CFTR targeting in epithelial cells

We used polarized and nonpolarized colonic cell lines (HT-29) to correlate CFTR function and expression with epithelial cell morphogenesis. Unpolarized cells express levels of CFTR mRNA and protein that are equivalent to those observed in polarized cells, and the extent of CFTR glycosylation is also similar. Despite these similarities in CFTR expression, the polarized cells secreted Cl in response tocAMP, but there was nocAMP-stimulated Cl conductance response in the unpolarized cells. In the polarized cells, CFTR is localized in the apical membrane domain, but in unpolarized cells the protein is retained at a perinuclear location. These findings indicate that a peripheral targeting mechanism, distal to the Golgi cisternae, controls the progression of N-linked glycoproteins like CFTR to the apical membrane. This targeting process does not become active until epithelial cells polarize. It may determine whether mutant forms of CFTR are targeted to the apical membrane.     

The porcine chloride channel calcium-activated family member pCLCA4a mirrors lung expression of the human hCLCA4

Pig models of cystic fibrosis (CF) have recently been established that are expected to mimic the human disease closer than mouse models do. The human CLCA (originally named chloride channels, calcium-activated) member hCLCA4 is considered a potential modifier of disease severity in CF, but its murine ortholog, mCLCA6, is not expressed in the mouse lung. Here, we have characterized the genomic structure, protein processing, and tissue expression patterns of the porcine ortholog to hCLCA4, pCLCA4a. The genomic structure and cellular protein processing of pCLCA4a were found to closely mirror those of hCLCA4 and mCLCA6. Similar to human lung, pCLCA4a mRNA was strongly expressed in porcine lungs, and the pCLCA4a protein was immunohistochemically detected on the apical membranes of tracheal and bronchial epithelial cells. This stands in sharp contrast to mouse mCLCA6, which has been detected exclusively in intestinal epithelia but not the murine lung. The results may add to the understanding of species-specific differences in the CF phenotype and support the notion that the CF pig model may be more suitable than murine models to study the role of hCLCA4.     

Establishment and characterization of a novel polarized MDCK epithelial cellular model for CFTR studies.

F508del is the most common mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that is responsible for the genetic disease Cystic Fibrosis (CF). It results in a major failure of CFTR to traffic to the apical membrane of epithelial cells, where it should function as a chloride (Cl-) channel. Most studies on localization, processing and cellular trafficking of wild-type (wt) and F508del-CFTR have been performed in non-epithelial cells. Notwithstanding, polarized epithelial cells possess distinctly organized and regulated membrane trafficking pathways. We have used Madin-Darby canine kidney (MDCK) type II cells (proximal tubular cells which do not express endogenous CFTR) to generate novel epithelial, polarized cellular models stably expressing wt- or F508del-CFTR through transduction with recombinant lentiviral vectors. Characterization of these cell lines shows that wt-CFTR is correctly processed and apically localized, producing a cAMP-activated Cl- conductance. In contrast, F508del-CFTR is mostly detected in itsimmature form, localized intracellularly and producing only residual Cl- conductance. These novel cell lines constitute bona fide models and significantly improved resources to investigate the molecular mechanisms of polarized membrane traffic of wt- and F508del-CFTR in the same cellular background. They are also useful to identify/validate novel therapeutic compounds for CF.     

Apical CFTR Expression in Human Nasal Epithelium Correlates with Lung Disease in Cystic Fibrosis

Introduction

Although most individuals with cystic fibrosis (CF) develop progressive obstructive lung disease, disease severity is highly variable, even for individuals with similar CFTR mutations. Measurements of chloride transport as expression of CFTR function in nasal epithelial cells correlate with pulmonary function and suggest that F508del-CFTR is expressed at the apical membrane. However, an association between quantitative apical CFTR expression in nasal epithelium and CF disease severity is still missing.

