1 Cellular Biology Laboratory, Winthrop-University Hospital, Mineola 11501; and 2 School of Medicine, State University of New York, Stony Brook, New York 11794
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ABSTRACT |
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Recently we demonstrated the induction of apoptosis by the addition of recombinant lipocalin-type prostaglandin D2 synthase (L-PGDS) to the culture medium of LLC-PK1 cells. Because protein kinase C (PKC) has been shown to be involved in the apoptotic process of various cell types, we examined the potential role of L-PGDS in phorbol 12-myristate 13-acetate (PMA)-induced apoptosis. We report here the enzymatic activation and phosphorylation of L-PGDS in response to phorbol ester in cell culture and the direct phosphorylation of recombinant L-PGDS by PKC in vitro. Treatment of cells with PMA or L-PGDS decreased phosphatidylinositol 3-kinase (PI3-K) activity and concomitantly inhibited protein kinase B (PKB/Akt) phosphorylation, which led to the hypophosphorylation and activation of Bad. In addition, hypophosphorylation of retinoblastoma protein was also observed in response to L-PGDS-induced apoptosis. Cellular depletion of L-PGDS levels by using an antisense RNA strategy prevented PI3-K inactivation by phorbol ester and inhibited caspase-3 activation and apoptosis. We conclude that phorbol ester-induced apoptosis is mediated by L-PGDS phosphorylation and activation by PKC and is accompanied by inhibition of the PI3-K/PKB anti-apoptotic signaling pathways.
lipocalin-type prostaglandin D2 synthase; phorbol
12-myristate 13-acetate; phosphatidylinositol 3-kinase; apoptosis; LLC-PK1; protein kinase C; -trace
protein
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INTRODUCTION |
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LIPOCALIN-TYPE
PROSTAGLANDIN D2 SYNTHASE (L-PGDS),
an isomerase that converts prostaglandin (PG)H2 into
PGD2, is a unique enzyme in that it also functions as a
transporter of small, lipophilic molecules such as bile salts and
retinol (26, 27). Recently we added the novel role of
apoptotic inducer to the list of L-PGDS protein functions
(13, 18). L-PGDS, which is overexpressed in
Escherichia coli, was added exogenously to
LLC-PK1 cells, where it caused a fivefold increase in
apoptosis. The apoptosis observed appeared to involve
activation of the caspase-3 pathway and presumably the L-PGDS
enzymatic end product,
15-deoxy--12,14PGJ2 (15-dPGJ2).
15-dPGJ2 is a natural peroxisome
proliferator-activated receptor-
(PPAR-
) ligand that is known to
induce apoptosis. Because the process was dependent on the
presence of calcium in the medium, we decided to investigate the role
of calcium-dependent protein kinase C (PKC) in L-PGDS-mediated apoptosis.
PKC refers to a family of serine and threonine kinases that have
traditionally been associated with the regulation of cell growth and
differentiation in response to a variety of stimuli (16).
More recently, members of the PKC family have been linked to cell death
and apoptosis of various cells (3, 17). For example, phorbol ester induces apoptosis in the renal
epithelial cell line LLC-PK1 (11), PKC
downregulation suppresses apoptotic signals in 3Y1 rat fibroblasts
(31), tyrosine phosphorylation of PKC
is essential for
its apoptotic effects (2), PKC
modulates thromboxane A2-mediated apoptosis in myocytes
(22), and PKC
knockout mice show a marked decrease in
cellular apoptosis and increased cell proliferation
(12). In one study of phorbol 12-myristate 13-acetate (PMA)-induced apoptosis (11), the
process seemed to be the result of a conflict between growth-retarding
signals elicited by PMA and growth-promoting signals stimulated by
serum. The conflict apparently led to DNA damage, which in turn led to apoptosis. When the conflict was prevented by serum starvation, apoptosis was suppressed.
The activation or inhibition of several other downstream signal transduction proteins by a variety of cytokines within a cell has been shown to alter the balance between cell proliferation and apoptosis. Classically, stimulation of the phosphatidylinositol 3-kinase (PI3-K) pathway and subsequent protein kinase B (PKB/Akt) phosphorylation have been shown to be anti-apoptotic. Inhibition of PI3-K phosphorylation by the stimulation of phosphoinositide-specific inositol polyphosphate 5-phosphatase IV inhibits PKB phosphorylation and leads to apoptotic cell death in human embryonic kidney cells (9). Similarly, phosphorylation of the retinoblastoma protein (pRb) is known to promote cellular proliferation and inhibit cellular apoptosis (29, 30). Conversely, proapoptotic events such as the activation of PKC but not extracellular signal-related kinase (ERK) have been demonstrated to play a role in vitamin E succinate-induced apoptosis of HL-60 cells (1).
