Cystic Fibrosis Transmembrane Conductance Regulator mRNA Research Articles

The RNA Binding Protein Tristetraprolin Contributes to CFTR mRNA Stability in Cystic Fibrosis.

Cystic Fibrosis (CF) is the most common inherited disorder and is characterized by an inflammatory phenotype. Here, we found that in bronchial epithelium reconstituted form lung tissue biopsies from patients with CF, the RNA-binding protein tristetraprolin (TTP), a key regulator of inflammation, is dysregulated in cells that strongly express cytokines and interleukins. TTP activity is regulated by extensive post-translational modifications, particularly phosphorylation. We found that in addition to mRNA downregulation, phosphorylated TTP (which cannot bind to mRNA) accumulated in CF cultures, suggesting that the imbalance in TTP phosphorylation status could contribute to the inflammatory phenotype in CF. We confirmed TTP destabilizing role on IL8 mRNA through its 3'UTR sequence in CF cells. We next demonstrated that TTP phosphorylation is mainly regulated by MK2 through activation of ERK, which also was hyperphosphorylated. TTP is considered a mRNA decay factor with some exception, and we present a new positive role of TTP in CF cultures. We determined that TTP binds to specific ARE motifs on the 3'UTR of mRNA sequences and also, for the first time, to the 3'UTR of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) where TTP binding stabilizes the mRNA level. This study identified new partners that can be targeted in CF and proposes a new way to control CFTR gene expression.

Cystic fibrosis cell models for high-throughput analysis and drug screening

Cystic fibrosis (CF) is a single-gene disorder that affects the lung, digestive system, and other organs. Mutations in the CF transmembrane conductance regulator (CFTR) gene are classified into several classes based on their pathogenic mechanism and clinical severity. The distinct and heterogeneous clinical behavior of each CF class and the respective CFTR mutations have made the development of a durable therapy for all CF patients extremely challenging. While the FDA-approved drug elexacaftor/tezacaftor/ivacaftor (Trikafta) benefits CF patients carrying at least one F508del mutation in CFTR, it's not effective for many CF patients carrying a variety of other CFTR mutations. To establish a better understanding of CF pathophysiology and aid the development of novel therapeutics for different classes of CF patients, we have created four CF-mutation-specific cell models that recapitulate respectively four distinct CF classes and disease phenotypes, as confirmed by sequencing, CFTR mRNA and protein quantification. The channel function of each cell model was first validated using a well-established FLIPR (Fluorescent Imaging Plate Reader) membrane potential assay and then assessed by the YFP-based functional assay. Integrated with a halide-sensitive fluorescent reporter, these CF cell models can be used for high-throughput drug screening, as demonstrated by a proof-of-concept study using Trikafta. These cell models have the potential to advance CFTR mutation-specific therapies thus addressing the unmet needs of CF patients with rare mutations.

Baicalin-aluminum complex on the regulation of IPEC-1 infected with enterotoxigenic Escherichia coli

Enterotoxigenic Escherichia coli (ETEC) is the main bacterial cause of diarrhea in weaned piglets. Baicalin-aluminum (BA) complex is the main active ingredient of Scutellaria baicalensis Georgi extracted-aluminum complex, which has been used to treat diarrhea in weaning piglets, however the underlying mechanism remains unclear. To investigate the effects of the BA complex on the regulation of porcine intestinal epithelial (IPEC-1) cells infected with ETEC, IPEC-1 cells were incubated with an ETEC bacterial strain at a multiplicity of infection of 1 for 6 h and then treated with different concentrations of the BA complex for 6 h. ETEC infection increased the levels of cAMP and cGMP, upregulated CFTR (cystic fibrosis transmembrane conductance regulator) mRNA, and downregulated NHE4 mRNA in IPEC-1 cells. Treatment with the BA complex inhibited ETEC adhesion and the production of cAMP and cGMP, reduced CFTR mRNA expression, and increased NHE4 mRNA expression. Overall, the BA complex weakened the adhesion of ETEC to IPEC-1 cells, and inhibited cAMP/cGMP-CFTR signaling in IPEC-1 cells.

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.

Quantitative Evaluation of CFTR Gene Expression: A Comparison between Relative Quantification by Real-Time PCR and Absolute Quantification by Droplet Digital PCR.

