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https://www.atsjournals.org/doi/10.1165/rcmb.2019-0442OC

https://www.ncbi.nlm.nih.gov/pubmed/33026825?dopt=Abstract

TMEM16A Mediated Mucus Production in Human Airway Epithelial Cells.

Am J Respir Cell Mol Biol. 2020 Oct 07;:

Authors: Cabrita I, Benedetto R, Wanitchakool P, Lerias J, Centeio R, Ousingsawat J, Schreiber R, Kunzelmann K

Abstract

TMEM16A is a Ca2+ activated chloride channel that was shown to enhance production and secretion of mucus in inflamed airways. It is, however, not clear whether TMEM16A is truly responsible for mucus production, or whether mucin and TMEM16A are upregulated independently during inflammatory airway diseases such as asthma and cystic fibrosis (CF). We examined this question using BCi-NS1 cells, a human airway basal cell line that maintains multipotent differentiation capacity, and the two human airway epithelial cell lines, Calu-3 and CFBE. The data demonstrate that exposure of airway epithelial cells to IL-8 and IL-13, two cytokines known to be enhanced in CF and asthma, respectively, leads to increase in mucus production. Expression of MUC5AC was fully dependent on expression of TMEM16A, as shown by siRNA-knockdown of TMEM16A. In addition, different inhibitors of TMEM16A attenuated IL-13 induced mucus production. Interestingly, in CFBE cells expressing F508delCFTR, IL-13 was unable to upregulate membrane expression of TMEM16A or Ca2+-activated whole cell currents. The regulator of TMEM16A, CLCA1, strongly augmented both Ca2+- and cAMP-activated Cl- currents in cells expressing wtCFTR, but failed to augment membrane expression of TMEM16A in F508delCFTR-expressing CFBE cells. The data confirm the functional relationship between CFTR and TMEM16A and suggest an impaired upregulation of TMEM16A by IL-13 or CLCA1 in cells expressing the most frequent CF-causing mutation F508delCFTR.

PMID: 33026825 [PubMed – as supplied by publisher]

PubMed:33026825

https://www.mdpi.com/2073-4425/11/10/1169/htm

https://www.ncbi.nlm.nih.gov/pubmed/33036232?dopt=Abstract

Detargeting Lentiviral-Mediated CFTR Expression in Airway Basal Cells Using miR-106b.

Genes (Basel). 2020 Oct 06;11(10):

Authors: Choi SH, Reeves RE, Romano Ibarra GS, Lynch TJ, Shahin WS, Feng Z, Gasser GN, Winter MC, Evans TIA, Liu X, Luo M, Zhang Y, Stoltz DA, Devor EJ, Yan Z, Engelhardt JF

Abstract

Lentiviral-mediated integration of a CFTR transgene cassette into airway basal cells is a strategy being considered for cystic fibrosis (CF) cell-based therapies. However, CFTR expression is highly regulated in differentiated airway cell types and a subset of intermediate basal cells destined to differentiate. Since basal stem cells typically do not express CFTR, suppressing the CFTR expression from the lentiviral vector in airway basal cells may be beneficial for maintaining their proliferative capacity and multipotency. We identified miR-106b as highly expressed in proliferating airway basal cells and extinguished in differentiated columnar cells. Herein, we developed lentiviral vectors with the miR-106b-target sequence (miRT) to both study miR-106b regulation during basal cell differentiation and detarget CFTR expression in basal cells. Given that miR-106b is expressed in the 293T cells used for viral production, obstacles of viral genome integrity and titers were overcome by creating a 293T-B2 cell line that inducibly expresses the RNAi suppressor B2 protein from flock house virus. While miR-106b vectors effectively detargeted reporter gene expression in proliferating basal cells and following differentiation in the air-liquid interface and organoid cultures, the CFTR-miRT vector produced significantly less CFTR-mediated current than the non-miR-targeted CFTR vector following transduction and differentiation of CF basal cells. These findings suggest that miR-106b is expressed in certain airway cell types that contribute to the majority of CFTR anion transport in airway epithelium.

PMID: 33036232 [PubMed – as supplied by publisher]

PubMed:33036232

https://www.mdpi.com/2073-4409/9/8/1845/htm

https://www.ncbi.nlm.nih.gov/pubmed/32781626?dopt=Abstract

Related Articles

Cystic Fibrosis Defective Response to Infection Involves Autophagy and Lipid Metabolism.

Cells. 2020 Aug 06;9(8):

Authors: Mingione A, Ottaviano E, Barcella M, Merelli I, Rosso L, Armeni T, Cirilli N, Ghidoni R, Borghi E, Signorelli P

