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J. Biol. Chem., Vol. 275, Issue 28, 21169-21176, July 14, 2000

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A Role for Protein Kinase C in the Inhibitory Effect of Epidermal Growth Factor on Calcium-stimulated Chloride Secretion in Human Colonic Epithelial Cells^*

Jimmy Y. C. Chow, Jorge M. Uribe, and Kim E. Barrett^§

From the Department of Medicine, University of California, San Diego, School of Medicine, San Diego, California 92103

Received for publication, March 15, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Epidermal growth factor (EGF) inhibits carbachol-induced chloride secretion in T₈₄ colonic epithelial cells and has been shown to activate phosphatidylinositol (PI) 3-kinase, leading to inhibition of a basolateral potassium conductance. We asked whether the inhibitory effect of EGF on secretion is due to activation of specific isoforms of protein kinase C (PKC) by PI 3-kinase. Western analysis revealed that PKC, , , , µ, /, and  were expressed in T₈₄ cells. Ro318220 (an inhibitor active against PKC, 10 µM) but not Gö6983 (an inhibitor active against PKC, 10 µM) reversed the inhibitory effect of EGF (100 ng/ml) on carbachol-stimulated chloride secretion. EGF induced the rapid translocation of PKC from the cytoplasm to the membrane. Wortmannin (50 µM) and LY294002 (20 nM), which are PI 3-kinase inhibitors that by themselves had no effect on PKC activity, significantly suppressed PKC translocation activated by EGF. LY294002 also reversed the inhibitory action of EGF on chloride secretion. PI (3,4)P₂ increased membrane-associated PKC and reduced carbachol-induced ⁸⁶Rb⁺ efflux. Antisense oligonucleotides against PKC decreased PKC mass and prevented the inhibitory effect of EGF on carbachol-induced ⁸⁶Rb⁺ efflux. Thus, the inhibitory effect of EGF on carbachol-induced chloride secretion involves the activation of PKC mediated by PI 3-kinase. Our findings contribute to the understanding of the cellular mechanisms that control chloride secretion.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Chloride secretion plays an important role in regulating water transport across epithelia in various organs (1, 2). Either oversecretion or undersecretion of chloride across epithelia can result in significant pathophysiological events, such as secretory diarrhea or cystic fibrosis, respectively. Therefore, efforts have been made to study chloride secretion at the cellular and subcellular levels to understand the underlying mechanisms involved.

The ion transport pathways comprising the chloride secretory mechanism of T₈₄ cells, a line of human colonic epithelial cells with a crypt cell phenotype, have been well defined (3, 4). Chloride is taken up across the basolateral membrane of the cells via a Na⁺/K⁺/2Cl cotransporter (NKCC1) and exits the cell across the apical membrane via chloride channels. At least some of these chloride channels are apparently identical to the cystic fibrosis transmembrane conductance regulator. Basolateral potassium channels allow for potassium recycling, whereas energy for the process is supplied by the activity of a basolateral Na⁺,K⁺-ATPase. It has been predicted that primary control for the overall transport process occurs at the level of apical chloride channels and basolateral potassium channels, in response to agonists that elevate levels of the positive second messengers for chloride secretion, cyclic nucleotides, or cytosolic calcium. In addition to positive regulation of this process, we have shown that signaling mechanisms intrinsic to the epithelium can also inhibit secretion. Thus, the muscarinic agonist, carbachol (CCh),¹ can initially activate secretion in a calcium-dependent fashion and then render cells refractory to additional stimulation. Further, growth factors such as epidermal growth factor (EGF) significantly inhibit secretion induced by calcium-dependent agonists, including CCh, without themselves serving as positive effectors of the transport mechanism.

The inhibitory pathway utilized by EGF also diverges from that activated by CCh. The CCh-evoked pathway is dependent on an increase in the messenger inositol (3,4,5,6)tetrakisphosphate, whereas that induced by EGF is not (5, 6). Rather, the inhibitory effect of EGF appears to be due to its ability to stimulate phosphatidylinositol (PI) 3-kinase with the production of 3-phosphorylated lipids (7). However, the downstream mediators involved, if any, were unknown. Efflux studies also indicated that EGF reduced calcium-stimulated basolateral ⁸⁶Rb⁺ but not apical ¹²⁵I efflux, suggesting that an effect of the growth factor on the basolateral potassium channel constitutes the target of the PI 3-kinase-dependent negative signaling pathway. Activation of protein kinase C, by pretreatment with 100 nM phorbol 12-myristate 13-acetate (PMA) also inhibits CCh-induced chloride secretion in T₈₄ epithelial cells. CCh has been shown to induce a transient increase in potassium conductance, which could be inhibited by PMA (8). It is possible that one or several isoforms of PKC could mediate the divergent inhibitory effect of EGF.

