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This article was retracted on February 15, 2001.
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 12 (December 15), 1999:
pp. 4067-4076
Distinct Roles of JNKs/p38 MAP Kinase and ERKs in Apoptosis and
Survival of HCD-57 Cells Induced by Withdrawal or Addition of
Erythropoietin
By
Rujiao Shan,
James O. Price,
William A. Gaarde,
Brett P. Monia,
Sanford B. Krantz, and
Zhizhuang Joe Zhao
From the Division of Hematology/Oncology, Department of Medicine,
Department of Pathology, Department of Veterans Affairs Medical
Service, and Vanderbilt-Ingram Cancer Center, Vanderbilt University,
Nashville, TN; and the Department of Molecular Pharmacology, Isis
Pharmaceuticals, Carlsbad, CA.
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ABSTRACT |
Erythropoietin (EPO), a major regulator of erythroid progenitor
cells, is essential for the survival, proliferation, and
differentiation of immature erythroid cells. To gain insight into the
molecular mechanism by which EPO functions, we analyzed the activation
of Jun N-terminal kinases (JNKs) and extracellular signal-regulated kinases (ERKs) in HCD-57 cells, a murine erythroid progenitor cell line
that requires EPO for survival and proliferation. Withdrawal of EPO
from the cell culture medium resulted in sustained activation of JNKs
plus p38 MAP kinase, and inactivation of ERKs, preceding apoptosis of
the cells. Addition of EPO to the EPO-deprived cells caused activation
of ERKs accompanied by inactivation of JNKs and p38 MAP kinase and
rescued the cells from apoptosis. Phorbol 12-myristate 13-acetate,
which activated ERKs by a different mechanism, also suppressed the
activation of JNKs and significantly retarded apoptosis of the cells
caused by withdrawal of EPO. Furthermore, MEK inhibitor PD98059, which
inhibited activation of ERKs, caused activation of JNKs, whereas
suppression of JNK expression by antisense oligonucleotides and
inhibition of p38 MAP kinase by SB203580 caused attenuation of the
apoptosis that occurs upon withdrawal of EPO. Finally, the activation
of JNKs and p38 MAP kinase and concurrent inactivation of ERKs upon
withdrawal of EPO were also observed in primary human erythroid
colony-forming cells. Taken together, the data suggest that activation
of ERKs promotes cell survival, whereas activation of JNKs and p38 MAP
kinase leads to apoptosis and EPO functions by controlling the dynamic
balance between ERKs and JNKs.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
ERYTHROPOIETIN (EPO), a glycoprotein of
34 kD produced by the mammalian kidney and liver in response to
hypoxia, is critical for survival, proliferation, and differentiation
of erythroid cells.1,2 EPO promotes cell viability by
preventing apoptosis of erythroid progenitor cells.3,4
However, the signal transduction mechanism by which this occurs is
still unclear. It is known that EPO transduces its signal through
interaction with the EPO receptor by activating multiple signaling
pathways, including the Ras/MAP kinase, JNK/p38 MAP kinase, JAK/STAT,
and PI-3 kinase signaling cascades.5 Among the various
signaling intermediates activated by EPO, extracellular
signal-regulated kinases (ERKs; also known as mitogen-activated protein
[MAP] kinases) have a crucial role in promoting cell proliferation
and differentiation, whereas Jun N-terminal kinases (JNKs; also known
as stress-activated protein kinases [SAPKs]) and p38 MAP kinase are
thought to be associated with apoptosis.6-9 ERKs, JNKs, and
p38 MAP kinase are structurally related, and all of them are activated
by phosphorylation of threonine and tyrosine. However, they are
activated by very different types of extracellular signals. ERKs are
activated by a variety of growth factors and hormones that promote cell
proliferation and differentiation. In contrast, JNKs and p38 MAP kinase
are activated by various cellular stresses, including inflammatory
cytokines, UV light, protein synthesis inhibitors, osmotic, heat and
chemical shock, and bacterial endotoxin. All of these stress-related
factors also induce apoptosis. Furthermore, activation of JNKs and p38
MAP kinase appears to counteract the activation of ERKs, and the
dynamic balance between growth factor-activated ERKs and
stress-activated JNK-p38 MAP kinase pathways is thought to be important
in determining whether a cell survives or undergoes
apoptosis.10 Because withdrawal of EPO leads to apoptosis
of erythroid progenitor cells, activation of JNKs and p38 MAP kinase
might play a very important role in this process. For this reason, we
analyzed here the activation of JNKs, p38 MAP kinase, and ERKs in
HCD-57 cells, a murine erythroid progenitor cell line that requires EPO
for survival and proliferation, and in human erythroid colony-forming
cells. We found that activation of JNKs and p38 MAP kinase plus
concurrent inhibition of ERKs, upon withdrawal of EPO, is critical for
induction of apoptosis, whereas activation of ERKs accompanied by
inhibition of JNKs and p38 MAP kinase is important for promoting cell survival.