In the present study, we investigated the molecular mechanism of L-PGDS-mediated apoptosis and the involvement of PKC. We report on the phosphorylation and enzymatic activation of L-PGDS by PKC in response to phorbol ester. L-PGDS phosphorylation, induced by PMA, was accompanied by decreases in PI3-K activity and PKB/Akt phosphorylation. This resulted in the hypophosphorylation and activation of Bad as well as the hypophosphorylation of pRb. Furthermore, the ability of phorbol ester to inhibit PI3-K and induce caspase-3 activation and apoptosis was lost in a cell line with depleted L-PGDS protein expression.
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EXPERIMENTAL PROCEDURES |
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Materials.
Cell culture reagents, antibiotics, antimycotics, fetal bovine serum,
Lipofectamine, geneticin (G418), and all media were purchased from Life
Technologies (Grand Island, NY). The -[33P]ATP (sp.
act., 3,000 Ci/mmol) and [32P]orthophosphate were
purchased from Dupont/New England Nuclear (Boston, MA). Electrophoresis
reagents were obtained from Bio-Rad (Richmond, CA). Bicinchoninic acid
protein assay reagent was purchased from Pierce (Rockford, IL). The PKC
inhibitor Gö-6976 was purchased from Calbiochem (San Diego, CA).
Restriction endonucleases, terminal deoxynucleotidyl
transferase-mediated dUTP nick-end labeling (TUNEL) assay reagents, and
PKC enzyme (a combination of
-,
-, and
-isoforms) were from
Roche Molecular Biochemicals (Indianapolis, IN). The caspase-3 activity
apoptotic detection kit was purchased from R&D (Minneapolis, MN).
Antibodies against actin, Akt, Bad, p85, p110, pRb, and ERK were all
purchased from Santa Cruz Biotechnology (Santa Cruz, CA). All
phospho-antibodies were purchased from Cell Signaling (Beverly, MA).
Antibody against L-PGDS and the PGD2-methoxime (MOX)
immunoassay kit were from Cayman Chemical (Ann Arbor, MI). The PKC
activator PMA was purchased from Biomol (Plymouth Meeting, PA).
Enhanced chemiluminescence reagent was from Amersham Pharmacia Biotech
(Piscataway, NJ). The mammalian expression vector pcDNA3 and
prokaryotic vector pRSET were purchased from Invitrogen (San Diego,
CA). All other reagents were purchased from Sigma Chemical (St. Louis, MO).
Cell culture. The pig kidney epithelial cell line LLC-PK1, a kind gift from Dr. Julia Lever (University of Texas, Houston), was maintained in DMEM/F-12 medium supplemented with 10% fetal calf serum, 50 U/ml penicillin, and 50 µg/ml streptomycin (Life Technologies, Gaithersburg, MD) at 37°C in 5% CO2. Transfected cells were maintained in this fashion with the addition of 200 µg/ml G418.
Metabolic labeling. LLC-PK1 cells were grown as described (see Cell culture), and at ~90% confluence, the cells were washed twice with phosphate-free DMEM and incubated for 1 h at 37°C. Next, the cells were labeled with [32P]orthophosphoric acid (0.1 mCi/ml) overnight and exposed to various agonists, which are detailed (see figure legends). The cells were rinsed three times with PBS that contained sodium vanadate (10 mM) and were immunoprecipitated.
Immunoprecipitation.
Cells were lysed in a buffer that contained 50 mM HEPES, pH 7.5, 2 mM
EDTA, 1% Triton X-100, 100 mM NaCl, 50 mM -glycerophosphate, 100 mM
NaF, 100 mM sodium pyrophosphate, 2 mM sodium orthovanadate, 2 µM
microcystin, and a cocktail of protease inhibitors. Equal amounts of
precleared protein lysate (usually between 500 and 1,000 µg) were
immunoprecipitated with the appropriate rabbit polyclonal antibody for
2 h at 4°C and were collected with protein A-Sepharose via
incubation overnight. The immunoprecipitated protein was either assayed
for kinase activity or it received immunoblot analysis.