Pertinent to this possibility, a screening study showed that calcium-independent PKC isoforms were activated by the lipid products of PI 3-kinase (9). Nakanishi et al. (10) showed that an atypical PKC isoform, PKC, was activated by the PI 3-kinase product phosphatidylinositol 3,4,5-trisphosphate. Furthermore, platelet-derived growth factor stimulated membrane translocation of the novel PKC isoform, PKC, in HepG2 cells, which was attributable to the ability of platelet-derived growth factor to activate PI 3-kinase (11). Therefore, the activity of PI 3-kinase may be important for the regulation of some PKC isoforms, especially those of the novel and atypical families. In the current study, our purpose was to examine whether the inhibitory effect of EGF on CCh-induced chloride secretion is due to the activation of novel and/or atypical isoforms of PKC. We also wanted to determine whether any activation of PKC is a consequence of PI 3-kinase activation.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- The following materials were obtained from the sources indicated: antisense and sense oligonucleotides for PKC, and serum- and antibiotic-free culture medium OPTI-MEM from Life Technologies, Inc.; Ro318220 and Gö3796 from Calbiochem (San Diego, CA); EGF from Genzyme (Cambridge, MA); Dulbecco's modified Eagle's medium/Ham's F-12 medium from JRH (Lenexa, KS); monoclonal antibodies directed against the PKC isoforms , , , , , µ, /, and  and positive controls (Jurkat, human fibroblast, and macrophage cell extracts) and goat anti-mouse horseradish peroxidase secondary antibody from Transduction Laboratories (Lexington, KY); Hybond ECL nitrocellulose membrane, Kodak x-ray films and ECL Plus detection kit from Amersham Pharmacia Biotech; L--phosphatidylinositol 3,4-bisphosphate from Alexis (San Diego, CA); wortmannin, LY294002, and L--phosphatidylinositol 3,4,5-trisphosphate from Biomol (Plymouth Meeting, PA); polyvinylidene difluoride membrane (PVDF) and cell culture membrane inserts (Millicell, 0.45-µm pore size mixed cellulose ester) from Millipore (Bedford, MA); ⁸⁶RbCl from NEN Life Science Products; Geneporter Transfection Reagent from Gene Therapy Systems (San Diego, CA); Me₂SO, Tween-20 (EIA grade), dithiothreitol, glycine, Tris, SDS, glycerol, bromphenol blue, sterile deionized water, leupeptin, phenylmethylsulfonyl fluoride, NaVO₄, and CCh from Sigma; blotting grade nonfat dry milk from Upstate Biotechnology (Lake Placid, NY); D_c protein assay kit, 7.5% polyacrylamide gels, and molecular mass markers from Bio-Rad; phosphatidylserine from Avanti Polar Lipids (Alabaster, AL); trypsin from Irvine Scientific (Santa Ana, CA); and newborn calf serum from Hyclone (Logan, UT). All other chemicals were of at least reagent grade and were obtained commercially.

Cell Culture-- Methods for the maintenance of T₈₄ cells for use in transepithelial electrolyte transport studies have been described previously (3). In brief, T₈₄ cells were grown in Dulbecco's modified Eagle's medium/Ham's F-12 medium with 5% newborn calf serum. For the measurement of chloride secretion and agonist-induced ⁸⁶Rb⁺ efflux, 2.5 × 10⁵ cells were seeded onto 12-mm cell culture membrane inserts. For experiments involving Western blotting, 10⁶ cells were seeded onto 30-mm inserts. Cells were cultured for 7-10 days to develop confluent monolayers prior to use. For experiments using oligonucleotides in the efflux assay, 2.5 × 10⁵ cells were plated per well into 24-well plates; these were used 3 days after plating when they were subconfluent.

Chloride Secretion-- Chloride secretion was measured as short circuit current (I_sc) across monolayers of T₈₄ cells, mounted in Ussing chambers (window area = 0.6 cm²) modified for use with cultured cells (3). I_sc (normalized to µA/cm²) was used to quantitate both basal transepithelial chloride secretion and that induced by calcium-dependent secretagogues. T₈₄ cells secrete chloride in response to various agonists, and the resulting changes in I_sc are wholly reflective of chloride secretion (12). I_sc measurements were carried out in Ringer's solution containing 140 mM Na⁺, 5.2 mM K⁺, 1.2 mM Ca²⁺, 0.8 mM Mg²⁺, 119.8 mM Cl, 25 mM HCO₃, 2.4 mM H₂PO₄, 0.4 mM HPO₄², and 10 mM glucose.

Treatment of Cells with Oligonucleotides-- Phosphorothioate oligonucleotides specific for PKC were purchased from Life Technologies, Inc. and used to treat cells as described by Liedtke and Cole (13) with slight modifications. Briefly, antisense oligonucleotides complementary to the translation initiation region of mRNA specific for human PKC, i.e. 5'-GGCTGGTACCATCACAAG-3', were used, whereas the sense oligonucleotide, 5'-CCGACCATGGTAGTGTTC-3', was taken as a control. Oligonucleotides were dissolved in sterile deionized water to a final concentration of 1 mM, aliquoted, and stored at 20 °C until ready for use. Oligonucleotides (6 µg), transfection reagent (21 µl), and OPTI-MEM (1 ml) were mixed and added to wells of confluent cell monolayers according to the manufacturer's instructions. The oligonucleotide incubation medium was replaced every 12 h for 48 h.

