Department of Biochemical Toxicology, School of Pharmaceutical Sciences, Showa University, Tokyo 142-8555, Japan
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ABSTRACT |
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Bufalin, an
Na+-K+-ATPase inhibitor, simultaneously
induced cell differentiation and apoptosis in human monocytic
leukemia THP-1 cells. In this study, we investigated the regulatory
role of protein kinase C (PKC) isozymes in bufalin-induced cell
differentiation and apoptosis. A PKC-specific but
isozyme-nonselective inhibitor, Ro-31-8220, and a cPKC selective
inhibitor, Gö-6976, caused significant attenuation of
bufalin-induced interleukin-1 (IL-1
) gene expression, a mature
monocytic marker, indicating that cPKC participates in the
bufalin-induced cell differentiation. On the other hand, cPKC
- and
nPKC
-defective THP-1/TPA cells displayed strong resistance to
the bufalin-induced DNA ladder formation. Rottlerin, an
nPKC
-specific inhibitor, partially attenuated preapoptotic effects
of bufalin, such as the limited proteolysis of nPKC
and
poly(ADP-ribose) polymerase and the cell staining by terminal
deoxynucleotidyl transferase-mediated dUTP nick end labeling,
suggesting that nPKC
is involved, at least in part, in
bufalin-induced apoptosis. In contrast, Gö-6976 and
rottlerin significantly augmented bufalin-induced apoptosis and
differentiation, respectively. The findings suggest that
bufalin-induced cell differentiation and apoptosis are
interlinked and that distinct PKC isozymes are involved in the fate of
the cell.
cell differentiation; apoptosis
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INTRODUCTION |
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BUFALIN, A SPECIFIC INHIBITOR of Na+-K+-ATPase, has been shown to induce leukemia cell differentiation (31) and apoptosis (26) under certain experimental conditions. It has been reported that bufalin utilizes the extracellular signal-regulated kinase (ERK) cascade for signal transduction, leading not only to cell differentiation (13) but also to apoptosis (25, 26). It is thus postulated that the ERK cascade may control bufalin-induced cell differentiation and apoptosis simultaneously. It is generally accepted that an increase in ERK activity counteracts apoptotic signals, including stress-activated mitogen-activated protein kinases (MAPKs), such as c-Jun NH2-terminal kinase (JNK) and p38, and family members of caspase-like proteases (14, 29). Nevertheless, explanations for the overlapping of responses and functions of these MAPKs between distinct stimuli (8, 10, 21) have been obscure. Therefore, bufalin induction of cell differentiation and apoptosis could be a valid experimental model to elicit precise roles of ERK and other MAPKs in cell death and survival.
We recently reported that a family of phospholipid-activated
serine/threonine protein kinases, protein kinase C (PKC), plays an
important role as an upper module governing the ERK protein kinase
cascade in the signal transduction leading to bufalin-mediated cell
differentiation (13). The findings prompted us to examine a function of PKC in bufalin-mediated apoptosis. A family of
mammalian PKC is classified into three subfamilies: conventional PKC
(cPKC), novel PKC (nPKC), and atypical PKC (aPKC). The cPKC subfamily is composed of -,
-, and
-isozymes, which are activated in a
manner dependent on Ca2+ and diacylglycerol. The nPKC
subfamily is composed of
-,
-, and
-isozymes, which require
diacylglycerol for the activity but are independent of
Ca2+. The aPKC subfamily is composed of
- and
/
-isozymes, which are independent of diacylglycerol and
Ca2+. Because of differences in tissue distribution,
subcellular localization, and substrate specificity, these PKC isozymes
are involved in diverse functions, including induction of cell
differentiation and apoptosis (22).
We previously reported that 12-O-tetradecanoylphorbol
13-acetate (TPA)-resistant THP-1 (THP-1/TPA) cells are capable of
inducing interleukin-1 (IL-1
) mRNA, a biochemical marker for
mature monocytes, in response to bufalin, similar to the parental THP-1
cells (20). In the present study, we show that THP-1/TPA
cells, in which cPKC
and nPKC
were deficient, are resistant to
bufalin-induced apoptosis. Using this model system in
conjunction with specific inhibitors, we demonstrate that cPKC and nPKC
have divergent roles in the bufalin-mediated cell differentiation and
apoptosis. Our data suggest that cPKC and nPKC direct cell
differentiation and apoptosis, respectively, and that these
pathways are coupled in deciding the fate of the cell.
