(Received for publication, February 28, 1997, and in revised form, May 2, 1997)
From the Bcl-2 is an intracellular membrane-associated
protein that prevents cell death induced by a variety of apoptotic
stimuli. A mechanism by which Bcl-2 exerts an anti-cell death effect
is, however, not fully understood. In the present study, Bcl-2
suppressed cell death of N18TG neuroglioma cells caused by various
apoptotic stresses, including etoposide, staurosporine, anisomycin, and ultraviolet irradiation. Concomitantly, Bcl-2 disrupted a signaling cascade to the c-Jun N-terminal kinase activation induced by the apoptotic stresses. Bcl-2 also prevented the etoposide-induced stimulation of MEKK1. Furthermore, overexpression of c-Jun N-terminal kinase antagonized the death-protective function of Bcl-2. These data
suggest that suppression of the c-Jun N-terminal kinase signaling pathway may be critical for Bcl-2 action.
Apoptosis is thought to be involved not only in normal
physiological processes, but also in pathogenesis of many diseases that
result from an imbalance between positive and negative regulators of
cell survival (1, 2). Numerous studies have demonstrated that Bcl-2 is
a typical positive regulator of cell survival (3). This 26-kDa
intracellular membrane-associated protein is capable of protecting
various cell types from experimentally induced cell death both in
vivo and in vitro (4, 5). For example, Bcl-2 rescues
cell death induced by a variety of stresses, including depletion of
trophic factors, anti-tumor drugs, oxygen free radicals, viral agents,
and heat shock as well as neuronal axotomy (4-7). Furthermore,
bcl-2 is thought to be a mammalian counterpart of ced-9, which acts to prevent cell death in
Caenorhabditis elegans (8). The molecular mechanism by which
Bcl-2 prevents cell death remains unknown, however. Interestingly, it
has been demonstrated recently that certain apoptotic stresses can
induce activation of the specific signaling systems such as
sphingomyelin or c-Jun N-terminal kinase
(JNK)1 pathways (9, 10). These findings
imply that Bcl-2 might suppress cell death through modulating
intracellular signaling cascades associated with apoptosis.
JNK, also termed stress-activated protein kinase (SAPK), is a new
member of the family of mammalian mitogen-activated protein (MAP)
kinases that mediate intracellular signals originated from diverse
extracellular stimuli, including growth factors, cytokines, or various
stresses (11). JNK is often activated through upstream protein kinases,
including JNKK and MEKK1 in response to a variety of cellular stresses
such as ionizing irradiation, alkylating chemicals, ultraviolet (UV)
light, or heat shock (12, 13). It is noteworthy that many stresses that
induce the stimulation of JNK can eventually cause cell death. In fact,
the stimulation of JNK was prerequisite for cell death under various
conditions, and a blockade of the JNK activation resulted in the
prevention of cell death (9, 10). These findings imply that JNK may mediate an intracellular signaling pathway leading to cell death.
In the present study, we investigated a possible mechanism for
anti-apoptotic action of Bcl-2. We observed that Bcl-2 blocked the
activation of the JNK signaling pathway by various apoptotic stresses
and that the blockade of JNK pathway might be associated with the cell
survival effect of Bcl-2.
N18TG cells were routinely
maintained in poly-D-lysine-coated plates containing
Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum. Cells were transfected with indicated expression vectors
by using LipofectAMINE (Life Technologies, Inc.). For bcl-2
transfection, cells were transfected with pcDNA3 (Invitrogen) or
pcDNA3 containing a full-length coding sequence of human
bcl-2. After 48 h of transfection, cultures were
maintained in the complete medium containing G418 (500 µg/ml) to
select neomycin-resistant cells. For JNK1 transfection, cells were
transfected with pCEP4 (Invitrogen) or pCEP4 containing JNK1 cDNA.
Stable cell lines were selected by adding hygromycin (200 units/ml) in
culture medium. Heterogeneous populations of the stably transfected
cells were used in this study to avoid any possible clonal
variations.
Cultured cells were exposed to etoposide
(10 µg/ml) or staurosporine (1 µg/ml) overnight or to anisomycin
(50 µg/ml) for 4 h. When indicated, cells were exposed to UV
light (40 Jm Cells were exposed to
10 µg/ml etoposide for 36 h, fixed with 4% paraformaldehyde,
permeablized with ice-cold ethanol, and stained with 50 ng/ml Hoechst
33258, as described previoiusly (15). Stained nuclei were observed and
photographed with a Zeiss Axiovert135 fluorescent microscope with
a × 40 objective.
Cells were exposed to the
indicated agents, harvested, and lysed with a buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM sodium chloride, 1 mM phenylmethylsulfonyl fluoride, 1% Nonidet P-40, 0.5%
deoxycholate, and 0.1% sodium dodecyl sulfate. Solubilized fractions
were immunoprecipitated with either a mouse monoclonal anti-JNK1
(PharMingen) or a rabbit polyclonal anti-MEKK1 antibody (Santa Cruz).
