|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 3 (August 1), 1999:
pp. 853-863
Requirement of Activation of JNK and p38 for Environmental
Stress-Induced Erythroid Differentiation and Apoptosis and of
Inhibition of ERK for Apoptosis
By
Yuka Nagata and
Kazuo Todokoro
From the Tsukuba Life Science Center, The Institute of Physical and
Chemical Research (RIKEN), Tsukuba, Ibaraki, Japan.
 |
ABSTRACT |
C-Jun amino terminal kinase/stress-activated protein kinases
(JNK/SAPK) and p38 subgroups of mitogen-activated protein kinases have
been suggested to play a critical role in apoptosis, cell growth,
and/or differentiation. We found that a short exposure of SKT6 cells,
which respond to erythropoietin (Epo) and induce erythroid
differentiation, to osmotic or heat shock induced transient activation
of JNK/SAPK and p38 and inactivation of ERK and resulted in erythroid
differentiation without Epo, whereas long exposure of the cells to
these stresses induced prolonged activation/inactivation of the same
kinases and caused apoptosis. Inhibition of JNK/SAPK and p38 resulted
in inhibition of stress-induced erythroid differentiation and
apoptosis. Inhibition of ERK had no effect on stress-induced erythroid
differentiation, but stimulated apoptosis. Activation of p38 and/or
JNK/SAPK for a short time caused erythroid differentiation without Epo,
although its prolonged activation induced apoptosis. Activation of ERK
suppressed stress-induced apoptosis. These results indicate that short
cellular stresses, inducing transient activation of JNK/SAPK and p38,
lead to cell differentiation rather than apoptosis. Furthermore,
activation of JNK/SAPK and p38 is required for both cell
differentiation and apoptosis, and the duration of their activation may
determine the cell fate, cell differentiation, and apoptosis. In
contrast, inactivation of ERK is required for stress-induced apoptosis
but not cell differentiation.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
C-JUN AMINO TERMINAL kinase (JNK) or
stress-activated protein kinases (SAPK),1,2 p38 MAP
kinase or cytokine suppressive anti-inflammatory drug binding
protein (p38),3,4 and classical mitogen-activated protein
(MAP) kinases (ERK) are subgroups of a large MAP kinase family. ERK is
a protein-serine/threonine kinase that is rapidly activated by a
variety of cell growth and differentiation stimuli5-7 and
plays a central role in mitogenic signaling.8 The p38 and
JNK/SAPK cascades are primarily activated by various environmental
stresses: osmotic shock, UV radiation, heat shock, x-ray radiation,
hydrogen peroxide, and protein synthesis inhibitors and by the
proinflammatory cytokines, tumor necrosis factor- (TNF-), and interleukin-1 (IL-1).1-4,9-14
These cellular stresses and proinflammatory cytokines induce apoptotic
cell death.13 Stimulation of Fas also induces activation of
p38 and JNK/SAPK.15,16 Thus, it has been suggested that
JNK/SAPK and p38 have a critical role in signal transduction of
apoptotic cell death.
Expression of a constitutively active mutant of MEK kinase-1 (MEKK1),
an upstream activator of JNK/SAPK, has been shown to induce apoptosis
in mouse fibroblasts.17 Similarly, expression of the
constitutively activated form of MKK3,18 which is an upstream kinase of p38, induced apoptosis in PC12 cells. Activation of
another upstream kinase of p38, MKK6, also induced apoptosis in Jurkat
T cells.19 Furthermore, overexpression of
ASK120 or TAK1,21 both of which are MAPK
kinases and activate both JNK/SAPK and p38, also caused apoptotic cell
death in COS7 cells and Xenopus embryos, respectively.
Conversely, cells expressing dominant-inhibitory mutants within the
JNK/SAPK pathway become highly resistant to stress-induced
apoptosis.13,18,22-24 Expression of dominant-negative SEK1,
which is an upstream kinase of JNK/SAPK, suppressed TNF--induced
apoptosis.13 The dominant-negative form of JNK1, but not of
p38, inhibited UV and  radiation-induced apoptosis.23
The dominant-negative mutants of c-Jun, SEK1 and MKK3,18
and anti-c-Jun antibody25 blocked apoptosis when nerve growth factor was removed in PC12 cell culture. Disruption of JNK3,
which is predominantly expressed in neurons, prevented neuronal apoptosis.26 These results support that JNK/SAPK and p38
play a vital part in apoptotic cell death.
There are conflicting reports concerning the involvement of JNK/SAPK
and p38 in apoptosis.27-31 Induction of JNK/SAPK activity by ligation of Fas was a result rather than a cause.27
TNF- receptor-mediated JNK/SAPK activation was not linked to
apoptosis.28 CD40 ligand, which is known to counteract
apoptosis in B cells, activates JNK/SAPK.29 Disruption of
SEK1 did not affect the induction of stress-induced apoptosis, but
instead protected thymocytes from Fas-mediated and CD3-mediated
apoptosis.30 TRAF2-deficient mice experienced severe
reduction of TNF--induced JNK/SAPK activation, but were highly
sensitive to TNF--induced apoptosis.31 These results
suggest that JNK/SAPK and p38 are not always involved in
stress-induced, TNF--induced, or Fas-induced apoptosis.
JNK/SAPK and p38 can also be activated by such mitogenic factors as
epidermal growth factor and phorbol esters11 and by T-cell
activation signaling.32 It was recently reported that various hematopoietic cytokines, interleukins, and colony-stimulating factors, which regulate hematopoietic cell growth, survival, and differentiation, activate not only classical MAP kinase ERK, but also
p38 and JNK/SAPK.33-37 It was further reported that T-cell proliferation in response to IL-2 and IL-7 requires p38
activation38 and that JNK/SAPK and p38 are required for
erythropoietin (Epo)-induced growth signal.39 Recently, we
also showed that Epo induces transient activation of JNK/SAPK, p38, and
ERK in SKT6 cells, which can respond to Epo and induce
hemoglobinization and that activation of JNK/SAPK and p38, but not ERK,
is required for Epo-induced erythroid differentiation of SKT6
cells.39 It was also shown that GTPase-deficient G proteins
induce persistent activation of JNK and PC12 cell differentiation, and
overexpression of c-Jun induces neurite outgrowth.40 In P19
embryonic carcinoma cells, ectopic expression of c-Jun leads to cell
differentiation,41 and overexpression of the dominant
negative form of JNK blocks the induction of endodermal differentiation
by retinoic acid.42 Differentiation of WEHI-3B(D+)
myelomonocytic leukemia cells is also induced by ectopic expression of
c-Jun.43 These reports indicate that JNK/SAPK and p38
pathways are involved in not only apoptotic, but also mitogenic and
differentiation signalings, at least, in some cell lineages.
To explore the roles of JNK/SAPK, p38, and ERK, we examined whether or
not environmental stresses such as osmotic or heat shock could mimic
Epo-induced erythroid differentiation of SKT6 cells. Surprisingly, we
found that a short exposure of the cells to these cellular stresses
induced transient activation of JNK/SAPK and p38 and transient
inactivation of ERK. This resulted in erythroid differentiation without
Epo stimulation, whereas long exposure to these stresses induced
persistent activation of JNK/SAPK and p38 and inactivation of ERK and
caused apoptotic cell death. Activation of JNK/SAPK and p38 was also
found to be required for stress-induced erythroid differentiation as
well as apoptotic cell death, and activation of p38 alone was
sufficient to induce erythroid differentiation to some extent without
any stimulation. Furthermore, inhibition of ERK was found to be
required for stress-induced apoptotic cell death but not erythroid
differentiation. We discuss here the possible roles of JNK/SAPK, p38,
and ERK during stress-induced cell differentiation and apoptotic cell death.
| |
MATERIALS AND METHODS |
Cell culture and stress-induced erythroid differentiation.
Epo-responsive mouse erythroleukemia SKT6 cells44 were
maintained in Ham F-12 medium supplemented with 10% fetal calf serum. SKT6 cells were induced to differentiate by the addition of 0.5 U/mL of
recombinant human Epo (2.6 × 105 U/mg; Kirin Brewery)
followed by incubation for 4.5 days, and hemoglobin-positive cells were
stained by 0.05% 2,7-diaminofluorene (DAF).39 Cells were
treated with osmotic shock (0.1 mol/L NaCl) or heat shock (42°C) for
1 hour, then cultured in normal culture medium with or without 0.5 U/mL
of Epo for 4.5 days, and hemoglobinized cells were counted.
