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Am J Physiol Gastrointest Liver Physiol 280: G828-G843, 2001;
0193-1857/01 $5.00
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Vol. 280, Issue 5, G828-G843, May 2001

Key role of PKC and Ca2+ in EGF protection of microtubules and intestinal barrier against oxidants

A. Banan, J. Z. Fields, Y. Zhang, and A. Keshavarzian

Departments of Internal Medicine (Division of Digestive Diseases), Pharmacology, and Molecular Biophysics and Physiology, Rush University Medical Center, Chicago, Illinois 60612


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Using monolayers of human intestinal (Caco-2) cells, we showed that growth factors (GFs) protect microtubules and barrier integrity against oxidative injury. Studies in nongastrointestinal cell models suggest that protein kinase C (PKC) signaling is key in GF-induced effects and that cytosolic calcium concentration ([Ca2+]i) is essential in cell integrity. We hypothesized that GF protection involves activating PKC and maintaining normal [Ca2+]i. Monolayers were pretreated with epidermal growth factor (EGF) or PKC or Ca2+ modulators before exposure to oxidants (H2O2 or HOCl). Oxidants disrupted microtubules and barrier integrity, and EGF protected from this damage. EGF caused rapid distribution of PKC-alpha , PKC-beta I, and PKC-zeta isoforms to cell membranes, enhancing PKC activity of membrane fractions while reducing PKC activity of cytosolic fractions. EGF enhanced 45Ca2+ efflux and prevented oxidant-induced (sustained) rises in [Ca2+]i. PKC inhibitors abolished and PKC activators mimicked EGF protection. Oxidant damage was mimicked by and potentiated by a Ca2+ ionophore (A-23187), exacerbated by high-Ca2+ media, and prevented by calcium removal or chelation or by Ca2+ channel antagonists. PKC activators mimicked EGF on both 45Ca2+ efflux and [Ca2+]i. Membrane Ca2+-ATPase pump inhibitors prevented protection by EGF or PKC activators. In conclusion, EGF protection of microtubules and the intestinal epithelial barrier requires activation of PKC signal transduction and normalization of [Ca2+]i.

tubulin; growth factor; monolayer barrier permeability; Caco-2 cells


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE GASTROINTESTINAL (GI) mucosal epithelium is an essential permeability barrier that normally restricts the passage of harmful proinflammatory molecules into the mucosa and systemic circulation (36). The loss of GI barrier integrity, in contrast, can lead to the penetration of normally excluded luminal compounds (e.g., bacterial endotoxin) into the mucosa and can result in the initiation and/or perpetuation of mucosal inflammation and injury. This damage has been implicated in a wide range of inflammatory illnesses including inflammatory bowel disease (IBD) where high levels of injurious oxidants are present (36, 41).

Accordingly, protection and maintenance of GI barrier function against oxidant insult are critical in preventing the sustained inflammation in these disorders. One protective strategy of tissues uses growth factors such as epidermal growth factor (EGF) and transforming growth factor (TGF)-alpha . These agents can protect the GI mucosal barrier against an array of insults under both in vivo and in vitro conditions (10, 12, 14, 23, 32, 38, 43, 45, 57, 59, 69, 73). For example, we previously demonstrated (10, 12, 14) that oxidants can cause loss of barrier integrity of monolayers of human intestinal (Caco-2) cells and that EGF or TGF-alpha prevents the development of monolayer hyperpermeability. However, the intracellular mechanisms underlying this protection remain unresolved.

We previously demonstrated (10, 14) that EGF and TGF-alpha protect the integrity of Caco-2 monolayers through the stabilization and remodeling of the microtubule cytoskeleton. We also showed a critical role for microtubule integrity in the maintenance of intestinal barrier integrity under in vitro (10, 13, 14) as well as in vivo (7, 8) conditions. This stabilizing effect appears to be a plausible mechanism for protection by growth factors because an intact microtubule cytoskeleton is required for the maintenance of cellular integrity, structure and architecture, and transport functions (9, 10, 11, 14, 17, 49, 72). Despite the critical importance of the microtubule cytoskeleton in intestinal barrier integrity, the intracellular signaling mechanisms through which EGF stabilizes the microtubules and intestinal barrier integrity remain elusive.

It has been proposed that Ca2+ is also important in maintaining mucosal barrier integrity and that high intracellular levels of this cation play a major role in promoting injury by various noxious agents including oxidants (11, 42, 75, 82, 83). Other studies using nonintestinal cellular models have suggested that protein kinase C (PKC) signaling is a key transduction pathway stimulated by growth factors (6, 18, 60, 68, 88, 92). In view of these considerations, we hypothesized that EGF protects the microtubule cytoskeleton and intestinal epithelial barrier by enhancing PKC signaling that, in turn, normalizes intracellular Ca2+ concentration ([Ca2+]i). The findings reported herein support this hypothesis. Thus the objectives of this study were to explore the interrelationships among PKC signal activation, microtubule integrity, epithelial barrier integrity, calcium homeostasis, and growth factor-mediated protection against oxidant insult under in vitro conditions.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell culture. Caco-2 cells (a human intestinal cell line) were obtained from American Type Culture Collection (Manassas, VA). The utility and characterization of this cell line were described previously (25, 31, 54). Experiments were performed in DMEM with and without fetal bovine serum.

Experimental design. In the first series of experiments, isotonic saline (control) or oxidant (H2O2 or HOCl; 0.1, 0.5, and 5 mM) was incubated with postconfluent monolayers of Caco-2 cells for 30 min. In the second series of experiments, monolayers were pretreated with EGF (human recombinant EGF, 1, 5, and 10 ng/ml) or saline for 10 min before exposure of monolayers to oxidant. All reagents were applied on the apical side of monolayers in DMEM unless otherwise indicated. We measured the effects of various agents alone or in combination on Caco-2 barrier integrity, microtubule stability, PKC signal, and calcium homeostasis. The concentrations of oxidants or EGF (10 ng/ml) used have been shown to be effective in our laboratory (10, 12, 14) as well as others (16, 62, 63, 89). EGF and all other chemicals were purchased from Sigma Chemical (St. Louis, MO).