Methods and Materials

Nasal epithelial cells from healthy individuals and individuals with CF between 12–18 years were obtained by nasal brushing. Apical CFTR expression was measured by confocal microscopy using CFTR mAb 596. Expression was compared between both groups and expression in CF nasal epithelial cells was associated with standardized pulmonary function (FEV₁%).

Results

The proportion of cells expressing apical CFTR in columnar epithelium is lower in CF compared to non-CF. The apical CFTR expression level was significantly correlated with FEV₁% in F508del homozygous subjects (r = 0.63, p = 0.012).

Conclusion

CFTR expression in nasal epithelial cells is lower in subjects with CF compared to healthy subjects. The proportion of cells expressing F508del-CFTR at the apical membrane is variable between subjects and is positively correlated with FEV₁% in F508del-CFTR homozygous subjects.     

Interleukin-13 interferes with CFTR and AQP5 expression and localization during human airway epithelial cell differentiation

Interleukin-13 (IL-13) is a central regulator of Th2-dominated respiratory disorders such as asthma. Lesions of the airway epithelial barrier frequently observed in chronic respiratory inflammatory diseases are repaired through proliferation, migration and differentiation of epithelial cells. Our work is focused on the effects of IL-13 in human cellular models of airway epithelial cell regeneration. We have previously shown that IL-13 altered epithelial cell polarity during mucociliary differentiation of human nasal epithelial cells. In particular, the cytokine inhibited ezrin expression and interfered with its apical localization during epithelial cell differentiation in vitro. Here we show that CFTR expression is enhanced in the presence of the cytokine, that two additional CFTR protein isoforms are expressed in IL-13-treated cells and that part of the protein is retained within the endoplasmic reticulum. We further show that aquaporin 5 expression, a water channel localized within the apical membrane of epithelial cells, is completely abolished in the presence of the cytokine. These results show that IL-13 interferes with ion and water channel expression and localization during epithelial regeneration and may thereby influence mucus composition and hydration.     

CFTR expression analysis in human nasal epithelial cells by flow cytometry

Rationale

Unbiased approaches that study aberrant protein expression in primary airway epithelial cells at single cell level may profoundly improve diagnosis and understanding of airway diseases. We here present a flow cytometric procedure to study CFTR expression in human primary nasal epithelial cells from patients with Cystic Fibrosis (CF). Our novel approach may be important in monitoring of therapeutic responses, and better understanding of CF disease at the molecular level.

Objectives

Validation of a panel of CFTR-directed monoclonal antibodies for flow cytometry and CFTR expression analysis in nasal epithelial cells from healthy controls and CF patients.

Methods

We analyzed CFTR expression in primary nasal epithelial cells at single cell level using flow cytometry. Nasal cells were stained for pan-Cytokeratin, E cadherin, and CD45 (to discriminate epithelial cells and leukocytes) in combination with intracellular staining of CFTR. Healthy individuals and CF patients were compared.

Measurements and Main Results

We observed various cellular populations present in nasal brushings that expressed CFTR protein at different levels. Our data indicated that CF patients homozygous for F508del express varying levels of CFTR protein in nasal epithelial cells, although at a lower level than healthy controls.

Conclusion

CFTR protein is expressed in CF patients harboring F508del mutations but at lower levels than in healthy controls. Multicolor flow cytometry of nasal cells is a relatively simple procedure to analyze the composition of cellular subpopulations and protein expression at single cell level.     

Applicability of different antibodies for the immunohistochemical localization of CFTR in respiratory and intestinal tissues of human and murine origin.

