From the Department of Biochemistry and Biophysics, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104
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ABSTRACT |
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Rat mesangial cells are normally resistant to
tumor necrosis factor- (TNF-
)-induced apoptosis. In this report
we show that the cells can be made susceptible to the apoptotic effect
of TNF-
when pretreated with actinomycin D, cycloheximide, or
vanadate. c-Jun N-terminal protein kinase (JNK) has been thought to
mediate apoptotic processes elicited by some stimuli, but its
involvement in TNF-
-induced apoptosis has been controversial. JNK
activation was investigated under conditions where the mesangial cells
were either resistant or susceptible to TNF-
-induced apoptosis.
TNF-
alone stimulated a single transient JNK activity peak. However, when the cells were pretreated with actinomycin D or cycloheximide, TNF-
stimulated a second sustained JNK activity peak. When the cells
were pretreated with the phosphatase inhibitor vanadate, TNF-
-induced JNK activation was greatly prolonged. In all three cases, a sustained JNK activation was associated with the initiation of
apoptosis. Our data suggest that a sustained activation of JNK induced
by these reagents may be associated with blocking the expression of a
phosphatase that inactivates JNK. Further studies reveal that the
expression of mitogen-activated protein kinase phosphatase-1 (MKP-1)
was induced by TNF-
, indicating that MKP-1 may be involved in
protecting the cells from apoptosis by preventing a prolonged
activation of JNK under normal conditions. Additional studies
showed that extracellular signal-regulated protein kinase activation
stimulated by TNF-
was unlikely to contribute to the resistance of
mesangial cells to TNF-
cytotoxicity.
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INTRODUCTION |
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Tumor necrosis factor-
(TNF-
)1 is a
multifunctional cytokine produced by many cell types. It elicits a wide
range of biological responses including cell proliferation,
differentiation, and apoptosis, depending on the cell type and its
state of differentiation (1). Most cells express specific receptors for
TNF-
. Two receptors have been characterized, designated as
TNF-
receptor 1 (TNFR1) and TNF-
receptor 2 (TNFR2). After
binding to these receptors, TNF-
elicits multiple signal
transduction pathways that regulate different cellular processes
(2).
Many tumor cells are sensitive to TNF--induced apoptosis, but normal
cells are usually resistant. Some cells undergo apoptosis only when
they are treated with TNF-
in the presence of other agents or when
the cells are damaged (3-5). The well characterized morphological
changes in the cells that undergo apoptosis include cell shrinkage,
cytoplasmic blebbing, and DNA digestion. Recently, activation of
interleukin 1-converting enzyme (ICE)-related proteases have been
implicated as the "executors of the cell death" at the onset stage
of the apoptotic process (6). However, the initial events in the signal
transduction pathway responsible for the later phases of cell death are
poorly understood. Among these signaling pathways, activation of
transcription factors c-Jun and c-Fos (components of the AP-1
transcription complex) and NF-
B are among the early cellular
responses. After phosphorylation, c-Fos and c-Jun form a heterodimer to
produce an active AP-1 transcription complex. Mitogen-activated protein
kinases (MAP kinase) are among the protein kinases that are responsible
for the phosphorylation of c-Fos and c-Jun (7). In mammalian cells,
three distinct subtypes have been identified in the MAP kinase family:
extracellular signal-regulated kinases (ERK), c-Jun N-terminal protein
kinases (JNK), and p38 kinases. JNK and p38 kinases are strongly
activated by extra- or intracellular stress and inflammatory cytokines
including TNF-
(8, 9). It is thought that activation of JNK and p38 kinases generally promotes an inhibition of cell growth or promotion of
cell death, whereas ERK is usually strongly activated by growth factors
and hormones that stimulate cell growth and is, therefore, involved in
the regulation of cell proliferation (9, 10). Strong and prolonged
activation of JNK has been reported in response to a variety of
stresses including UV light, ionizing irradiation, and hydrogen
peroxide, any of which can trigger apoptosis (11-13). Because TNF-
strongly activates JNK but only weakly stimulates ERK in many cells, it
is postulated that JNK activation is involved in TNF-
-induced
apoptosis (14-17). However, several reports show that activation of
JNK can be mediated through a noncytotoxic TRAF2 (TNF
receptor-associated factor 2 pathway initiated by TNF-
, which is not
linked to apoptosis (2, 18, 19). The role of the JNK pathway in
TNF-
-induced signaling, therefore requires further elucidation.