To further investigate the potential importance of the PKC signaling pathway in growth factor-mediated protection, in a third series of experiments monolayers were preincubated (10 min) with either a PKC activator or an inhibitor and then incubated with EGF before exposure to oxidant. PKC activators included a synthetic diacylglycerol (1-oleoyl-2-acetyl-sn-glycerol, OAG; 1, 50, and 100 µM) or a phorbol ester (12-O-tetradecanoylphorbol 13-acetate, TPA; 1, 30, and 60 nM) or its inactive analog (4alpha -phorbol 12,13-didecanoate, 4alpha -PDD; 20 nM) (11). PKC inhibitors included chelerythrine (1 µM) or bisindolylmalemide V (GF-109203X; 10 nM) or its inactive analog iGF-109203X. Controls were treated with vehicle (0.01% DMSO or 0.2% ethanol). We confirmed that these doses of PKC inhibitors were not toxic to cells.

In the fourth series of experiments, we investigated the role of alterations in [Ca2+]i on growth factor-mediated protection. Additional outcomes examined were [Ca2+]i and Ca2+ efflux. Monolayers were preloaded with the appropriate Ca2+ probe (fluo 3-AM or 45Ca2+), then preincubated for 10 min with EGF, OAG, or TPA, and finally exposed to oxidant for 30 min. Where indicated, monolayers were preincubated with either a membrane-bound Ca2+-ATPase pump inhibitor (vanadate or quercetine; 10 µM, 30 min) or a PKC inhibitor (as above) before the EGF, OAG, or TPA. Vehicle solution was 0.01% DMSO or 0.2% ethanol.

In the fifth series of experiments, we investigated the effects on monolayers of perturbations in extracellular and intracellular Ca2+. Caco-2 monolayers were preincubated (for 15 min unless otherwise indicated) with one of the following: 1) a Ca2+ ionophore (A-23187, 10 µM), 2) an antagonist of voltage-operated Ca2+ channels (VOCC; verapamil or nifedipine, 1 µM), 3) an antagonist of store-operated Ca2+ channels (SOCC; La3+, 25 µM), 4) an extracellular Ca2+ chelator (EGTA, 1 mM added immediately before the subsequent treatment), or 5) an intracellular Ca2+ chelator [1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA-AM); 20 µM, 30 min]. Experiments were performed in Ca2+-containing media (Hanks' balanced salt solution and HEPES; 1.8 mM), Ca2+-free media, or saline vehicle. Doses of each agent were previously shown to be effective under similar in vitro conditions (11, 42, 75, 79, 82, 83). Where indicated, cultures were then incubated with EGF and oxidant. Using similar protocols, EGF was replaced by OAG or TPA. Control cells were treated with vehicle.

In a sixth series of experiments, we isolated the monomeric and polymerized fractions of tubulin (structural protein of the microtubules), which was then analyzed using quantitative immunoblotting.

Immunofluorescent staining and high-resolution laser confocal microscopy of microtubules. Cells of monolayers were fixed, processed, and incubated with the primary antibody (monoclonal mouse anti-beta -tubulin) and then the secondary antibody (FITC-conjugated goat anti-mouse) as described previously (9, 17). After staining, single cells or clumps of two to three cells were observed with an argon laser (lambda  = 488 nm, NA 1.4; Zeiss). The cytoskeletal elements were examined in a blinded fashion for their overall morphology, orientation, and disruption as previously described (9-14).

Microtubule (tubulin) fractionation and quantitative immunoblotting of tubulin. Polymerized (S2) and monomeric (S1) fractions of tubulin were isolated and subjected to PAGE as previously described (9, 10, 14). To quantify the relative levels of tubulin, the optical density of the bands corresponding to immunoradiolabeled tubulin were measured with a laser densitometer.

Determination of barrier integrity. Permeability of monolayers was determined using a fluorescent marker, fluorescein sulfonic acid (FSA; 200 µg/ml, 478 Da), as described previously (10, 12, 84). After treatments, fluorescent signals from samples were quantitated by a fluorescence multiplate reader (excitation 485 nm, emission 530 nm; FL 600, Bio-Tek Instruments).

Measurement of PKC signal activity. PKC activity of the cytosolic and membrane fractions was assayed as described previously (11). Briefly, the cytosolic and detergent-solubilized (membrane) PKC fractions were collected and then processed for their ability to phosphorylate a synthetic peptide as described previously by others (56) and by us (11). Sample activity was corrected for protein concentration by the Bradford method (19), and PKC activity was expressed as picomoles per minute per milligram of protein.

Western immunoblotting of PKC isoforms. Membrane and soluble cell fractions were prepared as described in Measurement of PKC signal activity. After treatments, the distribution of PKC isoforms to membrane-associated fractions was assessed by immunoblotting and autoradiography (88). The PKC isoform-specific antibodies used for immunoblotting were as follows: mouse monoclonal anti-PKC-beta I, -PKC-beta II, -PKC-gamma , -PKC-delta , -PKC-epsilon , and -PKC-zeta (Santa Cruz Biotechnology, Santa Cruz, CA) at 0.2 µg/ml and mouse monoclonal anti-PKC-alpha (UBI, Lake Placid, NY) at 0.1 µg/ml (58). A horseradish peroxidase-conjugated goat anti-mouse antibody (1:3,000 dilution; Molecular Probes, Eugene, OR) was used as the secondary antibody.