The immunopellets were assayed for either JNK1 or MEKK1 activity, as
described previously (16). Either GST-c-Jun or GST-SEK1 fusion protein
was used as a substrate for either JNK1 or MEKK1, respectively. The
assay reaction mixture was subjected to electrophoresis on a 10%
polyacrylamide gel. The phosphorylated proteins were quantified by
autoradiography and densitometry. Protein concentrations were
determined by the BCA method (Pierce), as described in the
manufacture's protocol.
To investigate the mechanism of anti-cell death function of Bcl-2,
we transfected a mammalian expression vector containing bcl-2 or control vector into neuroglioma N18TG cells.
Expression of Bcl-2 protein was detected in Bcl-2-transfected cells,
but not in control cells (Fig. 1A). Next, we
examined the effect of etoposide on cell death in either control or
Bcl-2-expressing cells. Etoposide is an anti-cancer drug that inhibits
topoisomerase II (17). Ectopic expression of Bcl-2 in
bcl-2-transfected cells resulted in suppression of
etoposide-induced cell death, compared with control cells (Fig.
1B). We also found that etoposide induced apoptotic DNA
fragmentation in N18 control cells and that Bcl-2 prevented the
etoposide-induced DNA fragmentation (Fig. 1C). Bcl-2 also
exerts a death-protective effect in cells subjected to other apoptosis-inducing stresses, including staurosporine, ultraviolet (UV)
light, or anisomycin.2
The signaling mechanism for the anti-apoptotic action of Bcl-2 remains
unclear. The mammalian MAP kinases, which include JNK, extracellular
signal-regulated kinase (ERK), and p38 kinase, are parts of signal
transduction cascades leading to a variety of cellular events (11). In
particular, JNK has been shown to mediate intracellular signals leading
to apoptosis (9, 10, 18). All of the apoptotic stresses used in this
study, which included etoposide, staurosporine, UV light, and
anisomycin, could induce the stimulation of the JNK signaling pathway
in N18 control cells (Fig. 2A). Using these
apoptotic stresses, we investigated a possibility that Bcl-2 might
modulate the JNK signaling cascade. In Bcl-2-expressing cells,
etoposide had little effect, if at all, on JNK1 activity (Fig.
2A). Bcl-2 also suppressed the stimulation of JNK1
activities induced by staurosporine, UV light, and anisomycin (Fig.
2A). Immunoblot analysis indicated that the cellular level
of JNK1 protein was not changed by Bcl-2 expression (Fig.
2B). mRNA levels of endogenous JNK1 between control
cells and Bcl-2-expressing cells were also equivalent.2
These data suggest that Bcl-2 might disrupt the signaling cascade to
JNK1 stimulation initiated by cellular stresses. The JNK signaling cascade includes MEKK1, SEK/JNKK, and JNK (11). MEKK1 activates SEK/JNKK that, in turn, activates JNK1 (19). We, therefore, examined
the effect of Bcl-2 on the stress-induced stimulation of MEKK1. As
shown in Fig. 2C, Bcl-2 suppressed the etoposide-induced activation of MEKK. Taken together, these data indicate that the intracellular signaling machinery for the JNK stimulation is defective in Bcl-2-expressing cells, suggesting that the JNK signaling pathway may be downstream from the target(s) of Bcl-2 action.
If the suppression of JNK1 stimulation is a critical step for the
anti-apoptotic function of Bcl-2, overexpression of JNK1 could
antagonize the action of Bcl-2. To test this possibility, we
overexpressed JNK1 in Bcl-2-expressing cells by a stable transfection and named those cells N18-Bcl-2/JNK1. Treatment of N18-Bcl-2/JNK1 cells
with various apoptotic stresses resulted in stimulation of JNK1
activity (Fig. 3). This indicates that N18-Bcl-2/JNK1 cells, by overexpressing JNK1 protein, overcame the negative action of
Bcl-2 on the JNK signaling pathway. Next, we measured the viability of
N18 control, N18-JNK1, N18-Bcl-2, or N18-Bcl-2/JNK1 cells exposed to
etoposide, staurosporine, anisomycin, or UV light (Fig.
