Inhibition of apical Clminus /OHminus exchange activity in Caco-2 cells by phorbol esters is mediated by PKCepsilon

Seema Saksena, Ravinder K. Gill, Irfan A. Syed, Sangeeta Tyagi, Waddah A. Alrefai, K. Ramaswamy, and Pradeep K. Dudeja

Section of Digestive and Liver Diseases, Department of Medicine, University of Illinois at Chicago and West Side Department of Veterans Affairs Medical Center, Chicago, Illinois 60612


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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DISCUSSION
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The present studies were undertaken to examine the possible regulation of apical membrane Cl-/OH- exchanger in Caco-2 cells by protein kinase C (PKC). The effect of the phorbol ester phorbol 12-myristate 13-acetate (PMA), an in vitro PKC agonist, on OH- gradient-driven 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS)-sensitive 36Cl uptake in Caco-2 cells was assessed. The results demonstrated that PMA decreased apical Cl-/OH- exchanger activity via phosphatidylinositol 3-kinase (PI3-kinase)-mediated activation of PKCepsilon . The data consistent with these conclusions are as follows: 1) short-term treatment of cells for 1-2 h with PMA (100 nM) significantly decreased Cl-/OH- exchange activity compared with control (4alpha -PMA); 2) pretreatment of cells with specific PKC inhibitors chelerythrine chloride, calphostin C, and GF-109203X completely blocked the inhibition of Cl-/OH- exchange activity by PMA; 3) specific inhibitors for PKCepsilon (Ro-318220) but not PKCalpha (Go-6976) significantly blocked the PMA-mediated inhibition; 4) specific PI3-kinase inhibitors wortmannin and LY-294002 significantly attenuated the inhibitory effect of PMA; and 5) PI3-kinase activators IRS-1 peptide and phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] mimicked the effects of PMA. These findings provide the first evidence for PKCepsilon -mediated inhibition of Cl-/OH- exchange activity in Caco-2 cells and indicate the involvement of the PI3-kinase-mediated pathways in the regulation of Cl- absorption in intestinal epithelial cells.

phosphatidylinositol 3-kinase; protein kinase C epsilon; human intestine; chloride absorption; phorbol 12-myristate 13-acetate


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IN THE MAMMALIAN ILEUM and colon, the major mechanism of Na+ and Cl- uptake has been suggested to be via an electroneutral process involving the operation of the dual ion exchangers Na+/H+ and Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>(OH-) (36). In this regard, recent studies from our laboratory (3) characterized the luminal membrane Cl-/OH-(HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>) exchangers along the length of the human intestine. To date, however, the molecular nature of the intestinal apical membrane Cl-/OH-(HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>) exchanger is not very well established. Recent studies provided some evidence for the involvement of downregulated in adenoma (DRA) (17) and anion exchanger (AE)1 (38) in this transport process. Additionally, there have been very limited studies on the mechanism of regulation of the Cl-/OH-(HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>) exchanger.

Previous functional studies demonstrated that recombinant AE2-mediated Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange in Xenopus oocytes is activated by alkaline pH (23) and hypertonicity (24). Studies have also shown protein kinase C (PKC) to directly regulate the activity of Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchangers in isolated rat hepatocytes (6). Phorbol 12-myristate 13-acetate (PMA) was shown to inhibit the glucagon-induced stimulation of Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger in isolated rat hepatocytes by inhibiting intracellular cAMP accumulation in response to the hormone (4). A role of PKC was also suggested in the regulation of rat colonic electrolyte transport by inhibiting active NaCl absorption and stimulating electrogenic Cl- secretion (13). Studies from our laboratory (1) and by others (14) demonstrated the regulation of Na+/H+ exchanger (NHE) isoforms by various protein kinases. To date, however, almost nothing is known about the regulation of apical anion exchangers by PKC or any other signal cascades in the human intestine.

In this regard, phorbol esters, analogs of diacylglycerol (DAG, a lipid produced during membrane phosphatidylinositol turnover), activate PKC by translocating it from the cytosol to the membrane (34). A number of cellular studies showed phorbol esters to induce a variety of responses including effects on ionic channels, second messenger production, cell-cell communication, membrane transport, protein phosphorylation, cellular growth, morphology, differentiation, and transformation (34, 40, 48). PMA has also been shown to induce phosphatidylinositol 3-kinase (PI3-kinase) activity and to increase the levels of lipid products of PI3-kinase, phosphoinositides, in mouse epidermal JB6 cells (20). PI3-kinase signaling pathway has been suggested to play an important regulatory role in the translocation of insulin-regulatable glucose transporters (11, 48) and NHE (28, 45).