Measurement of [Ca2+]i. Alterations in [Ca2+]i were determined using the selective fluorescence Ca2+ indicator fluo 3-AM (Molecular Probes) as described previously (42, 85). Briefly, monolayers were incubated with fluo 3 for 60 min (final concentration of 4 µM), and the continuous fluorescent signals from samples were then quantitated by a fluorescence multiplate reader (FL 600, Bio-Tek Instruments) at 37°C, using excitation and emission wavelengths of 485 and 530 nm, respectively.

Measurement of 45Ca2+ efflux. Caco-2 cells were preloaded with 45Ca2+ (10 µCi/ml) for 1 h at 37°C and then incubated with the test agents. After centrifugation, radioactivity in the supernatant and in the suspension of lysed cells was determined by scintillation counting as described previously (11, 42).

Statistical analysis. Data are presented as means ± SE. Statistical analysis was performed using analysis of variance followed by Dunnett's multiple-range test (34). All experiments were carried out with a sample size of at least four to six observations per group. P values < 0.05 were deemed statistically significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Protection by EGF and PKC activators against oxidant-induced damage to intestinal monolayers. Preincubation of Caco-2 monolayers with EGF or PKC activators (OAG or TPA) before H2O2 dose-dependently and significantly attenuated both barrier hyperpermeability (increases in FSA clearance; Table 1 and Fig. 1) and microtubule cytoskeletal disruption (decreases in % normal microtubules; Table 1). The highest dose of EGF (10 ng/ml) provided complete (99%) protection. PKC activators provided slightly less (90%) protection. OAG protection (of permeability and of microtubules) was not significantly different from EGF protection. Thereafter, we used 10 ng/ml of EGF, 50 µM OAG, or 30 nM TPA. A biologically inactive phorbol ester (4alpha -PDD) did not protect (Fig. 1). Figure 1 also shows that preincubation with PKC inhibitors (chelerythrine or GF-109203X), but not an inactive analog (iGF-109203X), prevented the protective effects of EGF or PKC activators. Table 2 shows analogous effects for protection measured by percentage of cells showing a normal microtubule cytoskeleton. PKC activators and EGF had similar protective effects against monolayer barrier dysfunction caused by another oxidant, HOCl (Table 3).

                              
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  		Table 1.   Effect of protective agents on Caco-2 monolayer barrier integrity, microtubule cytoskeleton, and PKC activity



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  		Fig. 1.   Protective effect of agents that stimulate protein kinase C (PKC) signal activity on the epithelial (Caco-2 cell) barrier integrity in H2O2-exposed monolayers assessed by fluorescein sulfonic acid (FSA) clearance. Growth factor [epidermal growth factor (EGF), 10 ng/ml] or PKC activators [1-oleoyl-2-acetyl-sn-glycerol (OAG), 50 µM or 12-O-tetradecanoyl phorbol 13-acetate (TPA), 30 nM] were added to the monolayers 10 min before exposure to oxidant (H2O2, 5 mM). In select experiments, monolayers were pretreated with either a PKC inhibitor [chelerythrine (1 µM), GF-109203X (10 nM), or the inactive analog iGF-109203X] or a biologically inactive phorbol ester [4alpha -phorbol 12,13-didecanoate (4alpha -PDD), 20 nM]. Barrier integrity was calculated as apical to basolateral flux of FSA divided by the concentration of probe in the apical chamber, expressed as clearance. *P < 0.05 vs. vehicle (control); dagger P < 0.05 vs. H2O2; +P < 0.05 vs. EGF (or OAG or TPA) + H2O2; &P < 0.05 vs. corresponding GF-109203X + EGF (or OAG or TPA) + H2O2. n = 4-6/group in all experiments shown in Figs. 1-10.


                              
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  		Table 2.   Effects of activators or inhibitors of PKC on microtubule cytoskeletal integrity in Caco-2 monolayers


                              
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  		Table 3.   Effects of activators or inhibitors of PKC on HOCl-induced intestinal cell monolayer barrier disruption

We also assessed microtubule integrity by high-resolution laser scanning confocal microscopy. Control cells from untreated monolayers showed a normal and stellar distribution of the microtubule cytoskeleton originating from the microtubule organizer center (MTOC or perinuclear region) and radiating throughout the cytosol (Fig. 2A, a). Exposure of monolayers to H2O2 produced extensive fragmentation, disorganization, and collapse of the microtubule cytoskeleton (Fig. 2A, b). Preincubation with EGF prevented the disruption of microtubules (Fig. 2A, c). Figure 2, B and C, shows that PKC activators OAG (Fig. 2B, a) and TPA (Fig. 2C, a) had similar protective effects. Preincubation with PKC inhibitor, but not inactive iGF-109203X (Fig. 2, B, c and C, c, respectively), abolished protection of microtubules by OAG (Fig. 2B, b), TPA (Fig. 2C, b), or EGF (Table 2).


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  		Fig. 2.   The intracellular distribution of the microtubule cytoskeleton as shown by immunofluorescent staining in intestinal cells from monolayers. A: cell monolayers were treated with isotonic saline (control, a), 5 mM H2O2 (b), or EGF (10 ng/ml) and then H2O2 (c). B: cells were pretreated, before exposure to H2O2, with OAG (50 µM, a), PKC inhibitor GF-109203X and then OAG (b), or inactive analog iGF-109203X and then OAG (c). C: cells were preincubated, before H2O2, with TPA (30 nM, a), PKC inhibitor GF-109203X and TPA (b), or iGF-109203X and TPA (c). Microtubules in control cells (A, a) appear as a intact filamentous network that courses radially throughout the cytosol. In cells exposed to 5 mM H2O2 (without pretreatment), the microtubules appear disrupted, collapsed, and fragmented (A, b). In cells pretreated with EGF (A, c), normal microtubule architecture is highly preserved. Similarly, microtubule morphology in OAG (B, a)- or TPA (C, a)-pretreated cells appears intact and resembles the architecture detected in the iGF-109203X-preincubated group (B, c and C, c, respectively). In cells from monolayers pretreated with GF-109203X + OAG (B, b) or GF-109203X + TPA (C, b) before exposure to oxidant, a clear collapse of the microtubule cytoskeleton can be seen. Bar, 25 µm.