NF-B is a transcription factor present as a heterodimer complexed
with I-
B in the cytoplasm of unstimulated cells. Upon cell
stimulation, I-
B is phosphorylated and degraded, resulting in the
release of NF-
B, which is translocated to the nucleus where it
initiates transcription activity (20). TNF-
is known to induce
NF-
B activation. Initially, it was thought that NF-
B activation
was involved in apoptosis induced by TNF-
until more recent findings
suggested that activation of NF-
B by TNF-
stimulates the
synthesis of a survival factor, which protects the cell from apoptosis
(21-23). This hypothesis provides a plausible explanation for the
reason why some cells, notably primary cells, are resistant to
TNF-
-induced apoptosis. Nevertheless, the putative protective factor has not been identified, and the role of NF-
B activation in
the overall progress of apoptosis has not been resolved.
Mesangial cells are a prominent cell type in the glomerulus, and they
take part in the regulation of glomerular hemodynamics. They produce
inflammatory cytokines such as TNF- and are involved in the uptake
and clearance of immune complexes from the glomeruli by phagocytosis
(24). Cell death from apoptosis is prominent during the course of
various renal diseases as well as in the early stages of kidney
development. In the present study, the cytotoxic effect of TNF-
on
rat mesangial cells has been investigated. Our results show that
although TNF-
activated JNK, ERK, and NF-
B, it was unable, by
itself, to induce apoptosis. However, under conditions when JNK was
activated in a sustained fashion, the cells underwent apoptosis.
Of particular interest, our data suggest that a sustained activation of
JNK may be associated with a diminished amount or activity of a
phosphatase responsible for the dephosphorylation and
inactivation of JNK, thereby triggering apoptosis.
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EXPERIMENTAL PROCEDURES |
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Materials--
Recombinant TNF- was obtained from Chemicon
International Inc. (Temecula, CA). Anti-ERK2, anti-I-
B-
, and
anti-NF-
B antibodies were purchased from Santa Cruz Biotechnology
(Santa Cruz, CA). Anti-phospho-c-Jun (Ser-63), anti-phospho-JNK
antibodies were from New England Biolabs (Beverly, MA). Anti-c-Jun
antibodies were from Oncogene (Uniondale, NY), and anti-phospho-ERK was
from Promega (Madison, WI). Myelin basic protein (MBP) and PD098059 were purchased from Sigma. The terminal deoxynucleotidyl
transferase-mediated dUTP-biotin nick end-labeling (TUNEL) assay kit
was from Boehringer Mannheim.
Cell Culture and Cell Viability Assay-- Rat mesangial cells were isolated from male Sprague-Dawley rats under sterile conditions using the sieving technique as described (25). The cells were maintained in RPMI 1640 medium containing 20% fetal calf serum and 0.6 unit/ml insulin at 37 °C in a humidified incubator (5% CO2, 95% air). Cells from 5-20 passages were used. After the cells were grown to 80-90% confluence, they were made quiescent by incubation for 16-18 h in insulin-free RPMI 1640 medium containing 2% fetal calf serum.