Abstract

Cystic fibrosis (CF) is a hereditary disease, with 70% of patients developing a proteinopathy related to the deletion of phenylalanine 508. CF is associated with multiple organ dysfunction, chronic inflammation, and recurrent lung infections. CF is characterized by defective autophagy, lipid metabolism, and immune response. Intracellular lipid accumulation favors microbial infection, and autophagy deficiency impairs internalized pathogen clearance. Myriocin, an inhibitor of sphingolipid synthesis, significantly reduces inflammation, promotes microbial clearance in the lungs, and induces autophagy and lipid oxidation. RNA-seq was performed in Aspergillusfumigatus-infected and myriocin-treated CF patients’ derived monocytes and in a CF bronchial epithelial cell line. Fungal clearance was also evaluated in CF monocytes. Myriocin enhanced CF patients’ monocytes killing of A. fumigatus. CF patients’ monocytes and cell line responded to infection with a profound transcriptional change; myriocin regulates genes that are involved in inflammation, autophagy, lipid storage, and metabolism, including histones and heat shock proteins whose activity is related to the response to infection. We conclude that the regulation of sphingolipid synthesis induces a metabolism drift by promoting autophagy and lipid consumption. This process is driven by a transcriptional program that corrects part of the differences between CF and control samples, therefore ameliorating the infection response and pathogen clearance in the CF cell line and in CF peripheral blood monocytes.

PMID: 32781626 [PubMed – in process]

PubMed:32781626

https://www.ncbi.nlm.nih.gov/pubmed/32573948?dopt=Abstract

https://onlinelibrary.wiley.com/doi/abs/10.1002/ppul.24919

Highlights From the 2019 North American Cystic Fibrosis Conference.

Pediatr Pulmonol. 2020 Jun 23;:

Authors: Hoppe JE, Guimbellot J, Martiniano SL, Toprak D, Davis C, Daines CL, Muhlebach MS, Esther CR, Dellon EP

Abstract

The 33rd annual North American Cystic Fibrosis Conference (NACFC) was held in Nashville, Tennessee on October 31-November 2, 2019. Abstracts of presentations from the conference were published in a supplement to Pediatric Pulmonology 1 . This review briefly summarizes presentations in several major topic areas at the conference: pathophysiology and basic science of cystic fibrosis (CF) lung disease; clinical trials; clinical quality improvement; microbiology and treatment of infection; and transition, advanced lung disease and transplant, mental health and psychosocial concerns. The review is intended to highlight several areas and is not a comprehensive summary of the conference. Citations from the conference are by first author and abstract number or symposium number, as designated in the supplement. This article is protected by copyright. All rights reserved.

PMID: 32573948 [PubMed – as supplied by publisher]

PubMed:32573948

https://onlinelibrary.wiley.com/doi/abs/10.1002/ppul.24364

https://www.ncbi.nlm.nih.gov/pubmed/31715088?dopt=Abstract

Anti-inflammatories and mucociliary clearance therapies in the age of CFTR modulators.

Pediatr Pulmonol. 2019 Nov;54 Suppl 3:S46-S55

Authors: Perrem L, Ratjen F

Abstract

Cystic fibrosis (CF) is a genetic and life-limiting disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. This multi-system disease is characterized by progressive lung disease and pancreatic insufficiency amongst other manifestations. CFTR primarily functions as a chloride channel that transports ions across the apical membrane of epithelial cells but has other functions, including bicarbonate secretion and inhibition of sodium transport. Defective CFTR disrupts these functions, causing viscous and dehydrated mucus to accumulate, compromising the airway lumen and contributing to obstructive pulmonary disease. The combination of CFTR dysfunction, mucus obstruction, and infection drive an exaggerated and dysfunctional inflammatory response, which contributes to irreversible airway destruction and fibrosis. CFTR modulators, an exciting new class of drugs, increase the expression and/or function of CFTR variant protein and improve multiple clinical endpoints, such as lung function, pulmonary exacerbation rates, and nutritional status. However, these genotype-specific drugs are not universally available, the clinical response is variable, and lung function still declines over time when bronchiectasis is established. Consequently, even in the age of CFTR modulators, we must target other important aspects of the CF airway disease, such as inflammation and mucociliary clearance. This review highlights the mechanisms of inflammation and mucus accumulation in the CF lung and discusses anti-inflammatory and mucociliary clearance agents that are currently in development focusing on compounds for which clinical trial data have recently become available.

PMID: 31715088 [PubMed – in process]

PubMed:31715088

https://www.atsjournals.org/doi/10.1165/rcmb.2018-0314OC

https://www.ncbi.nlm.nih.gov/pubmed/31596601?dopt=Abstract

S-Nitrosylation of CHIP Enhances F508Del CFTR Maturation.

Am J Respir Cell Mol Biol. 2019 Oct 09;:

Authors: Zaman K, Knight J, Hussain F, Cao R, Estabrooks SK, Altawallbeh G, Holloway K, Jafri A, Sawczak V, Li Y, Getsy P, Sun F, Raffay T, Cotton C, Brodsky JL, Periasamy A, Lewis SJ, Gaston B