Treatment of T₈₄ Cells with Phospholipids-- Phosphatidylinositol 3,4-bisphosphate (PI (3,4)P₂) was first dissolved in Me₂SO at room temperature and then aliquoted at different concentrations in prewarmed Ringer's solution (37 °C). The final concentration of Me₂SO used as a vehicle was 0.01% (v/v). Phosphatidylinositol 3,4,5-trisphosphate (PI (3,4,5)P₃) was dissolved in chloroform containing 10 µM of carrier lipid (phosphatidylserine), blown dry with nitrogen, and then aliquoted accordingly in Ringer's solution. Both lipid preparations were rapidly sonicated for 15 min immediately prior to their application to cell monolayers and then incubated for 30 min.

Western Blotting-- T₈₄ cells were washed three times with Ringer's solution and allowed to equilibrate for 30 min at 37 °C. Cells were then stimulated as noted. The reaction was stopped by 3 washes with ice-cold phosphate-buffered saline. Hypotonic lysis buffer (4 °C, containing 1 mM NaVO₄, 1 µg/ml leupeptin, and 100 µg/ml phenylmethylsulfonyl fluoride, which was freshly added prior to use) was then added. The cells were incubated with gentle rocking at 4 °C for 30 min and then scraped from the filter supports on ice and further lysed by passage (five times) through a 27G needle. The lysate was then fractionated into soluble and particulate fractions in lysis buffer by centrifugation as described by Liedtke et al. (14). The supernatant was used as the cytosolic fraction, whereas the pellet was resuspended by vigorous vortex mixing in the same lysis buffer and then used as the membrane fraction. For experiments examining PKC isoforms in unfractionated cells, ice-cold lysis buffer was then added (consisting of phosphate-buffered saline, 1% Nonidet P-40, 1 mM NaVO₄, 1 µg/ml leupeptin, and 100 µg/ml phenylmethylsulfonyl fluoride), and the cells were incubated at 4 °C for 30 min. The cells were then scraped into microcentrifuge tubes, and the samples were centrifuged at 7200 × g for 10 min to remove insoluble material. The protein content in each sample was determined and adjusted. All samples were then resuspended in 2× gel loading buffer (50 mM Tris, pH 6.8, 2% SDS, 100 mM dithiothreitol, 0.2% bromphenol blue, 20% glycerol), boiled for 5 min, and then loaded onto a polyacrylamide gel for separation. Resolved proteins were transferred overnight at 4 °C onto a PVDF membrane. The membrane was then blocked with a 1% solution of skim milk in water for 1 h at room temperature, followed by further incubation with monoclonal antibodies to specific PKC isoforms (1:1000). After washing with Tris-buffered saline with 1% Tween (TBST), the anti-mouse horseradish peroxidase secondary antibody was applied to the membrane. After washing with TBST, the membrane was treated with a chemiluminescent solution according to manufacturer's instructions and exposed to x-ray film. Densitometric analysis of the blots was performed using a digital imaging system.

Measurement of Potassium Channel Opening-- A ⁸⁶Rb⁺ efflux technique was used to monitor the opening of basolateral potassium channels in response to different stimuli, as reported by Venglarik et al. (15) with modifications. For the PI (3,4)P₂ study, cells grown on permeable inserts were rinsed with warm Hanks' balanced salt solution (HBSS) containing 137.6 mM Na⁺, 146.3 mM Cl, 5.8 mM K⁺, 0.44 mM H₂PO₄, 0.34 mM HPO₄², 1 mM Ca²⁺, 1 mM Mg²⁺, 15 mM HEPES (pH 7.2), and 10 mM D-glucose. The cells were loaded for 30 min with ⁸⁶Rb⁺ (1 µCi/ml, added basolaterally) at 37 °C. Simultaneously, PI (3,4)P₂ (0, 10, 50 and 80 µM) was added on both sides. Cells were then subjected to three gentle rinses with HBSS to remove extracellular isotope. After the final rinse, fresh HBSS (300 µl) was added in individual wells of a cell culture plate. The buffer was maintained at 37 °C by placing the culture plates on a thermostatic heating block. The first three aliquots were used to establish a stable base line in efflux buffer only. The buffer was sequentially transferred to scintillation vial at 2-min intervals for the ⁸⁶Rb⁺ efflux assay. At various times, as indicated by the experimental design, the buffer was switched to a solution containing CCh. CCh was then continuously present for the remainder of the assay. At the end of the experiment, the inserts were immersed in scintillation fluid. For the antisense oligonucleotide study, cells were plated on 24-well plates instead of on inserts (13). Similar procedures were followed except that the radioactive counts remaining in the cells were extracted with 0.1 M nitric acid for 30 min at the end of the experiment. All samples were then assessed for their content of ⁸⁶Rb⁺ using open channel readings from a liquid scintillation counter (Beckman LS3180). The fraction of intracellular ⁸⁶Rb⁺ remaining in the cell layer during each time point was calculated from the sample and extract counts. Time-dependent rates of ⁸⁶Rb⁺ efflux were calculated as ln(⁸⁶Rb_t=1⁺/⁸⁶Rb_t=2⁺)/(t₁  t₂), where ⁸⁶Rb⁺ is the percentage of intracellular Rb⁺ at time t, and t₁ and t₂ are successive time points.