In the precision medicine era of cystic fibrosis (CF), therapeutic interventions, by the so-called modulators, target the cystic fibrosis transmembrane conductance regulator (CFTR) protein. The levels of targetable CFTR proteins are a main variable in the success of patient-specific therapy. In turn, the CFTR protein level depends, at least in part, on the level of CFTR mRNA. Many mechanisms can modulate the CFTR mRNA level, for example, transcriptional rate, stability of the mRNA, epigenetics, and pathogenic variants that can affect mRNA production and degradation. Independently from the causes of variable CFTR mRNA levels, their exact quantitative assessment is of great importance in CF. Methods with high analytical sensitivity, precision, and accuracy are mandatory for the quantitative evaluation aimed at the amelioration of the diagnostic, prognostic, and therapeutic aspects. This paper compares, for the first time, two CFTR gene expression quantification methods: a well-established method for the relative quantification of CFTR mRNA using a real-time PCR and an innovative method for its absolute quantification using a droplet digital PCR. No comprehensive methods for absolute CFTR quantification via droplet digital PCR have been published so far. The accurate quantification of CFTR expression at the mRNA level is a critical step for the personalized therapeutic approaches of CF.

Q1291H-CFTR molecular dynamics simulations and ex vivo theratyping in nasal epithelial models and clinical response to elexacaftor/tezacaftor/ivacaftor in a Q1291H/F508del patient.

Background: Cystic fibrosis (CF) is caused by a wide spectrum of mutations in the CF transmembrane conductance regulator (CFTR) gene, with some leading to non-classical clinical presentations. We present an integrated in vivo, in silico and in vitro investigation of an individual with CF carrying the rare Q1291H-CFTR allele and the common F508del allele. At age 56years, the participant had obstructive lung disease and bronchiectasis, qualifying for Elexacaftor/Tezacaftor/Ivacaftor (ETI) CFTR modulator treatment due to their F508del allele. Q1291H CFTR incurs a splicing defect, producing both a normally spliced but mutant mRNA isoform and a misspliced isoform with a premature termination codon, causing nonsense mediated decay. The effectiveness of ETI in restoring Q1291H-CFTR is largely unknown. Methods: We collected clinical endpoint measurements, including forced expiratory volume in 1s percent predicted (FEV1pp) and body mass index (BMI), and examined medical history. In silico simulations of the Q1291H-CFTR were compared to Q1291R, G551D, and wild-type (WT)-CFTR. We quantified relative Q1291H CFTR mRNA isoform abundance in patient-derived nasal epithelial cells. Differentiated pseudostratified airway epithelial cell models at air liquid interface were created and ETI treatment impact on CFTR was assessed by electrophysiology assays and Western blot. Results: The participant ceased ETI treatment after 3months due to adverse events and no improvement in FEV1pp or BMI. In silico simulations of Q1291H-CFTR identified impairment of ATP binding similar to known gating mutants Q1291R and G551D-CFTR. Q1291H and F508del mRNA transcripts composed 32.91% and 67.09% of total mRNA respectively, indicating 50.94% of Q1291H mRNA was misspliced and degraded. Mature Q1291H-CFTR protein expression was reduced (3.18% ± 0.60% of WT/WT) and remained unchanged with ETI. Baseline CFTR activity was minimal (3.45 ± 0.25μA/cm2) and not enhanced with ETI (5.73 ± 0.48μA/cm2), aligning with the individual's clinical evaluation as a non-responder to ETI. Conclusion: The combination of in silico simulations and in vitro theratyping in patient-derived cell models can effectively assess CFTR modulator efficacy for individuals with non-classical CF manifestations or rare CFTR mutations, guiding personalized treatment strategies and optimizing clinical outcomes.

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.

The Cystic Fibrosis Transmembrane Conductance Regulator Gene (CFTR) Is under Post-Transcriptional Control of microRNAs: Analysis of the Effects of agomiRNAs Mimicking miR-145-5p, miR-101-3p, and miR-335-5p.

