TGF-beta effects on epithelial ion transport and barrier: reduced Clminus secretion blocked by a p38 MAPK inhibitor

Kathryn Howe1, Jack Gauldie2, and Derek M. McKay1

1 Intestinal Disease Research Program and 2 Center for Gene Therapeutics, Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada L8N 3Z5


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Growth factors affect a variety of epithelial functions. We examined the ability of TGF-beta to modulate epithelial ion transport and permeability. Filter-grown monolayers of human colonic epithelia, T84 and HT-29 cells, were treated with TGF-beta (0.1-100 ng/ml, 15 min-72 h) or infected with an adenoviral vector encoding TGF-beta (Ad-TGFbeta ) for 144 h. Ion transport (i.e., short-circuit current, Isc) and transepithelial resistance (TER) were assessed in Ussing chambers. Neither recombinant TGF-beta nor Ad-TGFbeta infection affected baseline Isc; however, exposure to >= 1 ng/ml TGF-beta led to a significant (30-50%) reduction in the Isc responses to forskolin, vasoactive intestinal peptide, and cholera toxin (agents that evoke Cl- secretion via cAMP mobilization) and to the cell-permeant dibutyryl cAMP. Pharmacological analysis of signaling pathways revealed that the inhibition of cAMP-driven epithelial Cl- secretion by TGF-beta was blocked by pretreatment with SB-203580, a specific inhibitor of p38 MAPK, but not by inhibitors of JNK, ERK1/2 MAPK, or phosphatidylinositol 3'-kinase. TGF-beta enhanced the barrier function of the treated monolayers by up to threefold as assessed by TER; however, this event was temporally displaced from the altered Isc response, being statistically significant only at 72 h posttreatment. Thus, in addition to TGF-beta promotion of epithelial barrier function, we show that this growth factor also reduces responsiveness to cAMP-dependent secretagogues in a chronic manner and speculate that this serves as a braking mechanism to limit secretory enteropathies.

short-circuit current; T84 epithelia; growth factor; transepithelial resistance


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

A NUMBER OF ESSENTIAL FUNCTIONS are provided by the epithelial lining of the gastrointestinal (GI) tract. In addition to nutrient digestion/absorption and immune surveillance roles, the gut epithelium also secretes electrolytes; it is this vectorial electrolyte secretion that establishes a driving force for directed water movement. Excess water in the lumen can lead to diarrhea (essentially a protective process), which if recurrent or prolonged may result in dehydration and even death. Given the association of GI epithelia with the enteric nervous system, neighboring enteroendocrine cells, and the luminal contents, it is not surprising that nervous and endocrine input as well as bacteria and/or their products constitute the major regulators of epithelial function. Recent literature, however, particularly from in vitro studies, has shown that immune mediators including cytokines can also modify epithelial function (24).

Growth factors are cytokines that have been shown to have both mitogenic and nonmitogenic effects. Specifically, in terms of ion transport, treatment of colonic epithelial cell lines with transforming growth factor (TGF)-alpha or epidermal growth factor (EGF) resulted in decreased secretory responses to stimulants of chloride secretion (6, 39). TGF-beta is a multifunctional cytokine, the bioactivity of which can be grouped into three main properties: 1) regulation of cell growth and proliferation, 2) immunomodulation, and 3) stimulation of wound repair (epithelial restitution). Studies have shown that TGF-beta is upregulated in many diseases, including inflammatory bowel disease (IBD) (2), an enteropathy often characterized by perturbed water movement. Given that altered epithelial electrolyte transport can lead to aberrant water balance in the gut, that other growth factors have been shown to affect this process, and that TGF-beta is upregulated during enteric disease, the primary aim of this study was to determine the effect of TGF-beta on epithelial ion transport function.