The current studies were undertaken to determine 1) the possible regulation of Cl-/OH- exchange activity in apical membranes of Caco-2 cells, the human colonic adenocarcinoma cell line, by PMA and 2) the role of specific PKC isoform(s) and also the signal transduction pathways involved in this process. Our data demonstrate that PMA decreases the apical Cl-/OH- exchange activity in Caco-2 cells and provide evidence for the involvement of PI3-kinase and PKCepsilon -mediated pathways in the regulation of Cl- transport.


    MATERIALS AND METHODS
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Materials. 4,4'-Diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) was obtained from Sigma (St. Louis, MO). Radionuclide 35S-sulfuric acid and 36Cl were obtained from NEN Life Science Products (Boston, MA). Caco-2 cells were obtained from American Type Culture Collection (ATCC, Manassas, VA). PMA, 4alpha -phorbol 12-myristate 13-acetate (4alpha -PMA, inactive form), chelerythrine chloride, calphostin C, GF-109203X, wortmannin, LY-294002, and IRS-1 (Y608) peptide were obtained from Biomol (Plymouth Meeting, PA). Ro-318220, Go-6796, and phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] were obtained from Calbiochem (San Diego, CA). Affinity-purified rabbit polyclonal antibody against PKCepsilon and goat-anti-rabbit antibody conjugated to horseradish peroxidase (HRP) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). All other chemicals were of at least reagent grade and were obtained from Sigma or Fisher Scientific (Pittsburgh, PA).

Cell culture. Caco-2 cells obtained from ATCC were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 4.5 g/l glucose, 2 mM glutamine, 50 U/ml penicillin, 50 µg/ml streptomycin, 10 mM HEPES, 1% essential and nonessential amino acids, and 20% fetal bovine serum, pH 7.4, in 5% CO2-95% O2 at 37°C. For the uptake experiments, cells from between passages 20 and 25 were plated in 24-well plates at a density of 2 × 104 cells/ml. Confluent monolayers were then used for transport experiments on the 10th to 12th days after plating (i.e., 6-8 days after confluence). To study the effect of phorbol esters 4alpha -PMA and PMA on Cl-/OH- exchange activity, cells were rendered quiescent by serum removal overnight before study. Cells were then acutely exposed to 100 nM PMA for 15, 30, 60, and 120 min. In a separate set of experiments, cells were exposed to 1 µM PMA for 24 h. Control cells were treated with an equivalent amount of 4alpha -PMA (inactive form).

In a separate set of experiments, cells were pretreated with the specific PKC inhibitors chelerythrine chloride (2 µM), calphostin C (200 nM), and GF-109203X (50 nM) or specific PKCalpha isoform inhibitor Go-6976 (5-50 nM), specific PKCepsilon isoform inhibitor Ro-318220 (100 nM), or specific PI3-kinase inhibitors wortmannin (100 nM) and LY-294002 (5 µM) for 1 h before the addition of 4alpha -PMA and PMA (100 nM). These inhibitors were also coincubated along with 4alpha -PMA and PMA for another 1 h. In another set of experiments, cells were treated with PI3-kinase activators IRS-1 peptide (0.1-10 µM) and PI(3,4,5)P3 (10 µM) for 1 h. Before their application to the cell monolayers, IRS-1 peptide was dissolved in H2O and PI(3,4,5)P3 solution in DMSO was rapidly sonicated for ~5-10 min.