Effects of EGF on PKC activity and on intracellular translocation of PKC isoforms to membrane fractions. EGF or the PKC activators OAG or TPA dose-dependently increased PKC activity as shown in Table 1. Nonprotective doses did not significantly increase PKC activity. Figure 3 shows that pretreatment with the PKC inhibitors chelerythrine and GF-109203X (but not iGF-109203X) significantly inhibited the ability of EGF or the PKC activators to stimulate PKC activity associated with the membrane-bound fraction. The combination of OAG (or TPA) and growth factor elicited no additional effects on PKC activity compared with those evoked by individual agents alone, suggesting that both agents work through the same signal pathway.


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  		Fig. 3.   PKC activity of the membrane-bound fractions from Caco-2 monolayers after the treatments indicated. Monolayers were preincubated with EGF (10 ng/ml), OAG (50 µM), TPA (30 nM), 4alpha -PDD (20 nM), or vehicle before oxidant insult (5 mM H2O2). Where indicated, monolayers were pretreated with PKC inhibitors (chelerythrine or GF-109203X or inactive analog iGF-109203X) before protective agents. Data are means ± SE. *P < 0.05 vs. vehicle; dagger P < 0.05 vs. H2O2; +P < 0.05 vs. EGF (or OAG) + H2O2; &P < 0.05 vs. GF-109203X + EGF (or OAG or TPA) + H2O2.

Relative levels of PKC activity in both the cytosolic and membrane-bound fractions of Caco-2 monolayers are shown in Table 4. These data demonstrate that EGF-, OAG-, or TPA-induced PKC activation is caused by the soluble to membrane-bound translocation and/or shift of PKC. PKC inhibitors did not significantly affect the distribution (translocation) of PKC in the cytosolic and membrane fractions (Table 5), suggesting that the mechanisms of action of these inhibitors involve potentially more direct actions on PKC itself, such as inhibition of the catalytic domain of PKC enzymes (Fig. 3).

                              
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  		Table 4.   Relative PKC activity levels


                              
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  		Table 5.   The effect of PKC inhibitors and calcium regulating agents on PKC translocation in Caco-2 cell monolayers

To determine the PKC isoforms that are involved in protection, we assessed their intracellular distribution and their shift to the membrane-associated fractions when activated by treatments. Figure 4 shows that EGF promoted the distribution of PKC-alpha , PKC-beta I, and PKC-zeta isoforms into membrane fractions as shown by increases in the band density of respective isoforms. OAG and TPA caused distribution of PKC-alpha and PKC-beta I into membranes. Simultaneously, cytosolic cell fractions showed a decrease in these isoforms (not shown). These data on EGF-induced PKC isoform translocation to the membranes are consistent with the data shown earlier on PKC activity shift from the cytosolic to the membrane fractions.


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  		Fig. 4.   The effect of EGF and PKC activators on PKC isoform translocation to the membrane fractions of Caco-2 cells. Cells were preincubated with EGF (10 ng/ml) or control (vehicle) for 10 min. EGF increased the distribution of PKC-alpha (top, apparent mol mass ~80 kDa), PKC-beta I (middle, ~78 kDa), and PKC-zeta (bottom, ~72 kDa) to the membrane fractions indicating their activation. In select experiments, OAG (50 µM) or TPA (30 nM) replaced EGF. Membrane extracts (75 µg protein/lane) were analyzed by SDS-PAGE and Western blots using monoclonal anti-PKC isoform-specific primary antibody followed by horseradish peroxidase-conjugated secondary antibody. The region of each gel shown was between the Mr 67,000 and 93,000 prestained molecular weights run in adjacent lanes.

Role of Ca2+ homeostasis in EGF protection of microtubules and barrier integrity. We next evaluated the effects on barrier integrity by agents known to modulate Ca2+ homeostasis (Fig. 5). Switching to a high-Ca2+ (10 mM) medium significantly exaggerated H2O2-induced barrier dysfunction. In contrast, incubation of monolayers in Ca2+-free or low-Ca2+ (100 µM) medium or with an agent that chelates extracellular Ca2+ (EGTA) markedly prevented oxidant damage. It is to be noted that for periods up to 1.5 h barrier function was restored by the removal of Ca2+, but beyond 2 h low-Ca2+ medium disrupted barrier function (not shown). Moreover, known blockers (verapamil, nifedipine) of VOCC substantially attenuated H2O2-induced barrier dysfunction. Furthermore, preincubation of monolayers with an intracellular Ca2+ chelator (BAPTA-AM) or with a SOCC antagonist (La3+) significantly prevented loss of barrier integrity. Finally, a Ca2+ ionophore (A-23187) not only disrupted monolayer barrier integrity by itself but also exaggerated the effects of H2O2 on barrier dysfunction. Table 5 shows that these known calcium-modifying agents had no significant affect on PKC translocation.


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  		Fig. 5.   Alterations in epithelial monolayer barrier integrity as determined by FSA clearance after the addition of modulators that affect Ca2+ homeostasis. Caco-2 monolayers were exposed to oxidant (5 mM H2O2) or Ca2+ ionophore (A-23187) in 1.8 mM Ca2+-containing media. Where indicated, monolayers were preincubated, before exposure to oxidant, with an extracellular Ca2+ chelator (EGTA), an intracellular Ca2+ chelator (BAPTA-AM), a voltage-operated Ca2+ channel antagonist (VOCC; verapamil and nifedipine), or a store-operated Ca2+ channel blocker (SOCC; lanthanum). In other experiments, Ca2+-free, low-Ca2+ (100 µM) or high-Ca2+ (10 mM) media were used before oxidant. Clearance was determined as described in Fig. 1. *P < 0.05 vs. vehicle; dagger P < 0.05 vs. H2O2.

