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.     

Mechanism of chloride permeation in the cystic fibrosis transmembrane conductance regulator chloride channel

The cystic fibrosis transmembrane conductance regulator (CFTR) functions as a Cl- channel important in transepithelial salt and water transport. While there is a paucity of direct structural information on CFTR, much has been learned about the molecular determinants of the CFTR Cl- channel pore region and the mechanism of Cl- permeation through the pore from indirect structure-function studies. The first and sixth transmembrane regions of the CFTR protein play major roles in forming the channel pore and determining its functional properties by interacting with permeating Cl- ions. Positively charged amino acid side-chains are involved in attracting negatively charged Cl- ions into the pore region, where they interact briefly with a number of discrete sites on the pore walls. The pore appears able to accommodate more than one Cl- ion at a time, and Cl- ions bound inside the pore are probably sensitive to one another's presence. Repulsive interactions between Cl- ions bound concurrently within the pore may be important in ensuring rapid movement of Cl- ions through the pore. Chloride ion binding sites also interact with larger anions that can occlude the pore and block Cl- permeation, thus inhibiting CFTR function. Other ions besides Cl- are capable of passing through the pore, and specific amino acid residues that may be important in allowing the channel to discriminate between different anions have been identified. This brief review summarizes these mechanistic insights and tries to incorporate them into a simple cartoon model depicting the interactions between the channel and Cl- ions that are important for ion translocation.     

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.     

Interaction between calcium-activated chloride channels and the cystic fibrosis transmembrane conductance regulator

We investigated interactions between cystic fibrosis conductance regulator (CFTR) and endogenous Ca2+-activated Cl- channels (CaCC) in bovine pulmonary artery endothelium (CPAE). CPAE cells, which do not express CFTR, were transiently transfected with wild-type (WT) CFTR and the deletion mutant deltaF508 CFTR. Currents through CaCC were significantly reduced after expression of WT CFTR. This inhibition was increased by stimulation (isobutylmethylxanthine, forskolin) of CFTR in cells expressing WT CFTR. There were no such effects when deltaF508 mutant CFTR, which is retained in the endoplasmic reticulum, was expressed. It is concluded that CFTR and CaCC are functionally coupled probably through a direct channel-channel interaction.     

Expression of cystic fibrosis transmembrane conductance regulator in the human distal lung

The determination of the expression of cystic fibrosis transmembrane conductance regulator (CFTR) in the lung is essential for a full understanding of the normal lung physiology and the pathogenesis of the lung disease in cystic fibrosis (CF). However, studies on the expression of CFTR in the distal adult human lung have yielded conflicting results despite functional evidence of expression of CFTR in bronchiolar and alveolar epithelial cells. We used 2 high-affinity monoclonal anti-CFTR antibodies, MAb24-1 and MAb13-1, to determine the expression of CFTR in samples of bronchiolar and alveolar tissues obtained from the same non-CF individuals. CFTR immunostaining was detected in the epithelium of bronchiolar and alveolar tissues. The staining pattern was similar with both antibodies. In bronchioles, CFTR labeling was present mostly in ciliated cells; in alveoli, CFTR labeling was detected in both type I and type II cells. We conclude that CFTR is expressed in human bronchiolar and alveolar epithelial cells. The potential importance of CFTR expression in alveoli should be further investigated, particularly with respect to the CF lung disease and the physiology of the alveolar region.     