Abstract

S-Nitrosothiols (SNOs) are endogenous signaling compounds with a diverse spectrum of beneficial airway effects that are both cGMP-dependent and -independent. SNOs are present in healthy human airways, but levels are low in the airways of cystic fibrosis (CF) patients. Here, we evaluated the interactions of SNOs with molecular co-chaperone C-terminus Hsc70 interacting protein (CHIP)- an E3 ubiquitin ligase that targets improperly folded CFTR for degradation. CHIP was expressed in primary human bronchial epithelial and CFBE41o – cells expressing either wild type or F508del CFTR. Confocal microscopy and immunoprecipitation studies showed the cellular co-localization of CFTR and CHIP and showed that GSNO inhibits the CHIP-CFTR interaction. SNOs significantly reduced both the expression and activity of CHIP, leading to higher levels of both the immature and mature forms of F508del CFTR; in fact, inhibition of the expression and function of CHIP by SNOs not only improved CFTR maturation, but also increased CFTR stability at the cell membrane. GSNO treated cells also had more S-nitrosylated CHIP and less ubiquitinated CFTR than untreated cells, suggesting that S-nitrosylation of CHIP prevents CFTR ubiquitination by inhibiting CHIP’s E3 ubiquitin ligase function. Further, the exogenous SNOs S-nitrosoglutathione diethyl ester (GNODE) and S-nitro-N-acetylcysteine (SNOAC) increased CFTR expression at the cell surface. Following CHIP knockdown with siRNA duplexes specific for CHIP, F508del CFTR expression increased at the cell surface. We conclude that SNOs effectively reduce CHIP-mediated degradation of CFTR, resulting in increased F508del CFTR expression on the surface of airway epithelial cells.

PMID: 31596601 [PubMed – as supplied by publisher]

PubMed:31596601

https://www.mdpi.com/1422-0067/20/19/4933

https://www.ncbi.nlm.nih.gov/pubmed/31590401?dopt=Abstract

Related Articles

Transforming Growth Factor-β1 Selectively Recruits microRNAs to the RNA-Induced Silencing Complex and Degrades CFTR mRNA under Permissive Conditions in Human Bronchial Epithelial Cells.

Int J Mol Sci. 2019 Oct 05;20(19):

Authors: Mitash N, Mu F, Donovan JE, Myerburg MM, Ranganathan S, Greene CM, Swiatecka-Urban A

Abstract

Mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene lead to cystic fibrosis (CF). The most common mutation F508del inhibits folding and processing of CFTR protein. FDA-approved correctors rescue the biosynthetic processing of F508del-CFTR protein, while potentiators improve the rescued CFTR channel function. Transforming growth factor (TGF-β1), overexpressed in many CF patients, blocks corrector/potentiator rescue by inhibiting CFTR mRNA in vitro. Increased TGF-β1 signaling and acquired CFTR dysfunction are present in other lung diseases. To study the mechanism of TGF-β1 repression of CFTR, we used molecular, biochemical, and functional approaches in primary human bronchial epithelial cells from over 50 donors. TGF-β1 destabilized CFTR mRNA in cells from lungs with chronic disease, including CF, and impaired F508del-CFTR rescue by new-generation correctors. TGF-β1 increased the active pool of selected micro(mi)RNAs validated as CFTR inhibitors, recruiting them to the RNA-induced silencing complex (RISC). Expression of F508del-CFTR globally modulated TGF-β1-induced changes in the miRNA landscape, creating a permissive environment required for degradation of F508del-CFTR mRNA. In conclusion, TGF-β1 may impede the full benefit of corrector/potentiator therapy in CF patients. Studying miRNA recruitment to RISC under disease-specific conditions may help to better characterize the miRNAs utilized by TGF-β1 to destabilize CFTR mRNA.

PMID: 31590401 [PubMed – in process]

PubMed:31590401

https://www.sciencedirect.com/science/article/pii/S1357272519301992?via%3Dihub

https://www.ncbi.nlm.nih.gov/pubmed/31563560?dopt=Abstract

Inflammatory role of extracellular sphingolipids in Cystic Fibrosis.

Int J Biochem Cell Biol. 2019 Sep 26;:105622

Authors: Zulueta A, Peli V, Dei Cas M, Colombo M, Paroni R, Falleni M, Baisi A, Bollati V, Chiaramonte R, Del Favero E, Ghidoni R, Caretti A

Abstract

Ceramide is emerging as one of the players of inflammation in lung diseases. However, data on its inflammatory role in Cystic Fibrosis (CF) as part of the extracellular machinery driven by lung mesenchymal stem cells (MSCs)-derived extracellular vesicles (EVs) are missing. We obtained an in vitro model of CF-MSC by treating control human lung MSCs with a specific CFTR inhibitor. We characterized EVs populations derived from MSCs (ctr EVs) and CF-MSCs (CF-EVs) and analyzed their sphingolipid profile by LC-MS/MS. To evaluate their immunomodulatory function, we treated an in vitro human model of CF, with both EVs populations. Our data show that the two EVs populations differ for the average size, amount, and rate of uptake. CF-EVs display higher ceramide and dihydroceramide accumulation as compared to control EVs, suggesting the involvement of the de novo biosynthesis pathway in the parental CF-MSCs. Higher sphingomyelinase activity in CF-MSCs, driven by inflammation-induced ceramide accumulation, sustains the exocytosis of vesicles that export new formed pro-inflammatory ceramide. Our results suggest that CFTR dysfunction associates with an enhanced sphingolipid metabolism leading to the release of EVs that export the excess of pro-inflammatory Cer to the recipient cells, thus contributing to maintain the unresolved inflammatory status of CF.

