In vivo measurements of ion transport in long-living CF mice

The Cftr (Cystic Fibrosis Transmembrane Conductance Regulator) gene codes for an epithelial chloride (C1) channel essential for fluid secretion into the respiratory and gastrointestinal tract and from exocrine glands. Mice lacking CFTR function due to a disruption of Cftr exon 10 or exon 1 (Cftr (m1UNC/m1UNC) or Cftr(m1HSC/m1HFC) mice, respectively) generally suffer from severe gastrointestinal disease resulting in death shortly after birth or at the time of weaning. However, a subgroup of the Cftr(m1HSC/m1HSC) mice have been characterized which exhibit relatively mild intestinal pathology resulting in a noncompromised lifespan compared to the more severely affected Cftr(m1UNC/m1UNC) mice. We compared the ion transport capacity of the intestinal mucosa of the mildly and severely affected CF mice using the in vivo technique of rectal potential difference (PD) measurement and found that the net calcium-activated chloride conductance toward the lumen was much greater in the rectum of mildly affected mice than in the severely affected mice. Hence, the milder phenotype may be related to the expression of a factor which enhances the net calcium-activated chloride conductance into the lumen of the intestinal tract.     

Regulation of CFTR chloride channels by syntaxin and Munc18 isoforms

The cystic fibrosis gene encodes a cyclic AMP-gated chloride channel (CFTR) that mediates electrolyte transport across the luminal surfaces of a variety of epithelial cells. The molecular mechanisms that modulate CFTR activity in epithelial tissues are poorly understood. Here we show that CFTR is regulated by an epithelially expressed syntaxin (syntaxin 1A), a membrane protein that also modulates neurosecretion and calcium-channel gating in brain. Syntaxin 1A physically interacts with CFTR chloride channels and regulates CFTR-mediated currents both in Xenopus oocytes and in epithelial cells that normally express these proteins. The physical and functional interactions between syntaxin 1A and CFTR are blocked by a syntaxin-binding protein of the Munc18 protein family (also called n-Secl). Our results indicate that CFTR function in epithelial cells is regulated by an interplay between syntaxin and Munc18 isoforms.     

Cellular heterogeneity of CFTR expression and function in the lung: implications for gene therapy of cystic fibrosis

Cystic fibrosis (CF) has become a paradigm disorder for the clinical testing of gene therapies in the treatment of inherited disease. In recent years, efforts directed at gene therapy of CF have concentrated on improving gene delivery systems to the airway. Surrogate endpoints for complementation of CFTR dysfunction in the lung have been primarily dependent on correction of chloride transport abnormalities. However, it is now clear that the pathophysiology of CF airways disease is far more complex than can be solely attributed to altered chloride permeability. For example, in addition to functioning as a chloride channel, CFTR also has been implicated in the regulation of other apical membrane conductance pathways through interactions with the amiloride sensitive epithelial sodium channel (ENaC) and the outwardly rectifying chloride channel (ORCC). Superimposed on this functional diversity of CFTR is a highly regulated pattern of CFTR expression in the lung. This heterogeneity occurs at both the level of CFTR protein expression within different cell types in the airway and the anatomical location of these cells in the lung. Potential targets for gene therapy of CF include ciliated, non-ciliated, and goblet cells in the surface airway epithelium as well as submucosal glands within the interstitium of the airways. Each of these distinct cellular compartments may have functionally distinct roles in processes which affect the pathogenesis of CF airways disease, such as fluid and electrolyte balance. However, it is presently unclear which of these cellular targets are most pathophysiologic relevant with regard to gene therapy. Elucidation of the underlying mechanisms of CFTR function in the airway will allow for the rational design of gene therapy approaches for CF lung diseases. This review will provide a summary of the field's current knowledge regarding CFTR functional diversity in the airway and the implications of such diversity for gene therapies of CF lung disease.     