For cell viability assays, mesangial cells were grown in 12-well plates. The quiescent cells were treated with reagents for the indicated times. Uptake of neutral red dye was used as a measurement of cell viability (26). At the end of the incubations, the medium was removed, and the cells were incubated in Dulbecco's modified Eagle's medium with 2% fetal calf serum containing 0.001% neutral red for 90 min at 37 °C. The uptake of the dye by viable cells was terminated by removal of the media, washing the cells briefly with 1 ml 4% paraformaldehyde in phosphate-buffered saline, pH 7.4, and solubilizing the internalized dye with 1 ml of a solution containing 50% ethanol and 1% glacial acetic acid. The absorbances, which correlate with the amount of live cells, were determined at 540 nm. During the cell incubations cell morphology was also examined under a light microscope.Immunocytochemical Detection of Apoptosis and c-Jun Phosphorylation-- Cells grown on 25-mm glass coverslips in six-well plates were fixed with 4% paraformaldehyde in phosphate-buffered saline, pH 7.4, after treatment with various reagents as indicated. DNA strand breaks were identified using a modified TUNEL assay kit. Briefly, the fixed cells were treated with terminal deoxyribonucleotidyl transferase, which incorporates fluorescein-tagged nucleotides onto 3'-OH termini of fragmented DNA. This was then visualized by adding anti-fluorescein antibody conjugated with horseradish peroxidase followed by diaminobenzidine. The positively stained dark colored nuclei were analyzed under a light microscope. Phosphorylation of c-Jun was detected with anti-phospho-c-Jun antibodies essentially following the immunocytochemistry protocol provided by the manufacturer (New England Biolabs).
Cell Lysate Preparation-- The quiescent cells were treated with reagents for the indicated times, washed twice with ice-cold phosphate-buffered saline, and scraped into cell lysis buffer containing 50 mM Hepes, pH 7.5, 150 mM NaCl, 1 mM Na3VO4, 50 mM pyrophosphate, 100 mM NaF, 1 mM EGTA, 1.5 mM MgCl2, 1% Triton X-100, 10% glycerol, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride. The cells were incubated in lysis buffer for 30 min on ice with periodic vortexing and centrifuged at 15,000 × g for 15 min. The supernatant was designated as cell lysate. Protein concentration was determined by the method of Bradford using BSA as standard (27). The gelatinous pellets that contained nuclei and cytoskeleton components were extracted with SDS sample buffer and heated at 100 °C for 5 min. The extract was designated as crude nuclear fraction.
Protein Kinase Assays--
JNK activity was measured using a
solid phase kinase assay method. GST-c-Jun(1-79) (GST-Jun) fusion
protein was isolated from bacterial cells expressing pGEX-c-Jun plasmid
(kindly provided by Dr. J. K. Westwick). GST-Jun bound to
glutathione-agarose beads was used to affinity purify JNK. JNK activity
was determined using GST-Jun as substrate (28). Briefly, 100 µg of
cell lysate was incubated with 2 µg of GST-Jun-agarose beads at
4 °C for 2 h with rotation and centrifuged at 10,000 × g for 1 min. The beads were washed three times with washing
buffer (25 mM Hepes, pH 7.5, 50 mM NaCl, 0.1 mM EDTA, 2.5 mM MgCl2, 0.05% (v/v)
Triton X-100, 5 µg/ml aprotinin, 5 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 20 mM
-glycerophosphate, and 10 mM NaF). The beads were then
resuspended in 10 µl of kinase buffer containing as final
concentration 20 mM Hepes, pH 7.5, 10 mM
MgCl2, 1 mM Na3VO4, 20 mM
-glycerophosphate, 5 mM NaF, 10 µg/ml
aprotinin, 10 µg/ml leupeptin, 40 µM ATP, and 1 µCi
of [
-32P]ATP. After incubation at room temperature for
20 min, the reaction was terminated by adding SDS sample buffer
followed by heating at 100 °C for 3 min. The proteins were separated
on 12% SDS-PAGE, and the phosphorylated proteins were detected by
autoradiography. The kinase activity was quantified by scintillation
counting of the radioactivity in the phospho-GST-Jun bands excised from
the gels.
Western Blot Analysis-- The protein samples were subjected to SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were blocked with 5% non-fat dry milk in Tris-buffered saline containing 0.05% Tween 20 (TBST) and incubated with primary antibodies followed by horseradish peroxidase-conjugated secondary antibodies according to the manufacturer's instructions. The immunnoblots were visualized by an enhanced chemiluminescence (ECL) kit obtained from Amersham.