Using monolayers of the human T84 or HT-29 colonic epithelial cell lines as model epithelia, we have shown that exposure to TGF-beta leads to a ~30% decrease in epithelial secretory responses to stimuli that initiate cAMP-dependent Cl- secretion. This disruption of normal electrolyte transport events is temporally distinct from the ability of TGF-beta to increase epithelial barrier function and can be restored by treatment with SB-203580, an inhibitor of the p38 mitogen activated protein kinase (MAPK) signaling pathway.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell culture. The human colonic adenocarcinoma-derived T84 epithelial cell line was maintained in media containing equal volumes of Dulbecco's modified Eagle's medium (DMEM) and Ham's F-12 medium, supplemented with 10% (vol/vol) fetal calf serum, 1.5% (vol/vol) HEPES, and 2% (vol/vol) penicillin-streptomycin (all from Life Technologies, Grand Island, NY) at 37°C, 5% CO2. The HT-29cl.19A cell line (HT-29; a kind gift from Dr. J. A. Groot, University of Amsterdam) was maintained in DMEM supplemented with 5% (vol/vol) fetal calf serum, 0.1% vol/vol L-glutamine, 2% (vol/vol) penicillin-streptomycin, and 5% (vol/vol) sodium bicarbonate. Cells were seeded onto semipermeable filter supports (0.4-µm pore size; Costar, Cambridge, MA) with a surface area of 1 cm2 (106 cells) for physiological assessment and grown to confluence as determined by transepithelial resistance (TER; minimum 6 days growth).

Physiological assessment studies. Recombinant human TGF-beta (R&D Systems, Minneapolis, MN) was added to the basolateral compartment of the semipermeable filter supports at concentrations of 0.1, 1, 10, or 100 ng/ml. The basolateral surface of T84 cells was exposed to TGF-beta for 4, 8, 16, 24, and 72 h, at which point cells were mounted in specialized Ussing chambers (Precision Instrument Design, Tahoe City, CA) under voltage-clamped conditions as previously described (25), and short-circuit current (Isc) responses to known stimuli of Cl- secretion were measured. In other studies, T84 monolayers were exposed to TGF-beta for 15 min, then rinsed twice in fresh medium, and Isc responses examined 16 h later. Comparative studies examined Isc responses to EGF (10 or 100 ng/ml; R&D Systems) after 16 h of exposure. Acute experiments exposed T84 cells to TGF-beta for 30 min (1 or 10 ng/ml) while mounted in the Ussing chambers, followed by measurement of Isc responses. In all experiments, baseline Isc was obtained after 10 min of equilibration. Stimulated Cl- secretion was induced by forskolin (10-5 M), vasoactive intestinal peptide (VIP, 10-7 M), cholera toxin (10 µg/ml), and dibutyryl cAMP (200 µM) (all from Sigma Chemical, St. Louis, MO), and the maximal change in Isc (Delta Isc) was recorded. These secretagogues were chosen because of their known ability to elicit Cl- secretion via raising intracellular cAMP (13, 23, 44). TER (in Omega /cm2) of the T84 monolayers was recorded with chopstick electrodes and a voltmeter (Millipore, Bedford, MA) as a measure of paracellular permeability.

Adenoviral infection. Twenty-four hours after seeding (106 cells/ml on semipermeable filter supports), T84 cells were infected with replication-deficient adenovirus constructs encoding active TGF-beta [Ad-TGFbeta (35)] at a multiplicity of infection (MOI) of 10, 20, or 50 virus particles/cell. Sixteen hours later, T84 cells were rinsed twice with medium to remove any residual virus and cultured for 6 days, whereupon they were mounted in Ussing chambers for analysis of secretory responsiveness to forskolin. Changes in barrier function were monitored daily throughout the 6-day postinfection period by recording TER.

Epithelia infected with either 1) the deletion variant (empty vector) of the replication-deficient adenovirus [Ad-delete (16)] or 2) adenovirus encoding latent TGF-beta [Ad-latent TGFbeta (47)] were used as additional controls for these experiments. Infectivity of the adenovirus was verified in T84 cells by using virus expressing the marker genes beta -galactosidase or luciferase (27). Finally, synthesis of the active form of TGF-beta from Ad-TGFbeta -infected cells and noninfected control epithelia was determined by the presence of TGF-beta in the media at 24-h intervals postinfection (24-120 h) using ELISA (R&D Systems). When the experiments were completed, epithelial cells were trypsinized off the filter supports and viability was assessed with the trypan blue exclusion technique.