Data Analysis-- All data are expressed as the means for a series of n experiments ± S.E. Data were analyzed by one-way analysis of variance (ANOVA) followed by Student-Newman-Keul's post-hoc test or by Student's t tests for unpaired samples using GraphPad Prism 2.0 (San Diego, CA). p < 0.05 was considered statistically significant.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Expression of PKC Isoforms in T₈₄ Cells-- More than 11 isoforms of PKC have been identified (16). To further our understanding of the functions of different isoforms of PKC in T₈₄ cells, a screening study was performed using Western analysis of the isoforms expressed in whole cell extracts. T₈₄ cells contained proteins immunoreactive with antibodies to PKC (82 kDa) and  (80 kDa) in the conventional family, PKC (90 kDa), µ (115 kDa), and  (82 kDa) in the novel family and PKC/ (74 kDa/74 kDa) and  (72 kDa) in the atypical family (Fig. 1). PKC and  were not detected. On the basis of these data and prior findings that suggested that novel and atypical isoforms of PKC are downstream targets of PI 3-kinase (9), we selected PKC and PKC for further analysis, as representative examples of novel and atypical isoforms.

View larger version (12K): [in this window] [in a new window]   Fig. 1.   Western analysis of conventional (cPKC), atypical (aPKC), and novel (nPKC) PKC isoforms in T84 cells. Cell lysates were subjected to electrophoresis through 7.5% SDS-polyacrylamide gels and transferred to PVDF membranes. Membranes were probed with monoclonal antibodies specific for the PKC isoforms (, , , , , µ, , , , and /). Only those isoforms present in T84 cells (T) as assessed by molecular mass standards and positive control preparations run in parallel (rabbit brain (B) and HeLa cells (H)) are shown. PKC isoforms were detected through the use of an enhanced chemiluminescent methodology as described under "Experimental Procedures." The diamonds denote the predicted molecular masses of the different PKCs. These Western blots are representative of three similar experiments.

Effect of Ro318220 and Gö6983 on the Ability of EGF to Inhibit CCh-induced Chloride Secretion in T₈₄ Cells-- EGF is able to suppress the stimulatory effect of CCh on chloride secretion (7). To test whether PKC is involved in this effect of EGF, we used two PKC inhibitors, Ro318220 and Gö6983, which have been shown to inhibit differentially novel and atypical PKCs, namely  and  isoforms, respectively. The reported IC₅₀ of Gö6983 for PKC is 60 nM (17) and that of Ro318220 for PKC is 24-48 nM (18, 19), depending on the cell type studied. As shown in Fig. 2A, basolateral addition of EGF (100 ng/ml) significantly suppressed CCh-induced chloride secretion, as expected (5). Addition of Gö6983 (10 µM) alone to both basolateral and apical sides of the T₈₄ monolayers 15 min prior to CCh (100 µM) did not have an independent effect on chloride secretion. Gö6983 also failed to reverse the inhibitory effect of EGF on CCh-induced chloride secretion. In contrast, although it had no significant effect on CCh-induced chloride secretion alone, addition of Ro318220 significantly reversed the inhibitory effect of EGF on CCh-stimulated chloride secretion (Fig. 2B). These data suggest that PKC (or other isoforms sensitive to Gö6983) is unlikely to mediate the inhibitory effect of EGF on chloride secretion. Conversely, PKC, or other isoforms sensitive to Ro318220, was a candidate to mediate the inhibitory effect of EGF on chloride secretion. We therefore examined the role of PKC in more detail.

View larger version (22K): [in this window] [in a new window]   Fig. 2.   Effects of Gö6983 (A) and Ro318220 (B) on the inhibitory effect of EGF on CCh-induced chloride secretion in T84 cells. Monolayers on inserts were pretreated bilaterally with either Gö3968 (10 µM, A) or Ro318220 (10 µM, B) for 15 min prior to the basolateral addition of EGF (100 ng/ml). This was followed 15 min later by basolateral addition of CCh (100 µM). Control monolayers received CCh alone, CCh plus EGF, or CCh plus the relevant PKC inhibitor. Data are presented as the peak increases in Isc (Isc) induced by CCh. Data are the means ± S.E. for five experiments in the Gö6983 study and 7-13 experiments in the Ro318220 study. The asterisks denote responses that differ significantly from those induced by CCh alone. p < 0.05 by ANOVA with Student-Newman-Keul's post-hoc test.

Effect of EGF on PKC Activation in T₈₄ Cells-- Because one of the isoforms of PKC sensitive to Ro318220, PKC, has previously been shown to lie downstream of PI 3-kinase in other systems, we moved on to study whether EGF has any effect on the activation of PKC in T₈₄ cells. T₈₄ cell monolayers were incubated with EGF (100 ng/ml) for various times. EGF could activate PKC in the cells as early as 30 s after addition, as measured by translocation of the enzyme to the membrane fraction (Fig. 3). PKC translocation was maximal within 15 min of EGF addition and maintained for at least 1 h. PMA, included in these experiments as a positive control, also induced a marked translocation of PKC (Fig. 3). We concluded from these data that EGF likely causes activation of PKC in T₈₄ cells, with kinetics that correspond to those of the ability of the growth factor to inhibit chloride secretion (7).