S-oligos, inhibitors, and activators of MAP kinases.
Various concentrations of antisense, sense, or scrambled
phosphothioester oligonucleotides (S-oligos) (BEX, Tokyo, Japan), p38-specific inhibitor SB203580 (SmithKline Beacham, King of Prussia, PA) in dimethyl sulfoxide (DMSO), MEK-specific inhibitor PD98059 (Calbiochem, La Jolla, CA) in ethanol, or p38 and JNK/SAPK activators, anisomycin (Calbiochem)45 and C2-ceramide
(Calbiochem),46 were mixed in the culture medium before
stress treatment, and the effect on cell differentiation and apoptotic
cell death was examined. The S-oligos were added again 24 hours after
stress treatment. The antisense S-oligos used were described
previously.39 As control, the corresponding sense S-oligos
and scrambled S-oligos were used.
In vitro protein kinase assay.
Activity of ERK1, ERK2, JNK1, JNK2, or p38 was assayed in vitro as
described previously.35,36,39
Analysis of apoptotic cell death and DNA fragmentation.
Apoptotic cell death was measured by Tdt-mediated dUTP nick-end
labeling (TUNEL) methods as followed by the manufacturer's protocol
(fluorescein apoptosis detection system) (Promega, Madison, WI). DNA
fragmentation was assayed by the modified method of Sellins and
Cohen.47 SKT6 cells (1 × 106 cells) were
washed with phosphate-buffered saline and suspended in a lysis buffer
(10 mmol/L Tris-HCl, pH 7.5, 10 mmol/L ethylenediamine tetraacetate,
0.5% Triton X-100) for 10 minutes on ice. The suspension was
centrifuged at 16,000 rpm for 20 minutes and the fragmented DNA was
recovered from the supernatant. The supernatant was subjected to
digestion with ribonuclease A (0.5 mg/mL) for 1 hour at 37°C followed
by incubation with proteinase K (0.5 mg/mL) for 1 hour at 37°C. Equal
amounts of the precipitated DNA were separated by 1.8% agarose gel
electrophoresis and visualized by ethidium bromide staining.
Caspase-3 assay.
Caspase-3 activity was measured by CPP32 colorimetric assay kit
(Clontech, Palo Alto, CA). The cell extracts (100 µg protein) were
incubated at 37°C for 60 minutes with labeled specific substrate (DEVD-pNA) in reaction buffer. The colorimetry of the cleaved substrates was measured in a spectrophotometer at a wavelength of 405 nm. One unit is defined as the amount of enzyme required to cleave 1 pmol of the substrate at 37°C for 60 minutes under the standard
reaction conditions.
Plasmid construction.
An HindIII-XbaI fragment of
pcDNA3-flag-MKK6(Glu)48 was ligated to an
ecdysone-inducible mammalian expression vector pIND (Invitrogen,
Carlsbad, CA).49 The pIND/MKK6(Glu) and pVgRXR were
transfected into SKT6 cells. Several double resistant (Zeocin and G418)
clones were isolated, and the expression of MKK6(Glu) was induced by 2 µmol/L muristerone A, which was confirmed by antiflag-tag antibody
(M2). The hemagglutinin (HA) epitope-tagged constitutively active MKK1
( N3-S218E/S222D)50 was subcloned into the
KpnI/EcoRV sites in pCDNA3 and transfected into SKT6 cells. The expression of HA-MKK1 mutant was detected by immunoblotting with monoclonal antibody to HA.
| |
RESULTS |
A short exposure of SKT6 cells to heat or osmotic shock induces
erythroid differentiation without Epo.
In an attempt to determine the roles of JNK/SAPK and p38 in apoptotic
and cell differentiation signals, we examined whether environmental
stresses such as osmotic shock and heat shock could induce cell
differentiation rather than apoptotic cell death. Mouse erythroleukemia
SKT6 cells can respond to Epo and induce erythroid differentiation.
These cells are not dependent on Epo to proliferate and their
proliferation is neither enhanced nor impaired by the presence of Epo.
We found that a short exposure (less than 1 hour) of SKT6 cells, which
can be induced to differentiate into hemoglobinized cells in response
to Epo (Fig 1A, left two panels), to
osmotic shock (0.1 mol/L NaCl) or heat shock (42°C) induced erythroid
differentiation without Epo stimulation (Fig 1A, right two panels),
although the number of hemoglobinized cells was about one third of that
stimulated with Epo (Fig 1B, lanes 3 and 5). In the presence of Epo,
these stresses additively stimulated the Epo-induced erythroid
differentiation (Fig 1B, lanes 4 and 6) compared with Epo alone (Fig
1B, lane 2). The best conditions to induce erythroid differentiation,
but not to induce apoptotic cell death, were found to be with the
addition of 0.1 mol/L NaCl to the cells for 1 hour or incubation of the
cells at 42°C for 1 hour.
View larger version (71K):
[in this window]
[in a new window]
| Fig 1.
A short exposure of SKT6 cells to osmotic or heat shock
induces erythroid differentiation rather than apoptotic cell death. (A)
Left two panels: the hemoglobinized SKT6 cells stained with DAF after
culture with or without Epo for 4.5 days. Right two panels: the
hemoglobinized cells after treatment with 0.1 mol/L NaCl or 42°C for
1 hour, followed by incubation without Epo for 4.5 days. (B) The
relative differentiation of SKT6 cells. Cells treated with 0.1 mol/L
NaCl for 1 hour (lanes 3, 4), cells incubated at 42°C for 1 hour
(lanes 5, 6), or untreated cells (lanes 1, 2) were cultured with (lanes
2, 4, 6) or without (lanes 1, 3, 5) Epo for 4.5 days and stained. The
percentage of hemoglobinized cells in the presence of Epo is defined as
100%.
|
|
The majority of the cells treated with these cellular stresses for up
to 1 hour survived (Fig 2A), and neither
caspase-3 activation (Fig 2B, lanes 2 and 9) nor DNA fragmentation (Fig
2C, lanes 3 and 9) was observed. This was true even 24 hours after
release of these short stresses (Fig 2B, lanes 7 and 14; Fig 2C, lanes 6 and 12). However, a longer exposure of the cells to these stresses actually activated caspase-3 (Fig 2B, lanes 3 to 6 and 10 to 13) and
DNA fragmentation (Fig 2C, lanes 5 and 11), leading to apoptotic cell
death (Fig 2A).
View larger version (23K):
[in this window]
[in a new window]
| Fig 2.
(A) Cell death rate (%) after osmotic (left panel) or
heat (right panel) shock for various periods as indicated. (B)
Caspase-3 activity in cells treated with 0.1 mol/L NaCl (lanes 1 to 7)
or incubated at 42°C (lanes 8 to 14) for the periods indicated. Lanes
7 and 14: activity in the cells treated with NaCl (lane 7) or heat
(lane 14) for 1 hour followed by incubation for an additional 24 hours
under normal conditions. (C) DNA fragmentation of the cells treated
with 0.1 mol/L NaCl (left panel) or 42°C (right panel) for various
periods as indicated. Lanes 1 and 7: molecular weight marker. Lanes 6 and 12: cells treated with the stresses for 1 hour followed by a
further 24-hour incubation under normal conditions.
|
|
Transient activation of JNK/SAPK and p38 and inactivation of ERK by a
short osmotic or heat shock.
The activity of the MAP kinase family (JNK1, JNK2, p38, ERK1, and ERK2)
was measured during osmotic or heat shock-induced erythroid
differentiation of SKT6 cells. Cells were exposed to these cellular
stresses for 1 hour and released. In vitro kinase assays in the
immunoprecipitates with each specific antibody at the indicated time
point showed that the activities of JNK1 (Figs 3A and
B, left panels), JNK2 (Figs 3A and B,
right panels), and p38 (Figs 3C and D) were only weakly detected before
osmotic-treatment or heat-treatment (Figs 3A-D, lane 1), but rapidly
and dramatically increased within 15 minutes after stress treatment
(Figs 3A-D, lane 2). They remained at the maximum levels until stress
release (Figs 3A-D, lane 3), but returned to the basal levels within 3 hours thereafter (Figs 3A-D, lane 6).
View larger version (18K):
[in this window]
[in a new window]
| Fig 3.