Cystic fibrosis (CF) is caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR) protein, which has a major role as a chloride (Cl(-)) channel. Although perhaps all functions of CFTR are still not fully characterized, localization studies are necessary to understand the consequences of the more than 1000 mutations thus far identified. Our aim was to determine the histological localization of CFTR on respiratory and colon epithelia of human and murine origin with a panel of several antibodies produced against different CFTR epitopes, using an indirect immunofluorescence method. Our results on human tissues confirm the apical localization of CFTR in ciliated cells of the respiratory mucosa and show that in colon tissue CFTR is observed in both apical and basolateral membranes of epithelial cells from colon crypts. However, poor tissue preservation of colon biopsies after immunohistochemistry (IHC) raises doubts about the latter localization. Contrary to human, mouse colon epithelium (not biopsed) presents good tissue preservation and evidences many cylindrical surface cells with high apical expression of CFTR. For the antibodies' sensitivity, we demonstrate that MATG1061, 24-1, M3A7, and MPCT-1 give good results, allowing the histological localization of CFTR protein of both human and murine origin.     

Matrine modulates HSC70 levels and rescues ΔF508-CFTR

Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-dependent Cl(-) channel located in the plasma membrane, and its malfunction results in cystic fibrosis (CF), the most common lethal genetic disease in Caucasians. Most CF patients carry the deletion of Phe508 (ΔF508 mutation); this mutation prevents the delivery of the CFTR to its correct cellular location, the apical (lumen-facing) membrane of epithelial cells. Molecular chaperones play a central role in determining the fate of ΔF508-CFTR. In this report, we show that the Matrine, a quinolizidine alkaloid, downregulates the expression of the molecular chaperone HSC70 and increases the protein levels of ΔF508-CFTR in human alveolar basal epithelial cells (A549 cell line), stably transfected with a ΔF508-CFTR-expressing construct. Moreover, Matrine induced ΔF508-CFTR release from endoplasmic reticulum to cell cytosol and its localization on the cell membrane. Interestingly, downregulation of HSC70 resulted in increased levels of ΔF508-CFTR complexes with the co-chaperone BAG3 that in addition appeared to co-localize with the mutated protein on the cell surface. These results shed new light on ΔF508-CFTR interactions with proteins of the chaperones/co-chaperones system and could be useful in strategies for future medical treatments for CF.     

Murine CLCA5 is uniquely expressed in distinct niches of airway epithelial cells

The murine mCLCA5 protein is a member of the chloride channel regulators, calcium-activated (CLCA) family and is suspected to play a role in airway mucus cell differentiation. Although mCLCA5 mRNA was previously found in total lung extracts, the expressing cells and functions in the naive murine respiratory tract are unknown. Therefore, mCLCA5 protein expression was identified by immunohistochemistry and confocal laser scanning microscopy using entire lung sections of naive mice. Moreover, we determined mRNA levels of functionally related genes (mClca3, mClca5, Muc5ac and Muc5b) and quantified mCLCA5-, mCLCA3- and CC10-positive cells and periodic acid-Schiff-positive mucus cells in naive, PBS-treated or Staphylococcus aureus-infected mice. We also investigated mCLCA5 protein expression in Streptococcus pneumoniae and influenza virus lung infection models. Finally, we determined species-specific differences in the expression patterns of the murine mCLCA5 and its human and porcine orthologs, hCLCA2 and pCLCA2. The mCLCA5 protein is uniquely expressed in highly select bronchial epithelial cells and submucosal glands in naive mice, consistent with anatomical locations of progenitor cell niches. Under conditions of challenge (PBS, S. aureus, S. pneumoniae, influenza virus), mRNA and protein expression strongly declined with protein recovery only in models retaining intact epithelial cells. In contrast to mice, human and porcine bronchial epithelial cells do not express their respective mCLCA5 orthologs and submucosal glands had fewer expressing cells, indicative of fundamental differences in mice versus humans and pigs.     

Beta-oestradiol rescues DeltaF508CFTR functional expression in human cystic fibrosis airway CFBE41o- cells through the up-regulation of NHERF1.