Pharmacological inhibition of intracellular signaling. Physiological assessment experiments were conducted by using a single dose (10 ng/ml) and time (16 h) of TGF-beta exposure to T84 monolayers. T84 cells were pretreated with inhibitors of 1) p38 MAPK [1 h, 10 µM, SB-203580; Calbiochem, La Jolla, CA (18, 20, 33)]; 2) c-Jun NH2-terminal kinase (JNK) [30 min, 10 µM, SP-600125; Calbiochem (17)]; 3) extracellular signal-regulated kinase 1/2 (ERK1/2) signaling, via inhibition of MEK, the enzyme upstream of ERK1/2 [1 h, 25 µM, PD-98059; Calbiochem (4, 8)]; or 4) phosphatidylinositol 3' kinase (PI 3-K) [15 min, 20 µM, LY-294002; Sigma Chemical (42)]. TGF-beta was subsequently added to the basal compartment of the culture well (inhibitor not washed out) and, after a defined incubation time, the monolayers were mounted in Ussing chambers and Isc responses to forskolin were recorded. Pharmacological inhibition of the p38 MAPK signaling cascade in HT-29 cells was accomplished by pretreating monolayers with SB-203580 for 30 min (0.1-50 µM), subsequently followed by TGF-beta application (100 ng/ml, 24 h).

Statistical and data analysis. Data are normalized to time-matched controls (i.e., percentage of control response) and are presented as means ± SE. Data were analyzed using one-way ANOVA, and P < 0.05 was accepted as the level of statistical significance.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

TGF-beta reduces Isc responses to cAMP secretagogues. Treatment with TGF-beta (0.1-100 ng/ml, 4-72 h) did not significantly affect baseline Isc compared with naive time-matched controls [0.17 ± 0.17 vs. 0.33 ± 0.33 µA/cm2 for control and TGF-beta -treated (10 ng/ml, 16h), respectively, n = 6 monolayers from a representative experiment]. Addition of TGF-beta to the Ussing chamber for 30 min (0.1, 1, 10 ng/ml) had no effect on epithelial baseline Isc or Delta Isc evoked by forskolin (data not shown). Treatment with TGF-beta for 16 h did, however, cause a statistically significant decrease in Isc responses to forskolin; this was evident with >= 1 ng/ml (data not shown). Figure 1 shows that T84 cells exposed to TGF-beta (10 ng/ml) for 16 or 24 h demonstrated a statistically significant decrease in forskolin-induced Isc responses of ~30% compared with controls (P < 0.0001). This reduced responsiveness to forskolin was further enhanced by 72 h post-TGF-beta treatment, being on average only 50% of the magnitude of the response observed in time-matched control monolayers (P < 0.05 compared with 16 and 24 h of TGF-beta exposure). After 72 h of TGF-beta treatment, there was a significant increase in TER (1,405 ± 254 Omega /cm2 compared with control at 846 ± 204 Omega /cm2, P < 0.05, n = 9-12 monolayers) that was not observed with shorter TGF-beta exposure periods. In "washout" experiments, cells exposed to TGF-beta (10 ng/ml) for 16 h and subsequently rinsed free of TGF-beta also displayed a similar decrease in Isc responses to forskolin at 72 h posttreatment (40.6 ± 1.7% of control response, P < 0.0001, n = 3 monolayers), comparable to those seen after 72 h of persistent TGF-beta exposure (49.3 ± 7.5% of control responses, P < 0.0001, n = 9 monolayers). This washout treatment also resulted in increased TER (1,598 ± 48 Omega /cm2 vs. controls at 847 ± 20 Omega /cm2, P < 0.001, n = 3). T84 cells exposed to TGF-beta for 16 h at the highest dose used (i.e., 100 ng/ml) displayed a ~30% decrease in forskolin-induced increases in Isc, which was not statistically different from the inhibition of Isc observed with lower doses of cytokine (i.e., 10 ng/ml). Furthermore, additional washout experiments revealed that exposure to TGF-beta for 15 min was sufficient to induce diminished responsiveness to forskolin upon examination 16 h later (untreated controls 37.3 ± 4 vs. TGF-beta -treated cells 17 ± 2.1 µA/cm2. P < 0.001, n = 6-7 monolayers), in this case a 55% reduction in Delta Isc.


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Fig. 1.   Bar chart showing the time-dependent decrease in T84 epithelial Delta Isc (maximal change in short-circuit current) responses to forskolin (Fsk; 10-5 M) after exposure to TGF-beta (10 ng/ml). Values are percentages of control monolayer responses (*P < 0.05, **P < 0.001 compared with control; #P < 0.05 compared with 16- and 24-h TFG-beta exposure; n = 6-10 monolayers) and are expressed as means ± SE. Control Delta Isc responses to Fsk ranged from 40 to 80 µA/cm2.

