Non-CFTR chloride channels likely contribute to secretion in the murine small intestine

While most cystic fibrosis (CF) transmembrane conductance regulator (CFTR)-knockout animals die due to intestinal obstruction before or at the time of weaning, a subpopulation of these animals are long living and exhibit a milder phenotype. The decreased severity of intestinal disease in these mildly affected CF mice is related to the expression of non-CFTR genetic modifiers. The identity of these genetic modifiers is not known, but we hypothesize that they may complement CFTR function as a chloride channel in this tissue. To assess the contribution of non-CFTR chloride channels to chloride secretion across the small intestine of CF mice with mild disease, we measured the basal transepithelial potential difference across this tissue as well as the secretory response to agonists of the cAMP and the calcium-mediated signaling pathways. Chloride secretion across the small intestine of mildly affected CF mice was not stimulated by forskolin or by carbachol. The absence of CFTR is thus not compensated by the activity of a distinct, cAMP- or calcium-activated chloride channel at the apical surface of the intestinal epithelium. On the other hand, a basal chloride secretion across the intestinal epithelium was present in these animals, and we hypothesize that this activity may be linked to improved survival of these animals.     

Aminoglycoside suppression of a premature stop mutation in a Cftr-/- mouse carrying a human CFTR-G542X transgene

Cystic fibrosis (CF) is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Since approximately 5% of all mutant CF alleles are stop mutations, it can be calculated that approximately 10% of CF patients carry a premature stop mutation in at least one copy of the CFTR gene. Certain ethnic groups, such as the Ashkenazi Jewish population, carry a much higher percentage of CF stop mutations. Consequently, a therapeutic strategy aimed at suppressing this class of mutation would be highly desirable for the treatment of this common genetic disease. We have shown previously that aminoglycoside antibiotics can suppress premature stop mutations in the CFTR gene in a bronchial epithelial cell line [Nat Med (1997) 3:1280]. To address whether aminoglycosides can suppress a CFTR premature stop mutation in an animal model, we constructed a transgenic mouse with a null mutation in the endogenous CFTR locus (Cftr-/-) that also expressed a human CFTR-G542X cDNA under control of the intestinal fatty acid binding protein promoter. We then investigated whether the daily administration of the aminoglycoside antibiotics gentamicin or tobramycin could restore the expression of a detectable level of CFTR protein. Immunofluorescence staining of intestinal tissues from Cftr-/- hCFTR-G542X mice revealed that gentamicin treatment resulted in the appearance of hCFTR protein at the apical surface of the glands of treated mice. Weaker staining was also observed in the intestinal glands following tobramycin treatment. Short-circuit current measurements made on intestinal tissues from these mice demonstrated that a significant number of positive cAMP-stimulated transepithelial chloride current measurements could be observed following gentamicin treatment (P=0.008) and a near significant number following tobramycin treatment (P=0.052). When taken together, these results indicate that gentamicin, and to a lesser extent tobramycin, can restore the synthesis of functional hCFTR protein by suppressing the hCFTR-G542X premature stop mutation in vivo.     

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.     

Functional human CFTR produced by stable Chinese hamster ovary cell lines derived using yeast artificial chromosomes

The cystic fibrosis transmembrane conductance regulator gene (CFTR) encodes a transmembrane protein (CFTR) which functions in part as a cyclic adenosine monophosphate (cAMP)-regulated chloride channel. CFTR expression is controlled temporally and cell specifically by mechanisms that are poorly understood. Insight into CFTR regulation could be facilitated by the successful introduction of the entire 230 kb human CFTR and adjacent sequences into mammalian cells. To this end, we have introduced two different CFTR-containing yeast artificial chromosomes (YACs) (320 and 620 kb) into Chinese hamster ovary-K1 (CHO) cells. Clonal cell lines containing human CFTR were identified by PCR, and the genetic and functional analyses of one clone containing each YAC are described. Integration of the human CFTR-containing YACs into the CHO genome at a unique site in each cell line was demonstrated by fluorescence in situ hybridization (FISH). Southern blot analysis suggested that on the order of one copy of human CFTR was integrated per CHO cell genome. Fiber-FISH and restriction analysis suggested that CFTR remained grossly intact. Northern analysis showed full-length, human CFTR mRNA. Immunoprecipitation followed by phosphorylation with protein kinase demonstrated mature, glycosylated CFTR. Finally, chloride secretion in response to cAMP indicated the functional nature of the human CFTR. This study provides several novel results including: (i) functional human CFTR can be expressed from these YACs; (ii) CHO cells are a permissive environment for expression of human CFTR; (iii) the level of human CFTR expression in CHO cells is unexpectedly high given the lack of endogenous CFTR production; and (iv) the suggestion by Fiber-FISH of CFTR integrity correlates with functional gene expression. These YACs and the cell lines derived from them should be useful tools for the study of CFTR expression.      