35SO<UP><SUB>4</SUB><SUP>2<UP>−</UP></SUP></UP> and 36Cl- uptake. SO<UP><SUB>4</SUB><SUP>2−</SUP></UP> and Cl- uptake experiments were performed essentially as described by Olsnes et al. (35) with some modifications (2). Caco-2 cells were incubated with DMEM base medium containing 20 mM HEPES-KOH, pH 8.5, for 30 min at room temperature. The medium was removed, and the cells were rapidly washed with 1 ml of tracer-free uptake mannitol buffer containing 260 mM mannitol and 20 mM Tris-MES, pH 7.0. The cells were then incubated with the uptake buffer for a 2-min time period. For 35SO<UP><SUB>4</SUB><SUP>2−</SUP></UP> uptake studies, the uptake buffer was the mannitol buffer containing 0.4 µCi/ml of 35SO<UP><SUB>4</SUB><SUP>2−</SUP></UP> of sulfuric acid and 50 µM cold K2SO4. For 36Cl- uptake studies, the uptake buffer was the mannitol buffer containing 1.3 µCi of 36Cl- (2.7 mM) of hydrochloric acid per milliliter. The uptake was terminated by removing the buffer and washing the cells rapidly two times with 1 ml of ice-cold phosphate-buffered saline (PBS), pH 7.2. Finally, the cells were solubilized by incubation with 0.5 N NaOH for 4 h. The protein concentration was measured by the method of Bradford (9), and the radioactivity was measured by Packard Liquid Scintillation Analyzer TRI-CARB 1600-TR (Packard Instrument, Downers Grove, IL). The uptake values are expressed as picomoles per milligram per 2 minutes for SO<UP><SUB>4</SUB><SUP>2−</SUP></UP> and nanomoles per milligram of protein per 2 minutes for Cl-.

Subcellular fractionation. Caco-2 cells grown to confluence in 25-cm2 flasks (Corning Costar) were washed with ice-cold PBS three times and scraped into 400 µl of the cold homogenization buffer (HB) containing (in mM) 20 Tris · HCl, pH 7.5, 250 sucrose, 4 EDTA, and 2 EGTA with complete protease inhibitor cocktail tablets. The cells were homogenized on ice with 25 strokes of a glass tissue homogenizer. The resulting homogenate was ultracentrifuged at 59,000 rpm for 50 min at 4°C (Optima TLX ultracentrifuge; Beckman). The supernatant was designated the cytosolic fraction. The pellet was resuspended in 150 µl of the HB containing 0.5% (vol/vol) Triton X-100 by brief sonication and incubated on ice for 30 min. At the end of the incubation period, the samples were centrifuged at 14,000 rpm for 20 min at 4°C. The resulting supernatant was designated the membrane fraction.

Gel electrophoresis and Western blotting. Equal amounts (~75 µg/sample) of protein, as determined by the Bradford assay, were combined with Laemmli's sample buffer containing 5% (vol/vol) beta -mercaptoethanol and boiled for 5 min. Proteins were separated by electrophoresis on 8% SDS-PAGE gels and transblotted to nitrocellulose membranes. The protein-bound nitrocellulose membranes were first incubated for 1 h at room temperature in blocking buffer containing 1× PBS, 0.1% Tween 20, and 5% nonfat dry milk. Nitrocellulose membranes were then incubated with the polyclonal antibody specific to PKCepsilon (1:800 dilution) in the blocking buffer containing 1× PBS, 0.1% Tween 20, and 1% nonfat dry milk for 1 h at room temperature and rinsed for 30 min with a wash buffer containing 1× PBS and 0.1% Tween 20. Finally, the membranes were incubated with HRP-conjugated goat anti-rabbit IgG antibody (1:2,000 dilution) for 1 h at room temperature and washed for 45 min with agitation, during which the wash buffer was changed every 5 min. PKC bands were visualized with enhanced chemiluminescence (ECL) detection reagents.

Statistical analysis. Results are expressed as means ± SE. Each independent set represents means ± SE of data from at least nine wells used on three separate occasions. Student's t-test was used for statistical analysis. P < 0.05 was considered statistically significant.


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Acute and chronic effects of PMA. To examine the effect of PMA on Cl-/OH- or SO<UP><SUB>4</SUB><SUP>2−</SUP></UP>/OH- exchange activity, Caco-2 cells were serum deprived overnight and then incubated with 100 nM PMA or its inactive form (4alpha -PMA) at different time points and DIDS-sensitive Cl-/OH- and SO<UP><SUB>4</SUB><SUP>2−</SUP></UP>/OH- exchange activities were assessed as described in MATERIALS AND METHODS. As shown in Fig. 1, no change in Cl-/OH- exchange activity was observed over 15- and 30-min time periods, whereas incubation of Caco-2 cells with PMA for 1 h decreased the activity by ~50% compared with control (4alpha -PMA treated) and the inhibition remained about the same even at 2 h. However, similar treatment of Caco-2 cells with PMA (100 nM, 1 h) showed no significant change in SO<UP><SUB>4</SUB><SUP>2−</SUP></UP>/OH- exchange activity (1,626 ± 144 vs. 1,351 ± 87 pmol · mg-1 · 2 min-1 in PMA treated and 4alpha -PMA treated, respectively). Exposure to PMA (1 µM) for 24 h to downregulate PKC also did not alter Cl-/OH- exchange activity (data not shown).