We then evaluated the role of Ca2+ homeostasis in growth factor-induced protection against oxidant damage. Figures 6 and 7 show what happened when we preincubated monolayers with EGF, OAG, or TPA, with or without a Ca2+ ionophore or inhibitors of membrane Ca2+ pumps. The Ca2+ ionophore and each inhibitor of the membrane-bound Ca2+-ATPase pump (quercetine, vanadate) prevented protection by growth factor (Fig. 6A) and by OAG or TPA (Fig. 6B) against hyperpermeability and against microtubule instability (Fig. 7) in monolayers exposed to oxidant. Unlike Ca2+ ionophore (A-23187), Ca2+-ATPase inhibitors by themselves had no injurious effects on the microtubules or barrier integrity.


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  		Fig. 6.   Alterations of the protective effects of EGF (A) or PKC activators (B) by either Ca2+ ionophore (A-23187) or inhibitors of membrane-bound Ca2+-ATPase (quercetine and vanadate). Monolayers were preincubated with A-23187 or Ca2+-ATPase inhibitor in combination with EGF (10 ng/ml) or PKC activators (OAG, 50 µM; TPA, 30 nM), and then exposed to oxidant (5 mM H2O2). Where shown, monolayers were incubated with other calcium-modifying agents as indicated in Fig. 5. FSA clearance was determined as described in Fig. 1. *P < 0.05 vs. vehicle; dagger P < 0.05 vs. H2O2; +P < 0.05 vs. corresponding EGF (or OAG or TPA) + H2O2.



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  		Fig. 7.   Percentage of Caco-2 cells of monolayers exhibiting normal microtubule architecture as assessed by high-resolution laser confocal microscopy. Conditions as in Figs. 5 and 6. *P < 0.05 vs. vehicle; dagger P < 0.05 vs. H2O2; +P < 0.05 vs. EGF (or OAG or TPA) + H2O2; &P < 0.05 vs. H2O2 (high or low calcium) or H2O2 + EGTA (or BAPTA-AM, verapamil, or lanthanum).

Manipulation of Ca2+ homeostasis by the same agents that prevented loss of barrier integrity (Fig. 5) also prevented disruption of microtubule cytoskeleton as quantitated by the percentage of cells with normal microtubules assessed by laser confocal microscopy (Fig. 7). For instance, pretreatment of monolayers with a VOCC antagonist (nifedipine) significantly prevented microtubule instability: 74 ± 3% of cells showed normal microtubules vs. 25 ± 4% in H2O2-exposed monolayers.

To investigate the underlying cause of microtubule stability and/or instability, we performed quantitative Western immunoblotting of the polymerized tubulin pool (S2 fraction, index of stability) and monomeric tubulin pool (S1 fraction, index of disassembly) in response to various treatment regimes. Figure 8 shows that similar to H2O2, A-23187 caused a significant reduction in the S2 stable polymerized tubulin and an increase in the S1 monomeric tubulin, indicating depolymerization of the microtubules. High-Ca2+ (10 mM) medium, which had exacerbated loss of microtubule stability and barrier dysfunction (Figs. 6 and 7), also significantly exaggerated H2O2-induced microtubule disassembly by increasing tubulin depolymerization. In contrast, removal of Ca2+ (low-Ca2+ or Ca2+-free media, chelation of extracellular Ca2+ by EGTA or of intracellular Ca2+ by BAPTA-AM) maintained tubulin assembly, indicating microtubule stability. In additional experiments, VOCC blockers (verapamil, nifedipine) or a SOCC antagonist (La3+) also significantly prevented H2O2-induced microtubule depolymerization, as shown by enhancement of tubulin assembly. For instance, percent tubulin polymerization for monolayers pretreated with nifedipine was 60 ± 0.5% vs. 35 ± 0.4% for H2O2 alone and 66 ± 0.25% for vehicle.


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  		Fig. 8.   Quantitative immunoblotting analysis of the distribution of polymerized tubulin (S2, index of stability) and monomeric tubulin (S1, index of disassembly) in Caco-2 monolayers (A and B). Conditions were identical to those in Figs. 5-7. Percent polymerization of tubulin pool = [S2 divided by total tubulin pool (S2 + S1)]. *P < 0.05 vs. vehicle; dagger P < 0.05 vs. H2O2; +P < 0.05 vs. EGF (or OAG or TPA) + H2O2; &P < 0.05 vs. H2O2 (high or low calcium) or H2O2 + EGTA (or BAPTA-AM, verapamil, or lanthanum).

Furthermore, a Ca2+ ionophore and inhibitors of the membrane-bound Ca2+-ATPase pump (quercetine and vanadate) each prevented EGF (Fig. 8A)- and OAG or TPA (Fig. 8B)-induced tubulin assembly in monolayers exposed to oxidant, paralleling similar findings on both microtubules (Fig. 7) and monolayer barrier integrity (Fig. 6). In contrast, pretreatment of monolayers with the inactive 4alpha -PDD did not prevent tubulin disassembly in H2O2-exposed monolayers (37 ± 1% vs. 35 ± 0.4% for H2O2). Moreover, in monolayers incubated with H2O2, preincubation with a PKC inhibitor, GF-109203X, abolished the increase in tubulin assembly by EGF (31 ± 0.60%), OAG (34 ± 0.30%), or TPA (36 ± 0.45%). As expected, the inactive analog iGF-109203X was ineffective when preadministered before EGF (65 ± 0.40%), OAG (62 ± 0.34%), or TPA (61 ± 0.53%) and H2O2 insult.

