1,25-Dihydroxyvitamin D3 and TPA activate phospholipase D in Caco-2 cells: role of PKC-alpha

Sharad Khare, Marc Bissonnette, Beth Scaglione-Sewell, Ramesh K. Wali, Michael D. Sitrin, and Thomas A. Brasitus

Department of Medicine, University of Chicago, Chicago, Illinois 60637


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
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1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] and 12-O-tetradecanoylphorbol 13-acetate (TPA) both activated phospholipase D (PLD) in Caco-2 cells. GF-109203x, an inhibitor of protein kinase C (PKC) isoforms, inhibited this activation by both of these agonists. 1,25(OH)2D3 activated PKC-alpha , but not PKC-beta 1, -beta II, -delta , or -zeta , whereas TPA activated PKC-alpha , -beta 1, and -delta . Chronic treatment with TPA (1 µM, 24 h) significantly reduced the expression of PKC-alpha , -beta I, and -delta and markedly reduced the ability of 1,25(OH)2D3 or TPA to acutely stimulate PLD. Removal of Ca2+ from the medium, as well as preincubation of cells with Gö-6976, an inhibitor of Ca2+-dependent PKC isoforms, significantly reduced the stimulation of PLD by 1,25(OH)2D3 or TPA. Treatment with 12-deoxyphorbol-13-phenylacetate-20-acetate, which specifically activates PKC-beta I and -beta II, however, failed to stimulate PLD. In addition, the activation of PLD by 1,25(OH)2D3 or TPA was markedly reduced or accentuated in stably transfected cells with inhibited or amplified PKC-alpha expression, respectively. Taken together, these observations indicate that PKC-alpha is intimately involved in the stimulation of PLD in Caco-2 cells by 1,25(OH)2D3 or TPA.

calcitriol; phorbol esters; intracellular calcium; phospholipases; protein kinase C isoforms


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INTRODUCTION
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INCREASING EVIDENCE has accumulated over the past several years that phosphatidylcholine (PC)-specific phospholipase D (PLD) enzymes may play key roles in the transduction of extracellular signals involved in the regulation of a number of important cellular processes (4, 10, 14, 29). PLD hydrolyzes PC to generate phosphatidic acid (PA) and choline (14, 19). Increases in PA, via activation of PLD, have been implicated in the regulation of cellular proliferation, differentiation, inflammation, as well as cellular trafficking and secretion of proteins (19, 22, 29). PA can subsequently be hydrolyzed by phospholipase A2 to form the second messenger, lysophosphatidic acid (LPA) in some cell types (38) or can be dephosphorylated by phosphatidate phosphorylhydrolase to produce 1,2-diacylglycerol (DAG) (19), a lipid mediator that activates members of the Ca2+- and phospholipid-dependent protein kinase C (PKC) family (19).

The regulation of PLD activity in mammalian cells is complex. In many but not all cells, for example, stimulation of PLD by a variety of agonists appears to be dependent on the prior activation of phosphatidylinositol-specific phospholipase C (PI-PLC) (43) and mediated by a PKC-dependent process (2, 8, 12, 29, 33, 42, 44, 46). Moreover, depending on the cell type, the regulation of PLD by PKC may involve one or more isoforms of this serine-threonine protein kinase family (2, 8, 12, 29, 33, 42, 44, 46) and may occur via phosphorylation-dependent (29, 33) or -independent mechanisms (9, 29, 50).

In this regard, it has become increasingly clear that 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], the major active metabolite of vitamin D3, can rapidly activate PKC and induce a number of important cellular effects, in addition to its well-established role in the regulation of mineral metabolism (18, 58-60). We have previously shown, for example, that this secosteroid alters the proliferation of rat colonocytes (60), as well as the growth and differentiation of Caco-2 cells (18), a cell line derived from a human colonic adenocarcinoma (18). Studies by our laboratory, utilizing experimental models of colon cancer, have also demonstrated that alterations in the vitamin D status of rats can influence the process of colonic malignant transformation (51, 61).

In an attempt to further elucidate the roles of vitamin D3 in the physiological regulation and pathophysiological derangements of important cellular processes in the colon, our laboratory has studied the effects of 1,25(OH)2D3 and other metabolites and/or analogs of vitamin D3 in rat colonocytes and in Caco-2 cells over the past several years. We have shown, for example, that 1,25(OH)2D3 rapidly (seconds to minutes) stimulated membrane polyphosphoinositide hydrolysis, thereby, generating inositol 1,4,5-trisphosphate (IP3) and DAG, increased intracellular Ca2+ concentration ([Ca2+]i), as well as activated PKC-alpha , as assessed by its translocation from the cytosolic to particulate fraction using Western blotting techniques and its phosphorylation of the MARCKS protein, a known PKC substrate (6, 7, 60). We have also demonstrated that 1,25(OH)2D3 caused a significant increase in the particulate association, tyrosine phosphorylation and biochemical activation of PI-PLC-gamma indicating that this isoform of PI-PLC, at least in part, was responsible for the hydrolysis of membrane polyphosphoinositides induced by this secosteroid in these cells (27). Furthermore, activation of PI-PLC-gamma by this secosteroid required the stimulation of pp60c-src (27). More recently, in preliminary studies, our laboratory has found that 1,25(OH)2D3, in addition to the aforementioned transient increase in DAG levels caused by polyphosphoinositide hydrolysis, also induced a sustained increase in DAG derived from a non-PI membrane lipid pool in colonocytes (28). In a number of other cell types such sustained increases in DAG have been shown to result from PC hydrolysis by agonist-induced PLD activation.

In the present studies we examined whether 1,25(OH)2D3 or 12-O-tetradecanoylphorbol 13-acetate (TPA), a tumor-promoting phorbol ester previously shown to activate PKC and/or PLD in other cells, stimulated PLD activity in Caco-2 cells. Based on the aforementioned observations, it was also of interest to determine if the stimulation of PLD by one or both of these agents required the activation of a particular PKC isoform(s) present in these cells. The results of these studies serve as the basis for the present report.