RNA Isolation and Northern Blot Analysis-- Total RNA was isolated from mesangial cells using TRI-reagent (Molecular Research Center, Inc.) as recommended by the manufacturer. Northern blot analysis was performed as described previously (32). HindIII/BamHI fragment of pCEP4-MKP-1 (MAP kinase phosphatase-1) plasmid (kindly provided by Drs. N. Tonks and H. Sun) was used as a probe.
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RESULTS |
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Effect of TNF- on the Viability of Mesangial Cells in the
Presence or Absence of Actinomycin D and Cycloheximide--
Mesangial
cells are essentially insensitive to TNF-
cytotoxicity at a
concentration of 10 ng/ml (Fig. 1), which
is sufficient to induce apoptosis in susceptible cells (4, 17).
Prolonged incubation of mesangial cells with 10 ng/ml TNF-
for
48 h showed little deterioration of cell viability. However, when
the cells were pretreated with the transcription inhibitor actinomycin
D or the protein synthesis inhibitor cycloheximide, cell viability was
decreased dramatically (Fig. 1). The cells did not show demonstrable signs of apoptosis after they were treated with TNF-
, actinomycin D,
or cycloheximide alone. Cell death resulting from preincubation with
either actinomycin D or cycloheximide followed by TNF-
stimulation showed typical characteristics of apoptosis as examined under a
microscope and as determined by the TUNEL analysis. At the end of the
4-h incubation, more than 50% of the cells attached to the coverslips
were TUNEL staining-positive when the cells were pretreated with either
actinomycin D or cycloheximide followed by stimulation with TNF-
,
whereas under the same conditions only 5% of the cells were TUNEL
staining-positive when the cells were treated with TNF-
only. This
is the same as for the control experiments where the cells were not
treated with any reagent. These results indicate that mesangial cells
are normally resistant to TNF-
-induced apoptosis with this
resistance depending on de novo protein synthesis, a
property shown by other nontransformed cell types (3, 4).
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Activation of JNK by TNF- and the Effects of Actinomycin D and
Cycloheximide--
The JNK pathway has been implicated in the
regulation of apoptosis induced by various stimuli (12, 13, 33). Two
protein kinases with molecular masses of 54 and 46 kDa were activated by TNF-
as detected by an in-gel assay when GST-Jun was used as
substrate in mesangial cells. They were identified as JNK2 and JNK1,
respectively, based on their molecular weights. Their identities were
further confirmed by Western blot analysis using anti-phospho-JNK
antibodies that only recognized the activated JNK1 and JNK2 (data not
shown). To investigate if there was a correlation between JNK activity
and cell viability, the time course of JNK activation was determined in
cells after treatment with TNF-
. As shown in Fig.
2A, the activation of JNK by
TNF-
was very transient. Maximal activation was reached within 15 min, giving a 5-10 fold-stimulation, which diminished to the basal level by 30 min. However, when the cells were pretreated with actinomycin D or cycloheximide, a second JNK activation peak was detected. In the case of actinomycin D, the second activity peak started at 2 h and lasted at least for another hour with increased activity (Fig. 2B). In the case of cycloheximide, the second
activity peak started at 3 h after the addition of TNF-
with a
lower level of activation compared with the effect of actinomycin D
(Fig. 2C). In addition to inducing the second peak of JNK
activation, cycloheximide also potentiated the duration of the first
TNF-
-induced activity peak of JNK compared with TNF-
alone as a
control (Fig. 2A). The second activity peak of JNK was not
caused directly by actinomycin D or cycloheximide because neither
treatment had a significant effect on JNK activity (Figs. 2,
B and C, lower panels). Although it is
reported that cycloheximide by itself activates JNK in some cells (15),
its effect on mesangial cells was rather weak. A similar result was
also reported by Liu et al. in rat mesangial cells (34).