Cl- absorption across the thick ascending limb is not altered in cystic fibrosis mice. A role for a pseudo-CFTR Cl- channel

The cortical thick ascending limb (CTAL) absorbs Cl- via a Na+-K+-Cl- cotransport at the apical membrane and several Cl- channels at the basolateral membrane, including a 9-pS channel having several properties of the cystic fibrosis transmembrane conductance regulator (CFTR). Having checked that CFTR mRNA is present in the mouse CTAL, we investigated whether this channel is a CFTR molecule by applying the patch-clamp technique to CTALs microdissected from CFTR knockout mice (cftrm1Unc). The 9-pS channel was active in cell-attached patches from tubules of mice homozygous for the disrupted cftr gene [CFTR (-/-)] at the same frequency and with the same activity (NPo) as in normal [CFTR (+/+)] or heterozygous [CFTR (+/-)] mice. The conductive properties of the channel, studied on inside-out patches, were identical in CFTR (-/-), CFTR (+/+), and CFTR (+/-) tubules, as were the sensitivities to internal pH and internal ATP, two typical features of this channel. In addition, the Cl- absorption in isolated, microperfused CTALs and the Na+-K+-Cl- cotransport activity were identical in CFTR (-/-), CFTR (+/+), and CFTR (+/-) mice. These results show that the 9-pS Cl- channel is distinct from CFTR, and that the CFTR protein has no influence on the Cl- absorption in this part of the renal tubule.     

Expression of human lysozyme mRNA in the mammary gland of transgenic mice

Owing to its inherent antimicrobial effect and positive charge, the expression of human lysozyme in bovine milk could be beneficial by altering the overall microbial level and the functional and physical properties of the milk. We have used transgenic mice as model systems to evaluate the expression of human lysozyme containing fusion gene constructs in the mammary gland. Expression of human lysozyme was targeted to the mammary gland by using the 5' promoter elements of either the bovine beta (line B mice) or alpha s1 (line H mice) casein genes coupled to the cDNA for human lysozyme. Expression of human lysozyme mRNA was not found in mammary tissue from any of line B mice. Tissues were analysed from six lines of H mice and two, H6 and H5, were found to express human lysozyme mRNA in the mammary gland at 42% and 116%, respectively, of the levels of the endogenous mouse whey acidic protein gene. At peak lactation, female mice homozygous for the H5 and H6 transgene have approximately twice the amount of mRNA encoding human lysozyme as hemizygous animals. Expression levels of human lysozyme mRNA in the mammary gland at time points representing late pregnancy, early, peak and late lactation corresponded to the profile of casein gene expression. Human lysozyme mRNA expression was not observed in transgenic males, virgin females or in the kidney, liver, spleen or brain of lactating females. A very low level of expression of human lysozyme mRNA was observed in the salivary gland of line H5.     

New strategies for the treatment of colic: modifying the parent/infant interaction

Cystic fibrosis (CF) is caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Its product is a cyclic AMP-dependent Cl- channel, that is defective in CF. Since cAMP regulates the expression of many genes and since the 5'-flanking region of the CFTR gene contains cAMP response elements, we hypothesized that intracellular cAMP might modulate not only the cAMP-dependent Cl- channel CFTR, but also CFTR gene expression in epithelial cells. To accomplish this, we investigated Cl- secretion and CFTR-mRNA levels in HT-29 and T84 colon carcinoma epithelial cells before and after exposure to forskolin and 8-bromo-cAMP for 12 hr. While resting T84 cells increased Cl- secretion in response to forskolin strongly and immediately, HT-29 cells did not, although both cell lines showed highly increased Cl- efflux in response to A23187, a calcium ionophore. Interestingly, prolonged exposure to forskolin (12 hr) induced a clear decrease of CFTR-mRNA levels in T84 cells, but an increase of CFTR-mRNA levels in HT-29 cells, thus demonstrating different behaviour of CFTR gene regulation in different epithelial cells in response to intracellular cAMP. These results suggest that cells with an effective cAMP-dependent Cl- channel (CFTR) respond to prolonged stimulation of this channel with down-regulation of CFTR gene expression, while cells with no effective cAMP-dependent Cl--secretion respond with an up-regulation of CFTR gene expression.     