(1) Background: MicroRNAs are involved in the expression of the gene encoding the chloride channel CFTR (Cystic Fibrosis Transmembrane Conductance Regulator); the objective of this short report is to study the effects of the treatment of bronchial epithelial Calu-3 cells with molecules mimicking the activity of pre-miR-145-5p, pre-miR-335-5p, and pre-miR-101-3p, and to discuss possible translational applications of these molecules in pre-clinical studies focusing on the development of protocols of possible interest in therapy; (2) Methods: CFTR mRNA was quantified by Reverse Transcription quantitative Polymerase Chain Reaction (RT-qPCR). The production of the CFTR protein was assessed by Western blotting; (3) Results: The treatment of Calu-3 cells with agomiR-145-5p caused the highest inhibition of CFTR mRNA accumulation and CFTR production; (4) Conclusions: The treatment of target cells with the agomiR pre-miR-145-5p should be considered when CFTR gene expression should be inhibited in pathological conditions, such as polycystic kidney disease (PKD), some types of cancer, cholera, and SARS-CoV-2 infection.

Ionocyte-Specific Regulation of Cystic Fibrosis Transmembrane Conductance Regulator.

CFTR (cystic fibrosis transmembrane conductance regulator) is a tightly regulated anion channel that mediates chloride and bicarbonate conductance in many epithelia and in other tissues, but whether its regulation varies depending on the cell type has not been investigated. Epithelial CFTR expression is highest in rare cells called ionocytes. We studied CFTR regulation in control and ionocyte-enriched cultures by transducing bronchial basal cells with adenoviruses that encode only eGFP or FOXI1 (forkhead box I1) + eGFP as separate polypeptides. FOXI1 dramatically increased the number of transcripts for ionocyte markers ASCL3 (Achaete-Scute Family BHLH Transcription Factor 3), BSND, ATP6V1G3, ATP6V0D2, KCNMA1, and CFTR without altering those for secretory (SCGB1A1), basal (KRT5, KRT6, TP63), goblet (MUC5AC), or ciliated (FOXJ1) cells. The number of cells displaying strong FOXI1 expression was increased 7-fold, and there was no evidence for a broad increase in background immunofluorescence. Total CFTR mRNA and protein levels increased 10-fold and 2.5-fold, respectively. Ionocyte-enriched cultures displayed elevated basal current, increased adenylyl cyclase 5 expression, and tonic suppression of CFTR activity by the phosphodiesterase PDE1C, which has not been shown previously to regulate CFTR activity. The results indicate that CFTR regulation depends on cell type and identifies PDE1C as a potential target for therapeutics that aim to increase CFTR function specifically in ionocytes.

Lower Expression of CFTR Is Associated with Higher Mortality in a Meta-Analysis of Individuals with Colorectal Cancer.

Individuals with cystic fibrosis (CF), caused by biallelic germline mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), have higher risk and earlier onset of colorectal cancer (CRC). A subset of CRC patients in the non-CF population expresses low levels of tumoral CFTR mRNA which may also cause decreased CFTR activity. To determine the consequences of reduced CFTR expression in this population, we investigated association of tumoral CFTR expression with overall and disease-specific mortality in CRC patients. CFTR mRNA expression, clinical factors and survival data from 1177 CRC patients reported in the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus studies GSE39582 and GSE17538 were included. Log-transformed and z-normalized [mean = 0, standard deviation (SD) = 1] CFTR expression values were modeled as quartiles or dichotomized at the median. Univariate and multivariable Cox proportional hazards regression models were used to estimate hazard ratios (HR) and 95% confidence intervals (CI) for overall and disease-specific mortality in individual studies and meta-analyses. Analyses of each of the three individual datasets showed a robust association of decreased CFTR expression with increased mortality. In meta-analyses adjusted for stage at diagnosis, age and sex, CFTR expression was inversely associated with risk of overall death [pooled HR (95% CI): 0.70 (0.57-0.86)] and disease-specific death [pooled HR (95% CI): 0.68 (0.47-0.99)]. Associations did not differ by stage at diagnosis, age, or sex. Meta-analysis of overall death stratified by microsatellite instable (MSI) versus microsatellite stable (MSS) status indicated potential interaction between MSI/MSS status and CFTR expression, (p-interaction: 0.06). The findings from these three datasets support the hypothesis that low CFTR expression is associated with increased CRC mortality.