A short exposure of SKT6
cells to osmotic or heat shock induces transient activation of JNK/SAPK
and p38, but suppresses activity of ERK. SKT6 cells were treated with
osmotic shock (0.1 mol/L NaCl) (A, C, E) or heat shock (42°C) (B, D,
F) for 0, 15, or 60 minutes, and cultured for a further 0, 30, 60, or
180 minutes under normal conditions. (A and B) The JNK1 (left panels)
and JNK2 (right panels) activity in the immunoprecipitates at the
indicated time points after stress treatment was assayed with GST-c-Jun
as a substrate. Arrows indicate the phosphorylated GST-c-Jun. (C and D)
The p38 activity in the immunoprecipitates at the indicated time points
after stress treatment was assayed with GST-ATF-2 as a substrate.
Arrows indicate the phosphorylated GST-ATF-2. (E and F) The ERK1 (left
panels) or ERK2 (right panels) activity in the immunoprecipitates at
the indicated time points after stress treatment was assayed with major
basic protein (MBP) as a substrate. Arrows indicate the phosphorylated
MBP.
|
|
In contrast, the activities of ERK1 (Figs 3E and F, left panels) and
ERK2 (Figs 3E and F, right panels) were clearly detected before stress
treatment (Figs 3E and F, lane 1), but rapidly decreased with this
treatment (Figs 3E and F, lanes 2 and 3). They remained low until
stress release (Figs 3E and F, lane 3) had returned to the original
levels within 3 hours thereafter (Figs 3E and F, lane 6). In-gel kinase
assays confirmed the specific activation of JNK/SAPK and inactivation
of ERK (data not shown). These results indicate that a short exposure
of SKT6 cells to these cellular stresses induces transient activation
of JNK/SAPK as well as p38, but transiently suppresses the activity of ERK.
Inhibition of p38 results in a block of stress-induced erythroid
differentiation and delay of apoptotic cell death, and inhibition of
MEK stimulates stress-induced apoptotic cell death.
We next examined the effects of inhibition of these MAP kinases by
their specific inhibitors on stress-induced erythroid differentiation. The p38-specific inhibitor SB203580 strongly blocked both
osmotic-induced (Fig 4A, left panel, closed
circles) and heat-induced (Fig 4B, left panel, closed
circles) hemoglobinization of SKT6 cells in a dose-dependent manner.
SB203580 actually inhibited p38 activity as mentioned
previously.39 The MEK-specific inhibitor PD98059 did not
affect these stress-induced erythroid differentiation at all (Figs 4A
and B, right panels), although PD98059 (50 µmol/L) completely blocked
activation of both ERK1 and ERK2 as described.39 Neither
the inhibitor alone nor the solvents used to dissolve the inhibitors
(0.1% DMSO or 0.1% ethanol) affected cell differentiation (data not
shown). These results indicate that, at least, activation of p38,
although not ERK, is essential for stress-induced erythroid differentiation.
View larger version (23K):
[in this window]
[in a new window]
| Fig 4.
Activation of p38 but not MEK is required for
stress-induced erythroid differentiation. SKT6 cells mixed with various
concentrations of SB203580 (left panel) or PD98059 (right panel) were
treated by (A) osmotic or (B) heat shock for 1 hour, and the percentage
of hemoglobinized cells (%) was counted after 4.5 days. The percentage
of hemoglobinized cells after stress treatment without inhibitor is
defined as 100%. Values shown are the mean of five experiments.
|
|
The effects of these inhibitors on osmotic-induced and heat-induced
apoptotic cell death were also tested. Addition of SB203580 clearly
caused delay of osmotic-induced (Fig 5A,
left panel, open circles) and heat-induced (Fig 5B, left panel, open
circles) apoptotic cell death. In contrast, inhibition of ERK
activation by PD98059 clearly stimulated osmotic-induced (Fig 5A, right
panel) or heat-induced (Fig 5B, right panel) apoptotic cell death.
Neither the inhibitor alone (Figs 5A and B, closed circles) nor the
solvents used to dissolve the inhibitors (data not shown) affected cell
differentiation. Thus, activation of p38 and inhibition of ERK are
required for stress-induced apoptotic cell death.
View larger version (23K):
[in this window]
[in a new window]
| Fig 5.
Inhibition of p38 causes delay of stress-induced
apoptotic cell death, but inhibition of MEK stimulates stress-induced
apoptotic cell death. Cell death rate (%) after (A) osmotic or (B)
heat shock with ( ) or without ( ) SB203580 (left panel) or PD98059
(right panel) for various periods as indicated. Values shown are the
mean of five experiments.
|
|
Antisense S-oligos of p38 and JNK/SAPK block stress-induced erythroid
differentiation and cause delay of apoptosis, and those of ERK
stimulate stress-induced apoptotic cell death.
We also tested the effects of inhibition of these MAP kinases by their
specific antisense S-olios on stress-induced erythroid differentiation.
Antisense S-oligos of p38, JNK1, and JNK2 strongly inhibited the
osmotic-induced (Fig 6A) or heat-induced
(Fig 6B) hemoglobinization of SKT6 cells in a dose-dependent manner
(Figs 6A and B, middle three panels, closed circles). All of these
antisense S-oligos specifically inhibited the expression of each
corresponding kinase as described.39 In contrast, antisense
S-oligos of ERKs (commonly effective to both ERK1 and ERK2) did not
affect stress-induced erythroid differentiation at all (Figs 6A and B,
right end panels, closed circles). Neither scrambled S-oligos (Figs 6A
and B, left end panels) nor sense S-oligos (Figs 6A and B, open
circles) had any effect on it. Environmental stresses, thus, induce
erythroid differentiation without Epo through activation of JNK1, JNK2, and p38, but not ERK.
View larger version (30K):
[in this window]
[in a new window]
| Fig 6.
Activation of p38 and JNK/SAPK, but not ERK, is required
for stress-induced erythroid differentiation. SKT6 cells mixed with
various concentrations (0 to 30 µmol/L) of antisense S-oligos ()
or sense S-oligos () of p38, JNK1, JNK2, ERKs (common to both ERK1
and ERK2), or scrambled S-oligos were (A) osmotic shocked or (B) heat
shocked for 1 hour, and the percentage of hemoglobinized cells was
counted after 4.5 days. The percentage of hemoglobinized cells after
stress treatment without S-oligos is defined as 100%. Each
point represents the mean of five replicates.
|
|
Similarly, addition of antisense S-oligos of p38, JNK1, and JNK2 caused
delay of osmotic-induced (Fig 7A) and
heat-induced (Fig. 7B) apoptotic cell death (Figs 7A and B, middle
three panels, open circles). In contrast, addition of antisense
S-oligos of ERK-stimulated apoptotic cell death (Figs 7A and B, right
end panels). Neither scrambled S-oligos (Figs 7A and B, left end
panels) nor sense S-oligos (Figs 7A and B, closed circles) had any
effect on it. Taken together, we concluded that the activation of
JNK/SAPK and p38 plays a critical role in stress-induced erythroid
differentiation, and that activation of JNK/SAPK and p38 and the
inhibition of ERK are critical for induction of
stress-induced apoptotic cell death in SKT6 cells.
View larger version (32K):
[in this window]
[in a new window]
| Fig 7.
Inhibition of p38 or JNK/SAPK causes delay of
stress-induced apoptotic cell death, but inhibition of ERK stimulates
stress-induced apoptotic cell death. SKT6 cells mixed with 10 µmol/L
of antisense S-oligos () or sense S-oligos () of p38, JNK1, JNK2,
ERKs (common to both ERK1 and ERK2), or scrambled S-oligos were (A)
osmotic shocked or (B) heat shocked for 1 hour, and the cell death rate
(%) was measured at the indicated time points. Each point represents
the mean of five replicates.
|
|
Activation of p38 and/or JNK/SAPK induces erythroid differentiation.
The effect of chemical activators on p38 and/or JNK/SAPK on erythroid
differentiation without Epo stimulation was also examined. Incubation
of SKT6 cells for 1 hour with C2-ceramide (100 µmol/L), which is an analog of a stress-induced physiologic activator of p38 and
JNK/SAPK, ceramide,46 or with a protein biosynthesis inhibitor anisomycin (377 µmol/L), which is known to activate p38 and
JNK/SAPK and induce apoptosis,45 resulted in specific activation of p38 and JNK/SAPK (data not shown) and led to production of hemoglobinized cells without Epo stimulation (Fig
8A, lanes 3 and 4). Incubation of the cells
with these activators for a longer period or at higher concentrations
caused apoptotic cell death (data not shown). These data are consistent
with the previous finding that low concentrations of ceramide induced
HL60 cell differentiation.51
View larger version (26K):
[in this window]
[in a new window]
| Fig 8.