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Materials. The full-length cDNA encoding human PKC-alpha in pBluescript was obtained from Hug and co-workers (17). Eukaryotic expression vector pRC/CMV was purchased from In Vitrogen (San Diego, CA). Qiagen columns were supplied by Qiagen (Chatsworth, CA). Tricyclodecan-9-yl xanthate (D-609) was obtained from Bio-Mol Research Laboratories (Plymouth Meeting, PA). ICN Biochemical (Aurora, OH) provided propranolol hydrochloride. EDTA, EGTA, ATP, digitonin ammonium persulfate, beta -glycerophosphate, TEMED, and 2-mercaptoethanol were purchased from Sigma Chemical (St. Louis, MO). Electrophoretic grade acrylamide, bis-acrylamide, Tris, SDS, and prestained molecular weight markers were obtained from Bio-Rad (Richmond, CA). Kodak (Rochester, NY) supplied the X-OMAT AR film. The protease inhibitors 4-(2-aminoethyl)-benzene sulfonyl fluoride (AEBSF), aprotinin, and Ca2+-specific PKC isoform inhibitor Gö-6976 were obtained from Calbiochem (San Diego, CA). Upstate Biotechnology (Lake Placid, NY) provided monoclonal anti-PKC-alpha antibodies, whereas polyclonal anti-PKC-alpha , -beta I, -beta II, -delta , -epsilon , and -zeta antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Polyvinylidene difluoride membranes (Immobilon-P) were purchased from Millipore (Bedford, MA). The enhanced chemiluminescence (ECL) Western blotting protein detection kit, the peroxidase-coupled sheep anti-mouse and donkey anti-rabbit antibodies, [gamma -32P]ATP, and [3H]myristic acid were supplied by Amersham (Arlington Heights, IL). Phosphatidylbutanol (Pbt), PA, and DAG were purchased from Avanti Polar Lipids (Alabaster, AL). Phorbol esters, TPA, 12-deoxyphorbol-13-phenylacetate-20-acetate (DOPPA), and the PKC-inhibitor GF-109203x were obtained from LC Laboratories (Woburn, MA). 1alpha ,25-Dihydroxyvitamin D3 was purchased from Steroids Limited (Chicago, IL). The PKC-beta I- and -beta II-specific inhibitor, Lilly compound 379196, was kindly provided by Dr. Kirk Ways, Lilly Research Laboratories. PKC substrate peptide derived from myelin basic protein Ac-MBP4-14 and phosphocellulose disks were obtained from GIBCO BRL-Life Technologies (Gaithersburg, MD). Unless otherwise noted, all other reagents were obtained from Sigma Chemical or Fisher Scientific (Springfield, NJ) and were of the highest purity available.

Cell culture. Caco-2 cells, derived from a human colonic carcinoma, were cultured at 37°C in an atmosphere of 5% CO2-95% air as previously described by our laboratory (55). The cells from passages 21-40 were maintained in standard DMEM containing 4.5 g/l glucose, 2 mM L-glutamine, 50 U/ml penicillin, 50 µg/ml streptomycin, 2 µg/ml gentamicin, 10 mM HEPES, 1% essential and nonessential amino acids, and 20% fetal bovine serum (FBS), unless otherwise indicated. Cells, plated at 105 cells/ml, were subcultured in six-multiwell plates or grown in 50 or 150 cm2 flasks as appropriate. Experiments were carried out with cells at 70-80% confluency.

Plasmid construction, transfection, and expression of human PKC-alpha in Caco-2 cells. PKC-alpha was subcloned in sense or antisense orientations into the eukaryotic expression vector pRc/CMV as previously described by our laboratory (48). Caco-2 cells were transfected with 40 µg of the PKC-alpha constructs, or the pRc/CMV vector alone, by the calcium phosphate coprecipitation method. Clones of empty vector, sense and antisense transfections that have been previously described (48), were selected by G418 resistance.

PKC downregulation in Caco-2 cells. To examine the downregulation of specific PKC isoforms expressed in Caco-2 cells, TPA (1 µM final concentration) or vehicle (DMSO, 0.05%) was added to the cells. After 24 h, medium was removed and cells were lysed by boiling for 3 min in Laemmli SDS-stop buffer. Protein was measured by amidoblack staining of samples spotted on nitrocellulose with BSA as the standard (49).

Detection of PKC isoforms by Western blotting. SDS-treated samples (20 µg protein) were separated by SDS-PAGE using a 10% resolving gel and electroblotted to Immobilon-P membranes following the method of Towbin et al. (57). Homogenates (10 µg protein) from rat or human brain were included as positive controls where appropriate. Nonspecific binding of antibodies was blocked by incubating the blots with 5% nonfat dry milk in 50 mM Tris · HCl (pH 7.4), 150 mM NaCl with 0.05% Tween 20 (TBST) for 2 h. After blocking, the blots were incubated overnight at 4°C with isoform-specific antibodies, rabbit polyclonal anti-PKC-alpha , -beta I, -beta II, -delta or -zeta (0.2 µg/ml) or mouse monoclonal anti-PKC-alpha (0.1 µg/ml). After four washes in 10 ml TBST, the blots were incubated with 1:3,000 final dilutions of appropriate peroxidase-coupled secondary antibodies. The blots were washed four times in TBST, and the PKC isoforms were then detected by xerography on X-OMAT AR film using an ECL system as recommended by the manufacturer. The xerograms were quantified using a scanner (JX-3F6, Sharp Electronics, Mahwah, NJ), and IP lab gel software (Signal Analytics, Vienna, VA). Under the conditions of our immunologic detection, the ECL assay was linear between 5 and 40 µg of total protein loaded.

PKC isoform translocation in Caco-2 cells. To examine the translocation of PKC isoforms, Caco-2 cells grown in 50-cm2 flasks were washed and preincubated for 30 min at 37°C in 2 ml of HEPES buffer containing (in mM) 10 HEPES, pH 7.2, 140 NaCl, 5 KCl, 5 MgCl2, 2 CaCl2, and 10 D-glucose. Cells were pretreated with inhibitors or their appropriate vehicles for the indicated times and concentrations and then stimulated with 1,25(OH)2D3 (100 nM final concentration), TPA (200 nM final concentration), DOPPA (200 nM final concentration), or appropriate agonist vehicles. At the indicated times, as previously described by our laboratory (6), the cells were lysed in 0.7 ml of extraction buffer by four cycles of repeated freezing in a dry ice-ethanol bath followed by thawing in a 37°C water bath. The extraction buffer contained (in mM) 25 Tris, pH 7.6, 5 EGTA, 0.7 CaCl2 [free Ca2+ estimated to be <10 nM using the program CHELATOR (6)], 100 µM AEBSF, 10 µg/ml leupeptin, and 10 µg/ml aprotinin. The cells were further disrupted by four passages through a 25-gauge needle and fractionated into soluble and particulate components by centrifugation at 100,000 g for 30 min at 4°C in a Sorvall-RCM 120 EX ultracentrifuge. Fractions were immediately boiled in Laemmli SDS-stop buffer. Protein determinations and Western blotting detection of PKC isoforms in soluble and particulate fractions were carried out as previously described.