TGF-beta treatment (10 ng/ml, 16 h) also resulted in a significant diminution of epithelial responsiveness to cAMP-driven Cl- secretion initiated by VIP and cholera toxin (Fig. 2). Similarly, cells exposed to TGF-beta displayed reduced Delta Isc responses to the cell-permeant cAMP analog, dibutyryl cAMP (Fig. 2). In contrast to the effects of TGF-beta on Isc responses, T84 monolayers treated with EGF for 16 h (10 or 100 ng/ml) displayed Isc responses to forskolin that were not different from controls (Table 1).


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Fig. 2.   Bar chart showing reduced epithelial responses to cAMP secretagogues (Fsk, 10-5 M; VIP, 10-7 M; cholera toxin, 10 µg/ml) and a cell-permeant cAMP analog (dibutyryl cAMP, 200µM) in T84 monolayers exposed to TGF-beta (10 ng/ml, 16 h). Values are percentages of control monolayer responses (*P < 0.05 compared with control; n = 4-12 monolayers) and are expressed as means ± SE.


                              
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Table 1.   EGF-treated (16 h) T84 cells do not display reduced Isc responses to forskolin

In additional experiments, HT-29 epithelial cells treated with TGF-beta (100 ng/ml, 72 h) displayed reduced Isc responses to forskolin (control 118.7 ± 23.7 vs. TGF-beta -treated 64.3 ± 12.3 µA/cm2, P < 0.05, n = 3 monolayers). After 72 h of exposure, TGF-beta -treated HT-29 monolayers also exhibited a small, but significant, increase in TER compared with controls (control 287.7 ± 6.8 vs. TGF-beta 302.0 ± 3.1 Omega /cm2, P < 0.05, n = 3 monolayers).

Exposure to TGF-beta 1 delivered via adenoviral gene transfer causes decreased forskolin responsiveness. We sought to determine what effect chronic exposure to TGF-beta would have on epithelial function using adenoviral gene transfer of the biologically active form of TGF-beta . Cell infectivity was verified by use of adenoviral vectors encoding marker genes (Fig. 3A). Cells infected with the highest dose of virus (50 MOI) displayed no significant decrease in viability, as determined by the trypan blue exclusion technique (data not shown), and exhibited increased TGF-beta production compared with uninfected controls (e.g., 912.2 ± 202.4 pg/ml TGF-beta in supernatants collected 72 h after infection with Ad-TGFbeta vs. 217.9 ± 8.5 pg/ml in supernatants of naive controls, P < 0.05, n = 3). Assessment of Isc responses to forskolin 6 days post-Ad-TGFbeta infection revealed a statistically significant decrease in secretory responsiveness compared with naive controls and T84 cells infected with Ad-delete or Ad-latent TGFbeta (Fig. 3B). In addition, Ad-TGFbeta infection resulted in a statistically significant increase in TER by the end of the 6-day (144 h) postinfection period (Fig. 3C), which was first apparent 72 h postinfection (Fig. 3D). Similar to the experiments with recombinant TGF-beta , HT-29 cells infected with Ad-TGFbeta (50 MOI) displayed reduced Isc responses to forskolin 6 days postinfection (66 ± 18.7 compared with controls at 118.7 ± 23.7 µA/cm2, P < 0.05, n = 3).


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Fig. 3.   A: T84 monolayers infected with luciferase-encoding adenovirus (ad-luciferase) for 72 h at 10, 20, or 50 multiplicity of infection (MOI) display increased production of luciferase [30-50% of T84 cells showed positive beta -galactosidase staining after 72 h of infection with beta -galactosidase-encoding adenovirus (20 MOI), data not shown]. B and C: decreased epithelial Isc responses to Fsk (10-5 M; B) and the increase in transepithelial resistance (TER; C) following adenoviral infection with active TGF-beta (Ad-TGFbeta ; 10, 20, or 50 MOI, 144 h) but not inactive TGF-beta (Ad-latent TGFbeta ) or a deletion variant (Ad-delete) (both 50 MOI). Data are percentages of control monolayers (*P < 0.05 compared with untreated controls; n = 6-12) and are expressed as means ± SE. D: line graph from a representative experiment (n = 3 monolayers) depicting the change in TER over the 6-day postinfection (50 MOI) period as a percentage of uninfected control TER values (controls 500-2,000 Omega /cm2; n = 3-5 experiments; *P < 0.05 compared with controls), expressed as means ± SE.