Activation of p38 and/or JNK/SAPK induces erythroid
differentiation or apoptotic cell death to some extent, and activation
of ERK inhibits stress-induced apoptotic cell death. (A) Addition of
C2-ceramide or anisomycin for 1 hour induces erythroid
differentiation. (B) Transient expression of MKK6 (Glu) induces
erythroid differentiation. Lanes 1 and 2: transfectants of MKK6 (Glu)
in pIND expression vector incubated with or without Epo. Lanes 3 and 4:
transfectants incubated with or without muristerone A for 1 day. The
percentage of hemoglobinized cells was counted after 4.5 days. Each
value represents the mean of six independent clones. (C) A prolonged
MKK6 expression slightly causes apoptotic cell death. Lanes 1 and 2:
cell death rate of the transfectants with or without Epo. Lanes 3 and
4: cell death rate of the transfectants incubated with muristeron A for
4.5 days. (D) Expression of constitutively active MKK1 inhibits
stress-induced apoptosis. The transfectants constitutively expressing
MKK1 active mutant ( N3-S218E/S222D) () or mock-transfectants
() were treated with osmotic (left panel) or heat (right panel)
shock for various periods as indicated, and the cell death rate (%)
was measured. Values shown are the mean of five experiments.
|
|
Transient expression of constitutively active MKK6 causes erythroid
differentiation, although its prolonged expression slightly induces
apoptotic cell death.
We further examined whether the transient activation of p38 is enough
to induce erythroid differentiation without Epo stimulation and whether
the persistent activation of p38 induces apoptosis. Transfectants of a
constitutively active form of MKK6, MKK6 (Glu), which can specifically
activate p38,48 in the inducible expression vector pIND,
were hemoglobinized in response to Epo without MKK6 (Glu) expression
(Fig 8B, lane 2). Incubation of the transfectants with muristerone A
(and without Epo) for 1 day resulted in specific activation of p38
(data not shown), and led to production of hemoglobinized cells within
4.5 days (Fig 8B, lane 4) and no DNA fragmentation (data not shown). In
contrast, a longer exposure (4.5 days) of the cells to muristerone A
caused apoptotic cell death to some extent (Fig 8C, lane 4).
Transfection of vector alone had no effect on cell differentiation
(data not shown). Thus, the transient activation of p38 is sufficient
to induce some level of erythroid differentiation without Epo
stimulation, although its prolonged activation induces apoptotic cell
death to some extent.
Expression of MKK1 inhibits stress-induced apoptotic cell death.
Expression of constitutively active form of MKK1, a protein kinase that
phosphorylates and activates ERK, prevented the cells from induction of
apoptotic cell death by osmotic shock (Fig 8D, left panel, open
circles) and heat shock (Fig 8D, right panel, open circles) compared
with mock transfectants (Fig 8D, closed circles), demonstrating that
direct and selective activation of the ERK cascade suppresses apoptotic
cell death and leads to survival of SKT6 cells.
| |
DISCUSSION |
A model of environmental stress-induced erythroid differentiation and
apoptotic cell death in SKT6 cells is depicted in Fig 9. Epo induces transient activation of all
MAP kinase family, ERK, JNK/SAPK, and p38,35,36 which, in
turn, leads to erythroid differentiation. Actually, activation of
JNK/SAPK and p38, but not ERK, is required for Epo-induced erythroid
differentiation,39 and Epo-induced ERK activation may act
on cell survival. Short exposure of SKT6 cells to environmental stress
such as osmotic shock or heat shock, induces transient activation of
JNK/SAPK and p38 (Fig 3), which partly mimics Epo stimulation and
causes erythroid differentiation to some extent (Fig 1), whereas ERK activity is transiently suppressed (Fig 3). Inhibition of JNK/SAPK and/or p38, but not of ERK, strongly suppresses
Epo-induced39 and environmental stress-induced erythroid
differentiation (Figs 4 and 6). Activation of JNK/SAPK and/or p38
promotes erythroid differentiation to some extent (Fig 8). Thus,
JNK/SAPK and p38, but not ERK, was confirmed to be essential for
erythroid differentiation. A longer exposure of the cells to the
environmental stresses leads to persistent activation of JNK/SAPK and
p38 and inactivation of ERK (Fig 3), which finally induces apoptotic
cell death (Fig 2). Inhibition of JNK/SAPK and/or p38 causes delay of
stress-induced apoptotic cell death (Figs 5 and 7), but inhibition of
ERK stimulates stress-induced apoptosis (Figs 5 and 7). Furthermore,
activation of JNK/SAPK and/or p38 stimulates stress-induced apoptotic
cell death, although activation of ERK strongly suppresses it (Fig 8).
Thus, prolonged activation of JNK/SAPK and p38 as well as suppression
of ERK activity is required for stress-induced apoptotic cell death.
View larger version (24K):
[in this window]
[in a new window]
| Fig 9.
Model of stress-induced cell differentiation and
apoptotic cell death through MAPK family in SKT6 cells.
|
|
We have shown that environmental stresses for a short time induce
erythroid differentiation rather than apoptotic cell death without Epo
stimulation through p38 and JNK/SAPK, but not ERK, cascades in SKT6
cells, and that signal leading to cell differentiation are, at least in
part, shared with those leading to apoptotic cell death in SKT6 cells.
We also performed in vitro erythroid colony-formation assays of mouse
fetal liver cells of 13.5-day-old embryos in semisolid culture. The
fetal liver cells were treated with osmotic shock (0.1 mol/L NaCl for
30 minutes) or heat shock (40°C for 40 minutes) and cultured in
Methocell (Tokyo Chem., Tokyo, Japan) with or without Epo for 2 days. The milder stress treatment was used for these
primary cells because the conditions used for SKT6 cells caused
apoptotic cell death. The hemoglobinized colonies formed were counted.
It was clearly demonstrated that these environmental stresses actually
induced erythroid differentiation of primary mouse fetal liver cells
without Epo, whereas the numbers of erythroid colonies formed by
osmotic or heat treatment were about one third of those of Epo
stimulation (data not shown). Furthermore, human promyelocytic HL60
cells, which can be differentiated into macrophage-like and
granulocyte-like cells with 12-O-tetradecanoylphorbol-13-acetate and
DMSO, respectively, similarly differentiated into granulocyte-like cells by comparable stress treatment, and the differentiation was
blocked by inhibition of p38 (data not shown). Therefore, induction of
differentiation by these cellular stresses for a short time is not a
characteristic of SKT6 cells. The results obtained with SKT6 cells can
be more generalized in normal erythropoiesis.
We showed here that the prolonged and persistent activation of JNK/SAPK
and p38 and inactivation of ERK induced apoptotic cell death, while
transient activation of JNK/SAPK and p38 led to erythroid
differentiation of SKT6 cells. Thus, we concluded that JNK/SAPK and p38
serve an important function in both cell differentiation and apoptosis
and that duration of activation of these kinases may partly contribute
to determining the cell fate, cell differentiation, and apoptotic cell
death. Chen et al23 also reported that the T-cell
activation signals through CD28 induced a rapid and transient JNK1
activation in Jurkat T-cells, which in turn stimulated cell growth,
whereas radiation or UV caused delayed and persistent JNK1
activation, which led to apoptotic cell death. These results also
support our conclusion. The delay of JNK/SAPK activation may be caused
by the time needed for accumulation of irreparable damage to certain
threshold levels. The different timing and/or duration of JNK/SAPK and
p38 activation may alter the outcome, proliferation, differentiation,
or apoptosis. The prolonged JNK/SAPK and p38 activation after stress
treatment may be caused by the lack or low level of dual-specific
Thr/Tyr phosphatases, which dephosphorylate and inactivate the
phophorylated ERK, JNK/SAPK, and/or p38.51-55 Thus, the
prolonged and persistent activation of JNK/SAPK and p38 may overcome a
certain threshold level to trigger activation of the factors required
for apoptosis such as caspase family and specific DNases and/or to
induce inhibition of apoptosis inhibitors such as Bcl-2,
Bcl-XL, and Hsp family, which finally lead to apoptotic
cell death.
We show here that environmental stresses not only trigger activation of
JNK/SAPK and p38, but also concurrently suppresses ERK activity. The
inactivation of ERK together with activation of JNK/SAPK and p38 may be
critical for apoptosis. Xia et al18 reported that
concurrent activation of JNK/SAPK and p38 and inhibition of ERK induces
apoptosis, whereas activation of ERK prevents apoptosis in PC12 cells.