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MATERIALS AND METHODS |
Materials.
Recombinant human EPO was provided by Amgen, Inc (Thousand Oaks, CA).
MEK inhibitor PD98059 and rabbit phospho-specific antibodies against
JNKs (Thr183/Tyr185), ERKs (Tyr204), MKK3/6 (Ser189/207), and p38 MAP
kinase (Thr180/Tyr182) were purchased from New England Biolabs Inc
(Beverly, MA). Rabbit polyclonal anti-JNK antibody recognizing both
JNK1 and JNK2 and anti-p38 antibody were purchased from Santa Cruz
Biotechnology (Santa Cruz, CA). Rabbit anti-MAPK polyclonal antibody
recognized both ERK1 and ERK2 was raised in rabbits as previously
described.11 Phorbol 12-myristate 13-acetate (PMA) was
purchased from Sigma Chemical Co (St Louis, MO), and p38 MAP kinase
inhibitor SB203580 was from Calbiochem (La Jolla, CA).
Cell culture.
HCD-57 cells were maintained at 3 to 5 × 105 cells/mL
in Iscove's modified Dulbecco's medium (IMDM) supplemented with 20%
heat-inactivated fetal calf serum, 0.2 µmol/L 2-mercaptoethanol, and
2 U/mL recombinant human EPO in a 5% CO2 environment and
at 37°C. Human erythroid colony-forming cells were purified from
peripheral blood as previously reported.12 The cells were
expanded and maintained in IMDM supplemented with 20% fetal calf
serum, 1% deionized bovine serum albumin (BSA), 2 U/mL recombinant
human EPO, 100 ng/mL recombinant human stem cell factor (SCF), 50 U/mL
recombinant human interleukin-3, 10 µg/mL insulin,
10 4 mol/L 2-mercaptoethanol, 500 U/mL penicillin,
and 40 µg/mL streptomycin at 37°C in a high humidity 5%
CO2, 95% air incubator. Day-8 human erythroid
colony-forming cells were collected for study. The purity of the cells
was 80% ± 4.6% as determined by plasma clot assays.12 The EPO-free media used for starvation of HCD-57 and human erythroid colony-forming cells had the same compositions as the culture media,
except that EPO was omitted. For EPO starvation, normal growing cells
were spun down and washed twice with the EPO-free medium, followed by
further incubation in the same medium. In some cases, EPO (2 U/mL),
PMA, PD98059, and SB203580 were added to the starved cells, as detailed
in figure legends. For cell viability analyses, 4 cell replicates were
counted in the presence of 0.2% trypan blue, with at least 200 cells
counted for each sample.
Suppression of JNK1/2 by antisense oligonucleotides.
Antisense oligonucleotide 21861 for mouse JNK1/2 and control
oligonucleotide 101125 with a mismatched sequence are both mixed backbone (phosphodiester/phosphorothioate) 2'-methoxyethyl
chimera.13 Their sequences were
5'-CGGTAGGCTCGCTTAGCATG-3' and
5'-TGAGGCGTTAAGACGTTCAA-3' for the antisense and
control oligonucleotides, respectively. The oligonucleotides were
introduced into cells by lipofection. Briefly, 0.8 µL of
oligonucleotides at a concentration of 1 mmol/L was mixed with 10 µL
Lipofectin (1 mg/mL; Life Technologies, Inc, Grand Island, NY) and
incubated for 15 minutes at room temperature. The mixtures were then
added to HCD-57 cells (~7.5 × 105/mL) in 2 mL normal culture medium in 6-well plates. After 4.5 hours of
incubation at 37°C, the transfection medium was replaced with fresh
normal culture medium and cells were further cultured for 40 hours
before withdrawal of EPO from medium. After 1 to 4 days of EPO
starvation, cells were subjected to apoptosis analyses.
Western blotting analyses.
After the indicated periods of incubation, HCD-57 cells were collected,
washed with cold phosphate-buffered saline, and then lysed in buffer A
containing 20 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl, 1 mmol/L EDTA,
1 mmol/L EGTA, 1% Triton X-100, 2.5 mmol/L sodium pyrophosphate, 1 mmol/L  -glycerophosphate, 1 mmol/L Na3VO4, 1 µg/mL leupeptin, and 1 mmol/L phenylmethyl sulfonyl fluoride (PMSF).