Inhibition of p38 MAPK, but not JNK, ERK1/2 MAPK, or PI 3-K, reduced the TGF-beta effect on cAMP-mediated Isc. Pretreatment of HT-29 cells with SB-203580 (>= 1 µM), a potent inhibitor of p38 MAPK activity, 30 min before TGF-beta application completely restored the Isc responses to forskolin to control values (Fig. 4), whereas similarly treated T84 monolayers resulted in a significant, but only partial, improvement in Isc responses to forskolin (Table 2). Other signaling pathways in T84 epithelial cells did not appear to contribute to the diminished Isc, because pretreatment with the JNK inhibitor (SP-600125), ERK1/2 pathway inhibitor (PD-98059), and the PI 3-K inhibitor (LY-294002) did not inhibit the observed effect of TGF-beta on ion transport; in fact, exposure to PD-98059 or LY-294002 alone caused a significant decrease in Isc responses to forskolin ~16 h later (Table 2).


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Fig. 4.   Bar chart depicting a dose-response to the recovery of TGF-beta -induced (10 ng/ml, 24 h) diminished Isc responses to Fsk (10-5 M) in HT-29 epithelia pretreated with the p38 MAPK inhibitor SB-203580. Values are percentages of control responses to Fsk (*P < 0.05 compared with naive controls; n = 8-9 monolayers) and are expressed as means ± SE.


                              
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Table 2.   Pharmacological inhibition of p38, but not JNK, ERK1/2, or PI 3-K reduces TGF-beta inhibition of Delta Isc to forskolin in T84 epithelia


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

TGF-beta is a multifunctional peptide that affects cell proliferation and differentiation and has immunosuppressive properties. Using human enteric epithelial cell lines, we have shown that 1) exposure to TGF-beta significantly decreases cAMP-driven Cl- secretion; 2) this effect can be blocked by an inhibitor of p38 MAPK but not by pharmacological inhibitors of JNK, ERK1/2 MAPK, or PI 3-K activity; and 3) the increased barrier function caused by TGF-beta is temporally distinct (i.e., delayed) from the altered ion transport characteristics of the treated epithelium.

Ion transport is an important element of gut homeostasis. As the driving force for water movement, it facilitates surface hydration, which, if dysregulated, can result in debilitating diarrhea or constipation. TGF-beta had no effect on tonic epithelial ion transport (i.e., baseline Isc) but significantly reduced secretory responses to three cAMP-dependent secretagogues that operate via different mechanisms. This effect likely lies downstream of cAMP generation, because TGF-beta -treated cells stimulated with dibutyryl cAMP displayed similarly reduced Isc events. The responses to forskolin, VIP, cholera toxin, and dibutyryl cAMP were all reduced by a similar magnitude (i.e., ~30%), suggesting that although TGF-beta can dampen cAMP-driven Cl- secretion, the pathophysiology that would be associated with total blockade of Cl- secretion is avoided. The inhibition of secretory responsiveness required a minimum of 1 ng/ml TGF-beta and was statistically significant 16 h posttreatment; however, constant exposure to TGF-beta was not essential, because a 15-min exposure resulted in reduced Delta Isc to forskolin 16 h later. Higher doses of TGF-beta did not affect the time required to observe a reduced Delta Isc to forskolin, and they did not affect the magnitude of the response. This lack of a dose response has been noted for TGF-alpha inhibition of bradykinin-induced Delta Isc in HCA-7 colonocytes, although in this instance the Isc responses were reduced by 6 h posttreatment (6).

The effects of recombinant TGF-beta were reproduced in epithelia infected with adenovirus encoding the gene for the active form of TGF-beta ; diminished Delta Isc to forskolin were apparent 144 h postinfection (i.e., end of the experiment). However, unlike other growth factors, such as EGF and IGF, which induce acute effects on epithelial Delta Isc (39, 11), TGF-beta when added to T84 monolayers in Ussing chambers for 30 min had no effect on subsequent forskolin-evoked Delta Isc. Thus TGF-beta can be added to the list of cytokines that directly modulate epithelial ion transport (reviewed in Ref. 24). In addition, the effect of TGF-beta is set apart from those of other growth factors that rapidly affect ion transport; rather, the TGF-beta effect is reminiscent of IFN-gamma or IL-4 diminution of epithelial secretory responsiveness, which typically requires 24 h or longer to become apparent (1, 10).

