Department of Biochemical Toxicology, School of Pharmaceutical Sciences, Showa University, Tokyo 142-8555, Japan
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ABSTRACT |
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Treatment of human leukemia THP-1 cells
with bufalin, a specific inhibitor of
Na+-K+-ATPase, sequentially induces
c-fos and inflammatory cytokines interleukin-1
(IL-1
) and tumor necrosis factor-
(TNF-
) gene expressions
before the appearance of mature phenotypes of monocytic cells. In this
study we examined the signal transduction leading to bufalin-induced
gene expressions. Bufalin selectively activated extracellular
signal-regulated kinase (ERK), compared with other mitogen-activated
protein (MAP) kinase family members. Pretreatment of THP-1 cells with
PD-98059, an inhibitor of the ERK-kinase cascade, abolished
bufalin-induced c-fos and IL-1
gene expressions, indicating that the ERK-kinase cascade mediates the induction of inflammatory cytokines by bufalin. Inhibition of the
Na+/Ca2+ exchanger by KB-R7943 and of protein
kinase C (PKC) by Ro-31-8220 suppressed ERK activation and gene
expressions of c-fos and IL-1
. These findings suggest that
Na+-K+-ATPase inhibition by bufalin induces
calcium influx and thereby activates PKC and ERK. In cells treated with
an inhibitor of p38 MAP kinases, SB-203580, bufalin-mediated ERK
activation became persistent and the induction of IL-1
and TNF-
expressions was significantly augmented. These results suggest that
cross talk in bufalin-mediated ERK activation is negatively regulated
by endogenous p38 MAP kinase activations.
interleukin-1; sodium-potassium-adenosinetriphosphatase; cell
differentiation; extracellular signal-regulated kinase
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INTRODUCTION |
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BUFALIN IS ONE OF THE MAJOR active components of a traditional Chinese medicine called Senso or Ch'an Su that is prepared from toad venom extracts. Although the well-known pharmacological actions of bufalin are cardiotonic effects, bufalin has been shown to induce cell differentiation (23, 24, 36) and apoptosis (30-32) in human leukemia cells under different experimental conditions. Cells treated with bufalin in the absence of serum preferentially undergo apoptosis; meanwhile, cells survive and eventually express differentiated phenotypes in the presence of serum. Ouabain, a typical cardiac glycoside and a specific inhibitor of the Na+-K+-ATPase, shows similar effects on leukemia cells, but to a lesser extent (23). The effect of bufalin on cardiac myocytes is due to the inhibition of Na+-K+-ATPase and thereby disruption of the cation homeostasis. Analogously, several lines of evidence indicated that the induction of cell differentiation by bufalin is mediated by Na+-K+-ATPase inhibition (23).
The mitogen-activated protein (MAP) kinase superfamily of serine/threonine kinases has emerged as an important component of cellular signal transduction. The MAP kinase family members have been implicated in events necessary for proliferation, differentiation, apoptosis, and certain kinds of stress responses (14). Three MAP kinase families, extracellular signal-regulated kinases (ERK), p38 MAP kinases, and c-Jun NH2-terminal kinases (JNK), have been well characterized. These MAP kinases are activated by specific cascades responsible for certain stimuli and eventually induce a variety of cell responses (5, 12, 22).
It has been suggested that the excessive activation of the ERK-kinase cascade, which is generally known to play a role in cell survival, is an event necessary for bufalin-mediated apoptosis (30, 32). Such a hyperexcitation of the ERK-kinase cascade is due to the activation of an upper signal component Ras and the concomitant downregulation of the protein kinase A activity that may negatively regulate the Raf-1 function (30). However, signal transduction leading to bufalin-mediated cell differentiation has not been explored. We report here that ERK activation, similar to apoptosis, plays a critical role in the induction of differentiation of human monocytic leukemia THP-1 cells by bufalin. In addition, we show evidence that p38 MAP kinases and/or their downstream molecules may modulate ERK activity and eventually cell differentiation.
