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Blood, Vol. 96 No. 3 (August 1), 2000:
pp. 933-940
HEMATOPOIESIS
From the Department of Pharmacology/Toxicology, Medical College of
Virginia Campus, Richmond, VA; and the Department of Biology, Virginia
Commonwealth University, Richmond, VA.
Jun N-terminal kinase (JNK) and p38, members of the
mitogen-activated protein kinase family of serine/threonine
kinases, are activated as a result of cellular stress but may
also play a role in growth factor-induced proliferation and/or survival
or differentiation of many cells. A recent report has
implicated JNK and p38 in the induction of apoptosis in the
erythropoietin (EPO)-dependent erythroid cell line HCD57 following EPO
withdrawal, whereas our previously reported data did not support
a role for JNK in growth factor withdrawal-induced apoptosis in HCD57
cells. Therefore, further testing was done to see if JNK was
activated in EPO withdrawal-induced apoptosis; the study was extended
to p38 and characterized the effect of EPO on JNK and p38
activities. Treatment of HCD57 cells with EPO resulted in a
gradual and sustained activation of both JNK and p38 activity; these
activities decreased on EPO withdrawal. Transient activation of p42/p44
extracellular signal-related kinases (ERK) was also detected.
Inhibition of ERK activity inhibited proliferation in EPO-treated cells
but neither induced apoptosis nor activated JNK. Inhibition of p38
activity inhibited proliferation but did not protect HCD57 cells from
apoptosis induced by EPO withdrawal. Treatment of HCD57 cells with
tumor necrosis factor-alpha induced JNK activation but did not induce
apoptosis. These results implicate JNK, p38, and ERK in EPO-induced
proliferation and/or survival of erythroid cells but do not support a
role for JNK or p38 in apoptosis induced by EPO withdrawal from
erythroid cells.
(Blood. 2000;96:933-940)
The glycoprotein hormone erythropoietin (EPO) is the
primary regulator in the control of erythroid cell maturation. Cells at
the proerythroblast stage or colony-forming unit-erythroid stage of
differentiation depend on EPO for continued differentiation. Apoptosis
or programmed cell death occurs when EPO is withdrawn in
vitro.1-4 Recently, a family of serine-threonine protein
kinases that is structurally similar yet functionally distinct has been identified. These mitogen-activated protein kinases (MAPKs) fall into 4 distinct groups: the extracellular signal-related kinases (ERKs)5,6 the cJun-amino terminal kinases
(JNKs),7 p38 map kinase (p38),8 and
Erk5/BMK1.9 Although these kinases all represent the end of
pathways involving multiple serine-threonine kinases that are activated
in a cascade,10 they exhibit different physiological
effects on cell development. The ERK pathway is primarily associated
with promoting proliferation, whereas the role of the JNK and p38
pathways is more complex. Apoptosis-inducing agents, such as UV
irradiation,11 ion irradiation,12 growth factor
deprivation,13,14 and inflammatory cytokines such as tumor
necrosis factor The end result of activation of signal transduction cascades is often
the phosphorylation and activation (or deactivation) of transcription
factors. JNK and p38 activation results in the phosphorylation of their
substrates activator protein-1 (AP1) and activating transcription
factor-2 (ATF-2), respectively.27,28 AP1 comprises members
of the Fos and Jun families of proto-oncogene and binds to DNA in a
sequence-specific manner to activate or to repress
transcription.29 AP1 has long been associated with cell
cycle progression, tumor promotion, and proliferation. JNK phosphorylates the N-terminus of the cJun protein and increases its
transactivation potential. Of the 3 known Jun family members (cJun,30 JunB,31 and JunD32), cJun
is the only member that can serve as an efficient substrate for
JNK.33,34 The ATF family of transcription factors includes
ATF-1, ATF-2, ATF-3, ATF-4, and the cyclic AMP responsive binding
protein (CREB).35 Although ATF-2 is the only member of this
family known to be phosphorylated directly by p38, CREB is activated
indirectly in response to p38 activation via activation of the
ribosomal SK 6 kinases, which then activate CREB.36,37 JNK
and p38, therefore, can have multiple effects on transcription factor activation.
Recently, JNK and p38 have been implicated in the regulation of
erythroid proliferation and survival. JNK and p38 activation were
initially reported to be induced by EPO,20,38 and recent reports39,40 have suggested that p38 and JNK are necessary for the initiation of erythroid differentiation. Our laboratory has
previously reported on the role of AP1 and JNK in the regulation of
erythroid cell proliferation and apoptosis.41 By using the murine erythroleukemia cell line HCD57, we demonstrated that AP1 DNA-binding activity was induced in either the proliferative and growth
factor withdrawal states but that different AP1 factors were involved
in the two processes. cJun DNA-binding activity and JNK activity were
induced in the presence of EPO, whereas EPO withdrawal resulted in a
decrease in JNK activity and an increase in JunB DNA-binding activity.