Increased expression of glucagon-like peptide-1 and cystic fibrosis transmembrane conductance regulator in the ileum and colon in mouse treated with metformin.

Metformin, an oral medication, is prescribed to patients with type 2 diabetes mellitus. Although the efficacy, safety, and low economic burden of metformin on patients have long been recognized, approximately 5% of the patients treated with this drug develop severe diarrhea and discontinue the treatment. We previously reported that 1,000 mg·kg-1·day-1 of metformin induced diarrhea in diabetic obese (db/db) mice and wood creosote (traditional medication for diarrhea) ameliorated the symptoms. In this study, we attempted to elucidate the molecular mechanisms by which metformin induces diarrhea. Cystic fibrosis transmembrane conductance regulator (CFTR) is a key ion (chloride) channel in cyclic adenosine monophosphate (cAMP)-induced diarrhea. Metformin treatment increased bile flow (bile acids and bilirubin) in the ileum of mice. In addition, the treatment was accompanied by an increase in mRNA and protein levels of CFTR in the mucosa of the ileum and colon in both wild-type (C57BL/6J) and db/db mice. Glucagon-like peptide-1 (GLP-1), as well as cholic acid, induces CFTR mRNA expression in human colon carcinoma Caco-2 cells through cAMP signaling. Although wood creosote (10 mg/kg) ameliorated diarrhea symptoms, it did not alter the mRNA levels of Glp-1 or Cftr. Similar to overeating, metformin upregulated GLP-1 and CFTR expression, which may have contributed to diarrhea symptoms in mice. Although we could not identify db/db mouse-specific factors associated with metformin-induced diarrhea, these factors may modulate colon function. Wood creosote may not interact with these factors but ameliorates diarrhea symptoms.

Abstract A027: The oncogenic mutation BRAF-V600E is associated with colorectal cancers expressing low levels of CFTR mRNA

Abstract Background and purpose: The cystic fibrosis transmembrane conductance regulator (CFTR) gene is a tumor suppressor in colorectal cancer (CRC). People with cystic fibrosis, caused by biallelic germline mutations in CFTR, have a higher risk developing CRC. We also found that loss of CFTR is implicated in sporadic CRC. In a study of 90 persons diagnosed with stage II CRC, the 23% of patients with lowest CFTR expression had 30% lower disease-free survival at 3 years. The cause of decreased CFTR expression in this group is not known. We analyzed the TCGA COADREAD study to identify associations between low expression of CFTR and cancer-causing genetic alterations. Of these alterations, only BRAF-V600E mutation correlated with low expression of CFTR. The BRAF-V600E mutation is found in ~10% of CRC and is associated with global promoter hypermethylation leading to down regulation of tumor suppressor genes. Accordingly, our hypothesis was that BRAF-V600E mutation contributes to low expression of CFTR via CFTR promoter DNA methylation. Methods: In the TCGA COADREAD database 283 primary colorectal cancers diagnosed at stages II, III and IV had full information needed for this study: CFTR mRNA expression, somatic mutation, overall survival (OS), and DNA methylation status. OS was visualized using Kaplan-Meier analysis. Association between CFTR expression and methylation was determined by Pearson correlation coefficient (PCC) analysis. Associations between mutations and CFTR expression or methylation were determined by Point Biserial correlation coefficient (PBS) analysis. Results: Kaplan-Meier analysis of the 283 cases showed that overall survival was worse in the 25% of cases with lowest CFTR expression (p=0.035). The BRAF-V600E mutation was significantly associated with lower CFTR expression (PBS r=-0.38, p<0.0001) and 81% of BRAF V600E mutations were found in the 25% of cases with lowest CFTR expression. In contrast mutations in APC and KRAS showed a positive association with CFTR expression (PBS r=0.25, p<0.0001; PBS r=0.13, p=0.028 respectively). The CFTR promoter is hypermethylated in several cancers and BRAF-V600E mutation is associated with aberrant promoter DNA methylation. We examined DNA methylation at 9 CpG sites in the CFTR promoter and found that low CFTR expression significantly associated with increased DNA methylation at these 9 sites (PCC for average of 9 sites r= -0.56, p=<0.001). The association between BRAF-V600E mutation and CFTR methylation was modest but significant (PBS r=0.16, p=0.0063). Conclusions: BRAF-V600E mutation was significantly associated with CRC cases expressing low levels of CFTR, a subset with poor overall survival, as was CFTR promoter methylation. BRAF-V600E showed a modest association with CFTR promoter methylation and so may play a role in down-regulation of CFTR expression. However, association of BRAF-V600E with CFTR promoter methylation was modest compared to its association with CFTR expression suggesting that BRAF-V600E mutation may play additional roles in CRC tumors expressing low levels of CFTR. Citation Format: Patricia Scott, Anna Prizment, Rahul Bhattacharya, Zachary Blankenheim, Nathan Pankratz, Timothy Starr, Robert Cormier. The oncogenic mutation BRAF-V600E is associated with colorectal cancers expressing low levels of CFTR mRNA [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr A027.