Upon identifying that TGF-beta exerts an effect on epithelial ion transport, a number of mechanistic issues arose. One option was to investigate the physiological changes mediated by TGF-beta , such as the possible change in amount, location, or activity of the apical Cl- channel, CFTR. Indeed, growth factor regulation of specific ion channels (3, 34) and cytokine modulation of CFTR gene expression have been reported (7, 9, 30). Alternatively, the TGF-beta signaling pathway leading to the decreased secretory response could be addressed. Data from many cell types show that TGF-beta binding to its surface receptor causes mobilization of classic MAPKs and a unique series of molecules designated SMADs, and these signaling cascades regulate specific aspects of the biological effects of TGF-beta (29, 37, 41, 46). Little is known, however, of the TGF-beta signaling pathways in epithelia in general (36), with a distinct lack of data relevant to enteric epithelial cells.

Using a pharmacological approach, we found that the TGF-beta effects on ion transport were reduced in two cell lines by pretreatment with the established inhibitor of the p38 MAPK pathway, SB-203580. This is the first time p38 MAPK has been implicated in governing a TGF-beta function in gut epithelia, and these findings are complementary to a recent report showing that hyperosmolar stress-induced reduction in colonic CFTR mRNA was blocked by inhibition of p38 MAPK activity (5). Whereas low-dose SB-203580 (i.e., 1 µM) completely restored the TGF-beta -induced diminished ion transport events in HT-29 cells, higher doses of the pharmacological inhibitor only partially corrected the defect in T84 epithelia. This finding indicates cell line-specific differences and, in the case of T84 cells, suggests either reduced sensitivity to SB-203580 or that other signaling pathways participate in the TGF-beta effect. Thus we adopted the same pharmacological approach to assess the putative involvement of other signaling molecules in TGF-beta modulation of ion transport. JNK is likely activated in tandem with p38 MAPK in response to TGF-beta (22, 43) and can affect the Na+-K+-2Cl- transporter (19). Interference with the activity of this transporter would impact on apical Cl- secretion events. Furthermore, high-dose SB-203580 (i.e., 10-20 µM) may inhibit JNK activity (45), yet use of a reputedly specific inhibitor of JNK activity did not ameliorate the TGF-beta -induced diminution of T84 Delta Isc to forskolin. ERK, the third member of the MAPK family, is mobilized in a variety of cell types by TGF-beta (14, 15, 28): pharmacological inhibition of MEK, the enzyme upstream of ERK, did not affect the decreased Delta Isc induced by TGF-beta . Finally, use of an established inhibitor of PI 3-K activity excluded the involvement of this ubiquitous signaling molecule in the TGF-beta -induced perturbation of cAMP-driven Cl- secretion. The latter observation contrasts with the diminished carbachol-induced increases in Isc in T84 cells caused by EGF that are PI 3-K sensitive (40). In this context, we observed that TGF-beta did not consistently alter carbachol-elicited increases in Isc in T84 or HT-29 epithelia (personal observation).

Exposure to the MEK and PI 3-K inhibitors alone reduced T84 Delta Isc to forskolin (Table 2), suggesting that longer inhibition of ubiquitous signaling pathways may affect multiple energy-dependent processes such as epithelial ion transport. The inhibition of forskolin-induced Delta Isc by LY-294002 appears to contradict the work of Dickson et al. (12). The discrepancy may be due to the fact that in the latter study, the epithelium was exposed to LY-294002 in Ussing chambers for <= 30 min and Isc responses were immediately assessed. Indeed, when those investigators inhibited PI 3-K via wortmannin for 24 h, they observed an ~50% decrease in forskolin-stimulated Isc, which is compatible with the data in the current study.

