1 Department of Medicine, University of Chicago, Chicago, Illinois 60637; and 2 Department of Medicine, University of California, San Diego, California 92093-0652
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ABSTRACT |
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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- (PKC-
) 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-
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-
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-
, 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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IN THE ACCOMPANYING PAPER we have demonstrated that
protein kinase C- (PKC-
) 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-
(PI-PLC-
) (25). Moreover, as shown in these studies (25), prior activation of PI-PLC-
was required for the activation of PKC-
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-, 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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METHODS |
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Materials.
Upstate Biotechnology (Lake Placid, NY) provided monoclonal
anti-PKC-, 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, [
-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-.
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-
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
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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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RESULTS |
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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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Src activation is involved in stimulation of PLD by
1,25(OH)2D3 but
not by TPA.
Prior studies have shown that coupling of
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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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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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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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- 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-
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-
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-
to the membrane fraction of cells in a sustained manner, via a
Ca2+-independent mechanism,
because PKC-
, 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-
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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DISCUSSION |
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PKC- 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-
, 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-
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 -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-.
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-
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
- or
- subunits of this
protein(s) are involved in the activation of
pp60c-src by
1,25(OH)2D3.
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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, -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-. 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-
, 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-
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- 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- 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.
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ACKNOWLEDGEMENTS |
---|
We thank Lynn Kaczmarz for excellent secretarial assistance.
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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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