4A). N18-Bcl-2 cells, which expressed the
Bcl-2 protein, were more resistant to the apoptotic stresses used in
the study, compared with the other cells. However, N18-Bcl-2/JNK1
cells, which expressed both recombinant Bcl-2 and JNK1 proteins, were
as sensitive as control N18 cells to cell death induced by the
apoptotic stresses. These data demonstrated that overexpression of JNK1
in Bcl-2-expressing cells counteracted the anti-cell death function of
Bcl-2. As shown in Fig. 4B, Bcl-2 also prevented DNA
fragmentation in cells treated with the apoptotic stresses, and
this effect of Bcl-2 was negated by the overexpression of JNK1,
resulting in the induction of DNA fragmentation by apoptotic stimuli in
N18-Bcl-2/JNK1 cells. We also examined morphological changes of
apoptotic nuclei with Hoechst 33258 stain after cells were exposed to
10 mM etoposide (Fig. 4, C-F). Treatment of N18
cells with etoposide caused nuclear fragmentation (Fig. 4D),
and the etoposide-induced nuclear breakdown was prevented by Bcl-2
(Fig. 4E). Overexpression of JNK1, however, counteracted the
protective effect of Bcl-2 on the etoposide-induced nuclear
fragmentation (Fig. 4F). These data strongly suggest that sustained activation of JNK1 may override the anti-apoptotic function of Bcl-2, leading to cell death.
Overexpression of JNK1 counteracts the
anti-apoptotic function of Bcl-2. Cultured cells were exposed to
etoposide (10 µg/ml), staurosporine (1 µg/ml), UV light (40 Jm
MAP kinase signaling pathways are involved in a variety of cellular
events, including cell growth, differentiation, and development (20).
JNK, a member of the MAP kinase family, is often stimulated in response
to many cellular stresses (11-13). Furthermore, JNK is thought to
mediate an intracellular signal for stress-activated apoptosis (9, 10).
Bcl-2 prevents apoptosis induced by a variety of cytotoxic
stimuli. The mechanism by which Bcl-2 prevents apoptosis is, however,
still unclear, even though Bcl-2 has been proposed to function as an
anti-oxidant or free-radical scavenger, or to modulate calcium efflux
through the endoplasmic reticulum in some experimental models (21-23).
We report here that various stresses which induce the stimulation of
JNK activity can cause cell death and that Bcl-2 prevents both cell
death and JNK stimulation induced by those stresses. Moreover,
overexpression of JNK1 counteracted the anti-apoptotic function of
Bcl-2 in response to the apoptotic stresses. Interestingly, a recent
study with PC12 cells also reported that JNK activation induced by
nerve growth factor withdrawal was blocked by Bcl-2 (24). Although a
detailed mechanism by which Bcl-2 blocks JNK activation is not clear
yet, we found that Bcl-2 disrupted the stimulation of MEKK1, an
upstream protein kinase in JNK signaling pathway (19). It suggests that
Bcl-2 might act on its cellular target(s) that might be involved in the
stimulation of MEKK, which can activate the JNK pathway. A precise
mechanism by which overexpression of JNK1 overcomes the antagonistic
effect of Bcl-2 on the stress-induced stimulation of endogenous JNK1 is
not clear yet. One possibility could be that it might result from a
dose effect of the overexpressed JNK1 protein. That is, JNK1 activity
could be enhanced to a significant level in JNK1-overexpressing cells
in response to apoptotic stresses, if Bcl-2 could not completely block
the MEKK/JNK1 signaling pathway. Another possibility could be that
there might exist alternative pathways for stress-induced stimulation
of JNK1. In this regard, it is noteworthy that JNK1 signaling pathway
can be also regulated through other MAPKKKs such as ASK1 (25),
independent of MEKK1. In any event, our findings in this study strongly
suggest that the JNK signaling pathway may be downstream from the
target(s) of Bcl-2 action. Notably, overexpression of
Bcl-XL, a functional analog of Bcl-2 (26), in N18TG cells
also resulted in the suppression of the JNK stimulation after treatment
of cells with etoposide or the other stresses used in this
study.2 Therefore, it appears that suppression of the JNK
pathway may be crucial for the anti-cell death function of both Bcl-2
and Bcl-XL. Our findings presented in this study provide
new insights into the mechanism by which Bcl-2 and its related proteins
regulate cell death and survival.
We thank Drs. R. J. Davis, J. Woodgett, M. Karin, and S. J. Korsmeyer for clones and plasmids and Dr. W. A. Toscano, Jr. for critical reading of the manuscript.
Cell Biology and ¶ Molecular Genetics
Laboratories,
Graduate School of
Biotechnology,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
Cell Culture and Transfections
2), then further incubated overnight. After
treatment with the indicated agents, cells were harvested, and lysed
with a solution containing 20 mM EDTA, 0.8% sodium lauryl
sarcosine, 100 mM Tris-HCl, pH 8.0. Cell lysates were
treated with RNase A, RNase T1, and proteinase K, as described
previously (14). Fragmented DNA samples were separated by
electrophoresis on 1.5% agarose gel and visualized with ethidium
bromide.
Fig. 1.