Agent specificity must be considered in any pharmacological study. Although accepted as a specific p38 MAPK inhibitor, SB-203580 has recently been shown to block the activity of ALK5, a kinase that phosphorylates SMAD3 (21). Thus involvement of SMAD3 in TGF-beta inhibition of Delta Isc to forskolin is a possibility. We have been unable to convincingly and consistently show increased amounts of phosphorylated p38 MAPK in nuclear or whole cell extracts of serum-starved TGF-beta -treated epithelial cells, or elevated p38 MAPK activity (data not shown). This may be due to 1) the relatively high constitutive phospho-p38 MAPK found in control extracts; 2) the possibility that there may in fact be only very subtle increases in activated p38 MAPK, because TGF-beta causes only a partial ablation of the Isc response; or 3) the TGF-beta effect on ion transport is via a specific p38 MAPK isoform (i.e., alpha , beta , gamma , or delta ), the increases of which are masked by constitutive expression of the other isoforms. Indeed, in light of the data from Laping et al. (21), a finding of increased phospho-p38 MAPK in TGF-beta -treated epithelia would be suggestive, but not unequivocal proof, of involvement of this enzyme in the altered ion transport. Thus, although we have ruled out JNK, ERK, and PI 3-K, definitive statements regarding the intracellular signals that mediate the TGF-beta effect on enteric epithelial ion transport require the development of p38 MAPK and SMAD3 knockout (and preferably inducible knockout) cell lines suitable for the analysis of vectorial ion transport.

Recombinant or adenovirally delivered TGF-beta was shown to increase TER, an accepted index of the paracellular permeability pathway. This finding is in accordance with studies where TGF-beta was shown to increase epithelial barrier function and/or preserve barrier integrity compromised by inflammatory cytokines (26, 31, 32). Intriguingly, we found that the effects of TGF-beta on barrier function were temporally distinct from those on ion transport: TER was, unlike Isc, unaltered by 16-24 h post-TGF-beta treatment. This divergence in the timing of TGF-beta effects on two of the primary roles of the enteric epithelium implies utilization of different or additional signaling pathways in the modulation of epithelial barrier and ion transport. Research directed toward understanding the structural basis for the TGF-beta -induced increased TER, the signaling events underlying this process, and definition of the mechanism(s) responsible for the temporal separation of enhancement of the epithelial barrier and diminished cAMP-driven Cl- secretion are required. As noted, TGF-beta can antagonize IFN-gamma -mediated disruption of the barrier function of T84 monolayers (32). Because IFN-gamma can reduce TER within 24 h of exposure and TGF-beta -induced increases in TER are not apparent at this time (i.e., 24 h), it is suggested that the TGF-beta inhibition of IFN-gamma -induced decreases in TER occurs via a distinct mechanism, that is, inhibition of the IFN-gamma signaling cascade. The reciprocal event (i.e., IFN-gamma interference of TGF-beta SMAD signaling) has been shown (38).

In conclusion, irrespective of the intracellular signaling mechanism, exposure of model gut epithelia to TGF-beta not only led to the expected increase in TER (i.e., at 72 h posttreatment) but also resulted in diminished responsiveness to cAMP-dependent secretagogues 16 h posttreatment. Prolonged exposure to TGF-beta (using adenoviral vectors encoding the gene for the active form of TGF-beta ) maintained the Cl- secretion abnormality. The temporal separation of TGF-beta modulation of epithelial ion transport and barrier functions adds to our appreciation of the complexity and spectrum of biological activities coordinated by this pleiotropic cytokine. Finally, we speculate that in addition to its ability to maintain or enhance epithelial barrier function, TGF-beta may reduce pathophysiological complications resulting from excess water movement into the gut lumen by limiting cAMP-driven Cl- secretion.


    ACKNOWLEDGEMENTS

We thank J. Lu and I. Gunawan for technical assistance.


    FOOTNOTES

K. Howe has been a recipient of an Ontario Graduate Scholarship in Science and Technology (2000) and a Natural Sciences and Engineering Research Council Studentship (2001-2003). D. M. McKay is a Canadian Institutes of Health Research (CIHR) Scholar (1998-2003). This work was funded by CIHR Grant MT-13421 (to D. M. McKay).

Address for reprint requests and other correspondence: D. M. McKay, Intestinal Disease Research Program, McMaster Univ., HSC-3N5C, 1200 Main St. West, Hamilton, Ontario, Canada L8N 3Z5 (E-mail: mckayd{at}mcmaster.ca).

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.

August 14, 2002;10.1152/ajpcell.00414.2001

Received 31 July 2001; accepted in final form 1 August 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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