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Fig. 1.   Time course of phorbol 12-myristate 13-acetate (PMA) treatment on OH- gradient-stimulated 36Cl- uptake in Caco-2 cells. Caco-2 cells, 5-7 days after confluence, were serum starved overnight and then treated with 100 nM PMA (filled bars) for 15-, 30-, 60-, and 120-min time periods. Control cells received 100 nM inactive 4alpha -PMA (open bars). Cells were incubated for 30 min at 25°C in HEPES-KOH medium adjusted to pH 8.5. The cells were then washed and incubated with uptake buffer, pH 7.0, containing 36Cl- for 2 min (as described in MATERIALS AND METHODS). Results are means ± SE of 6-9 determinations. *P < 0.05 compared with control (4alpha -PMA).

Effect of PKC inhibitors on ability of PMA to inhibit DIDS-sensitive Cl- uptake in Caco-2 cells. To confirm that the PMA-mediated effect on Cl-/OH- exchange activity was mediated via PKC activation, the effects of specific PKC inhibitors on PMA-mediated changes in Cl-/OH- exchange activity were examined. Overnight serum-deprived cells were pretreated with specific PKC inhibitors for 1 h before addition of PMA or 4alpha -PMA (100 nM), followed by coincubation for another 1 h. As shown in Fig. 2, pretreatment of cells with the specific PKC inhibitors, for example, chelerythrine chloride (2 µM; Fig. 2A), calphostin C (200 nM; Fig. 2B), or GF-109203X (50 nM; Fig. 2C) completely abolished the PMA-mediated inhibition of Cl-/OH- exchange activity in Caco-2 cells. These results suggested that the observed decrease in Cl-/OH- exchange activity involved PKC activation.


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Fig. 2.   Effect of specific inhibitors of protein kinase C (PKC) on the inhibitory effect of PMA on Cl-/OH- exchange activity in Caco-2 cells. Overnight serum-starved postconfluent cells were pretreated with specific PKC inhibitors chelerythrine chloride (Chel Cl, 2 µM; A), calphostin C (Calp C, 200 nM; B), or GF-109203X (GF, 50 nM; C) for 1 h before the addition of PMA (100 nM), followed by coincubation with PMA (100 nM) for another 1 h, and 36Cl- uptake was measured for 2 min (as described in MATERIALS AND METHODS). Results are expressed as % of control and represent means ± SE of 6-9 determinations. *P < 0.05 compared with control (4alpha -PMA). Control values: 1.55 ± 0.30 (A), 1.71 ± 0.49 (B), and 1.93 ± 0.16 (C) nmol · mg-1 · 2 min-1.

Effect of isoform-specific inhibitors of PKC on PMA-induced inhibition of Cl-/OH- exchange activity in Caco-2 cells. To test which particular PKC isoform is involved in this effect of PMA, we used two PKC inhibitors, Go-6976 and Ro-318820, which have been shown to differentially inhibit classic and novel PKCs, namely, alpha - and epsilon -isoforms, respectively. The reported IC50 of Go-6976 for PKCalpha is 5 nM (30) and that of Ro-318220 for PKCepsilon is 24-48 nM (26) depending on the cell type studied. As shown in Fig. 3A, Go-6976 (5 nM; 1 h before PMA addition, followed by coincubation for another 1 h) failed to block the inhibitory effect of PMA on Cl-/OH- exchange activity. Similar results were obtained with 10 and 50 nM concentrations of Go-6976 (data not shown). However, Ro-318220 (100 nM; 1 h before PMA addition, followed by coincubation for another 1 h) significantly attenuated the inhibitory effect of PMA on Cl-/OH- exchange activity in Caco-2 cells (Fig. 3B). The data suggest that PKCalpha or other isoforms sensitive to Go-6976 do not appear to mediate the inhibitory effect of PMA on Cl- uptake. Conversely, the PKCepsilon isoform, sensitive to Ro-318220, was observed as the potential candidate to mediate the inhibitory effect of PMA on Cl- uptake.


