Mucin glycosylation and sulphation in airway epithelial cells is not influenced by cystic fibrosis transmembrane conductance regulator expression

Abnormalities in mucus properties and clearance make a major contribution to the pathology of cystic fibrosis (CF). Our aim was to test the hypothesis that the defects in CF mucus are a direct result of mutations in the CF transmembrane conductance regulator (CFTR) protein. We evaluated a single mucin molecule MUC1F/5ACTR that carries tandem repeat sequence from MUC5AC, a major secreted airway mucin, in a MUC1 mucin vector. To establish whether the presence of mutant or normal CFTR directly influences the O-glycosylation and sulphation of mucins in airway epithelial cells, we used the CFT1-LC3 (DeltaF508 CFTR mutant) and CFT1-LCFSN (wild-type CFTR corrected) human airway epithelial cell lines. MUC1F/5ACTR mucin was immunoprecipitated, centricon purified, and O-glycosylation was evaluated by Matrix-assisted laser desorption ionization and electrospray tandem mass spectrometry to determine the composition of different carbohydrate structures. Mass spectrometry data showed the same O-glycans in both CFTR mutant and wild-type CFTR corrected cells. Metabolic labeling assays were performed to evaluate gross glycosylation and sulphation of the mucins and showed no significant difference in mucin synthesized in six independent clones of these cell lines. Our results show that the absence of functional CFTR protein causes neither an abnormality in mucin O-glycosylation nor an increase in mucin sulphation

Ets homologous factor (EHF) has critical roles in epithelial dysfunction in airway disease

The airway epithelium forms a barrier between the internal and external environments. Epithelial dysfunction is critical in the pathology of many respiratory diseases, including cystic fibrosis. Ets homologous factor (EHF) is a key member of the transcription factor network that regulates gene expression in the airway epithelium in response to endogenous and exogenous stimuli. EHF , which has altered expression in inflammatory states, maps to the 5' end of an intergenic region on Chr11p13 that is implicated as a modifier of cystic fibrosis airway disease. Here we determine the functions of EHF in primary human bronchial epithelial (HBE) cells and relevant airway cell lines. Using EHF ChIP followed by deep sequencing (ChIP-seq) and RNA sequencing after EHF depletion, we show that EHF targets in HBE cells are enriched for genes involved in inflammation and wound repair. Furthermore, changes in gene expression impact cell phenotype because EHF depletion alters epithelial secretion of a neutrophil chemokine and slows wound closure in HBE cells. EHF activates expression of the SAM pointed domain-containing ETS transcription factor, which contributes to goblet cell hyperplasia. Our data reveal a critical role for EHF in regulating epithelial function in lung disease

Differential contribution of cis -regulatory elements to higher order chromatin structure and expression of the CFTR locus

Higher order chromatin structure establishes domains that organize the genome and coordinate gene expression. However, the molecular mechanisms controlling transcription of individual loci within a topological domain (TAD) are not fully understood. The cystic fibrosis transmembrane conductance regulator (CFTR) gene provides a paradigm for investigating these mechanisms. CFTR occupies a TAD bordered by CTCF/cohesin binding sites within which are cell-type-selective cis-regulatory elements for the locus. We showed previously that intronic and extragenic enhancers, when occupied by specific transcription factors, are recruited to the CFTR promoter by a looping mechanism to drive gene expression. Here we use a combination of CRISPR/Cas9 editing of cis-regulatory elements and siRNA-mediated depletion of architectural proteins to determine the relative contribution of structural elements and enhancers to the higher order structure and expression of the CFTR locus. We found the boundaries of the CFTR TAD are conserved among diverse cell types and are dependent on CTCF and cohesin complex. Removal of an upstream CTCF-binding insulator alters the interaction profile, but has little effect on CFTR expression. Within the TAD, intronic enhancers recruit cell-type selective transcription factors and deletion of a pivotal enhancer element dramatically decreases CFTR expression, but has minor effect on its 3D structure

Ets homologous factor regulates pathways controlling response to injury in airway epithelial cells

Ets homologous factor (EHF) is an Ets family transcription factor expressed in many epithelial cell types including those lining the respiratory system. Disruption of the airway epithelium is central to many lung diseases, and a network of transcription factors coordinates its normal function. EHF can act as a transcriptional activator or a repressor, though its targets in lung epithelial cells are largely uncharacterized. Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq), showed that the majority of EHF binding sites in lung epithelial cells are intergenic or intronic and coincide with putative enhancers, marked by specific histone modifications. EHF occupies many genomic sites that are close to genes involved in intercellular and cell–matrix adhesion. RNA-seq after EHF depletion or overexpression showed significant alterations in the expression of genes involved in response to wounding. EHF knockdown also targeted genes in pathways of epithelial development and differentiation and locomotory behavior. These changes in gene expression coincided with alterations in cellular phenotype including slowed wound closure and increased transepithelial resistance. Our data suggest that EHF regulates gene pathways critical for epithelial response to injury, including those involved in maintenance of barrier function, inflammation and efficient wound repair

A genome-wide analysis of open chromatin in human tracheal epithelial cells reveals novel candidate regulatory elements for lung function

Distal cell-type-specific regulatory elements may be located at very large distances from the genes that they control and are often hidden within intergenic regions or in introns of other genes. The development of methods that enable mapping of regions of open chromatin genome wide has greatly advanced the identification and characterisation of these elements