In situ assay of PKC activation. For assay of in situ PKC phosphorylating activity, Caco-2 cells were plated in 24-multiwell plates. Cells were pretreated with inhibitors or their appropriate vehicles for the indicated times and concentrations as noted in the legend of Fig. 11. In other experiments to examine the effects of PKC downregulation, cells were treated for 24 h with 1 µM TPA and then stimulated with 1,25(OH)2D3 (100 nM final concentration) or TPA (100 nM final concentration). After aspiration of the medium, PKC activation was assayed by adding 100 µl permeabilization-kinase assay buffer to the cells, which contained 137 mM NaCl, 5.4 mM KCl, 0.3 mM Na2HPO4, 0.4 mM K2HPO4, 1 mg/ml glucose, 20 mM HEPES, 10 mM MgCl2, 25 mM beta -glycerophosphate, 50 µg/ml digitonin, 100 µM [gamma -32P]ATP (500 counts per minute/pmol), 2.5 mM CaCl2, 5 mM EGTA (pH 7.2), and 50 µM PKC substrate peptide AC-MBP4-14 (20). After a 10-min incubation, 10 µl of 25% (wt/vol) TCA were added and 20 µl from each well were spotted onto a phosphocellulose disk, washed with 1% (vol/vol) concentrated phosphoric acid in water, and radioactivity was assessed by scintillation counting as previously described (20).

PLD and DAG measurements. For assays of DAG levels and PLD activity, Caco-2 cells were metabolically labeled with [3H]myristic acid (1 µCi/ml) for 24 h in medium containing 0.5% FBS. In preliminary experiments under these conditions, 80% of total radioactivity incorporated into cells was found in the PC fraction. After removal of the radiolabeling medium, cultures were rinsed twice with serum-free medium and equilibrated in fresh medium at 37°C for 1 h. Cells were treated with 1,25(OH)2D3 or TPA at the indicated concentrations, and for the indicated times, with or without 30 nM 1-butanol. For determination of DAG levels, the reaction was rapidly stopped by the addition of 0.6 ml of methanol-6 N HCl (50:2) as previously described (5).

In the presence of a primary alcohol, PLD catalyzes a transphosphatidylation reaction in which the phosphatidyl moiety of the substrate phospholipid is transferred to the alcohol, thereby producing the corresponding phosphatidylalcohol (4, 10). This unique property has been widely used to identify and characterize PLD activity in many cell systems (4, 10). In preliminary studies, 30 nM 1-butanol added to cells metabolically labeled with [3H]myristic acid was found to produce maximal [3H]Pbt levels (data not shown). This concentration of 1-butanol was therefore utilized in the present experiments. For determination of PLD activity in the presence of 1-butanol, the transphosphatidylation reaction was stopped with ice-cold HClO4 (4% vol/vol), and Pbt was quantified as previously described (37). Briefly, the precipitate was collected by centrifugation and washed once with Ca2+-free Hanks' buffer, and the lipids were extracted with chloroform and methanol. Extraction of lipids was performed according to procedures described by Billah et al. (5), with minor modifications (52, 53). Cells or precipitates were rapidly treated with 0.6 ml of methanol-6 N HCl (50:2). Lipids were extracted by the addition of 0.6 ml of chloroform. Phase separation was obtained by adding 200 µl of 1 M NaCl. The organic phase was re-extracted with 0.6 ml of 0.35 M NaCl and 0.2 ml of methanol-6 N HCl (50:1) and recovered, dried under nitrogen, and solubilized in chloroform-methanol (9:1). DAG was separated by TLC (Silica gel 60A plates) using as a solvent system hexane-diethylether-methanol-glacial acetic acid (90:20:3:2) as previously described (5). PA and Pbt, the products of the PLD reaction in the absence or presence of butanol, respectively, were separated on HP-TLC plates (Whatman LHP-K), using the organic phase of a mixture of iso-octane-ethylacetate-acetic acid-water (5:11:2:10). Appropriate standards were applied to each plate for quantitation. After the spots were localized by exposure to iodine vapor, the appropriate area of each of the samples, comigrating with the marking standard, was scrapped into a scintillation vial and radioactivity was assessed by scintillation counting.

Statistics. Data are expressed as percent above control (means ± SE). Differences between treatments were evaluated by ANOVA (Dunnett's test) and considered significant for P < 0.05.


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1,25(OH)2D3 and TPA each stimulate PC-PLD in a time- and concentration-dependent manner in Caco-2 cells. As shown in Fig. 1A, as assessed by [3H]Pbt formation, the addition of 1,25(OH)2D3 (100 nM final concentration) to intact Caco-2 cells metabolically labeled with [3H]myristic acid, caused a detectable increase in PLD activity within 5 min, which continued to increase for at least 30 min. As indicated in Fig. 1B, this effect of 1,25(OH)2D3 on PLD activity was dose dependent, with 100 nM 1,25(OH)2D3 producing a greater effect than 50 nM. In keeping with the hydrolytic activity of PLD in the absence of butanol 1,25(OH)2D3 also stimulated the formation of PA and DAG in these cells (Fig. 2). Moreover, preincubation of these cells with propranolol (500 µM final concentration), before the addition of 1,25(OH)2D3 (100 nM for 20 min), significantly inhibited the accumulation of DAG and increased the levels of PA (Fig. 2), consistent with the ability of propranolol to inhibit the activity of phosphatidate phosphorylhydrolase in other cells (31).