BACKGROUND INFORMATION: CF (cystic fibrosis) is a disease caused by mutations within the CFTR (CF transmembrane conductance regulator) gene. The most common mutation, DeltaF508 (deletion of Phe-508), results in a protein that is defective in folding and trafficking to the cell surface but is functional if properly localized in the plasma membrane. We have recently demonstrated that overexpression of the PDZ protein NHERF1 (Na(+)/H(+)-exchanger regulatory factor 1) in CF airway cells induced both a redistribution of DeltaF508CFTR from the cytoplasm to the apical membrane and the PKA (protein kinase A)-dependent activation of DeltaF508CFTR-dependent chloride secretion. In view of the potential importance of the targeted up-regulation of NHERF1 in a therapeutic context, and since it has been demonstrated that oestrogen treatment increases endogenous NHERF1 expression, we tested the hypothesis that oestrogen treatment can increase NHERF1 expression in a human bronchiolar epithelial CF cell line, CFBE41o(-), with subsequent rescue of apical DeltaF508CFTR chloride transport activity. RESULTS: We found that CFBE41o(-) cells do express ERs (oestrogen receptors) in the nuclear fraction and that beta-oestradiol treatment was able to significantly rescue DeltaF508CFTR-dependent chloride secretion in CFBE41o(-) cell monolayers with a peak between 6 and 12 h of treatment, demonstrating that the DeltaF508CFTR translocated to the apical membrane can function as a cAMP-responsive channel, with a significant increase in chloride secretion noted at 1 nM beta-oestradiol and a maximal effect observed at 10 nM. Importantly, knock-down of NHERF1 expression by transfection with siRNA (small interfering RNA) for NHERF1 inhibited the beta-oestradiol-dependent increase in DeltaF508CFTR protein expression levels and completely prevented the beta-oestradiol-dependent rescue of DeltaF508CFTR transport activity. CONCLUSIONS: These results demonstrate that beta-oestradiol-dependent up-regulation of NHERF1 significantly increases DeltaF508CFTR functional expression in CFBE41o(-) cells.     

The role of regulated CFTR trafficking in epithelial secretion

The focus of this review is the regulated trafficking of the cystic fibrosis transmembrane conductance regulator (CFTR) in distal compartments of the protein secretory pathway and the question of how changes in CFTR cellular distribution may impact on the functions of polarized epithelial cells. We summarize data concerning the cellular localization and activity of CFTR and attempt to synthesize often conflicting results from functional studies of regulated endocytosis and exocytosis in CFTR-expressing cells. In some instances, findings that are inconsistent with regulated CFTR trafficking may result from the use of overexpression systems or nonphysiological experimental conditions. Nevertheless, judging from data on other transporters, an appropriate cellular context is necessary to support regulated CFTR trafficking, even in epithelial cells. The discovery that disease mutations can influence CFTR trafficking in distal secretory and recycling compartments provides support for the concept that regulated CFTR recycling contributes to normal epithelial function, including the control of apical CFTR channel density and epithelial protein secretion. Finally, we propose molecular mechanisms for regulated CFTR endocytosis and exocytosis that are based on CFTR interactions with other proteins, particularly those whose primary function is membrane trafficking. These models provide testable hypotheses that may lead to elucidation of CFTR trafficking mechanisms and permit their experimental manipulation in polarized epithelial cells.     

Naturally Occurring Deletion Mutants of the Pig-Specific,Intestinal Crypt Epithelial Cell Protein CLCA4b without Apparent Phenotype

The human CLCA4 (chloride channel regulator, calcium-activated) modulates the intestinal phenotype of cystic fibrosis (CF) patients via an as yet unknown pathway. With the generation of new porcine CF models, species-specific differences between human modifiers of CF and their porcine orthologs are considered critical for the translation of experimental data. Specifically, the porcine ortholog to the human CF modulator gene CLCA4 has recently been shown to be duplicated into two separate genes, CLCA4a and CLCA4b. Here, we characterize the duplication product, CLCA4b, in terms of its genomic structure, tissue and cellular expression patterns as well as its in vitro electrophysiological properties. The CLCA4b gene is a pig-specific duplication product of the CLCA4 ancestor and its protein is exclusively expressed in small and large intestinal crypt epithelial cells, a niche specifically occupied by no other porcine CLCA family member. Surprisingly, a unique deleterious mutation of the CLCA4b gene is spread among modern and ancient breeds in the pig population, but this mutation did not result in an apparent phenotype in homozygously affected animals. Electrophysiologically, neither the products of the wild type nor of the mutated CLCA4b genes were able to evoke a calcium-activated anion conductance, a consensus feature of other CLCA proteins. The apparently pig-specific duplication of the CLCA4 gene with unique expression of the CLCA4b protein variant in intestinal crypt epithelial cells where the porcine CFTR is also present raises the question of whether it may modulate the porcine CF phenotype. Moreover, the naturally occurring null variant of CLCA4b will be valuable for the understanding of CLCA protein function and their relevance in modulating the CF phenotype.     

