1,25-Dihydroxyvitamin D3 but not TPA activates PLD in Caco-2 cells via pp60c-src and RhoA

Sharad Khare1, Marc Bissonnette1, Ramesh Wali1, Susan Skarosi1, Gerry R. Boss2, Friederike C. von Lintig2, Beth Scaglione-Sewell1, Michael D. Sitrin1, and Thomas A. Brasitus1

1 Department of Medicine, University of Chicago, Chicago, Illinois 60637; and 2 Department of Medicine, University of California, San Diego, California 92093-0652


    ABSTRACT
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DISCUSSION
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In the accompanying paper [Khare et al., Am. J. Physiol. 276 (Gastrointest. Liver Physiol. 39): G993-G1004, 1999], activation of protein kinase C-alpha (PKC-alpha ) was shown to be involved in the stimulation of phospholipase D (PLD) by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and 12-O-tetradecanoylphorbol 13-acetate (TPA) in Caco-2 cells. Monomeric or heterotrimeric G proteins, as well as pp60c-src have been implicated in PLD activation. We therefore determined whether these signal transduction elements were involved in PLD stimulation by 1,25(OH)2D3 or TPA. Treatment with C3 transferase, which inhibits members of the Rho family of monomeric G proteins, markedly diminished the ability of 1,25(OH)2D3, but not TPA, to stimulate PLD. Brefeldin A, an inhibitor of ADP-ribosylation factor proteins, did not, however, significantly reduce the stimulation of PLD by either of these agents. Moreover, 1,25(OH)2D3, but not TPA, activated pp60c-src and treatment with PP1, a specific inhibitor of the pp60c-src family, blocked the ability of 1,25(OH)2D3 to activate PLD. Pretreatment of cells with pertussis toxin (PTx) markedly reduced the stimulation of PLD by either agonist. PTx, moreover, inhibited the stimulation of pp60c-src and PKC-alpha by 1,25(OH)2D3. PTx did not, however, block the membrane translocation of RhoA induced by 1,25(OH)2D3 or inhibit the stimulation of PKC-alpha by TPA. These findings, taken together with those of the accompanying paper, indicate that although 1,25(OH)2D3 and TPA each activate PLD in Caco-2 cells in part via PKC-alpha , their stimulation of PLD differs in a number of important aspects, including the requirement for pp60c-src and RhoA in the activation of PLD by 1,25(OH)2D3, but not TPA. Moreover, the requirement for different signal transduction elements by 1,25(OH)2D3 and TPA to induce the stimulation of PLD may potentially underlie differences in the physiological effects of these agents in Caco-2 cells.

calcitriol; phorbol esters; pertussis toxin; G proteins; signal transduction


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INTRODUCTION
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REFERENCES

IN THE ACCOMPANYING PAPER we have demonstrated that protein kinase C-alpha (PKC-alpha ) is intimately involved in the activation of phosphatidylcholine-phospholipase D (PC-PLD) by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and 12-O-tetradecanoylphorbol 13-acetate (TPA) in Caco-2 cells (24a). In addition to PKC, both monomeric and heterotrimeric guanine nucleotide binding proteins (G proteins) have been implicated in the regulation of PLD activity in other cell types (12, 16, 18, 19, 24, 28, 31-33, 39, 44). Guanosine 5'-O-(3-thiotriphosphate), for example, was shown to activate PLD in isolated membranes or permeabilized cells, suggesting the involvement of one or both types of these G proteins in this phenomenon (26). Members of the Rho family and ADP-ribosylation factor (Arf) monomeric GTPases, as well as pertussis toxin (PTx)-sensitive and -insensitive heterotrimeric G proteins, have in fact been shown to be involved in the activation of PLD (26, 27).

Other studies have demonstrated that PLD can be stimulated in response to growth factors, acting via tyrosine kinase receptors (2, 9, 20, 45). Increases in PLD activity have also recently been reported in cells transformed by v-ras and v-src oncogenes (10, 21), as well as by the activation of the nonreceptor tyrosine kinase, pp60c-src (23). In this regard, our laboratory has recently shown that 1,25(OH)2D3, but not TPA, activated pp60c-src in colonocytes, which in turn induced a significant increase in the tyrosine phosphorylation, plasma membrane association, and biochemical activation of phosphoinositide-specific phospholipase C-gamma (PI-PLC-gamma ) (25). Moreover, as shown in these studies (25), prior activation of PI-PLC-gamma was required for the activation of PKC-alpha by 1,25(OH)2D3 in Caco-2 cells.

Based on these considerations, in the present studies it was therefore of interest to determine whether, in addition to PKC-alpha , one or more monomeric or heterotrimeric G proteins, as well as pp60c-src and/or p21ras influenced the activation of PLD by 1,25(OH)2D3 or TPA in Caco-2 cells. Because, as previously noted, 1,25(OH)2D3 also appeared to activate PLD, at least in part by a PKC-independent mechanism, we were particularly interested in determining whether this secosteroid required one or more of the aforementioned signal transduction elements to stimulate PLD (24a). The results of these experiments, as well as a discussion of their potential biological significance, serve as the basis for this report.


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Materials. Upstate Biotechnology (Lake Placid, NY) provided monoclonal anti-PKC-alpha , anti-Src antibodies, and a Src peptide substrate derived from p34cdc-2. Polyclonal anti-RhoA and anti-Ras Y13-259 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Polyvinylidene difluoride membranes (Immobilon-P) were purchased from Millipore (Bedford, MA). Amersham (Arlington Heights, IL) provided the enchanced chemiluminescence Western blotting protein detection kit, the peroxidase-coupled sheep anti-mouse and donkey anti-rabbit antibodies, [gamma -32P]ATP, and [3H]myristic acid. PKC substrate peptide derived from myelin basic protein, Ac-MBP4-14, and phosphocellulose disks were obtrained from GIBCO BRL-Life Technologies (Gaithersburg, MD). Phosphatidylbutanol (Pbt) was purchased from Avanti Polar Lipids (Alabaster, AL). PP1 was provided by Pfizer Central Research (Gorton, CT). Brefeldin A and protein A- and G-agarose were purchased from Sigma Chemical (St. Louis, MO). Fura 2-acetoxymethyl ester (fura 2-AM) was obtained from Molecular Probes (Eugene, OR). PTx, cholera toxin, and C3 transferase were purchased from List Biological Laboratories (Campbell, CA). Unless otherwise noted, all other reagents were obtained from Sigma Chemical or Fisher Scientific (Springfield, NJ) and were of the highest purity available.

