Resistance to TNF-
cytotoxicity can be achieved through
different signaling pathways in rat mesangial cells
Yan-Lin
Guo,
Baobin
Kang, and
John R.
Williamson
Department of Biochemistry and Biophysics, School of Medicine,
University of Pennsylvania, Philadelphia, Pennsylvania 19104
 |
ABSTRACT |
We reported previously that Ro-318220 blocked expression of
mitogen-activated protein kinase phosphatase-1 (MKP-1) induced by tumor
necrosis factor-
(TNF-
) and subsequently caused apopotosis in
mesangial cells (Y.-L. Guo, B. Kang, and J. R. Williamson. J. Biol. Chem. 273: 10362-10366,
1998). These data support our hypothesis that a TNF-
-inducible
phosphatase may be responsible for preventing sustained activation of
c-Jun NH2-terminal protein kinase
(JNK) and consequent cell death in these cells (Y.-L. Guo, K. Baysal,
B. Kang, L.-J. Yang, and J. R. Williamson. J. Biol. Chem. 273: 4027-4034, 1998). In this study, we
investigated the involvement of protein kinase C (PKC) in regulation of
MKP-1 expression in mesangial cells together with effects on viability.
Although originally characterized as a PKC inhibitor, Ro-318220
inhibited TNF-
-induced MKP-1 expression through a mechanism other
than blocking the PKC pathway. Furthermore, inhibition of the PKC
pathway neither significantly affected TNF-
-induced MKP-1 expression nor made cells susceptible to toxic effect of TNF-
. Thus PKC activation is not essential for cells to achieve the resistance to
TNF-
cytotoxicity displayed by normal mesangial cells. However, activation of PKC by phorbol 12-myristate 13-acetate (PMA) dramatically increased cellular resistance to the apoptotic effect of TNF-
. Coincidentally, PMA stimulated MKP-1 expression and suppressed JNK
activation. Therefore, PMA-induced MKP-1 expression may contribute to
the protective effect of PMA. These results provide a mechanistic explanation for previous documentation that PKC activation can rescue
some cells from apopotosis.
apoptosis; mitogen-activated protein kinase; mitogen-activated
protein kinase phosphatase; c-Jun
NH2-terminal protein kinase; tumor
necrosis factor-
 |
INTRODUCTION |
TUMOR NECROSIS FACTOR-
(TNF-
) is a polypeptide
cytokine that can elicit a wide range of biological responses depending
on the cell type and the state of differentiation (3, 39). One of these
responses is the induction of apoptosis or programmed cell death in
some cells (27). Although certain tumor cells, virus-infected cells, or
damaged cells are sensitive to TNF-
-induced apopotosis, many normal
cells are usually resistant (24, 35). Therefore, TNF-
plays an
important role in determining cell viability under physiological and
pathological conditions.
The current hypothesis to explain how TNF-
can selectively kill some
cells while not being harmful to others implies the operation of two
opposite signaling pathways: a preexisting destructive (apoptotic)
pathway and an inducible protective (survival) pathway. Interactions
between the two pathways and particularly the balance of their
activities will determine whether the cells survive or die (24).
Recently, significant progress has been made in clarifying the
mechanism of apoptosis induced by TNF-
. However, the identities of
the protective factors induced by TNF-
and their mechanisms of
action remain elusive. TNF-
activation of extracellular
signal-regulated protein kinase (ERK) and nuclear factor-
B (NF-
B)
has been proposed to counteract the cytotoxicity of the apoptotic
pathway in certain cells (1, 37, 42) but not in others (10, 20). Some of the putative protective factors so far identified include a manganous superoxide dismutase (41), a zinc finger protein (29), and
members of the Bcl-2 family of proteins (11, 19). Given the complexity
of TNF-
signaling pathways, it is apparent that different protective
factors may exert their antiapoptotic effects through different
mechanisms and may act at different stages of the apoptotic process in
a cell type- and stimulus-dependent manner.