View larger version (24K): [in this window] [in a new window]   Fig. 3.   Time course of PKC translocation in T84 cells after EGF (100 ng/ml) addition. T84 cell monolayers on inserts were stimulated for various times as shown with EGF. A monolayer stimulated with PMA (1 µM) served as a positive control. The cells were then lysed and fractionated into membrane and cytosol fractions. The presence of PKC in these fractions was determined as described under "Experimental Procedures." The data are from a single experiment representative of six similar experiments.

Effects of Wortmannin and LY294002 on EGF-induced PKC Activation and CCh-stimulated Chloride Secretion in T₈₄ Cells-- Our previous work has shown that PI 3-kinase is involved in the inhibitory action of EGF on CCh-induced chloride secretion. To determine whether PKC is a downstream effector of PI 3-kinase, two inhibitors of PI 3-kinase, wortmannin and LY294002, were used. Wortmannin has been shown previously to reverse the inhibitory effect of EGF on calcium-induced chloride secretion in T₈₄ cells (7). Here, we found that wortmannin (100 nM) also suppressed EGF-induced translocation of PKC, although a lower concentration of wortmannin (50 nM) had no effect (Fig. 4). LY294002, a more specific PI 3-kinase inhibitor, which acts through a mechanism distinct from that of wortmannin (20, 21), also significantly reduced the translocation of PKC induced by EGF (Fig. 5). Interestingly, the effects of LY294002 was most pronounced at the lowest concentration tested (20 µM), and 100 µM LY294002 was less effective at inhibiting the effect of EGF on PKC translocation. In sum, these data further confirm our hypothesis that PKC is a downstream effector of PI 3-kinase in T₈₄ cells stimulated with EGF. Moreover, LY294002 (20 µM) also reversed the inhibitory effect of EGF on chloride secretion, at a dose that blocked PKC translocation induced by EGF (Fig. 6).

View larger version (28K): [in this window] [in a new window]   Fig. 4.   Effect of wortmannin (Wort) on the ability of EGF to cause PKC translocation in T84 cells. Monolayers on inserts were pretreated bilaterally with wortmannin (50 or 100 nM) for 20 min prior to the addition of EGF to the basolateral side for 15 min. Cells were then lysed, and the membrane fraction was subjected to electrophoresis, transferred to a PVDF membrane, and subsequently probed with a monoclonal antibody against PKC. The data were quantitated by image analysis, are expressed as arbitrary units, and are the means ± S.E. for 5-8 experiments. *, significantly different from no addition, p < 0.05; ++, significantly different from EGF alone, p < 0.01, by ANOVA with Student-Newman-Keul's post-hoc test.

View larger version (29K): [in this window] [in a new window]   Fig. 5.   Effect of LY294002 on the ability of EGF to cause PKC translocation in T84 cells. Monolayers on inserts were pretreated bilaterally with LY294002 (20, 40, or 100 µM) for 20 min prior to the addition of EGF (100 ng/ml) to the basolateral side for 15 min. Cells were then lysed, and the membrane fractions were subjected to electrophoresis, transferred onto a PVDF membrane, and subsequently probed with monoclonal antibody against PKC. The data were quantitated by image analysis and are the means ± S.E. for six experiments. *, significantly different from no addition, p < 0.05; +, p < 0.05; ++, p < 0.01, significantly different from EGF alone by ANOVA with Student-Newman-Keul's post-hoc test.

View larger version (37K): [in this window] [in a new window]   Fig. 6.   Effect of LY294002 on the inhibitory action of EGF on CCh-induced chloride secretion in T84 cells. Monolayers on inserts were mounted in Ussing chambers and pretreated bilaterally with LY294002 (20 µM) for 15 min prior to the basolateral addition of EGF (100 ng/ml). This was followed 15 min later by basolateral addition of CCh (100 µM). Controls received CCh alone, CCh plus EGF, or CCh plus LY294002. The data are the means ± S.E. for four experiments and are expressed as the peak increases in Isc (Isc) induced by CCh addition. Asterisks denote responses that differ significantly from those induced by CCh alone. *, p < 0.05; ***, p < 0.001. Plus signs denote responses that differ significantly from those induced by CCh plus EGF. ++, p < 0.01 by ANOVA with Student-Newman-Keul's post-hoc test.

Effects of PI (3,4)P₂ and PI (3,4,5)P₃ on PKC Activation and CCh-stimulated ⁸⁶Rb⁺ Efflux in T₈₄ Cells-- The products of PI 3-kinase activity include the 3-phosphorylated lipids PI (3,4)P₂ and PI (3,4,5)P₃. It has been shown previously that a cell-permeable form of PI (3,4,5)P₃ is able to suppress CCh-induced chloride secretion (22). Furthermore, treatment of T₈₄ cells with EGF led to a large, rapid, and sustained elevation in the levels of PI (3,4)P₂ and PI (3,4,5)P₃ (7). However, the mechanism whereby these lipids might inhibit chloride secretion was not known. We hypothesized this could be due to their ability to activate PKC in T₈₄ cells. To test this, we preincubated T₈₄ cells with either of these lipids at different concentrations for 30 min. Fig. 7 showed that PI (3,4)P₂ induced the activation of PKC in a dose-dependent manner. The same doses of PI (3,4)P₂ also significantly suppressed CCh-induced ⁸⁶Rb⁺ efflux (Fig. 8). However, PI (3,4,5)P₃ did not seem to have any effect on the activity of PKC at concentrations up to 80 µM (data not shown). Nevertheless, the data with PI (3,4)P₂, at least, are consistent with the hypothesis that translocation of PKC via PI 3-kinase activation can be linked to the inhibition of a basolateral potassium channel.