In contrast, a recent report by Shan et al.42 has
implicated JNK and p38 in the induction of apoptosis induced by EPO
withdrawal in HCD57 cells. Their report suggested a
reciprocal relationship between p42/p44 ERK activation and JNK
activation similar to that observed in growth factor-deprived PC12
neuronal cells.14 To clarify our position, we present here further studies into the role of JNK, ERK, and p38 in erythroid proliferation and initiation of apoptosis in HCD57 cells. These studies
confirm our previous contention that JNK and cJun activities are not
associated with the induction of apoptosis in HCD57 cells but are
instead associated with EPO-induced proliferation. In addition, p38
activation appears to participate in EPO-dependent proliferation but
not in apoptosis induced by EPO withdrawal.
HCD57 cells
Materials
Cell culture HCD57(R) cells were cultured in Iscove modified Dulbecco medium (IMDM) (Life Technologies Inc, Gaithersburg, MD), 25% fetal calf serum (Hyclone, Logan, UT), and 10 µg/mL gentamicin (Life Technologies Inc) at 37°C in a 5% CO2 environment and maintained in 1 U EPO/mL media. HCD57(K) cells were cultured in the same media with the exception that 30% fetal calf serum was used. For each time point, 2.5 × 106 HCD57 cells were used. For EPO-deprivation studies, the cells were washed 3 times in media and incubated in the absence of EPO for the times indicated in the figure legends. For EPO-induced proliferation studies, HCD57(R) cells were washed 3 times and incubated for 18 hours in the above media minus EPO. The cells were then treated with 10 U EPO/mL for the times indicated in the figure legends. For studies with the MEK inhibitor PD98059 and the p38 inhibitor SB203580, cells were washed 3 times to remove all EPO from the cells and then cultured in media containing EPO, EPO + DMSO, EPO + 50 µmol/L PD98 058, EPO + 20 µmol/L SB203580, or no additional growth factor for the times indicated in the figure legends. Cell viability was determined by counting several hundred cells on a hemocytometer in the presence of 0.2% trypan blue. For TNF- studies, HCD57(R) cells were washed 3 times to remove all EPO from the cells and then cultured in media
containing no EPO for 18 hours. The cells were then treated with
various concentrations of TNF-, 10 SCF/mL or 1 U EPO/mL as indicated in the figure legends.
Western blot analysis Following treatment of the cells under the different conditions, HCD57(R) or HCD57(K) cells were harvested and lysed immediately in sample buffer (0.05 mol/L Tris, pH = 8, 2% sodium dodecyl sulfate, 0.1% bromophenol blue, 10% glycerol, and 10% -mercaptoethanol) and sonicated for 10 seconds each to shear the genomic DNA. Equal volumes (40 µL) of sample were electrophoresed on a 10% acrylamide SDS-PAGE gel (for JNK, AKT, and ERK blots) or a 12% acrylamide SDS-PAGE gel (for the p38 blots) and transferred to nitrocellulose. The
blots were blocked for 1 hour in TBST buffer (25 mmol/L Tris, pH = 7.8, 125 mmol/L NaCl, and 0.25% Tween-20) containing 5% nonfat milk and then incubated in primary antibody overnight at 4°C
in TBST buffer containing 5% bovine serum albumin (BSA). The blots were washed in TBST buffer, and specific reactive proteins were detected by using enhanced chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ). The blot was then stripped as previously described46 and re-probed successively with
phospho-specific antibodies as indicated in the figures.
To ensure equal loading of proteins, the blots were last probed with
antibodies that recognize both the phosphorylated and nonphosphorylated
forms of JNK-1, ERK1, or p38 (as indicated in the figure legends).
Molecular weights are indicated in kilodaltons (kD). For the in vitro
kinase assays, total cell extracts were immunoprecipitated as
previously described with anti-JNK-1 and subjected to an in vitro
kinase assay as previously described41 using 1 µg
GST-Jun fusion protein as a substrate (a generous gift from Dr
Paul Dent). In the case of the kinase assays in Figures 1 and 3,
following electrophoresis of the samples on a 10% acrylamide SDS/PAGE
gel, the proteins were transferred to nitrocellulose and exposed to
autoradiography 18 hours at  80°C with an intensifying screen
to visualize phosphorylated GST-Jun. The blots were then probed with
the polyclonal anti-JNK1 antibody to ensure equal loading of proteins.
