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Am J Physiol Cell Physiol 294: C1362-C1370, 2008. First published April 9, 2008; doi:10.1152/ajpcell.00256.2007
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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

Induction of Na+/K+/2Cl– cotransporter expression mediates chronic potentiation of intestinal epithelial Cl– secretion by EGF

Fiona O'Mahony, Ferial Toumi, Magdalena S. Mroz, Gail Ferguson, and Stephen J. Keely

Department of Molecular Medicine, Royal College of Surgeons in Ireland, Dublin, Ireland

Submitted 14 June 2007 ; accepted in final form 5 April 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Alterations in EGF receptor (EGFR) signaling occur in intestinal disorders associated with dysregulated epithelial transport. In the present study, we investigated a role for the EGFR in the chronic regulation of intestinal epithelial secretory function. Epithelial Cl– secretion was measured as changes in short-circuit current (Isc) across voltage-clamped monolayers of T84 cells in Ussing chambers. Acute treatment of T84 cells with EGF (100 ng/ml, 15 min) chronically enhanced Isc responses to a broad range of secretagogues. This effect was apparent within 3 h, maximal by 6 h, and sustained for 24 h after treatment with EGF. The Na+/K+/2Cl– cotransporter (NKCC1) inhibitor bumetanide (100 µM) abolished the effect of EGF, indicating increased responses are due to potentiated Cl– secretion. Neither basal nor agonist-stimulated levels of intracellular Ca2+ or PKA activity were altered by EGF, implying that the effects of the growth factor are not due to chronic alterations in levels of second messengers. EGF increased the expression of NKCC1 with a time course similar to that of its effects on Cl– secretion. This effect of EGF was maximal after 6 h, at which time NKCC1 expression in EGF-treated cells was 199.9 ± 21.9% of that in control cells (n = 21, P < 0.005). EGF-induced NKCC1 expression was abolished by actinomycin D, and RT-PCR analysis demonstrated EGF increased expression of NKCC1 mRNA. These data increase our understanding of mechanisms regulating intestinal fluid and electrolyte transport and reveal a novel role for the EGFR in the chronic regulation of epithelial secretory capacity through upregulation of NKCC1 expression.

epidermal growth factor; epithelium; chloride secretion; ion transport

THE TRANSPORT OF WATER across intestinal epithelial cells is an important process that contributes to whole body fluid and electrolyte homeostasis and maintains appropriate hydration of the mucosal surface. However, dysregulated epithelial fluid secretion is a common feature of many intestinal disorders and can lead to the clinical manifestation of diarrhea, the leading cause of mortality in third-world countries and a huge financial burden to healthcare in the United States (31). In the intestine, fluid secretion is driven predominantly by the active secretion of Cl– (6). Cl– secretion is, in turn, promoted by an array of blood-borne, neural and immunologically derived substances that act at cell surface G protein-coupled receptors (GPCRs). Two subfamilies of GPCRs are particularly important in promoting intestinal secretion: GqPCRs, which elevate intracellular Ca2+, and GsPCRs, which stimulate the production of cAMP. In turn, increases in second messenger levels alter the activity of the transport proteins comprising the epithelial secretory pathway. At the molecular level, the pathway of epithelial secretion has been quite well elucidated (7). The energy for the process is derived from a basolateral Na+/K+-ATPase pump that, with each cycle, pumps 3 Na+ from the cell in return for 2 K+. The activity of this pump creates chemical gradients for Na+ and Cl– uptake via basolateral Na+/K+/2Cl– cotransporters (NKCC1), which carry 1 Na+, 1 K+, and 2 Cl– into the cell. Since K+ is recycled through channels in the basolateral membrane, the concerted activity of these basolateral transport proteins specifically accumulates Cl– within the cell, thereby creating a gradient for its efflux when apical channels open. The best described of these Cl– channels is the cAMP-dependent CFTR, but others are known to exist.