View larger version (51K): [in this window] [in a new window]   Fig. 7.   Effect of PI (3,4)P2 on PKC translocation in T84 cells. Monolayers on inserts were pretreated bilaterally with PI (3,4)P2 (10, 30, 50, and 80 µM) for 30 min. Cells were then lysed, and the membrane fractions were subjected to electrophoresis, transferred onto a PVDF membrane, and subsequently probed with monoclonal antibody against PKC. A depicts a single representative experiment. In B, the data were quantitated by image analysis and are the means ± S.E. for three experiments. *, significantly different from 0 µM, p < 0.05 by ANOVA with Student-Newman-Keul's post-hoc test.

View larger version (33K): [in this window] [in a new window]   Fig. 8.   Effect of PI (3,4)P2 on CCh-induced 86Rb+ efflux in T84 cells. Monolayers on inserts were pretreated bilaterally with PI (3,4)P2 (10, 50, and 80 µM) and basolaterally with 86RbCl (1 µCi/ml) for 30 min. The data are the means ± S.E. for 11 experiments and are expressed as the peak increases in the rate of 86Rb+ efflux induced by CCh (100 µM) addition. Asterisks denote responses that differ significantly from those induced by CCh alone. *, p < 0.05; **, p < 0.01, by ANOVA with Student-Newman-Keul's post-hoc test.

Effects of Antisense Oligonucleotides on PKC Levels and the Inhibitory Effect of EGF on CCh-stimulated ⁸⁶Rb⁺ Efflux in T₈₄ Cells-- Using a pharmacological approach (Ro318220 and Gö6983), it was inferred that PKC participates in the ability of EGF to inhibit CCh-induced chloride secretion in T₈₄ cells. To verify this conclusion, antisense oligonucleotides against PKC were used. Cells were cultured in the presence of antisense oligonucleotides to PKC for 48 h. Optimal inactivation of PKC in T₈₄ cells was obtained with antisense oligonucleotide at a concentration of 6 µg/ml, resulting in a reduction in total PKC mass of 59% (Fig. 9A). This was also accompanied by a reversal of the inhibitory effect of EGF on CCh-induced ⁸⁶Rb⁺ efflux (Fig. 10). The sense oligonucleotide, on the other hand, did not significantly reduce the amount of PKC in T₈₄ cells (Fig. 9B) nor reverse the effect of EGF on CCh-stimulated ⁸⁶Rb⁺ efflux (Fig. 10). In control experiments, the transfection reagent used did not adversely affect CCh-induced ⁸⁶Rb⁺ efflux in T₈₄ cells (efflux rate, 0.088 ± 0.005 versus 0.085 ± 0.008 min¹, n = 3, not significant by Student's t test). Overall, these results indicate that PKC mediates the effect of EGF on potassium channel opening.

View larger version (30K): [in this window] [in a new window]   Fig. 9.   Effects of antisense oligonucleotide (A) and sense oligonucleotide (B) pretreatment on the mass of PKC in lysates of T84 cells. Control subconfluent cells on plastic received Geneporter alone (21 µl), Geneporter plus antisense oligonucleotide (3 or 6 µg/ml, A), or Geneporter plus sense oligonucleotide (3 or 6 µg/ml, B) for 48 h. The cells were then lysed, and the cell lysates were subjected to electrophoresis through a 7.5% SDS-polyacrylamide gel and transferred to a PVDF membrane. Membranes were probed with monoclonal antibodies specific for PKC. PKC was detected through the use of an enhanced chemiluminescent methodology as described under "Experimental Procedures." The upper panels show representative Western blots. The lower panels show total PKC expressed as a percentage of levels in cells treated with Geneporter alone and are the means ± S.E. for three experiments. Asterisks denote a value that differs significantly than cells treated with Geneporter alone; p < 0.01 by ANOVA with Student-Newman-Keul's post-hoc test.

View larger version (36K): [in this window] [in a new window]   Fig. 10.   Effects of sense and antisense PKC oligonucleotide pretreatment on the inhibitory effect of EGF on CCh-induced 86Rb+ efflux in T84 cells. Subconfluent cells on plastic were pretreated with Geneporter alone (21 µl), Geneporter plus sense oligonucleotide (6 µg/ml), and Geneporter plus antisense oligonucleotide (6 µg/ml) for 48 h. The cells were incubated with 86RbCl (1 µCi/ml) for 30 min prior to assay. The cells were then treated with CCh (100 µM) with or without pretreatment with EGF (100 ng/ml) as shown in the figure. The data are the means ± S.E. for 17 experiments and are expressed as the peak increases in 86Rb+ efflux induced by CCh (100 µM) addition. Asterisks denote responses that differ significantly from those induced by CCh alone. ***, significantly different from CCh alone, p < 0.001; ++, significantly different from values in cells with antisense pretreatment, p < 0.01 by ANOVA with Student-Newman-Keul's post-hoc test.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Although the mechanisms responsible for initiating and maintaining chloride secretory responses in colonic epithelial cells have been well worked out, the mechanisms for inhibiting or terminating such responses are relatively unexplored. It has been predicted that primary control for the overall transport process rests at the level of apical chloride channels and basolateral potassium channels. Therefore, it is important to understand the regulators of signaling pathways that impinge on these ion channels.

