Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, C61
Ca2+-activated Cl- channel expression and currents are upregulated by epidermal growth factor receptor activation in colonic epithelial cells
M. S. Mroz1, S. J. Keely1
1. RCSI, Dublin, Ireland.
Enhanced activation of the epidermal growth factor receptor (EGFR) occurs in a number of intestinal disorders associated with dysregulated epithelial transport, including infectious diseases and inflammatory bowel diseases. Furthermore, recent studies have implicated the novel Ca2+-dependent Cl- channel (CaCC), TMEM16A, in the pathogenesis of infectious diarrhoea. We have previously shown that EGF chronically potentiates colonic epithelial secretory function, an effect which in vivo would contribute to the onset of diarrhoea. Thus, in the present study we investigated a potential role for CaCCs in mediating chronic, prosecretory effects of EGF. T84 cell monolayers were mounted in Ussing chambers and the activity of transport proteins was measured as changes in short-circuit current. Protein expression/phosphorylation was measured by RT-PCR and western blotting. Data are presented as mean ± SEM and were statistically analysed by ANOVA or students t-tests as appropriate. As previously reported, acute treatment with EGF (100 ng/ml; 15 min) increased Cl- secretory responses to the Ca2+ -dependent agonist, carbachol (CCh; 100 µM) by 187 ± 15 % (n = 53; p < 0.001 ) when measured 24 h later. Acute treatment with EGF did not chronically alter CCh-induced mobilization of intracellular Ca2+ (n = 9). Under conditions that isolate apical Cl- currents through CaCC, EGF potentiated CCh-induced responses to 173 ± 25% of those in control cells (n = 26; p < 0.01). Furthermore, we found that mRNA and protein expression of the CaCC, transmembrane protein 16A, (TMEM16A), was increased by EGF to 256 ± 38% (n = 7; p < 0.01) and 297 ± 46% (n = 9, p < 0.001) of control levels, respectively. EGF-potentiation of Cl- secretory responses was inhibited by the general protein kinase C inhibitor, GF109203X (5 μM), but were unaltered by the PKCα/β inhibitor, Gö6976 (1 μM) and the PKCε translocation inhibitor (200 μM) (n = 5). In contrast, EGF-induced increases in CaCC currents and TMEM16A expression (n = 4) were significantly reduced in the presence of the PKCδ inhibitor, rottlerin (20 μM). Furthermore, EGF significantly increased phosphorylation of PKCδ(n = 10), but not PKCα (n = 3), with a maximal effect occurring 1 h after treatment. Finally, the PI3-K inhibitor, LY290042 (25 μM) inhibited the effects of EGF on Cl- secretion (n = 6), CaCC currents (n = 7), TMEM16A expression (n = 5) and PKCδ activation (n = 5). Ca2+-dependent Cl- conductances, likely mediated by TMEM16A, are chronically upregulated in response to EGFR activation in colonic epithelial cells through a mechanism involving PKCδ and PI3-K. Further elucidation of the molecular mechanisms involved may yield new targets for development of drugs to treat epithelial transport disorders.
Where applicable, experiments conform with Society ethical requirements