A sustained activation of c-Jun
NH2-terminal protein kinase (JNK)
has been shown to be an apoptotic signal in many cells (6, 38). In our
previous study (12), we found that rat mesangial cells are normally
resistant to TNF-
-induced apoptosis. However, they can be made
susceptible to the apoptotic effect of TNF-
when pretreated with
cycloheximide or vanadate. We proposed that a TNF-
-induced
mitogen-activated protein kinase phosphatase-1 (MKP-1) was responsible
for suppressing a prolonged activation of JNK in these cells, thereby
protecting them from apopotosis. This hypothesis is supported by our
recent finding that blockade of the TNF-
-induced expression of MKP-1
by Ro-318220 caused a sustained activation of JNK and resulted in
apopotosis (13). A similar conclusion was reached independently of our
work by Franklin et al. (9), who demonstrated that conditional
expression of MKP-1 protected ultraviolet-induced apoptosis by
inhibiting JNK activity in U937 cells. Because Ro-318220 was originally
characterized as a protein kinase C (PKC) inhibitor (40), it was of
interest to investigate whether it exerted its effect through
inhibition of the PKC pathway. PKC activation has been implicated in
the regulation of TNF-
-initiated cellular processes (17, 23, 31,
32). In several studies, it was found that activation of PKC could
protect some cells from apoptosis, but the mechanisms involved are
unknown (17, 28). In this study, we extend our investigation of the
mechanisms by which Ro-318220 inhibits TNF-
-induced MKP-1 and
provide some insight into how the PKC pathway may be involved in
protecting cells from apoptosis.
 |
MATERIALS AND METHODS |
Materials.
Recombinant TNF-
was obtained from Chemicon International (Temecula,
CA). Anti-c-Fos antibodies, anti-PKC-
, -
, and -
antibodies, and anti-MKP-1 antibodies were purchased from Santa Cruz
Biotechnology (Santa Cruz, CA). Anti-c-Jun antibodies were from New
England Biolabs (Beverly, MA). Anti-PKC-
, -
, -
, -
, and -
antibodies were from Transduction Laboratories (Lexington, KY).
Ro-318220 (bisindolylmaleimide IX) and GF-109203X (bisindolylmaleimide
I) were from LC Laboratories (San Diego, CA). Phorbol 12-myristate 13-acetate (PMA) was purchased from Sigma (St. Louis, MO).
Cell culture.
Rat mesangial cells were isolated from male Sprague-Dawley rats under
sterile conditions using the sieving technique described previously
(21). The cells were maintained in RPMI 1640 medium containing 20% FCS
and 0.6 U/ml insulin at 37°C in a humidified incubator (5%
CO2 and 95% air). Cells from
passages
5-20
were used. After the cells were grown to 80-90% confluence, they
were incubated for 16-18 h in insulin-free RPMI 1640 medium
containing 2% FCS before the experiments.
Cell viability assay.
For cell viability assays, mesangial cells were grown in 12-well
plates. They were treated with reagents for the times indicated. Uptake
of neutral red dye was used as a measurement of cell viability (12). At
the end of the incubations, the medium was removed and the cells were
incubated in DMEM containing 2% FCS and 0.001% neutral red for 90 min
at 37°C. The uptake of the dye by viable cells was terminated by
removing the medium, washing the cells briefly with 1 ml of 4%
paraformaldehyde in PBS (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. The cell morphology was also examined under
a light microscope periodically during the cell treatment.
Immunocytochemical detection of apoptosis and c-Jun
phosphorylation.
For apoptosis analysis, cells grown on 25-mm glass coverslips in
six-well plates were fixed with 4% paraformaldehyde in PBS after
treatment with various reagents as indicated. DNA strand breaks were
identified using a TUNEL assay kit (Boehringer Mannheim). Briefly, the
fixed cells were treated with terminal deoxyribonucleotidyl transferase, which incorporates fluorescein-tagged nucleotides onto
3'-OH termini of fragmented DNA. After incubation with terminal deoxyribonucleotidyl transferase, the same coverslips were washed briefly with PBS and used for the detection of phospho-c-Jun. Positively stained nuclei were detected with Texas red-conjugated secondary antibodies as described previously (13). Fluorescein-tagged apoptotic nuclei and Texas red-labeled nuclei were visualized using
fluorescence microscopy.
Immunocytochemical detection of PKC translocation.
Cells grown on 25-mm glass coverslips in six-well plates were fixed
with 4% paraformaldehyde in PBS after treatment with PMA or TNF-
as
indicated. PKC-
was immunolocalized using its specific antibodies
and visualized with Texas red-conjugated secondary antibodies using
fluorescence microscopy following the immunocytochemistry protocol as
described (13).
Preparation of whole cell lysate.
The cells were treated with reagents for the times indicated, washed
twice with ice-cold PBS, 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 (PMSF). The cells were incubated for 10 min on ice and then lysed by sonication (25 pulses, output control 3)
using a Branson sonicator and centrifuged at 15,000 g for 15 min. The supernatant was
designated whole cell lysate. Protein concentration was determined by
the method of Bradford (5) using BSA as standard.
Preparation of cytosol and particulate fractions.