Samples containing 20 µg of total proteins were separated on 10%
sodium dodecyl sulfate (SDS)-polyacrylamide gels and electrically
transferred to polyvinylidene difluoride (PVDF) membranes. The
membranes were blocked with 5% nonfat dry milk and then probed with
various primary antibodies overnight at 4°C. After washing 3 times
for 10 minutes each with a washing buffer, the membranes were incubated
with horseradish peroxidase-conjugated second antibodies, and the
antibody complexes were visualized by using the ECL method (Amersham
Life Science Inc, Arlington Heights, IL).
Activity assay of JNKs.
The enzymatic activity of JNKs was determined by using the JNK assay
kit from New England Biolabs Inc (Beverly, MA). Briefly, cells were
lysed in buffer A as described above. Cell extracts, containing 250 µg total proteins, were incubated overnight at 4°C with the
N-terminal c-Jun (1-89) fusion protein bound to glutathione-Sepharose beads. The N-terminal 89 amino acid segment of c-Jun contains a
high-affinity binding site for JNKs just N-terminal to the 2 phosphorylation sites, Ser63 and Ser73. It can selectively pull down
JNKs from the cell lysates. After washing twice with buffer A and once
with a kinase buffer (25 mmol/L Tris, pH 7.5, 5 mmol/L -glycerolphosphate, 2 mmol/L dithiothreitol [DTT],
0.1 mmol/L Na3VO4, and 10 mmol/L
MgCl2) to remove nonspecifically bound proteins, the beads
were resuspended in the kinase assay buffer. The kinase reaction was
performed by adding 100 µmol/L ATP to the suspension. Phosphorylation of c-Jun was measured by Western blot analyses with a
phospho-specific c-Jun antibody that specifically detects Ser63-phosphorylated c-Jun, a site important for c-JUN-dependent transcriptional activity.
Activity assay of p38 MAP kinase.
Cell extracts as obtained above were subjected to immunoprecipitation
with regular anti-p38 MAP kinase antibody at 4°C for 4 hours. The
beads were washed twice with cell lysis buffer and then twice with
kinase buffer (20 mmol/L MOPS, pH 7.2, 2 mmol/L EGTA, 20 mmol/L
MgCl2, 0.1 mmol/L sodium orthovanadate, 1 mmol/L DTT, and
0.1% Triton X-100). This was followed by the addition of 2 µg of
glutathione-S-transferase (GST)-ATF-2 (1-96) fusion protein (amino
terminal domain corresponding to amino acids 1-96 of ATF; Santa Cruz
Biotechnology) in 20 µL of kinase buffer supplemented with 30 µmol/L cold ATP and 5 µCi of [ -32P] ATP. After 20 minutes of incubation at 30°C, the reactions were terminated by
mixing with SDS gel sample buffer and boiling. The samples were
resolved by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiographed.
Detection of apoptosis of HCD-57 cells.
Apoptosis of HCD-57 cells was analyzed by using the in situ cell death
detection kit from Boehringer Mannheim (Indianapolis, IN). The method
is essentially based on the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) technique, and it
can detect apoptosis at very early stages. In brief, cells were fixed
with 4% paraformaldehyde for 30 minutes at room temperature, permeablized with 0.1% Triton X-100/0.1% sodium citrate for 2 minutes
on ice, and then labeled with fluorescein dUTP for 1 hour at 37°C.
The cells were then analyzed with a FACScan analyzer (Becton Dickinson,
San Jose, CA) with standard configuration or centrifuged onto glass
slides followed by photography with a fluorescent microscope. For the
FACScan analyses, Listmode data were analyzed offline with WinList
software (Verity Software House, Inc, Topssham, ME). Baseline apoptosis
was set at 3% to 6%, and the experimental effect was measured against
this background.
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RESULTS |
Withdrawal of EPO causes activation of JNKs and p38 MAP kinase
preceding apoptosis of HCD-57 cells.