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EXPERIMENTAL PROCEDURES |
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Materials. Bufalin was isolated from chloroform extracts of a
Chinese toad venom preparation (Senso, Shibata Pharmaceutical, Tokyo,
Japan) by repetitive chromatography on silica gel and a LiChroprep
RP-18 Lobar column (Merck, Darmstadt, Germany), as described previously
(23). PD-98059 and Ro-31-8220 were purchased from Calbiochem (La Jolla,
CA). -Hydroxyfarnesyl phosphonic acid (
-HFPA) was purchased from
Cayman (Ann Arbor, MI). KB-R7943 was kindly provided by H. Nakagawa
(Kanebo, Osaka, Japan). SB-203580 was a gift from SmithKline Beecham
(King of Prussia, PA). Polyclonal anti-ERK, anti-phospho-p38 MAP
kinase, anti-phospho-JNK, and monoclonal anti-phospho-ERK antibodies
for immunoblot analysis were purchased from New England Biolabs
(Beverly, MA). [
-32P]ATP and
[
-32P]dCTP were from The Institute of
Isotopes of the Hungarian Academy of Sciences, Hungary. Other chemicals
were of the highest grade commercially available.
Cell culture. THP-1 cells (28) were obtained from Riken Cell Bank (Tsukuba, Japan). Cells were maintained in an RPMI-1640 medium supplemented with 10% heat-inactivated fetal calf serum, 20 mM HEPES, 0.2% sodium bicarbonate, and penicillin-streptomycin. Cells were seeded at 1 × 105 cells/ml and maintained in continuous logarithmic growth in a humidified 5% CO2 atmosphere at 37°C. To determine cell adhesion, cells attached to the substrate were washed twice with PBS and treated with 0.25% trypsin/0.02% EDTA for counting. Phagocytic activity was determined by measuring the cells that engulfed carboxylate latex particles (average diameter, 0.75 µm, 2.5% solids-latex; Polysciences, Warrington, PA). Cells were incubated with latex particles (2 µl/ml) at 37°C for 5 h. After washing twice with PBS, cells containing more than five latex particles were scored as phagocytic cells.
Northern blot analysis. Total RNA was isolated from the
cells by acid guanidinium thiocyanate-phenol-chloroform extraction as
described by Chomczynski and Sacchi (2). Northern blot analysis was
carried out as described previously (24). Probes used were the 1.7-kb
EcoR I/Pst I fragment of a human c-fos cDNA
purified from pSPT-fos cDNA plasmid (29) (JCRB), the 1.1-kb
Pst I insert of a human interleukin-1 (IL-1
) cDNA
purified from IL-1 X-14 plasmid (21) and the 0.82-kb EcoR I
insert of a human tumor necrosis factor-
(TNF-
) cDNA purified
from pUC-RI-4 large plasmid (26) (American Type Culture Collection),
and the 0.5-kb insert of a rat glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) cDNA purified from GD5 plasmid (20).
Immunoblot analysis. Cells were lysed in boiling SDS-sample buffer. Denatured proteins were separated on a 10% polyacrylamide gel and transferred to a polyvinylidene difluoride membrane (Pall Biosupport Division, Port Washington, NY) at 120 mA for 1 h. The membrane was incubated with 0.2% casein-based I-Block (Tropix, Bedford, MA) dissolved in 20 mM Tris-HCl (pH 7.6) containing 137 mM NaCl and 0.1% Tween 20 (TTBS) for 1 h at room temperature, washed with TTBS (3 × 15 min), and incubated for 1 h with primary antibody dissolved in the blocking solution overnight at 4°C. After washing, the membrane was incubated for 1 h with respective horseradish peroxidase-linked secondary antibodies. Immunoreactive proteins were detected by the enhanced chemiluminescence system (Amersham-Pharmacia Biotech, Buckinghamshire, UK).
Immunocomplex kinase assay. Cells (2 × 106) were washed with ice-cold PBS containing 50 mM NaF, 30 mM Na4P2O7, and 100 µM
Na3VO4 and lysed in 0.5 ml of the lysis buffer
[150 mM NaCl, 10 mM Tris-HCl (pH 7.4), 5 mM EDTA, 50 mM NaF, 30 mM Na4P2O7, 100 µM
Na3VO4, 50 µg/ml
4-(2-aminoethyl)-benzenesulfonyl fluoride, 2 µg/ml leupeptin, and 1%
Triton X-100] for 30 min at 0°C. The cell lysate was
centrifuged at 15,000 rpm for 15 min. The supernatant (400 µl) was
incubated with 2 µg of anti-ERK antibodies (Santa Cruz) for 1 h at
4°C and 20 µl of protein A-agarose for an additional 2 h at
4°C. The immunocomplex was washed twice with 1 ml of the lysis
buffer containing 0.5 M NaCl and twice with 1 ml of the kinase buffer
(20 mM Tris-HCl, pH 7.6, 10 mM MgCl2, 2 mM
MnCl2, 1 mM dithiothreitol, 100 µM
Na3VO4, 1 mM EGTA). The kinase reaction was
carried out with 10 µl of the kinase buffer, 2 µg of myelin basic
protein (MBP), and 5 µCi of [-32P]ATP at
30°C for 15 min. The reaction was stopped by the 2× SDS sample buffer and boiling for 5 min. Phosphorylated MBP was separated by 13% SDS-PAGE, fixed, dried, and analyzed with a Fuji BAS 3000 image
analyzer (Fuji, Kanagawa, Japan).