Intracellular calcium concentrations under disruptive and protective conditions. To further evaluate the role of Ca2+ in our Caco-2 cell model, we used the Ca2+-sensitive dye fluo 3 (Fig. 9A) to monitor [Ca2+]i. Monolayers exposed to H2O2 (5 mM shown) exhibited a significant and rapid increase in [Ca2+]i within 2 min (peak = 455 ± 11 nM vs. 124 ± 10 nM for controls) followed by a gradual decrease. [Ca2+]i remained significantly elevated at 30 min (304 ± 15 nM). H2O2 at 0.1 (peak [Ca2+]i = 171 ± 10 nM) and 0.5 (225 ± 8 nM) mM resulted in a significant but less marked rise in [Ca2+]i than 5 mM H2O2. Monolayers treated with a protective dose of EGF (10 ng/ml) alone also had a rapid initial rise in [Ca2+]i, which returned to normal by 10 min. Monolayers pretreated with EGF (10 ng/ml) and subsequently exposed to H2O2 had a similar early transient rise in [Ca2+]i extending to 2 min, but unlike H2O2 alone, this was followed by a rapid and significant decline in [Ca2+]i (136 ± 12 nM) that was similar to control levels (127 ± 9 nM). A nonprotective dose of EGF (1 ng/ml; EGF alone = 140 ± 15 nM) in combination with H2O2 (EGF + H2O2 = 312 ± 18 nM at 30 min) did not normalize [Ca2+]i. Effects on [Ca2+]i statistically similar to those of EGF were observed when monolayers were pretreated with the PKC activators OAG and TPA (Fig. 9B). As expected, inactive phorbol ester 4alpha -PDD did not normalize [Ca2+]i against H2O2 insult (442 ± 16 nM at 10 min and 296 ± 19 nM at 30 min).


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  		Fig. 9.   Intracellular Ca2+ alterations as determined by the Ca2+-sensitive fluorescent probe fluo 3-AM. Cells preloaded with fluo 3-AM were preincubated with a Ca2+-ATPase inhibitor (vanadate or quercetine) or a PKC inhibitor (chelerythrine and/or GF-109203X or inactive analog iGF-109203X) and then exposed to H2O2 (5 mM) in the presence or absence of pretreatment with EGF (A) or OAG or TPA (B). *P < 0.05 vs. vehicle (control); dagger P < 0.05 vs. H2O2; +P < 0.05 vs. EGF (or OAG or TPA) + H2O2.

Preincubation of monolayers (Fig. 9, A and B) with membrane Ca2+-ATPase pump inhibitors (quercetine or vanadate) or PKC inhibitors (chelerythrine and/or GF-109203X) abrogated EGF (Fig. 9A)- and OAG or TPA (Fig. 9B)-induced normalization of [Ca2+]i. [Ca2+]i levels remained elevated during the entire 30-min observation period. Interestingly, neither PKC inhibitors nor Ca2+-ATPase inhibitors by themselves had any significant effects on baseline calcium levels.

Calcium efflux under disruptive and protective conditions. Because Ca2+-ATPase blockers prevented EGF-induced protection and maintained [Ca2+]i at high levels, we surmised that Ca2+ efflux is a key mechanism for growth factor-induced protective effects on the normalization of [Ca2+]i and the maintenance of both microtubules and barrier function. Indeed, direct measurement of Ca2+ efflux from monolayers prelabeled with 45Ca2+ (Fig. 10; data shown for 10-min observation period) showed that protective doses of EGF (Fig. 10A) and PKC activators (Fig. 10B) markedly and significantly increased Ca2+ efflux. Inhibitors of the membrane Ca2+-ATPase pump (quercetine, vanadate) or inhibitors of PKC, at doses that prevent EGF protection, abolished this increase in efflux. Also, H2O2 modestly but significantly reduced Ca2+ efflux, which may partly explain the increase in [Ca2+]i induced by this agent.


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  		Fig. 10.   Effect on 45Ca2+ efflux from Caco-2 monolayers by EGF (A), PKC activators (OAG or TPA or inactive 4 alpha -PDD) (B), PKC inhibitors (chelerythrine and/or GF-109203 X or the iGF-109203 X), or Ca2+-ATPase inhibitors (quercetine or vanadate). After preincubation with these agents monolayers were exposed to oxidant (5 mM H2O2). *P < 0.05 vs. vehicle. dagger P < 0.05 vs. H2O2. +P < 0.05 vs. EGF (or OAG or TPA) + H2O2.

Finally, we did not observe any significant Ca2+ efflux in monolayers pretreated with the nonprotective doses of 1 ng/ml EGF (49 ± 4%), 10 µM OAG (51 ± 3%), or 1 nM TPA (48 ± 2%) compared with vehicle (48 ± 3%), paralleling the lack of effects of these doses on FSA clearance (barrier function), microtubule integrity, PKC activity, and [Ca2+]i.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Investigating the mechanisms by which growth factors protect intestinal cells and the intestinal permeability barrier against oxidant-induced damage, as the current study has done, is clinically important because free radical damage results in a "leaky gut" that is thought to be one of the underlying mechanisms in IBD (14, 36, 41). We focused on mechanisms involving the microtubule cytoskeleton for two reasons: 1) because it is well established that maintaining an intact microtubule cytoskeleton is essential for the integrity of barrier permeability as well as normal cellular function and structure of enterocytes (8-10, 13, 14, 31), and 2) because our recent studies (10, 14) found that maintaining an intact microtubule cytoskeleton is necessary for protection by growth factors. On the basis of the results from the current study, we conclude that the ability of EGF to prevent oxidant-induced microtubule disruption and barrier leakiness in our model is mediated by enhanced PKC activity and normalization of Ca2+ homeostasis. To our knowledge, this is the first time this mechanism has been ascribed to the defense and repair of GI epithelial cells.

We felt particularly confident in drawing this conclusion because it was supported by several independent lines of evidence. First, pretreatment of intestinal monolayers with EGF, which prevents oxidant-induced hyperpermeability, simultaneously increases PKC activity and evokes a cascade of changes that are consistent with the proposed mechanism. EGF activates specific PKC isoforms, increases the proportion of cells showing a normal microtubule architecture, increases the size of the polymerized tubulin pool while decreasing the size of the monomeric tubulin pool, normalizes cytosolic Ca2+, and increases Ca2+ efflux.