Immunoblot analysis. Culture plates were washed four times with ice-cold PBS, which was followed by the addition of cell lysis buffer that contained 50 mM Tris · HCl, pH 7.6, 2.0 mM EDTA, 2.0 mM EGTA, 1.0% SDS, 1.0 mM benzamidine, 2.0 mM phenylmethylsulfonyl fluoride (PMSF), and 10 µg/ml each of leupeptin, aprotinin, antipain, soybean trypsin inhibitor, and pepstatin A. When phosphorylation was detected, PBS and cell-lysis buffer contained 2 mM sodium vanadate and 1 µm microcystin at 4°C. Plates were scraped, and the cell lysate was sonicated and centrifuged at 2,000 g for 5 min. Typically, 50 µg of protein was mixed with Laemmli sample buffer (that contained 0.1% bromphenol blue, 1.0 M NaH2PO4, pH 7.0, 50% glycerol, and 10% SDS) and boiled for 5 min before it was loaded onto an SDS-PAGE gel. The separated proteins were transferred to polyvinylidene difluoride (PVDF) membrane and probed with the proper antibodies before detection via enhanced chemiluminescence reagent and subsequent autoradiography were performed. The intensity of the signal was quantitated by densitometric analysis of the autoradiograms.
PKC activity assay. In vitro PKC activity was measured as described by Kitano et al. (10). Briefly, the 50-µl reaction mixture contained 20 mM Tris · HCl, pH 7.5, 10 mM MgCl2, 0.5 mM CaCl2, 5 mM dithiothreitol, 1.5 µg L-PGDS substrate where indicated, mixed micelles (0.31 mg/ml phosphatidylserine, 60 µg/ml diolein, 0.03% Triton X-100 in 20 mM Tris · HCl, pH 7.5, sonicated separately), and 0.25 mU/ml PKC enzyme diluted in 20 mM Tris · HCl, pH 7.5, 0.5 mM EDTA, with 0.5 mM EGTA. The reaction was initiated by the addition of 5 µM [33P]ATP (10 µCi/µl) and was incubated at 30°C for 30 min. After termination of the reaction with 1.5 µl of 10 mM ATP, 25 µl of the reaction mixture was removed and 5× Laemmli gel buffer was added. The sample was heated to 95°C for 5 min, separated on a 12% SDS-PAGE gel, and transferred to PVDF membrane; the dried membrane was exposed to film overnight.
Assay of L-PGDS enzymatic activity.
L-PGDS enzymatic activity was determined using a PGD2-MOX
enzyme immunoassay kit. Cells were treated as described and lysed by
freeze-thawing in 0.1 M Tris · HCl (pH 7.5) and 1 mM PMSF. Equal amounts of whole-cell lysates were used as the enzyme
source. The reaction mixture contained 0.1 M
Tris · HCl (pH 7.5), 1.0 mM -mercaptoethanol,
40 µM PGH2 as the substrate, and cell extracts in a final
volume of 50 µl. The reaction was initiated by addition of the
substrate to the reaction mixture, incubated for 1 min at 25°C, and
terminated by heating the mixture to 100°C for 10 min. The resulting
PGD2 product in the reaction mixture was quantitated according to the manufacturer's instructions.
PI3-K activity assay. Anti-p110 and anti-p85 antibodies were used to immunoprecipitate PI3-K activity from 500 µg of precleared lysate. Assays were performed as previously described (20).
Apoptotic activity. Apoptosis was quantitated by either TUNEL staining as previously described (13, 18) or by measurement of caspase-3 activity as per the manufacturer's procedure.
Construction, transfection, and selection of stable L-PGDS antisense cell lines. LLC-PK1 cells were the parent strain used for transfection with the mammalian expression system vector pcDNA3. L-PGDS depletion was accomplished by inserting the L-PGDS gene in the antisense orientation within pcDNA3 and stable clones selected with 2.0 mg/ml G418. Independent clones were picked up with cloning disks and were screened for the absence of L-PGDS protein expression via immunoblot analysis of cell extracts after 10 days of culturing using L-PGDS antibody as described previously (19). Screening with L-PGDS antibody led to the identification of 8 clones out of 200 G418-resistant clones that lacked L-PGDS protein. Two clones that exhibited a >90% depletion of L-PGDS protein were used for all of the experiments. Control cells were transfected with the empty expression vector.