cGMP stimulation of cystic fibrosis transmembrane conductance regulator Cl- channels co-expressed with cGMP-dependent protein kinase type II but not type Ibeta

In order to investigate the involvement of cGMP-dependent protein kinase (cGK) type II in cGMP-provoked intestinal Cl- secretion, cGMP-dependent activation and phosphorylation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels was analyzed after expression of cGK II or cGK Ibeta in intact cells. An intestinal cell line which stably expresses CFTR (IEC-CF7) but contains no detectable endogenous cGK II was infected with a recombinant adenoviral vector containing the cGK II coding region (Ad-cGK II) resulting in co-expression of active cGK II. In these cells, CFTR was activated by membrane-permeant analogs of cGMP or by the cGMP-elevating hormone atrial natriuretic peptide as measured by 125I- efflux assays and whole-cell patch clamp analysis. In contrast, infection with recombinant adenoviruses expressing cGK Ibeta or luciferase did not convey cGMP sensitivity to CFTR in IEC-CF7 cells. Concordant with the activation of CFTR by only cGK II, infection with Ad-cGK II but not Ad-cGK Ibeta enabled cGMP analogs to increase CFTR phosphorylation in intact cells. These and other data provide evidence that endogenous cGK II is a key mediator of cGMP-provoked activation of CFTR in cells where both proteins are co-localized, e. g. intestinal epithelial cells. Furthermore, they demonstrate that neither the soluble cGK Ibeta nor cAMP-dependent protein kinase are able to substitute for cGK II in this cGMP-regulated function.     

Complementation of null CF mice with a human CFTR YAC transgene

We have made transgenic mice carrying a 320 kb YAC with the intact human cystic fibrosis transmembrane regulator (CFTR) gene. Mice that only express the human transgene were obtained by breeding with Cambridge null CF mice. One line has approximately two copies of the intact YAC. Mice carrying this transgene and expressing no mouse cftr appear normal and breed well, in marked contrast to the null mice, where 50% die by approximately 5 days after birth. The chloride secretory responses in these mice are as large or larger than in wild-type tissues. Expression of the transgene is highly cell type specific and matches that of the endogenous mouse gene in the crypt epithelia throughout the gut and in salivary gland tissue. However, there is no transgene expression in some tissues, such as the Brunner's glands, where it would be expected. Where there are differences between the mouse and human pattern of expression, the transgene follows the mouse pattern. We have thus defined a cloned fragment of DNA which directs physiological levels of expression in many of the specific cells where CFTR is normally expressed.     

Antimitotic and tubulin-interacting properties of vinflunine, a novel fluorinated Vinca alkaloid