Actually, we also observed here that inhibition of ERK clearly
stimulates stress-induced apoptotic cell death and that activation of
ERK prevents SKT6 cells from stress-induced apoptotic cell death. Thus,
JNK/SAPK and p38 cascades may also act in an opposite way from ERK in
SKT6 cells. The dynamic balance between MAP kinases and phosphates,
and/or ERK and JNK/SAPK-p38 may also contribute to determine the cell
fate, whether the cells undergo proliferation, differentiation, or
apoptosis. The target molecules of these MAP kinases for apoptotic cell
death as well as cell differentiation have to be identified to further
understand the molecular mechanism of apoptosis and cell differentiation.
Inhibition of either JNK/SAPK or p38 suppressed stress-induced
differentiation as well as apoptosis in SKT6 cells. However, the
caspase-3 specific inhibitor, DEVD-CHO, caused delay of stress-induced apoptosis, but did not affect stress-induced erythroid differentiation (data not shown). The activation of caspase-3 appears to be essential for the apoptotic process and occurs after irreversible commitment to
cell death. As shown in Fig 2B, caspase-3 was activated between 1 hour
and 6 hours after stress treatments, suggesting that the commitment to
cell death took place during this period. DNA fragmentation occurred
around 24 hours after stress treatments; most of the cells fell into
irreversible crisis at around 36 hours. Therefore, differentiation and
cell death processes appear to occur in the following order: activation
of JNK/SAPK and p38 and inactivation of ERK, commitment of
differentiation or cell death, caspase-3 activation, DNA fragmentation,
and finally total cell death.
The level of stress-induced differentiation did not reach to that of
Epo-stimulated differentiation, indicating that transient activation of
only JNK/SAPK and p38 may not be enough for full erythroid
differentiation. We noticed that ERK activation inhibits stress-induced
apoptotic cell death but has nothing to do with erythroid
differentiation. It has been described, however, that JAK-STAT
signaling pathway may be involved in Epo-induced erythroid differentiation.56,57 Therefore, activation of p38 and
JNK/SAPK is required for erythroid differentiation, but other
independent signaling pathways such as JAK-STAT pathway may also play a
role in full erythroid differentiation.
| |
ACKNOWLEDGMENT |
We thank R.J. Davis for MKK6 (Glu), N.G. Ahn for MKK1 constructs, E. Nishida for discussion, J.S. Lee for SB203580, and N. Takahashi and J. Iita for technical assistance.
| |
FOOTNOTES |
Submitted January 27, 1999; accepted April 6, 1999.
Supported in part by a Special Grant for Promotion of Research from The
Institute of Physical and Chemical Research (RIKEN), and grants from
the Ministry of Education, Science and Culture of Japan, from the
Uehara Memorial Foundation, and from the Suzuken Memorial Foundation.
Address reprint requests to Kazuo Todokoro, PhD, Tsukuba Life
Science Center, The Institute of Physical and Chemical Research
(RIKEN), 3-1, Koyadai, Tsukuba, Ibaraki 305-0074, Japan; e-mail:
todokoro{at}rtc.riken.go.jp.
| |
REFERENCES |
1.
Derijard B, Hibi M, Wu I-H, Barrett T, Su B, Deng T, Karin M, Davis RJ:
JNK1: A protein kinase stimulated by UV light and Ha-Ras that binds and phosphorylates the c-Jun activation domain.
Cell
76:1025, 1994[Medline]
[Order article via Infotrieve]
2.
Kyriakis JM, Banerjee P, Nikolakaki E, Dai T, Rubie EA, Ahmad MF, Avruch J, Woodgett JR:
The stress-activated protein kinase subfamily of c-Jun kinases.
Nature
369:156, 1994[Medline]
[Order article via Infotrieve]
3.
Han J, Lee JD, Bibbs L, Ulevitch RJ:
A MAP kinase targeted by endotoxin and hyperosmolarity in mammalian cells.
Science
265:808, 1994[Abstract/Free Full Text]
4.
Rouse J, Cohen P, Trigon S, Morange M, Alonso-Llamazares A, Zamanillo D, Hunt T, Nebreda AR:
A novel kinase cascade triggered by stress and heat shock that stimulates MAPKAP kinase-2 and phosphorylation of the small heat shock proteins.
Cell
78:1027, 1994[Medline]
[Order article via Infotrieve]
5.
Ray LB, Sturgill TW:
Rapid stimulation by insulin of a serine/threonine kinase in 3T3-L1 adipocytes that phosphorylates microtubule-associated protein 2 in vitro.
Proc Natl Acad Sci USA
84:1502, 1987[Abstract/Free Full Text]
6.
Nishida E, Gotoh Y:
The MAP kinase cascade is essential for diverse signal transduction pathways.
Trends Biochem Sci
18:128, 1993[Medline]
[Order article via Infotrieve]
7.
Marshall CJ:
Specificity of receptor tyrosine kinase signaling: Transient versus sustained extracellular signal-regulated kinase activation.
Cell
80:179, 1995[Medline]
[Order article via Infotrieve]
8.
Pages G, Lenormand P, L'Allemain G, Chambard JC, Meloche S, Poyssegur J:
Mitogen-activated protein kinase p42mapk and p44mapk are required for fibroblast proliferation.
Proc Natl Acad Sci USA
90:8319, 1993[Abstract/Free Full Text]
9.
Freshney NW, Rawlinson L, Guesdon F, Jones E, Cowley S, Hsuan J, Saklatvala J:
Interleukin-1 activates a novel protein kinase cascade that results in the phosphorylation of Hsp27.
Cell
78:1039, 1994[Medline]
[Order article via Infotrieve]
10.
Galcheva-Gargova Z, Derijard B, Wu IH, Davis RJ:
An osmosensing signal transduction pathway in mammalian cells.
Science
265:806, 1994[Abstract/Free Full Text]
11.
Raingeaud J, Gupta S, Rogers J, Dickens M, Han J, Ulevitch R, Davis RJ:
Pro-inflammatory cytokines and environmental stress cause p38 mitogen-activated protein kinase activation by dual phosphorylation on tyrosine and threonine.
J Biol Chem
270:7420, 1995[Abstract/Free Full Text]
12.
Sluss HK, Barrett T, Derijard B, Davis RJ:
Signal transduction by tumor necrosis factor mediated by JNK protein kinases.
Mol Cell Biol
14:8376, 1994[Abstract/Free Full Text]
13.
Verheij M, Bose R, Lin XH, Yao B, Jarvis WD, Grant S, Birrer MJ, Szabo E, Zon LI, Kyriakis JM, Haimovitz-Friedman A, Fuks Z, Kolesnick RN:
Requirement for ceramide-initiated SAPK/JNK signaling in stress-induced apoptosis.
Nature
380:75, 1996[Medline]
[Order article via Infotrieve]
14.
Wang XZ, Ron D:
Stress-induced phosphorylation and activation of the transcription factor CHOP (GADD153) by p38 MAP Kinase.
Science
272:1347, 1996[Abstract]
15.
Goillot E, Raingeaud J, Ranger A, Tepper RI, Davis RJ, Harlow E, Sanchez I:
Mitogen-activated protein kinase-mediated Fas apoptotic signaling pathway.
Proc Natl Acad Sci USA
94:3302, 1997[Abstract/Free Full Text]
16.
Juo P, Kuo CJ, Reynolds SE, Konz RF, Raingeaud J, Davis RJ, Biemann H-P, Blenis J:
Fas activation of the p38 mitogen-activated protein kinase signalling pathway requires ICE/CED-3 family proteases.
Mol Cell Biol
17:24, 1997[Abstract]
17.
Johnson NL, Gardner AM, Diener KM, Lange-Carter CA, Gleavy J, Jarpe MB, Minden A, Karin M, Zon LI, Johnson G:
Signal transduction pathways regulated by mitogen-activated/extracellular response kinase kinase kinase induce cell death.
J Biol Chem
271:3229, 1996[Abstract/Free Full Text]
18.
Xia Z, Dickens M, Raingeaud J, Davis RJ, Greenberg ME:
Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis.
Science
270:1326, 1995[Abstract/Free Full Text]
19.
Huang S, Jiang Y, Li Z, Nishida E, Mathias P, Lin S, Ulevitch RJ, Nemerow GR, Han J:
Apoptosis signaling pathway in T cells is composed of ICE/Ced-3 family proteases and MAP kinase kinase 6b.
Immunity
6:739, 1997[Medline]
[Order article via Infotrieve]
20.
