Cystic Fibrosis Transmembrane Conductance Regulator Transcripts Research Articles

A promoter-dependent upstream activator augments CFTR expression in diverse epithelial cell types

The cystic fibrosis transmembrane conductance regulator (CFTR) gene encodes an anion-selective channel found in epithelial cell membranes. Mutations in CFTR cause cystic fibrosis (CF), an inherited disorder that impairs epithelial function in multiple organs. Most men with CF are infertile due to loss of intact genital ducts. Here we investigated a novel epididymis-selective cis-regulatory element (CRE), located within a peak of open chromatin at -9.5 kb 5′ to the CFTR gene promoter. Activation of the -9.5 kb CRE alone by CRISPRa had no impact on CFTR gene expression. However, CRISPRa co-activation of the -9.5 kb CRE and the CFTR gene promoter in epididymis cells significantly augmented CFTR mRNA and protein expression when compared to promoter activation alone. This increase was accompanied by enhanced chromatin accessibility at both sites. Furthermore, the combined CRISPRa strategy activated CFTR expression in other epithelial cells that lack open chromatin at the -9.5 kb site and in which the locus is normally inactive. However, the -9.5 kb CRE does not function as a classical enhancer of the CFTR promoter in transient reporter gene assays. These data provide a novel mechanism for activating/augmenting CFTR expression, which may have therapeutic utility for mutations that perturb CFTR transcription.

Putting bicarbonate on the spot: pharmacological insights for CFTR correction in the airway epithelium.

Introduction: Cystic fibrosis (CF) is caused by defective Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) proteins. CFTR controls chloride (Cl-) and bicarbonate (HCO3 -) transport into the Airway Surface Liquid (ASL). We investigated the impact of F508del-CFTR correction on HCO3 - secretion by studying transepithelial HCO3 - fluxes. Methods: HCO3 - secretion was measured by pH-stat technique in primary human respiratory epithelial cells from healthy subjects (WT) and people with CF (pwCF) carrying at least one F508del variant. Its changes after CFTR modulation by the triple combination VX445/661/770 and in the context of TNF-α+IL-17 induced inflammation were correlated to ASL pH and transcriptional levels of CFTR and other HCO3 - transporters of airway epithelia such as SLC26A4 (Pendrin), SLC26A9 and NBCe1. Results: CFTR-mediated HCO3 - secretion was not detected in F508del primary human respiratory epithelial cells. It was rescued up to ∼ 80% of the WT level by VX-445/661/770. In contrast, TNF-α+IL-17 normalized transepithelial HCO3 - transport and increased ASL pH. This was related to an increase in SLC26A4 and CFTR transcript levels. VX-445/661/770 induced an increase in pH only in the context of inflammation. Effects on HCO3 - transport were not different between F508del homozygous and F508del compound heterozygous CF airway epithelia. Conclusion: Our studies show that correction of F508del-CFTR HCO3 - is not sufficient to buffer acidic ASL and inflammation is a key regulator of HCO3 - secretion in CF airways. Prediction of the response to CFTR modulators by theratyping should take into account airway inflammation.

Features of CFTR mRNA and implications for therapeutics development.

Cystic fibrosis (CF) is an autosomal recessive disease impacting ∼100,000 people worldwide. This lethal disorder is caused by mutation of the CF transmembrane conductance regulator (CFTR) gene, which encodes an ATP-binding cassette-class C protein. More than 2,100 variants have been identified throughout the length of CFTR. These defects confer differing levels of severity in mRNA and/or protein synthesis, folding, gating, and turnover. Drug discovery efforts have resulted in recent development of modulator therapies that improve clinical outcomes for people living with CF. However, a significant portion of the CF population has demonstrated either no response and/or adverse reactions to small molecules. Additional therapeutic options are needed to restore underlying genetic defects for all patients, particularly individuals carrying rare or refractory CFTR variants. Concerted focus has been placed on rescuing variants that encode truncated CFTR protein, which also harbor abnormalities in mRNA synthesis and stability. The current mini-review provides an overview of CFTR mRNA features known to elicit functional consequences on final protein conformation and function, including considerations for RNA-directed therapies under investigation. Alternative exon usage in the 5'-untranslated region, polypyrimidine tracts, and other sequence elements that influence splicing are discussed. Additionally, we describe mechanisms of CFTR mRNA decay and post-transcriptional regulation mediated through interactions with the 3'-untranslated region (e.g. poly-uracil sequences, microRNAs). Contributions of synonymous single nucleotide polymorphisms to CFTR transcript utilization are also examined. Comprehensive understanding of CFTR RNA biology will be imperative for optimizing future therapeutic endeavors intended to address presently untreatable forms of CF.