PLD assay. Caco-2 cells were plated in 25-cm2 flasks, grown to 70% confluency, and scrape-loaded with C3 transferase according to the method of Malcolm et al. (32). Briefly cells were scraped in 500 µl of scrape-loading buffer (in mM: 10 Tris · HCl, pH 7.2, 114 KCl, 25 NaCl, 5.5 MgCl2) either alone or in the presence of 5 µg/ml C3 transferase, using a rubber policeman. Equal volumes of cell suspensions were then plated in six-well plates and metabolically labeled overnight with [3H]myristic acid (1 µCi/ml) in medium containing 0.5% fetal bovine serum (FBS). Cultures were rinsed twice with serum-free medium and equilibrated in fresh medium at 37°C for 1 h. Cells were then treated with 1,25(OH)2D3 (100 nM final concentration) or TPA (200 nM final concentration) for 20 min, and PLD activity was measured as described previously (24a). In some experiments, cells were preincubated with vehicle or brefeldin A (50 µg/ml), an antagonist of Arf proteins, for 30 min and then stimulated with agonists. To examine the role of pp60c-src in PLD stimulation, [3H]myristic acid-labeled cells were pretreated with PP1 (10 µM) or vehicle (DMSO) for 15 min and then stimulated with 1,25(OH)2D3 (100 nM final concentration) for 20 min. Pbt, the transphosphatidylation product of the PLD reaction, was measured as described (24a). In some experiments, Caco-2 cells were pretreated either with PTx (100 ng/ml, 18 h) or cholera toxin (100 ng/ml, 2 h) and then stimulated with agonists.

RhoA expression and translocation in Caco-2 cells. To examine the expression and translocation of RhoA, Caco-2 cells were grown in 25-cm2 flasks. At 70-80% confluency cells were preincubated for 30 min in HEPES buffer containing (in mM) 10 HEPES, pH 7.2, 140 NaCl, 5 KCl, 5 MgCl2, 2 CaCl2, and 10 glucose. Cells were then stimulated with 1,25(OH)2D3 (100 nM final concentration) or TPA (200 nM final concentration) for the indicated times, lysed in extraction buffer containing 25 mM Tris, pH 7.6, 5 mM EGTA, 10 µg/ml aprotinin, and 10 µM 4-(2-aminoethyl)-benzene sulfonyl fluoride (AEBSF) and fractionated into soluble and particulate components by ultracentrifugation as described earlier (24a). Particulate fractions were immediately boiled in Laemmli SDS-buffer, and RhoA was detected by Western blotting using rabbit polyclonal anti-RhoA (0.1 µg/ml) as described (24a). To examine the total expression of RhoA, cells were scrape-loaded with C3 transferase or vehicle and plated overnight in medium containing 0.5% FBS. Cellular proteins were then solubilized in SDS-Laemmli buffer, and RhoA was detected in total cell lysates by Western blotting as previously described.

pp60c-src Immunoprecipitation and kinase assay. Cells were stimulated with vehicle or 1,25(OH)2D3 (100 nM final concentration) or TPA (200 nM final concentration) for the indicated times and rapidly lysed in modified RIPA buffer containing 50 mM Tris · HCl, pH 7.4, 1% nonidet P-40 (NP-40), 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM sodium vanadate, 100 µM AEBSF, 10 µg/ml leupeptin, and 10 µg/ml aprotinin. Lysates were precleared with 50 µl of protein A-agarose beads and centrifugated at 14,000 rpm in a microcentrifuge for 10 min. The soluble fraction (500 µg protein, 1 mg/ml) was incubated with 2 µg of anti-pp60c-src monoclonal antibodies (Upstate Biotechnology, 05-184) for 2 h. Immune complexes were collected by incubation for 12 h with 50 µl of a 50% mixture of protein A- and protein G-agarose and 5 µg goat anti-mouse antibodies, followed by centrifugation. Immunoprecipitates were washed three times in the extraction buffer and once with buffer containing 50 mM Tris · HCl, pH 7.5, and 10 mM MgCl2.

Immunoprecipitates of pp60c-src were assayed for kinase activity at 30°C for 20 min by 32P-incorporation into a pp60c-src peptide substrate derived from p34cdc-2, cdc-2 (6-20)NH2, as previously described by our laboratory (25). The kinase buffer contained 50 mM Tris · HCl, pH 7.5, 10 mM MgCl2, 50 µM ATP (2 µCi/assay), and 200 µM p34cdc-2 peptide substrate. The reactions were terminated by spotting the supernatant on P81 phosphocellulose disks and washed three times in 75 mM phosphoric acid, and the radioactivity was quantified by Cherenkov counting. Parallel immunoprecipitates were probed for pp60c-src abundance to ensure comparable kinase mass in samples from control and agonist-treated cells.

In other experiments, Caco-2 cells were pretreated with PTx (100 ng/ml, 18 h) and then stimulated with 1,25(OH)2D3 (100 nM final concentration) for 1 min. Then pp60c-src was immunoprecipitated and kinase activity was assayed as previously described.