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Fig. 3.   Effect of specific inhibitors of PKC isoforms (alpha  and epsilon ) on the inhibitory effect of PMA on Cl-/OH- exchange activity in Caco-2 cells. Overnight serum-starved postconfluent cells were pretreated with PKCalpha inhibitor GO-6976 (5 nM; A) or PKCepsilon inhibitor RO-318220 (100 nM; B) for 1 h before addition of PMA (100 nM), followed by coincubation with PMA (100 nM) for another 1 h, and 36Cl- uptake was measured for 2 min (as described in MATERIALS AND METHODS). Results are expressed as % of control and represent means ± SE of 6-9 determinations. *P < 0.05 compared with control (4alpha -PMA). Control values: 1.36 ± 0.09 (A) and 1.06 ± 0.04 (B) nmol · mg-1 · 2 min-1.

Effect of PI3-kinase inhibitors wortmannin and LY-294002 on PMA-mediated inhibition of Cl-/OH- exchange activity in Caco-2 cells. PKCepsilon , the isoform sensitive to RO-318220, was previously shown to lie downstream of PI3-kinase in signal transduction cascades in other systems (10). It was therefore of interest to determine whether PI3-kinase pathway is involved in the PMA-mediated inhibition of Cl-/OH- exchange process in Caco-2 cells. Wortmannin (100 nM; 1 h before PMA addition, followed by coincubation for another 1 h) suppressed the PMA-mediated inhibition of Cl-/OH- exchange activity (Fig. 4A). LY-294002 (5 µM), a more specific PI3-kinase inhibitor that acts through a mechanism distinct from that of wortmannin (47), also significantly attenuated the inhibitory effect of PMA on the Cl-/OH- exchange process in Caco-2 cells (Fig. 4B). These findings implicate the activation of PI3-kinase and subsequent activation of its downstream effector PKCepsilon in the effects of PMA on apical Cl-/OH- exchange process in Caco-2 cells.


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Fig. 4.   Effect of specific inhibitors of phosphatidylinositol 3-kinase (PI3-kinase) on the inhibitory effect of PMA on Cl-/OH- exchange activity in Caco-2 cells. Overnight serum-starved postconfluent cells were pretreated with specific PI3-kinase inhibitors wortmannin (Wort, 100 nM; A) or LY-294002 (LY, 5 µM; B) for 1 h before addition of PMA (100 nM), followed by coincubation with PMA (100 nM) for another 1 h, and 36Cl- uptake was measured for 2 min (as described in MATERIALS AND METHODS). Results are expressed as % of control and represent means ± SE of 6-9 determinations. *P < 0.05 compared with control (4alpha -PMA). Control values: 1.63 ± 0.36 (A) and 0.92 ± 0.12 (B) nmol · mg-1 · 2 min-1.

Effect of PI3-kinase activator IRS-1 peptide on inhibition of Cl-/OH- exchange activity in Caco-2 cells. We next examined whether the PI3-kinase activator IRS-1 peptide could mimic the effects of PMA on Cl-/OH- exchange activity. IRS-1 peptide, a tyrosine-phosphorylated peptide, activates the PI3-kinase enzyme by binding to its SH2 domain. IRS-1 peptide (0.1-10 µM, 1 h) significantly decreased Cl-/OH- exchange activity in a dose-dependent manner (Fig. 5). These results suggest that IRS-1 peptide could mimic the effects of PMA on the Cl-/OH- exchange process in Caco-2 cells.


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Fig. 5.   Effect of IRS-1 peptide on the inhibition of Cl-/OH- exchange activity in Caco-2 cells. Overnight serum-starved postconfluent cells were treated with PI3-kinase activator IRS-1 peptide (0.1-10 µM) for 1 h, and 36Cl- uptake was measured for 2 min (as described in MATERIALS AND METHODS). Results are expressed as % of control and represent means ± SE of 3-6 determinations. *P < 0.05 compared with control. Control value: 1.39 ± 0.22 nmol · mg-1 · 2 min-1.

Effect of PI3-kinase activator PI(3,4,5)P3 on inhibition of Cl-/OH- exchange activity in Caco-2 cells. The products of PI3-kinase activity include the 3-phosphorylated lipids phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2] and PI(3,4,5)P3. It was shown previously that PMA can induce PI3-kinase activity and increase the levels of PI(3,4)P2 and PI(3,4,5)P3 (second messengers) in mouse epidermal JB6 cells (21). Hence, to further establish the role of PI3-kinase in PMA-mediated inhibition of Cl-/OH- exchange activity, Caco-2 cells were incubated with 10 µM of PI(3,4,5)P3 for 1 h. PI(3,4,5)P3 significantly decreased the Cl-/OH- exchange activity by ~50% (Fig. 6). These findings further confirm that the PMA-mediated inhibition of Cl-/OH- exchange activity involves the activation of PI3-kinase, which in turn can activate PKCepsilon .