The EGF receptor (EGFR, or ErbB1) is a critical regulator of epithelial function. This receptor, a member of the ErbB family of receptor tyrosine kinases, is expressed on the basolateral side of intestinal epithelia, where it has long been known to regulate cell growth and differentiation in response to growth factors such as EGF and TGF-{alpha} (15). However, in recent years, it has become appreciated that growth factors acting at the EGFR can also influence a number of other important aspects of epithelial function, including barrier function (4, 5), restitution (20, 35, 38), and fluid and electrolyte transport. EGF acutely promotes intestinal Na+ absorption (22, 23, 28) and also has the ability to inhibit Cl– secretion (40). In vivo, such actions would tend to promote fluid absorption, thereby restoring normal balance in conditions where malabsorption and/or hypersecretion occur. Indeed, altered levels of EGFR ligands and EGFR kinase activity are known to occur in intestinal disorders associated with diarrhea, most notably inflammatory bowel disease (IBD) (1, 2, 12, 32, 34), suggesting a role for the EGFR in the pathogenesis of, or recovery from, such conditions. Furthermore, it has been found that manipulation of EGFR-dependent signaling mechanisms appears to be beneficial in treating intestinal inflammation in both animal models and human subjects (18, 34, 39), while EGFR inhibitors used in chemotherapy are often dose limited by their tendancy to cause diarrhea (9, 30). Such observations suggest that EGFR-dependent signaling pathways might prove to be useful targets in the development of agents for treatment of intestinal transport disorders.

However, before we can hope to exploit EGFR-dependent signaling in the treatment of disease, a greater understanding of the role that the receptor plays in regulating epithelial function is required. Notably, while previous studies have demonstrated that EGF can acutely alter intestinal epithelial secretion, the more long-term consequences of EGFR activation on epithelial secretory capacity are unknown. Thus, using the T84 cell line as a model of the the intestinal epithelium, we investigated a potential role for the EGFR in the chronic regulation of intestinal epithelial Cl– secretion.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture. The colonic epithelial cell line T84 was cultured on petri dishes in DMEM-Hams F-12 media (1:1) (Sigma Chemical) supplemented with 5% newborn calf serum (HyClone, Logan, UT). Cells were cultured in an atmosphere of 5% CO2 at 37°C with medium changes every 3–4 days. For Ussing chamber/voltage-clamp experiments, ~5 x 105 cells were seeded onto 12-mm Millicel-HA Transwells (Millipore, Bedford, MA). For Western blot experiments, ~106 cells were seeded onto 30-mm Millicell-HA Transwells. Cells seeded onto Millicell filters were cultured for 10–15 days before being used. Under these conditions, T84 cells develop the polarized phenotype of native epithelial cells and are widely considered to be among the best models for reductionist studies of epithelial secretion currently available.

Electrophysiological measurements. After being cultured for 10–15 days on filter supports, T84 cell monolayers were washed in serum-free medium and allowed to equilibrate for 30 min. Cells were then treated on the basolateral side with EGF at various concentrations and for various periods of time as noted in the figures. After 24 h, EGF-treated monolayers were mounted in Ussing chambers (aperture = 0.6 cm2), voltage clamped to zero potential difference, and monitored for changes in short-circuit current ({Delta}Isc). Under such conditions, secretagogue-induced changes in Isc across T84 monolayers are wholly reflective of changes in electrogenic Cl– secretion (10). Isc measurements were carried out in Ringer solution containing (in mM) 140 Na+, 5.2 K+, 1.2 Ca2+, 0.8 Mg2+, 119.8 Cl–, 25 HCO3–, 2.4 HPO42–, and 10 glucose. Results were normalized and expressed as {Delta}Isc (in µA/cm2).

Western blot analysis. T84 cell monolayers were washed (for 3 times) in serum-free medium and allowed to equilibrate for 30 min at 37°C. Cells were then treated with EGF for 15 min, after which cells were washed and allowed to recover for 24 h in serum-free medium. Monolayers were then washed (twice) with ice-cold PBS and lysed in ice-cold lysis buffer (500 µl) (consisting of 1% Triton X-100, 1 mM NaVO4, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 µg/ml antipain, 1 mM NaF, 1 mM EDTA, and 100 µg/ml PMSF in PBS). Cells were then incubated at 4°C for 45 min, scraped into microcentrifuge tubes, and centrifuged at 12,000 rpm for 10 min. After centrifugation, the pellet was discarded, and samples were adjusted so that they contained equal amounts of protein. Samples were then mixed with 2x gel loading buffer [50 mM Tris (pH 6.8), 2% SDS, 100 mM DTT, 0.2% bromophenol blue, and 20% glycerol] and boiled for 3 min, and proteins separated by SDS-PAGE. Separated proteins were transferred to a polyvinylidene difluoride membrane, after which the membrane was washed in 1% blocking buffer for 30 min, followed by an incubation with the appropriate dilution of primary antibody in 1% blocking buffer for 60 min. This was followed by washing (for 5 times) in Tris-buffered saline with 1% Tween (TBST). Horseradish peroxidase-conjugated secondary antibodies were then added to the membrane in 1% blocking buffer and allowed to incubate for an additional 30 min. After being further washed (5 times) in TBST, immunoreactive proteins were detected using an ECL detection kit (Amersham Lifesciences) and exposure of the membrane to X-ray film. Quantitation of protein phosphorylation was determined by densitometry using Scion image software.