Translocation of PKC-alpha . Caco-2 cells were pretreated with PTx (100 ng/ml, 18 h) or vehicle and then stimulated with 1,25(OH)2D3 (100 nM final concentration), TPA (200 nM final concentration), or appropriate vehicles for the indicated times. Particulate and soluble fractions were prepared, and PKC-alpha in particulate fractions was detected by Western blotting as described earlier (24a).

In situ PKC assay. Caco-2 cells were pretreated with PTx (100 ng/ml, 18 h) or vehicle and then stimulated with agonists. PKC-phosphorylating activity was assayed in situ using Ac-MBP as substrate as described previously (24a).

Measurement of [Ca2+]i in Caco-2 cells. Caco-2 cells were pretreated with PTx (100 ng/ml) or vehicle for 18 h and then loaded with the Ca2+-sensitive fluorescence dye fura 2-AM (2 µM final concentration) at 37°C for 30 min. After brief trypsin digestion, the extracellular fura 2-AM was removed by washing four times in HEPES buffer as previously described (3). The fluorescence responses in the fura 2-loaded Caco-2 cells were analyzed using a SLM-4800 C spectrofluorometer (SLM Instruments, Urbana, IL). Cells were maintained in a continuously stirred quartz cuvette and stimulated with 1,25(OH)2D3 (100 nM final concentration), TPA (200 nM final concentration), or appropriate vehicles for 5 min. Fluorescence was measured at excitation wavelengths of 340 or 380 nm, with emission at 505 nm. Signals from the fluorometer were collected and analyzed on an IBM-PC with SLM spectrum processor version 3.2. The intracellular Ca2+ concentration ([Ca2+]i) was calculated according to Grynkiewicz et al. (14) as previously described by our laboratory (3).

Measurements of Ras-bound GTP and GDP. Caco-2 cells were plated and allowed to grow overnight in serum-containing medium. After washing and incubation in serum-free medium for 72 h, cells were treated with 1,25(OH)2D3 (100 nM final concentration) or vehicle for the indicated times. Cells were rapidly scraped at 4°C, collected by centrifugation, and stored at -70°C until assay. Cells were extracted by shaking for 10 min in ice-cold RIPA buffer (10 mM HEPES, pH 7.4, 2 mM MgCl2, 30 mM NaCl, 1% NP-40, 0.5 mM phenylmethylsulfonyl fluoride, and 10 µg/ml each of aprotinin, leupeptin, and pepstatin). The extracts were centrifuged at 10,000 g for 5 min, and the supernatants were divided into two aliquots, which were incubated with either 3 µg of the anti-Ras antibody Y13-259 (Santa Cruz Biotechnology) or 3 µg of rat IgG (Cappel, Malvern, PA). To each aliquot, goat anti-rat IgG and protein G-agarose were added followed by NaCl, SDS, and deoxycholate to final concentrations of 500 mM, 0.05%, and 0.5%, respectively. The samples were shaken gently for 1 h at 4°C, and then the immunoprecipitates were washed four times in RIPA buffer containing 500 mM NaCl, 0.05% SDS, and 0.5% deoxycholate and two times in 20 mM Tris-phosphate buffer, pH 7.4. To elute the Ras-bound guanine nucleotides, the washed immunoprecipitates were resuspended in 30 µl of 5 mM Tris-phosphate, pH 7.4, 2 mM dithiothreitol, 2 mM EDTA (TED buffer) and heated to 100°C for 3 min and then cooled on ice and centrifuged at 10,000 g for 2 min. The immunoprecipitates were washed with an additional 15 µl of TED buffer, which was combined with the first 30 µl of TED. Eluted GTP was then quantitatively converted to ATP using NDP kinase and ADP with the ATP measured in the luciferase-luciferin system involving coupled reactions
GTP + ADP   <AR><R><C>NDP kinase</C></R><R><C><LIM><OP><ARROW>→</ARROW></OP></LIM></C></R><R><C></C></R></AR>   GDP + ATP
ATP + luciferin  <AR><R><C>luciferase</C></R><R><C><LIM><OP><ARROW>→</ARROW></OP></LIM></C></R><R><C></C></R></AR>  oxyluciferin + AMP + pyrophosphate + &ggr; (light)
To measure GDP, the nucleotide was converted to GTP, using pyruvate kinase and phosphoenolpyruvate
GDP + phospho<IT>enol</IT>pyruvate   <AR><R><C>pyruvate kinase</C></R><R><C><LIM><OP><ARROW>→</ARROW></OP></LIM></C></R><R><C></C></R></AR>   GTP + pyruvate
GTP was then quantified as previously described. The reaction mixture (15 µl), containing 50 mM glycine, pH 7.8, 10 mM dithiothreitol, 8 mM MgSO4, 50 µM phosphoenolpyruvate, 3 mU pyruvate kinase, and 5 µl of sample or GDP standard was incubated for 30 min at 30°C. The amounts of GDP and GTP in the samples were determined from standard curves prepared with each set of samples, and the data were expressed as femtomoles of GTP or GDP per microgram DNA or milligram protein in the cell lysate. Each of these assays was sensitive to 1 fmol of GTP or GDP, respectively, and was performed as described previously (43). DNA was measured by a standard fluorescence method using the fluorescent dye bis-benzimidazole, and protein was measured by the Bradford assay (6).

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


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Members of Rho family of proteins modulate ability of 1,25(OH)2D3 but not TPA to activate PLD. As noted earlier, members of the Rho family of monomeric G proteins, particularly RhoA, have been shown to modulate the effects of various agonists on the activity of PLD in several cell types (13, 18, 32, 44). Caco-2 cells were therefore treated with a C3 transferase from Clostridium botulinum, which ADP-ribosylates and thereby inactivates members of the Rho family (33), to determine if Rho proteins were involved in the activation of PLD by either 1,25(OH)2D3 or TPA. As shown in Fig. 1A, pretreatment of Caco-2 cells with C3 transferase markedly reduced the ability of 1,25(OH)2D3 to activate PLD, whereas it had no significant effect on the TPA-induced stimulation of PLD.