Cystic fibrosis (CF) lung disease has been linked to multiple primary defects in airway epithelia caused by a dysfunctional cystic fibrosis transmembrane conductance regulator (CFTR) gene. These defects include altered Cl- and Na+ permeability as well as intracellular defects in glycoprotein processing. This apparent diversity in CFTR function is reflected in the complex patterning of CFTR expression in airway epithelia. Such complexities present challenges in the design of CF gene therapies that are capable of reconstituting the endogenous patterns of CFTR gene expression in appropriate target cells. Using a human bronchial xenograft model of the CF airway, we have evaluated the efficacy of recombinant adenoviral and cationic liposome-mediated gene transfer to correct Cl- permeability and mucous sulfation defects found in CF lung disease. Results from these studies demonstrated a clear vector-specific complementation profile for these two defects that was dependent on the type of cell transduced and the level of transgene expression. Single-dose administration of recombinant adenovirus effectively transduced high levels of CFTR transgene expression in 11 +/- 1% of epithelial cells and was capable of correcting cAMP-induced changes in Cl- permeability to 91 +/- 14% that seen in non-CF airways. However, this level of transgene expression was incapable of reversing defects in mucous sulfation due to the lack of efficient targeting to goblet cells. In contrast, cationic liposome-mediated delivery of CFTR encoding plasmids to CF airways achieved extremely low levels of transgene expression with insignificant correction (7.4 +/- 2.4%) of cAMP-induced Cl- permeability. This low level of transgene expression, however, efficiently reduced mucous sulfation to levels seen in non-CF airways. Differences in the complementation profiles of these two vectors in correcting Cl- permeability and mucous sulfation defects mirror the ability of recombinant adenovirus and liposomes to reconstitute only certain features of the endogenous distribution and abundance of CFTR protein expression. Such findings suggest that the level of intracellular CFTR required to facilitate proper glycoprotein processing may be much lower than that needed to mediate bulk Cl- flow across the airway epithelium. In summary, these data present the first example by which two different vector systems can efficiently complement independent primary defects associated with a single dysfunctional gene.     

Lack of beta-amyloidosis in transgenic mice expressing low levels of familial Alzheimer''s disease missense mutations

Point mutations within the beta-amyloid precusor protein (beta-APP) gene known to segregate with Alzheimer's disease in certain families were introduced into human beta-APP cDNAs and expressed under the control of a neuron-specific enolase (NSE) promoter in mice. The transgenic animals exhibited transgene expression predominantly in neocortex and hippocampus where the levels were maximally 1.3-fold of those of wild-type mouse beta-APP. Quantitative immunoblot analysis in homozygous mice carrying different missense mutations showed slightly increased alpha-secretory processing. In V7171 mice compared to nontransgenic mice there was more alpha-secretory beta-APP (beta-APPsec) in cortex/hippocampus, less in cerebellum, and no difference in midbrain/brain stem. In none of the transgenic animals tested was a 4 kDa amyloid fragment detected by Western blotting of brain extracts, immunohistochemistry, or by 125I-A beta-binding onto brain sections. No glial reaction was observed. Behavioral analysis of mice carrying the V7171 mutation showed no appreciable deficit in comparison to wild-type mice. Together, these data suggest that low levels of expression of mutated beta-APP in 10-12-month-old transgenic mouse brains result in slightly more beta-APPsec, and are insufficient to induce amyloidogenic processing and AD-like pathology.     

Transgenic models in renal tubular physiology

Animal transgenesis has proven to be useful for physiological as well as physiopathological studies. Besides the classical approach based on the random integration of a DNA construct in the mouse genome, gene targeting can be achieved using totipotent embryonic stem (ES) cells for targeted transgenesis. Transgenic mice are then derived from the transgenic ES cells. This allows the introduction of null mutations in the genome (so-called knock-out) or the control of the transgene expression by the endogenous regulatory sequences of the gene of interest (so-called knock-in). Development of these transgenic animals leads to a better understanding of the cellular function of many genes or to the generation of animal models for human diseases. The purpose of this short review is to describe animal models in renal tubular physiopathology. Recent progresses will allow the generation of animal models with conditional expression of the transgene of interest or with a conditional gene mutation. This permits spatial and temporal control of the expression of the transgene or of the mutation. This should allow the generation of models suitable for physiological analysis or closer to disease state.     

Cystic fibrosis transmembrane conductance regulator is an epithelial cell receptor for clearance of Pseudomonas aeruginosa from the lung

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride ion channel, but its relationship to the primary clinical manifestation of CF, chronic Pseudomonas aeruginosa pulmonary infection, is unclear. We report that CFTR is a cellular receptor for binding, endocytosing, and clearing P. aeruginosa from the normal lung. Murine cells expressing recombinant human wild-type CFTR ingested 30-100 times as many P. aeruginosa as cells lacking CFTR or expressing mutant DeltaF508 CFTR protein. Purified CFTR inhibited ingestion of P. aeruginosa by human airway epithelial cells. The first extracellular domain of CFTR specifically bound to P. aeruginosa and a synthetic peptide of this region inhibited P. aeruginosa internalization in vivo, leading to increased bacterial lung burdens. CFTR clears P. aeruginosa from the lung, indicating a direct connection between mutations in CFTR and the clinical consequences of CF.     