Downregulated in adenoma and putative anion transporter are regulated by CFTR in cultured pancreatic duct cells

The mechanism of the pancreatic ductal HCO secretion defect in cystic fibrosis (CF) is not well defined. However, a lack of apical Cl(-)/HCO exchange may exist in CF. To test this hypothesis, we examined the expression of Cl(-)/HCO exchangers in cultured pancreatic duct epithelial cells with physiological features prototypical of CF [CFPAC-1 cells lacking a functional CF transmembrane conductance regulator (CFTR)] or normal duct cells (CFPAC-1 cells transfected with functional wild-type CFTR, CFPAC-WT). Cl(-)/HCO exchange activity, assayed with the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein in cells grown on coverslips, increased about twofold in cells transfected with functional CFTR. This correlated with increased apical (36)Cl influx in cells expressing functional CFTR and grown on permeable support. Northern hybridizations indicated the induction of downregulated in adenoma (DRA) in cells expressing functional CFTR. The expression of putative anion transporter PAT1 also increased significantly in cells expressing functional CFTR. DRA was detected at high levels in native mouse pancreas by Northern hybridization and localized to the apical domain of the duct cells by immunohistochemical studies. In conclusion, CFTR upregulates DRA and PAT1 expression in cultured pancreatic duct cells. We propose that the pancreatic HCO secretion defect in CF patients is partly due to the downregulation of apical Cl(-)/HCO exchange activity mediated by DRA (and possibly PAT1).     

Pseudomonas aeruginosa inhibits endocytic recycling of CFTR in polarized human airway epithelial cells

The most common mutation in the CFTR gene in individuals with cystic fibrosis (CF), F508, leads to the absence of CFTR Cl^– channels in the apical plasma membrane, which in turn results in impairment of mucociliary clearance, the first line of defense against inhaled bacteria. Pseudomonas aeruginosa is particularly successful at colonizing and chronically infecting the lungs and is responsible for the majority of morbidity and mortality in patients with CF. Rescue of F508-CFTR by reduced temperature or chemical means reveals that the protein is at least partially functional as a Cl^– channel. Thus current research efforts have focused on identification of drugs that restore the presence of CFTR in the apical membrane to alleviate the symptoms of CF. Because little is known about the effects of P. aeruginosa on CFTR in the apical membrane, whether P. aeruginosa will affect the efficacy of new drugs designed to restore the plasma membrane expression of CFTR is unknown. Accordingly, the objective of the present study was to determine whether P. aeruginosa affects CFTR-mediated Cl^– secretion in polarized human airway epithelial cells. We report herein that a cell-free filtrate of P. aeruginosa reduced CFTR-mediated transepithelial Cl^– secretion by inhibiting the endocytic recycling of CFTR and thus the number of WT-CFTR and F508-CFTR Cl^– channels in the apical membrane in polarized human airway epithelial cells. These data suggest that chronic infection with P. aeruginosa may interfere with therapeutic strategies aimed at increasing the apical membrane expression of F508-CFTR. cystic fibrosis     

Defective CFTR increases synthesis and mass of sphingolipids that modulate membrane composition and lipid signaling