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Fig. 1.   1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] stimulates phospholipase D (PLD) in a time- and concentration-dependent manner. Caco-2 cells, metabolically labeled with [3H]myristic acid, were incubated with 1,25(OH)2D3 or vehicle (0.05% ethanol), and then lipids were extracted. [3H]phosphatidylbutanol (Pbt), the transphosphatidylation product of PLD, was resolved and quantified as described in METHODS. A: time course for PLD activation. Cells were incubated with 100 nM 1,25(OH)2D3 or vehicle for indicated times. B: 1,25(OH)2D3 dose dependence for PLD activation. Cells were incubated with indicated concentrations of secosteroid or vehicle for 20 min. Data are expressed as means ± SE; n = 3 independent experiments, each in triplicate. * P < 0.05 compared with vehicle-treated cells.


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Fig. 2.   Effect of 1,25(OH)2D3 on 1,2-diacylglycerol (DAG) and phosphatidic acid (PA) production in Caco-2 cells. [3H]myristic acid-labeled cells were treated with 1,25(OH)2D3 (100 nM, 20 min) or vehicle. In addition, in some experiments, cells were preincubated with propranolol (500 µM, 30 min) and then treated with 1,25(OH)2D3 or vehicle as noted. Lipids were extracted and [3H]DAG (open bars) or [3H]PA (solid bars) was resolved and quantified as described in METHODS (n = 3 independent experiments, each in triplicate) * P < 0.05 compared with cells treated with 1,25(OH)2D3 alone.

Treatment of cells with TPA (200 nM final concentration) caused a detectable increase in PLD activity as early as 5 min, which was maximal by 20 min, with no further increase up to 30 min (Fig. 3A). As shown in Fig. 3B, the effect of TPA on activation of PLD, like that of 1,25(OH)2D3, was dose dependent, and TPA at a final concentration of 200 nM maximally stimulated PLD activity. Based on these findings, unless otherwise indicated, all subsequent experiments were performed for 20 min after the in vitro addition of 100 nM 1,25(OH)2D3 or 200 nM TPA to Caco-2 cells.


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Fig. 3.   12-O-tetradecanoylphorbol 13-acetate (TPA) stimulates PLD in a time- and concentration-dependent manner. Cells, prelabeled with [3H]myristic acid, were treated with TPA or vehicle (0.05% DMSO). [3H]Pbt was assayed as described in METHODS. A: time course of TPA-stimulated PLD activity. Cells were incubated with 200 nM TPA or vehicle for indicated times. B: dose dependence for PLD activation by TPA. Cells were incubated with indicated concentrations of TPA or vehicle (DMSO) for 20 min (n = 3 independent experiments each in triplicate). * P < 0.05 compared with vehicle-treated cells.

Prior studies in other cell types have demonstrated that agonists that stimulate PC-PLD may also stimulate PC-PLC and/or PI-PLC (4, 14, 43, 44). To determine whether 1,25(OH)2D3 or TPA not only activated PC-PLD but also activated PC-PLC, Caco-2 cells were preincubated for 30 min with D-609 (50 µg/ml final concentration), a specific inhibitor of PC-PLC (26, 40), and then treated with 1,25(OH)2D3 or TPA for 20 min. Preincubation of Caco-2 cells with D-609 failed to inhibit the rise in DAG levels in these cells induced by 1,25(OH)2D3 or TPA (data not shown), indicating that these agents did not activate PC-PLC.

As previously noted, our laboratory had demonstrated that 1,25(OH)2D3 rapidly (seconds to minutes) activated PI-PLC-gamma in Caco-2 cells, generating inositol trisphosphate (IP3) and DAG (58). This effect was, however, transient with levels of both IP3 and DAG, returning to baseline values by 2 min, although DAG levels appeared to increase again by 5 min (58). In the present experiments, IP3 levels were found not to be increased in these cells from 5-30 min after the in vitro addition of 1,25(OH)2D3 (data not shown).

Broad-spectrum inhibitor of PKC isoforms blocks stimulation of PLD by 1,25(OH)2D3 or TPA. In view of previous studies that have demonstrated that PKC may mediate the activation of PLD by a variety of agonists (11, 21, 24, 25, 30, 32, 37, 43), including TPA (2, 30, 32, 37, 41, 44), cells were preincubated with a potent broad-spectrum inhibitor of PKC isoforms (56), the bisindolylmaleimide GF-109203x (5 µM final concentration) for 3 h and then treated with 1,25(OH)2D3 or TPA for 20 min. As shown in Fig. 4, GF-109203x significantly inhibited the stimulation of PLD by this secosteroid or TPA. Because GF-109203x inhibits both Ca2+-dependent and -independent isoforms of PKC (56), these findings indicated that one or more isoforms of this family of kinases present in Caco-2 cells (Fig. 7A) appeared to mediate the activation of PLD by these agents.


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Fig. 4.   GF-109203x blocks stimulation of PLD by 1,25(OH)2D3 or TPA. [3H]myristic acid-labeled Caco-2 cells were stimulated with 1,25(OH)2D3 (100 nM) or TPA (200 nM) for 20 min (open bars). In addition, in some experiments cells were preincubated with GF-109203x (5 µM, 3 h) and then stimulated with these agonists (solid bars) as noted. Lipids were then extracted, and [3H]Pbt was quantified (n = 3 independent experiments, each in triplicate). * P < 0.05 compared with cells incubated with 1,25(OH)2D3 or TPA alone.

To investigate the specific PKC isoforms involved in PLD activation by these agents, we performed several additional experiments to assess 1) the activation of specific PKC isoforms by these agents, 2) the effect of downregulation of specific PKC isoforms induced by chronic phorbol ester treatment on PLD stimulation by these agents, 3) the role(s) of Ca2+ on PLD stimulation by modulation of extracellular Ca2+ or by use of an inhibitor specific for the Ca2+-dependent PKC isoforms, and 4) the effects of inhibited or amplified PKC-alpha protein expression on this process, using stably transfected Caco-2 cells.