Cell surface biotinylation. The protocol used was based on one previously described (14). Following treatment, cells were washed three times in ice-cold PBS. Freshly prepared biotinylation buffer [1 mg/ml sulfo-NHS-biotin (Pierce) in PBS] was added to the basolateral side of the cells. Cells were then incubated at 4°C for 15 min on a rotating platform, after which the buffer was removed and replaced with a second fresh aliquot. After an additional 15-min incubation, cells were washed twice with PBS and then incubated with a quenching reagent (100 mM glycine in PBS). Cells were then washed with PBS and lysed in Triton lysis buffer for 45 min on ice. The lysate was centrifuged at 14,000 rpm for 6 min, and the protein concentration of the supernatant was determined and normalized. Samples were then precipitated on a rotator overnight at 4°C with 100 µl of streptavidin-agarose beads (Pierce). The beads were then washed three times in lysis buffer, and 40 µl of 2x Laemmli buffer (Sigma-Aldrich) were added. Samples were boiled at 95°C for 5 min and subjected to SDS-PAGE analysis. NKCC1 was detected by Western blot analysis as described above.

Intracellular Ca2+ imaging. After pretreatment with EGF (100 ng/ml, 15 min), T84 cells grown on glass coverslips were allowed to recover for 24 h in serum-free medium. Cells were then washed and loaded with 5 µM fura-2 AM [dissolved in 0.01% Pluronic F-127 plus 0.1% DMSO in physiological salt solution (140 mM NaCl, 5 mM KCl, 1 mM CaCl2, 10 mM D-glucose, and 10 mM HEPES-trimethylamine; pH 7.4)] at room temperature for 30 min. Coverslips were then washed and mounted in a perfusion chamber on a Nikon microscope stage. Cells were perfused with normal physiological salt solution for 5 min before the addition of carbachol (CCh; 100 µM) to the perfusing solution. The ratio of fura-2 fluorescence with excitation at 340 or 380 nm was measured every 3 s, and images were captured using an intensified charge-coupled device camera (ICCD200) and a MetaFluor Imaging System (Molecular Devices, Sunnyvale, CA).

RNA preparation and RT-PCR. After pretreatment with EGF (100 ng/ml, 15 min), T84 cell monolayers were allowed to recover for 24 h in serum-free medium. Total RNA was then isolated from the cells using a Qiagen RNeasy kit. RNA was treated for genomic DNA contamination using the Turbo DNA Free kit (Ambion). cDNA was synthesized using the Improm II reverse transcriptase kit (Promega), and cDNA was quantified and normalized before being loading into RT-PCR mixes. Products were amplified by cycle limitation RT-PCR using Go-Taq polymerase. Primers were obtained from MWG Biotechnology. NKCC1 was amplified using the following primers: forward 5'-ACA ATG GCG AAT GGT GAC T-3' and reverse 5'-CAT GGG GTT ACT TTT TGG TTA C-3'. The RT-PCR product was analyzed on a 2% 1x TAE agarose gel and imaged using a UV light source.

PKA activation assay. Cells were pretreated with EGF (100 ng/ml) and then allowed to recover for 24 h in serum-free medium. After this time, cells were washed and allowed to equilibrate for 30 min in Ringer solution. Cells were then stimulated with forskolin (FSK; 10 µM) for 5 min, after which they were lysed. Samples were normalized according to protein content, and PKA activity was measured using a commercially available kit (PepTag Assay, Promega). This kit detects PKA-mediated phosphorylation of a substrate peptide by virtue of the tendency of the phosphorylated substrate to migrate toward the cathode and the nonphosphorylated substrate to the anode.