Protein assay. Protein content of cell extracts was determined using bicinchoninic acid (24).
Statistics. Results are expressed as means ± SE of at least three independent experiments, each of which was performed in duplicate at different times. A paired Student's t-test was used to compare the basal vs. treated preparations, and an ANOVA was used to compare the mean values between treatments. A value of P < 0.05 was considered significant.
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RESULTS |
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Chronic PMA treatment induces apoptosis via PKC.
Previously, we demonstrated the induction of apoptosis by
chronic exposure of LLC-PK1 cells to L-PGDS
(13). Similarly, Lee and Rosson (11)
had demonstrated phorbol ester-induced apoptosis in this cell
line when serum was present. We decided to study whether the
L-PGDS-induced apoptosis that we had observed previously was
linked to PKC. Consequently, we treated cells with PMA in the presence
and absence of the PKC inhibitor compound Gö-6976, which
selectively inhibits the PKC and PKC
isoforms, to test whether
PKC activation induced apoptosis. Figure
1 demonstrates that chronic PMA exposure
results in a twofold induction of apoptosis, which is
significantly inhibited by the PKC inhibitor compound Gö-6976.
Similar data using the TUNEL assay for apoptotic quantification were generated (data not shown).
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PMA increases L-PGDS phosphorylation and enzymatic activity.
Because it appeared clear that the PKC pathway had a role in
L-PGDS-induced apoptosis in LLC-PK1 cells, and
subsequent amino acid-sequence analysis of the L-PGDS protein (using
the Prosite program) revealed three potential PKC phosphorylation
sites, we decided to investigate whether PKC could phosphorylate L-PGDS and possibly modulate its enzymatic activity. When
32P-labeled cells were stimulated with 100 nM PMA,
immunoprecipitation of L-PGDS reveled a threefold induction of L-PGDS
phosphorylation after chronic exposure to PMA for 15 h in the
presence of serum (Fig. 2A, lane
3). Figure 2B
shows no change in the relative amount of L-PGDS protein from
all treatments.
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Cells that express L-PGDS antisense mRNA resist PMA-induced
caspase-3 activation and apoptosis.
Because we observed that PMA-induced apoptosis was accompanied
by L-PGDS phosphorylation and enzymatic activation, we decided to
confirm the role of L-PGDS in PMA/PKC-induced apoptosis by creating a cell line that overexpresses antisense L-PGDS mRNA to
decrease L-PGDS protein expression. More than 200 clones were screened
and assayed for the reduction in L-PGDS protein synthesis. Figure
5A illustrates a Western blot
analysis of two stable clones with L-PGDS protein expression inhibited
by fivefold (lanes 2 and 3). Lane 1 represents wild-type LLC-PK1 cells, and lane 4 is a transfected cell line that harbors the empty expression vector. The reductions in L-PGDS levels were not due to loading errors as is
demonstrated by the equal expression of actin among all cell lines
(Fig. 5B). When the antisense cells were exposed to 100 nM
PMA for 15 h, there was an almost complete loss of the ability of
PMA to induce apoptosis as measured by TUNEL assay (Fig.
5C) and caspase-3 activity (Fig. 5D). These data
lend further support to the role of L-PGDS in phorbol ester-induced
apoptosis.
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Chronic treatment with PMA or L-PGDS inhibits PI3-K signaling.