PKC-alpha mediates activation of PLD by 1,25(OH)2D3 and TPA. Prior studies by our laboratory, utilizing Western blotting techniques with specific antibodies for PKC isoforms, have demonstrated that Caco-2 cells possessed both the Ca2+-dependent PKC isoforms, PKC-alpha , -beta I, and -beta II, as well as the Ca2+-independent PKC isoforms, PKC-delta and -zeta (1, 6). In an attempt to determine the specific isoforms of PKC involved in mediating the stimulation of PLD by 1,25(OH)2D3 or TPA, Caco-2 cells were treated with each of these agents for 1-30 min to determine which isoform(s) of PKC these agents activated, as assessed by isoform translocation from the cytosolic to particulate fraction of these cells, using Western blotting techniques. In agreement with prior studies from our laboratory (6), 1,25(OH)2D3 (100 nM final concentration) rapidly but transiently (1-2 min) activated PKC-alpha , but not PKC-beta I, -beta II or -delta (Fig. 5). As can be seen in Fig. 6, TPA (200 nM final concentration), however, not only induced the particulate association of PKC-alpha but also that of PKC-beta I and -delta , but not PKC-beta II within 2.5 min of addition, an effect that persisted for as long as 30 min. It should be noted that prior studies in our laboratory have demonstrated that PKC-zeta was unresponsive to 1,25(OH)2D3 or TPA in Caco-2 cells (6).


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Fig. 5.   1,25(OH)2D3 causes increased association of protein kinase C (PKC)-alpha with particulate fraction of Caco-2 cells. Caco-2 cells were treated with 1,25(OH)2D3 (100 nM) or vehicle for indicated times in minutes (controls, C, were at 0 and 30 min). Particulate fractions were prepared by subcellular fractionation as described in METHODS. A: Western blots of particulate PKC isoform abundance. Cellular proteins (20 µg/lane) were separated by electrophoresis on 10% SDS-polyacrylamide gels and electrotransferred onto nitrocellulose membranes, which were immunoblotted with monoclonal anti-PKC-alpha or polyclonal PKC-beta I, -beta II or -delta antibodies. Immunoblots are representative of 3 independent experiments. B: quantitative Western blotting of PKC-alpha translocation. Western blots of 3 independent experiments were quantified, and data are expressed as particulate percentage of total PKC-alpha immunoreactivity. SE was <10% in all cases and was omitted to simplify figure. * P < 0.05 for 60 and 90 s compared with control.


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Fig. 6.   TPA acutely activates PKC-alpha , -beta I, and -delta in Caco-2 cells. Cells were treated with TPA (200 nM) or vehicle for indicated times. After cell lysis, particulate and soluble fractions were prepared as described in METHODS. A: Western blot of particulate abundance of specific PKC isoforms. Each fraction (20 µg protein/lane) was analyzed by 10% SDS-polyacrylamide gel electrophoresis and Western blotting using isoform-specific antibodies. Immunoblots are representative of 3 independent experiments. B: quantitative Western blotting of particulate fractions of PKC-alpha , -beta I, and -delta . After scanning densitometry, data are expressed as particulate percentage of total isoform-specific PKC immunoreactivity. For each of isoforms PKC-alpha , -beta I, and -delta translocation to particulate fraction was significant for times 2.5-30 min. SE was <10% in all cases and was omitted to simplify figure. PKC-beta II did not change with TPA (data not shown).

To further address the potential role of these PKC isoforms in the activation of PLD by 1,25(OH)2D3 or TPA, Caco-2 cells were chronically treated with TPA (1 µM final concentration) for 24 h, and the protein expression of each of the five isoforms of PKC present in these cells was examined. As shown in Fig. 7A, after 24 h of exposure to TPA, the total protein expression of PKC-alpha , -beta I, and -delta was reduced by 95, 40, and 95%, respectively, whereas neither the expression of PKC-beta II or -zeta was changed (data not shown), in agreement with previous studies (6, 54).


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Fig. 7.   Chronic TPA treatment inhibits acute PLD stimulation by 1,25(OH)2D3 or TPA. PKC was downregulated by chronic TPA treatment of Caco-2 cells with TPA (1 µM) or vehicle (DMSO) for 24 h. A: Western blots of PKC isoforms. Cells were lysed by boiling in SDS buffer, and PKC isoforms were detected by Western blotting as described in Fig. 6. Quantitation of total PKC-alpha , -beta I, and -delta immunoreactivity is expressed as percentage of control (DMSO treated) cells (open bars). TPA-treated samples are shown with solid bars. Inset: Western blots of total isoform-specific PKC expression, representative of 3 independent experiments. B: agonist-stimulated PLD activity. Caco-2 cells, pretreated with 1 µM TPA (solid bars) or DMSO (open bars) for 24 h, were stimulated for 20 min with 100 nM 1,25(OH)2D3 or 200 nM TPA. Lipids were extracted and [3H]Pbt was quantified as described in METHODS (n = 3 independent experiments each in triplicate). * P < 0.05 compared with cells not treated with chronic TPA.

Based on these findings, we next examined the ability of 1,25(OH)2D3 or TPA to acutely activate PLD after chronic TPA treatment of these cells, i.e., when the protein expression of PKC-alpha , -beta I, and -delta were significantly downregulated. As shown in Fig. 7B, under these experimental conditions the stimulation of PLD by 1,25(OH)2D3 or TPA was reduced by 70 and 90%, respectively. Taken together, these findings suggested that PKC-alpha is the predominant isoform involved in the activation of PLD by 1,25(OH)2D3, whereas, separately or in combination, PKC-alpha , -beta I, and/or -delta may be involved in the activation of PLD by TPA.

Because PKC-alpha and -beta I are Ca2+-dependent isoforms, whereas PKC-delta is a Ca2+-independent isoform, the effects of extracellular Ca2+ on the stimulation of PLD by 1,25(OH)2D3 or TPA were examined. Caco-2 cells were incubated in medium containing 2 mM Ca2+ or in medium nominally free of Ca2+ (containing 2 mM EGTA with no added Ca2+) and treated acutely with 1,25(OH)2D3 or TPA. It bears emphasis that previous studies by our laboratory utilizing the Ca2+-sensitive fluorescent dye fura 2 showed that the [Ca2+]i levels in these cells were markedly reduced under the aforementioned Ca2+-free conditions (58). As shown in Fig. 8A, in the absence of extracellular Ca2+ the ability of 1,25(OH)2D3 or TPA to stimulate PLD was markedly diminished compared with their ability to activate PLD in the presence of extracellular Ca2+.