Statistical analysis. All data are expressed as means ± SE for a series of n experiments. Student t-tests were used to compare paired data. One-way ANOVA with the Student-Neuman-Keuls posttest was used when three or more groups of data were compared. P values of ≤0.05 were considered to be statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
EGF chronically potentiates Cl– secretion across colonic epithelial cells. As a starting point for our experiments, we first confirmed previous reports demonstrating that EGF exerts acute antisecretory actions on colonic epithelial cells (40). We found that treatment of voltage-clamped T84 cells with EGF (100 ng/ml) for 15 min acutely inhibited subsequent responses to a prototypical Ca2+-dependent secretagogue, CCh (100 µM) (Fig. 1A). Responses to CCh in EGF-pretreated cells were reduced to 20.0 ± 2.7% of those in control cells (n = 6, P < 0.001). Having confirmed that EGF exerts acute antisecretory effects, we next examined the consequences of long-term exposure to the growth factor on the ability of T84 cells to elicit secretory responses. Cell monolayers were pretreated basolaterally with EGF (100 ng/ml) in serum-free medium for a period of 24 h. After this time, cells were washed of the growth factor and mounted in Ussing chambers, and Isc responses to CCh (100 µM) were measured. We found that in cells pretreated with EGF, subsequent responses to CCh were significantly potentiated (Fig. 1B). Control responses to CCh were 24.0 ± 2.7 µA/cm2 compared with 52.2 ± 3.2 µA/cm2 in EGF-pretreated cells (n = 11, P < 0.001). Under similar conditions, Isc responses to the cAMP-dependent agonist FSK (10 µM) were also increased with a maximal control response of 52.8 ± 5.5 µA/cm2 compared with 73.8 ± 5.2 µA/cm2 in EGF-pretreated cells (n = 11, P < 0.001; Fig. 1C). In control experiments, we found that the incubation of T84 cells in serum-free medium alone did not alter their ability to evoke secretory responses. Responses to CCh (100 µM) in cells starved of serum for 24 h were 105.8 ± 9.6% of those in cells that were maintained in serum-containing medium (n = 4).


Figure 1
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  		Fig. 1. Chronic treatment with EGF enhances carbachol (CCh)- and forskolin (FSK)-induced short-circuit current (Isc) responses in T84 cells. A: monolayers of T84 cells were mounted in Ussing chambers and, after a 10-min equilibration period, were treated basolaterally with EGF (100 ng/ml). After a further 15 min, Isc responses to CCh (100 µM) were measured (n = 6). B and C: T84 cell monolayers were pretreated with EGF (100 ng/ml) for 24 h. After being washed, cells were mounted in Ussing chambers, and subsequent Isc responses to CCh (100 µM, n = 11; B) and FSK (10 µM, n = 11; C) were measured. Data are expressed as means ± SE of changes in Isc ({Delta}Isc) to CCh or FSK added at time 0.

 
Further analysis revealed that exposure of the cells to EGF for periods as short as 5 min were sufficient to induce potentiation of agonist-induced Isc responses when measured 24 h later (Fig. 2A) and that EGF was maximally effective in the concentration range of 30–100 ng/ml (Fig. 2B). Unless otherwise noted, all subsequent experiments were conducted by measuring agonist-induced secretory responses 24 h after acute (15 min) stimulation with EGF (100 ng/ml). Under these conditions, EGF slightly, but significantly, increased basal Isc but did not alter baseline values of resistance or potential difference (Table 1). Under these treatment conditions, we also found that, similar to its effects on the M3 receptor agonist CCh, EGF also chronically potentiated responses to the Gq protein-coupled H1 receptor agonist histamine (100 µM), thapsigargin (TG; 2 µM), which elevates intracellular Ca2+ in a receptor-independent manner, and heat-stable enterotoxin of Esherichia coli (STa; 50 nM), which stimulates secretion through a cGMP-dependent signaling pathway (Fig. 3, A–D). Responses to histamine, TG, and STa were 159.4 ± 13.9% (n = 5, P < 0.005), 221.6 ± 36.2% (n = 6, P < 0.05), and 128.8 ± 5.7% (n = 3, P < 0.05) of those in control cells, respectively. To verify that the effects of EGF in potentiating Isc responses were due to increased Cl– secretion, we used bumetanide to block NKCC1, the primary uptake pathway for Cl– in intestinal epithelia. We found that the addition of bumetanide (100 µM) to the basolateral bathing solution completely abolished the ability of EGF to potentiate CCh-induced secretory responses (Fig. 3E).


Figure 2
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  		Fig. 2. Acute EGF chronically potentiates CCh-stimulated Isc responses in a concentration-dependent manner in T84 cells. A: cells were pretreated with EGF (100 ng/ml) for various periods of time (5 min to 2 h) and then allowed to recover in serum-free medium for 24 h before subsequent Isc responses to CCh (100 µM) were measured in Ussing chambers. Data are expressed as means ± SE of percentages of control responses to CCh (n = 6–11, as represented by numbers within the bars). *P < 0.05; **P < 0.01; ***P < 0.005. B: cells were pretreated with EGF at various concentrations for 15 min and then allowed to recover in serum-free medium for 24 h before subsequent Isc responses to CCh (100 µM) were measured. Data are expressed as means ± SE of {Delta}Isc (n = 6). *P < 0.05; **P < 0.01.