Second, these effects of EGF are selectively mimicked by PKC activators (OAG and TPA) or agents known to affect Ca2+ homeostasis. Third, the protective effects of EGF or PKC activators are attenuated by specific PKC inhibitors. Fourth, agents that are known to dysregulate Ca2+ homeostasis worsen oxidant-induced cytoskeletal or barrier disruption or induce disruption themselves. Fifth, our previous work (10, 14) shows a significant correlation between barrier disruption and microtubule disruption, and the current study shows additional significant correlations: protection against barrier disruption and PKC activity (r = 0.89, P < 0.05), protection against barrier disruption and [Ca2+]i (r = 0.95, P < 0.05), protection against barrier disruption and Ca2+ efflux (r = 0.98, P < 0.05), protection against microtubule disruption and PKC activity (r = 0.90, P < 0.05), protection against microtubule disruption and [Ca2+]i (r = 0.97, P < 0.05), protection against microtubule disruption and Ca2+ efflux (r = 0.96, P < 0.05), and increase in PKC and in Ca2+ efflux (r = 0.88, P < 0.05).

Looking further into the mechanisms of EGF protection, our study suggests that not all PKC isoforms and not all Ca2+ pathways are equally important. Our studies on redistribution of PKC to membrane fractions suggest that alpha -, beta I-, and zeta -isoforms of PKC are activated by EGF. Consistent with these findings, it is known that PKC profoundly affects cellular functions in nonintestinal cell types (3-5, 22, 26, 28, 32, 35, 47, 48, 50, 71, 74, 78, 88, 94), and several studies show that growth factors (e.g., EGF) activate PKC in several cell types including the epithelium (6, 18, 88, 92). For example, EGF caused translocation of both PKC-alpha and PKC-beta I into membranes in canine gastric cells (88) and caused PKC-alpha membrane association in mammary epithelial cultures (18). Moreover, PKC has been suggested to be a mediator of EGF-induced alterations in the actin component of the cytoskeleton in HeLa and corneal endothelial cells (22, 39) and of EGF-mediated inhibition of canine parietal cell function (88).

In our studies, OAG and TPA only induced activation of classic PKC-alpha and PKC-beta I. This finding is not surprising because studies in non-GI models (i.e., fibroblasts) showed that these PKC activators can only induce activation of classic PKC isoforms (33). Our current studies using an acute model of PKC activation are also consistent with previous reports in which acute administration of a low dose of a PKC activator (TPA) caused rapid activation of PKC in parietal cells and subsequently inhibited cell secretions via PKC-mediated processes (18). Also, within 5-15 min after exposure to TPA (0.1 µM), there were rapid PKC-mediated effects on the enhancement of cell migration concomitant with rapid reorganization of the actin component of the cytoskeleton in corneal endothelial cells (39). In other non-GI cells (e.g., fibroblasts), after 15 min in TPA, PKC redistributed to cell membranes and was associated with rapid cytoskeletal remodeling (33, 86, 93). In further support of our findings are recent studies proposing that diacylglycerol apparently modulates intestinal epithelial (Caco-2) permeability (5, 48). The effects of PKC activation in cellular models are complex and, we acknowledge, vary with different experimental settings and cell types (5, 26, 48). In our studies, PKC inhibitors did not affect PKC translocation (Table 5). Our PKC activity measurements (Fig. 3), however, show that these inhibitors prevent PKC activation and that the inhibitory effects of PKC inhibitors in our studies were probably caused by their direct inhibition of the catalytic domain of PKC as suggested by others (6, 68, 78, 92).

[Ca2+]i in eukaryotic cells is exquisitely balanced, and derangement of [Ca2+]i homeostasis can lead to the disruption of many cell functions including the cell's cytoskeletal network (37, 40, 76). Our calcium experiments, which used a wide variety of known calcium-modifying agents, were all consistent with the idea that decreases in [Ca2+]i and enhanced efflux of Ca2+ are particularly important in EGF protection. This conclusion is supported by several findings. EGF enhanced Ca2+ efflux and normalized [Ca2+]i. Protection by EGF was significantly blunted by Ca2+ ionophore and membrane Ca2+ pump inhibitors. Additionally, manipulations that lowered extracellular or intracellular Ca2+ (e.g., EGTA, BAPTA-AM, low Ca2+, or VOCC and SOCC Ca2+ channel antagonists) protected against oxidant-induced disruption. Maneuvers that raised extracellular or intracellular Ca2+ levels (e.g., high-Ca2+ media) had the opposite effect, potentiating oxidant-induced cytoskeletal injury and barrier dysfunction. It is to be noted that the effects of these known Ca2+-modifying agents did not involve alterations in PKC translocation in our model because these agents did not significantly change PKC distribution (Table 5). Furthermore, EGF prevented the sustained rise in [Ca2+]i that is induced by oxidants concomitantly with maintaining intact microtubules, tubulin assembly, and barrier integrity.

Although the precise mechanism through which changes in Ca2+ homeostasis mediate protection by EGF is not established, our findings suggest that EGF elicits its protective effects by PKC-induced changes in [Ca2+]i. EGF or PKC activators abolished the prolonged elevation of [Ca2+]i that was induced by oxidants and normalized [Ca2+]i; these effects were abrogated by PKC inhibitors. Our study also suggests that maintaining Ca2+ homeostasis through Ca2+ efflux is an important mechanism through which EGF protects. The fact that membrane Ca2+-ATPase inhibitors, Ca2+ ionophores, and PKC inhibitors prevented EGF protection supports the proposed mechanism. Our proposed mechanism is consistent with previous studies in non-GI cells in which phorbol 12-myristate 13-acetate (a PKC activator) enhanced Ca2+ efflux via a proposed PKC-dependent extrusion mechanism in human T lymphocytes, osteoblasts, and neuronal cells (3, 4, 90, 94). Another study showed that TPA reduced the amplitude of Ca2+ influx in human lung epithelial cells, suggesting stimulation of Ca2+ efflux (46).

