The novel role of epidermal growth factor (EGF) in the regulation of ion channels in the calu-3 submucosal cell line

Clements, Craig (2012) The novel role of epidermal growth factor (EGF) in the regulation of ion channels in the calu-3 submucosal cell line. Doctoral thesis, University of East Anglia.

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Cystic fibrosis transmembrane conductance regulator (CFTR) is a cell membrane bound chloride ion channel regulated by cyclic AMP-dependent phosphorylation and levels of intracellular ATP. Mutations in this channel, such as the common deletion of phenylalanine at residue 508 (CFTRΔF508), leads to a decrease in chloride transport seen in the disease condition cystic fibrosis (CF). The mutant CFTR is not processed in the normal way and consequently not delivered to the cell membrane. Currently, the effect of growth factors such as epidermal growth factor (EGF) on ion transport in the airway has not been previously researched and is consequently unknown. Therefore the aim of this thesis is to determine (i) if EGF has an effect on ion transport in the submucosal cell line Calu-3, (ii) what the mechanisms are behind this, and (iii) if the effect of EGF was due to induction of gelatinase activity or a transactivation process. Functional investigations looking at ion transport were carried out by using short circuit current. This technique was complemented by traditional molecular biology techniques such as RT-PCR, Western blotting, flow cytometry and gelatin zymography. The level of EGF, a potent inducer of gelatinases, is known to be elevated in the lungs during tissue repair in CF. Calu-3 cells preincubated with EGF on the basolateral membrane increased initial current at one hour via a EGFR-PI3K-PKC-δ-KCNN4/KCNQ1 signalling pathway. Similarly, preincubation with EGF also decreased forskolin induced short circuit current compared to untreated monolayers at 1 to 3 hours, with a recovery at 24 hours. The decreases were found to be dependent on the activation of KCNQ1 since chromanol 293B, a specific inhibitor for KCNQ1, restored the short circuit current back to untreated levels. Stimulation of the β2 adrenergic receptors with salbutamol were not reduced using metalloproteinase inhibitor, GM-6001 and EGFR inhibitor, AG1478. This suggested that stimulation of β2 adrenergic receptors does not lead to transactivation of EGFR via activation of sheddases and the release of EGF ligand. β3 adrenergic receptors are present in Calu-3, but produce negligible currents when stimulated. It was concluded that EGF induced potassium channel activation led to a change in chloride driving force. This activation of potassium channels has previously been linked to wound repair in the airway during disease. The implications of this study suggest that manipulation of the EGF signalling pathway and / or potassium channel activity in the lungs may be beneficial in disease conditions such as CF for increasing chloride transport.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Medicine and Health Sciences > Norwich Medical School
Depositing User: Users 2593 not found.
Date Deposited: 02 Feb 2015 15:13
Last Modified: 26 Mar 2015 15:52


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