Downstream Alternate Start Site Allows N-Terminal Nonsense Variants to Escape NMD and Results in Functional Recovery by Readthrough and Modulator Combination.

Genetic variants that introduce premature termination codons (PTCs) have remained difficult to therapeutically target due to lack of protein product. Nonsense mediated mRNA decay (NMD) targets PTC-bearing transcripts to reduce the potentially damaging effects of truncated proteins. Readthrough compounds have been tested on PTC-generating variants in attempt to permit translation through a premature stop. However, readthrough compounds have not proved efficacious in a clinical setting due to lack of stable mRNA. Here, we investigate N-terminal variants in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which have been shown to escape NMD, potentially through a mechanism of alternative translation initiation at downstream AUG codons. We hypothesized that N-terminal variants in CFTR that evade NMD will produce stable transcript, allowing CFTR function to be restored by a combination of readthrough and protein modulator therapy. We investigate this using two cell line models expressing CFTR-expression minigenes (EMG; HEK293s and CFBEs) and primary human nasal epithelial (NE) cells, and we test readthrough compounds G418 and ELX-02 in combination with CFTR protein modulators. HEK293 cells expressing the variants E60X and L88X generate CFTR-specific core glycosylated products that are consistent with downstream translation initiation. Mutation of downstream methionines at codons 150 and 152 does not result in changes in CFTR protein processing in cells expressing L88X-CFTR-EMG. However, mutation of methionine at 265 results in loss of detectable CFTR protein in cells expressing E60X, L88X, and Y122X CFTR-EMGs, indicating that downstream translation initiation is occurring at the AUG codon at position M265. In HEK293 stable cells harboring L88X, treatment with readthrough compounds alone allows for formation of full-length, but misfolded CFTR protein. Upon addition of protein modulators in combination with readthrough, we observe formation of mature, complex-glycosylated CFTR. In CFBE and NE cells, addition of readthrough ELX-02 and modulator therapy results in substantial recovery of CFTR function. Our work indicates that N-terminal variants generate stable CFTR transcript due to translation initiation at a downstream AUG codon. Thus, individuals with CF bearing 5′ nonsense variants that evade NMD are ideal candidates for treatment with clinically safe readthrough compounds and modulator therapy.

Biphasic regulation of CFTR expression by ENaC in epithelial cells: The involvement of Ca2+-modulated cAMP production