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Fig. 1.   Rho is involved in stimulation of phospholipase D (PLD) by 1,25-dihydroxyvitamin D [1,25(OH)2D3] but not by 12-O-tetradecanoylphorbol 13-acetate (TPA). A: effect of C3 transferase on agonist-stimulated PLD activity. Caco-2 cells were metabolically labeled with [3H]myristic acid and scrape-loaded with (solid bars) or without (open bars) C3 transferase (10 µg/ml) as described in METHODS and then stimulated with 1,25(OH)2D3 (100 nM) or TPA (200 nM) for 20 min. Lipids were extracted, and [3H]phosphatidylbutanol (Pbt) was quantified (n = 3 independent experiments, each in triplicate; * P < 0.05 compared with cells without C3 transferase treatment). B: quantitative Western blotting of 1,25(OH)2D3-stimulated RhoA translocation. Caco-2 cells were treated with 1,25(OH)2D3 (100 nM) or vehicle for indicated times in minutes. Particulate fractions were prepared by subcellular fractionation as described in METHODS. Cellular proteins from particulate fraction (20 µg/lane) were separated by electrophoresis on 10% SDS-polyacrylamide gels and electrotransferred onto nitrocellulose membranes, which were immunoblotted with polyclonal anti-RhoA antibodies. Western blots of 2 independent experiments were quantified by scanning densitometry, and data were expressed as particulate RhoA percent above control. Note control values were not significantly different at 0 and 30 min. C: effect of C3 transferase treatment on RhoA expression. Caco-2 cells were scrape-loaded with medium alone or with C3 transferase (10 µg/ml) and plated overnight. Cells were lysed by boiling in SDS buffer, and RhoA was detected by Western blotting as described. Immunoblots are representative of 2 independent experiments.

Recent studies have indicated that lysophosphatidic acid (LPA), an activator of PLD, causes the translocation of RhoA from the cytosolic to the particulate fraction of cells (32). Furthermore, in this system, C3 transferase reduced the protein expression of RhoA by an as yet unclear mechanism (32). In Caco-2 cells we therefore investigated the effect of 1,25(OH)2D3 on RhoA translocation. We also examined the effect of C3 transferase on RhoA expression in these cells because this toxin blocked PLD stimulation by this secosteroid. As shown in Fig. 1B, in response to the in vitro addition of 1,25(OH)2D3, RhoA rapidly (1-10 min), but transiently, translocated to the particulate fraction of Caco-2 cells. In addition, C3 transferase markedly reduced the protein expression of RhoA in these cells (Fig. 1C).

Because Arf proteins have also been shown to be involved in the stimulation of PLD by a number of agonists in a variety of cells (7, 33), we next asked whether these small monomeric G proteins were also required for the activation of PLD by 1,25(OH)2D3 or TPA in Caco-2 cells. To address this issue, cells were preincubated with brefeldin A, an inhibitor of Arf proteins (7), and then treated with 1,25(OH)2D3 (100 nM final concentration), TPA (200 nM final concentration), or appropriate vehicles for 20 min, and PLD activity was measured. As shown in Fig. 2, under these conditions, this Arf antagonist failed to significantly inhibit the activation of PLD by either of these agonists.


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Fig. 2.   ADP-ribosylation factor proteins are not involved in activation of PLD. [3H]myristic acid-labeled Caco-2 cells were preincubated with brefeldin A (50 µg/ml, 30 min, solid bars) or vehicle (open bars) and then stimulated with 100 nM 1,25(OH)2D3 or 200 nM TPA for 20 min. Lipids were extracted, and [3H]Pbt was quantified as described in METHODS (n = 3 independent experiments, each in triplicate).

Src activation is involved in stimulation of PLD by 1,25(OH)2D3 but not by TPA. Prior studies have shown that coupling of alpha 2A/D-adrenergic receptors to PLD stimulation was regulated by the nonreceptor tyrosine kinase pp60c-src (23). In addition, as noted earlier, our laboratory has recently demonstrated that 1,25(OH)2D3, but not TPA, activated pp60c-src in rat colonocytes (25). We therefore determined whether the activation of PLD by 1,25(OH)2D3 or TPA involved this nonreceptor tyrosine kinase in Caco-2 cells. As shown in Fig. 3A, 1,25(OH)2D3 activated pp60c-src, as assessed by this secosteroid's ability to increase the phosphorylation of the pp60c-src substrate, p34cdc-2, by immunoprecipitated pp60c-src. In contrast to these findings with 1,25(OH)2D3, TPA, however, failed to activate pp60c-src (Fig. 3A). Moreover, in agreement with prior studies by our laboratory in rat colonocytes (25), neither TPA nor 1,25(OH)2D3 altered the protein expression of pp60c-src (data not shown).


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Fig. 3.   pp60c-src is activated by 1,25(OH)2D3 but not TPA and required for PLD stimulation by this secosteroid. A: pp60c-src activity. Caco-2 cells were incubated with 1,25(OH)2D3 (100 nM) or TPA (200 nM) for indicated times and then lysed in extraction buffer. pp60c-src was subsequently isolated by immunoprecipitation, and kinase activity was determined by 32P incorporation into peptide derived from p34cdc-2 as described in METHODS (mean of 2 independent experiments, each in duplicate). B: effect of pp60c-src inhibition on PLD stimulation by 1,25(OH)2D3. [3H]myristic acid-labeled cells were preincubated with PP1 (10 µM, 15 min) or vehicle and then stimulated with 1,25(OH)2D3 (100 nM) for 20 min. Lipids were extracted, and [3H]Pbt was quantified as described in METHODS (n = 3 independent experiments, each in triplicate; * P < 0.05 compared with the cells not treated with PP1).

