Epidermal growth factor chronically upregulates Ca(2+)-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.

Figure 1. EGF chronically potentiates Ca²⁺-dependent Cl⁻ conductances in colonic epithelial cells

Monolayers of T₈₄ cells were acutely treated with EGF (basolateral; 100 ng ml⁻¹; 15 min) and 24 h later were mounted in Ussing chambers for measurements of transepithelial Cl⁻ secretion or isolated apical Cl⁻ currents. A, representative tracing showing Cl⁻ secretory responses to basolateral addition of CCh and apical addition of FSK in control or EGF-pretreated cells. The right panel shows mean changes in secretory responses to CCh (n= 53) and FSK (n= 59) in control and EGF-pretreated cells. B, under conditions to isolate cAMP-dependent Cl⁻ conductances, EGF pretreatment did not alter I^Cl(apical) responses to FSK (10 μm; n= 5). C, under conditions to isolate Ca²⁺-dependent Cl⁻ conductances EGF significantly potentiated I_CaCC responses to CCh (100 μm; n= 26). Asterisks denote statistically significant differences from control cells that were not pretreated with EGF. **P < 0.01; ***P < 0.001.

Figure 2. EGF induces TMEM16A expression in colonic epithelial cells

A, monolayers of T₈₄ cells were treated with EGF (basolateral; 100 ng ml⁻¹; 15 min) and at various times (2, 4, 6 and 24 h) afterwards TMEM16A mRNA expression was measured by real-time PCR. B–C, CFTR and TMEM16A protein expression was investigated 24 h after EGF pretreatment by Western blotting. CFTR protein migrated as a band with a molecular weight of 170 kDa and TMEM16A as a doublet of bands at 114 kDa. EGF pretreatment significantly increased TMEM16A mRNA (n= 4) (A) and protein expression (n= 9) (B), without altering levels of CFTR protein expression (n= 8) (C). D, cells were pretreated with EGF (100 ng ml⁻¹; 15 min) and after 24 h were mounted in Ussing chambers under conditions for measuring Ca²⁺-dependent Cl⁻ conductances. Cells were then apically treated with the TMEM16A blocker, T16A_inh-A01 (10 μm), and I_CaCC responses to CCh (100 μm) were measured. T16Ainh-A01 abolished the potentiating effects of EGF on CCh-induced I_CaCC responses (n= 7). *P < 0.05 **P < 0.01; ***P < 0.001.

Figure 3. PKCδ mediates EGF-induced increases in Ca²⁺-dependent Cl⁻ secretion

A–D, monolayers of T₈₄ cells were treated with EGF (basolateral; 100 ng ml⁻¹; 15 min) alone or in presence of a general PKC inhibitor, GF109203X (GF) (5 μm; n= 6) (A), a PKCɛ translocation inhibitor (TI) (200 μm; n= 5) (B), PKCα/β inhibitor, Gö6976 (1 μm; n= 6) (C), or a PKCδ inhibitor, rottlerin (20 μm; n= 4) (D). All pharmacological inhibitors were added bilaterally for 30 min before EGF treatment. After 24 h monolayers were mounted in Ussing chambers and Cl⁻ secretory responses to basolateral CCh (100 μm) were measured. Results are expressed as mean ± SEM percentage control I_sc responses to CCh in cells not treated with EGF. E, monolayers of T₈₄ cells were treated with basolateral EGF (100 ng ml⁻¹; 15 min) and at various times afterwards PKCα or PKCδ phosphorylation was measured by Western blotting. Densitometric analysis of several experiments is shown with results expressed as mean ± SEM percentage control PKCα or PKCδ expression in untreated cells. Since Western blots for PKCδ revealed 2 closely migrating bands that may represent splice variants of the protein, both of these bands were included in densitometric analyses. EGF stimulated phosphorylation of PKCδ (n= 3 − 10) but not PKCα (n= 3). Representative blots are shown in the right panel. *P < 0.05; **P < 0.01.

Figure 4. PKCδ mediates EGF-induced increases in Ca²⁺-dependent Cl⁻ conductances and TMEM16A expression

A, monolayers of T₈₄ cells were acutely treated with basolateral EGF (100 ng ml⁻¹; 15 min) alone or in presence of the PKCδ inhibitor, rottlerin (20 μm; bilateral addition). Cells were pretreated with rottlerin for 30 min before addition of EGF. After 24 h cell monolayers were mounted in Ussing chambers under conditions to isolate Ca²⁺-dependent Cl⁻ conductances. Data are expressed as mean ± SEM percentage control I_CaCC responses to basolateral CCh (100 μm). Rottlerin reversed EGF potentiation of CCh-stimulated CaCC conductances (n= 4). B, monolayers of T₈₄ cells were acutely treated with EGF alone or in the presence of rottlerin (20 μm). Cells were then lysed and TMEM16A expression measured by Western blotting. The lower panel shows densitometric analysis of 5 experiments with data expressed as mean ± SEM percentage TMEM16A expression in cells not treated with EGF. Rottlerin abolished EGF-induced increases in TMEM16A expression. **P < 0.01.

Figure 5. PI3K mediates EGF-induced increases in Ca²⁺-dependent Cl⁻ conductances, TMEM16A expression and PKCδ activation

A and B, monolayers of T₈₄ cells were acutely treated with basolateral EGF (100 ng ml⁻¹; 15 min) alone or in the presence of the PI3K inhibitor, LY294002 (LY) (25 μm; bilateral addition). Cells were pretreated with LY290042 for 30 min before addition of EGF. After 24 h cell monolayers were mounted in Ussing chambers for measurements of Cl⁻ secretion (A) or Ca²⁺-dependent Cl⁻ conductances (I_CaCC) stimulated by basolateral CCh (100 μm) (B). Data are expressed as mean ± SEM percentage control I_sc responses to CCh in cells not treated with EGF (A) or I_sc response to CCh (B). C, TMEM16A mRNA expression was measured by RT-PCR 4 h after EGF treatment in the absence or presence of LY290042 (n= 4). D, TMEM16A protein expression was measured by Western blotting 24 h after EGF treatment in the absence or presence of LY290042 (n= 5). E, PKCδ phosphorylation was measured 1 h after EGF treatment in the absence or presence of LY290042 (n= 5). *P < 0.05; **P < 0.01.