Department of Medicine, University of Chicago, Chicago, Illinois 60637
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ABSTRACT |
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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-, but not
PKC-
1,
-
II, -
, or -
, whereas TPA
activated PKC-
, -
1, and
-
. Chronic treatment with TPA (1 µM, 24 h) significantly reduced
the expression of PKC-
, -
I,
and -
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-
I and
-
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-
expression, respectively. Taken
together, these observations indicate that PKC-
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-, 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-
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-
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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METHODS |
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Materials.
The full-length cDNA encoding human PKC- 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,
-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-
antibodies, whereas polyclonal anti-PKC-
, -
I,
-
II, -
, -
, and -
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,
[
-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). 1
,25-Dihydroxyvitamin
D3 was purchased from Steroids
Limited (Chicago, IL). The
PKC-
I- and
-
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- in Caco-2 cells.
PKC-
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-
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-, -
I,
-
II, -
or -
(0.2 µg/ml)
or mouse monoclonal anti-PKC-
(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 -glycerophosphate,
50 µg/ml digitonin, 100 µM
[
-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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RESULTS |
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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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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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PKC- 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-
, -
I, and
-
II, as well as the Ca2+-independent PKC isoforms,
PKC-
and -
(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-
, but not
PKC-
I,
-
II or -
(Fig.
5). As can be seen in Fig.
6, TPA (200 nM final concentration),
however, not only induced the particulate association of PKC-
but
also that of PKC-
I and -
, but not PKC-
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-
was unresponsive to 1,25(OH)2D3
or TPA in Caco-2 cells (6).
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Changes in expression of PKC- 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-
, 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-
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-
, 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-
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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PKC-independent activation of PLD by
1,25(OH)2D3.
The aforementioned studies clearly establish the role of PKC,
particularly PKC-, 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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DISCUSSION |
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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-, and/or PKC-
I or
-
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-
. Moreover, in a cell-free
system (36), PKC-
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-
-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- and -
(6). Furthermore, only PKC-
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-
and -
, also possessed
PKC-
I,
-
II, and -
(1, 48). In the
present experiments, several lines of evidence indicated that PKC-
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-
I and
-
II) or -independent isoforms
(PKC-
and -
) 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-
and markedly diminished in cells with inhibited
expression of this PKC isoform. Taken together, these findings indicate
that PKC-
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-
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-. After this
treatment, the expression of PKC-
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-
-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- is the predominant PKC
isoform involved in the activation of PLD by TPA. TPA was found to
acutely activate PKC-
, -
I,
and -
, but not PKC-
II or
-
in Caco-2 cells. Although chronic TPA treatment (1 µM, 24 h)
markedly reduced the expression of PKC-
(~95%), as well as that
of PKC-
(~95%) and -
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-
or -
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-
,
in the stimulation of PLD by TPA. Moreover, the failure of DOPPA, a
specific activator of PKC-
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-
I in
this biochemical event. These findings, taken together with our studies
in transfected Caco-2 cells demonstrating that overexpression of
PKC-
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-
plays a major role in
this phorbol ester-induced phenomenon in Caco-2 cells.
The exact mechanism(s) by which the activation of PKC- 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-
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--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-
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-
-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.
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ACKNOWLEDGEMENTS |
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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 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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