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.     

Human-specific cystic fibrosis transmembrane conductance regulator antibodies detect in vivo gene transfer to ovine airways

A panel of 11 human cystic fibrosis transmembrane conductance regulator (hCFTR) antibodies were tested in ovine nasal, tracheal, and bronchial epithelial brushings. Two of these, G449 (polyclonal) and MATG1104 (monoclonal), recognized hCFTR but did not cross react with endogenous sheep CFTR. This specificity allows immunologic detection of hCFTR expressed in gene transfer studies in sheep against the background of endogenous ovine CFTR, thus enhancing the value of the sheep as a model animal in which to study CFTR gene transfer. Studies on mixed populations of human and sheep nasal epithelial cells showed that detection of hCFTR by these two antibodies was possible even at the lowest proportion of human cells (1:100). The hCFTR gene was delivered in vivo by local instillation using polyethylenimine-mediated gene transfer to the ventral surface of the ovine trachea and hCFTR mRNA and protein levels scored in a blinded fashion. Despite abundant hCFTR mRNA expression, the number of cells expressing hCFTR protein detectable by G449 was low (approximately 0.006-0.05%). Immunohistochemistry for hCFTR in animals treated by whole-lung aerosol demonstrated positive cells in sections of tracheal epithelium and in distal conducting airways. The strategic use of hCFTR-specific antibodies supports the utility of the normal sheep as a model for hCFTR gene transfer studies.     

Engraftment of donor-derived epithelial cells in multiple organs following bone marrow transplantation into newborn mice

Bone marrow-derived cells (BMDCs) can engraft as epithelial cells throughout the body, including in the lung, liver, and gastrointestinal (GI) tract following transplantation into lethally irradiated adult recipients. Except for rare disease models in which marrow-derived epithelial cells have a survival advantage over endogenous cells, the currently attained levels of epithelial engraftment of BMDCs are too low to be of therapeutic benefit. Here we tested whether the degree of bone marrow to epithelial engraftment would be higher if bone marrow transplantation (BMT) were performed on 1-day-old mice, when tissues are undergoing rapid growth and remodeling. BMT into newborn mice after multiple different regimens allowed for robust hematopoietic engraftment, as well as the development of rare donor-derived epithelial cells in the GI tract and lung but not in the liver. The highest epithelial engraftment (0.02%) was obtained in mice that received a preparative regimen of two doses of busulfan in utero. When BMDCs were transplanted into myelosuppressed newborn mice that lacked expression of the cystic fibrosis transmembrane conductance regulator (CFTR) protein, the chloride channel that is not functional in patients with cystic fibrosis, the engrafted mice showed partial restoration of CFTR channel activity, suggesting that marrow-derived epithelial cells in the GI tract were functional. However, BMT into newborn mice, regardless of the myeloablative regimen used, did not increase the number of bone marrow-derived epithelial cells over that which occurs after BMT into lethally irradiated adult mice.     

Role of oxygen availability in CFTR expression and function

The cystic fibrosis transmembrane conductance regulator (CFTR) serves a pivotal role in normal epithelial homeostasis; its absence leads to destruction of exocrine tissues, including those of the gastrointestinal tract and lung. Acute regulation of CFTR protein in response to environmental stimuli occurs at several levels (e.g., ion channel phosphorylation, ATP hydrolysis, apical membrane recycling). However, less information is available concerning the regulatory pathways that control levels of CFTR mRNA. In the present study, we investigated regulation of CFTR mRNA during oxygen restriction, examined effects of hypoxic signaling on chloride transport across cell monolayers, and related these findings to a possible role in the pathogenesis of chronic hypoxic lung disease. CFTR mRNA, protein, and function were robustly and reversibly altered in human cells in relation to hypoxia. In mice subjected to low oxygen in vivo, CFTR mRNA expression in airways, gastrointestinal tissues, and liver was repressed. CFTR mRNA expression was also diminished in pulmonary tissues taken from hypoxemic subjects at the time of lung transplantation. Environmental factors that induce hypoxic signaling regulate CFTR mRNA and epithelial Cl(-) transport in vitro and in vivo.     