The regulatory interaction between two typical epithelial ion channels, cystic fibrosis transmembrane conductance regulator (CFTR) and the epithelial sodium channel (ENaC), for epithelial homeostasis has been noted, although the underlying mechanisms remain unclear. Here, we report that in a human endometrial epithelial cell line (ISK), shRNA-based stable knockdown of ENaC produced a biphasic effect: a low (∼23%) degree of ENaC knockdown resulted in significant increases in CFTR mRNA and protein levels, CFTR-mediated Cl− transport activity as well as intracellular cAMP concentration, while a higher degree (∼50%) of ENaC knockdown did not further increase but restored CFTR expression and cAMP levels. The basal intracellular Ca2+ level of ISK cells was lowered by ENaC knockdown or inhibition in a degree-dependent manner. BAPTA-AM, an intracellular Ca2+ chelator that lowers free Ca2+ concentration, elevated cAMP level and CFTR mRNA expression at a low (5 µM) but not a high (50 µM) dose, mimicking the biphasic effect of ENaC knockdown. Moreover, KH-7, a selective inhibitor of soluble adenylyl cyclase (sAC), abolished the CFTR upregulation induced by low-degree ENaC knockdown or Ca2+ chelation, suggesting the involvement of sAC-driven cAMP production in the positive regulation. A luciferase reporter to indicate CFTR transcription revealed that all tested degrees of ENaC knockdown/inhibition stimulated CFTR transcription in ISK cells, suggesting that the negative regulation on CFTR expression by the high-degree ENaC deficiency might occur at post-transcription stages. Additionally, similar biphasic effect of ENaC knockdown on CFTR expression was observed in a human bronchial epithelial cell line. Taken together, these results have revealed a previously unidentified biphasic regulatory role of ENaC in tuning CFTR expression involving Ca2+-modulated cAMP production, which may provide an efficient mechanism for dynamics and plasticity of the epithelial tissues in various physiological or pathological contexts.

Carbon nanoparticles adversely affect CFTR expression and toxicologically relevant pathways

Cystic fibrosis is an autosomal recessive disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) that can lead to terminal respiratory failure. Ultrafine carbonaceous particles, which are ubiquitous in ambient urban and indoor air, are increasingly considered as major contributors to the global health burden of air pollution. However, their effects on the expression of CFTR and associated genes in lung epithelial cells have not yet been investigated. We therefore evaluated the effects of carbon nanoparticles (CNP), generated by spark-ablation, on the human bronchial epithelial cell line 16HBE14o− at air–liquid interface (ALI) culture conditions. The ALI-cultured cells exhibited epithelial barrier integrity and increased CFTR expression. Following a 4-h exposure to CNP, the cells exhibited a decreased barrier integrity, as well as decreased expression of CFTR transcript and protein levels. Furthermore, transcriptomic analysis revealed that the CNP-exposed cells showed signs of oxidative stress, apoptosis and DNA damage. In conclusion, this study describes spark-ablated carbon nanoparticles in a realistic exposure of aerosols to decrease CFTR expression accompanied by transcriptomic signs of oxidative stress, apoptosis and DNA damage.

Krüppel-Like Factor 5 Regulates CFTR Expression Through Repression by Maintaining Chromatin Architecture Coupled with Direct Enhancer Activation

Single cell RNA-sequencing has accurately identified cell types within the human airway that express the Cystic Fibrosis Transmembrane Conductance regulator (CFTR) gene. Low abundance CFTR transcripts are seen in many secretory cells, while high levels are restricted to rare pulmonary ionocytes. Here we focus on the mechanisms coordinating basal CFTR expression in the secretory compartment. Cell-selective regulation of CFTR is achieved within its invariant topologically associating domain by the recruitment of cis-regulatory elements (CREs). CRE activity is coordinated by cell-type-selective transcription factors. One such factor, Krüppel-Like Factor 5 (KLF5), profoundly represses CFTR transcript and protein in primary human airway epithelial cells and airway cell lines. Here we reveal the mechanism of action of KLF5 upon the CFTR gene. We find that depletion or ablation of KLF5 from airway epithelial cells changes higher order chromatin structure at the CFTR locus. Critical looping interactions that are required for normal gene expression are altered, the H3K27ac active chromatin mark is redistributed, and CTCF occupancy is modified. However, mutation of a single KLF5 binding site within a pivotal airway cell CRE abolishes CFTR expression. Hence, KLF5 has both direct activating and indirect repressive effects, which together coordinate CFTR expression in the airway.

A Hyperglycemic Microenvironment Inhibits Tendon-to-Bone Healing through the let-7b-5p/CFTR Pathway.