 

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  		Table 1. Effects of EGF on basal electrical parameters of T84 cells

 

Figure 3
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  		Fig. 3. Acute EGF chronically induces a generalized enhanced secretory phenotype in T84 cells. Cells were pretreated with EGF (100 ng/ml) for 15 min and then allowed to recover for 24 h in serum-free medium. Cells were then mounted in Ussing chambers, and subsequent Isc responses to CCh (100 µM, n = 14; A), histamine (100 µM, n = 5; B), thapsigargin (TG; 2 µM, n = 6; C), or heat-stable enterotoxin of Esherichia coli (STa; 50 nM, n = 3; D) were measured. Data are expressed as means ± SE of {Delta}Isc to agonists added at time 0. E: cells were treated with EGF (100 ng/ml) for 15 min, and, 24 h later, Isc responses to CCh (100 µM) were measured either in the absence or presence of basolateral bumetanide (Bum; 100 µM). Data are expressed as means ± SE of {Delta}Isc (n = 5). **P < 0.01 compared with cells stimulated with CCh alone; ###P < 0.01 compared with EGF-pretreated cells.

 
TGF-{alpha}, but not heregulin, mimics the chronic potentiating effects of EGF on colonic epithelial secretory responses. We next examined whether ligands of ErbB receptors, other than EGFR, had a similar capability to chronically potentiate epithelial secretory capacity. In these experiments, cells were acutely (15 min) stimulated with the EGFR (ErbB1) agonists EGF or TGF-{alpha} or with the ErbB3 agonist heregulin (HRG) (all at concentrations of 100 ng/ml). After 24 h, cells were mounted in Ussing chambers, and the subsequent secretory responses to CCh (100 µM) were measured. We found that only TGF-{alpha}, but not HRG, mimicked the effects of EGF in chronically potentiating CCh-induced Isc responses (Fig. 4A).


Figure 4
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  		Fig. 4. Potentiating effects of EGF on CCh-induced secretory responses in T84 cells are mimicked by TGF-{alpha} and mediated by a soluble factor. A: cells were pretreated with EGF (100 ng/ml), TGF-{alpha} (100 ng/ml), or heregulin (HRG; 100 ng/ml) for 15 min and then allowed to recover for 24 h in serum-free medium. Monolayers were then mounted in Ussing chambers, and Isc responses to CCh (100 µM) were measured. Data are expressed as means ± SE of {Delta}Isc (n = 7). **P < 0.01 compared with control responses to CCh. B: cells were pretreated with EGF (100 ng/ml, 15 min) and then allowed to recover for 24 h in serum-free medium, after which Isc responses to CCh were measured. Conditioned media from these control and EGF-pretreated cells were then added to the basolateral side of virgin T84 cell monolayers, and, after 24 h of incubation, Isc responses to CCh were measured. Data are expressed as percentages of control (C) Isc responses to CCh (n = 8). *P < 0.05; **P < 0.01.

 
Chronic effects of EGF are mediated by release of a soluble factor from epithelial cells. In further experiments, we examined if EGF exerts its chronic actions on Cl– secretion through direct actions on the epithelium or whether it acts indirectly through the release of an autocrine factor. Basolateral media from control monolayers or those acutely treated with EGF (100 ng/ml, 15 min) were collected 24 h after stimulation and then incubated with virgin monolayers for a further 24 h, after which Isc responses to CCh were measured. We found that treatment with EGF-conditioned media mimicked the effects of EGF itself in potentiating Isc responses to CCh (Fig. 4B).

EGF does not chronically alter agonist-induced accumulation of second messengers in colonic epithelial cells. We next analyzed if the effects of EGF in chronically potentiating epithelial secretory responses might be due to alterations in the ability of secretagogues to evoke changes in intracellular levels of second messengers. We first examined the effects of EGF on CCh-induced increases in intracellular Ca2+ concentration ([Ca2+]i). T84 cells, grown on glass coverslips, were stimulated with EGF (100 ng/ml) for 15 min and then allowed to recover for 24 h in serum-free medium. Cells were then loaded with fura-2 AM, and CCh-induced changes in [Ca2+]i were measured. We found that in cells pretreated with EGF, the ability of CCh to elevate levels of intracellular Ca2+ was not altered compared with control cells (Fig. 5A). In further experiments, we analyzed the effects of EGF on basal and FSK-stimulated PKA activity. After acute stimulation with EGF, cells were lysed, and cytosolic PKA activity was measured using a commercially available kit. We found that neither basal nor FSK-stimulated PKA activity was altered by EGF pretreatment (Fig. 5B).