Incomplete rescue of cystic fibrosis transmembrane conductance regulator deficient mice by the human CFTR cDNA

We have used a mouse model to study the ability of human CFTR to correct the defect in mice deficient of the endogenous protein. In this model, expression of the endogenous Cftr gene was disrupted and replaced with a human CFTR cDNA by a gene targeted 'knock-in' event. Animals homozygous for the gene replacement failed to show neither improved intestinal pathology nor survival when compared to mice completely lacking CFTR. RNA analyses showed that the human CFTR sequence was transcribed from the targeted allele in the respiratory and intestinal epithelial cells. Furthermore, in vivo potential difference measurements showed that basal CFTR chloride channel activity was present in the apical membranes of both nasal and rectal epithelial cells in all homozygous knock-in animals examined. Ussing chamber studies showed, however, that the cAMP-mediated chloride channel function was impaired in the intestinal tract among the majority of homozygous knock-in animals. Hence, failure to correct the intestinal pathology associated with loss of endogenous CFTR was related to inefficient functional expression of the human protein in mice. These results emphasize the need to understand the tissue- specific expression and regulation of CFTR function when animal models are used in gene therapy studies.      

The cystic fibrosis transmembrane conductance regulator is not a base transporter in isolated duodenal epithelial cells

Duodenal epithelial bicarbonate secretion has previously been shown to be greatly impaired in mice deficient of the cystic fibrosis transmembrane conductance regulator (CFTR). It has been proposed that transmembranal bicarbonate transport occurs through the CFTR channel itself. In the present study, the transport of acid/base equivalents across the plasma membrane of proximal duodenal epithelial cells from CFTR deficient mice was compared with that of cells from normal littermates. Mixed epithelial cells from both villi and crypts were isolated from proximal duodenum and intracellular pH was assessed by cuvette-based fluorescence spectrometry using the pH sensitive dye 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein. The steady state intracellular pH, the acid extrusion rate and the alkaline extrusion rate were unaffected by CFTR deficiency in the presence of CO(2)/HCO(-)(3). Forskolin had no effect on acid extrusion or alkaline extrusion rates. In control experiments without CO(2)/HCO(-)(3), the intrinsic buffering capacities, the steady state intracellular pH and the acid extrusion rates were equivalent in the cells from CFTR deficient mice and normal littermates. The results are consistent with a model where acid/base transport is almost exclusively mediated by the previously described transporters in the murine duodenum (i.e. Na+/H+ exchange, Cl(-)/HCO(-)(3). exchange and Na+:HCO(-)(3). cotransport). There were no evidence for significant CFTR dependent HCO(-)(3). transport in proximal duodenal epithelial cells of mixed villus and crypt origin.     

Expression of cystic fibrosis transmembrane conductance regulator during early human embryo development

Formation of the blastocyst is one of the first morphological changes in early embryonic development. Ion transport has been shown to be crucial for blastocoele cavity formation and expansion, although the mechanisms that underlie this process are presently unknown. As a transmembrane Cl(-) channel, the cystic fibrosis transmembrane conductance regulator (CFTR) may participate in ion transport and early blastocoele formation. CFTR mRNA was detected throughout preimplantation embryo development and in the unfertilized oocyte. Immunocytochemistry disclosed the presence of CFTR protein from the 8-cell stage, reaching maximum immunoreactivity at early blastocyst stage embryos. Patch clamp electrophysiology of morulae and blastocysts demonstrated typical CFTR Cl(-) channel activities in the apical membrane of trophectoderm cells. Thus CFTR is expressed both at mRNA and protein levels in human morulae and blastocysts, and functions as a cAMP-regulated apical membrane Cl(-) channel. These data suggest that CFTR may contribute to blastocoele formation in the early human embryo.     

囊性纤维变性跨膜调节器基因突变对男性生殖的影响

1　ＣＦＴＲ基因突变及主要症状囊性纤维变性跨膜调节器 (ｃｙｓｔｉｃｆｉｂｒｏｓｉｓｔｒａｎｓ ｍｅｍｂｒａｎｅｃｏｎｄｕｃｔａｎｃｅｒｅｇｕｌａｔｏｒ,ＣＦＴＲ)基因于 1989年被发现 ,位于人的第 7染色体上。其编码的蛋白是一种膜蛋白 ,这种跨膜蛋白是一个Ｃｌ- 离子通道蛋白 ,受ｃＡＭＰ的调控。正常的ＣＦＴＲ由 14 80个氨基酸组成 ,含有两个结构区 ,每个结构区含有 6个跨膜片段[1] ,由称为Ｒ -结构域的多肽链连接 ,Ｒ -结构域上有大量的磷酸化位点 ,还有两个核苷酸结合位点(ｎｕｃｌｅｏｔｉｄｅｂｉｎｄｉｎｇｆｏｌｄｓ ,Ｎ…     