Background Tendon-to-bone healing is a difficult process in treatment of rotator cuff tear (RCT). In addition, diabetes is an important risk factor for poor tendon-to-bone healing. Therefore, we investigated the specific mechanisms through which diabetes affects tendon-to-bone healing by regulating the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). Methods Tendon-derived stem cells (TDSCs) were extracted from rats after which their proliferative capacities were evaluated by the MTT assay. The expression levels of CFTR and tendon-related markers were determined by qRT-PCR. Then, bioinformatics analyses and dual luciferase reporter gene assays were used to identify miRNAs with the ability to bind CFTR mRNA. Finally, CFTR was overexpressed in TDSCs to validate the specific mechanisms through which the high glucose microenvironment inhibits tendon-to-bone healing. Results The high glucose microenvironment downregulated mRNA expression levels of tendon-related markers and CFTR in TDSCs cultured with different glucose concentrations. Additionally, bioinformatics analyses revealed that let-7b-5p may be regulated by the high glucose microenvironment and can regulate CFTR levels. Moreover, a dual luciferase reporter gene assay was used to confirm that let-7b-5p targets and binds CFTR mRNA. Additional experiments also confirmed that overexpressed CFTR effectively reversed the negative effects of the hyperglycaemic microenvironment and upregulation of let-7b-5p on TDSC proliferation and differentiation. These findings imply that the hyperglycemic microenvironment inhibits CFTR transcription and, consequently, proliferation and differentiation of TDSCs in vitro by upregulating let-7b-5p. Conclusions A hyperglycemic microenvironment inhibits TDSC proliferation in vitro via the let-7b-5p/CFTR pathway, and this is a potential mechanism in diabetes-induced poor tendon-to-bone healing.

Mesenchymal Stem Cell exosome delivered Zinc Finger Protein activation of cystic fibrosis transmembrane conductance regulator

Cystic fibrosis is a genetic disorder that results in a multi‐organ disease with progressive respiratory decline which leads to premature death. Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene disrupts the capacity of the protein to function as a channel, transporting chloride ions and bicarbonate across epithelial cell membranes. Small molecule treatments targeted at potentiating or correcting CFTR have shown clinical benefits, but are only effective for a small percentage of individuals with specific CFTR mutations. To overcome this limitation, we engineered stromal‐derived mesenchymal stem cells (MSC) and HEK293 cells to produce exosomes containing a novel CFTR Zinc Finger Protein fusion with transcriptional activation domains VP64, P65 and Rta to target the CFTR promoter (CFZF‐VPR) and activate transcription. Treatment with CFZF‐VPR results in robust activation of CFTR transcription in patient derived Human Bronchial Epithelial cells (HuBEC). We also find that CFZF‐VPR can be packaged into MSC and HEK293 cell exosomes and delivered to HuBEC cells to potently activate CFTR expression. Connexin 43 appeared to be required for functional release of CFZF‐VPR from exosomes. The observations presented here demonstrate that MSC derived exosomes can be used to deliver a packaged zinc finger activator to target cells and activate CFTR. The novel approach presented here offers a next‐generation genetic therapy that may one day prove effective in treating patients afflicted with Cystic fibrosis.

The CFTR Mutation c.3453G > C (D1152H) Confers an Anion Selectivity Defect in Primary Airway Tissue that Can Be Rescued by Ivacaftor.

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene variant, c.3453G > C (D1152H), is associated with mild Cystic Fibrosis (CF) disease, though there is considerable clinical variability ranging from no detectable symptoms to lung disease with early acquisition of Pseudomonas aeruginosa. The approval extension of ivacaftor, the first CFTR modulator drug approved, to include D1152H was based on a positive drug response of defective CFTR-D1152H chloride channel function when expressed in FRT cells. Functional analyses of primary human nasal epithelial cells (HNE) from an individual homozygous for D1152H now revealed that while CFTR-D1152H demonstrated normal, wild-type level chloride conductance, its bicarbonate-selective conductance was impaired. Treatment with ivacaftor increased this bicarbonate-selective conductance. Extensive genetic, protein and functional analysis of the nasal cells of this D1152H/D1152H patient revealed a 90% reduction of CFTR transcripts due to the homozygous presence of the 5T polymorphism in the poly-T tract forming a complex allele with D1152H. Thus, we confirm previous observation in patient-derived tissue that 10% normal CFTR transcripts confer normal, wild-type level chloride channel activity. Together, this study highlights the benefit of patient-derived tissues to study the functional expression and pharmacological modulation of CF-causing mutations, in order to understand pathogenesis and therapeutic responses.