Based on these observations, we preincubated Caco-2 cells with PP1, a highly specific inhibitor of the c-Src family of nonreceptor tyrosine kinases (25) and then treated these cells with 1,25(OH)2D3. As shown in Fig. 3B, PP1 significantly inhibited the activation of PLD by 1,25(OH)2D3 but not by TPA (data not shown), indicating that pp60c-src was involved in the stimulation of PLD by this secosteroid.

PLD stimulation by 1,25(OH)2D3 is independent of p21ras. Prior studies have shown that 1) pp60c-src can activate p21ras (30), 2) p21ras can stimulate PLD (21, 22), and 3) Rho proteins can be downstream effectors of p21ras (47). Because 1,25(OH)2D3 stimulated pp60c-src activity and RhoA translocation, it was of interest to assess whether 1,25(OH)2D3 could also activate p21ras in Caco-2 cells. After secosteroid treatment of these cells, we immunoprecipitated Ras proteins using the pan Ras antibody Y13-259, and then measured GTP and GDP bound to these proteins by a highly sensitive luciferase-based method as we have previously described (43).

In this regard, 1,25(OH)2D3 (100 nM final concentration) failed to activate p21ras, as assessed by the activation ratio of GTP to GTP + GDP bound to p21ras, 1-10 min after the administration of this secosteroid to Caco-2 cells incubated in serum-free medium (Table 1). In contrast to these findings, as shown in Table 1 and in agreement with others (42), the addition of serum for 10 min to serum-deprived cells significantly increased the activation ratio of p21ras. It should be noted that 1,25(OH)2D3 also failed to activate p21ras in cells incubated in the presence of serum (data not shown).

                              
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Table 1.   Effect of 1,25(OH)2D3 on p21ras activation in Caco-2 cells

PTx-sensitive heterotrimeric G proteins are involved in regulation of PLD by 1,25(OH)2D3 and TPA. In view of previous studies that indicated that, in addition to monomeric G proteins, heterotrimeric G proteins may also be involved in the regulation of agonist-induced stimulation of PLD activity (12, 24, 28, 31), it was of interest to pretreat Caco-2 cells with either PTx (100 ng/ml, 18 h) or cholera toxin (100 ng/ml, 2 h) to examine their effects on 1,25(OH)2D3- or TPA-induced PLD activation. As shown in Fig. 4, PTx, but not cholera toxin, significantly inhibited the ability of each of these agonists to stimulate PLD.


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Fig. 4.   Pertussis toxin (PTx) but not cholera toxin blocks stimulation of PLD by 1,25(OH)2D3 or TPA. Caco-2 cells were metabolically labeled with [3H]myristic acid and pretreated with vehicle (open bars), PTx (100 ng/ml, 18 h, solid bars), or cholera toxin (100 ng/ml, 2 h, hatched bars) and then stimulated with 1,25(OH)2D3 (100 nM) or TPA (200 nM) for 20 min. 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 toxin).

To assess the ordering of the aforementioned signal transduction components involved in PLD stimulation, we asked whether pretreatment of Caco-2 cells with PTx could block the activation of pp60c-src by 1,25(OH)2D3 and/or inhibit the ability of this secosteroid to induce the translocation of PKC-alpha and/or RhoA to the particulate fraction of these cells. As shown in Fig. 5A, PTx significantly blocked the stimulation of pp60c-src by 1,25(OH)2D3. In addition, PTx significantly limited the ability of this secosteroid, but not TPA, to induce the translocation of PKC-alpha from the cytosolic to particulate fraction of Caco-2 cells (Fig. 5B). In agreement with these latter findings, moreover, PTx significantly inhibited the ability of 1,25(OH)2D3, but not TPA, to stimulate PKC biochemical activity, as assessed by an in situ PKC phosphorylation assay (17, 46) (Fig. 6). In contrast, PTx had no significant inhibitory effect on the translocation of RhoA induced by 1,25(OH)2D3 in these cells (Fig. 5C).


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Fig. 5.   PTx inhibits stimulation of c-Src and protein kinase C-alpha (PKC-alpha ) but not RhoA translocation induced by 1,25(OH)2D3 in Caco-2 cells. A: pp60c-src activity. Caco-2 cells were pretreated with vehicle or PTx (100 ng/ml, 18 h) and then stimulated with 1,25(OH)2D3 (100 nM) for 1 min. pp60c-src was then immunoprecipitated, and kinase activity was determined by 32P incorporation into peptide as described in METHODS. Data represent means ± SE of 3 independent experiments, each in triplicate. B: PKC-alpha translocation. Caco-2 cells were pretreated with vehicle or PTx (100 ng/ml, 18 h) and then stimulated with 1,25(OH)2D3 (100 nM) or TPA (200 nM) for indicated times in minutes (controls, C, at 0 and 30 min). Particulate fractions were prepared by ultracentrifugation as described in METHODS. Cellular proteins (20 µg/lane) were resolved, and PKC-alpha was detected by Western blotting using monoclonal anti-PKC-alpha antibodies. Immunoblots are representative of 2 independent experiments. C: RhoA translocation to particulate fraction. Caco-2 cells were pretreated with vehicle or PTx (100 ng/ml, 18 h) and then stimulated with 1,25(OH)2D3 (100 nM) for indicated times in minutes (controls, C, at 0 and 30 min). Particulate fractions were prepared by subcellular fractionation as described in METHODS. RhoA was detected in particulate fraction by Western blotting using polyclonal anti-RhoA antibodies. Immunoblots are representative of 2 independent experiments.