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RESULTS |
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Differentiation of THP-1 cells by bufalin. Bufalin induced
growth arrest (Fig. 1A) and the
concomitant increase in the number of adherent and phagocytic cells in
a time-dependent (Fig. 1C) and dose-dependent (Fig. 1A)
manner in human monocytic THP-1 cells. Bufalin at a concentration of 30 nM also induced the sustained c-fos gene expression in THP-1
cells observed as early as 6 h, and lasting at least up to 48 h after
treatment (Fig. 1C). In addition, expressions of IL-1 and
TNF-
, inflammatory cytokines that were produced by mature monocytes,
increased (Fig. 1C) in conjunction with functional markers of
monocytic differentiation such as phagocytosis (Fig. 1A) and
cell adhesion (Fig. 1, A and B). Therefore, we utilized
c-fos, IL-1
, and TNF-
gene expressions as markers of cell
differentiation in the following study.
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Effect of the inhibitor of
Na+/Ca2+
exchanger.
Bufalin binds to the myocyte Na+-K+-ATPase and
inhibits its pumping function, thus leading to transient
accumulation of intracellular Na+. The
Na+/Ca2+-exchange system functions in its
reverse mode to decrease cytoplasmic Na+ and
concomitantly to increase Ca2+ concentration in myocardial
cells. These bufalin-mediated changes in the cation concentration are
thought to be the basis of its primary action on cardiac muscle. We
asked whether the influx of calcium ion by the
Na+/Ca2+ exchanger was involved in the
machinery of bufalin-mediated differentiation of THP-1 cells. KB-R7943
is a recently developed specific inhibitor for the exchanger and blocks
the calcium influx more potently than the efflux (11, 33). Pretreatment
of cells with KB-R7943 at a concentration sufficient to block the
Na+/Ca2+ exchanger (34) inhibited
bufalin-mediated c-fos and IL-1 gene expressions to about
50% of the control (Fig. 2,
A and B). These results suggest that influxed
Ca2+ may function as a second messenger in bufalin-mediated
signal transduction. To ensure this notion, we utilized calcium
ionophore A-23187. The forced increase in the calcium concentration in
cells resulted in a dose-dependent increase in IL-1
and TNF-
expressions (Fig. 2C). However, the ionophore did not induce
functional differentiation markers, such as cell adhesion and
phagocytosis (data not shown).
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DISCUSSION |
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The known biochemical action of bufalin is the inhibition of
Na+-K+-ATPase activity. Bufalin induces an
increase in the intracellular Na+ concentration and a
concomitant influx of Ca2+ by the action of the
Na+/Ca2+exchanger. These mechanisms are the
basis of the positive inotropic effect of cardiotonic steroids on
isolated heart preparations (35); bufalin has been shown to have a
similar effect (8). On the other hand, our previous studies have
demonstrated that differentiation-inducing activities of cardiotonic
steroids correlated well with the Na+-K+-ATPase
inhibitory abilities in human leukemia cells (23). In addition,
murine-derived leukemia cells display much less susceptibility to the
action of bufalin than human cells (23). Such species-dependent differences are related to the lower binding affinity between cardiotonic steroids and the -subunit of murine
Na+-K+-ATPase (9, 18). Furthermore,
ouabain-resistant THP-1 cells are less susceptible to bufalin (23),
indicating that the primary target of the cardiotonic steroid in cell
differentiation is Na+-K+-ATPase. Although
there is no direct evidence indicating that inhibition of
Na+-K+-ATPase is involved in bufalin-mediated
apoptosis, it is postulated that the enzyme inhibition could also be a
primary action (30). It has been reported that an excessive activation
of the ERK-kinase cascade may function during bufalin induction of
apoptosis (30); however, signal transductions that elicit cell
differentiation are still obscure. In this study we thus attempted to
clarify the downstream module of Na+-K+-ATPase
inhibition by using selective inhibitors for signal transducers.