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) that affect protein structure and channel function. CFTR, localized in the apical membrane within cholesterol and sphingomyelin rich regions, is an ABC transporter that functions as a chloride channel. Here, we report that expression of defective CFTR (ΔF508CFTR or decreased CFTR) in human lung epithelial cell lines increases sphingolipid synthesis and mass of sphinganine, sphingosine, four long-chain saturated ceramide species, C16 dihydroceramide, C22, C24, C26-ceramide, and sphingomyelin, and decreases mass of C18 and unsaturated C18:1 ceramide species. Decreased expression of CFTR is associated with increased expression of long-chain base subunit 1 of serine-palmitoyl CoA, the rate-limiting enzyme of de novo sphingolipid synthesis and increased sphingolipid synthesis. Overexpression of ΔF508CFTR in bronchoalveolar cells that do not express CFTR increases sphingolipid synthesis and mass, whereas overexpression of wild-type CFTR, but not of an unrelated ABC transporter, ABCA7, decreases sphingolipid synthesis and mass. The data are consistent with a model in which CFTR functions within a feedback system that affects sphingolipid synthesis and in which increased sphingolipid synthesis could reflect a physiological response to sequestration of sphingolipids or altered membrane structure.     

Internalization of cationized ferritin by isolated pancreatic acinar cells

The internalization of cationized ferritin (CF) was studied in isolated pancreatic acinar cells in vitro. Horseradish peroxidase (HRP) was used in conjunction with CF to compare internalization of soluble-phase and membrane-bound tracers. The mode of internalization of CF was dependent upon tracer concentration and origin of the plasma membrane (apical vs. lateral-basal). At the lower tracer concentrations (0.19 and 0.38 mg/ml), internalization from the apical cell surface occurred via small vesicles. The tracer then appeared in multivesicular bodies, in tubules, and in irregular membrane-bound structures. After 15 min, CF particles were seen in many small vesicles near the Golgi apparatus, but not in the Golgi saccules. In contrast, at the lateral-basal cell surface the CF particles tended to form clusters. These clusters were more pronounced at higher CF concentrations (0.76 and 1.5 mg/ml) and were associated with elongated cellular processes, which seemed to engulf CF accumulations in a phagocytic manner. Once internalized, CF was found primarily in large irregular structures which appeared to migrate slowly toward the nucleus, reaching a juxtanuclear position after approximately 30 min. CF was observed in lysosomes after 30-45 min and by 90 min most of the CF was confined to large vacuoles and to trimetaphosphatase-positive lysosomes. Similar routes were observed when cells were double-labeled with CF and HRP, where endocytic structures showed co-localization of both tracers. The results of this study indicate the importance of the Golgi region in the intracellular sorting of internalized apical membrane. Furthermore, this work confirms the presence of distinct endocytic pathways at the apical and lateral-basal cell surfaces.     

Expression of MAL and MAL2, two elements of the protein machinery for raft-mediated transport, in normal and neoplastic human tissue

Polarized transport of lipids and proteins to the apical and basolateral membrane subdomains is essential for the functioning of epithelial cells. Apical transport is mediated by a direct route from the Golgi and an indirect route, referred to as transcytosis, involving the transport of the protein to the basolateral membrane followed by its internalization and subsequent transcellular transport to the apical subdomain. MAL and MAL2 have been demonstrated to be essential components of the machinery for the direct and indirect routes, respectively. Herein, we review the range of expression of MAL and MAL2 in normal human tissue and compare it with that of neoplastic tissue. Our analysis provides insight into the potential use of MAL- and MAL2-mediated pathways in many types of epithelial cells as well as in nonepithelial cells. In addition, the specific alterations in MAL and/or MAL2 expression observed in specific types of carcinoma provides a basis to understand the loss of the polarized phenotype that frequently accompanies the neoplastic transformation process. This points out potential applications of MAL and MAL2 as markers for tumor characterization.     