The CFTR Amplifier Nesolicaftor Rescues TGF-β1 Inhibition of Modulator-Corrected F508del CFTR Function.

Highly effective cystic fibrosis transmembrane conductance regulator (CFTR) modulators have led to dramatic improvements in lung function in many people with cystic fibrosis (PwCF). However, the efficacy of CFTR modulators may be hindered by persistent airway inflammation. The cytokine transforming growth factor-beta1 (TGF-β1) is associated with worse pulmonary disease in PwCF and can diminish modulator efficacy. Thus, strategies to augment the CFTR response to modulators in an inflammatory environment are needed. Here, we tested whether the CFTR amplifier nesolicaftor (or PTI-428) could rescue the effects of TGF-β1 on CFTR function and ciliary beating in primary human CF bronchial epithelial (CFBE) cells. CFBE cells homozygous for F508del were treated with the combination of elexacaftor/tezacaftor/ivacaftor (ETI) and TGF-β1 in the presence and absence of nesolicaftor. Nesolicaftor augmented the F508del CFTR response to ETI and reversed TGF-β1-induced reductions in CFTR conductance by increasing the expression of CFTR mRNA. Nesolicaftor further rescued the reduced ciliary beating and increased expression of the cytokines IL-6 and IL-8 caused by TGF-β1. Finally, nesolicaftor augmented the F508del CFTR response to ETI in CFBE cells overexpressing miR-145, a negative regulator of CFTR expression. Thus, CFTR amplifiers, but only when used with highly effective modulators, may provide benefit in an inflamed environment.

High expression of CFTR in cumulus cells from mature oocytes is associated with high-quality of oocyte and subsequent embryonic development.

The purpose of this study was to explore the association of expression of cystic fibrosis transmembrane conductance regulator (CFTR) in cumulus cells (CCs) from mature oocytes with oocyte quality and embryonic development. A total of 338 infertile women who underwent ovarian stimulation cycle of oocyte retrieval in Zhejiang University School of Medicine were retrospectively enrolled in this study. The relative mRNA expression levels of CFTR, bone morphogenetic protein 15 (BMP15), and growth differentiation factor 9 (GDF9) in CCs were detected by qPCR technology. ROC curve was applied for the diagnosis of oocyte maturation. The serum levels of anti-Müllerian hormone (AMH), E2, follicle-stimulating hormone (FSH), luteinizing hormone (LH), and androstenedione were measured. Oocyte maturation rate, fertilization rate, cleavage rate, high-quality embryo formation rate, and implantation rate after embryo transfer were also determined. The mRNA expression levels of CFTR in CCs were significantly increased in metaphase II (MII) oocytes compared to that in metaphase I (MI) or germinal vesicle (GV) oocytes. The ROC curve analysis illustrated that CFTR mRNA expression could efficiently discriminate MII oocytes from MI or GV oocytes (AUC = 0.954), and revealed that 0.695 RQU is the optimal cut-off value for diagnosis. So the cut-off value of 2-ΔΔCT = 0.70 was used to divide the patients into two groups: low- (n = 114) and high-CFTR group (n = 224). The mRNA expression of CFTR in CCs was positively correlated with the antral follicular count (AFC), number of oocytes retrieved, number of MII oocytes, serum E2 level on hCG day, and BMP15 and GDF9 expression in CCs. Under continuous stimulation with the same dose of recombinant follicle-stimulating hormone (rFSH), the number of follicles, average recovered oocytes, recovered oocytes, MII oocytes, as well as the oocyte recovery rate, fertilization rate, oocyte cleavage rate, high-quality embryo formation rate, and implantation rate were decreased in patients with lower CFTR. This study suggests that CFTR expression in CCs is associated with the developmental potential of human oocytes.