HCD-57 cells require EPO for survival. When cultured in the absence of
EPO, the cells ceased proliferating, and by 24 hours of EPO starvation,
60% of the cells were dead due to apoptosis (Fig 1A). After 72 hours, essentially no
cells survived. Although this phenomenon has been well documented, the
signal transduction initiated by withdrawal of EPO, which leads to
apoptosis, is not clear. Because activation of JNKs has been implicated
in apoptosis of cells in other systems,10 we analyzed the
activity of JNKs in this process. First, we used a phospho-specific
antibody that specifically recognizes Thr183/Tyr185 phosphorylated JNKs
to determine the activation of the enzymes. As shown in Fig 1B, when
HCD-57 cells were cultured in EPO-free medium, sustained and enhanced phosphorylation of JNKs was observed. Increased phosphorylation of JNKs
(JNK1 and JNK2) was seen at 4 hours, and it reached a plateau at 16 hours. The magnitude of activation of the JNKs caused by deprivation of
EPO was comparable to that seen when HCD-57 cells were exposed to UV
light, a well-known stimuli of the JNK pathway (data not shown). After
24 hours, the phosphorylation decreased, which was probably due to the
fact that significant cell death occurred. To check whether EPO
starvation affects the expression level of JNKs, Western blotting was
performed with regular anti-JNK antibody that recognizes both
phosphorylated and nonphosphorylated JNKs. As shown in Fig 1B, no
significant change occurred in the JNK protein levels. These data
indicate that the increased anti-phospho-JNK reactivity is due to
increased phosphorylation of protein. Similar experiments demonstrated
that EPO starvation caused a decrease in phosphorylation of ERKs,
indicating inactivation of ERKs. Because dual phosphorylation of JNKs
at Thr183/Tyr185 is essential for kinase activity, phosphorylation at
this site is an excellent marker of JNK activity.7-9 To
confirm JNK activation, we performed an in vitro kinase assay by using a c-Jun N-terminal fusion protein as a substrate. As expected, the
kinase activity of JNKs in cell extracts was consistent with the
phosphorylation of JNKs determined by using the phospho-specific antibody (Fig 1B, bottom panel). We further analyzed the activation of
p38 MAP kinase and its upstream activator MKK3/6. As expected, withdrawal of EPO also caused activation of both enzymes, as
demonstrated by phospho-specific anti-p38 and anti-MKK3/6 antibodies
(Fig 1C). For both MKK3/6 and p38 MAP kinase, the activation appeared
at 4 hours and peaked at 16 hours. Kinase activity assay of p38 MAP kinase with recombinant ATF-2 as a substrate further verified the
results (Fig 1C, bottom panel). Together, the data suggest that
apoptosis of HCD-57 cells upon withdrawal of EPO is preceded by
sustained activation of JNKs and p38 MAP kinase that might be
responsible for the apoptosis.
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| Fig 1.
Withdrawal of EPO produces activation of JNKs and p38 MAP
kinase preceding apoptosis of HCD-57 cells. HCD-57 cells (2 × 107/mL) were incubated in EPO-free medium for the indicated
periods of time and then either were labeled with fluorescein-dUTP for
flow cytometry analyses (A) or were lysed in buffer A for JNK and p38
MAPK activation assays (B and C) as described in Materials and Methods.
(A) Flow cytometry analyses of apoptotic HCD-57 cells. The left peak
represents normal growing cells, whereas the right peak corresponds to
apoptotic cells, and the percentage of cells that undergo apoptosis is
indicated. (B and C; top 4 panels) Cell extracts (20 µg) were
resolved on 10% SDS-PAGE, transferred to PVDF membranes, and probed
with phospho-specific and regular antibodies against ERK1/2, JNK1/2,
p38 MAPK, and MKK3/6 as indicated. (B and C; bottom panels) Cell
extracts (containing 250 µg of total proteins) were incubated with an
N-terminal c-Jun (1-89) fusion protein bound to glutathione Sepharose
beads for JNK kinase activity assay or with anti-p38 MAP kinase
antibody for p38 MAPK activity assays with ATF-2 as a substrate.
Phosphorylation of c-Jun and ATF-2 was determined by Western blotting
with the phospho-specific c-Jun antibody and 32P
autoradiography, respectively.
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Addition of EPO produces activation of ERKs, inactivation of JNKs and
p38 MAP kinase, and prevention of apoptosis.
ERKs are major transducers of EPO signaling, and activation of ERKs by
EPO has been well documented.14-18 In conjunction with our
EPO starvation study described above, in which the cells were EPO-starved but serum-fed, we added EPO back to the EPO-starved cells
and analyzed ERKs by using a phospho-specific antibody. Withdrawal of
EPO from culture medium caused a significant decrease in the
phosphorylation of ERKs, although a basal phosphorylation of ERKs
remained even after 16 hours of EPO starvation (see Fig 1B), which
might be attributed to the presence of serum. Upon addition of EPO to
EPO-starved cells, a marked increase in phosphorylation of ERKs was
obtained by 8 to 16 hours after the addition of EPO, and at 24 hours,
the level of phosphorylation went back to the level obtained with cells
grown in the presence of EPO (Fig 2A, upper
panels). Western blotting analyses with regular anti-ERKs antibody
showed that the protein levels of ERKs were not affected by the
addition of EPO. Our previous study has shown that increased phosphorylation of ERKs parallels an increase in kinase activity of the
enzymes, because the phosphorylation is required for
activation.18 Therefore, the data indicated activation of
ERKs after addition of EPO to EPO-starved cells. It should be noted
that this slow but sustained activation of ERKs by EPO is different
from the rapid and transient activation of ERKs obtained with
serum-starved cells, as reported previously.14-18
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| Fig 2.