CFTR Expression Analysis for Subtyping of Human Pancreatic Cancer Organoids.

Background Organoid cultures of human pancreatic ductal adenocarcinoma (PDAC) have become a promising tool for tumor subtyping and individualized chemosensitivity testing. PDACs have recently been grouped into different molecular subtypes with clinical impact based on cytokeratin-81 (KRT81) and hepatocyte nuclear factor 1A (HNF1A). However, a suitable antibody for HNF1A is currently unavailable. The present study is aimed at establishing subtyping in PDAC organoids using an alternative marker. Methods A PDAC organoid biobank was generated from human primary tumor samples containing 22 lines. Immunofluorescence staining was established and done for 10 organoid lines for cystic fibrosis transmembrane conductance regulator (CFTR) and KRT81. Quantitative real-time PCR (qPCR) was performed for CFTR and HNF1A. A chemotherapeutic drug response analysis was done using gemcitabine, 5-FU, oxaliplatin, and irinotecan. Results A biobank of patient-derived PDAC organoids was established. The efficiency was 71% (22/31) with 68% for surgical resections and 83% for fine needle aspirations. Organoids could be categorized into the established quasimesenchymal, exocrine-like, and classical subtypes based on KRT81 and CFTR immunoreactivity. CFTR protein expression was confirmed on the transcript level. CFTR and HNF1A transcript expression levels positively correlated (n = 10; r = 0.927; p = 0.001). PDAC subtypes of the primary tumors and the corresponding organoid lines were identical for most of the cases analyzed (6/7). Treatment with chemotherapeutic drugs revealed tendencies but no significant differences regarding drug responses. Conclusions Human PDAC organoids can be classified into known subtypes based on KRT81 and CFTR immunoreactivity. CFTR and HNF1A mRNA levels correlated well. Furthermore, subtype-specific immunoreactivity matched well between PDAC organoids and the respective primary tumor tissue. Subtyping of human PDACs using CFTR might constitute an alternative to HNF1A and should be further investigated.

Cell-Selective Regulation of CFTR Gene Expression: Relevance to Gene Editing Therapeutics.

The cystic fibrosis transmembrane conductance regulator (CFTR) gene is an attractive target for gene editing approaches, which may yield novel therapeutic approaches for genetic diseases such as cystic fibrosis (CF). However, for gene editing to be effective, aspects of the three-dimensional (3D) structure and cis-regulatory elements governing the dynamic expression of CFTR need to be considered. In this review, we focus on the higher order chromatin organization required for normal CFTR locus function, together with the complex mechanisms controlling expression of the gene in different cell types impaired by CF pathology. Across all cells, the CFTR locus is organized into an invariant topologically associated domain (TAD) established by the architectural proteins CCCTC-binding factor (CTCF) and cohesin complex. Additional insulator elements within the TAD also recruit these factors. Although the CFTR promoter is required for basal levels of expression, cis-regulatory elements (CREs) in intergenic and intronic regions are crucial for cell-specific and temporal coordination of CFTR transcription. These CREs are recruited to the promoter through chromatin looping mechanisms and enhance cell-type-specific expression. These features of the CFTR locus should be considered when designing gene-editing approaches, since failure to recognize their importance may disrupt gene expression and reduce the efficacy of therapies.

Subacute TGFβ expression drives inflammation, goblet cell hyperplasia, and pulmonary function abnormalities in mice with effects dependent on CFTR function.