Expression of cystic fibrosis transmembrane conductance regulator in human endometrium

BACKGROUND: As a cAMP-regulated Cl- channel, cystic fibrosis transmembrane conductance regulator (CFTR) plays a critical role in the active secretion of electrolytes and fluid in epithelial cells. Women with CFTR gene mutations are less fertile, generally assumed to be due to cervical factors. However, there is little known about CFTR protein expression in human endometrium and its possible roles in reproduction. METHODS AND RESULTS: CFTR protein and mRNA levels in human endometrium were analysed using immunohistochemical and in situ hybridization methods, respectively. Significant expression of CFTR protein was only seen in the glandular cells from late proliferative to all secretory phases, consistent with western blot analysis. High levels of CFTR mRNA were present only around the ovulatory period. In cultured glandular cells, the production of CFTR protein and mRNA was stimulated by estradiol and inhibited by progesterone. A forskolin-activated Cl- current in endometrial epithelial cells with a linear I-V relationship was detected by the whole-cell patch-clamp technique. CONCLUSIONS: (i) CFTR mRNA and protein were localized in human endometrial epithelial cells and the amounts varied in a cyclic manner; (ii) CFTR expression in cultured glandular cells was up- and downregulated by estradiol and progesterone, respectively; and (iii) CFTR in human endometrium functions as a cAMP-activated Cl- channel.     

Inactivation of the murine cftr gene abolishes cAMP-mediated but not Ca2+-mediated secretagogue-induced volume decrease in small-intestinal crypts

The cellular volume of crypts isolated from 2- to 3-week-old mouse small intestine has been measured to assess the capacity of the epithelial cells to respond to secretagogues. Vasoactive intestinal polypeptide (VIP) or carbachol, respectively cAMP- and calcium-mediated secretagogues, produced a reduction crypt volume attributed to KCl loss through channels activated by the agonists. Consistent with the participation of separate chloride channels, 4,4-diisothiocyanatostilbene-2,2-disulphonic acid (DIDS) blocked the carbacholbut not the VIP-induced volume decrease, whilst gl- ibenclamide abolished the VIP effect without affecting the carbachol-induced volume decrease. Animals homozygous for a disrupted cftr gene, introduced by gene targeting, were also used as the source for crypt isolation. In these CFTR(-/-) crypts, VIP failed to elicit any reduction in cellular volume, while the response to carbachol was indistinguishable from that seen in crypts from age-matched control animals. These results are consistent with murine CFTR being a cAMP-activated chloride channel inhibited by glibenclamide and resistant to DIDS. A separate chloride conductance activated by calcium mobilization in small-intestinal crypts appears to be independent of CFTR.     

Human huntingtin derived from YAC transgenes compensates for loss of murine huntingtin by rescue of the embryonic lethal phenotype

Huntington disease (HD) is caused by expansion of a CAG trinucleotide repeat in exon 1 of a novel gene. The HD protein (huntingtin) plays a critical role in early embryonic development since homozygous targeted disruption of the murine HD gene results in embryonic lethality by day 7.5. To rescue this phenotype by transgene based huntingtin expression it is therefore essential to express the protein early enough in development in the appropriate cells. Since YAC based transgenes are known to be regulated in an appropriate temporal and tissue-specific manner, we sought to rescue the embryonic lethality by breeding YAC transgenic mice expressing human huntingtin with mice heterozygous for the targeted disruption. We generated viable offspring homozygous for the disrupted murine HD gene but expressing human huntingtin derived from the YAC. This result clearly shows that YAC transgene based expression of huntingtin occurs prior to 7.5 days gestation. Additionally, we show that human huntingtin expression in YAC transgenic mice follows an identical tissue distribution and subcellular localisation pattern as that of the murine endogenous protein and that expression levels of 2-3 times endogenous can be achieved. This shows that human huntingtin under the influence of its native promoter, despite differences to the murine protein, is functional in a murine background and can compensate for loss of the murine protein. These results show that YAC transgenic approaches are a particularly promising route to producing an animal model for disorders associated with CAG expansion.      

Indispensable function for embryogenesis, expression and regulation of the nonspecific form of the 5-aminolevulinate synthase gene in mouse

The first step of heme biosynthesis in animals is catalyzed by 5-aminolevulinate synthase (ALAS), which controls heme supply in various tissues. To clarify the roles that the nonspecific isoform of ALAS (ALAS-N) plays in vivo , we prepared a green fluorescent protein (GFP) knock-in mouse line in which the Alas1 gene (encoding ALAS-N) is replaced with a gfp gene. We found that mice bearing a homozygous knock-in allele ( Alas1 ^GFP/GFP) were lethal by embryonic day 8.5, demonstrating that ALAS-N is essential for early embryogenesis. Fluorescence microscopic and flow cytometric analyses of heterozygous mouse ( Alas1 ^+/GFP) tissues showed that the Alas1 expression level differs substantially in tissues; Alas1 is highly expressed in testis Leydig cells, exocrine glands (including submandibular and parotid glands), endocrine glands (such as adrenal and thyroid glands) and hematopoietic lineage cells (including neutrophils and eosinophils). Quantitative analyses of GFP mRNA and ALAS-N mRNA in various tissues of Alas1 ^+/GFP mice suggested that the destabilization of ALAS-N mRNA was not uniform in the various tissues. These results thus lay bare that elaborate control of the endogenous heme supply operates in various mouse tissues through regulation of the ALAS-N expression level and that this control is essential for heme homeostasis in animals.     