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Activation of JNK by TNF- and the Effect of Protein Phosphatase
Inhibitors--
From the above data it appeared that a sustained JNK
activation was necessary for the induction of apoptosis, whereas
activation of JNK by TNF-
alone in mesangial cells was too transient
to initiate the apoptotic process. To determine whether a sustained JNK
activation is able to render the cells susceptible to TNF-
cytotoxicity using other approaches, mesangial cells were pretreated with two protein phosphatase inhibitors, vanadate and okadaic acid,
which are known to inhibit the tyrosine phosphatases and type 1/type 2A
serine/threonine phosphatases, respectively. These treatments
presumably would inactivate protein phosphatases that dephosphorylate
JNK, thereby prolonging the activation of JNK by TNF-
. As shown in
Fig. 4, whereas pretreatment of the cells with okadaic acid (Fig. 4B) slightly potentiated the
duration of JNK activation stimulated by TNF-
, pretreatment of the
cells with vanadate (Fig. 4C) dramatically prolonged
TNF-
-induced JNK activation. Control experiments showed that okadaic
acid alone had little effect on JNK activity, but vanadate alone
activated JNK about 2-fold. These results indicate that inactivation of JNK occurs mainly by a vanadate-sensitive tyrosine phosphatase.
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Effect of TNF- on the Viability of Mesangial Cells in the
Presence or Absence of Phosphatase Inhibitors--
To investigate
whether there was a correlation between the duration of JNK activation
and cell viability, the mesangial cells were incubated with TNF-
in
the presence of okadaic acid and vanadate. As shown in Fig.
6, preincubation of the cells with okadaic acid followed by stimulation with TNF-
had no effect on cell
viability. In contrast, pretreatment of the cells with vanadate
followed by TNF-
stimulation dramatically increased cell death.
Cells that were treated with vanadate alone also resulted in
significant cell death (Fig. 6). It should be noted that vanadate alone
also caused JNK activation at a low level (~3-fold) but in a
sustained manner, in conjunction with a sustained phosphorylation of
c-Jun (Fig. 5). Cells from parallel experiments were examined for
apoptosis by the TUNEL analysis. Under the conditions tested, only the
cells treated with vanadate or vanadate plus TNF-
showed the
morphological characteristic of apoptosis. At the end of the 4-h
incubation period, about 25% of the cells attached to the coverslips
were TUNEL staining-positive when the cells were treated with vanadate
alone, and 35% of the cells were TUNEL staining-positive when treated
with vanadate and TNF-
. Only 5-7% of the cells were TUNEL
staining-positive when the cells were treated with okadaic acid alone
or in combination with TNF-
. These results indicate that cell death
resulted from apoptosis and was paralleled by a sustained activation of
JNK and c-Jun phosphorylation state.
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Activation of ERK by TNF- and the Effect of Protein Phosphatase
Inhibitors--
It is known that TNF-
activates both JNK and ERK,
but generally JNK is activated more strongly than ERK, as reported in a number of cell types (14, 15, 36). TNF-
activated both ERK1 and ERK2
as determined by Western blot analysis using antibodies that only
recognized the active ERK1 and ERK2. The effect of TNF-
on ERK2
activation in mesangial cells is shown in Fig.
7. The activation of ERK2 was transient
with the activity peaking at 15 min followed by a decline to the basal
level after 30-60 min. Similar kinetics were obtained when ERK2
activation was measured by the gel shift assay or when the activation
of ERK2 was quantified using the kinase assay method, in which ERK2 was
immunoprecipitated and its activity determined using MBP as substrate
(Fig. 7). The maximum activation of ERK2 at 15 min was 3-5 fold above
basal levels (Fig. 7A), whereas the maximum activation of
JNK was 8-10-fold (Fig. 2A). The effects of protein
phosphatase inhibitors on ERK2 activity showed a pattern similar to
that for JNK with okadaic acid having little effect and vanadate
profoundly prolonging ERK2 activation, as judged by the gel shift assay
(Fig. 7B).