PMID: 31563560 [PubMed – as supplied by publisher]

PubMed:31563560

https://www.ncbi.nlm.nih.gov/pubmed/31524632?dopt=Abstract

IL-1β dominates the promucin secretory cytokine profile in cystic fibrosis.

J Clin Invest. 2019 Sep 16;:

Authors: Chen G, Sun L, Kato T, Okuda K, Martino MB, Abzhanova A, Lin JM, Gilmore RC, Batson BD, O’Neal YK, Volmer AS, Dang H, Deng Y, Randell SH, Button B, Livraghi-Butrico A, Kesimer M, Ribeiro CM, O’Neal WK, Boucher RC

Abstract

Cystic fibrosis (CF) lung disease is characterized by early and persistent mucus accumulation and neutrophilic inflammation in the distal airways. Identification of the factors in CF mucopurulent secretions that perpetuate CF mucoinflammation may provide strategies for novel CF pharmacotherapies. We show that IL-1β, with IL-1α, dominated the mucin prosecretory activities of supernatants of airway mucopurulent secretions (SAMS). Like SAMS, IL-1β alone induced MUC5B and MUC5AC protein secretion and mucus hyperconcentration in CF human bronchial epithelial (HBE) cells. Mechanistically, IL-1β induced the sterile α motif-pointed domain containing ETS transcription factor (SPDEF) and downstream endoplasmic reticulum to nucleus signaling 2 (ERN2) to upregulate mucin gene expression. Increased mRNA levels of IL1B, SPDEF, and ERN2 were associated with increased MUC5B and MUC5AC expression in the distal airways of excised CF lungs. Administration of an IL-1 receptor antagonist (IL-1Ra) blocked SAMS-induced expression of mucins and proinflammatory mediators in CF HBE cells. In conclusion, IL-1α and IL-1β are upstream components of a signaling pathway, including IL-1R1 and downstream SPDEF and ERN2, that generate a positive feedback cycle capable of producing persistent mucus hyperconcentration and IL-1α and/or IL-1β-mediated neutrophilic inflammation in the absence of infection in CF airways. Targeting this pathway therapeutically may ameliorate mucus obstruction and inflammation-induced structural damage in young CF children.

PMID: 31524632 [PubMed – as supplied by publisher]

PubMed:31524632

https://www.jci.org/articles/view/131652

https://www.ncbi.nlm.nih.gov/pubmed/31524633?dopt=Abstract

Revealing the molecular signaling pathways of mucus stasis in cystic fibrosis.

J Clin Invest. 2019 Sep 16;:

Authors: Birket SE, Rowe SM

Abstract

Mucus obstruction is a hallmark of cystic fibrosis (CF) airway disease, leading to chronic infection, dysregulated inflammation, and progressive lung disease. As mucus hyperexpression is a key component in the initiation and perpetuation of airway obstruction, the triggers underlying mucin release must be identified and understood. In this issue of the JCI, Chen et al. sought to delineate the mechanisms that allow IL-1α/IL-1β to perpetuate the mucoinflammatory environment characteristic of the CF airway. The authors demonstrated that IL-1α and IL-1β stimulated non-CF human bronchial epithelial (HBE) cells to upregulate and secrete both MUC5B and MUC5AC in a dose-dependent manner, an effect that was neutralized by the inhibition of the IL-1α/IL-1β receptor (IL-1R1). Further experiments using mouse models and excised lung tissue identified contributors that drive a vicious feedback cycle of hyperconcentrated mucus secretions and persistent inflammation in the CF airway, factors that are likely at the nidus of progressive lung disease.

PMID: 31524633 [PubMed – as supplied by publisher]

PubMed:31524633

https://www.ncbi.nlm.nih.gov/pubmed/31501480?dopt=Abstract

https://www.nature.com/articles/s41598-019-46237-1

G3BP1, p62 and USP10 could be therapeutic targets for ubiquitinated protein aggregation disorders, including cystic fibrosis (CF)

Related Articles

G3BP1 inhibits ubiquitinated protein aggregations induced by p62 and USP10.

Sci Rep. 2019 Sep 09;9(1):12896

Authors: Anisimov S, Takahashi M, Kakihana T, Katsuragi Y, Kitaura H, Zhang L, Kakita A, Fujii M

Abstract

The aberrant accumulation of ubiquitinated protein aggregates in cells plays a critical role in the pathogenesis of several degenerative diseases, including Parkinson disease (PD) and cystic fibrosis (CF). In this study, we found that Ras GTPase-activating protein-binding protein 1 (G3BP1) inhibits ubiquitinated protein aggregations induced by p62 and USP10 in cultured cells. p62 is a ubiquitin receptor, and p62 and its binding partner USP10 have been shown to augment ubiquitinated protein aggregation. G3BP1 interacted with p62 and USP10 and inhibited p62/USP10-induced protein aggregation. The G3BP1 inhibition of protein aggregations targeted two aggregation-prone proteins, α-synuclein and CFTR-ΔF508, which are causative factors of PD and CF, respectively. G3BP1 depletion increased the amounts of ubiquitinated α-synuclein and CFTR-ΔF508 protein. A proteasome reporter indicated that G3BP1 depletion inhibits the proteasome activity. We herein present evidence that G3BP1, p62 and USP10 together control ubiquitinated protein toxicity by controlling both ubiquitination and aggregation. Taken together, these results suggest that G3BP1, p62 and USP10 could be therapeutic targets for ubiquitinated protein aggregation disorders, including PD and CF.