Localization studies of rare missense mutations in cystic fibrosis transmembrane conductance regulator (CFTR) facilitate interpretation of genotype-phenotype relationships

We have been investigating the functional consequences of rare disease-associated amino acid substitutions in the cystic fibrosis transmembrane conductance regulator (CFTR). Mutations of the arginine residue at codon 1070 have been associated with different disease consequences; R1070P and R1070Q with "severe" pancreatic insufficient cystic fibrosis (CF) and R1070W with "mild" pancreatic sufficient CF or congenital bilateral absence of the vas deferens. Intriguingly, CFTR bearing each of these mutations is functional when expressed in nonpolarized cells. To determine whether R1070 mutations cause disease by affecting CFTR localization, we created polarized Madin Darby canine kidney (MDCK) cell lines that express either wild-type or mutant CFTR from the same genomic integration site. Confocal microscopy and biotinylation studies revealed that R1070P was not inserted into the apical membrane, R1070W was inserted at levels reduced from wild-type while R1070Q was present in the apical membrane at levels comparable to wild-type. The abnormal localization of CFTR bearing R1070P and R1070W was consistent with deleterious consequences in patients; however, the profile of CFTR R1070Q was inconsistent with a "severe" phenotype. Reanalysis of 16 patients with the R1070Q mutation revealed that 11 carried an in cis nonsense mutation, S466X. All 11 patients carrying the complex allele R1070Q-S466X had severe disease, while 4 out of 5 patients with R1070Q had "mild" disease, thereby reconciling the apparent discrepancy between the localization studies of R1070Q and the phenotype of patients bearing this mutation. Our results emphasize that localization studies in relevant model systems can greatly assist the interpretation of the disease-causing potential of rare missense mutations.     

Reduced IL-10 secretion by CD4+ T lymphocytes expressing mutant cystic fibrosis transmembrane conductance regulator (CFTR)

Expression of the CFTR protein is thought to be physiologically important only in exocrine epithelial cells. However, chronic respiratory inflammation and infection remain unexplained phenomena in disease pathogenesis. Non-transformed, antigen-responsive CD4⁺ T cells cloned from healthy controls and CF patients homozygous or heterozygous for the δF508 mutation transcribed CFTR mRNA and expressed immunoreactive cytoplasmic CFTR protein. T cell clones (TCC) from controls and CF patients displayed equivalent Ca²⁺-mediated Cl⁻ current; however, TCC from patients with CF but not controls displayed defective cAMP-mediated Cl⁻ current. Although CF-derived TCC preserved mitogen and antigen proliferative responses and specificity to tetanus toxoid epitopes, they selectively secreted ≈ 45% less IL-10 compared with control TCC after activation with concanavalin A (Con A) (624 ± 101 versus 1564 ± 401 pg/ml per 10⁶ cells, respectively; P = 0.04) or anti-CD3/phorbol ester (5148 ± 1634 versus 11 788 ± 2390 pg/ml; P = 0.05). This difference was independent of atopy. Secretion of interferon-gamma, IL-2, and IL-4 was comparable in CF and control TCC after both forms of activation, while IL-5 was reduced in CF TCC following anti-CD3/phorbol myristate acetate (PMA) but not after Con A. We conclude that expression of mutant CFTR in human TCC is accompanied by ion channel dysfunction characteristic of the CF phenotype, and is accompanied by a reduction in IL-10 secretion after polyclonal activation. It is possible that disruption of IL-10-mediated anti-inflammatory homeostasis may contribute to early onset sustained inflammation in CF airways.