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Fig. 8.   Role of Ca2+ in PLD stimulation. [3H]myristic acid-labeled Caco-2 cells were treated with indicated agents, and lipids were then extracted and [3H]Pbt was quantified as described in METHODS (n = 3 independent experiments, each in triplicate). A: effect of chelation of extracellular Ca2+ on PLD stimulation. Cells were incubated for 20 min with 100 nM 1,25(OH)2D3 or 200 TPA in presence of buffer containing 2 mM Ca2+ (open bars) or 2 mM EGTA (solid bars). * P < 0.05 compared with cells incubated in 2 mM Ca2+. B: effect of PKC downregulation on PLD stimulation by ionomycin. Cells were preincubated with TPA (chronic TPA, 1 µM, 24 h) or DMSO and then stimulated with ionomycin (400 nM, 20 min). * P < 0.05 compared with cells treated with ionomycin alone. C: effect of inhibition of Ca2+-dependent PKC isoforms on PLD stimulation. Cells were preincubated with Gö-6976 (2 µM, 3 h, solid bars) or vehicle (open bars) and then stimulated with 100 nM 1,25(OH)2D3 or 200 nM TPA for 20 min. * P < 0.05 compared with cells not treated with Gö-6976.

The inhibition of PLD stimulation by Ca2+ chelation may reflect a direct Ca2+ requirement for PLD enzyme activity and/or the effect of limiting the activity of Ca2+-dependent PKC isoforms possibly required for PLD activation. In this regard, treatment of these cells with the Ca2+ ionophore ionomycin (400 nM, 20 min) stimulated PLD activity. As shown in Fig. 8B, chronic TPA treatment (1 µM, 24 h), however, significantly inhibited the ability of this ionophore to stimulate PLD. It should also be noted that basal PLD activity was not affected by chronic TPA treatment of these cells (data not shown). These observations suggest that increased intracellular Ca2+ alone is insufficient to activate PLD after downregulation of phorbol ester-sensitive PKC isoforms. To further clarify the identity(ies) of the PKC isoform(s) potentially involved in the activation of PLD by 1,25(OH)2D3 or TPA, we next utilized the indolocarbazole Gö-6976, a specific inhibitor of Ca2+-dependent isoforms of PKC (34). As shown in Fig. 8C, preincubation of cells with 2 µM Gö-6976 markedly reduced the ability of 1,25(OH)2D3 (by 80%) or TPA (by 95%) to stimulate PLD.

Taken together, these observations indicated that PKC-alpha was involved in the activation of PLD by 1,25(OH)2D3, whereas PKC-alpha and possibly PKC-beta I, another Ca2+-dependent PKC isoform, but not PKC-delta , were required for the activation of this enzyme by TPA. To determine whether PKC-beta I was involved in the TPA-induced stimulation of PLD, we therefore incubated Caco-2 cells for 20 min with DOPPA (200 nM final concentration), a phorbol ester previously shown to specifically activate PKC-beta I and -beta II but not other isoforms of PKC (47). In agreement with prior studies in other cell types (47), DOPPA was found to activate PKC-beta I and -beta II but not other PKC isoforms present in Caco-2 cells as assessed by their translocation to the particulate fraction (Fig. 9A). DOPPA, however, failed to activate PLD (Fig. 9B), indicating that PKC-beta I was unlikely to be involved in the stimulation of PLD by TPA. Finally, a cell-permeant specific inhibitor of the PKC-beta isoforms (23), Lilly compound 379196 failed to alter TPA-induced stimulation of PLD (Fig. 9B), suggesting that PKC-alpha , but not -beta I, is the predominant PKC isoform mediating the actions of TPA on PLD. Similarly, pretreatment with Lilly compound 379196 failed to block PLD stimulation in response to 1,25(OH)2D3 (data not shown).


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Fig. 9.   12-Deoxyphorbol-13-phenylacetate-20-acetate (DOPPA) activates PKC-beta I but not PLD. A: Western blots of TPA- and DOPPA-induced PKC-alpha and beta I translocation. Caco-2 cells were treated with TPA (200 nM) or DOPPA (200 nM) or vehicle (DMSO, control) for 20 min. After cell lysis particulate fractions were prepared and PKC isoform expression in each fraction was detected as described in METHODS. B: phorbol ester-induced PLD activation. [3H]myristic acid-labeled Caco-2 cells were stimulated with TPA (200 nM) or DOPPA (200 nM) for 20 min. In some samples, before TPA treatment, cells were preincubated with Lilly compound 379196 (50 nM, 30 min). Lipids were then extracted, and [3H]Pbt was quantified as described in METHODS. Lilly compound 379196 alone had no effect on PLD activity (data not shown; n = 3 independent experiments, each in triplicate). * P < 0.05 compared with vehicle-treated cells.

Changes in expression of PKC-alpha modulate the ability of 1,25(OH)2D3 and TPA to stimulate PLD in Caco-2 cells. Our laboratory has recently established and characterized several clones of Caco-2 cells stably transfected with sense or antisense cDNA for PKC-alpha , which overexpress (3-fold increased) or underexpress (95% decreased), respectively, this isoform (48). Moreover, these clones displayed no significant alterations in the expression of the other nontargeted isoforms of PKC present in these cells (48). We therefore utilized these transfected cells to determine whether alterations in the expression of PKC-alpha affected the ability of 1,25(OH)2D3 or TPA to stimulate PLD. As shown in Fig. 10, Caco-2 sense transfectants, which overexpressed PKC-alpha , had a significant increase in their ability to activate PLD by either 1,25(OH)2D3 or TPA compared with empty vector transfected Caco-2 cells. In contrast, in antisense transfected cells with inhibited PKC-alpha expression, there was a marked reduction in the ability of either of these agents to stimulate PLD compared with their control (empty vector transfected) counterparts.


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Fig. 10.   Amplified PKC-alpha expression in Caco-2 cells enhances PLD activation by 1,25(OH)2D3 and TPA. Caco-2 cells were stably transfected with pRC/CMV vector alone (hatched bars) or pRC/CMV containing sense (open bars) or antisense (solid bars) cDNA coding for PKC-alpha . Cells were labeled with [3H]myristic acid and then treated with 1,25(OH)2D3 (100 nM) or TPA (200 nM) for 20 min. [3H]Pbt was extracted and quantified as described in METHODS (n = 3 independent experiments, each in triplicate). * P < 0.05 compared with agonist-treated empty vector transfected cells.