PMID: 31501480 [PubMed – in process]

PubMed:31501480

https://www.cysticfibrosisjournal.com/article/S1569-1993(19)30868-9/fulltext

https://www.ncbi.nlm.nih.gov/pubmed/31474496?dopt=Abstract

Related Articles

Whole-blood transcriptomic responses to lumacaftor/ivacaftor therapy in cystic fibrosis.

J Cyst Fibros. 2019 Aug 29;:

Authors: Kopp BT, Fitch J, Jaramillo L, Shrestha CL, Robledo-Avila F, Zhang S, Palacios S, Woodley F, Hayes D, Partida-Sanchez S, Ramilo O, White P, Mejias A

Abstract

BACKGROUND: Cystic fibrosis (CF) remains without a definitive cure. Novel therapeutics targeting the causative defect in the cystic fibrosis transmembrane conductance regulator (CFTR) gene are in clinical use. Lumacaftor/ivacaftor is a CFTR modulator approved for patients homozygous for the CFTR variant p.Phe508del, but there are wide variations in treatment responses preventing prediction of patient responses. We aimed to determine changes in gene expression related to treatment initiation and response.

METHODS: Whole-blood transcriptomics was performed using RNA-Seq in 20 patients with CF pre- and 6 months post-lumacaftor/ivacaftor (drug) initiation and 20 non-CF healthy controls. Correlation of gene expression with clinical variables was performed by stratification via clinical responses.

RESULTS: We identified 491 genes that were differentially expressed in CF patients (pre-drug) compared with non-CF controls and 36 genes when comparing pre-drug to post-drug profiles. Both pre- and post-drug CF profiles were associated with marked overexpression of inflammation-related genes and apoptosis genes, and significant under-expression of T cell and NK cell-related genes compared to non-CF. CF patients post-drug demonstrated normalized protein synthesis expression, and decreased expression of cell-death genes compared to pre-drug profiles, irrespective of clinical response. However, CF clinical responders demonstrated changes in eIF2 signaling, oxidative phosphorylation, IL-17 signaling, and mitochondrial function compared to non-responders. Top overexpressed genes (MMP9 and SOCS3) that decreased post-drug were validated by qRT-PCR. Functional assays demonstrated that CF monocytes normalized calcium (increases MMP9 expression) concentrations post-drug.

CONCLUSIONS: Transcriptomics revealed differentially regulated pathways in CF patients at baseline compared to non-CF, and in clinical responders to lumacaftor/ivacaftor.

PMID: 31474496 [PubMed – as supplied by publisher]

PubMed:31474496

https://royalsocietypublishing.org/doi/10.1098/rsif.2019.0187

https://www.ncbi.nlm.nih.gov/pubmed/31455163?dopt=Abstract

Related Articles

Thickness of the airway surface liquid layer in the lung is affected in cystic fibrosis by compromised synergistic regulation of the ENaC ion channel.

J R Soc Interface. 2019 Aug 30;16(157):20190187

Authors: Olivença DV, Fonseca LL, Voit EO, Pinto FR

Abstract

The lung epithelium is lined with a layer of airway surface liquid (ASL) that is crucial for healthy lung function. ASL thickness is controlled by two ion channels: epithelium sodium channel (ENaC) and cystic fibrosis (CF) transmembrane conductance regulator (CFTR). Here, we present a minimal mathematical model of ENaC, CFTR and ASL regulation that sheds light on the control of ENaC by the short palate lung and nasal epithelial clone 1 (SPLUNC1) protein and by phosphatidylinositol 4,5-biphosphate (PI(4,5)P2). The model, despite its simplicity, yields a good fit to experimental observations and is an effective tool for exploring the interplay between ENaC, CFTR and ASL. Steady-state data and dynamic information constrain the model’s parameters without ambiguities. Testing the hypothesis that PI(4,5)P2 protects ENaC from ubiquitination suggests that this protection does not improve the model results and that the control of the ENaC opening probability by PI(4,5)P2 is sufficient to explain all available data. The model analysis further demonstrates that ASL at the steady state is sensitive to small changes in PI(4,5)P2 abundance, particularly in CF conditions, which suggests that manipulation of phosphoinositide metabolism may promote therapeutic benefits for CF patients.

PMID: 31455163 [PubMed – in process]

PubMed:31455163

https://www.fasebj.org/doi/10.1096/fj.201901050R

https://www.ncbi.nlm.nih.gov/pubmed/31450978?dopt=Abstract

Related Articles

CK19 stabilizes CFTR at the cell surface by limiting its endocytic pathway degradation.

