTR protein may have additional consequences for the cellular function other than the abnormal cAMP-mediated Cl- secretion.

Two cystic fibrosis transmembrane conductance regulator mutations have different effects on both pulmonary phenotype and regulation of outwardly rectified chloride currents.

Cystic fibrosis (CF), a disorder of electrolyte transport manifest in the lungs, pancreas, sweat duct, and vas deferens, is caused by mutations in the CF transmembrane conductance regulator (CFTR). The CFTR protein has been shown to function as a cAMP-activated chloride channel and also regulates a separate protein, the outwardly rectifying chloride channel (ORCC). To determine the consequence of disease-producing mutations upon these functions, mutant CFTR was transiently expressed in Xenopus oocytes and in human airway epithelial cells lacking functional CFTR. Both G551D, a mutation that causes severe lung disease, and A455E, a mutation associated with mild lung disease, altered but did not abolish CFTR's function as a chloride channel in Xenopus oocytes. Airway epithelial cells transfected with CFTR bearing either A455E or G551D had levels of chloride conductance significantly greater than those of mock-transfected and lower than those of wild-type CFTR-transfected cells, as measured by chloride efflux. A combination of channel blockers and analysis of current-voltage relationships were used to dissect the contribution of CFTR and the ORCC to whole cell currents of transfected cells. While CFTR bearing either mutation could function as a chloride channel, only CFTR bearing A455E retained the function of regulating the ORCC. These results indicate that CF mutations can affect CFTR functions differently and suggest that severity of pulmonary disease may be more closely associated with the regulatory rather than chloride channel function of CFTR.