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Fig. 6.   PTx blocks ability of 1,25(OH)2D3 but not TPA to stimulate PKC phosphorylating activity in situ. Caco-2 cells, plated in 24-multiwell plates, were grown to 70% confluence. Cells were preincubated with vehicle or PTx (100 ng/ml) for 18 h and then treated with 1,25(OH)2D3 (100 nM) or TPA (200 nM) for 10 min in presence of permeabilizing phosphorylation reaction mixture containing digitonin, [gamma -32P]ATP, and PKC-specific substrate derived from myelin basic protein (Ac-MBP) as described in METHODS. Each value represents means ± SE of 3 separate experiments. * P < 0.05 compared with 1,25(OH)2D3 alone.

Because PKC-alpha , like several other PKC isoforms, is a phorbol ester receptor (37), it was conceivable that although PTx did not block the ability of TPA to induce the membrane translocation of PKC-alpha it may still have inhibited the activity of this PKC isoform. As shown in Fig. 6, as assessed by an in situ PKC assay (17, 46), pretreatment of cells with PTx, however, did not significantly inhibit the activation of PKC by TPA in these cells. These latter findings therefore taken together with the failure of PTx to inhibit the TPA-induced translocation of PKC-alpha indicate that PTx did not block TPA-stimulated PLD by inhibiting this PKC isoform.

Pretreatment of Caco-2 cells with PTx reduces increase in [Ca2+]i induced by 1,25(OH)2D3. In prior studies, 1,25(OH)2D3 has been shown by our laboratory to increase [Ca2+]i in Caco-2 cells (48) and in rat colonocytes (49), via inositol 1,4,5-trisphosphate (IP3)-mediated release of Ca2+ from intracellular stores, as well as by increasing the influx of this divalent cation across the plasma membrane(s) of these cells. Because PKC-alpha is a Ca2+-dependent isoform and previous studies have demonstrated that PTx can block the increases of [Ca2+]i induced by some agents (8, 34, 41), it was of interest to determine whether this toxin inhibited the activation of PKC-alpha induced by 1,25(OH)2D3 by blocking Ca2+ mobilization by this secosteroid. As shown in Fig. 7, in agreement with our prior studies (48), 1,25(OH)2D3 increased [Ca2+]i, an effect that was significantly inhibited by pretreatment of these cells with PTx. These observations are consistent with previous studies by our laboratory (3) in which 1,25(OH)2D3 was found to transiently increase the translocation of PKC-alpha to the membrane fraction of these cells, at least in part by releasing Ca2+ from intracellular stores via the short-lived induction of IP3. In contrast to these findings, TPA alone or after pretreatment with PTx did not increase [Ca2+]i in Caco-2 cells (Fig. 7). These findings are consistent with the ability of phorbol esters such as TPA to induce the translocation of PKC-alpha to the membrane fraction of cells in a sustained manner, via a Ca2+-independent mechanism, because PKC-alpha , like other PKC isoforms, is a phorbol ester receptor (37). It bears emphasis, however, that these results indicate that even the transient activation of PKC-alpha by 1,25(OH)2D3, like the long-lived activation of this PKC isoform by TPA, can lead to the sustained stimulation of PLD.


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Fig. 7.   PTx blocks 1,25(OH)2D3-induced increases in intracellular Ca2+ concentration ([Ca2+]i). Caco-2 cells were pretreated with PTx (100 ng/ml) for 18 h and then treated with 1,25(OH)2D3 (100 nM), TPA (200 nM), or appropriate vehicle for 5 min, and intracellular Ca2+ levels were measured using cell permeant Ca2+-sensitive fluorescent dye, fura 2-acetoxymethyl ester as described in METHODS. Basal Ca2+ was 150 ± 10 nM and was not different with PTx treatment alone. Each value represents means ± SE of 3 separate preparations. * P < 0.05, compared with control (no treatment). dagger  P < 0.05 compared with 1,25(OH)2D3 alone.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

PKC-alpha has been shown to be involved in the activation of PC-PLD by 1,25(OH)2D3 and TPA in Caco-2 cells (24a). The present studies demonstrate, however, that the activation of PLD by 1,25(OH)2D3, and TPA, while both involving PKC-alpha , differ in a number of other signal transduction components involved in the regulation of this enzyme(s). In this regard, prior studies in other cell types employing a variety of agonists, including tumor-promoting phorbol esters, have shown that PLD regulation may involve members of the Rho family and/or Arf monomeric G proteins (16, 18, 32, 33, 39, 44). We therefore utilized a C3 transferase of C. botulinum, which ADP ribosylates and inactivates Rho proteins (33), to determine whether these small monomeric G proteins might be involved in the regulation of PC-PLD activity by 1,25(OH)2D3 or TPA in Caco-2 cells. We were particularly interested in the Rho family of monomeric G proteins in view of prior studies in HL-60 cells, which have shown that both PKC-alpha and RhoA were concomitantly involved in TPA-stimulated PC-PLD activation in a cell-free system containing isolated membranes (39). In contrast to these findings in HL-60 cells, whereas C3 transferase almost totally abolished the stimulatory effect of 1,25(OH)2D3 on PLD in Caco-2 cells, this toxin did not affect the ability of TPA to activate PLD. It did, however, significantly block the ability of 1,25(OH)2D3 to activate PLD, as well as reduce the expression of RhoA. Moreover, in agreement with prior studies in Rat1 fibroblasts using LPA, another agonist of PLD (32), 1,25(OH)2D3 rapidly but transiently induced the translocation of RhoA from the soluble to the particulate fraction of Caco-2 cells. It is, however, unclear from the present studies whether the C3 transferase-induced inhibition of 1,25(OH)2D3-stimulated PLD was due to its reduction in total RhoA expression and/or to the decrease in RhoA translocation induced by this secosteroid. These findings do, however, indicate that RhoA, as well as perhaps other members of the Rho family of monomeric G proteins, are involved in the activation of PLD by 1,25(OH)2D3, but not by TPA, in these cells. In addition, the failure of brefeldin A, an inhibitor of guanine nucleotide exchange on Arf proteins (35), to significantly affect the ability of 1,25(OH)2D3 or TPA to stimulate PLD would indicate that these latter monomeric G proteins are unlikely to be required for the stimulation of PLD by either of these agonists in Caco-2 cells. In keeping with these findings in Caco-2 cells, recent studies in 1321N1 human astrocytoma cells also found that Arf was not involved in agonist stimulation of PLD (35).