Detection of apoptosis of HCD57 cells Apoptosis of HCD57(R) and HCD57(K) cells was detected by using flow cytometry analysis of propidium iodide-stained cells. Following cell treatment, HCD57 cells were fixed in 70% ethanol overnight at 4°C. The cells were then washed in phosphate buffered saline (PBS) and stained overnight in 3 mmol/L NaCitrate, 2 µmol/L propidium iodide, and 50 µg/mL RNAse A at 4°C in the dark. The cells were then collected, washed once in 1X PBS, and analyzed by using the FACScan flow cytometer (Becton Dickinson, Rutherford, NJ). Cells containing sub-G0/G1 DNA indicative of apoptosis were gated and shown as a percentage of the total number of cells.
JNK and p38 activities decrease on EPO withdrawal in HCD57 cells To test if JNK and/or p38 are activated following EPO withdrawal to induce apoptosis, we utilized in vitro assay of JNK activity and activation specific antibodies for JNK and p38. Two variants of the HCD57 cell line were used in the following studies. One cell line, which we have designated HCD57(K), undergoes rapid apoptosis within 24 hours of EPO withdrawal. The other cell line, designated HCD57(R), undergoes apoptosis more slowly and does not exhibit significant apoptosis until 48 to 72 hours following EPO withdrawal. We have previously shown that removing EPO from HCD57(R) cells by using an EPO-neutralizing antibody resulted in decreased JNK activity over a 24-hour period (Figure 5 of reference 41). To ensure that there was no activation of JNK in the period of time from 24 to 96 hours that may correlate with the later activation of apoptosis in this cell line, we repeated our in vitro kinase assay of EPO-deprived HCD57(R) cells over a 96-hour period. Figure 1A shows that JNK activity was observed in the presence of EPO and was no longer detectable 24 hours following EPO withdrawal. JNK activity was also not detected at later time points. To further confirm this observation of a loss of JNK activity in EPO-deprived HCD57(R) cells, whole cell extracts were subjected to Western blot analysis by using an antibody specific to the active form of JNK (phospho-JNK antibody; Figure 1B). JNK1 and JNK2 activation were detected in the presence of EPO but decreased greatly on EPO withdrawal. These results confirmed our earlier observation that JNK activity decreased on EPO withdrawal and did not correlate with the initiation of apoptosis in HCD57(R) cells.
JNK and p38 are activated by EPO in HCD57 cells
Inhibition of ERK activity does not induce JNK activity or apoptosis in HCD57 cells ERK and JNK have been shown to have opposing effects on apoptosis induced by growth factor withdrawal in PC12 cells.14 In this study, both suppression of ERK activity and activation of JNK correlated with the induction of apoptosis. To determine if the inhibition of ERK activity affected JNK activity or induced apoptosis in our system, HCD57(R) cells were treated for 48 hours in EPO in the absence or the presence of 50 µmol/L PD98059, a potent inhibitor of MEK, which activates ERK1 and ERK2. JNK activity was assessed by in vitro kinase activity of JNK1/2 immunoprecipitates. As we observed in the experiments outlined above, JNK activity was undetectable in the absence of EPO but high in the presence of EPO (Figure 3A, lanes 1 and 2). JNK activity was unaffected by treatment with the PD98059 (Figure 3A, lane 4). Western blot analysis of samples isolated in parallel to the JNK immunoprecipitates using an ERK phospho-specific antibody revealed that ERK activation was completely suppressed in the PD98059 treated cells, indicating that the inhibitor was functional (Figure 3B, lane 4). By counting the cells with the use of trypan blue exclusion, we determined that the number of cells was dramatically decreased in the PD98059-treated cells (Figure 3C). We then stained these cells with propidium iodide and analyzed the stained cells by using flow cytometry to look for DNA fragmentation indicative of apoptosis. The results of this flow cytometry are shown in Figure 3D. Whereas cells cultured in the absence of EPO showed an increase in the number of apoptotic cells (Figure 3D, top left panel), cells treated with the PD98059 inhibitor in the presence of EPO showed no evidence of DNA fragmentation (Figure 3D, lower right panel), even though the cell number was decreased compared with control cells (Figure 3C). It appears, therefore, that inhibition of ERK activity inhibited proliferation of HCD57 cells, but it induced neither JNK phosphorylation nor apoptosis.
