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

Department of Pediatrics, Duke University Medical Center, Durham, North Carolina 27710, USA.
The FASEB Journal (Impact Factor: 5.7). 09/2005; 19(10):1374-6. DOI: 10.1096/fj.04-2675fje
Source: PubMed

ABSTRACT 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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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. 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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. 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