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Fig. 6.   Effect of phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] on the inhibition of Cl-/OH- exchange activity in Caco-2 cells. Overnight serum-starved postconfluent cells were treated with PI(3,4,5)P3 (10 µM) for 1 h, and 36Cl- uptake was measured for 2 min (as described in MATERIALS AND METHODS). Results are expressed as % of control and represent means ± SE of 3-6 determinations. *P < 0.05 compared with control. Control value: 0.96 ± 0.02 nmol · mg-1 · 2 min-1.

Time course of translocation of PKCepsilon in Caco-2 cells after PMA addition. In light of the data of PKC isoform inhibitors presented above indicating that PKCepsilon may be the major isoform responsible for the regulation of the Cl-/OH- exchanger in Caco-2 cells, we further characterized the time course of PKCepsilon membrane translocation on PMA addition. With Western blot analysis, it is clear that an increase in PKCepsilon density in the membrane fraction was apparent as early as 15 min after PMA (100 nM) addition and continued to increase up to at least 1 h (Fig. 7). The translocation/activation of PKCepsilon within 1 h of PMA treatment is consistent with our functional data showing inhibition of Cl-/OH- exchange activity in Caco-2 cells.


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Fig. 7.   Time course of membrane translocation of PKCepsilon in Caco-2 cells in response to PMA and effect of PI3-kinase inhibitors. Overnight serum-starved postconfluent cells were treated with 100 nM of PMA for 15-, 30-, and 60-min time periods. Untreated control cells received 100 nM of inactive 4alpha -PMA. To study the effect of wortmannin and LY-294002 on the ability of PMA to cause PKCepsilon translocation in Caco-2 cells, overnight serum-starved postconfluent cells were pretreated with the PI3-kinase inhibitors wortmannin (WM, 100 nM) and LY-294002 (LY, 5 µM) for 1 h before addition of PMA for 1 h. The cytosolic and membrane fractions were subjected to SDS-PAGE and probed with specific PKCepsilon antibody to examine subcellular distribution (A). C, cytosol; M, membrane. Data were quantitated by densitometric analysis and expressed as arbitrary units (AU) and represent means ± SE of 3 determinations (B). *P < 0.05 compared with untreated control (4alpha -PMA); **P < 0.05 compared with PMA (60 min) alone.

Effect of LY-294002 and wortmannin on PMA-induced PKCepsilon activation in Caco-2 cells. Previous studies showed that PI3-kinase can activate novel PKCs (30, 31). PI3-kinase activity was also found to be involved in the inhibitory action of EGF on carbachol (CCh)-induced Cl- secretion (46). To determine whether PKCepsilon is a downstream effector of PI3-kinase, specific inhibitors of PI3-kinase, LY-294002 (5 µM) and wortmannin (100 nM), were used. We found that both inhibitors inhibited the PMA-induced translocation of PKCepsilon by ~40% (Fig. 7). These data further confirm our findings that PKCepsilon is a downstream effector of PI3-kinase in the PMA-mediated inhibition of Cl-/OH- exchange activity in Caco-2 cells.

Effect of IRS-1 peptide on PMA-induced PKCepsilon activation in Caco-2 cells. To further substantiate our findings that PI3-kinase is upstream to PKCepsilon , we used the PI3-kinase activator IRS-1 peptide. IRS-1 peptide (1 µM, 1 h) significantly induced the translocation/activation of PKCepsilon from the cytosol to the membrane fractions (Fig. 8) compared with the control. These findings suggest that IRS-1 peptide could mimic the effects of PMA on PKCepsilon translocation and further confirm that PI3-kinase is the upstream effector of PKCepsilon in the PMA-mediated inhibition of Cl-/OH- exchange activity in Caco-2 cells.