Several findings suggest that the proposed mechanism for EGF protection is generalizable. First, in the present study, we observed similar effects when HOCl was the oxidant rather than H2O2. This is consistent with reports (41) that both oxidants are known to be elaborated by activated neutrophils at sites of inflammation and that both appear to be important in IBD. Second, in previous studies (10, 12, 14) we reported similar protective effects for TGF-alpha , a structurally similar growth factor elaborated by intestinal mucosa that appears to act through the same EGF receptor. Indeed, other studies have shown that it is not possible to convincingly disassociate the biological activities of EGF and TGF-alpha in any cell population, including GI epithelium (32, 59, 77). It is therefore reasonable to assume that most biological effects of TGF-alpha will also be produced by EGF. Regarding protection of intestinal tissue, the only major difference between EGF and TGF-alpha appears to be their site of origin. Whereas TGF-alpha is elaborated directly by the intestinal epithelium (32), EGF found in the intestine is normally synthesized elsewhere. For instance, several previous studies showed that salivary EGF as well as EGF contained within secretions of Brunner's glands and exocrine pancreas are the major source of intestinal EGF and that they play a key role in protection of small and large bowel (15, 59, 61, 65). For example, EGF prevented damage to the intestinal epithelium in an animal model of colitis (IBD), similar to our oxidant model of IBD in vitro (15, 61). EGF also prevented Clostridium difficile toxin-induced epithelial damage to human colon in vitro (70). Although the relative contributions of these two growth factors to in vivo defense and repair of the intestinal mucosal barrier remain to be fully established, our study already suggests that targeting this protective mechanism in new drug development studies may be worthwhile, either by enhancing the signaling pathways of endogenous growth factors or by introducing exogenous agents that are EGF and/or TGF-alpha mimetics. Another key step will be to establish that this putative mechanism for intestinal barrier protection by growth factors is operative in vivo in animals and humans, an idea that has already received some support (2, 15, 30, 44, 51-53, 55, 61, 64, 66, 67, 70, 80, 91). For instance, Wright et al. (91) demonstrated the existence of EGF-like immunoreactivity in a novel cell lineage derived from intestinal stem cells in the injured intestinal mucosa in the instances of IBD and peptic ulcer disease. Overall, the preponderance of evidence suggests that our EGF model is a relevant model for studying the intracellular mechanism of intestinal protection.

An apparent limitation of our conclusion that PKC signal activation is a major mediator of EGF-induced protection is that protection of barrier integrity against H2O2 insult by PKC agonists (OAG, TPA) was slightly less (92% and 85%, respectively) compared with EGF (98%). However, not all PKC isoforms were activated by OAG and TPA as they were by EGF. Thus it appears that the greater protection by EGF is due to its ability to activate additional PKC isoforms. An alternative interpretation is that as much as 10-15% of protection may be due to non-PKC pathways. A similar pattern was found for protection against HOCl. Also, although it is reasonable to state that increases in PKC and decreases in [Ca2+]i are necessary components of the protective mechanism for EGF, they may not be entirely sufficient; other molecular mechanisms may also be involved. Some studies (20, 24, 27, 29) have suggested that protection by growth factors may involve stimulation of ornithine decarboxylase (ODC) and subsequent synthesis of essential polyamine compounds. For instance, EGF enhances intestinal repair against ethanol injury, and this effect is mediated, in part, by ODC because an irreversible inhibitor of this enzyme, alpha -difluoromethyl ornithine, significantly prevented protection (20). Moreover, EGF can modulate the expression of the rate-limiting enzyme ODC, which is a key step in the biosynthesis of polyamines (24, 29, 51). Specifically, EGF not only enhances the intestinal mucosal expression of ODC but also increases the levels of another polyamine regulatory enzyme, diamine oxidase, in Caco-2 cells. A recent series of studies, including our own (7, 8), showed that polyamines contribute, at least in part, to the repair of GI injury (51, 87). We also showed (7, 8) that polyamines are important for cytoskeletal reorganization and healing under in vivo conditions. Thus the ODC-polyamine system could provide an additional component to the mechanism through which EGF protects the intestinal mucosa. Future studies will be needed to determine whether there are interactions between PKC signal enhancement and normalization of Ca2+ homeostasis by EGF and EGF-induced changes in the ODC-polyamine system.

In summary, our studies demonstrate that EGF protects the intestinal monolayer barrier against oxidant damage by preventing damage and disruption of the microtubule cytoskeleton, and the current study shows that activation of PKC signaling and normalization of intracellular Ca2+ appear to be key mechanisms. Although the total protective mechanism for growth factors remains to be established, on the basis of the current report and our previous work (9-14) there is already a rationale for considering development of certain new drug targets for IBD. In particular, inhibitors (e.g., selective antioxidants) could be developed that target those reactions that have been shown by our studies to be required for oxidative-induced damage. Alternatively, agents could be developed that enhance or mimic the protective effects of growth factors against oxidant-induced insult such as occurs under inflammatory conditions.


    ACKNOWLEDGEMENTS

This work was supported in part by a grant from Rush University Medical Center.


    FOOTNOTES

Portions of this work were presented at Research Forum of the annual meeting of the American Gastroenterological Association in San Diego, CA, 2000 (10a).

Address for reprint requests and other correspondence: A. Banan, Rush Univ. Medical Center, Division of Digestive Diseases, 1725 W. Harrison, Suite 206, Chicago, IL 60612 (E-mail:ali_banan{at}rush.edu).

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.

Received 6 June 2000; accepted in final form 21 November 2000.


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ABSTRACT
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MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Gastrointest Liver Physiol 280(5):G828-G843
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