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EXPERIMENTAL PROCEDURES |
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Materials. TPA was purchased from Sigma Chemical (St. Louis, MO). Ro-31-8220, Gö-6976, and rottlerin were purchased from Calbiochem (La Jolla, CA). Monoclonal anti-PKC antibodies for immunoblot analysis and polyclonal anti-poly(ADP-ribose) polymerase (PARP) antibodies were purchased from Transduction Laboratories (Lexington, KY) and Wako Chemicals (Osaka, Japan), respectively. LY-379196 was a gift from Eli Lilly (Indianapolis, IN). Apoptosis Screening Kit was purchased from Wako Chemicals. Other chemicals were of the highest grade commercially available.
Cell culture. THP-1 cells (24) were obtained from Riken Cell Bank (Tsukuba, Japan). THP-1/TPA cells (20) were maintained in medium containing 100 nM TPA and were cultivated without TPA for 1 wk before use. The cells were cultivated as reported previously (20).
Northern blot analysis.
Total RNA was isolated from cells by acid guanidinium
thiocyanate-phenol-chloroform extraction as described by Chomczynski and Sacchi (1). Northern blot analysis was carried out as
described previously (20). Probes used were the 1.1-kb
PstI insert of a human IL-1 cDNA purified from IL-1 X-14
plasmid (17) (American Type Culture Collection) and the
0.5-kb insert of a glyceraldehyde 3-phosphate dehydrogenase (GAPDH)
cDNA purified from GD5 plasmid (16).
DNA fragmentation. Cells were washed with PBS and lysed in 10 mM Tris · HCl (pH 7.4) containing 10 mM EDTA and 0.5% (wt/vol) Triton X-100 for 10 min at 4°C. After centrifugation at 15,000 rpm for 5 min, the supernatant was incubated with 0.2 mg/ml RNase A at 50°C for 60 min and then with 0.2 mg/ml proteinase K at 37°C for 30 min. After precipitation with isopropanol, samples were subjected to electrophoresis on a 1% (wt/vol) agarose gel in 40 mM Tris acetate (pH 7.5) containing 1 mM EDTA for 60 min at 100 V. DNA was visualized by ethidium bromide staining.
Quantification of bufalin-induced apoptosis. Apoptotic cells were quantified by a modified terminal deoxynucleotidyl transferase-mediated dUPT nick end labeling (TUNEL) method according to the manufacturer's instructions (Wako). Briefly, fixed cells in 96-well microplates were incubated with terminal deoxynucleotidyl transferase and fluorescein-labeled dUTP at 37°C for 30 min and then with peroxidase-conjugated anti-fluorescein antibodies at 37°C for 30 min. The peroxidase reaction was carried out with hydrogen peroxide and o-phenylenediamine as substrates and measured at 492 nm.
Immunoblot analysis. Cells were lysed in the boiling SDS-sample buffer (62.5 mM Tris · HCl, pH 6.8, 5% 2-mercaptoethanol, 2% SDS, 10% glycerol, and 0.025% bromphenol blue). Denatured proteins were separated on a polyacrylamide gel (8%) and transferred to a polyvinylidene difluoride membrane (Pall Biosupport Division, Port Washington, NY) at 120 mA for 1 h with a semidry blotting apparatus. The membrane was incubated with 0.2% casein-based I-Block (Tropix, Bedford, MA) dissolved in 25 mM Tris · HCl, pH 7.4, containing 137 mM NaCl, 2.68 mM KCl, and 0.1% Tween 20 (TTBS) for 1 h at room temperature, washed with TTBS (3 times for 15 min each), and incubated for 1 h with primary antibody dissolved in the blocking solution overnight at 4°C. After it was washed with TTBS, the membrane was incubated for 1 h with the horseradish peroxidase-linked secondary antibodies. Immunoreactive proteins were detected by the enhanced chemiluminescence system (Amersham-Pharmacia Biotech, Buckinghamshire, UK).
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RESULTS |
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Responses of THP-1 and THP-1/TPA cells to bufalin.
A low concentration (30 nM) of bufalin induces differentiation of THP-1
cells characterized by continuous c-fos and egr-1 expressions (20), induction of IL-1 transcripts (Fig.
1A), and functional markers
for mature monocytes, such as phagocytosis and substrate adherence
(13). A higher concentration of bufalin (100 nM) also
induced IL-1
mRNA, but to a lesser extent (Fig. 1A).
Treatment of THP-1 cells with 100 nM bufalin induced DNA ladder
formation, a typical characteristic of apoptotic cell death. Bufalin at 30 nM also induced DNA fragmentation, but to a lesser extent
(Fig. 1B). We reported that inhibition of MAPK/ERK kinase (MEK), an upper kinase of ERK, abrogates IL-1
induction by bufalin (13). DNA ladder formation by bufalin was significantly
suppressed by pretreatment of cells with a specific MEK inhibitor,
U-0126 (3) (Fig. 1B). Bufalin induced IL-1
gene expression in THP-1/TPA cells to an extent similar to that in
THP-1 cells (20) (Fig. 1A). However, apparently
no ladder formation was observed when THP-1/TPA cells were exposed to
100 nM bufalin (Fig. 1B).