Inhibition of p38 activity inhibits proliferation but not induction of apoptosis The activation of p38 in HCD57 cells in response to EPO suggests a role for p38 in EPO-induced proliferation or survival. To determine if p38 contributes to either proliferation or survival of these cells, we treated HCD57(K) cells with the specific p38 inhibitor SB20850 and assessed both cell proliferation and apoptosis. We found that treatment of HCD57(K) cells with the SB20850 inhibitor for 72 hours in the presence of EPO suppressed proliferation (Figure 4A, 72 hours + SB). Treatment with the inhibitor in the absence of EPO for 24 hours, however, did not suppress apoptosis (Figure 4B, No EPO + SB). This result supports the hypothesis that p38 activity contributes to EPO-induced proliferation and not to the induction of apoptosis.
TNF- has been shown to activate JNK in a number of systems.
Depending on the system, this activation may induce proliferation or
apoptosis. We were interested in whether TNF- could activate JNK in
HCD57 cells and if so, whether this JNK activation induced apoptosis.
We found that HCD57(R) cells treated with exogenous TNF- in the
absence of EPO resulted in JNK activation after 1 hour (Figure
5A, lane 3); JNK activation was still
detected 24 hours following TNF- treatment (Figure
5A, lane 7, and 5B, lane 4). An assessment
of the DNA content of cells incubated in TNF- for 24 hours, however,
revealed that no DNA degradation indicative of apoptosis was detected
greater than that induced by the removal of EPO. Therefore, although
treatment with TNF- can clearly induce JNK activation, it does not
induce apoptosis in HCD57(R) cells.
It is clear from the literature that AP1 and JNK may exhibit proliferative, pro-survival, or pro-apoptotic effects, depending on the cell type. In neuronal cells for instance, cJun phosphorylation and JNK activation appear to be critical for the induction of apoptosis during both growth factor deprivation- and stress-induced apoptosis.53-55 We have examined the possibility that JNK activation may play a role in the induction of apoptosis induced by EPO deprivation in EPO-dependent erythroid cells. We have previously reported that AP1 DNA binding increased on EPO withdrawal of HCD57 cells; however, we observed that JNK activity was high in the presence of EPO and disappeared on EPO withdrawal41 (and Figure 1 of this report). This result led us to explore the role of the specific AP1 family members in the induction of apoptosis. We determined that JunB was present in the AP1 complex when EPO was withdrawn, whereas cJun was present in the AP1 complex in the presence of EPO but not when EPO was absent. Taken together, these results implicated cJun and JNK in the proliferation and/or survival and JunB in the initiation of apoptosis of HCD57 cells.
We thank Dr Amy Lawson and Dr Haifeng Bao for their helpful discussions regarding this manuscript.
Submitted February 14, 2000; accepted March 31, 2000.
Supported by grant R01DK39781 (S.T.S.) from the National Institutes of Health, a grant from the American Heart Association (S.M.J.-H.), and grant IN-105 from the American Cancer Society (J.J.R.).
Reprints: Stephen T. Sawyer, Department of Pharmacology/Toxicology, PO Box 980613, Richmond, VA 23298; e-mail: ssawyer{at}hsc.vcu.edu.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
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Top
Abstract
Introduction
-32P-ATP and
GST-Jun fusion protein as a substrate and transferred to
nitrocellulose. Following exposure to show the phosphorylated GST-Jun
(upper arrow), the proteins were immunoblotted with goat anti-JNK-1 to
visualize JNK proteins (lower arrow). (B) Whole cell lysates were
immunoblotted with an anti-phospho-JNK antibody (upper panel). The blot
was then stripped and reprobed with anti-JNK1 to ensure equal loading
of proteins (arrow, lower panel). (C) Whole cell lysates were
immunoblotted with an anti-phospho-p38 antibody (upper panel). The blot
was then stripped and reprobed with anti-phospho-ERK antibody (middle
panel) to detect phosphorylated ERK (arrow, middle panel) and p38
(lower panel) to ensure equal loading of proteins (arrow, lower panel).
(D) HCD57(K) cells were deprived of EPO for the times indicated. Whole
cell lysates were immunoblotted with anti-phospho-JNK antibody (top
panel). Arrows indicated phosphorylated JNK-1 and -2 proteins. The blot
was then stripped and reprobed with anti-phospho-p38 antibody to detect
phosphorylated p38 (arrow, middle panel) and anti-JNK1 to ensure equal
loading of proteins (arrow, lower panel).
induces JNK activity but does not induce apoptosis in
HCD57 cells