FASEB J. 2019 Aug 26;:fj201901050R

Authors: Hou X, Wu Q, Rajagopalan C, Zhang C, Bouhamdan M, Wei H, Chen X, Zaman K, Li C, Sun X, Chen S, Frizzell RA, Sun F

Abstract

Protein interactions that stabilize the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) at the apical membranes of epithelial cells have not yet been fully elucidated. We identified keratin 19 (CK19 or K19) as a novel CFTR-interacting protein. CK19 overexpression stabilized both wild-type (WT)-CFTR and Lumacaftor (VX-809)-rescued F508del-CFTR (where F508del is the deletion of the phenylalanine residue at position 508) at the plasma membrane (PM), promoting Cl- secretion across human bronchial epithelial (HBE) cells. CK19 prevention of Rab7A-mediated lysosomal degradation was a key mechanism in apical CFTR stabilization. Unexpectedly, CK19 expression was decreased by ∼40% in primary HBE cells from homogenous F508del patients with CF relative to non-CF controls. CK19 also positively regulated multidrug resistance-associated protein 4 expression at the PM, suggesting that this keratin may regulate the apical expression of other ATP-binding cassette proteins as well as CFTR.-Hou, X., Wu, Q., Rajagopalan, C., Zhang, C., Bouhamdan, M., Wei, H., Chen, X., Zaman, K., Li, C., Sun, X., Chen, S., Frizzell, R. A., Sun, F. CK19 stabilizes CFTR at the cell surface by limiting its endocytic pathway degradation.

PMID: 31450978 [PubMed – as supplied by publisher]

PubMed:31450978

https://www.nature.com/articles/s41598-019-48105-4

https://www.ncbi.nlm.nih.gov/pubmed/31413336?dopt=Abstract

A new type of mouse has been developed that will advance in vivo testing of new CF therapies

Related Articles

A BAC Transgene Expressing Human CFTR under Control of Its Regulatory Elements Rescues Cftr Knockout Mice.

Sci Rep. 2019 Aug 14;9(1):11828

Authors: Gawenis LR, Hodges CA, McHugh DR, Valerio DM, Miron A, Cotton CU, Liu J, Walker NM, Strubberg AM, Gillen AE, Mutolo MJ, Kotzamanis G, Bosch J, Harris A, Drumm ML, Clarke LL

Abstract

Small-molecule modulators of cystic fibrosis transmembrane conductance regulator (CFTR) biology show promise in the treatment of cystic fibrosis (CF). A Cftr knockout (Cftr KO) mouse expressing mutants of human CFTR would advance in vivo testing of new modulators. A bacterial artificial chromosome (BAC) carrying the complete hCFTR gene including regulatory elements within 40.1 kb of DNA 5′ and 25 kb of DNA 3′ to the gene was used to generate founder mice expressing hCFTR. Whole genome sequencing indicated a single integration site on mouse chromosome 8 (8qB2) with ~6 gene copies. hCFTR+ offspring were bred to murine Cftr KO mice, producing hCFTR+/mCftr- (H+/m-) mice, which had normal survival, growth and goblet cell function as compared to wild-type (WT) mice. Expression studies showed hCFTR protein and transcripts in tissues typically expressing mCftr. Functionally, nasal potential difference and large intestinal short-circuit (Isc) responses to cAMP stimulation were similar in magnitude to WT mice, whereas small intestinal cAMP ΔIsc responses were reduced. A BAC transgenic mouse with functional hCFTR under control of its regulatory elements has been developed to enable the generation of mouse models of hCFTR mutations by gene editing for in vivo testing of new CF therapies.

PMID: 31413336 [PubMed – in process]

PubMed:31413336

https://www.mdpi.com/2218-273X/9/8/365

https://www.ncbi.nlm.nih.gov/pubmed/31416173?dopt=Abstract

Related Articles

Neutrophil Extracellular Trap Formation: Physiology, Pathology, and Pharmacology.

Biomolecules. 2019 Aug 14;9(8):

Authors: Ravindran M, Khan MA, Palaniyar N

Abstract

Neutrophil extracellular traps (NETs), a unique DNA framework decorated with antimicrobial peptides, have been in the scientific limelight for their role in a variety of pathologies ranging from cystic fibrosis to cancer. The formation of NETs, as well as relevant regulatory mechanisms, physiological factors, and pharmacological agents have not been systematically discussed in the context of their beneficial and pathological aspects. Novel forms of NET formation including vital NET formation continue to be uncovered, however, there remain fundamental questions around established mechanisms such as NADPH-oxidase (Nox)-dependent and Nox-independent NET formation. Whether NET formation takes place in the tissue versus the bloodstream, internal factors (e.g. reactive oxygen species (ROS) production and transcription factor activation), and external factors (e.g. alkaline pH and hypertonic conditions), have all been demonstrated to influence specific NET pathways. Elements of neutrophil biology such as transcription and mitochondria, which were previously of unknown significance, have been identified as critical mediators of NET formation through facilitating chromatin decondensation and generating ROS, respectively. While promising therapeutics inhibiting ROS, transcription, and gasdermin D are being investigated, neutrophil phagocytosis plays a critical role in host defense and any therapies targeting NET formation must avoid impairing the physiological functions of these cells. This review summarizes what is known in the many domains of NET research, highlights the most relevant challenges in the field, and inspires new questions that can bring us closer to a unified model of NET formation.