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.

Mechanism of Xiaoqinglong Decoction and Qingqi Huatan Pills in improving pathological airway mucus based on nuclear factor-κB/microRNA-494 signaling regulation of mucin 5AC and cystic fibrosis transmembrane conductance regulator

To observe the effects of Xiaoqinglong Decoction and Qingqi Huatan Pills on interleukin-1β (IL-1β)-induced mucushypersecretion model of human airway epithelial H292 cellsand related molecules of nuclear factor-κB/microRNA-494(NF-κB/miR-494) signaling pathway, and to explore the mechanism of the two medicines in improving pathological airway mucus. Methyl thiazolyl tetrazolium (MTT) colorimetric method was used to detect the effects of different concentrations of Xiaoqinglong Decoction and Qingqi Huatan Pills on the activity of H292 cellsinduced by IL-1β, and the appropriate concentration was selected for subsequent experiments. Cells were randomly divided into blank group, IL-1β model group (5 μg/L IL-1β), NF-κB inhibitor pyrrolidinedithiocarbamate (PDTC) group (5 μg/L IL-1β+100 μmol/L PDTC), Xiaoqinglong Decoction (5 μg/L IL-1β+1 000 mg/L Xiaoqinglong Decoction) and Qingqi Huatan Pill group (5 μg/L IL-1β+1 000 mg/L Qingqi Huatan Pills). 5 μg/L IL-1β was used to induce H292 cells for 24 hours to establish a model of airway epithelial mucus hypersecretion. Enzyme linked immunosorbent assay (ELISA) method was used to detect the levels of mucin 5AC (MUC5AC), tumor necrosis factor-α (TNF-α) and IL-8 and the synthesis of intracellular MUC5AC and cystic fibrosis transmembrane conductance regulator (CFTR). Real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the expression levels of MUC5AC mRNA, CFTR mRNA, miR-494. Western blotting was used to detect protein expression of key proteins (p65) and NF-κB inhibitors (IκB) in NF-κB signaling pathway. Xiaoqinglong Decoction and Qingqi Huatan Pills with the concentration of 1 000 mg/L were selected for the follow-up experiment. Compared with the blank group, the levels of MUC5AC, TNF-α and IL-8 were significantly increased in the model group, intracellular MUC5AC protein content and mRNA expression were also significantly increased, intracellular CFTR protein content and mRNA expression were significantly decreased, and intracellular p65 protein expression was significantly up-regulated, the expression of IκB protein was significantly down-regulated, and the expression of miR-494 was significantly increased. Compared with the model group, the levels of MUC5AC, TNF-α and IL-8 were significantly reduced in PDTC group, Xiaoqinglong Decoction group and Qingqi Huatan Pill group, intracellular MUC5AC protein content and mRNA expression were also significantly decreased, and intracellular p65 protein expression was significantly down-regulated, and IκB protein expression was significantly up-regulated, miR-494 expression was significantly reduced. Intracellular CFTR protein content and mRNA expression were significantly increased in both PDTC group and Qingqi Huatan Pill group. Compared with the PDTC group, the level of TNF-α in the Xiaoqinglong Decoction group was significantly increased (ng/L: 22.77±3.14 vs. 11.09±3.37, P < 0.05),the content and mRNA expression of CFTR and IκB protein expression was significantly decreased [CFTR protein (ng/L): 97.38±6.62 vs. 227.04±19.48, CFTR mRNA (2-ΔΔCt): 0.99±0.08 vs. 1.21±0.08, IκB/β-actin: 1.69±0.11 vs. 2.00±0.18, all P < 0.05], the level of TNF-α in Qingqi Huatan Pill group was significantly higher (ng/L: 19.08±3.71 vs. 11.09±3.37, P < 0.05). Compared with Xiaoqinglong Decoction group, the protein content and mRNA expression of CFTR and IκB protein expression in Qingqi Huatan Pill group were significantly increased [CFTR protein (ng/L): 235.01±22.71 vs. 97.38±6.62, CFTR mRNA (2-ΔΔCt): 1.32±0.15 vs. 0.99±0.08, IκB/β-actin: 1.94±0.16 vs. 1.69±0.11, all P < 0.05]. The effect of Xiaoqinglong Decoctionin improving the hypersecretion of mucus in the airway epithelium may be related to the inhibition of NF-κB/miR-494 inflammatory signal-mediated MUC5AC hypersecretion, while the effect of Qingqi Huatan Pills may be related to the inhibition of NF-κB/miR-494 inflammatory signal-mediated MUC5AC hypersecretion and CFTR dysfunction. Therefore, the difference in the mechanism of the two treatments of airway pathological mucus is mainly in the regulation of CFTR mRNA and protein.