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PKC isozymes in THP-1 and THP-1/TPA cells.
THP-1 cells expressed -,
-, and
-isozymes of cPKC and the
-isozyme of nPKC (Fig. 2). THP-1/TPA
cells also expressed
- and
-isozymes of cPKC to an extent similar
to the parental cells; however, cPKC
and nPKC
virtually
disappeared in the resistant cells (Fig. 2). Other isozymes were
detected very weakly or not at all in THP-1 and THP-1/TPA cells (data
not shown).
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Effect of PKC inhibitors on bufalin-induced cell differentiation.
We previously showed that pretreatment of THP-1 cells with
Ro-31-8220, a specific but isozyme-nonselective PKC inhibitor
(7), reduces bufalin-induced IL-1 gene expression
(13). Gö-6976, which selectively inhibits cPKC
isozymes (18), was also effective; bufalin-induced IL-1
gene expression was inhibited concentration dependently and apparently
cancelled at 3 µM Gö-6976 (Fig.
3A). A suppressive effect of
the PKC inhibitors on bufalin-induced IL-1
expression was also seen
in THP-1/TPA cells to an extent similar to that observed in THP-1 cells
(Fig. 3B). Interestingly, bufalin-induced IL-1
gene
expression was significantly enhanced by rottlerin, an nPKC
-specific
inhibitor (6), in a concentration-dependent manner. The
nPKC
inhibitor at 2 µM augmented the bufalin-induced level of
IL-1
transcripts by approximately fivefold (Fig. 3C). However, as expected, rottlerin showed no effect on bufalin-induced IL-1
gene expression in PKC
-deficient THP-1/TPA cells (Fig. 3D).
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Effect of PKC inhibitors on bufalin-induced apoptosis.
THP-1 cells pretreated with PKC inhibitors followed by 100 nM bufalin
were subjected to analysis of apoptotic changes. Bufalin induced
limited proteolysis of 78-kDa nPKC and 116-kDa PARP, which is known
to be catalyzed by caspase-like proteases (2, 23),
resulting in the appearance of the 43- and 85-kDa fragments, respectively. Pretreatment with Gö-6976 significantly augmented limited proteolysis of PKC
and PARP (Fig.
4A). The cPKC inhibitor also
accelerated bufalin-induced DNA fragmentation (Fig. 4B) and the TUNEL-based cell staining (Fig. 4C). However, such an
augmented effect was not observed when cells were pretreated with
Ro-31-8220 (Fig. 4B). On the other hand, pretreatment
with rottlerin suppressed the bufalin-induced level of proteolytic
products of nPKC
and PARP (Fig. 4A). In addition,
rottlerin attenuated nucleosomal cleavage as determined by TUNEL
staining (Fig. 4C). However, LY-379196, a specific PKC
inhibitor (9), did not affect the preapoptotic proteolysis (data not shown).
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DISCUSSION |
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Bufalin preferentially induced THP-1 cells undergoing cell
differentiation at a low concentration (30 nM) and apoptosis at a high concentration (100 nM). On the other hand, 30 nM bufalin also
induced DNA fragmentation, but to a lesser degree than 100 nM bufalin.
In addition, 100 nM bufalin induced IL-1 expression, but to a lesser
degree than 30 nM bufalin. These results indicate that bufalin induces
cell differentiation and apoptosis simultaneously. It has been
suggested that the ERK cascade plays a major role in the cellular
signal transduction involved in the induction of cell differentiation
(13) and apoptosis (26) by bufalin, which are generally considered to be opposite responses
(29). The present study also demonstrated that the
specific inhibitor for the ERK cascade inhibited bufalin-induced DNA
fragmentation. The query how the same molecule controls differentiation
and apoptosis has arisen from these observations. One possible
resolution for the issue would be that distinct upper signals utilize
the ERK cascade, resulting in separate responses. It is suggested that scaffold or adaptor proteins may function in such a regulation (28, 30). On the basis of this hypothesis, we demonstrated in the present study a role of PKC isozymes in bufalin-mediated cell
differentiation and apoptosis.