In agreement with prior studies by our laboratory in rat colonocytes (25), 1,25(OH)2D3, but not TPA, was also found to stimulate pp60c-src activity in Caco-2 cells. Furthermore, PP1, a specific inhibitor of members of the pp60c-src family of nonreceptor tyrosine kinases (25), markedly reduced the activation of PLD by 1,25(OH)2D3 but not by this phorbol ester. These findings indicate that the stimulation of PLD by 1,25(OH)2D3, but not by TPA, also involves the activation of pp60c-src in Caco-2 cells. Furthermore, these observations suggest that other members of this nonreceptor tyrosine kinase family, besides pp60c-src, are not involved in the activation of PLD by TPA in these cells.

Prior studies in other cell types have demonstrated that stimulation of pp60c-src subsequently led to the tyrosine phosphorylation of Shc, thereby inducing the formation of complexes of Shc, Grb2 and Sos, a guanine nucleotide exchange factor, which, in turn, activated p21ras (30). Moreover, activation of p21ras has been shown to stimulate PLD (21, 22). It was therefore possible that 1,25(OH)2D3 might activate p21ras, via stimulation of pp60c-src, and thereby activate PLD. This possibility, however, appears unlikely because we were unable to demonstrate p21ras activation by this secosteroid.

In agreement with several (12, 31) but not all studies (28) employing various agonists in other cells, pretreatment of Caco-2 cells with PTx, but not cholera toxin, significantly inhibited the actions of 1,25(OH)2D3 or TPA to stimulate PLD. In this regard, it should be noted that pretreatment of chick myoblasts with PTx has also been shown to inhibit the activation of PLD by 1,25(OH)2D3 (36). Although to our knowledge PTx has not been found to affect the stimulation of PLD by TPA in other cells, this toxin has been shown to block the potentiative effect of TPA on the f-Met-Leu-Phe-induced stimulation of PLD in human neutrophils (38). PTx-sensitive G proteins are inactivated by ADP ribosylation of their alpha -subunits and include members of the Gi and Go families (29). The present findings therefore indicate that one or more of these PTx-sensitive G proteins appear to be involved in the regulation of PLD by each of these agents in Caco-2 cells.

To further define the role of these PTx-sensitive heterotrimeric G proteins in the stimulation of PLD by 1,25(OH)2D3, we examined the effects of PTx on the ability of this secosteroid to translocate RhoA to the particulate fraction of these cells and to activate pp60c-src and PKC-alpha . The results of these studies demonstrated that pretreatment of Caco-2 cells with PTx failed to significantly inhibit the particulate translocation of RhoA by 1,25(OH)2D3. In contrast to these findings, however, PTx blocked both the activation of pp60c-src and PKC-alpha by this secosteroid in these cells. In addition, PTx partially but significantly blocked the increase in [Ca2+]i induced by 1,25(OH)2D3. These findings, taken together with the results of the accompanying paper (24a), as well as those of prior studies by our laboratory on the effects of 1,25(OH)2D3 in isolated colonocytes (4), suggest a schema depicted in Fig. 8 for the signal transduction events involved in the stimulation of PLD by 1,25(OH)2D3 in Caco-2 cells. Because PTx significantly inhibited 1,25(OH)2D3-induced pp60c-src activation, which is required for PLD stimulation by this secosteroid, this would place the PTx-sensitive heterotrimeric G proteins involved in this phenomenon upstream of pp60c-src. The exact identity(ies) of these G protein is, however, currently unknown. Moreover, it is not clear whether the alpha - or beta gamma - subunits of this protein(s) are involved in the activation of pp60c-src by 1,25(OH)2D3.


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Fig. 8.   Proposed schema of 1,25(OH)2D3 and TPA stimulation of PLD in Caco-2 cells. Note our results suggest that G proteins downstream of 1,25(OH)2D3 and TPA are likely different.

Recent studies by Luttrell et al. (30) are, however, of interest in this regard because they demonstrated that, on stimulation of COS-7 cells by LPA, a known PLD agonist, beta gamma -subunits, derived from PTx-sensitive G proteins, activated pp60c-src. The mechanism of this activation, while currently unclear, may involve binding of the SH2 domains of pp60c-src to phosphatidylinositol 3,4,5-trisphosphate, a product of phosphatidylinositol 3-kinase (30). Dikic et al. (11) have also recently shown that activation of PTx-sensitive or -insensitive G protein-coupled receptors in PC-12 cells stimulated in response to either LPA or bradykinin, or to TPA, respectively, induced the tyrosine phosphorylation of the Ca2+- and PKC-dependent tyrosine protein kinase, Pyk2, a member of the focal adhesion kinase family of integrin receptor-associated tyrosine kinases (11). Tyrosine phosphorylation of Pyk2, in turn, led to its binding to the SH2 domain of pp60c-src and subsequent activation of pp60c-src (11). Whether 1,25(OH)2D3 can activate pp60c-src via one or both of these pathways involving PTx-sensitive G proteins in Caco-2 cells and thereby stimulate PC-PLD is unknown at this time and will require further study.