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Fig. 8.   Effect of IRS-1 peptide on membrane PKCepsilon in Caco-2 cells. Overnight serum-starved postconfluent cells were treated with PI3-kinase activator IRS-1 peptide (1 µM) for 1 h. The cytosolic and membrane fractions were subjected to SDS-PAGE and probed with specific PKCepsilon antibody to examine subcellular distribution (A). Data were quantitated by densitometric analysis and expressed as arbitrary units and represent means ± SE of 3 determinations (B). *P < 0.05 compared with control.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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Our data demonstrate a novel pathway for the regulation of Cl-/OH- exchange by PMA occurring via activation of a PI3-kinase and the involvement of a downstream calcium-independent PKC isoform, namely, PKCepsilon .

PKC was previously shown to directly regulate the activity of the Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger in isolated rat hepatocytes, in which phorbol esters inhibit the stimulation of the Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger induced by dibutyryl cAMP or HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> (6). A role of PKC has also been suggested in the regulation of rat colonic electrolyte transport by inhibiting active NaCl absorption and stimulating electrogenic Cl- secretion (13). Activation of PKC by pretreatment with 100 nM PMA has also been shown to inhibit CCh-induced Cl- secretion in T84 cells (39). Studies on the regulation of NHE3 by PMA also showed that the PMA-induced acute inhibition of endogenous NHE3 in Caco-2 cells is mediated by PKC (25). Our data, complementing the above studies of inhibition of NHE3 by PMA, showed that short-term treatment of Caco-2 cells with PMA at 100 nM for 1 h resulted in a significant decrease in Cl-/OH- exchange activity. Parallel studies were also carried out with recently demonstrated SO<UP><SUB>4</SUB><SUP>2−</SUP></UP>/OH- anion exchange activity in our laboratory (2) in Caco-2 cells to examine whether PMA effects were specific for Cl-/OH- exchange. The results demonstrated that, in contrast to the inhibition of Cl-/OH- exchange, SO<UP><SUB>4</SUB><SUP>2−</SUP></UP>/OH- exchange activity remained unaltered by PMA treatment, suggesting that the effects of PMA were specific to the Cl-/OH- exchanger.

The PMA-mediated inhibition of Cl-/OH- exchange activity was completely blocked by specific inhibitors of PKC, chelerythrine chloride (2 µM), calphostin C (200 nM), and GF-109203X (50 nM), thereby confirming the involvement of PKC in PMA-mediated inhibition of Cl-/OH- exchange activity in Caco-2 cells. Because PMA-induced acute inhibition of Cl-/OH- exchange activity is mediated by PKC, it is thus possible that one or several isoforms of PKC could mediate the divergent inhibitory effect of PMA. Thus PKCs are grouped into three major classes, conventional PKC isoforms (alpha , beta 1, beta 2, gamma ), novel PKCs (delta , epsilon , eta , theta ), and atypical PKCs (xi , lambda ) (22). Conventional PKCs are activated by DAG in a Ca2+-dependent manner. In contrast, activation of novel PKCs is Ca2+ independent (40). In addition to the natural activation, conventional and novel PKCs are also activated with high specificity by PMA (8). For this reason, PMA is often used in the study of mechanisms of conventional and novel PKC activation and their function (12).