Figure 5
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  		Fig. 5. EGF does not chronically alter basal or agonist-stimulated second messenger levels in T84 cells. A: cells were pretreated with EGF (100 ng/ml, 15 min), and, 24 h later, mobilization of intracellular Ca2+ in response to CCh (100 µM) was measured by fura-2 fluorescence. Data are expressed as mean fluorescence ratios at 340 and 380 nM (n = 6 coverslips for each condition). B: cells were pretreated with EGF (100 ng/ml, 15 min), and basal or FSK (10 µM)-stimulated PKA activity was measured using a nonradioactive PepTag assay kit. Top, upon electrophoretic separation, the phosphorylated peptide substrate migrated toward the cathode (+), as indicated by the arrow. Bottom, densitometric analysis of 4 similar experiments. Data are expressed as fold changes over basal PKA activity. **P < 0.01 compared with control unstimulated cells.

 
EGF increases NKCC1 surface expression in colonic epithelial cells. We next analyzed the effects of EGF on NKCC1 expression, the key entry pathway for Cl– in epithelial cells. T84 cell monolayers were stimulated with EGF (100 ng/ml, 15 min). After 24 h, cells were lysed, and the lysates probed by Western blot analysis with antibodies against NKCC1 or the {alpha}-subunit of the Na+/K+-ATPase pump. We found that in EGF-pretreated cells, levels of NKCC1 protein expression were significantly increased. In contrast, treatment with the growth factor did not alter the expression of the {alpha}-subunit of the Na+/K+-ATPase pump (Fig. 6, A and B). In further experiments, we compared the time course over which EGF induces NKCC1 expression with that with which it potentiates epithelial responses to secretagogues. Increases in the expression of NKCC1 were apparent within 3 h, were almost maximal by 6 h, and remained elevated for at least 24 h after acute stimulation with EGF (Fig. 6C). Cells that were acutely pretreated with EGF initially showed a decrease in their ability to evoke secretory responses to CCh. However, this inhibitory effect was no longer apparent after 3 h, and, within 6 h after EGF treatment, responses to CCh were potentiated to almost a similar degree as those observed after 24 h (Fig. 6D). In further experiments using a cell surface biotinylation approach, we found that increases in cellular NKCC1 were accompanied by increases in the expression of the protein at the cell surface (Fig. 6E).


Figure 6
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  		Fig. 6. EGF increases Na+/K+/2Cl– cotransporter (NKCC1) protein expression in T84 cells. A: cells were stimulated with EGF (100 ng/ml, 15 min), and, 24 h later, cells were lysed and analyzed for NKCC1 and Na+/K+-ATPase expression by Western blot analysis. B: densitometric analysis of several similar experiments (n = 6). ***P < 0.001 compared with control untreated cells. C: cells were treated with EGF (100 ng/ml) for 15 min, and, at various times afterward, cells were lysed and analyzed for the expression of NKCC1 by Western blot analysis. D: densitometric analysis of several similar experiments (n = 3–4 for each time point) compared with the time course of the effects of acute EGF (100 ng/ml) on CCh-stimulated Isc responses (n = 4). E: cells were stimulated with EGF (100 ng/ml) for the times indicated, after which they were analyzed for basolateral NKCC1 surface expression by biotin labeling (n = 5). *P < 0.05; **P < 0.01 compared with control cells.

 
Finally, we examined if EGF induction of NKCC1 protein expression is due to effects at the level of gene transcription. Pretreatment of the cells with the inhibitor of transcription actinomycin D abolished the effects of the growth factor on NKCC1 protein expression (Fig. 7A). Furthermore, RT-PCR analysis revealed that acute treatment with EGF induced a time-dependent accumulation of NKCC1 mRNA with a maximal response occurring 2–4 h after treatment with the growth factor (Fig. 7B).


