ErbB2 activity is required for airway epithelial repair following neutrophil elastase exposure

Duke University, Durham, North Carolina, United States
The FASEB Journal (Impact Factor: 5.04). 09/2005; 19(10):1374-6. DOI: 10.1096/fj.04-2675fje
Source: PubMed


In cystic fibrosis and chronic bronchitis, airways are chronically injured by exposure to neutrophil elastase (NE). We sought to identify factors required for epithelial repair following NE exposure. Normal human bronchial epithelial cells were treated with NE (50 nM, 22 h) or control vehicle. Following NE treatment, we found a marked and sustained decrease in epithelial proliferation as detected by Ki67 immunostaining. 3H-thymidine incorporation was also initially depressed but increased over 72 h in NE-treated cells, which suggests that DNA synthesis constitutes an early repair process following NE exposure. We hypothesized that ErbB2 receptor tyrosine kinase, a regulator of cancer cell proliferation, was required for epithelial DNA synthesis following NE exposure. Immediately following NE treatment, by flow cytometry analysis, we found a decrease in ErbB2 surface expression. Protein levels of the full-length 185 kD ErbB2 receptor significantly decreased following NE treatment and smaller ErbB2-positive bands, ranging in size from 23 to 40 kD, appeared, which suggests that NE caused ErbB2 degradation. By real-time RT-PCR analysis, we found no change in ErbB2 mRNA expression following NE treatment, which suggests that changes in ErbB2 protein levels were regulated at the post-translational level. Following NE treatment, full-length 185 kD ErbB2 levels increased to pretreatment levels, correlating with the increase in thymidine incorporation during the same time period. Importantly, inhibition of ErbB2 activity with AG825 (5 microM) or Herceptin (3.1 microM), an ErbB2-neutralizing antibody, blocked thymidine incorporation only in NE-treated cells. These results suggest ErbB2 is a critical factor for epithelial recovery following NE exposure.

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Available from: James C Bonner, Oct 07, 2015
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    • "Several studies reported that ErbB1 – ErbB4 and their ligands , including EGF , AREG , and NRG activity , are required for airway epithelial cell proliferation and repair ( Britsch , 2007 ; Dammann et al . , 2003 ; Fischer et al . , 2005 ; Lemjabbar et al . , 2003 ; Nielsen , 1989 ; O ' Donnell et al . , 2004 ; in rewieved Brechbuhl et al . , 2014 ; Finigan et al . , 2011 ; Manzo et al . , 2012 ) . There - fore , the presence of EGFRs , together with their ligands , in the basal and ciliated cells of bronchial epi - thelium in the quail lung throughout the post - h"
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    ABSTRACT: The purpose of this study is to determine the possible changes in the localization of the four Epidermal Growth Factor Receptors and three ligands in quail lungs from the first day of hatching until the 125th after hatching using immunohistochemical methods. Immunohistochemical results demonstrated that four EGFRs and their ligands are chiefly located in the cytoplasm of cells. Additionally, ErbB4, AREG, and NRG1 are localized to the nucleus and nucleolus, but EGF is present in the nucleolus. ErbB2 was also found in the cell membrane. In the epithelium of secondary bronchi, the goblet cells only exhibited ErbB1 and ErbB2, whereas the basal and ciliated cells exhibited EGFRs and ligands immunoreactivity. The atrial granular cells displayed moderate levels of ErbB1-ErbB3 and EGF and strong levels of ErbB4, AREG, and NRG1 immunoreactivity. While the squamous atrial cells and squamous respiratory cells of air capillaries and endothelial cells of blood capillaries exhibited moderate to strong ErbB2, ErbB4, AREG, and NRG1 immunoreactivity, they had negative or weak ErbB1, ErbB3, and EGF immunoreactivity. The expression levels of ErbB2-ErbB4, EGF, AREG, and NRG1 were also detected in fibroblasts. Although ErbB2 was highly expressed in the bronchial and vascular smooth muscle cells, weak expression of ErbB1, ErbB3, AREG and EGF and moderate expression of ErbB4 and NRG1 were observed. Macrophages were only negative for ErbB1. In conclusion, these data indicate that the EGFR-system is functionally active at hatching, which supports the hypothesis that the members of EGFR-system play several cell-specific roles in quail lung growth after hatching. Microsc. Res. Tech., 2015. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.