Accumulated evidence has indicated that the transient increase in
intracellular calcium concentration leads leukemia cells to undergo
cell differentiation (25). Under such experimental conditions,
activation of the ERK-kinase cascade and the concomitant increase in
the c-fos expression have been observed (19). As demonstrated
in this study, KB-R7943 that inhibits the
Na+/Ca2+ exchanger-operated calcium influx
suppressed bufalin induction of c-fos and IL-1 expressions,
suggesting that the increased calcium mobilization contributes (if it
is not entirely responsible) to the bufalin induction of cell
differentiation. Induction of IL-1
and TNF-
expressions by calcium ionophore further supports the idea that a
calcium influx may trigger bufalin-mediated cellular signals. On the
other hand, A-23187 did not induce phagocytosis and cell-substrate
adherence, suggesting that the transient increase in intracellular
Ca2+ is an event important to the terminal differentiation
of THP-1 cells. Similar effects of A-23187 on THP-1 cells have been
reported previously (10).
Increased calcium ion may activate various signal transducers. The
classical type of the PKC (cPKC, cPKC
1, cPKC
2, and cPKC
) is
the well-demonstrated class of signal module activated by increased Ca2+ (25). These PKC isoforms have been shown to be
involved in cell proliferation, differentiation, and apoptosis. This
study demonstrated that the submicromolar range of Ro-31-8220, which preferentially inhibits the cPKC rather than other types of PKC (13),
suppressed the expression of differentiation markers induced by
bufalin, suggesting involvement of cPKC in the bufalin-mediated signal
transduction. It has been reported that PKC directly interacts with and
activates c-Raf and, as a consequence, transduces the signal to the
ERK-kinase cascade (15). The transient activation of ERK by bufalin was
sensitive to KB-R7943 and Ro-31-8220. Moreover, PD-98059, which
inhibits association of c-Raf and MEK, was capable of blocking bufalin
induction of c-fos and IL-1
gene expressions, indicating
that Na+-K+-ATPase inhibition may lead to
sequential activation of cPKC
c-Raf
MEK
ERK.
The fact that inhibition of the Ras function by
-HFPA showed
virtually no effect on bufalin induction of c-fos and IL-1
expressions further supports the assumption described above. Other MAP
kinase family members, p38 MAP kinases and JNK, seem to be less
sensitive to bufalin as determined by phosphospecific antibodies,
indicating that bufalin preferentially and selectively activates the
ERK-kinase cascade among the MAP kinase families during cell differentiation.
Meanwhile, the basal phosphorylation state of p38 MAP kinases was relatively high compared with ERK or JNK. That SB-203580 significantly augmented bufalin induction of the ERK activation and cytokine expressions was surprising and suggests that the basal activity of the p38 MAP kinases modulates either ERK dephosphorylation or the upstream module of the ERK-kinase cascade. In addition, p38 MAP kinase activities could negatively regulate THP-1 cells to undergo differentiation. To our knowledge, such a synergistic activation of ERK by the p38 MAP kinase inhibitor has not been reported to date. Molecular mechanisms governing the cross talk between the ERK and the p38 MAP kinase cascades during bufalin-mediated cell differentiation and apoptosis are currently under investigation.
In conclusion, our data show that Na+-K+-ATPase inhibition by bufalin induces activation of the ERK-kinase cascade, an event necessary for differentiation of THP-1 cells. Our data also suggest that the Na+/Ca2+ exchanger-operated transient Ca2+ influx sequentially mobilizes PKC, the ERK-kinase cascade, c-fos, and other specific gene expressions. It is noteworthy that bufalin utilizes the ERK-kinase cascade as a central signal module during not only cell differentiation but also apoptosis. Further studies regarding cross talk between MAP kinases could elucidate machinery deciding cell differentiation and apoptosis.
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ACKNOWLEDGEMENTS |
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We thank Suzanne Knowlton for critical reading of the manuscript and K. Makino and K. Mizuno for technical assistance in part of this study.
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FOOTNOTES |
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This work was supported in part by a Grant-in-Aid from the Ministry of Education, Science, Sports and Culture of Japan.
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: M. Kurosawa, Dept. of Biochemical Toxicology, Showa Univ. School of Pharmaceutical Sciences, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan (E-mail: kuromasa{at}pharm.showa-u.ac.jp).
Received 11 August 1999; accepted in final form 12 October 1999.
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