Figure 7
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  		Fig. 7. EGF-induced NKCC1 expression occurs at the level of gene transcription. A: cells were pretreated with the inhibitor of transcription actinomycin D (AD; 250 ng/ml, 30 min) prior to stimulation with EGF (100 ng/ml, 15 min). Cells were maintained in serum-free medium containing AD. After 24 h, cells were lysed, and NKCC1 expression was measured by Western blot analysis (n = 3). *P < 0.05 compared with control cells; #P < 0.05 compared with EGF-pretreated cells. B: cells were pretreated with EGF (100 ng/ml) for 15 min. After various time points (1–24 h), cells were harvested, and mRNA was isolated. RT-PCR was performed with primers specific for NKCC1 or GADPH. NKCC1 expression was quantified by densitometry and normalized to GADPH expression (n = 3). ***P < 0.001 compared with control unstimulated cells.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present study increases our knowledge of the role of the EGFR in the regulation of intestinal epithelial secretion. Our data show that, in addition to its previously reported acute antisecretory actions (40, 41), over more prolonged periods of time, exposure to EGF enhances epithelial responsiveness to a broad range of secretagogues acting through Ca2+, cAMP, and cGMP-dependent signaling pathways. The effects of EGF are apparent within 3–6 h and sustained for at least 24 h. Furthermore, potentiation of agonist-induced responses by EGF was abolished in the presence of bumetanide, indicating that the effects of the growth factor are due to enhanced Cl– secretion. In vivo, such actions would be accompanied by increased fluid secretion into the intestinal lumen in response to secretory stimuli.

The EGFR, or ErbB1, is a member of the ErbB family of receptor tyrosine kinases, of which there are three other known members: ErbB2, ErbB3, and ErbB4. We (25) have previously found ErbB2 and ErbB3 to be expressed in T84 cells, whereas ErbB4 appears to be absent. Our present data show that while another EGFR ligand, TGF-{alpha}, mimicked the chronic actions of EGF on epithelial secretory responsiveness, HRG, an ErbB3 agonist, was without effect. Thus, it appears that it is specifically EGFR activation and not that of other members of the ErbB family that has the capability to chronically upregulate epithelial secretion. This is interesting in light of our previous findings demonstrating that HRG, like EGF, exerts acute antisecretory effects on intestinal epithelial cells and that both growth factors exert their actions through a common phosphatidylinositol 3-kinase (PI3K)-mediated pathway (25, 41). Thus, it is likely that the chronic effects of EGF in enhancing epithelial secetion are transduced by signaling mechanisms distinct from those that mediate its acute antisecretory effects. The identity of the signaling mechanism(s) involved is currently under investigation and multiple possibilities exist, since, in addition to PI3K, EGF activates multiple effector proteins in colonic epithelia, including MAPKs, PLC-{gamma}, NF-{kappa}B, and PKC (4, 5, 11, 17, 20, 26, 27). Interestingly, our present data suggest that EGF ultimately exerts its actions on epithelial secretion by an indirect mechanism that involves the release of a soluble factor from the epithelium that then presumably acts in an autocrine fashion to promote responsiveness to secretagogues. The identity of this autocrine factor is currently unknown but is the subject of ongoing study on our laboratory.

We hypothesized that EGF might exert its chronic actions on secretory responses by altering the ability of agonists to mobilize intracellular second messengers. However, this does not appear to be the case since pretreatment with EGF did not increase either intracellular Ca2+ responses to CCh or activation of PKA by FSK. Rather, our data suggest that the effects of EGF are mediated at the fundamental level of transport protein expression. Induction of NKCC1 protein expression by EGF was apparent within 3 h, almost maximal by 6 h, and sustained for 24 h after treatment with the growth factor. Furthermore, increases in total cellular NKCC1 protein expression were accompanied by increases in cell surface expression of the protein as measured by cell surface biotinylation. Thus, the time course of increased NKCC1 expression temporally correlates with its effects in potentiating secretory responses and, since NKCC1 is the protein responsible for loading colonic epithelial cells with Cl–, increases in its expression would be expected to enhance epithelial secretory capacity. Our data also indicate that the effects of EGF in increasing NKCC1 expression are specific since it did not alter the expression of the {alpha}-subunit of the Na+/K+-ATPase pump. Experiments are currently underway to examine the effects of EGF on the expression of other components of the epithelial Cl– secretory pathway, and initial data suggest that the growth factor also increases expression of CFTR Cl– channels and both KCNN4 and KCNQ1 K+ channels (unpublished observations). Future studies will aim to more comprehensively characterize the effects of EGF on the expression of these transport proteins.