Losartan ameliorates TGF-β1-induced CFTR dysfunction and improves correction by cystic fibrosis modulator therapies.

Highly effective modulator therapies dramatically improve the prognosis for those with cystic fibrosis (CF). The triple combination of elexacaftor, tezacaftor, and ivacaftor (ETI) benefits many, but not all, of those with the most common F508del mutation in the CF transmembrane conductance regulator (CFTR). Here, we showed that poor sweat chloride concentration responses and lung function improvements upon initiation of ETI were associated with elevated levels of active TGF-β1 in the upper airway. Furthermore, TGF-β1 impaired the function of ETI-corrected F508del-CFTR, thereby increasing airway surface liquid (ASL) absorption rates and inducing mucus hyperconcentration in primary CF bronchial epithelial cells in vitro. TGF-β1 not only decreased CFTR mRNA, but was also associated with increases in the mRNA expression of TNFA and COX2 and TNF-α protein. Losartan improved TGF-β1–mediated inhibition of ETI-corrected F508del-CFTR function and reduced TNFA and COX2 mRNA and TNF-α protein expression. This likely occurred by improving correction of mutant CFTR rather than increasing its mRNA (without an effect on potentiation), thereby reversing the negative effects of TGF-β1 and improving ASL hydration in the CF airway epithelium in vitro. Importantly, these effects were independent of type 1 angiotensin II receptor inhibition.

Targeted quantitation of CFTR protein expression in vivo using immunoprecipitation &amp; parallel reaction monitoring tandem mass spectrometry

Abstract Background The cystic fibrosis transmembrane conductance regulator (CFTR) protein is a low-abundance membrane protein. The dysfunction of CFTR protein is the fundamental cause of cystic fibrosis (CF), a fatal genetic disease. In recent years, the novel messenger RNA (mRNA)-based therapy shows high potential to treat CF disease, by delivering CFTR mRNA into lung epithelial cells to generate fully functional CFTR replacement protein. To evaluate mRNA drug efficacy, a targeted quantitative proteomics method is needed to estimate the expression level of mRNA encoded CFTR protein. Methods In this paper, a method combining membrane protein extraction, immunoprecipitation (IP), and nanoLC-MS/MS for quantifying CFTR in lung tissue samples was reported for the first time. Absolute quantification was performed by constructing a standard curve by spiking recombinant human CFTR protein in mouse lung tissue matrix. Results This method was qualified, with good linearity of standard curve and lower limit of quantification of human CFTR at 1.4 pg per mg tissue. The coefficient of variation of back calculated concentration of all standards and their back-calculation errors were &lt; 20%. The CFTR expression level in mouse lungs dosed with mRNA encapsulated lipid nanoparticle (LNP), and the endogenous level in wild type human lungs were measured successfully. Conclusions The result demonstrated high sensitivity, precision and accuracy of this proteomics method for quantifying low-abundance CFTR protein in lung tissue sample.