Sheep Models of F508del and G542X Cystic Fibrosis Mutations Show Cellular Responses to Human Therapeutics

Cystic Fibrosis (CF) is a genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. The F508del and G542X are the most common mutations found in US patients, accounting for 86.4% and 4.6% of all mutations, respectively. The F508del causes deletion of the phenylalanine residue at position 508 and is associated with impaired CFTR protein folding. The G542X is a nonsense mutation that introduces a stop codon into the mRNA, thus preventing normal CFTR protein synthesis. Here, we describe the generation of CFTRF508del/F508del and CFTRG542X/G542X lambs using CRISPR/Cas9 and somatic cell nuclear transfer (SCNT). First, we introduced either F508del or G542X mutations into sheep fetal fibroblasts that were subsequently used as nuclear donors for SCNT. The newborn CF lambs develop pathology similar to CFTR−/− sheep and CF patients. Moreover, tracheal epithelial cells from the CFTRF508del/F508del lambs responded to a human CFTR (hCFTR) potentiator and correctors, and those from CFTRG542X/G542X lambs showed modest restoration of CFTR function following inhibition of nonsense-mediated decay (NMD) and aminoglycoside antibiotic treatments. Thus, the phenotype and electrophysiology of these novel models represent an important advance for testing new CF therapeutics and gene therapy to improve the health of patients with this life-limiting disorder

Nucleosome occupancy reveals regulatory elements of the CFTR promoter

Access to regulatory elements of the genome can be inhibited by nucleosome core particles arranged along the DNA strand. Hence, sites that are accessible by transcription factors may be located by using nuclease digestion to identify the relative nucleosome occupancy of a genomic region. In order to define novel cis regulatory elements in the ∼2.7-kb promoter region of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, we define its nucleosome occupancy. This profile reveals the precise positions of nucleosome-free regions (NFRs), both cell-type specific and others apparently unrelated to CFTR-expression level and offer the first high-resolution map of the chromatin structure of the entire CFTR promoter in relevant cell types. Several of these NFRs are strongly bound by nuclear factors in a sequence-specific manner, and directly influence CFTR promoter activity. Sequences within the NFR1 and NFR4 elements are highly conserved in many human gene promoters. Moreover, NFR1 contributes to promoter activity of another gene, angiopoietin-like 3 (ANGPTL3), while NFR4 is constitutively nucleosome-free in promoters genome wide. Conserved motifs within NFRs of the CFTR promoter also show a high level of protection from DNase I digestion genome-wide, and likely have important roles in the positioning of nucleosome core particles more generally

ICAM-1 and CD44 expression in human bronchial epithelium and the role of CD44 isoforms in cell adhesion, migration, and repair\dShih-hsing Leir.

Mechanical damage of confluent epithelial cells induced the expression of CD44 on the cells up to 500 &mu;m from the wound edge and for up to 48 hours. Before cell confluence, the expression of CD44 at low cell densities was significantly higher than in confluent cultures, while ICAM-1 was lower. IFNγ and TNFα co-stimulation increased ICAM-1 expression significantly, while little change was seen in CD44. IL-1β and IL-4 induced the expression of CD44s, CD44v3 and CD44v9. Individual primary epithelial cells expressed several CD44 isoforms, while CD44 isoforms were undetectable in columnar epithelial cells from human airway. Down-regulation of v8-v9-v10 isoforms and small decreases of v6-v7-v8-v9-v10 and a v3-containing isoforms were seen in the cells with cytokine treatments and mechanical damage. In addition, the function of CD44 was investigated in cell adhesion and migration. Cytokines induced a CD44s-dependent cell adhesion to hyaluronic acid (HA). IFNγ-induced cell binding to HA without increasing the level of cell surface CD44 indicated that other mechanisms are involved in the modulation of CD44/HA binding. CD44 antibodies inhibited cell migration and demonstrated that CD44 plays an important role in cell migration, and may be associated with the repair processes of bronchial epithelium. I have found increased CD44 protein expression or changes in the alternative splicing of CD44 isoforms during the repair of epithelial damage. This study describes the sub-cellular expression and regulation of ICMA-1 and CD44, and provides some indication of functions of these CAMs in human bronchial epithelial cells.</p

Inflammatory cytokines can enhance CD44-mediated airway epithelial cell adhesion independently of CD44 expression

In airways, the cell surface molecule CD44 is upregulated on bronchial epithelial cells in areas of damage. We have shown that a blocking standard CD44 (CD44s) antibody caused a 77% (± 19%) inhibition of cell migration at 3 h after mechanical damage and decreased epithelial cell repair of cells grown on cell culture filter inserts. With the use of primary human bronchial epithelial cells and the bronchial epithelial cell line 16HBE 14o-, a CD44s antibody inhibited &gt;95% (P &lt; 0.01) of cell binding to hyaluronic acid (HA). The cytokines TNF-?, IFN-?, IL-1?, and IL-4 stimulated a 2- to 3.5-fold increase in CD44-dependent cell binding to HA. IFN-? treatment did not increase CD44 expression as assessed by flow cytometry, although phorbol myristate acetate treatment did. This indicates that IFN-?-induced cell binding to HA did not require increased CD44 expression. These data indicate that CD44 is important for bronchial epithelial cell binding to HA and that cytokines known to be expressed in inflammation can increase HA binding independently of the level of CD44 expression