The PI3-K pathway is traditionally perceived as anti-apoptotic. For
example, several growth factors that inhibit apoptosis also
stimulate the activation of PI3-K. Zhou et al. (32)
demonstrated that activation of PI3-K by the inhibition of Na,K-ATPase
is cytoprotective in LLC-PK1 cells. More importantly,
inhibitors of PI3-K such as wortmannin and LY-294002 inhibit PKB/Akt
phosphorylation and have been shown to enhance apoptosis when
added to LLC-PK1 cells as measured by the activation of
caspase-3 and -9 (8, 25). We found similar results in our
system, where 100 nM wortmannin caused a twofold increase in the
apoptotic index (apoptotic indexes: control, 5%; wortmannin,
12%). Because PI3-K is considered to be a classic antiapoptotic
pathway that is activated for cell survival, the effects of chronic
treatment with either PMA or recombinant L-PGDS on PI3-K activity were
investigated. Cells incubated with 100 nM PMA or 50 µg/ml L-PGDS for
15 h were immunoprecipitated with anti-p85 antibody and then
assayed for PI3-K enzymatic activity in p85 immunoprecipitates. As
shown in Fig. 6A (lane
3), exposure of LLC-PK1 cells to PMA resulted in a
marked decrease in the amount of phosphatidylinositol
3,4,5-trisphosphate (PIP3) formed. Treatment with
PKC inhibitor prevented the PMA-induced decrease in PI3-K activity and
restored PIP3 formation (Fig. 6A, lane
4). More importantly, PMA failed to inhibit PI3-K activity in
L-PGDS-depleted cells (Fig. 6A, lane 7). Treatment of
LLC-PK1 cells with recombinant L-PGDS also inhibited PI3-K
enzymatic activity (Fig. 6B). The reduction in PI3-K
activity in L-PGDS-treated cells was due to a marked reduction in the
association of the p110 catalytic subunit with the p85 regulatory
subunit (Fig. 6C). The levels of the p85 PI3-K subunit were
not altered due to L-PGDS treatment (Fig. 6D).
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PMA- or L-PGDS-induced inhibition of PI3-K is accompanied by Akt
inactivation and Bad activation.
Because PI3-K enzymatic activities were decreased in PMA- and
L-PGDS-treated cells, we decided to study whether substrates downstream
of PI3-K and related to apoptosis were also affected. Therefore, the effects of PMA and L-PGDS on PKB/Akt and Bad
phosphorylation were determined. Chronic incubation with PMA or L-PGDS
for 15 h resulted in a three- or sixfold decrease in Akt
phosphorylation, respectively (Fig. 7A, compare lanes
1 and 3 or 5 and
6), whereas Akt protein levels
remained constant. The level of Bad phosphorylation similarly decreased
by 50% with exposure to either PMA or L-PGDS (Fig. 7B,
compare lanes 1 and 3 or 5 and
6). The effects of PMA on Akt dephosphorylation as well as
Bad dephosphorylation could be prevented by pretreatment with
Gö-6976 (Fig. 7, A and B, lane 4).
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L-PGDS inhibits pRb phosphorylation but has no effect on ERK
phosphorylation.
Phorbol ester-induced apoptosis of LLC-PK1 cells
has been associated with the hypophosphorylation of pRb
(11). We decided to investigate the effect of L-PGDS on
pRb levels and phosphorylation. When 50 µg/ml L-PGDS was added to
cells for 15 h, there was a 2.5-fold decrease in pRb
phosphorylation (Fig. 8A). The
level of ERK phosphorylation, however, was not affected (Fig.
8B).
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DISCUSSION |
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In the present study, we demonstrate that chronic exposure of LLC-PK1 cells to phorbol ester leads to the phosphorylation and enzymatic activation of L-PGDS and appears to be mediated via PKC. Although not conclusive, this interpretation is supported by the in vitro data, the 32P-labeled L-PGDS immunoprecipitation rates in response to chronic PMA exposure, and the potential PKC phosphorylation sites found within the L-PGDS amino acid sequence. In addition, mutations within several of the PKC phosphorylation site consensus sequences resulted in an L-PGDS protein with limited ability to induce apoptosis when added to cells exogenously (data not shown). Enzymatic activation of L-PGDS in response to chronic PMA treatment is consistent with the work of Mahmud et al. (14), who have demonstrated the activation of hematopoietic PGDS activity in response to phorbol esters in human megakaryoblastic cells.
L-PGDS, when added to cells at high levels, is only able to efficiently induce apoptosis when serum is present in the culture medium. We found similar results with PMA, where the full enzymatic activation of L-PGDS and the induction of apoptosis were found only after chronic exposure in the presence of serum. It is quite possible that only under these conditions are so-called "growth conflicts" in place, which allow for the induction of apoptosis. The effect of serum on PMA-induced apoptosis has been studied previously (29, 30), and it was concluded that the conflict between pRb-dependent growth signals and caspase activity is required for PKC-signaled apoptosis. Similarly, Lee and Rosson (11) demonstrated that only in the presence of serum was apoptosis induced in LLC-PK1 renal cells after chronic exposure to PMA. A requirement for serum to facilitate apoptosis seems logical, because selective programmed cell death, apoptosis, is usually observed under conditions where serum is plentiful and some cells need to continue to proliferate. An example is embryogenesis, where cells are both proliferating and undergoing apoptosis. Alternatively, there may be a threshold of PGD2 products that is needed for the apoptotic signal to proceed, and these products could be limited by the PGH2 substrate. This phenomenon requires further examination.
