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

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

Impact of the F508del mutation on ovine CFTR, a Cl- channel with enhanced conductance and ATP-dependent gating

Cross-species comparative studies are a powerful approach to understand the epithelial Cl- channel cystic fibrosis transmembrane conductance regulator (CFTR), which is defective in the genetic disease cystic fibrosis (CF). Here, we investigate the single-channel behaviour of ovine CFTR and the impact of the most common CF mutation, F508del-CFTR, using excised inside-out membrane patches from transiently transfected CHO cells. Like human CFTR, ovine CFTR formed a weakly inwardly rectifying Cl- channel regulated by PKA-dependent phosphorylation, inhibited by the open-channel blocker glibenclamide. However, for three reasons, ovine CFTR was noticeably more active than human CFTR. First, single-channel conductance was increased. Second, open probability was augmented because the frequency and duration of channel openings were increased. Third, with enhanced affinity and efficacy, ATP more strongly stimulated ovine CFTR channel gating. Consistent with these data, the CFTR modulator phloxine B failed to potentiate ovine CFTR Cl- currents. Like its impact on human CFTR, the F508del mutation caused a temperature-sensitive folding defect, which disrupted ovine CFTR protein processing and reduced membrane stability. However, the F508del mutation had reduced impact on ovine CFTR channel gating in contrast to its marked effects on human CFTR. We conclude that ovine CFTR forms a regulated Cl- channel with enhanced conductance and ATP-dependent channel gating. This phylogenetic analysis of CFTR structure and function demonstrates that subtle changes in structure have pronounced effects on channel function and the consequences of the CF mutation F508del. This article is protected by copyright. All rights reserved

Collagen XV Inhibits Epithelial to Mesenchymal Transition in Pancreatic Adenocarcinoma Cells

<div><p>Collagen XV (COLXV) is a secreted non-fibrillar collagen found within basement membrane (BM) zones of the extracellular matrix (ECM). Its ability to alter cellular growth <i>in vitro</i> and to reduce tumor burden and increase survival <i>in vivo</i> support a role as a tumor suppressor. Loss of COLXV during the progression of several aggressive cancers precedes basement membrane invasion and metastasis. The resultant lack of COLXV subjacent to the basement membrane and subsequent loss of its interactions with other proteins in this zone may directly impact tumor progression. Here we show that COLXV significantly reduces invasion of pancreatic adenocarcinoma cells through a collagen I (COLI) matrix. Moreover, we demonstrate that epithelial to mesenchymal transition (EMT) in these cells, which is recapitulated <i>in vitro</i> by cell scattering on a COLI substrate, is inhibited by over-expression of COLXV. We identify critical collagen-binding surface receptors on the tumor cells, including the discoidin domain receptor 1 (DDR1) and E-Cadherin (E-Cad), which interact with COLXV and appear to mediate its function. In the presence of COLXV, the intracellular redistribution of E-Cad from the cell periphery, which is associated with COLI-activated EMT, is inhibited and concurrently, DDR1 signaling is suppressed. Furthermore, continuous exposure of the pancreatic adenocarcinoma cells to high levels of COLXV suppresses endogenous levels of N-Cadherin (N-Cad). These data reveal a novel mechanism whereby COLXV can function as a tumor suppressor in the basement membrane zone.</p></div

Collagen XV inhibits phosphorylation of Pyk2 and suppresses N-Cadherin.

<p>Vector-only (BxVC3, BxVC4, BxVC5, BxVC10) and COLXV-expressing clones (Bx15.5, Bx15.14, Bx15.23, Bx15.24) were grown on COLI coated plates (A, B,); plastic and COLI (C) or plastic alone (F) and lysed after 48 h. Western blots probed with antibodies specific for A) total FAK or pFAK; B) total Pyk2 or pPyk2; C, F) N-Cad; are shown. ß-tubulin provides an estimate of total protein. Expression of COLXV slightly enhances pFAK relative to FAK (though this is not statistically significant, p = 0.0913), but depresses pPyk2 relative to Pyk2 (* p = 0.0447) and suppresses N-Cadherin levels. Blots were quantified by integrating data acquired with the Lasso tool of Adobe Photoshop (C, D, E, F) and analyzed by students t test (E, *p<0.01). Each clone was analyzed at least 3 times for FAK, pFAK, Pyk2, pPyk2 and N-Cad expression.</p

E-Cadherin is stabilized at the cell periphery in collagen XV expressing cells.

<p>Confocal microscopy with an antibody specific for the extracellular domain of E-Cad (green) and nuclei stained with DAPI (blue). BxVC1 vector clone and Bx15.23 COLXV clone grown on plastic A) or COLI B). E-Cad is most abundant at the cell surface in both clones on plastic. On COLI, E-Cad moves from the cell periphery into the cytoplasm in BxVC1, but this redistribution is inhibited in the presence of COLXV (BX15.23). C) EEA1 (red) is found in the endoplasmic reticulum (ER)/Golgi zone of the cells grown on plastic, while E-Cad is at the cell periphery. D) After relocation of E-Cad on COLI, EEA1 colocalizes with E-Cad (white arrowheads) in BxVC1 cells but not Bx15.23 cells. Images are representative of several clones. E) Flow cytometry after staining cells with an E-Cad antibody shows increased cell-surface expression of E-Cad in cells with COLXV (Bx15.5 and 15.24) in comparison to vector controls (BxVC4 and BxVC1). All experiments performed a minimum of 3 times with consistent results.</p

Collagen XV inhibits scatter of BxPC-3 cells on a collagen I substrate and interacts directly with DDR1 and E-Cadherin.

<p>A) Vector control (BxVC3) and COLXV expressing (Bx15.1, Bx15.14) clones are shown grown on plastic substrate and on COLI coated substrate. Phase contrast microscopy, all panels 100X magnification. 3 vector clones and 5 COLXV expressing clones (2 with low expression and 3 with high expression) were analyzed at least 3 times and the results were consistent. B) Immunoprecipitation (IP) of DDR1 from vector clone (BxVC3) and COLXV clone (Bx15.14), followed by probing of western blots of the IP material with antibodies specific for COLXV, DDR1 and E-Cad. C) IP of FLAG-tagged COLXV (FCOLXV) from the BxF15.3 clone using the M2 antibody. D) IP of COLXV from clone Bx15.3 with M2 after depletion of DDR1 with a specific siRNA (DDR1i) or transfection of scrambled control siRNA (Scrbi). Depletion of DDR1 reduced the amount of DDR1, but not E-Cad, interacting with COLXV. E) IP of COLXV from clone BxF15.3 with M2 after depletion of E-Cad with a specific siRNA (E-Cadi) or transfection of scrambled control siRNA (Scrbi). Depletion of E-Cad reduced the amounts interacting with COLXV. All experiments were performed a minimum of 3 times with consistent results.</p

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

Ets homologous factor (EHF) is an Ets family tran-scription 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 uncharac-terized. Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq), showed that the major-ity of EHF binding sites in lung epithelial cells are intergenic or intronic and coincide with putative en-hancers, marked by specific histone modifications. EHF occupies many genomic sites that are close to genes involved in intercellular and cell–matrix adhe-sion. RNA-seq after EHF depletion or overexpression showed significant alterations in the expression of genes involved in response to wounding. EHF knock-down also targeted genes in pathways of epithe-lial development and differentiation and locomotory behavior. These changes in gene expression coin-cided with alterations in cellular phenotype includ-ing slowed wound closure and increased transep-ithelial resistance. Our data suggest that EHF regu-lates gene pathways critical for epithelial response to injury, including those involved in maintenance of barrier function, inflammation and efficient wound repair