Date of Award

4-2011

Document type

Thesis

Degree Name

MSc by research (Master of Science by research)

First Supervisor

Dr Stephen Keely

Keywords

Digestive System, Gastrointestinal Tract, Intestinal Absorption, Epidermal Growth Factor

Abstract

Background: The epidermal growth factor receptor (EGFR) plays a critical role in regulating transport processes in the intestine. Since EGFR signalling can become dysregulated in several conditions of intestinal disease, the receptor may contribute to disturbances in fluid and electrolyte transport often associated with such disorders. Several previous studies have focused on investigating acute actions of EGF on epithelial transport function. Here, we sought to elucidate molecular mechanisms involved in mediating chronic actions of EGF through investigating effects of the growth factor on CI- secretion, expression and activity of transport proteins and signalling mechanisms involved in its actions.

Methods: TB4 cell monolayers were mounted in Ussing chambers and CI- secretion was measured as changes in short-circuit current. Gene and protein expressionlphosphorylation was measured by RT-PCR, confocal microscopy and western blotting. Surface protein expression was determined by cell surface biotinylation. lntracellular ca2' levels were measured by Fura-2 fluorescence and cAMP levels by ELlSA assays.

Results: Acute treatment with EGF (100 nglml; 15 minutes) increased CI- secretory responses to the ca2+ and CAMP-dependent agonists, carbachol (CCh; 100 pM) and forskolin (FSK; 10 pM) when measured 24 hours later. Acute treatment with EGF did not chronically alter CCh-induced induced elevations in intracellular ca2' but significantly potentiated FSK-induced cAMP accumulation. The effects of EGF are mediated by a heat-labile soluble factor. Treatment with EGF did not affect the membrane expression or activity of KCNN4, KCNQI, the ~ a ' - K'-ATPase or CFTR. In contrast, under conditions that specifically isolate apical CI- currents through ca2'-activated channels (CaCC), EGF potentiated CCh-induced responses. Furthermore, mRNA and protein expression of the CaCC, transmembrane protein 16A (TMEMI 6A), was increased by EGF TMEM16A expression and CaCC currents were inhibited by the phosphatidylinositol 3-kinase inhibitor, LY290042 (25 pM). A general protein kinase C inhibitor, GF109203X (5 pM), also inhibited EGF-induced potentiation of CI- secretion. However, while the PKCaIP inhibitor, Go6976 (1 pM) and a PKCE translocation inhibitor (200 pM) were without effect, EGF-induced increases in CI- secretion, CaCC currents and TMEM16A expression were significantly reduced in the presence of the PKCG inhibitor, rottlerin (20 pM). Furthermore, EGF significantly increased phosphorylation of PKCG, but not PKCa, with a maximal effect occurring 1 hour after treatment. Moreover, the P13-K inhibitor, LY290042 (25 pM) inhibited the effects of EGF on PKC6 activation. Inhibition of extracellular regulated kinase 112 with PD98059 (25 pM) abolished EGF-induced increases in ca2'- and CAMP-stimulated CI- secretion, whereas Src kinase inhibition with PP2 (20 pM) and p38 kinase inhibition with SB203580 (10 pM) inhibited effects of the growth factor on CAMP-stimulated CI- secretion only.

Conclusions: EGF chronically enhances epithelial secretory capacity through a mechanism that involves P13K and PKCG-dependent induction of the novel CaCC, TMEM16A. Our results suggest that targeting EGFR-dependent signalling pathways represents a good approach for development of new drugs to treat intestinal transport disorders.

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Comments

A thesis submitted to the Royal College of Surgeons for the degree of Master of Science, 2010.