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Effects of PD098059 on the Activation of JNK and ERK by
TNF---
Whereas the JNK pathway has been implicated in
pro-apoptotic processes, the ERK pathway has been thought to be
anti-apoptotic (10). Activation of the ERK pathway has been shown to
suppress apoptosis induced by various agents including TNF-
, but
selectively blocking ERK activation greatly potentiates the apoptotic
process (10, 37). To test the possibility that ERK activation may contribute to the resistance of mesangial cells to TNF-
cytotoxicity, PD098059, a compound known to inhibit ERK kinase
specifically, thereby blocking ERK activation (38), was used to block
ERK activation by TNF-
. As shown in Fig.
8, pretreatment of mesangial cells with
30 µM PD098059 effectively abolished the activation of
ERK induced by TNF-
, but JNK activity was not affected. Control experiments showed that PD098059 alone had no effect on ERK or JNK
activity. These results are in agreement with its reported effect on
ERK and JNK activities in other cells (39). Additionally, neither cell
viability nor morphology of the mesangial cells was affected by
pretreatment with PD098059 compared with cells that were treated with
TNF-
alone. The above results indicate that ERK activation
stimulated by TNF-
is unlikely to contribute to the resistance of
the mesangial cells to TNF-
cytotoxicity.
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Activation of NF-B by TNF-
in the Presence and Absence of
Protein Phosphatase Inhibitors--
Recent evidence favors the
hypothesis that activation of NF-
B initiates transcription of a gene
encoding a factor that protects the cells from apoptosis (21-23).
Although our primary hypothesis is that a sustained activation of JNK
caused by inhibition of a JNK phosphatase with vanadate is the primary
factor involved in TNF-
-induced apoptosis, it is possible that
treatment of the cells with vanadate may also interfere with the
activation of NF-
B by TNF-
. This may have the effect of blocking
the synthesis of the protective factor, thereby rendering the cells
susceptible to TNF-
cytotoxicity. To investigate this possibility,
the effect of vanadate on the activation of NF-
B by TNF-
was
determined by Western blot analysis using anti-I-
B
antibodies.
The degradation of I-
B
was used as an indirect indication of
NF-
B activation (40). As illustrated in Fig.
9A, TNF-
treatment induced
an initial decrease of I-
B
, which was observed after 15 min but then returned to starting levels after 2 h. This biphasic response pattern has also been seen in other cells (40). Pretreatment of the
cells with either okadaic acid or vanadate failed to block the
degradation of I-
B
induced by TNF-
, with okadaic acid
pretreatment actually showing some potentiation of the TNF-
effect
(Fig. 9B). The activation of NF-
B was further confirmed
by immunocytochemistry using antibodies against the 65-kDa subunit of
NF-
B. In all cases, the 65-kDa subunit translocated from the cytosol
to the nucleus,2 indicating
that vanadate and okadaic acid did not block the activation of NF-
B
induced by TNF-
. Based on these observations, it is likely that
vanadate exerts its effect through the inhibition of phosphatases
rather than interfering with the NF-
B activation induced by
TNF-
.
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Induction of MKP-1 mRNA by TNF---
Our results thus far
suggest that a sustained activation of JNK induced by TNF-
in the
presence of actinomycin D and cycloheximide may be associated with
blocking the expression of a phosphatase that inactivates JNK. To
investigate if TNF-
could elicit the synthesis of such a
phosphatase, we investigated the gene expression of MKP-1 in rat
mesangial cells. MKP-1 is the most prominent and best characterized
member of MKP family which has been shown to inactivate JNK in
vitro and in vivo (41-43). As shown in Fig.
10, the mRNA of MKP-1 was strongly
induced by TNF-
at the same time, which coincided with the
inactivation of JNK (Fig. 2). This result strongly supports our
hypothesis that a TNF-
-inducible phosphatase may be responsible for
protecting mesangial cells from apoptosis by suppressing a prolonged
activation of JNK. MKP-1 could be the phosphatase or one of such
phosphatases.
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DISCUSSION |
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Mesangial cells are insensitive to TNF--induced apoptosis under
conditions that will induce apoptosis in TNF-
susceptible cells.