EGF inhibits calcium-activated chloride secretion evoked by a variety of agonists, including CCh and histamine (23). The inhibitory effect of EGF on CCh-induced chloride secretion appears to be due to its ability to activate PI 3-kinase to produce 3-phosphorylated lipids (7). Furthermore, EGF reduced Ca²⁺-stimulated ⁸⁶Rb⁺ but not ¹²⁵I efflux, indicating its influence on basolateral potassium channels (24). Previous work had shown that activation of PKC by PMA also inhibited CCh-induced chloride secretion in T₈₄ cells, that CCh induces a transient increase in basolateral potassium conductance, and that pretreatment with PMA inhibited this potassium conductance (8). The possibility of a link between PKC activation and the regulation of a potassium conductance under the influence of PI 3-kinase was thus raised.

PI 3-kinase has been shown in other systems to activate novel and atypical PKCs (9). Incubation of HepG2 cells with platelet-derived growth factor led to the translocation of PKC via the activation of PI 3-kinase (20). PKC was also activated by phosphatidylinositol 3,4,5-trisphosphate (10). Thus, PI 3-kinase may be an important regulator of novel and atypical PKCs. We therefore examined whether EGF can activate novel and/or atypical isoforms of PKC in T₈₄ cells, and if so, whether the inhibitory effect of EGF on CCh-induced chloride secretion can be attributed to the activation of either or both of these isoforms of PKC. We also wanted to examine whether activation of PKC is a result of PI 3-kinase activation.

The current study provides the connection between chloride secretion regulation by EGF and activation of PI 3-kinase by implicating a downstream calcium-independent PKC isoform, namely PKC. T₈₄ cells express seven PKC isoforms representative of three major classes of PKC isoforms (Fig. 1). Ro318220, but not Gö6983, completely reversed the inhibitory effect of EGF on CCh-induced chloride secretion (Fig. 2), suggesting that PKC but not PKC is likely to play a pivotal role in the inhibitory regulation of chloride secretion. Induction of PKC translocation by EGF (Fig. 3) further substantiates its role in the inhibition of chloride secretion.

Previous findings showed that the ability of EGF to inhibit CCh-induced chloride secretion was completely reversed by the PI 3-kinase inhibitor, wortmannin (7). Wortmannin is a somewhat nonspecific inhibitor of PI 3-kinase. Indeed PI 4-kinase, myosin light chain kinase, phospholipase A₂, and phospholipase D have all been shown to be inhibited by wortmannin (25-28). To exclude the possibilities of nonspecificity, LY294002, a more specific PI 3-kinase inhibitor, was used in the present study. LY294002 reversed the inhibitory effect of EGF on chloride secretory responses to CCh in the present study (Fig. 6). Furthermore, both wortmannin and LY294002 reduced EGF-promoted PKC translocation in T₈₄ cells (Figs. 4 and 5).

PKC activation has been related to the inhibition of a basolateral potassium channel in T₈₄ cells (8). Furthermore, in other cells platelet-derived growth factor activates PKC secondary to the activation of PI 3-kinase. In fact, Tsien et al. (22) have demonstrated that a chemically modified PI (3,4,5)P₃ (one of the PI 3-kinase products produced in T₈₄ cells in response to EGF (7)), significantly suppressed both CCh-induced chloride secretion in Ussing chambers and CCh-induced ⁸⁶Rb⁺ efflux in T₈₄ cells. Thus, PKC could be a downstream effector of PI 3-kinase. However, PI (3,4,5)P₃ was unexpectedly unable to stimulate translocation of PKC in our study, whereas PI (3,4)P₂ significantly activated PKC (Fig. 7). The latter phospholipid also inhibited CCh-induced ⁸⁶Rb⁺ efflux (Fig. 8). This apparent discrepancy might be attributable to the fact that PI (3,4,5)P₃ was previously shown to activate PKC in a cell-free system (9). In addition, the concentration and the chemical nature of PI (3,4,5)P₃ studied may have contributed to our failure to detect a stimulatory effect of the lipid on PKC. Tsien et al. (22) used a chemically modified PI (3,4,5)P₃ at a concentration of 200 µM to achieve an inhibitory effect on chloride secretion in T₈₄ cells. The unmodified lipid is likely to be poorly permeable, because there are three phosphate groups attached, and the resulting more polar and bulky phospholipid may be unable readily to penetrate the cytoplasmic membrane. Nevertheless, the data with PI (3,4)P₂, at least, suggest that EGF mediates its inhibitory effect on chloride secretion via the activation of PI 3-kinase, which in turn stimulates PKC via its lipid products.