PKC-independent activation of PLD by 1,25(OH)2D3. The aforementioned studies clearly establish the role of PKC, particularly PKC-alpha , in the stimulation of PLD by 1,25(OH)2D3. The persistence of some PLD residual activity (20-30%) induced by this secosteroid, however, in cells pretreated with GF-109203x, Gö-6976, or chronically exposed to phorbol ester, suggested that there may also be a PKC-independent mechanism involved in the stimulation of PLD by this secosteroid. To further address this issue, we therefore determined whether the concentrations of these PKC inhibitors used in the present studies indeed inhibited the ability of 1,25(OH)2D3 to activate PKC, as assessed by PKC substrate phosphorylation activity in situ. As assessed by this in situ assay and as shown in Fig. 11, both GF-109203x and Gö-6976 abolished the secosteroid-stimulated phosphorylation of a PKC-specific substrate at inhibitor concentrations shown to block only 70-80% of PLD activation by 1,25(OH)2D3. As also shown in Fig. 11, chronic TPA treatment of these cells was found to abolish the ability of this secosteroid to stimulate PKC phosphorylating activity.


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Fig. 11.   Chronic phorbol ester treatment and PKC inhibitors block ability of 1,25(OH)2D3 to stimulate PKC phosphorylating activity in situ. Caco-2 cells, plated in 24-multiwell plates, were grown to 70% confluence. Cells were preincubated with 5 µM GF-109203x or 2 µM Gö-6976 or their appropriate vehicles for 3 h. In chronic TPA experiments to downregulate PKC, cells were preincubated for 24 h in DMEM containing 0.5% serum and 1 µM TPA or vehicle (DMSO, control). After preincubation with chronic TPA or PKC inhibitors or appropriate vehicles, cells were treated with 100 nM 1,25(OH)2D3 in presence of permeabilizing phosphorylation reaction mixture containing digitonin, [gamma -32P]ATP, and PKC-specific substrate peptide derived from myelin basic protein (Ac-MBP) as described in METHODS. 32P-labeled Ac-MBP was then measured by scintillation counting, and data were expressed as percentage increase above control. * P < 0.05 compared with cells treated with 1,25(OH)2D3 alone. Note that 1,25(OH)2D3-stimulated PKC activity in presence of GF-109203x, Gö-6976, or after chronic TPA treatment was not different from that of unstimulated cells.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The present studies demonstrate for the first time that 1,25(OH)2D3 and TPA, in time- and concentration-dependent manners, activated PC-PLD in Caco-2 cells. These findings are in keeping with the results of prior studies in chick myoblasts and rat skeletal muscle in which 1,25(OH)2D3 activated PLD (16, 39), as well as those in several noncolonic cell types in which TPA activated this enzyme (2, 9, 37).

Prior studies in intact cells and/or in cell-free systems have also demonstrated that one or more isoforms of the PKC family of phospholipid-dependent, serine-threonine kinases can mediate the activation of PLD by phorbol esters or by a variety of other agonists (2, 8, 12, 19, 24, 30, 32, 33, 36, 42-44, 46, 50). The majority of these studies moreover have implicated the Ca2+-dependent PKC isoforms, PKC-alpha , and/or PKC-beta I or -beta II, in these activation events (2, 12, 13, 24, 30, 32, 33, 36, 42-44). In contrast to these findings, McKinnon and Parker (35) showed that phorbol esters not only failed to activate PLD in COS-1 cells but actually inhibited this enzymatic activity via activation of PKC-alpha . Moreover, in a cell-free system (36), PKC-alpha in the presence of ATP was recently shown to phosphorylate purified rat PLD and inhibit its catalytic activity. In other studies, the in vitro administration of TPA, as well as other agonists, was found to stimulate PLD activity in rat renal mesangial cells by a PKC-epsilon -dependent mechanism (46). Taken together, these findings indicated that, depending on the agonist and cell type, both Ca2+-dependent and -independent isoforms of PKC may regulate PC-PLD activity but in a complex manner.

In prior studies, our laboratory has shown that Caco-2 cells possessed PKC-alpha and -zeta (6). Furthermore, only PKC-alpha was found to be activated by 1,25(OH)2D3 or TPA in these cells (6). More recently, however, utilizing newly available and more specific antibodies in conjunction with Western blotting techniques, we have shown that Caco-2 cells, in addition to PKC-alpha and -zeta , also possessed PKC-beta I, -beta II, and -delta (1, 48). In the present experiments, several lines of evidence indicated that PKC-alpha was the principal isoform of PKC involved in the activation of PLD by 1,25(OH)2D3 or TPA. In this regard, 1,25(OH)2D3 was found to specifically activate this Ca2+-dependent PKC isoform, as assessed by its translocation from cytosol to particulate fraction but not the other Ca2+-dependent (PKC-beta I and -beta II) or -independent isoforms (PKC-delta and -zeta ) present in these cells. In keeping with this finding, both the removal of extracellular Ca2+ from the medium, as well as preincubation of Caco-2 cells with Gö-6976, a specific inhibitor of Ca2+-dependent PKC isoforms, markedly reduced the ability of 1,25(OH)2D3 to activate PLD. Moreover, the ability of 1,25(OH)2D3 to activate PLD was significantly enhanced in Caco-2 cells overexpressing PKC-alpha and markedly diminished in cells with inhibited expression of this PKC isoform. Taken together, these findings indicate that PKC-alpha was the principal PKC isoform involved in the stimulation of PLD by 1,25(OH)2D3. As previously shown by our laboratory (7), 1,25(OH)2D3 activates PKC-alpha via increases in DAG and [Ca2+]i generated by polyphosphoinositide hydrolysis in these cells. Prior activation of PI-PLC, observed within 2 min, therefore appears to be required for the stimulation of PLD, which occurs 5 min after the addition of this secosteroid to these cells.