Several hypotheses have been proposed to explain how normal cells
achieve this cellular resistance to TNF-
. First, TNF-
may
activate an anti-apoptotic signaling pathway that counteracts the
cytotoxicity of the apoptotic pathway (37, 44). Second, TNF-
may
elicit the synthesis of a protective factor, causing the cells to
become insensitive to TNF-
cytotoxicity (21, 23). Third, the
apoptotic pathway inducible by TNF-
may not be sufficiently activated to initiate the apoptotic process. However, to date none
of these hypotheses has been fully established. The present investigation was initiated to gain some insight into how mesangial cells achieve their resistance to TNF-
cytotoxicity.
Based on our current understanding of the roles of the different
subtypes of MAP kinases in the regulation of growth and development, it
has been proposed that the balance and/or threshold level of activation
of one pathway versus the other, e.g. ERK
versus JNK/p38 kinase, may determine whether a cell is going
to proliferate or undergo apoptosis (45). For example, in PC12 cells,
concurrent activation of JNK/p38 kinase pathways and inhibition of the
ERK pathway induces apoptosis, whereas direct and selective activation of the ERK pathway prevents apoptosis (10). In L929 cells, fibroblast growth factor-2 suppresses TNF--induced apoptosis by causing an
activation of ERK, and this effect could be reversed by inhibiting the
ERK pathway (37). The present study shows that in mesangial cells, ERK
was activated in a manner similar to the activation of JNK by TNF-
,
although the magnitude of its activation was lower than that of JNK
(3-fold versus 8-fold). However, when ERK activation by
TNF-
was selectively inhibited by pretreatment of mesangial cells
with PD098059, cell viability was not affected. This result argues
against the possibility that activation of ERK by TNF-
contributed
to the resistance of mesangial cells to TNF-
cytotoxicity.
Therefore, the anti-apoptotic effect attributable to activation of the
ERK pathway seems to be dependent on cell type and stimulus.
An obligatory role of the JNK pathway in apoptosis has been best
documented in various stress-induced apoptosis models (11, 13, 17, 46).
The duration of JNK activation is thought to be a critical factor
determining cell proliferation or apoptosis with transient activation
leading to cell proliferation or differentiation and prolonged JNK
activation causing apoptosis (11). In mesangial cells, the fact that
activation of JNK by TNF- lasted less than 15 min implies that it
may not be activated long enough to initiate the apoptotic process.
Interestingly, when the cells were treated with actinomycin D or
cycloheximide after stimulation with TNF-
, a second strong
activation of JNK was observed, which coincided with the onset of
apoptosis. The above result indicates that the JNK pathway may play
a role in TNF-
-mediated apoptosis only if the activating stimulus is
sufficiently prolonged. This observation resembles a similar pattern
observed between JNK activation and cell death in Fas-induced apoptosis
in a neuroblastoma cell line. Goillot et al.(47) found that
JNK activation induced by Fas was biphasic. The transient first
activity peak was detected at 15 min, then the activity decreased to
basal level followed by a second peak starting 2 h after
stimulation. The second activation peak of JNK was critical for the
initiation of the apoptotic process. In our experiments with mesangial
cells, no second activity peak of JNK was detected within 3 h of
treatment with TNF-
alone. The simplest and most likely
interpretation of these results is that under normal conditions the
extent of JNK activation is constrained in some manner, possibly by
activation of a JNK phosphatase. It is hypothesized that actinomycin D
or cycloheximide blocks the expression of a phosphatase that is
responsible for limiting the activation of JNK. According to this
conjecture, the second activity peak of JNK would only be detectable
when the induction of the phosphatase was blocked. This speculation is
further supported by results obtained from pretreatment of the cells
with vanadate. Vanadate inhibits both protein tyrosine phosphatases and
members of the MKP family, and its addition to mesangial cells resulted in a sustained activation of JNK by TNF-
. Thus in each situation so
far investigated, when JNK activation was sustained, the cells underwent apoptosis. These results suggest that a meaningful
correlation exists between the duration of JNK activation and cell
death.