Regulation of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) by the Nuclear Bile Acid Receptor, Farnesoid X Receptor

Introduction and AimsCFTR, a Cl‐ channel important in regulating intestinal fluid and electrolyte secretion, is implicated in the pathogenesis of a number of intestinal disorders. We have previously shown that bile acids, acting via the nuclear receptor, farnesoid X receptor (FXR), inhibit colonic epithelial CFTR expression. Dietary phytochemicals have been reported to have the capacity to modulate FXR signalling. Here, we set out to investigate mechanisms underlying FXR regulation of CFTR expression, and the potential for therapeutically targeting the receptor with dietary phytochemicals.MethodsMonolayers of T84 colonic epithelial cells were treated with the FXR agonist, GW4064 (5 µM) in the absence or presence of a plant phytochemical, designated here as KFS1 (5 µM). mRNA and protein expression were measured by qPCR and Western blotting. FXR binding to the CFTR promoter was investigated by chromatin immunoprecipitation (ChIP). Electrophysiological studies of T84cells were conducted in Ussing chambers. Studies on human and murine enteroids were carried out with ethical approval from Johns Hopkins University School of Medicine Institutional Review Board and the Institutional Review Board of the University of California San Diego, respectively.ResultsTreatment of T84 monolayers with GW4064 downregulated CFTR mRNA to 0.51 ± 0.06 fold after 12 hrs (n = 12; p &lt; 0.001) and protein levels to 0.28 ± 0.06 fold after 48 hrs, compared to controls (n = 8; p &lt; 0.001). Studies in Ussing chambers showed that GW4064 treatment for 48 hrs inhibited Cl‐ secretory responses to the Ca2+‐ and cAMP‐dependent agonists, carbachol (CCh; 100mM) and forskolin (FSK; 10mM), by 79.9 ± 7.5 % and 74.2 ± 8.9 %, respectively. Transcriptomic analysis of human colonic enteroids revealed FXR to be robustly expressed in secretory (crypt‐like) cells and that its activation also induced CFTR downregulation. FXR activation did not alter expression or phosphorylation of the p65 subunit of NF‐κB, or inhibit its translocation to the nucleus. ChIP‐qPCR analysis appears to show enhanced binding of FXR to the CFTR promoter upon GW4064 treatment. The phytochemical, KFS1 (5 mM; 24hrs), upregulated FXR mRNA and protein expression in T84 cells and enhanced GW4064‐induced downregulation of CFTR mRNA by 0.28 ± 0.05 fold (n = 8; p &lt;0.01) and protein by 0.25 ± 0.11 fold (n = 4) after 24 hours. Similarly, KFS1 significantly upregulated FXR mRNA expression in murine colonic epithelial enteroids (2.3 ± 0.2; n = 4; p &lt; 0.01) and enhanced GW4064‐induced downregulation of CFTR mRNA by 0.5 ± 0.1 fold (n = 4; p &lt; 0.05) compared to GW4064 alone. Finally, KFS1 enhanced FXR inhibition of agonist‐induced Cl‐ secretory responses across T84 cells mounted in Ussing chambers.ConclusionFXR regulates colonic epithelial CFTR expression and function by a mechanism which appears to involve direct binding of FXR to the CFTR promoter. By virtue of their ability to upregulate FXR expression, and thereby enhance its antisecretory actions, plant extracts containing KFS1 have excellent potential to be developed as FXR‐targeted nutraceuticals for the treatment and prevention of intestinal disease.

CFTR mRNAs with nonsense codons are degraded by the SMG6-mediated endonucleolytic decay\xa0pathway

Approximately 10% of cystic fibrosis patients harbor nonsense mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene which can generate nonsense codons in the CFTR mRNA and subsequently activate the nonsense-mediated decay (NMD) pathway resulting in rapid mRNA degradation. However, it is not known which NMD branches govern the decay of CFTR mRNAs containing nonsense codons. Here we utilize antisense oligonucleotides targeting NMD factors to evaluate the regulation of nonsense codon-containing CFTR mRNAs by the NMD pathway. We observe that CFTR mRNAs with nonsense codons G542X, R1162X, and W1282X, but not Y122X, require UPF2 and UPF3 for NMD. Furthermore, we demonstrate that all evaluated CFTR mRNAs harboring nonsense codons are degraded by the SMG6-mediated endonucleolytic pathway rather than the SMG5-SMG7-mediated exonucleolytic pathway. Finally, we show that upregulation of all evaluated CFTR mRNAs with nonsense codons by NMD pathway inhibition improves outcomes of translational readthrough therapy.