Epidermal growth factor chronically upregulates Ca2+‐dependent Cl− conductance and TMEM16A expression in intestinal epithelial cells

Abstract

Dysregulated epithelial fluid and electrolyte transport is a common feature of many intestinal disorders. However, molecular mechanisms that regulate epithelial transport processes are still poorly understood, thereby limiting development of new therapeutics. Previously, we showed that epidermal growth factor (EGF) chronically enhances intestinal epithelial secretory function. Here, we investigated a potential role for altered expression or activity of apical Cl(−) channels in mediating the effects of EGF. Cl(−) secretion across monolayers of T(84) colonic epithelia was measured as changes in short-circuit current. Protein expression/phosphorylation was measured by RT-PCR and Western blotting. Under conditions that specifically isolate apical Ca(2+)-activated Cl(−) channel (CaCC) currents, EGF pretreatment (100 ng ml(−1) for 15 min) potentiated carbachol (CCh)-induced responses to 173 ± 25% of those in control cells, when measured 24 h later (n = 26; P < 0.01). EGF-induced increases in CaCC currents were abolished by the transmembrane protein 16A (TMEM16A) inhibitor, T16A(inh)-A01 (10 μm). Furthermore, TMEM16A mRNA and protein expression was increased by EGF to 256 ± 38% (n = 7; P < 0.01) and 297 ± 46% (n = 9, P < 0.001) of control levels, respectively. In contrast, EGF did not alter CFTR expression or activity. EGF-induced increases in Cl(−) secretion, CaCC currents and TMEM16A expression were attenuated by a PKCδ inhibitor, rottlerin (20 μm), and a phosphatidylinositol 3-kinase (PI3K) inhibitor, LY290042 (25 μm). Finally, LY290042 inhibited EGF-induced phosphorylation of PKCδ. We conclude that EGF chronically upregulates Ca(2+)-dependent Cl(−) conductances and TMEM16A expression in intestinal epithelia by a mechanism involving sequential activation of PI3K and PKCδ. Therapeutic targeting of EGF receptor-dependent signalling pathways may provide new approaches for treatment of epithelial transport disorders.