Ichijo H, Nishida E, Irie K, ten Dijke P, Saitoh M, Moriguchi T, Takagi M, Matsumoto K, Miyazono K, Gotoh Y:
Induction of apoptosis by ASK1, a mammalian MAPKKK that activated SAPK/JNK and p38 signaling pathways.
Science
275:90, 1997[Abstract/Free Full Text]
21.
Shibuya H, Iwata H, Masuyama N, Gotoh Y, Yamaguchi K, Irie K, Matsumoto K, Nishida E, Ueno N:
Role of TAK1 and TAB1 in BMP signaling in early Xenopus development.
EMBO J
17:1019, 1998[Medline]
[Order article via Infotrieve]
22.
Butterfield L, Storey B, Maas L, Heasley LE:
c-Jun NH2-terminal kinase regulation of the apoptotic response of small cell lung cancer cells to ultraviolet radiation.
J Biol Chem
272:10110, 1997[Abstract/Free Full Text]
23.
Chen YR, Wang X, Templeton D, Davis RJ, Tan TH:
The role of c-Jun N-terminal kinase (JNK) in apoptosis induced by ultraviolet C and gamma radiation. Duration of JNK activation may determine cell death and proliferation.
J Biol Chem
271:31929, 1996[Abstract/Free Full Text]
24.
Zanke BW, Boudreau K, Rubie E, Winnett E, Tibbles LA, Zon L, Kyriakis J, Liu F-F, Woodgett JR:
The stress-activated protein kinase pathway mediates cell death following injury induced by cis-platinum, UV irradiation or heat.
Curr Biol
6:606, 1996[Medline]
[Order article via Infotrieve]
25.
Ham J, Babij C, Whitfield J, Pfarr CM, Lallemand D, Yaniv M, Rubin LL:
A c-Jun dominant negative mutant protects sympathetic neurons against programmed cell death.
Neuron
14:927, 1995[Medline]
[Order article via Infotrieve]
26.
Yang DD, Kuan CY, Whitmarsh AJ, Rincon M, Zheng TS, Davis RJ, Rakic P, Flavell RA:
Absence of excitotoxicity-induced apoptosis in the hippocampus of mice lacking the Jnk3 gene.
Nature
389:865, 1997[Medline]
[Order article via Infotrieve]
27.
Lenczowski JM, Dominguez L, Eder AM, King LB, Zacharchuk CM, Ashwell JD:
Lack of a role for Jun Kinase and AP-1 Fas-induced apoptosis.
Mol Cell Biol
17:170, 1997[Abstract]
28.
Liu ZG, Hsu H, Goeddel DV, Karin M:
Dissection of TNF receptor 1 effector functions: JNK activation is not linked to apoptosis while NF- B activation prevents cell death.
Cell
87:565, 1996[Medline]
[Order article via Infotrieve]
29.
Berberich I, Shu G, Siebelt F, Woodgett JR, Kyriakis JM, Clark EA:
Cross-linking CD40 on B cells preferentially induces stress-activated protein kinases rather than mitogen-activated protein kinases.
EMBO J
15:92, 1996[Medline]
[Order article via Infotrieve]
30.
Nishina H, Fischer KD, Radvanyi L, Shahinian A, Hakem R, Rubie EA, Bernstein A, Mak TW, Woodgett JR, Penninger JM:
Stress-signalling kinase Sek1 protects thymocytes from apoptosis mediated by CD95 and CD3.
Nature
385:350, 1997[Medline]
[Order article via Infotrieve]
31.
Yeh WC, Shahinian A, Speiser D, Kraunus J, Billia F, Wakeham A, de la Pompa JL, Ferrick D, Hum B, Iscove N, Ohashi P, Rothe M, Goeddel DV, Mak TW:
Early lethality, functional NF-B activation and increased sensitivity to TNF-induced cell death in TRAF2-deficient mice.
Immunity
7:715, 1997[Medline]
[Order article via Infotrieve]
32.
Su B, Jacinto E, Hibi M, Kallunki T, Karin M, Ben-Neriah Y:
JNK is involved in signal integration during costimulation of T lymphocytes.
Cell
77:727, 1994[Medline]
[Order article via Infotrieve]
33.
Foltz IN, Schrader JW:
Activation of the stress-activated protein kinases by multiple hematopoietic growth factors with the exception of interleukin-4.
Blood
89:3092, 1997[Abstract/Free Full Text]
34.
Foltz IN, Lee JC, Young PR, Schrader JW:
Hemopoietic growth factors with the exception of interleukin-4 activate the p38 mitogen-activated protein kinase pathway.
J Biol Chem
272:3296, 1997[Abstract/Free Full Text]
35.
Nagata Y, Nishida E, Todokoro K:
Activation of JNK signaling pathway by erythropoietin, thrombopoietin, and interleukin-3.
Blood
89:2664, 1997[Abstract/Free Full Text]
36.
Nagata Y, Moriguchi T, Nishida E, Todokoro K:
Activation of p38 MAP kinase pathway by erythropoietin and interleukin-3.
Blood
90:929, 1997[Abstract/Free Full Text]
37.
Rausch O, Marshall CJ:
Tyrosine 763 of the murine granulocyte colony-stimulating factor receptor mediates Ras-dependent activation of the JNK/SAPK mitogen-activated protein kinase pathway.
Mol Cell Biol
17:1170, 1997[Abstract]
38.
Crawley JB, Rawlinson L, Lali FV, Page TH, Saklatvala J, Foxwell BM:
T cell proliferation in response to interleukins 2 and 7 requires p38 MAP kinase activation.
J Biol Chem
272:15023, 1997[Abstract/Free Full Text]
39.
Nagata Y, Takahashi N, Davis RJ, Todokoro K:
Activation of p38 MAP kinase and JNK but not ERK is required for erythropoietin-induced erythroid differentiation.
Blood
92:1859, 1998[Abstract/Free Full Text]
40.
Heasley LE, Storey B, Fanger GR, Butterfield L, Zamarripa J, Blumberg D, Maue RA:
GTPase-deficient G alpha 16 and G alpha q induce PC12 cell differentiation and persistent activation of cJun NH2-terminal kinases.
Mol Cell Biol
16:648, 1996[Abstract]
41.
de Groot RP, Kruyt FA, van der Saag PT, Kruijer W:
Ectopic expression of c-jun leads to differentiation of P19 embryonal carcinoma cells.
EMBO J
9:1831, 1990[Medline]
[Order article via Infotrieve]
42.
Jho EH, Davis RJ, Malbon CC:
c-Jun amino-terminal kinase is regulated by Galpha12/Galpha13 and obligate for differentiation of P19 embryonal carcinoma cells by retinoic acid.
J Biol Chem
272:24468, 1997[Abstract/Free Full Text]
43.
Li J, King I, Satorelli AC:
Differentiation of WEHI-3B D+ myelomonocytic leukemia cells induced by ectopic expression of the protooncogene c-jun.
Cell Growth Differ
5:743, 1994[Abstract]
44.
Todokoro K, Kanazawa S, Amanuma H, Ikawa Y:
Specific binding of erythropoietin to its receptor on responsive mouse erythroleukemia cells.
Proc Natl Acad Sci USA
84:4126, 1987[Abstract/Free Full Text]
45.
Hazzalin CA, Le Panse R, Cano E, Mahadevan LC:
Anisomycin selectively desensitizes signalling components involved in stress kinase activation and fos and jun induction.
Mol Cell Biol
18:1844, 1998[Abstract/Free Full Text]
46.
Westwick JK, Bielawska AE, Dbaibo G, Hannun YA, Brenner DA:
Ceramide activates the stress-activated protein kinases.
J Biol Chem
270:22689, 1995[Abstract/Free Full Text]
47.
Sellins KS, Cohen JJ:
Gene induction by -irradiation leads to DNA fragmentation in lymphocytes.
J Immunol
139:3199, 1987[Abstract]
48.
Zechner D, Thuerauf DF, Hanford DS, McDonough PH, Glembotski CC:
A role for the p38 mitogen-activated protein kinase pathway in myocardial cell growth, sarcomeric organization, and cardiac-specific gene expression.
J Cell Biol
139:115, 1997[Abstract/Free Full Text]
49.
No D, Yao TP, Evans RM:
Ecdysone-inducible gene expression in mammalian cells and transgenic mice.
Proc Natl Acad Sci USA
93:3346, 1996[Abstract/Free Full Text]
50.