Cystic fibrosis transmembrane conductance regulator-associated ATP release is controlled by a chloride sensor

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that is defective in cystic fibrosis, and has also been closely associated with ATP permeability in cells. Using a Xenopus oocyte cRNA expression system, we have evaluated the molecular mechanisms that control CFTR-modulated ATP release. CFTR-modulated ATP release was dependent on both cAMP activation and a gradient change in the extracellular chloride concentration. Activation of ATP release occurred within a narrow concentration range of external Cl- that was similar to that reported in airway surface fluid. Mutagenesis of CFTR demonstrated that Cl- conductance and ATP release regulatory properties could be dissociated to different regions of the CFTR protein. Despite the lack of a need for Cl- conductance through CFTR to modulate ATP release, alterations in channel pore residues R347 and R334 caused changes in the relative ability of different halides to activate ATP efflux (wtCFTR, Cl > Br; R347P, Cl > Br; R347E, Br > Cl; R334W, Cl = Br). We hypothesize that residues R347 and R334 may contribute a Cl- binding site within the CFTR channel pore that is necessary for activation of ATP efflux in response to increases of extracellular Cl-. In summary, these findings suggest a novel chloride sensor mechanism by which CFTR is capable of responding to changes in the extracellular chloride concentration by modulating the activity of an unidentified ATP efflux pathway. This pathway may play an important role in maintaining fluid and electrolyte balance in the airway through purinergic regulation of epithelial cells. Insight into these molecular mechanisms enhances our understanding of pathogenesis in the cystic fibrosis lung.     

Targeted replacement of normal and mutant CFTR sequences in human airway epithelial cells using DNA fragments

Recent studies have reported that mutant genomic cystic fibrosis (CF) transmembrane conductance regulator ( CFTR ) sequences can be corrected in transformed CF airway epithelial cell lines by targeted replacement with small fragments of DNA with wild-type sequence. To determine if the observed genotype modification following small fragment homologous replacement (SFHR) was limited to transformed CF cell lines, further studies were carried out in both transformed and non-transformed primary normal airway epithelial cells. The endogenous genotype of these normal cell lines was modified following liposome or dendrimer transfection using DNA fragments with DeltaF508 CFTR sequence (488 nt, complementary single strands) designed to also contain a unique restriction enzyme cleavage site (Xho I). Replacement at the appropriate genomic locus by exogenous DeltaF508 CFTR DNA and its expression as mRNA was demonstrated by PCR amplification of genomic DNA and mRNA-derived cDNA as well as Xho I digestion of the PCR products. These studies show that SFHR occurs in both transformed and non-transformed primary human airway epithelial cells and indicate that single base substitution (the silent mutation giving rise to the Xho I site) and deletion or insertion of at least three consecutive bases can be achieved in both normal and CF epithelial cells. Furthermore, these studies reiterate the potential of SFHR as a strategy for a number of gene targeting applications, such as site-specific mutagenesis, development of transgenic animals, development of isogenic cell lines and for gene therapy.     