The addition of EPO to EPO-deprived cells produces
activation of ERKs, inactivation of JNKs and p38 MAPK, and rescue of
HCD-57 cells from apoptosis. (A and B) After 4 hours of EPO-starvation,
HCD-57 cells were cultured in complete medium containing 2 U/mL EPO for
the indicated periods of time. Cells were lysed for Western blot
analyses with the indicated antibodies (A) or cell growth was measured
as an index of cell viability (B). ( ) Cells were cultured in the
complete medium with EPO; ( ) cells were EPO-starved for 4 hours and
then were cultured in EPO-containing medium; ( ) cells were
continually incubated in EPO-free medium. (C and D) After 16 hours of
EPO-starvation, HCD-57 cells were cultured in complete medium
containing 2 U/mL EPO for 24 hours and then placed in EPO-free medium
and incubated for another 16 hours. Analyses of JNK1/2, p38 MAPK, and
ERK1/2s (C) and assays of apoptosis (D) were performed as described in
Fig 1. Lane C in (C) denotes control cells grown in the presence of
EPO.
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The addition of EPO to EPO-starved cells also resulted in inactivation
of JNKs and p38 MAP kinase, as demonstrated by the decreased
phosphorylation of the enzymes (Fig 2A, lower panels). The decrease in
phosphorylation of JNKs essentially coincided with the increase in
phosphorylation of ERKs. At 16 hours, the phosphorylation of JNKs
declined to a basal level equivalent to that observed with cells
cultured in EPO-containing medium. Western blotting analysis with
regular anti-JNKs and anti-p38 MAP kinase antibodies showed that the
levels of the proteins were not affected by addition of EPO. It should
be noted that a slight increase in the phosphorylation of JNKs and p38
MAP kinase was observed at 1 hour after the addition of
EPO. This might suggest a transient activation of JNKs and p38 MAP
kinase by EPO. This is consistent with the results observed with
serum-starved cells reported by others.19 The physiological
significance of this transient activation of JNKs is not known. The
transient nature of the activation makes it differ from the sustained
activation of the enzyme caused by withdrawal of EPO and should have a
different physiological meaning.
To analyze the consequence of the activation of ERKs and suppression of
JNKs and p38 MAP kinase induced by addition of EPO, we measured the
viability of cells. As shown in Fig 2B, the addition of EPO resulted in
a marked proliferation of the cells. Furthermore, fluorescent flow
cytometric analyses demonstrated that apoptosis of cells was inhibited
(Fig 2D).
To further show the correlation between activation of ERKs and
inactivation of JNKs and p38 MAP kinase, we removed EPO from culture
medium 24 hours after readdition of EPO to 16-hour EPO-starved cells
(Fig 2C and D). As expected, JNKs and p38 MAP kinase were reactivated,
and ERKs were reinactivated. This was accompanied by apoptosis of the
cells. Together, these results suggest that EPO promotes proliferation
and prevents apoptosis of cells by activating ERKs and suppressing JNKs
and p38 MAP kinase, thereby providing further evidence that ERKs and
JNKs have distinctly different roles in cell growth.
Withdrawal of EPO produces activation of JNKs and p38 MAP kinase and
inactivation of ERKs in human erythroid colony-forming cells.
The study described above was performed with HCD-57 cells, an immortal
cell line. Because the use of primary erythroid progenitor cells would
make the study more physiologically relevant, we used day-8 human
erythroid colony-forming cells purified from human peripheral blood
according to a well-established method previously described.12 The purity of these cells, which are primarily colony-forming units-erythroid (CFU-E), was 80% ± 4.6% based on the ability of cells to form erythroid colonies in plasma clot assays.
The data are shown Fig 3, and as observed
with HCD-57 cells, withdrawal of EPO caused activation of JNKs and p38
MAP kinase that reached a plateau at 16 hours. Accompanying this was a
decreased phosphorylation of ERKs. These data indicate that withdrawal
of EPO also produces activation of JNKs and p38 MAP kinase and
inactivation of ERKs in human primary erythroid colony-forming cells.
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| Fig 3.
Withdrawal of EPO produces activation of JNKs and p38
MAPK and inactivation of ERKs in human primary erythroid colony-forming
cells. Purifed day-8 human erythroid colony-forming cells, which are
mainly CFU-E, were EPO-starved for the indicated periods of time. Cell
extractions and Western blot analyses were performed as described in
Figs 1 and 2.
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PMA induces activation of ERKs and inhibition of JNKs and retards
cell apoptosis.