It appears that PKC and PKC
1 are the most
relevant isoforms involved with L-PGDS phosphorylation. This notion is supported by the data involving the indolocarbazole inhibitor Gö-6976, which selectively inhibits these PKC isozymes
(15). The fact that these are the classic
calcium-dependent isoforms is consistent with the calcium dependence we
have previously observed with L-PGDS-induced apoptosis. Other
forms, i.e., PKC
and PKC
, which have been shown to induce
apoptosis in certain cell lines, appear to involve p38
mitogen-activated protein kinase (MAPK) and operate independently of
the PI3-K pathway (5, 15, 21-23). We also observed no
change in ERK phosphorylation in response to L-PGDS. It is interesting
to note that selenocompounds, which are known to inhibit L-PGDS
enzymatic activity (6) and L-PGDS-induced apoptosis (13), have been shown to preferentially
inhibit the calcium-dependent isoforms of PKC (4). In
addition, bile salts, which are transported by L-PGDS in vivo, have
been shown to activate PKC and induce apoptosis in hepatocytes
(7). The possibility of other members of the MAPK family
such as c-Jun NH2-terminal kinase and p38 MAPK
participating in L-PGDS-mediated apoptosis needs to be examined
so that we can more clearly define the role of the MAPK pathway in this
process. We are currently investigating these kinases.
The molecular mechanism of L-PGDS-induced apoptosis appears to be at least in part via the inhibition of the PI3-K pathway and the subsequent inhibition of the phosphorylation of Bad and pRb. Usui et al. (28) have demonstrated a link between pRb phosphorylation and PI3-K signaling. These authors studied the effect of insulin on "clonal expansion" during adipocyte differentiation. Clonal expansion in response to insulin was accompanied by the hyperphosphorylation of pRb protein via the PI3-K pathway. These processes were inhibited by the addition of PI3-K inhibitors and rapamycin but not MAPK kinase inhibitors.
The activation of cellular phosphatases in response to PMA-induced and
PKC-mediated L-PGDS phosphorylation is a plausible synergistic
mechanism by which PI3-K activity is inhibited and apoptosis is
induced. Kisseleva et al. (9) demonstrated the inhibition
of PKB/Akt phosphorylation in kidney cells that express phosphoinositide-specific inositol polyphosphate 5-phosphatase IV, and
this resulted in increased apoptosis. One could imagine that
besides inhibiting PKB/Akt, L-PGDS phosphorylation could activate
similar phosphatases (e.g., protein phosphatase 2A) that are involved
with apoptotic signaling. The activity of certain phosphatases as
well as the involvement of other kinases (i.e., glycogen synthase
kinase-3) are currently under investigation.
Data presented in this study indicate that the induction of
apoptosis by phorbol ester is associated with the
phosphorylation and enzymatic activation of L-PGDS by PKC, the results
of which include the inhibition of PI3-K activity and subsequent
hypophosphorylation of the downstream targets Bad and pRb. Figure
9 is a schematic that summarizes the
effects of L-PGDS activation. These results indicate that phorbol
ester-induced apoptosis in LLC-PK1 cells is
mediated at least in part by L-PGDS phosphorylation by PKC, and the
mechanism may involve the inhibition of the PI3-K/PKB anti-apoptotic signaling pathways.
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ACKNOWLEDGEMENTS |
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We thank Dr. Najma Begum for assistance with the manuscript revisions.
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FOOTNOTES |
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This work was supported in part by a Grant-in-Aid Award from the American Heart Association Heritage Affiliate, a Career Development Award from the American Diabetes Association, and Winthrop-University Hospital.
Address for reprint requests and other correspondence: L. Ragolia, Cellular Biology Laboratory, Winthrop-Univ. Hospital, 222 Station Plaza North, Suite 505-B, Mineola, NY 11501 (E-mail: lragolia{at}winthrop.org).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published September 11, 2002;10.1152/ajpcell.00247.2002
Received 29 May 2002; accepted in final form 28 August 2002.
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