Cystic fibrosis (CF) produces variable lung disease phenotypes that are, in part, independent of the CF transmembrane conductance regulator ( CFTR) genotype. Transforming growth factor-β (TGFβ) is the best described genetic modifier of the CF phenotype, but its mechanism of action is unknown. We hypothesized that TGFβ is sufficient to drive pathognomonic features of CF lung disease in vivo and that CFTR deficiency enhances susceptibility to pathological TGFβ effects. A CF mouse model and littermate controls were exposed intratracheally to an adenoviral vector containing the TGFβ1 cDNA (Ad-TGFβ), empty vector, or PBS only. Studies were performed 1 wk after treatment, including lung mechanics, collection of bronchoalveolar lavage fluid, and analysis of lung histology, RNA, and protein. CF and non-CF mice showed similar weight loss, inflammation, goblet cell hyperplasia, and Smad pathway activation after Ad-TGFβ treatment. Ad-TGFβ produced greater abnormalities in lung mechanics in CF versus control mice, which was uniquely associated with induction of phosphoinositide 3-kinase and mitogen-activated protein kinase signaling. CFTR transcripts were reduced, and epithelial sodium channel transcripts were increased in CF and non-CF mice, whereas the goblet cell transcription factors, forkhead ortholog A3 and SAM-pointed domain-containing ETS-like factor, were increased in non-CF but not CF mice following Ad-TGFβ treatment. Pulmonary TGFβ1 expression was sufficient to produce pulmonary remodeling and abnormalities in lung mechanics that were associated with both shared and unique cell signaling pathway activation in CF and non-CF mice. These results highlight the multifunctional impact of TGFβ on pulmonary pathology in vivo and identify cellular-response differences that may impact CF lung pathology.

A transcription factor network represses CFTR gene expression in airway epithelial cells.

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause the inherited disorder cystic fibrosis (CF). Lung disease is the major cause of CF morbidity, though CFTR expression levels are substantially lower in the airway epithelium than in pancreatic duct and intestinal epithelia, which also show compromised function in CF. Recently developed small molecule therapeutics for CF are highly successful for one specific CFTR mutation and have a positive impact on others. However, the low abundance of CFTR transcripts in the airway limits the opportunity for drugs to correct the defective substrate. Elucidation of the transcriptional mechanisms for the CFTR locus has largely focused on intragenic and intergenic tissue-specific enhancers and their activating trans-factors. Here, we investigate whether the low CFTR levels in the airway epithelium result from the recruitment of repressive proteins directly to the locus. Using an siRNA screen to deplete ∼1500 transcription factors (TFs) and associated regulatory proteins in Calu-3 lung epithelial cells, we identified nearly 40 factors that upon depletion elevated CFTR mRNA levels more than 2-fold. A subset of these TFs was validated in primary human bronchial epithelial cells. Among the strongest repressors of airway expression of CFTR were Krüppel-like factor 5 and Ets homologous factor, both of which have pivotal roles in the airway epithelium. Depletion of these factors, which are both recruited to an airway-selective cis-regulatory element at -35 kb from the CFTR promoter, improved CFTR production and function, thus defining novel therapeutic targets for enhancement of CFTR.

The phospholipid flippase ATP8B1 mediates apical localization of the cystic fibrosis transmembrane regulator

Progressive familial intrahepatic cholestasis type 1 (PFIC1) is caused by mutations in the gene encoding the phospholipid flippase ATP8B1. Apart from severe cholestatic liver disease, many PFIC1 patients develop extrahepatic symptoms characteristic of cystic fibrosis (CF), such as pulmonary infection, sweat gland dysfunction and failure to thrive. CF is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel essential for epithelial fluid transport. Previously it was shown that CFTR transcript levels were strongly reduced in livers of PFIC1 patients. Here we have investigated the hypothesis that ATP8B1 is important for proper CFTR expression and function.We analyzed CFTR expression in ATP8B1-depleted intestinal and pulmonary epithelial cell lines and assessed CFTR function by measuring short-circuit currents across transwell-grown ATP8B1-depleted intestinal T84 cells and by a genetically-encoded fluorescent chloride sensor. In addition, we studied CFTR surface expression upon induction of CFTR transcription.We show that CFTR protein levels are strongly reduced in the apical membrane of human ATP8B1-depleted intestinal and pulmonary epithelial cell lines, a phenotype that coincided with reduced CFTR activity. Apical membrane insertion upon induction of ectopically-expressed CFTR was strongly impaired in ATP8B1-depleted cells.We conclude that ATP8B1 is essential for correct apical localization of CFTR in human intestinal and pulmonary epithelial cells, and that impaired CFTR localization underlies some of the extrahepatic phenotypes observed in ATP8B1 deficiency.