It bears emphasis, however, that the present studies, utilizing chronic administration of TPA to downregulate PKC isoforms in these cells, suggests that 1,25(OH)2D3 may also activate PLD by a mechanism independent of PKC-alpha . After this treatment, the expression of PKC-alpha was reduced by 95% as was the ability of acute TPA treatment to stimulate PLD. In contrast, the decrease in the ability of 1,25(OH)2D3 to activate PLD in these PKC-downregulated cells was only 70%. This discrepancy indicates that this secosteroid may also stimulate PLD via a PKC-alpha -independent mechanism. 1,25(OH)2D3 may, for example, activate PLD via stimulation of a PKC isoform not yet identified in these cells, which cannot be downregulated by chronic treatment with TPA. Alternatively, this secosteroid may be acting via a mechanism not involving PKC. Although further studies will be necessary to clarify this issue, we currently favor the latter possibility because 1,25(OH)2D3 stimulation of PLD was reduced by only 70% in cells pretreated with GF-109203x, an inhibitor of both Ca2+-dependent and -independent isoforms of PKC, and with Gö-6976, an inhibitor of Ca2+-dependent PKC isoforms. Moreover, the complete inhibition of PKC biochemical activation by 1,25(OH)2D3 after incubation with these inhibitors, as well as with chronic phorbol ester treatment of Caco-2 cells, strongly supports such a PKC-independent mechanism.

The present studies also indicate that PKC-alpha is the predominant PKC isoform involved in the activation of PLD by TPA. TPA was found to acutely activate PKC-alpha , -beta I, and -delta , but not PKC-beta II or -zeta in Caco-2 cells. Although chronic TPA treatment (1 µM, 24 h) markedly reduced the expression of PKC-delta (~95%), as well as that of PKC-alpha (~95%) and -beta I (~40%), concomitant with a significant reduction in the ability of this phorbol ester to acutely stimulate PLD, we believe it is highly unlikely that PKC-delta or -beta I are involved in the activation of this enzyme by TPA. The ability of TPA to stimulate PLD in these cells, as in the case of 1,25(OH)2D3, required a Ca2+-dependent isoform of PKC, as evidenced by the requirement for extracellular Ca2+, as well as by the significant inhibition of PLD stimulation by TPA when cells were preincubated with Gö-6976, a specific inhibitor of the Ca2+-dependent PKC isoforms. These latter observations do not support a role for the Ca2+-independent isoform, PKC-delta , in the stimulation of PLD by TPA. Moreover, the failure of DOPPA, a specific activator of PKC-beta isoforms to stimulate PLD, and of the Lilly compound 379196, a specific inhibitor of these PKC isoforms, to significantly block the activation of PLD by TPA also provides evidence against a role for PKC-beta I in this biochemical event. These findings, taken together with our studies in transfected Caco-2 cells demonstrating that overexpression of PKC-alpha markedly enhanced the ability of TPA to activate PLD, whereas the inhibited expression of this isoform markedly reduced TPA's stimulation of this enzyme, indicate that PKC-alpha plays a major role in this phorbol ester-induced phenomenon in Caco-2 cells.

The exact mechanism(s) by which the activation of PKC-alpha by 1,25(OH)2D3 or TPA leads to PLD stimulation in Caco-2 cells remains to be elucidated. Because, however, both GF-109203x and Gö-6976 markedly reduced the stimulation of PLD by these agents and, as shown in the present studies, as well as by others (34, 56), concomitantly inhibited the phosphorylating activity of PKC, it would appear that PLD activation by PKC-alpha may occur via a phosphorylation-dependent mechanism. This will, however, require further study.

Prior studies by our laboratory (61) and others (3, 45) have demonstrated that vitamin D3 metabolites-analogs prevent the development of tumors in experimental models of colonic carcinogenesis. Although the mechanism(s) involved in the chemopreventive actions of these secosteroids remains in large part unclear, our laboratory has demonstrated that 1,25(OH)2D3 deceases the proliferation and increases the differentiation of Caco-2 cells (18). The present observations that 1,25(OH)2D3 activates PLD in these cells via a PKC-alpha -dependent mechanism are therefore of considerable interest in view of prior studies that have shown that 1) increases in the activity and/or expression of PKC-alpha are intimately involved in the regulation of proliferation and differentiation of Caco-2 cells (1, 48) and 2) PKC-mediated activation of PLD can regulate these important processes in other cell types (15). Taken together, these present and prior observations therefore suggest that the effects of 1,25(OH)2D3 on Caco-2 cell growth and/or differentiation may involve the PKC-alpha -mediated activation of PLD, which may moreover be involved in the chemopreventive actions of vitamin D3 analogs in colonic carcinogenesis. Further studies, however, will be necessary to address these important issues.

In addition to PKC, monomeric and heterotrimeric guanine nucleotide binding proteins (G proteins), as well as nonreceptor tyrosine kinases, such as pp60c-src, have recently been shown to regulate the stimulation of PC-PLD by a variety of agonists in other cell types. In the accompanying study (26a), we have therefore examined the potential roles of these signal transduction elements in the activation of PC-PLD by 1,25(OH)2D3 and TPA in Caco-2 cells.


    ACKNOWLEDGEMENTS

We thank Lynn Kaczmarz for excellent secretarial assistance.


    FOOTNOTES

These studies were supported in part by the Samuel Freedman Cancer Laboratory, as well as the National Institute of Diabetes and Digestive and Kidney Diseases Grants P30-DK-42086 (Digestive Diseases Research Core Center, T. A. Brasitus), P30-DK-26678 (Clinical Nutrition Research Center, M. D. Sitrin), 5T32-DK-07074-20 (B. Scaglione-Sewell), and DK-39573 (T. A. Brasitus, M. D. Sitrin, and M. Bissonnette) and the National Cancer Institute Grants CA-36745 (T. A. Brasitus and M. Bissonnette) and CA-69532 (M. Bissonnette).

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: T. A. Brasitus, Dept. of Medicine, MC 4076, Univ. of Chicago Hospitals & Clinics, 5841 S. Maryland Ave., Chicago, IL 60637 (E-mail: tbrasitu{at}medicine.bsd.uchicago.edu).

Received 10 August 1998; accepted in final form 7 December 1998.


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Am J Physiol Gastroint Liver Physiol 276(4):G993-G1004
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