Regardless of the mechanism(s) involved in how PTx-sensitive G proteins activate pp60c-src in response to 1,25(OH)2D3, based on recent studies in our laboratory in isolated colonocytes (4) and in Caco-2 cells (25), it would appear that the activation of pp60c-src by 1,25(OH)2D3 thereby induces the tyrosine phosphorylation, translocation to the plasma membrane, and biochemical activation of PI-PLC-gamma . The activation of this PI-specific phospholipase, in turn, causes the breakdown of membrane polyphophoinositides, generating IP3 and 1,2-diacylgylcerol, thereby increasing intracellular Ca2+ and activating PKC-alpha , with subsequent stimulation of PLD in these cells (Fig. 8). In keeping with this contention, as noted earlier, PTx was shown to not only block the stimulation of pp60c-src by 1,25(OH)2D3 but also the activation of PKC-alpha by this secosteroid as assessed by the translocation of this PKC isoform from the soluble to particulate fraction of Caco-2 cells, as well as by an in situ assay of PKC kinase activity. The effect of PTx on secosteroid-stimulated PLD activity via this Ca2+-dependent PKC isoform may therefore at least in part be due to this ability of the toxin to inhibit the increase in [Ca2+]i caused by 1,25(OH)2D3 in Caco-2 cells. It bears emphasis that pretreatment of several cell types with PTx has also been shown to significantly reduce the increase in [Ca2+]i induced by a variety of agonists (8, 34, 41).

In addition, RhoA and/or other members of the Rho family of monomeric G proteins, but not Arf proteins, are also involved in the stimulation of PLD by 1,25(OH)2D3 in Caco-2 cells. However, because PTx did not inhibit the translocation of RhoA to the particulate fraction of Caco-2 cells induced by 1,25(OH)2D3, it would appear that the involvement of RhoA in this phenomenon is distinct from the PTx-sensitive G protein-pp60c-src-PKC-alpha pathway. As suggested in other cells, this RhoA-mediated response may involve the direct or indirect interaction of this monomeric protein(s) with PLD (32) (Fig. 8), but this will require further study. Similarly, the mechanism(s) involved in membrane association of RhoA induced by 1,25(OH)2D3 is also not clear at this time. In this regard, however, based on recent observations in a cell-free system derived from human neutrophils (5), 1,25(OH)2D3 may cause the translocation of RhoA to the membrane fraction of Caco-2 cells via interactions with an as yet unknown but apparently membrane-associated guanine nucleotide exchange factor(s), but this issue will also require further study.

Regardless of the mechanisms involved in these aforementioned 1,25(OH)2D3-mediated events involving RhoA, however, the present studies indicate that they do not involve the activation of p21ras, an upstream effector of RhoA in some cell types (47). Moreover, the results of the current experiments also indicate that the involvement of Rho proteins in the stimulation of PLD by 1,25(OH)2D3 may at least in part be responsible for the PKC-independent activation of PLD by this secosteroid (24a). Alternatively, RhoA activation by 1,25(OH)2D3 may be upstream of a PTx-sensitive step. It bears emphasis, however, that, whereas 1,25(OH)2D3 can use either the RhoA pathway or the pp60c-src pathway to activate PLD (Fig. 8), the Rho protein antagonist, C3 transferase, as well as either PTx or the pp60c-src antagonist PP1, can nearly completely ablate the stimulation of PLD by this secosteroid. These observations would therefore indicate that both pathways appear to be required for the ability of 1,25(OH)2D3 to activate PLD in Caco-2 cells, such that if one of these pathways is blocked the other is not sufficient for PLD activation.

Although one or more PTx-sensitive G proteins appear to be involved in the activation of PLD by both TPA and 1,25(OH)2D3, the finding that TPA, in contrast to 1,25(OH)2D3, fails to activate pp60c-src indicates that these agonists likely utilize different G proteins. Moreover, TPA, like 1,25(OH)2D3, does not require Arf protein(s) for its activation of PLD but, unlike 1,25(OH)2D3, also does not require Rho proteins for this effect (Fig. 8). Pretreatment of Caco-2 cells with PTx, moreover, only modestly reduced (30%) the ability of this phorbol ester to directly stimulate PKC phosphorylating activity in these cells. In contrast to our findings with respect to 1,25(OH)2D3, TPA, in the presence or absence of PTx, did not influence [Ca2+]i in these cells. Taken together, these findings indicate that PTx inhibits the ability of TPA to activate PLD by an as yet to be identified mechanism, which is at a step distal to its activation of PKC-alpha and, moreover, is Ca2+-independent. Further studies to address the exact mechanism(s) involved in this inhibitory effect of PTx on PLD stimulation by this phorbol ester will therefore be of interest. Because several human isoforms of PLD have been described (16), it will also be of interest to identify the specific isoforms activated by these agonists. The differential regulation of PLD observed in the present studies may reflect PLD isoform-specific activation by these agents.

The products of PLD have been implicated in the regulation of a number of important physiological processes, including proliferation, differentiation, inflammation, cellular trafficking of proteins, involving the formation of Golgi-coated vesicles, and protein secretion, as well as in pathological processes such as malignant transformation (2, 9, 26, 27). Our laboratory has previously shown that the in vitro addition of 1,25(OH)2D3 to Caco-2 cells reduced their proliferation and enhanced their differentiation (15). This is of particular interest because these latter actions of 1,25(OH)2D3 may at least in part be involved in the prevention of colonic tumors by this secosteroid, as well as by other metabolites-analogs of vitamin D3 previously reported in experimental models of colonic carcinogenesis (1, 40, 50). In this regard, as noted earlier (24a), it is possible that 1,25(OH)2D3 mediates these alterations in cellular proliferation and differentiation by activation of PLD via the pathways elucidated in the present studies. Moreover, it is also apparent from these studies that 1,25(OH)2D3 and TPA differ in their signaling pathways with respect to their activation of PLD. These latter differences are of interest in view of the known tumor promotional effects of phorbol esters, such as TPA, in other experimental systems, although the potential effects of phorbol esters on the promotion of colon cancer have not to our knowledge been examined. Further studies to explore the potential roles of the activation of PLD by 1,25(OH)2D3 or TPA in these processes will therefore be of interest.


    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 by 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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DISCUSSION
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Am J Physiol Gastroint Liver Physiol 276(4):G1005-G1015
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