No single pharmacological agent is completely specific for its target. Ro318220 is also a selective inhibitor of calcium-, diterpine-, and phorbol ester-activable PKC isoforms  and  (18). It also inhibits some PKC-related kinases, such as PRK-1 or PKN (1, 21, 22), increases the activity of JNK1, stimulates c-Jun expression in Rat-1 fibroblasts (32), and potentiates the effect of EGF on phospholipase D activity (33). In addition, Ro318220 and Gö6983 were shown to inhibit PKC, , and  (17, 34). Thus, conclusions drawn based on the use of Ro318220 could only be speculative. To further verify the role of PKC, an antisense oligonucleotide approach was employed.

Down-regulation of PKC using antisense oligonucleotide provided convincing evidence for a role of PKC in inhibition of CCh-induced chloride secretion. Treatment of T₈₄ cells with antisense oligonucleotides to PKC for 48 h markedly reduced the mass of PKC (Fig. 9A). Moreover, a major finding of this study is that these antisense oligonucleotides to PKC also potently reversed the inhibitory action of EGF on CCh-induced potassium channel opening (Fig. 10). The corresponding sense oligonucleotides were completely without effect on either parameter (Figs. 9B and 10). These data provide convincing evidence that PKC mediates the inhibitory effect of EGF on chloride secretion.

Chloride secretion induced by CCh in T₈₄ cells could be a consequence of the potassium channel opening on the basolateral side. In fact, CCh induces a transient increase in basolateral potassium conductance, and pretreatment with PMA inhibits the agonist-dependent potassium conductance in T₈₄ cells. Boland and Jackson (35) have also shown that protein kinase C inhibited voltage-gated potassium channel function in Xenopus oocytes. These data, in sum, clearly indicate that PKC may be linked to the regulation of potassium conductances. Moreover, activation of PKC isoforms may have regulatory effects on other electrolyte and nonelectrolyte transporters. Long term PMA treatment reduces cystic fibrosis transmembrane conductance regulator mRNA with a concomitant inhibition of cystic fibrosis transmembrane conductance regulator chloride channel activity and induces a cytosol-to-membrane translocation of PKC in human liver epithelial BC1 cells (36), and PKC specifically regulates the function of cystic fibrosis transmembrane conductance regulator in Calu-3 cells (13). PKC was also found to be necessary for inhibition of vasopressin-stimulated sodium transport in rabbit cortical collecting duct cells (37). Furthermore, it regulates basolateral endocytosis of NKCC1 in T₈₄ cells via effects on F-actin and the cytoskeleton and on a membrane-bound myristoylated alanine-rich protein kinase C substrate (38). Certainly, effects of PKC on endocytosis could account for the ability of EGF to inhibit chloride secretion, by retrieving basolateral transport proteins, including potassium channels, and thereby reducing secretory capacity. K⁺ channel opening was also previously shown to be regulated by the cytoskeleton (39).

In conclusion, the present study demonstrates that PKC modulates the overall chloride secretory response in T₈₄ cells by interacting directly or indirectly with a basolateral K⁺ channel. We also showed that PKC is a downstream effector of PI 3-kinase activated by EGF. PKC plays a central role linking activation of the EGF receptor to the overall regulation of chloride secretion. Although molecular cloning has demonstrated the existence of multiple PKC isoforms, the majority of these isoforms have not yet been well characterized as to their in vivo functions in cell types of the gastrointestinal tract (40, 41). This study, at least, demonstrated the importance and the underlying mechanisms of the PI 3-kinase pathway in the regulation of chloride secretion in colonic epithelial cells. In addition, understanding such mechanisms may lead to an ability to interfere with chloride secretion in patients with diarrhea in a more targeted fashion in the future.

	ACKNOWLEDGEMENT

We are grateful to Glenda Wheeler for assistance with manuscript preparation.

	FOOTNOTES

^* This work was supported by NIDDK, National Institutes of Health Grant DK28305 (to K. E. B.).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.

Recipient of a Predoctoral Fellowship, supported by the Institutional Training Grant in Digestive Diseases DK07202 while a student in the Biomedical Sciences Ph.D. program of UCSD School of Medicine.

^§ Chair of the Biomedical Sciences Ph.D. Program of UCSD School of Medicine. To whom correspondence should be addressed: Univ. of California, San Diego Medical Center, Division of Gastroenterology, 8414, 200 West Arbor Dr., San Diego, CA 92103-8414. Tel.: 619-543-3726; Fax: 619-543-6969; E-mail: kbarrett@ucsd.edu.

Published, JBC Papers in Press, May 4, 2000, DOI 10.1074/jbc.M002160200

	ABBREVIATIONS

The abbreviations used are: CCh, carbachol; EGF, epidermal growth factor; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; PI, phosphatidylinositol; PI (3, 4)P₂, PI 3,4-bisphosphate; PI (3, 4,5)P₃, PI 3,4,5-trisphosphate; PVDF, polyvinylidene difluoride; HBSS, Hanks' balanced salt solution; ANOVA, analysis of variance.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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