Activation of ERKs coincided with inactivation of JNKs, and vice versa.
One is initiated by addition of EPO, whereas the other is caused by
withdrawal of EPO. To prove that these 2 events are correlated rather
than isolated, we treated EPO-starved cells with a protein kinase C
activator, phorbol ester PMA. This notorious activator of ERKs
activates ERKs in a Ras-independent manner.6,7 As shown in
Fig 4A, when 50 nmol/L PMA was added to
4-hour EPO-starved cells, a slow activation of ERKs was observed with
phosphorylation of ERKs, reaching a peak at 16 hours and decreasing
thereafter. The level of the maximum activation of ERKs was slightly
higher than that obtained with control cells that were grown in the
presence of EPO. As observed with the addition of EPO, activation of
ERKs was accompanied by the inactivation of JNKs. Phosphorylation of JNKs was reduced to the basal level at 24 hours. Because PMA-induced activation of ERKs and suppression of JNKs does not involve EPO, the
data suggest that the ERK and JNK pathways may counteract each other,
and this might involve direct participation of the ERKs and JNKs
themselves. Additional data suggested that PMA also had a similar
effect on activation of p38 MAP kinase (data not shown).
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| Fig 4.
PMA produces activation of ERKs, inactivation of JNKs,
and inhibition of cell apoptosis. HCD-57 cells were cultured in an
EPO-free medium for 4 hours, followed by the addition of 50 nmol/L PMA.
Cells were cultured further for the indicated periods of time before
either being lysed for Western blot analyses with the indicated
antibodies or analyzed for apoptosis. Lane C denotes control cells
grown in the presence of EPO.
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We further analyzed the effects of PMA on the apoptosis of these cells.
As expected, apoptosis of the cells was significantly inhibited at 24 hours of culture by the addition of PMA (Fig 4B). More than 50%
inhibition of apoptosis was observed at 24 hours. However, this effect
was different from that observed with the addition of EPO. The addition
of PMA failed to rescue the cells from apoptosis completely, and
significant apoptosis occurred at 48 hours of incubation, even after
continued addition of fresh PMA every 24 hours. This correlated with
the decreased activation of ERKs after 24 hours of incubation. This
might be attributed to desensitization of protein kinase C by prolonged
PMA treatment. The data indicate that activation of ERKs participates
in the inhibition of apoptosis. Expression of constitutively active
ERKs should help to prove this. By showing that activation of ERKs leads to prevention of apoptosis of HCD-57 cells, this study further supports the notion that activation of ERKs has an essential role in
the expansion of erythroid progenitor cells, as proposed in our
previous studies.18
MEK inhibitor PD98059 inhibited activation of ERKs and caused
activation of JNKs.
To further define the interplay between ERKs and JNKs, we inhibited
activation of ERKs by treating HCD-57 cells with MEK inhibitor PD98059.
PD98059 is a potent inhibitor of MEK1, and it also inhibits MEK2.
Because MEK1 and MEK2 are directly upstream of ERKs in the MAP kinase
activation pathway, inhibition of MEK1/2 results in inhibition of ERKs.
As shown in Fig 5, treatment of normal
growing HCD-57 cells with 100 µmol/L PD98059 caused significant
inhibition of ERKs within 1 hour. Paralleling this was the activation
of JNKs as indicated by increased protein phosphorylation of the enzymes. Furthermore, PD98059 also caused a significant inhibition of
cell growth and a decrease in the number of viable cells (data not
shown). This provides further evidence that inactivation of ERKs causes
activation of JNKs.
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| Fig 5.
MEK inhibitor PD98059 inhibited activation of ERKs and
caused activation of JNKs. Normal growing HCD-57 cells were incubated
with 100 µmol/L PD98059 for the indicated periods of time. Cell
extractions and Western blot analyses were performed as described in
Figs 1 and 2.
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Suppression of JNK1/2 expression and inhibition of p38 MAP kinase by
SB203580 retarded apoptosis of cells.
The data described above showed a strong correlation between activation
of JNK1/2 and p38 MAP kinase and induction of apoptosis of HCD-57 cells
upon EPO withdrawal. To further show that activation of the kinases is
responsible for apoptosis of the cells, we inhibited JNK1/2 expression
by antisense oligonucleotides and p38 MAP kinase activity by specific
inhibitor SB203580. As shown in the inset of
Fig 6A, the antisense oligonucleotide of
JNK1/2 specifically suppressed expression of JNK1/2, compared
with the oligonucleotide with a mismatched sequence, whereas it
had no effect on the expression of ERK1/2 and p38 MAP kinase. As
expected, suppression of JNK1/2 significantly inhibited apoptosis of
HCD-57 cells in the absence of EPO, as shown by flow cytometric assays
(Fig 6A) and by fluorescent cell staining (Fig 6B). The effects were
most significant after 2 days of EPO starvation and showed 50%
inhibition. Similarly, addition of SB203580 totally inactivated p38 MAP
kinase (Fig 7A, inset) and caused an even
stronger inhibition of apoptosis (Fig 7). These data provide direct
evidence suggesting that activation of JNK1/2 and p38 MAP kinase is
responsible for apoptosis of HCD57 cells upon withdrawal of EPO.