While previous work has shown that NKCC1 activity is acutely regulated by phosphorylation and membrane trafficking (13, 14), there is little information regarding the mechanisms that regulate its cellular abundance. Indeed, there is a general lack of information regarding the regulation of epithelial function at the fundamental level of transport protein expression. This represents an important gap in our understanding of the molecular mechanisms of epithelial secretion, since factors that regulate transport protein expression are likely to be important in setting the basal tone of epithelial secretory capacity in both normal and pathological circumstances. Present data suggest that the regulation of NKCC1 abundance is likely to be multifactorial. Previous studies, also using T84 colonic epithelial cells, have identified short-chain fatty acids (SCFAs) as regulators of colonic NKCC1 expression (33). The predominant colonic SCFA, butyrate, decreases NKCC1 expression, thereby reducing epithelial secretory capacity. A recent study (24) has also demonstrated that NKCC1 is regulated by cellular O2 supply, with decreases in its expression occurring under hypoxic conditions. Thus, our present knowledge suggests that the expression of colonic epithelial secretory responses at any given time will be determined by luminal concentrations of SCFAs, tissue oxygen supply, and levels of mucosal EGFR ligands. Each of these factors can be altered in conditions of disease, and consequent changes in NKCC1 expression are likely to be important in resetting the basal tone of secretory capacity under such conditions. Based on the observations that EGF-induced NKCC1 expression is abolished by actinomycin D and that EGF increases NKCC1 mRNA abundance, our present data suggest that enhancement of secretagogue-induced responses by EGF is likely mediated at the level of enhanced NKCC1 gene transcription. The transcriptional mechanisms involved remain to be investigated, as does the possibility that posttranslational modifications may also play a role in NKCC1 expression, as they do with other components of the Cl– secretory mechanism (8).

The role of the EGFR as a critical regulator of intestinal epithelial ion and fluid transport is gradually emerging. Acutely, EGFR ligands inhibit Cl– secretion and promote Na+ and nutrient absorption (3, 16, 22, 29, 36, 40, 42, 44). On the other hand, in addition to its long-term effects in promoting agonist-induced secretory responses described here, EGF also upregulates epithelial absorptive function by increasing the expression of transport proteins involved in Na+ absorption, namely, Na+/H+ exhanger isoform 2 (43). Thus, it seems likely that alterations in the levels of EGFR ligands in conditions of disease will contribute to dysregulated transport associated with such conditions. With this in mind, it is interesting to note that EGFR ligands and kinase activity are altered in intestinal inflammation and that EGF itself is effective in the treatment of IBD in both animal models and human patients (1, 2, 12, 18, 19, 32, 34). However, at present, it is difficult to explain the physiological, or pathophysiological, relevance of the opposing acute and chronic effects of EGF on epithelial secretory function. However, drawing on findings from both our and other laboratories, a hypothetical model can be developed. Conditions that bring about epithelial injury and disruption of barrier function are known to induce local accumulation of growth factors such as EGF and TGF-{alpha} (15, 34, 35). Previous work has shown that the immediate effect of EGF on colonic epithelial cells is to inhibit secretory capacity (40). This might be advantageous under conditions of epithelial damage since it would allow diversion of cellular energy from costly transport processes to be used in restitutive mechanisms. Indeed, treatment of epithelial cells with EGFR ligands rapidly promotes epithelial wound healing (20, 21, 37, 38). Over a period of several hours, as the epithelial barrier is healed, one might then expect that enhanced secretion would be beneficial as it would serve to hydrate the newly repaired epithelial barrier and to flush the immediate area of cellular debris and other noxious substances. In future studies, we aim to test this hypothesis by examining NKCC1 expression in a model of epithelial restitution.

In summary, our study demonstrates a novel role for EGF, and related growth factors, in the chronic upregulation of epithelial secretory capacity. In vivo, such actions would serve to promote intestinal fluid secretion. The effects of EGF appear to be mediated by enhanced expression of NKCC1, a key component of the Cl– secretory pathway, and future studies will investigate signaling and transcriptional mechanisms involved. Our study underlines the critical role of the EGFR in regulating intestinal transport function and suggests that, in the future, manipulation of EGFR-dependent signaling mechanisms may prove useful in the treatment of dysregulated fluid and electrolyte transport associated with intestinal disease.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by a Senior Investigator Award from the Crohn's and Colitis Foundation of America and by an Investigator Program Award from the Science Foundation of Ireland.


   ACKNOWLEDGMENTS
 
We greatly appreciate the kind gift of rabbit anti-NKCC1 antibody from Dr. Christian Lytle (University of California, Riverside, CA).


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. J. Keely, Dept. of Molecular Medicine, RCSI Education and Research Centre, Beaumont Hospital, Dublin 9, Ireland (e-mail: skeely{at}rcsi.i.e)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
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