    Microscopy Research and Technique 07/2015; 78(9). DOI:10.1002/jemt.22544 · 1.15 Impact Factor
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    • "Further, the ability of SMC/Muc4 to alter ErbB2 localization in polarized human colon carcinoma CACO-2 cells has been demonstrated, indicating a strong physical association between the two molecules [78]. In an elegant study, ErbB2 activation was ascertained for epithelial cell repair following NE exposure [79]. In a similar study, NE treatment significantly enhanced MUC4 (the ligand for ErbB2) in bronchial epithelia cells in-vitro [55]. "
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    ABSTRACT: Recent studies have identified MUC4 mucin as a ligand for activation of ErbB2, a receptor tyrosine kinase that modulates epithelial cell proliferation following epithelial damage in airways of asthmatics. In this study, we investigated the potential role of IL-4, one of the Th2 inflammatory cytokines persistent in asthmatic airways, in regulating MUC4 expression using a cell line NCI-H650. Real time PCR analysis was performed to determine concentration and time dependent effects of IL-4 upon MUC4 expression. Nuclear run on experiments were carried out to explore potential transcriptional modulation. Western blotting experiments using a monoclonal antibody specific to ASGP-2 domain of MUC4 were performed to analyze MUC4 glycoprotein levels in plasma membrane fractions. To analyze potential signal transduction cascades, IL-4 treated confluent cultures were co-incubated, separately with a pan-JAK inhibitor, a JAK-3 selective inhibitor or a MEK-1, 2 (MAPK) inhibitor at various concentrations before MUC4 transcript analysis. Corresponding transcription factor activation was tested by western blotting using a monoclonal p-STAT-6 antibody. MUC4 levels increased in a concentration and time specific fashion reaching peak expression at 2.5 ng/ml and 8 h. Nuclear run on experiments revealed transcriptional enhancement. Corresponding increases in MUC4 glycoprotein levels were observed in plasma membrane fractions. Pan-JAK inhibitor revealed marked reduction in IL-4 stimulated MUC4 levels and JAK3 selective inhibitor down-regulated MUC4 mRNA expression in a concentration-dependent fashion. In accordance with the above observations, STAT-6 activation was detected within 5 minutes of IL-4 stimulus. No effect in MUC4 levels was observed on using a MAPK inhibitor. These observations signify a potential role for IL-4 in MUC4 up-regulation in airway epithelia.
    Respiratory research 02/2006; 7(1):39. DOI:10.1186/1465-9921-7-39 · 3.09 Impact Factor
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    ABSTRACT: Cystic Fibrosis (CF), the most common fatal genetic disorder, is due to mutations of the cystic fibrosis transmembrane regulator protein (CFTR). Loss of functional CFTR leads to mucus thickening with the consequences of chronic pulmonary infection and inflammation, the most common fatal complication of this disorder. This thesis aimed to examine one aspect of the pathophysiology of CF, the potential role of glutathione (GSH) and oxidant stress. CFTR is permeable to GSH, at least in vitro, where evidence of the ability of CFTR to transport GSH is strong. Furthermore, extracellular GSH is diminished in CF airway lining fluid and it has been hypothesized that deficiency of CFTR leads to higher intracellular GSH coupled with lower extracellular GSH. This would be consistent with the observation of low extracellular GSH in CF patients and increased resistance to apoptosis of CF cell lines, where falling intracellular GSH concentrations ([GSH]) are required to initiate apoptosis. In addition to CFTR, other channels transport GSH, including Multi-drug Resistance Protein 1 (MRP1) which has structural and functional homology to CFTR. Higher expression of MRP1 has been associated with fewer symptoms in CF patients, suggesting that MRP1 may contribute to CF pathophysiology. We therefore set out to study GSH at the cellular level, with or without functional CFTR, in an airway epithelial cell model. Given the importance of oxidant stress in CF, we tested whether cells were more susceptible to stress in the absence of CFTR function. We hypothesized that lack of CFTR function would increase intracellular and decrease extracellular [GSH]. We also hypothesized that MRP1 function would influence intracellular and extracellular [GSH]. Blockade of CFTR function in Calu-3 cells failed to alter either intra- or extracellular [GSH], independent of oxidant stress conditions, suggesting the channel was not a determinant of [GSH]. However, inhibition of other anion channels did increase intracellular [GSH], suggesting an alternate mechanism for regulating cellular [GSH]. In contrast to CFTR, inhibition of MRP1 increased intracellular [GSH], suggesting a role for