PMID: 31416173 [PubMed – in process]

PubMed:31416173

https://www.cysticfibrosisjournal.com/article/S1569-1993(19)30829-X/fulltext

https://www.ncbi.nlm.nih.gov/pubmed/31416774?dopt=Abstract

Related Articles

The impact of CFTR modulator therapies on CF airway microbiology.

J Cyst Fibros. 2019 Aug 12;:

Authors: Rogers GB, Taylor SL, Hoffman LR, Burr LD

Abstract

Major historical advances in cystic fibrosis (CF) respiratory clinical care, including mechanical airway clearance and inhaled medications, have aimed to address the consequences of cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction. In contrast, CFTR modulator therapies instead target the underlying protein defect that leads to CF lung disease. The extent to which these therapies might reduce susceptibility to chronic lung infections remains to be seen. However, by improving airway clearance, reducing the requirement for antibiotics, and in some cases, through direct antimicrobial effects, CFTR modulators are likely to result in substantial changes in CF airway microbiology. These changes could contribute substantially to the clinical benefit associated with modulator therapies, as well as providing an important indicator of treatment efficacy and residual pathophysiology. Indeed, the widespread introduction of modulator therapies might require us to re-consider our models of CF airway microbiology.

PMID: 31416774 [PubMed – as supplied by publisher]

PubMed:31416774

https://www.cysticfibrosisjournal.com/article/S1569-1993(19)30826-4/fulltext

https://www.ncbi.nlm.nih.gov/pubmed/31401006?dopt=Abstract

The role of endothelial cells in cystic fibrosis.

J Cyst Fibros. 2019 Aug 07;:

Authors: Declercq M, Treps L, Carmeliet P, Witters P

Abstract

Cystic fibrosis (CF) is an autosomal recessive disease caused by the loss of function of the cystic fibrosis transmembrane conductance regulator (CFTR) protein which primarily acts as a chloride channel. CFTR has mainly been studied in epithelial cells although it is also functional and expressed in other cell types including endothelial cells. The present review summarizes current knowledge on the role of the endothelium in CF. More specifically, this review highlights the role of endothelial cells in CF in acting as a semipermeable barrier, as a key regulator of angiogenesis, coagulation, the vascular tone and the inflammatory responses. It could contribute to different aspects of the disease including cardiovascular symptoms, excessive blood vessel formation, pulmonary and portal hypertension and CF-related diabetes. Despite the important role of vascular endothelium in many biological processes, it has largely been under investigated in CF.

PMID: 31401006 [PubMed – as supplied by publisher]

PubMed:31401006

https://www.frontiersin.org/articles/10.3389/fimmu.2019.01789/full

https://www.ncbi.nlm.nih.gov/pubmed/31428093?dopt=Abstract

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Metabolic Reprograming of Cystic Fibrosis Macrophages via the IRE1α Arm of the Unfolded Protein Response Results in Exacerbated Inflammation.

Front Immunol. 2019;10:1789

Authors: Lara-Reyna S, Scambler T, Holbrook J, Wong C, Jarosz-Griffiths HH, Martinon F, Savic S, Peckham D, McDermott MF

Abstract

Cystic Fibrosis (CF) is a recessive genetic disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR mutations cause dysregulation of channel function with intracellular accumulation of misfolded proteins and endoplasmic reticulum (ER) stress, with activation of the IRE1α-XBP1 pathway that regulates a subset of unfolded protein response (UPR) genes. This pathway regulates a group of genes that control proinflammatory and metabolic responses in different immune cells; however, the metabolic state of immune cells and the role of this pathway in CF remain elusive. Our results indicate that only innate immune cells from CF patients present increased levels of ER stress, mainly affecting neutrophils, monocytes, and macrophages. An overactive IRE1α-XBP1 pathway reprograms CF M1 macrophages toward an increased metabolic state, with increased glycolytic rates and mitochondrial function, associated with exaggerated production of TNF and IL-6. This hyper-metabolic state, seen in CF macrophages, is reversed by inhibiting the RNase domain of IRE1α, thereby decreasing the increased glycolic rates, mitochondrial function and inflammation. Altogether, our results indicate that innate immune cells from CF patients are primarily affected by ER stress. Moreover, the IRE1α-XBP1 pathway of the UPR is responsible for the hyper-metabolic state seen in CF macrophages, which is associated with the exaggerated inflammatory response. Modulating ER stress, metabolism and inflammation, by targeting IRE1α, may improve the metabolic fitness of macrophages, and other immune cells in CF and other immune-related disorders.

PMID: 31428093 [PubMed – in process]

PubMed:31428093

https://www.frontiersin.org/articles/10.3389/fimmu.2019.01784/full

https://www.ncbi.nlm.nih.gov/pubmed/31417560?dopt=Abstract

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The CXCL12/CXCR4 Signaling Axis Retains Neutrophils at Inflammatory Sites in Zebrafish.