Increased cystic fibrosis transmembrane conductance regulator (CFTR) expression in the human hydrosalpinx

BACKGROUND: Hydrosalpinx (HSP), characterized by abnormal fluid accumulation in the Fallopian tube, is one of the main causes of infertility in women; however, the mechanism underlying the formation of hydrosalpinx fluid (HF) remains elusive. The present study investigated the possible involvement of cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-dependent chloride channel, in the pathogenesis of hydrosalpinx. METHODS: Masson's trichrome staining was used to characterize epithelial transformation in human HSP; RT-PCR, immunohistochemistry and immunofluorescence staining were used for CFTR expression and localization. RESULTS: Masson's trichrome staining showed areas of epithelial transformation, focally attenuated and pseudostratified. Immunostaining showed enhanced CFTR immunoreactivity in the focally attenuated and pseudostratified areas of HSP epithelium. RT-PCR revealed that CFTR expression in HSP was significantly greater than that in normal Fallopian tubes. CONCLUSIONS: These results indicate that HSP epithelium undergoes epithelial transformation with elevated CFTR expression, which may lead to increased transepithelial electrolyte and fluid secretion resulting in HF formation. The present findings may lead to the development of new treatment strategies for infertile patients with HSP.     

Cystic fibrosis F508del patients have apically localized CFTR in a reduced number of airway cells

Present state of knowledge, mostly based on heterologous expression studies, indicates that the cystic fibrosis transmembrane conductance regulator (CFTR) protein bearing the F508del mutation is misprocessed and mislocalized in the cytoplasm, unable to reach the cell surface. Recently, however, it was described that protein levels and localization are similar between F508del and wild-type CFTR in airway and intestinal tissues, but not in the sweat glands. In this study, we used immunocytochemistry with three different anti-CFTR antibodies to investigate endogenous CFTR expression and localization in nasal epithelial cells from F508del homozygous patients, F508del carriers, and non-CF individuals. On average, 300 cells were observed per individual. No significant differences were observed for cell type distributions among CF, carrier, and non-CF samples; epithelial cells made up approximately 80% to 95% of all cells present. CFTR was detected mostly in the apical region (AR) of the tall columnar epithelial (TCE) cells, ciliated or nonciliated. By confocal microscopy analysis, we show that the CFTR apical region-staining does not overlap with either anti-calnexin (endoplasmic reticulum), anti-p58 (Golgi), or anti-tubulin (cilia) stainings. The median from results with three antibodies indicate that the apical localization of CFTR happens in 22% of TCE cells from F508del homozygous patients with CF (n = 12), in 42% of cells from F508del carriers (n = 20), and in 56% of cells from healthy individuals (n = 12). Statistical analysis indicates that differences are significant among all groups studied and for the three antibodies (p < 0.05). These results confirm the presence of CFTR in the apical region of airway cells from F508del homozygous patients; however, they also reveal that the number of cells in which this occurs is significantly lower than in F508del carriers and much lower than in healthy individuals. These findings may have an impact on the design of novel pharmacological strategies aimed at circumventing the CF defect caused by the F508del mutation.     

Down-regulation of volume-sensitive Cl- channels by CFTR is mediated by the second nucleotide-binding domain

Transient expression of wild-type human cystic fibrosis transmembrane conductance regulator (CFTR) in HEK293T cells resulted in a profound decrease in the amplitude of volume-sensitive outwardly rectifying Cl- channel (VSOR) current without changing the single-channel amplitude. This effect was not mimicked by expression of the DeltaF508 mutant of CFTR, which did not reach the plasma membrane. The VSOR regulation by CFTR was not affected by G551D mutation at first nucleotide-binding domain (NBD1), which is known to impair CFTR interaction with the outwardly rectifying chloride channel, ORCC, epithelial amiloride-sensitive Na-channel, ENaC, and renal potassium channel, ROMK2. The CFTR-VSOR interaction was insensitive to the deletion mutation, DeltaTRL, which is known to impair CFTR-PDZ domain binding. In contrast, the G1349D mutant, which impairs ATP binding at NBD2, effectively abolished the down-regulatory effect of CFTR. Furthermore, the K1250M mutation at the Walker A motif and the D1370N mutation at the Walker B motif, both known to impair ATP hydrolysis at NBD2, completely abolished the VSOR regulation by CFTR. Thus, we conclude that an ATP-hydrolysable conformation of NBD2 is essential for the regulation of the VSOR by the CFTR protein, and that VSOR is a first channel regulated by CFTR through its NBD2.