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| Fig 6.
Antisense oligonucleotides specifically suppress JNK1/2
expression and retard apoptosis of HCD-57 cells in the absence of EPO.
Antisense and control oligonucleotides for JNK1/2 were introduced into
HCD-57 cells by lipofection as described in Materials and Methods. EPO
starvation was started 44 hours after cell transfection. Expression
levels of JNK1/2, p38 MAPK, and ERK1/2 were determined by Western
blotting analyses 74 hours after EPO starvation (inset in the left
panel). Apoptosis was determined by flow cytometric assays after 1 to 4 days of EPO withdrawal (left panel) and by fluorescent cell staining
after 2 days (right panel). The photograph was taken with 400×
magnification. Open bars and the top photo on right, EPO-containing
medium; reverse-slashed bars and the second photo on right, EPO-free
medium; horizontal bars and the third photo, EPO-free medium plus
antisense oligonucleotide; and slashed bars and the bottom photo on
right, EPO-free medium plus control oligonucleotide. Bright cell images
indicate apoptosis.
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| Fig 7.
SB203580 inhibits p38 MAPK activity and retards apoptosis
of HCD-57 cells in the absence of EPO. Cells were
pretreated with 10 µmol/L SB203580 for 4 hours in normal
growth medium before starvation with EPO-free medium supplemented with
the same concentration of the inhibitor. The inset in the top panel
shows p38 MAP kinase activity after 2 days of EPO starvation, as
determined by using ATF-2 as a substrate. Apoptosis was determined by
flow cytometric assays after 1 to 4 days of EPO withdrawal (top panel)
and by fluorescent cell staining after 2 days (bottom panel). The
photograph was taken with 400× magnification. The stock solution of
SB203580 was made in water. Open bars and bottom, left photo,
EPO-containing medium; reverse-slashed bars and the bottom, middle
photo, EPO-free medium; and slashed bars and the bottom,
right photo, EPO- free medium plus 10 µmol/L SB203580.
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DISCUSSION |
The present study has shown a molecular mechanism for the crucial role
of EPO in the growth of erythroid progenitor cells. The presence of EPO
induces activation of ERKs and suppression of JNKs and p38 MAP kinase,
whereas withdrawal of EPO from the cell culture medium causes sustained
activation of JNKs and p38 MAP kinase and inactivation of ERKs.
Activation of ERKs promotes cell growth, whereas activation of JNKs and
p38 MAP kinase is associated with apoptosis. Therefore, EPO appears to
promote cell growth and prevent apoptosis by regulating the dynamic
balance between ERK and JNK/p38 MAP kinase activities. However, it
should be noted that the activation of JNKs and ERKs observed in this study is somewhat different from previous reports. First, we observed a
slow but prolonged activation of JNKs and p38 MAP kinase upon withdrawal of EPO, whereas a rapid and transient activation of JNKs was
observed after the addition of EPO to serum-starved
cells.14-18 The prolonged activation of JNKs correlates
with the onset of apoptosis produced by withdrawal of EPO. The rapid
and transient activation upon addition of EPO may have a different
physiological meaning. In fact, a recent study indicates that
activation of p38 MAP kinase and JNKs is required for EPO-induced
erythroid differentiation.20 Secondly, we observed a slow
and sustained activation of ERKs induced by EPO, whereas previous
studies reported a rapid and transient activation. This is probably
caused by the different ways in which the cells were starved. In the
current study, cells were deprived of EPO but remained in serum
containing medium, whereas in the previous studies, the cells were
deprived of serum. Serum starvation brings cells to a quiescent state
that may produce a different response.
Our study suggests that ERKs counteract JNKs and p38 MAP kinase.