this channel in regulating intracellular [GSH]. Paradoxically however, extracellular [GSH] was higher after administration of the MRP1 antagonist MK-571. In addition, we also observed higher CFTR activity with use of MK-571. These findings suggest that CFTR could be indirectly responsible for the changes in [GSH] through interaction with MRP1. CFTR blockade in the presence of MK-571 administration returned extracellular [GSH] to normal, which reinforces a role for CFTR in the extracellular [GSH] increases. Administration of either MK-571 or Montelukast increased both wild-type and deltaF508 CFTR expression and function in the BHK cell model. Montelukast may have beneficial effects in restoring CFTR expression and function, which if confirmed in other models and in vivo, may pave the way for future therapeutic treatments for CF patients. La fibrose kystique (FK) est causée par des mutations dans la protéine CFTR (pour « Cystic fibrosis transmembrane conductance regulator »). La perte de fonction de CFTR mène à l’épaississement des muqueuses des voies respiratoires et l’avènement d’infections pulmonaires chroniques avec réponse inflammatoire, la cause primaire de mortalité chez les personnes affligées de FK. Cette thèse va examiner une partie de la pathophysiologie de la FK, celle du rôle du glutathion (GSH) et du stress oxydatif. Le CFTR est perméable au GSH et les données expérimentales in vitro suggèrent que le CFTR puisse transporter le GSH. De plus, le GSH extracellulaire est diminué dans le liquide de surface recouvrant les voies respiratoires des patients avec la FK et de cela découle l’hypothèse que la déficience en CFTR mène à une augmentation du GSH intracellulaire, avec une diminution concomitante du GSH extracellulaire. Cette hypothèse s’alignerait bien avec le bas taux de GSH extracellulaire observé chez les patients affectés par la FK et la résistance accrue contre l’apoptose vue dans les lignées cellulaires de la FK. Cette résistance découle du fait que la cellule a besoin d’une diminution de concentration de GSH ([GSH]) intracellulaire pour entamer le processus d’apoptose. À part du CFTR, il existe d’autres canaux membranaires qui transportent le GSH, tels que le Multi-drug Resistance Protein 1 (MRP1). Ce canal a une forte homologie structurelle et fonctionnelle avec le CFTR et son expression se trouve liée avec moins de symptômes cliniques chez les patients atteints de FK. Ces observations suggèrent que le MRP1 pourrait jouer un rôle important dans l’évolution de la physiopathologie de la maladie. Notre but est d’étudier l’état du GSH du point de vue de la cellule, avec ou sans stress oxydatif, dans une lignée de cellules épithéliales des voies respiratoires. Considérant l’importance du stress oxydatif dans la FK, il a fallu déterminer si les cellules étaient plus susceptibles au stress en absence du fonctionnement du CFTR. On a mis devant l’hypothèse que le manque de fonctionnement du CFTR amène à l’augmentation de [GSH] interne avec diminution de [GSH] externe. De plus, nous croyions que le fonctionnement du MRP1 aurait des effets sur le [GSH] intracellulaire ou le [GSH] extracellulaire.En utilisant un bloqueur de canal visant le CFTR, nous n’avons pas observé de changement dans le [GSH] intracellulaire ou extracellulaire, chez les cellules Calu-3, avec ou sans stress oxydatif. Ces observations suggéraient que le CFTR n’était pas impliqué dans la régulation du [GSH]. Cependant, nous avons observé une augmentation du [GSH] intracellulaire avec les traitements utilisant les bloqueurs de canaux anioniques, ce qui amenait la possibilité qu’un autre mécanisme soit impliqué dans la régulation du [GSH]. Contrairement à l’inhibition de la fonction du CFTR, le blocage de MRP1 causait une augmentation du [GSH] intracellulaire, apportant au canal un rôle possible dans la régulation du GSH. De façon paradoxale, le [GSH] extracellulaire augmentait avec l’utilisation de MK-571, bloqueur de MRP1. De plus, nous avons constaté une augmentation de l’activité de CFTR avec l’utilisation de MK-571. Ces observations prises ensemble suggéraient que CFTR pouvait être impliqué de façon indirecte dans les changements de [GSH], à travers une interaction avec MRP1. En bloquant CFTR et MRP1 ensemble nous avons constaté que le [GSH] extracellulaire était revenu au niveau de base, ce qui venait appuyer un rôle pour le CFTR dans l’augmentation du [GSH] extracellulaire. Par après, nous avons constaté qu’un traitement de MK-571 ou de Montelukast, utilisant le modèle cellulaire BHK, causait une augmentation de l’expression et de la fonction du CFTR normal ainsi que du CFTR deltaF508. Le Montelukast pourrait alors avoir des effets bénéfiques sur le rétablissement de l’expression et de fonction de CFTR ce qui pourra ouvrir la porte à de nouvelles thérapies.
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