Mansour SJ, Matten WT, Hermann AS, Candia JM, Rong S, Fukasawa K, Vande Woude GF, Ahn NG:
Transformation of mammalian cells by constitutively active MAP kinase.
Science
265:966, 1994[Abstract/Free Full Text]
51.
Okazaki T, Bielawska A, Bell RM, Hannun Y:
Role of ceramide as a lipid mediator of 1,25-dihydroxyvitamin D3-induced HL-60 cell differentiation.
J Biol Chem
265:15823, 1990[Abstract/Free Full Text]
52.
Chu Y, Solski PA, Khosravi-Far R, Der CJ, Kelly K:
The mitogen-activated protein kinase phosphatases PAC1, MKP-1, and MKP-2 have unique substrate specificities and reduced activity in vivo toward the ERK2 sevenmaker mutation.
J Biol Chem
271:6497, 1996[Abstract/Free Full Text]
53.
Franklin CC, Srikanth S, Kraft AS:
Conditional expression of mitogen-activated protein kinase phosphatase-1, MKP-1, is cytoprotective against UV-induced apoptosis.
Proc Natl Acad Sci USA
95:3014, 1998[Abstract/Free Full Text]
54.
Liu Y, Gorospe M, Yang C, Holbrook NJ:
Role of mitogen-activated protein kinase phosphatase during the cellular response to genotoxic stress. Inhibition of c-Jun N-terminal kinase activity and AP-1-dependent gene activation.
J Biol Chem
270:8377, 1995[Abstract/Free Full Text]
55.
Ward Y, Gupta S, Jensen P, Wartmann M, Davis RJ, Kelly K:
Control of MAP kinase activation by the mitogen-induced threonine/tyrosine phosphatase PAC1.
Nature
367:651, 1994[Medline]
[Order article via Infotrieve]
56.
Gregory RC, Jiang N, Todokoro K, Crouse J, Pacifici RE, Wojchowski DM:
Erythropoietin receptor and STAT5-specificpathways promote SKT6 cell hemoglobinization.
Blood
92:1104, 1998[Abstract/Free Full Text]
57.
Chida D, Miura O, Yoshimura A, Miyajima A:
Role of cytokine signaling molecules in erythroid differentiation of mouse fetal liver hematopoietic cells: Functional analysis of signaling molecules by retrovirus-mediated expression.
Blood
93:1567, 1999[Abstract/Free Full Text]
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
Y. S. Kim, G. B. Park, H.-K. Lee, H. Song, I.-H. Choi, W. J. Lee, and D. Y. Hur
Cross-Linking of B7-H1 on EBV-Transformed B Cells Induces Apoptosis through Reactive Oxygen Species Production, JNK Signaling Activation, and fasL Expression
J. Immunol.,
November 1, 2008;
181(9):
6158 - 6169.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Bose and K. B. Udupa
Erythropoietin enhancement of rat pancreatic tumor cell proliferation requires the activation of ERK and JNK signals
Am J Physiol Cell Physiol,
August 1, 2008;
295(2):
C394 - C405.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Miyake, T. Utsugisawa, J. Flygare, T. Kiefer, I. Hamaguchi, J. Richter, and S. Karlsson
Ribosomal Protein S19 Deficiency Leads to Reduced Proliferation and Increased Apoptosis but Does Not Affect Terminal Erythroid Differentiation in a Cell Line Model of Diamond-Blackfan Anemia
Stem Cells,
February 1, 2008;
26(2):
323 - 329.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
W. Aerbajinai, J. Zhu, Z. Gao, K. Chin, and G. P. Rodgers
Thalidomide induces {gamma}-globin gene expression through increased reactive oxygen species mediated p38 MAPK signaling and histone H4 acetylation in adult erythropoiesis
Blood,
October 15, 2007;
110(8):
2864 - 2871.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. DiMascio, C. Voermans, M. Uqoezwa, A. Duncan, D. Lu, J. Wu, U. Sankar, and T. Reya
Identification of Adiponectin as a Novel Hemopoietic Stem Cell Growth Factor
J. Immunol.,
March 15, 2007;
178(6):
3511 - 3520.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. Sangerman, M. S. Lee, X. Yao, E. Oteng, C.-H. Hsiao, W. Li, S. Zein, S. F. Ofori-Acquah, and B. S. Pace
Mechanism for fetal hemoglobin induction by histone deacetylase inhibitors involves {gamma}-globin activation by CREB1 and ATF-2
Blood,
November 15, 2006;
108(10):
3590 - 3599.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Rubiolo, D. Piazzolla, K. Meissl, H. Beug, J. C. Huber, A. Kolbus, and M. Baccarini
A balance between Raf-1 and Fas expression sets the pace of erythroid differentiation
Blood,
July 1, 2006;
108(1):
152 - 159.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
W.-H. Wang, G. Gregori, R. L. Hullinger, and O. M. Andrisani
Sustained Activation of p38 Mitogen-Activated Protein Kinase and c-Jun N-Terminal Kinase Pathways by Hepatitis B Virus X Protein Mediates Apoptosis via Induction of Fas/FasL and Tumor Necrosis Factor (TNF) Receptor 1/TNF-{alpha} Expression
Mol. Cell. Biol.,
December 1, 2004;
24(23):
10352 - 10365.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G. Bandyopadhyay, T. Biswas, K. C. Roy, S. Mandal, C. Mandal, B. C. Pal, S. Bhattacharya, S. Rakshit, D. K. Bhattacharya, U. Chaudhuri, et al.
Chlorogenic acid inhibits Bcr-Abl tyrosine kinase and triggers p38 mitogen-activated protein kinase-dependent apoptosis in chronic myelogenous leukemic cells
Blood,
October 15, 2004;
104(8):
2514 - 2522.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. A. Bachelor and G. T. Bowden
Ultraviolet A-induced Modulation of Bcl-XL by p38 MAPK in Human Keratinocytes: POST-TRANSCRIPTIONAL REGULATION THROUGH THE 3'-UNTRANSLATED REGION
J. Biol. Chem.,
October 8, 2004;
279(41):
42658 - 42668.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. M. Jacobs-Helber and S. T. Sawyer
Jun N-terminal kinase promotes proliferation of immature erythroid cells and erythropoietin-dependent cell lines
Blood,
August 1, 2004;
104(3):
696 - 703.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y. Murayama, J.-i. Miyagawa, K. Oritani, H. Yoshida, K. Yamamoto, O. Kishida, T. Miyazaki, S. Tsutsui, T. Kiyohara, Y. Miyazaki, et al.
CD9-mediated activation of the p46 Shc isoform leads to apoptosis in cancer cells
J. Cell Sci.,
July 1, 2004;
117(15):
3379 - 3388.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Q.-S. Zhu, L. J. Robinson, V. Roginskaya, and S. J. Corey
G-CSF-induced tyrosine phosphorylation of Gab2 is Lyn kinase dependent and associated with enhanced Akt and differentiative, not proliferative, responses
Blood,
May 1, 2004;
103(9):
3305 - 3312.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. Baeza-Raja and P. Munoz-Canoves
p38 MAPK-induced Nuclear Factor-{kappa}B Activity Is Required for Skeletal Muscle Differentiation: Role of Interleukin-6
Mol. Biol. Cell,
April 1, 2004;
15(4):
2013 - 2026.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. Secchiero, E. Melloni, M. Heikinheimo, S. Mannisto, R. Di Pietro, A. Iacone, and G. Zauli
TRAIL regulates normal erythroid maturation through an ERK-dependent pathway
Blood,
January 15, 2004;
103(2):
517 - 522.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Uddin, J. Ah-Kang, J. Ulaszek, D. Mahmud, and A. Wickrema
Differentiation stage-specific activation of p38 mitogen-activated protein kinase isoforms in primary human erythroid cells
PNAS,
January 6, 2004;
101(1):
147 - 152.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Pervin, R. Singh, W. A. Freije, and G. Chaudhuri
MKP-1-Induced Dephosphorylation of Extracellular Signal-Regulated Kinase Is Essential for Triggering Nitric Oxide-Induced Apoptosis in Human Breast Cancer Cell Lines: Implications in Breast Cancer
Cancer Res.,
December 15, 2003;
63(24):
8853 - 8860.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T.-S. Chang, M. J. Kim, K. Ryoo, J. Park, S.-J. Eom, J. Shim, K. I. Nakayama, K. Nakayama, M. Tomita, K. Takahashi, et al.
p57KIP2 Modulates Stress-activated Signaling by Inhibiting c-Jun NH2-terminal Kinase/Stress-activated Protein Kinase
J. Biol. Chem.,
November 28, 2003;
278(48):
48092 - 48098.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Bhattacharya, R. M. Ray, M. J. Viar, and L. R. Johnson
Polyamines are required for activation of c-Jun NH2-terminal kinase and apoptosis in response to TNF-{alpha} in IEC-6 cells
Am J Physiol Gastrointest Liver Physiol,
November 1, 2003;
285(5):
G980 - G991.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Guillard, S. Chretien, A.-S. Pelus, F. Porteu, O. Muller, P. Mayeux, and V. Duprez
Activation of the Mitogen-activated Protein Kinases Erk1/2 by Erythropoietin Receptor via a Gi Protein beta gamma -Subunit-initiated Pathway
J. Biol. Chem.,
March 21, 2003;
278(13):
11050 - 11056.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Refsnes, P. E Schwarze, J. A Holme, and M. Laeg
Fluoride-induced apoptosis in human epithelial lung cells (A549 cells): role of different G protein-linked signal systems
Human and Experimental Toxicology,
March 1, 2003;
22(3):
111 - 123.