CFTR regions containing duodenum specific DNase I hypersensitive sites drive expression in intestinal crypt cells but not in fibroblasts

We have investigated CFTR specific intestinal expression by transfection assays in mouse cultured fibroblasts and transimmortalized intestinal crypt m-ICc12 cells using the beta-galactosidase gene linked to rat CFTR non-coding regions. Two constructs were studied, one encompassing a 5.3 kb region 5' to the gene where numerous duodenum-specific DNase I hypersensitive sites (DHSs) were previously mapped and the other including a 1.3 kb 3' region in which novel DHSs had been identified. In transient transfection assays, transgenes were expressed in m-ICc12 cells but not in fibroblasts. In m-ICc12 cells, the pattern of expression of the chromosomally integrated transgenes paralleled the endogenous expression of CFTR and beta-galactosidase activity was detected in cells containing villin and forming domes. Thus, a 6.6 kb region encompassing 5' and 3' non-coding parts of rat CFTR is able to drive specific expression of a reporter gene in cultured mouse intestinal cells having kept a crypt phenotype.     

Mutant (delta F508) cystic fibrosis transmembrane conductance regulator Cl- channel is functional when retained in endoplasmic reticulum of mammalian cells

Cystic fibrosis is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), a plasma membrane-localized chloride channel. Some mutations in CFTR, including one which affects most patients (delta F508-CFTR), prevent CFTR from exiting the endoplasmic reticulum (ER) where it is synthesized. To examine whether normal and mutant CFTRs function as chloride channels when they reside in the ER, the patch clamp technique was used to measure currents in the outer membrane of nuclei isolated from mammalian cells expressing CFTR. Both delta F508-CFTR as well as CFTR were revealed to function as cAMP-regulated chloride channels in native ER membrane. These results represent the first demonstrations of functional activity of CFTR in the biosynthetic pathway and suggest that conformational changes in the mutant protein, although recognized by ER-retention mechanisms, do not necessarily affect CFTR chloride channel properties, which may have implications for pathophysiology and therapeutic interventions in cystic fibrosis.     

Purified cystic fibrosis transmembrane conductance regulator (CFTR) does not function as an ATP channel

The gene mutated in cystic fibrosis codes for the cystic fibrosis transmembrane conductance regulator (CFTR). Previously, we provided definitive evidence that CFTR functions as a phosphorylation-regulated chloride channel in our planar lipid bilayer studies of the purified, reconstituted protein. Recent patch-clamp studies have lead to the suggestion that CFTR may also be capable of conducting ATP or inducing this function in neighboring channels. In the present study, we assessed the ATP channel activity of purified CFTR and found that the purified protein does not function as an ATP channel in planar bilayer studies of single channel activity nor in ATP flux measurements in proteoliposomes. Hence, CFTR does not possess intrinsic ATP channel activity and its putative role in cellular ATP transport may be indirect.     

Efficient expression of CFTR function with adeno-associated virus vectors that carry shortened CFTR genes

Adeno-associated virus (AAV)-based vectors have been shown to be effective in transferring the cystic fibrosis gene (CFTR) into airway epithelial cells in animal models and in patients. However, the level of CFTR gene expression has been low because the vector cannot accommodate the CFTR gene together with a promoter. In this study, we described a strategy to reduce the size of the CFTR cDNA to allow the incorporation of an effective promoter with the CFTR gene into AAV vectors. We engineered and tested 20 CFTR mini-genes containing deletions that were targeted to regions that may contain nonessential sequences. Functional analyses showed that four of the shortened CFTRs (one with combined deletions) retained the function and the characteristics of a wild-type CFTR, as measured by open probability, time voltage dependence, and regulation by cAMP. By using an AAV vector with a P5 promoter, we transduced these short forms of CFTR genes into target cells and demonstrated high levels of CFTR expression. We also demonstrated that smaller AAV/CFTR vectors with a P5 promoter expressed the CFTR gene more efficiently than larger vectors or a vector in which CFTR gene was expressed from the AAV inverted terminal repeat sequence. The CFTR mini-gene with combined deletions was packaged into AAV virions more efficiently, generated higher titers of transducing virions, and more effectively transferred CFTR function into target cells. These new vectors should circumvent the limitations of AAV vector for CFTR expression. Our strategy also may be applicable to other genes, the sizes of which exceed the packaging limit of an AAV vector.