The best model described to date for TNF receptor family-induced
apoptosis is that once the ligand binds to the receptor
(e.g. TNF- to the TNF receptor or Fas ligand to Fas), the
cytosolic part of the receptor recruits the "death domain-containing
proteins" TRADD (TNFR-associated
death domain protein) or FADD
(Fas-associated death
domain protein), which in turn activates a cascade of ICE proteases which executes the apoptotic process (2, 5). However, this
model does not include an involvement of JNK in the apoptotic pathway
activated by TNF-
or the Fas ligand. Schievella et al. (48) found that ERK and JNK activation induced by TNF-
could operate
through a pathway other than TRAF2. They identified a novel death
domain protein, MADD, which interacts with the TNF-
receptor and
activates both ERK and JNK. This finding provides evidence that the JNK
pathway is potentially involved in apoptosis. Similarly, a
Fas-binding protein, Daxx, has been characterized. It interacts with
the Fas death domain and activates JNK as well as the apoptotic process
(49). These findings provide the missing link between JNK activation
and apoptosis in the TNF-
receptor and Fas signaling pathways. It is
apparent that more than one pathway initiated by the TNF-
receptor
family can lead to cell death. It has been proposed, for instance, that
JNK activation could lead to another ICE protease cascade in addition
to the earlier characterized FADD-FLICE-ICE cascade (49).
Our findings suggest a novel explanation that may account for the fact
that mesangial cells protect themselves from TNF- cytotoxicity under
normal conditions. This proposed mechanism entails the induction of
phosphatase by TNF-
, which contributes toward protecting the cells
from TNF-
-induced apoptosis by suppressing a sustained JNK
activation in a way similar to the way MKPs cause inactivation of JNK
stimulated by stresses. It has been shown that some MKPs are inducible
by various stresses that activate JNK (41-43). If this mechanism is
involved, the duration of JNK activation would be regulated by MKP
through a feedback mechanism. Therefore, induction of MKP to inactivate
JNK serves as a protective mechanism against stress damage caused by
various agents (41, 42, 50). The TNF-
-inducible MKP may play a
similar role in protecting the cell from TNF-
cytotoxicity. This
hypothesis is strongly supported by the fact that the expression of
MKP-1 was induced by TNF-
in mesangial cells at the same time as the
inactivation of JNK. Therefore, we conclude that MKP-1 induced by
TNF-
may at least partially be responsible for protecting mesangial
cells from apoptosis by suppressing a prolonged activation of JNK.
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ACKNOWLEDGEMENTS |
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We thank Dr. J. K. Westwick for providing GST-c-Jun plasmid, Drs. N. Tonks and H. Sun for providing pCEP4-MKP-1 plasmid, and Dr. M. Peng for assistance in Northern blot analysis.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants DK-15120 and DK-48493 and by an American Diabetes Association career development award (to L-J. Y.).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.
Recipient of National Institutes of Health Training Grant
DK-07314.
§ Present address: Dept. of Pathology, Albert Einstein College of Medicine, New York, NY 10461.
¶ To whom correspondence should be addressed: Dept. of Biochemistry and Biophysics, University of Pennsylvania, 601 Goddard Laboratories, 37th and Hamilton Walk, Philadelphia, PA 19104. Tel.: 215-898-8785; Fax: 215-898-9918; E-mail: johnrwil{at}mail.med.upenn.edu.
1
The abbreviations used are: TNF-, tumor
necrosis factor-
; ICE, interleukin 1-converting enzyme; NF-
B,
nuclear factor-
B; MAP kinase, mitogen-activated protein kinase; ERK,
extracellular signal-regulated protein kinase; JNK, c-Jun N-terminal
protein kinase; I-
B, inhibitor of nuclear factor-
B; MBP, myelin
basic protein; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling; GST, glutathione
S-transferase; PAGE, polyacrylamide gel electrophoresis;
MKP, mitogen-activated protein kinase phosphatase.
2 Y.-L. Guo, K. Baysal, and J. R. Williamson, unpublished results.
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REFERENCES |
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