In addition to our previous observations that inhibition of PKC by
Ro-31-8220 sequentially attenuates the bufalin-mediated ERK
activation, c-fos induction, and IL-1 gene expression
(13), the present study demonstrated that the
cPKC-specific inhibitor Gö-6976 was also effective in the
expression of the monocytic marker. These results forcefully suggest
that PKC, in particular cPKC, plays an important role as an upper
module of the ERK cascade in bufalin-induced cell differentiation. It
has been known that cPKC can directly interact with Raf-1 and, thereby,
activate the ERK cascade (12). We have shown that a
recently established specific inhibitor of the
Na+/Ca2+ exchanger, KB-R7943 (27),
suppressed ERK activation and c-fos and IL-1
gene
expressions (13). Taken together, inhibition of
Na+-K+-ATPase by bufalin may sequentially
induce an increase in Ca2+ concentration via the
Na+/Ca2+ exchanger, activation of
Ca2+-dependent cPKC and the ERK cascade, and the gene
expressions needed for monocytic differentiation, such as
c-fos and egr-1.
The present study demonstrated that cPKC- and nPKC
-deficient
THP-1/TPA cells displayed strong resistance to bufalin-induced apoptosis, although these cells retained the ability to
differentiate in response to bufalin. Similar observations that the
TPA-resistant cells, such as the HL-525 variant of HL-60 cells and the
TUR variant of U-937 cells, acquired resistance to tumor necrosis
factor-
- and drug-induced apoptosis have been reported
(15, 19). Because these variant cells have been reported
to be deficient in cPKC
gene expression, it has been suggested that
the PKC isozyme is involved in apoptosis mediated by certain
inducers. On the other hand, nPKC
is proteolytically activated by
caspase-3-like proteases, along with execution of apoptosis
(2). Findings that the forced expression of the
catalytically active 43-kDa fragment of nPKC
induces apoptotic
changes in transfectants (4) have suggested that increased
activity of nPKC
contributes to apoptosis (5). These findings prompted us to examine which PKC isozyme, cPKC
or
nPKC
, is involved in bufalin-mediated apoptosis. The
cPKC-selective and cPKC
-specific inhibitors, Gö-6976 and
LY-379196, respectively, showed no inhibitory effect on bufalin-induced
nPKC
and PARP proteolysis and DNA fragmentation. However, nPKC
inhibition by rottlerin suppressed the preapoptotic proteolysis and
TUNEL staining. These results suggest that nPKC
promotes early
biochemical changes leading to the bufalin-mediated apoptosis.
On the other hand, rottlerin failed to suppress the bufalin-induced DNA
fragmentation (data not shown), indicating that nPKC
is involved, at
least in part, but not sufficient for bufalin-mediated apoptosis.
During the course of this study, we were surprised to find that
rottlerin accelerated the bufalin-induced IL-1 gene expression. A
possible interpretation of these findings is that rottlerin attenuates
bufalin-induced apoptosis, resulting in more cells surviving
and expressing IL-1
. Findings that rottlerin was not effective in
nPKC
-deficient THP-1/TPA cells in regard to bufalin induction of
IL-1
support the idea that inhibition of apoptosis by
suppression of nPKC
activity enhances expression of the cell differentiation marker. However, cells treated with 30 nM bufalin for
48 h displayed >80% viability (20), indicating that
IL-1
expressions in rottlerin-rescued cells cannot fully explain the significant effect of the nPKC
inhibitor. Therefore, it is also possible that nPKC
negatively regulates the bufalin-induced cell differentiation. Because rottlerin did not enhance the bufalin-induced ERK activation (not shown), nPKC
is likely to regulate downstream signals of the ERK cascade. Although we have not identified the target
molecule of nPKC
, it may lie in upstream signals that confer the
activation of c-fos and egr-1 transcription, the
expressions of which are essential for cell differentiation along the
monocytic lineage (11). Meanwhile, Gö-6976
significantly reinforced the bufalin-induced DNA fragmentation and the
proteolytic cleavage of nPKC
and PARP. Such effects of the
cPKC-specific inhibitor were abrogated in the case of the
isozyme-nonselective inhibitor Ro-31-8220, suggesting that cPKC
negatively regulates other PKC signals that are involved in
bufalin-mediated apoptosis. It is also possible that inhibition
of cell differentiation by the cPKC inhibitor relatively enhances
bufalin-mediated apoptosis.
On the basis of results presented in this study, we propose that cPKC
and nPKC are involved in the signal transduction leading to
bufalin-induced cell differentiation and apoptosis,
respectively (Fig. 5). In addition, we
suggest that bufalin-mediated cell differentiation and
apoptosis are coupled to each other and that the distinct PKC
isozymes direct the fate of individual cells.
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FOOTNOTES |
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Address for reprint requests and other correspondence: S. Numazawa, Dept. of Biochemical Toxicology, School of Pharmaceutical Sciences, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan (E-mail: numazawa{at}pharm.showa-u.ac.jp).
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.
Received 13 July 2000; accepted in final form 6 October 2000.
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