Targeted CFTR gene disruption with zinc-finger nucleases in human intestinal epithelial cells induces oxidative stress and inflammation

Cystic fibrosis (CF) is a multisystemic pathology caused by mutations of the CF transmembrane conductance regulator (CFTR) gene. ObjectivesAs the intestine harbors the greatest number of CFTR transcripts after birth and since CFTR plays a role in glutathione transport, we hypothesized that CFTR deletion might produce oxidative stress (OxS) and inflammation in CF intestinal epithelial cell. MethodsCFTR gene was abrogated in Caco-2/15 enterocytes through the zinc-finger nuclease system. Their oxidative and inflammatory characteristics were appreciated under basal conditions and after the treatment with the pro-oxidant iron-ascorbate (Fe/Asc) complex and pro-inflammatory lipopolysaccharide (LPS). ResultsIntestinal epithelial cells with CFTR knockout spontaneously exhibited an increased lipid peroxidation level, reflected by malondialdehyde overproduction and reduced antioxidant defense characterized by low enzymatic activities of glutathione peroxidase and catalase. CFTR silencing also resulted in elevated protein expression of pro-inflammatory tumor necrosis Factor-α, interleukin-6, cyclooxygenase-2, and the transcription factor nuclear factor-κB. Moreover, exaggerated OxS and inflammation processes occurred in CFTR−/− cells in response to the addition of Fe/Asc and LPS, respectively. ConclusionsIntestinal Caco-2/15 cells with CFTR deletion, display innate oxidative and inflammatory features while being more sensitive to pro-oxidant and pro-inflammatory stimuli. These two pathophysiological processes could be implicated in CF-related intestinal disorders.

Epigenetics in Cystic Fibrosis: Epigenetic Targeting of a Genetic Disease

Cystic fibrosis (CF) is a deadly genetic disease that affects the lungs and digestive system. A mutation in the CF transmembrane conductance regulator (CFTR) gene is the cause of the disease. How epigenetics contributes to CFTR expression is still poorly understood. Epigenetics is a mechanism that alters gene expression without changing the underlying DNA sequence. Epigenetic mechanisms include DNA methylation and histone modification. Both mechanisms have been implicated in CFTR gene regulation. Here we review epigenetic regulation of CFTR transcription while discussing potential epigenetic targeting strategies including DNA methyltransferase, histone deacetylase, and histone methyltransferase and demethylase inhibition. Because of the reversibility of epigenetics, targeting epigenetic mechanisms has been an attractive therapeutic approach. However, epigenetic targeting of CF disease is still at its infant stage.

Tgf-beta downregulation of distinct chloride channels in cystic fibrosis-affected epithelia.