Cross-talk between different signal transduction pathways is a basic
regulatory mechanism in cells. A well-known example is the
downregulation of the ERK activation pathway by cyclic AMP-dependent
protein kinase, which can phosphorylate Raf-1, thereby inhibiting Raf-1
activity.21,22 A similar type of cross-talk could take
place between the ERK and JNK/p38 MAP kinase pathways. Activation of
ERKs by EPO through interaction with the EPO receptor has been
extensively studied. One pathway that leads to activation is the
classic SHC/Grb2-dependent Ras/Raf-1 pathway, and the other is the
SHC/Grb2-independent PI-3 kinase pathways.14-18 Both
pathways involve activation of MEK, which then phosphorylates ERKs. How EPO withdrawal triggers the JNK and p38 MAP kinase pathways is not well
understood. In the JNK pathway, there is a kinase cascade sequentially
involving MEKK1 and MEK-4 (also termed SEK1, MKK4, or
JNKK).8,9 MEK-4 directly phosphorylates and activates JNKs.
Because the pathways leading to activation of ERKs and JNKs consist of
multiple signaling components, most of which are regulated by protein
phosphorylation, there are multiple ways for the cross-talk to take place.
Other suggestive evidence for the counteracting effects between the 2 pathways is that one pathway can upregulate the terminating signal of
the other pathway. Activated by upstream dual specific kinases, ERKs,
JNKs, and p38 MAP kinase are all inactivated by dual-specific protein
phosphatases.23,24 It is likely that the ERK and JNK/p38
MAP kinase pathways downregulate each other by turning on the
expression of the phosphatases, which will act on the other. MKP-1, an
immediate early gene product that is upregulated by stress related
factors and growth factor, was identified as the ERK
phosphatase,25 although it also acts on JNKs. Enzymes specifically dephosphorylating ERKs or JNKs might exist, but this has
not been completely determined.
Apoptosis serves as a major mechanism for the precise regulation of
cell numbers and as a defense mechanism to remove unwanted and
potentially dangerous cells.26,27 It can be initiated by withdrawal of growth factors and stress conditions, as well as stimulation with tumor necrosis factor or Fas ligand. The apoptosis pathway involves multiple components, and a central element of the
pathway is the Bcl-2 family of proteins.28-30 The
antiapoptotic members of the family, including Bcl-2 and
Bcl-XL, act upstream of the execution caspases, thus
preventing their proteolytic processing into active killers. In
contrast, the proapoptotic members of the family, such as BAD, form
heterodimers with Bcl-2, thereby inhibiting the antiapoptosis activity
of the latter. Phosphorylation of the Bcl-2 family proteins has been
well documented.31-37 Although phosphorylation of the
antiapoptotic members may both augment and suppress
activity,31-34 phosphorylation of BAD by protein kinase B
(Akt) causes BAD to lose its binding ability to Bcl-2, because the
phosphorylated BAD is sequestered in the cytosol by 14-3-3 protein.35-37 Because the Bcl-2 family proteins have
multiple phosphorylation sites, it can be postulated that multiple
protein kinases are involved in the regulation of these proteins. In
fact, phosphorylation of Bcl-2 and Bcl-XL by both ERKs and
JNKs has been observed, although the effects on its antiapoptosis
activity are not well understood.31-34 This suggests that
ERKs and JNKs act upstream of Bcl-2 family proteins. Because certain
caspases are able to activate MEKK-1 and Mst1 by specific
cleavages,38,39 caspase activation can lead to activation
of JNKs, which, in turn, activate additional caspases, comprising a
positive feedback loop. Raf-1 provides another link between the MAP
kinase activation pathways and the apoptosis pathway. Raf-1, a major
component of the ERK activation pathway, is also a major player in
prevention of apoptosis. It has been shown that Bcl-2 can target Raf-1
to mitochondria and that active Raf-1 fused with targeting sequences
from an outer mitochondrial membrane protein protected cells from
apoptosis.40 Finally, MAP kinases are mediators of signal
transduction from the cell surface to the nucleus. One nuclear target
of these MAP kinase signaling pathways is the transcription factor AP-1
that has been implicated in the induction of apoptosis in cells in response to stress factors and growth factor withdrawal.41
A recent study demonstrated that AP1 is necessary for the induction of
apoptosis after hormone withdrawal from the EPO-dependent erythroid cell line HCD-57.42 We think that this may be related to
activation of JNKs.
| |
FOOTNOTES |
Submitted November 5, 1998; accepted August 6, 1999.
Supported by a Veterans Health Administration Merit Review Grant (to
S.B.K.), National Institutes of Health (NIH) Grants No. DK-15555 and 2 T32-DK-07186 (to S.B.K.), HL-57393 and CA75218 (to Z.J.Z.), and
CA-68485 (to Vanderbilt-Ingram Cancer Center). R.S. is an Ortho Biotech
Hematology Fellow.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Sanford B. Krantz, MD, or Zhizhuang Joe
Zhao, PhD, 547, MRB II, 2220 Pierce Ave, Nashville, TN 37232-6305;
e-mail: joe.zhao{at}mcmail.vanderbilt.edu.
| |
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ABSTRACT
INTRODUCTION