Beta-oestradiol rescues DeltaF508CFTR functional expression in human cystic fibrosis airway CFBE41o- cells through the up-regulation of NHERF1

Background information. CF (cystic fibrosis) is a disease caused by mutations within the CFTR (CF transmembrane conductance regulator) gene. The most common mutation, ΔF508 (deletion of Phe‐508), results in a protein that is defective in folding and trafficking to the cell surface but is functional if properly localized in the plasma membrane. We have recently demonstrated that overexpression of the PDZ protein NHERF1 (Na⁺/H⁺‐exchanger regulatory factor 1) in CF airway cells induced both a redistribution of ΔF508CFTR from the cytoplasm to the apical membrane and the PKA (protein kinase A)‐dependent activation of ΔF508CFTR‐dependent chloride secretion. In view of the potential importance of the targeted up‐regulation of NHERF1 in a therapeutic context, and since it has been demonstrated that oestrogen treatment increases endogenous NHERF1 expression, we tested the hypothesis that oestrogen treatment can increase NHERF1 expression in a human bronchiolar epithelial CF cell line, CFBE41o⁻, with subsequent rescue of apical ΔF508CFTR chloride transport activity. Results. We found that CFBE41o⁻ cells do express ERs (oestrogen receptors) in the nuclear fraction and that β‐oestradiol treatment was able to significantly rescue ΔF508CFTR‐dependent chloride secretion in CFBE41o⁻ cell monolayers with a peak between 6 and 12 h of treatment, demonstrating that the ΔF508CFTR translocated to the apical membrane can function as a cAMP‐responsive channel, with a significant increase in chloride secretion noted at 1 nM β‐oestradiol and a maximal effect observed at 10 nM. Importantly, knock‐down of NHERF1 expression by transfection with siRNA (small interfering RNA) for NHERF1 inhibited the β‐oestradiol‐dependent increase in ΔF508CFTR protein expression levels and completely prevented the β‐oestradiol‐dependent rescue of ΔF508CFTR transport activity. Conclusions. These results demonstrate that β‐oestradiol‐dependent up‐regulation of NHERF1 significantly increases ΔF508CFTR functional expression in CFBE41o⁻ cells.     

Gastric mucosal cell homeostatic physiome. Critical role of ER-initiated membranes restitution in the fidelity of cell function renewal.

Homeostatic cell physiology is preserved through the fidelity of the cell membranes restitution. The task is accomplished through the assembly of the precisely duplicated segments of the cell membranes, and transport to the site of their function. Here we examined the mechanism that initiates and directs the restitution of the intra- and extracellular membranes of gastric mucosal cell. The homeostatic restitution of gastrointestinal epithelial cell membrane components was investigated by studying the lipidomic processes in endoplasmic reticulum (ER) and Golgi. The biomembrane lipid synthesis during the formation of transport vesicles in the systems containing isolated organelle and the cell-specific cytosol (Cyt) from rat gastric mucosal epithelial cells was assessed. The results revealed that lipids of ER transport vesicle and the transmembrane and intravesicular cargo are delivered en bloc to the point of destination. En bloc delivery of proteins, incorporated into predetermined in ER lipid environment, ensures fidelity of the membrane modification in Golgi and the restitution of the lipid and protein elements that are consistent with the organelle and the cell function. The mechanism that maintains apical membrane restitution is mediated through the synthesis of membrane segments containing ceramide (Cer). The Cer-containing membranes and protein cargo are further specialized in Golgi. The portion of the vesicles destined for apical membrane renewal contains glycosphingolipids and phosphatidylinositol 3-phosphate. The vesicles containing phosphatidylinositol 4-phosphate are directed to endosomes. Our findings revealed that the preservation of the physiological equilibrium in cell structure and function is attributed to (1) a complete membrane segment synthesis in ER, (2) its transport in the form of ER-transport vesicle to Golgi, (3) the membrane components-defined maturation of lipids and proteins in Golgi, and (4) en bloc transfer of the new segment of the membrane to the cell apical membrane or intracellular organelle.