Bcl-2 prevents cell death induced by
etoposide. N18TG cells were transfected with either a control
vector or a vector containing bcl-2 and named either N18 or
N18-Bcl-2 cells, respectively. A, immunoblot of Bcl-2 in
cells expressing Bcl-2 (N18-Bcl-2) and control cells
(N18). B, viability of either control or
Bcl-2-expressing cells in exposure to 10 µg/ml etoposide, as measured
by trypan blue exclusion. C, apoptotic DNA
fragmentation induced by etoposide. Either control cells
(Control) or Bcl-2-expressing cells (Bcl-2) were exposed to
10 µg/ml etoposide (E) for 18 h. DNA was extracted and DNA fragments were resolved on a 1.5% agarose gel.
[View Larger Version of this Image (16K GIF file)]
Fig. 2.
Bcl-2 suppresses the JNK1 stimulation induced
by cytotoxic stresses. A, either N18 control cells
(control) or Bcl-2-expressing cells (Bcl-2) were
exposed to etoposide (10 µg/ml), staurosporine (1 µg/ml), or
anisomycin (50 µg/ml) for 1 h or to UV irradiation (40 Jm2). Cells were harvested, lysed, and subjected to
immunoprecipitation with mouse anti-JNK1 monoclonal antibody. The
immunocomplex JNK1 was assayed by phosphorylating a substrate,
GST-c-Jun. Phosphorylated proteins were visualized by SDS-PAGE and
autoradiography. B, immunoblot of JNK1 in N18 control cells
(N18) and Bcl-2-expressing cells (N18-Bcl-2).
C, either control or Bcl-2-expressing cells were exposed to
10 µg/ml etoposide for 40 min. Cells were collected and subjected to
immunoprecipitation with rabbit polyclonal anti-MEKK1 antibody. The
immunopellets were assayed for MEKK1 activity, as described under
"Experimental Procedures."
[View Larger Version of this Image (33K GIF file)]
Fig. 3.
Overexpression of JNK1 in Bcl-2-expressing
cells overrides the suppressive effect of Bcl-2 on JNK1 activity.
JNK1 activity was stimulated by exposing either Bcl-2-transfected
(N18-Bcl-2) or Bcl-2/JNK1-transfected (N18-Bcl-2/JNK1) cells to
etoposide (10 µg/ml), staurosporine (1 µg/ml), anisomycin (50 µg/ml) for 1 h or to UV irradiation (40 Jm2) and
was assayed for phosphorylating GST-c-Jun, as for Fig. 2.
[View Larger Version of this Image (23K GIF file)]
Fig. 4.
2), or anisomycin (50 µg/ml). N18, control N18TG
cells; N18-JNK1, cells transfected with JNK1 gene; N18-Bcl-2; cells
transfected with bcl-2; N18-Bcl-2/JNK1, cells co-transfected
with bcl-2 and JNK1 gene. A, viability of cells
after treatment with apoptotic agents. Cultured cells were exposed to
etoposide or staurosporine overnight or anisomycin for 4 h. For UV
experiments, cells were exposed to 40-Jm
2 UV light, then
further incubated overnight. After treatment of cells with the
indicated agents, percentage of viability was determined by trypan blue
exclusion. B, DNA fragmentation induced by apoptotic agents.
Either N18-Bcl-2 cells (Bcl-2) or N18-Bcl-2/JNK1 cells (Bcl-2/JNK1) were exposed to etoposide (E),
staurosporine (S), anisomycin (A), or UV light
(U), the same as for A. DNA fragmentation was visualized, the same as for Fig. 1.
C-F, staining of apoptotic cells with Hoechst 33258. Cultured cells were incubated without (C) or with 10 µg/ml
etoposide (D-F) for 36 h, and morphological changes in
cells stained with Hoechst 33258 were examined and photographed with a
Zeiss Axiovert135 fluorescence microscope with a × 40 objective.
C and D, N18 control cells; E,
N18-Bcl-2 cells; F, N18-Bcl-2/JNK1 cells.
[View Larger Version of this Image (34K GIF file)]
*
This work was supported in part by the Korean Science
Foundation 95-0403-94-3 (to Y. J. O.).The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
**
To whom correspondence should be addressed: Cell Biology
Laboratory, Hanhyo Institute of Technology, 461-6, Jeonmin-dong, Yuseong-ku, Taejon 305-390, Korea. Tel.: 82-42-866-9122/9132; Fax:
82-42-866-9129.
1
The abbreviations used are: JNK, c-Jun
N-terminal kinase; SAPK, stress-activated protein kinase; MAP kinase,
mitogen-activated protein kinase; ERK, extracellular signal-regulated
kinase; SEK, SAPK kinase; JNKK, JNK kinase; MEKK, MEK kinase.
2
J. Park and E.-J. Choi, unpublished
observations.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.