Front Immunol. 2019;10:1784

Authors: Isles HM, Herman KD, Robertson AL, Loynes CA, Prince LR, Elks PM, Renshaw SA

Abstract

The inappropriate retention of neutrophils at inflammatory sites is a major driver of the excessive tissue damage characteristic of respiratory inflammatory diseases including COPD, ARDS, and cystic fibrosis. The molecular programmes which orchestrate neutrophil recruitment to inflammatory sites through chemotactic guidance have been well-studied. However, how neutrophil sensitivity to these cues is modulated during inflammation resolution is not understood. The identification of neutrophil reverse migration as a mechanism of inflammation resolution and the ability to modulate this therapeutically has identified a new target to treat inflammatory disease. Here we investigate the role of the CXCL12/CXCR4 signaling axis in modulating neutrophil retention at inflammatory sites. We used an in vivo tissue injury model to study neutrophilic inflammation using transgenic zebrafish larvae. Expression of cxcl12a and cxcr4b during the tissue damage response was assessed using in situ hybridization and analysis of RNA sequencing data. CRISPR/Cas9 was used to knockdown cxcl12a and cxcr4b in zebrafish larvae. The CXCR4 antagonist AMD3100 was used to block the Cxcl12/Cxcr4 signaling axis pharmacologically. We identified that cxcr4b and cxcl12a are expressed at the wound site in zebrafish larvae during the inflammatory response. Following tail-fin transection, removal of neutrophils from inflammatory sites is significantly increased in cxcr4b and cxcl12a CRISPR knockdown larvae. Pharmacological inhibition of the Cxcl12/Cxcr4 signaling axis accelerated resolution of the neutrophil component of inflammation, an effect caused by an increase in neutrophil reverse migration. The findings of this study suggest that CXCR4/CXCL12 signaling may play an important role in neutrophil retention at inflammatory sites, identifying a potential new target for the therapeutic removal of neutrophils from the lung in chronic inflammatory disease.

PMID: 31417560 [PubMed – in process]

PubMed:31417560

https://link.springer.com/article/10.1007%2Fs00018-019-03211-4

https://www.ncbi.nlm.nih.gov/pubmed/31327045?dopt=Abstract

Discovering the chloride pathway in the CFTR channel.

Cell Mol Life Sci. 2019 Jul 20;:

Authors: Farkas B, Tordai H, Padányi R, Tordai A, Gera J, Paragi G, Hegedűs T

Abstract

Cystic fibrosis (CF), a lethal monogenic disease, is caused by pathogenic variants of the CFTR chloride channel. The majority of CF mutations affect protein folding and stability leading overall to diminished apical anion conductance of epithelial cells. The recently published cryo-EM structures of full-length human and zebrafish CFTR provide a good model to gain insight into structure-function relationships of CFTR variants. Although, some of the structures were determined in the phosphorylated and ATP-bound active state, none of the static structures showed an open pathway for chloride permeation. Therefore, we performed molecular dynamics simulations to generate a conformational ensemble of the protein and used channel detecting algorithms to identify conformations with an opened channel. Our simulations indicate a main intracellular entry at TM4/6, a secondary pore at TM10/12, and a bottleneck region involving numerous amino acids from TM1, TM6, and TM12 in accordance with experiments. Since chloride ions entered the pathway in our equilibrium simulations, but did not traverse the bottleneck region, we performed metadynamics simulations, which revealed two possible exits. One of the chloride ions exits includes hydrophobic lipid tails that may explain the lipid-dependency of CFTR function. In summary, our in silico study provides a detailed description of a potential chloride channel pathway based on a recent cryo-EM structure and may help to understand the gating of the CFTR chloride channel, thus contributing to novel strategies to rescue dysfunctional mutants.

PMID: 31327045 [PubMed – as supplied by publisher]

PubMed:31327045

https://www.dovepress.com/lumacaftor-ivacaftor-in-the-treatment-of-cystic-fibrosis-design-develo-peer-reviewed-fulltext-article-DDDT

https://www.ncbi.nlm.nih.gov/pubmed/31409974?dopt=Abstract

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Lumacaftor-ivacaftor in the treatment of cystic fibrosis: design, development and place in therapy.

Drug Des Devel Ther. 2019;13:2405-2412

Authors: Connett GJ

Abstract

Lumacaftor-ivacaftor is a combination of two small molecule therapies targeting the basic defect in cystic fibrosis (CF) at a cellular level. It is a precision medicine and its effects are specific to individuals with two copies of the p.Phe508del gene mutation. The drug combination works by restoring functioning CF transmembrane conductance regulator (CFTR) protein in cell surface membranes and was the first CFTR modulator licensed for the homozygous p.Phe508del genotype. The drug is a combination of a CFTR corrector and potentiator. Lumacaftor, the corrector, works by increasing the trafficking of CFTR proteins to the outer cell membrane. Ivacaftor, the potentiator, works by enabling the opening of what would otherwise be a dysfunctional chloride channel. In vivo lumacaftor-ivacaftor improves Phe508del-CFTR activity in airways, sweat ducts and intestine to approximately 10-20% of normal CFTR function with greater reductions in sweat chloride levels in children versus adults. Its use results in a modest improvement in lung function and a decreased rate of subsequent decline. Perhaps more importantly, those treated report increased levels of well-being and their rate of respiratory exacerbations is significantly improved. This review traces the development and use of this combination of CFTR modulators, the first licensed drug for treating the homozygous p.Phe508del CF genotype at the intracellular level by correcting the protein defect.

PMID: 31409974 [PubMed – in process]

PubMed:31409974