[Abstract]
[PDF]
|
|
|
|
|
|
|
O. Witt, S. Monkemeyer, G. Ronndahl, B. Erdlenbruch, D. Reinhardt, K. Kanbach, and A. Pekrun
Induction of fetal hemoglobin expression by the histone deacetylase inhibitor apicidin
Blood,
March 1, 2003;
101(5):
2001 - 2007.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. M. Jacobs-Helber, K.-h. Roh, D. Bailey, E. N. Dessypris, J. J. Ryan, J. Chen, A. Wickrema, D. L. Barber, P. Dent, and S. T. Sawyer
Tumor necrosis factor-alpha expressed constitutively in erythroid cells or induced by erythropoietin has negative and stimulatory roles in normal erythropoiesis and erythroleukemia
Blood,
January 15, 2003;
101(2):
524 - 531.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. R. Vlahakis, A. Villasis-Keever, T. Gomez, M. Vanegas, N. Vlahakis, and C. V. Paya
G Protein-Coupled Chemokine Receptors Induce Both Survival and Apoptotic Signaling Pathways
J. Immunol.,
November 15, 2002;
169(10):
5546 - 5554.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G. M. Jenkins, L. A. Cowart, P. Signorelli, B. J. Pettus, C. E. Chalfant, and Y. A. Hannun
Acute Activation of de Novo Sphingolipid Biosynthesis upon Heat Shock Causes an Accumulation of Ceramide and Subsequent Dephosphorylation of SR Proteins
J. Biol. Chem.,
November 1, 2002;
277(45):
42572 - 42578.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. M.-Y. Ho, M. H.-H. Nguyen, J. K. Dierov, K. M. Badger, B. K. Beattie, P. Tartaro, R. Haq, B. W. Zanke, M. P. Carroll, and D. L. Barber
TEL-JAK2 constitutively activates the extracellular signal-regulated kinase (ERK), stress-activated protein/Jun kinase (SAPK/JNK), and p38 signaling pathways
Blood,
July 30, 2002;
100(4):
1438 - 1448.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. Haq, A. Halupa, B. K. Beattie, J. M. Mason, B. W. Zanke, and D. L. Barber
Regulation of Erythropoietin-induced STAT Serine Phosphorylation by Distinct Mitogen-activated Protein Kinases
J. Biol. Chem.,
May 3, 2002;
277(19):
17359 - 17366.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
V. L. Gabai and M. Y. Sherman
Molecular Biology of Thermoregulation: Invited Review: Interplay between molecular chaperones and signaling pathways in survival of heat shock
J Appl Physiol,
April 1, 2002;
92(4):
1743 - 1748.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. A. Bradham, E. Hatano, and D. A. Brenner
Dominant-negative TAK1 induces c-Myc and G0 exit in liver
Am J Physiol Gastrointest Liver Physiol,
November 1, 2001;
281(5):
G1279 - G1289.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Chen and A. J. Sytkowski
Erythropoietin Activates Two Distinct Signaling Pathways Required for the Initiation and the Elongation of c-myc
J. Biol. Chem.,
October 12, 2001;
276(42):
38518 - 38526.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J.-I. Park, H.-S. Choi, J.-S. Jeong, J.-Y. Han, and I.-H. Kim
Involvement of p38 Kinase in Hydroxyurea-induced Differentiation of K562 Cells
Cell Growth Differ.,
September 1, 2001;
12(9):
481 - 486.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. Chakravortty, Y. Kato, T. Sugiyama, N. Koide, M. M. Mu, T. Yoshida, and T. Yokochi
Inhibition of p38 Mitogen-Activated Protein Kinase Augments Lipopolysaccharide-Induced Cell Proliferation in CD14-Expressing Chinese Hamster Ovary Cells
Infect. Immun.,
February 1, 2001;
69(2):
931 - 936.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Q.-B. She, A. M. Bode, W.-Y. Ma, N.-Y. Chen, and Z. Dong
Resveratrol-induced Activation of p53 and Apoptosis Is Mediated by Extracellular- Signal-regulated Protein Kinases and p38 Kinase
Cancer Res.,
February 1, 2001;
61(4):
1604 - 1610.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
T. Ikuta, S. Ausenda, and M. D. Cappellini
Mechanism for fetal globin gene expression: Role of the soluble guanylate cyclase-cGMP-dependent protein kinase pathway
PNAS,
February 1, 2001;
(2001)
41599798.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
S Kojima, M Hatano, S Okada, T Fukuda, Y Toyama, S Yuasa, H Ito, and T Tokuhisa
Testicular germ cell apoptosis in Bcl6-deficient mice
Development,
January 1, 2001;
128(1):
57 - 65.
[Abstract]
[PDF]
|
|
|
|
|
|
|
S. M. Jacobs-Helber, J. J. Ryan, and S. T. Sawyer
JNK and p38 are activated by erythropoietin (EPO) but are not induced in apoptosis following EPO withdrawal in EPO-dependent HCD57 cells
Blood,
August 1, 2000;
96(3):
933 - 940.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y. Kashii, M. Uchida, K. Kirito, M. Tanaka, K. Nishijima, M. Toshima, T. Ando, K. Koizumi, T. Endoh, K.-i. Sawada, et al.
A member of Forkhead family transcription factor, FKHRL1, is one of the downstream molecules of phosphatidylinositol 3-kinase-Akt activation pathway in erythropoietin signal transduction
Blood,
August 1, 2000;
96(3):
941 - 949.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. S. Iordanov, J. M. Paranjape, A. Zhou, J. Wong, B. R. G. Williams, E. F. Meurs, R. H. Silverman, and B. E. Magun
Activation of p38 Mitogen-Activated Protein Kinase and c-Jun NH2-Terminal Kinase by Double-Stranded RNA and Encephalomyocarditis Virus: Involvement of RNase L, Protein Kinase R, and Alternative Pathways
Mol. Cell. Biol.,
January 15, 2000;
20(2):
617 - 627.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
L. T. Lam, C. Ronchini, J. Norton, A. J. Capobianco, and E. H. Bresnick
Suppression of Erythroid but Not Megakaryocytic Differentiation of Human K562 Erythroleukemic Cells by Notch-1
J. Biol. Chem.,
June 23, 2000;
275(26):
19676 - 19684.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y. Alsayed, S. Uddin, N. Mahmud, F. Lekmine, D. V. Kalvakolanu, S. Minucci, G. Bokoch, and L. C. Platanias
Activation of Rac1 and the p38 Mitogen-activated Protein Kinase Pathway in Response to All-trans-retinoic Acid
J. Biol. Chem.,
February 2, 2001;
276(6):
4012 - 4019.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Ikuta, S. Ausenda, and M. D. Cappellini
Mechanism for fetal globin gene expression: Role of the soluble guanylate cyclase-cGMP-dependent protein kinase pathway
PNAS,
February 13, 2001;
98(4):
1847 - 1852.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y. Morita, T. Naka, Y. Kawazoe, M. Fujimoto, M. Narazaki, R. Nakagawa, H. Fukuyama, S. Nagata, and T. Kishimoto
Signals transducers and activators of transcription (STAT)-induced STAT inhibitor-1 (SSI-1)/suppressor of cytokine signaling-1 (SOCS-1) suppresses tumor necrosis factor alpha -induced cell death in fibroblasts
PNAS,
May 9, 2000;
97(10):
5405 - 5410.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
TOP
ABSTRACT
INTRODUCTION