RationaleThe cystic fibrosis transmembrane conductance regulator (CFTR) and Calcium-activated Chloride Conductance (CaCC) each play critical roles in maintaining normal hydration of epithelial surfaces including the airways and colon. TGF-beta is a genetic modifier of cystic fibrosis (CF), but how it influences the CF phenotype is not understood.ObjectivesWe tested the hypothesis that TGF-beta potently downregulates chloride-channel function and expression in two CF-affected epithelia (T84 colonocytes and primary human airway epithelia) compared with proteins known to be regulated by TGF-beta.Measurements and Main ResultsTGF-beta reduced CaCC and CFTR-dependent chloride currents in both epithelia accompanied by reduced levels of TMEM16A and CFTR protein and transcripts. TGF-beta treatment disrupted normal regulation of airway-surface liquid volume in polarized primary human airway epithelia, and reversed F508del CFTR correction produced by VX-809. TGF-beta effects on the expression and activity of TMEM16A, wtCFTR and corrected F508del CFTR were seen at 10-fold lower concentrations relative to TGF-beta effects on e-cadherin (epithelial marker) and vimentin (mesenchymal marker) expression. TGF-beta downregulation of TMEM16A and CFTR expression were partially reversed by Smad3 and p38 MAPK inhibition, respectively.ConclusionsTGF-beta is sufficient to downregulate two critical chloride transporters in two CF-affected tissues that precedes expression changes of two distinct TGF-beta regulated proteins. Our results provide a plausible mechanism for CF-disease modification by TGF-beta through effects on CaCC.

Fine Characterization of the Recurrent c.1584+18672A>G Deep-Intronic Mutation in the Cystic Fibrosis Transmembrane Conductance Regulator Gene

Splicing mutations account for approximately 12% of the 1,890 cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations described in cystic fibrosis (CF). However, their impact on pre-mRNA processing frequently remains unclear. An interesting opportunity to study CFTR transcripts in vivo involves the use of RNA from nasal brushings. Through this approach we previously identified a deep-intronic mutation (c.1584+18672A>G) that activates a 104-base pair (bp) out-of-frame pseudoexon by creating a donor splice site. The screening of 230 patients with CF identified c.1584+18672A>G in three additional individuals, demonstrating that it is a recurrent, and potentially overlooked, mutation among Italian patients. Haplotype analysis suggests that it originated from at least two independent events. To characterize the mutation further, a genomic region, including the activated pseudoexon and surrounding intronic sequences, was cloned into an expression vector and transfected into HeLa cells. RT-PCR analysis identified two alternative splicing products, produced by the activation of two different cryptic acceptor splice sites. One included the 104-bp pseudoexon (78.7% of transcripts), and the other led to the inclusion of a 65-bp pseudoexon (21.3% of mRNAs). The allele-specific measurement of wild-type and aberrant splicings from the nasal-brushing RNA of the three probands with genotype F508del/c.1584+18672A>G demonstrated: (1) a low level of pseudoexon inclusion in the F508del transcript (not containing the splicing mutation); (2) residual wild-type splicing in the c.1584+18672A>G mRNA; (3) the degradation of aberrant transcripts; and (4) the relative strength of the different cryptic splice sites. Interestingly, the residual wild-type splicing detected in transcripts bearing the c.1584+18672A>G mutation correlates well with the milder clinical phenotype of patients.

Molecular and Functional Analysis of the Large 5′ Promoter Region of CFTR Gene Revealed Pathogenic Mutations in CF and CFTR-Related Disorders

Patients with cystic fibrosis (CF) manifest a multisystemic disease due to mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR); despite extensive testing of coding regions, a proportion of CF alleles remains unidentified. We studied 118 patients with CF and CFTR-related disorders, most with one or both unknown mutations after the scanning of CFTR coding regions, and a non-CF control group (n = 75) by sequencing the 6000-bp region at the 5' of the CFTR gene. We identified 23 mutations, of which 9 were novel. We expressed such mutations in vitro using four cell systems to explore their functional effect, relating the data to the clinical expression of each patient. Some mutations reduced expression of the gene reporter firefly luciferase in various cell lines and may act as disease-causing mutations. Other mutations caused an increase in luciferase expression in some cell lines. One mutation had a different effect in different cells. For other mutations, the expression assay excluded a functional role. Gene variants in the large 5' region may cause altered regulation of CFTR gene expression, acting as disease-causing mutations or modifiers of its clinical phenotype. Studies of in vitro expression in different cell systems may help reveal the effect of such mutations.