Previous studies demonstrated that both PKC and PI3-kinase play important roles in many different cell regulatory pathways as well as in a broad range of biological effects (19, 42). Several studies suggested that PMA can induce PI3-kinase activity (27, 33, 43) and increase the level of PI(3,4,5)P3 and PI(3,4)P2, lipid products of PI3-kinase (second messengers), as well as having a markedly synergistic effect with insulin on PI3-kinase activation in mouse epidermal JB6 cells (21). Earlier studies of Toker et al. (44) showed that the calcium-independent PKC isoforms were activated by the lipid products of PI3-kinase. Therefore, in the current study, we also attempted to examine the PKC isotype(s) involved in the regulation of Cl-/OH- exchange activity in Caco-2 cells by PMA. Our results showed that Ro-318220, but not Go-6976, completely blocked the inhibitory effect of PMA on Cl-/OH- exchange activity, suggesting that PKCepsilon , but not PKCalpha , was likely to play a pivotal role in the inhibitory effect on Cl- uptake by PMA. The results are well correlated with the Western blot analysis, showing that PMA treatment (100 nM, 1 h) resulted in the translocation of PKCepsilon from the cytosol to the membrane fractions in Caco-2 cells. In this regard, PI3-kinase has also been shown to activate novel PKCs in other systems (31). For example, PI3-kinase was found to be associated with PKCepsilon in a human hematopoietic cell line and platelets (15) and incubation of HepG2 cells with platelet-derived growth factor led to the translocation of PKCepsilon via the activation of PI3-kinase (30). However, in contrast, Huang et al. (20) showed that the effect of PMA-induced PI3-kinase activity was mediated by the novel PKCepsilon in mouse epidermal JB6 cells, suggesting that the PKCepsilon effect was upstream to PI3 kinase. In our system, the current data also indicate a link between PKCepsilon and PI3-kinase in Caco-2 cells and suggest PI3-kinase to be the upstream effector of PKCepsilon , as both wortmannin (100 nM) and LY-294002 (5 µM) blocked the inhibitory effect of PMA on Cl-/OH- exchange activity. In this regard, wortmannin is a somewhat nonspecific inhibitor of PI3-kinase, as it was also shown to inhibit phosphatidylinositol 4-kinase (PI 4-kinase), myosin light chain kinase, phospholipase A2, and phospholipase D (32). In contrast, LY-294002 is a more specific PI3-kinase inhibitor and therefore was also used to exclude the possibilities of nonspecific effects of wortmannin. Our data showed similar results with both inhibitors. Thus, in our study, the inhibitory effect of wortmannin on PMA-induced activation of PKCepsilon favors an essential role of the second messengers of PI3-kinase pathway and suggests a downstream position of PKCepsilon related to PI3-kinase. Our data are also in accordance with the previous findings of Chow et al. (10) showing that the ability of EGF to inhibit CCh-induced Cl- secretion in T84 cells was completely reversed by the PI3-kinase inhibitor wortmannin and that PKCepsilon acts as a downstream effector to PI3-kinase in T84 cells stimulated with EGF. Moreover, we have also shown the increased translocation of PKCepsilon from the cytosol to the membrane fractions in Caco-2 cells in the presence of the PI3-kinase activator IRS-1 peptide. Thus our results with the PI3-kinase activators IRS-1 peptide and PI(3,4,5)P3 further confirm that the observed effects of PMA on the Cl-/OH- exchange activity occur via PI3-kinase, which in turn activates PKCepsilon , rather than a direct activation of PKCepsilon by PMA.

Several earlier studies showed that agents like phorbol esters and CCh that increase PKC activity decrease Vmax of NHE3 in the Chinese hamster lung fibroblast cell line PS120 (45), the rabbit gall bladder epithelium (41), and the opossum kidney cell line (5). Because the inhibition occurs via decrease in Vmax and is observed within hours, this type of regulation might theoretically be achieved by a decrease in the number of active molecules at the membrane due to endocytic retrieval from and/or exocytic insertion into the plasma membrane and/or rapid degradation by changes in the turnover number of individual exchanger molecules. Multiple plasma membrane transport proteins (7, 16, 29, 37, 48) were also shown to be regulated, at least in part, by cellular redistribution (endocytic retrieval from and/or exocytic insertion into the plasma membrane). PKC was reported to generally stimulate apical but not basolateral endocytosis in Caco-2 cells (18). Hence, in our study, certainly effects of PKCepsilon via PI3-kinase pathway on endocytosis could account for the ability of PMA to inhibit Cl-/OH- exchange activity in Caco-2 cells by retrieving apical membrane transporter molecules to the cytoplasm. Future studies will address this important question regarding the mechanism of Cl-/OH- exchange attenuation.

In conclusion, the present studies demonstrate for the first time that PMA inhibits the Cl-/OH- exchange process in Caco-2 cells via the activation of PI3-kinase, which in turn stimulates PKCepsilon via its lipid products. Future studies to elucidate the detailed signal transduction pathways and molecular mechanism of this Cl- transport regulation would be important to better understand the role of PKC in intestinal electrolyte transport.


    ACKNOWLEDGEMENTS

These studies were supported by the Department of Veterans Affairs and National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-54016 (P. K. Dudeja), DK-33349 (K. Ramaswamy), and DK-09930 (W. A. Alrefai).


    FOOTNOTES

Address for reprint requests and other correspondence: P. K. Dudeja, Univ. of Illinois at Chicago, Medical Research Service (600/151), Chicago VA West Side Division, 820 South Damen Ave., Chicago, IL 60612 (E-mail: pkdudeja{at}uic.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.

July 24, 2002;10.1152/ajpcell.00473.2001

Received 4 October 2001; accepted in final form 17 July 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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