The cells were treated with 1 µM PMA or 10 ng/ml TNF-
for the
times indicated. The cells were washed twice with ice-cold PBS and
scraped into a buffer containing 25 mM Tris · HCl (pH 7.5), 2 mM EDTA, 1 mM dithiothreitol (DTT), 10% glycerol, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 1 mM PMSF. The cells were
incubated for 10 min on ice and then lysed by sonication as described
above. The cell lysate was centrifuged for 5 min at 500 g. The pellet containing the cell
debris was discarded. The supernatant was centrifuged at 100,000 g for 30 min at 4°C. The
supernatant was designated the cytosolic fraction. The pellet was
extracted with a buffer containing 25 mM Tris · HCl
(pH 7.5), 2 mM EDTA, 1 mM DTT, 10% glycerol, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM PMSF, and 1% Triton X-100 for 30 min on ice.
The extract was centrifuged at 100,000 g for 30 min at 4°C. The
supernatant, which contained solubilized membranes, was designated the
particulate fraction.
Western blot analysis.
The protein samples were subjected to SDS-PAGE and transferred onto
nitrocellulose membranes. The membranes were blocked with 5% nonfat
dry milk in Tris-buffered saline containing 0.05% Tween 20 and
incubated with primary antibodies followed by horseradish peroxidase-conjugated secondary antibodies according to the
manufacturer's instructions with some modifications. The immunoblots
were visualized by an enhanced chemiluminescence kit obtained from
Amersham Pharmacia Biotech.
 |
RESULTS |
Role of PKC in TNF-
- and PMA-induced MKP-1
expression.
PKC is a protein kinase family that is composed of >10 isoforms. They
are classified into three subgroups: conventional PKC isoforms (cPKC)
that are activated by Ca2+,
phosphatidylserine, and diacylglycerol or PMA; novel PKC isoforms (nPKC) that are activated by diacylglycerol or PMA but not by Ca2+, and atypical PKC isoforms
(aPKC) that are insensitive to
Ca2+ and PMA but are activated by
phosphatidylserine (26). Two approaches were used to investigate
whether Ro-318220 inhibited TNF-
-induced MKP-1 expression by
inhibiting the PKC pathway and to assess the potential involvement of
PKC in the expression of MKP-1. First, the cells were treated with PMA
for 18 h. This long-term incubation will cause a sustained activation
of PMA-sensitive PKC isoforms (cPKC and aPKC) and will eventually
result in their degradation, a phenomenon known as PKC downregulation
(26). In the second approach, before stimulation with TNF-
, the
cells were pretreated with GF-109203X, a close structural analog of
Ro-318220, which has been shown to be a potent but less specific
inhibitor of different PKC isoforms. This treatment acutely inhibits
total PKC activity, including aPKC activity (2, 8, 25). As shown in
Fig.
1A, whereas Ro-318220 totally blocked TNF-
-induced expression of MKP-1,
inhibition by GF-109203X or downregulation of PKC by PMA only showed a
slight inhibitory effect. In addition to MKP-1, TNF-
also induced
the expression of c-Fos, which was totally blocked by either Ro-318220
or GF-109203X (Fig. 1B).

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Fig. 1.
Effects of Ro-318220 (Ro), GF-109203X (GF), and protein kinase C (PKC)
downregulation on tumor necrosis factor- (TNF- )-induced
expression of mitogen-activated protein kinase phosphatase-1 (MKP-1)
and c-Fos. A: cells were stimulated
with 10 ng/ml TNF- for 30 min or were pretreated with 5 µM
Ro-318220 or 5 µM GF-109203X for 30 min and then stimulated with 10 ng/ml TNF- for 30 min. For PKC downregulation, cells were incubated
with 1 µM phorbol 12-myristate 13-acetate (PMA) for 18 h, medium was
changed to fresh starvation medium (RPMI 1640 containing 2% FCS), and
then cells were stimulated with TNF- for 30 min.
B: cells were treated with TNF- for
30 or 60 min or with combinations of TNF- and Ro-318220 or
GF-109203X as described for A. Whole
cell lysate was subjected to SDS-PAGE and transferred to nitrocellulose
membranes. MKP-1 and c-Fos were identified by Western blot analysis
using specific antibodies.
|
|
PMA is one of the best-studied PKC activators and has been shown to
induce the expression of MKP-1 in some cell types (4, 22). This is also
the case in mesangial cells, as shown in Fig. 2. However, in contrast to the expression
of MKP-1 induced by TNF-
, PMA-induced expression of MKP-1 was
totally inhibited by both Ro-318220 and GF-109203X (Fig.
2A). The effect of PMA on the
expression of c-Fos was also examined (Fig.
2B). It strongly stimulated c-Fos
expression, which was also blocked by both Ro-318220 and GF-109203X.
These results indicate that Ro-318220 and GF-109203X were equally
effective in inhibiting the effect of PMA, which was most likely
mediated by the PKC pathway.

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Fig. 2.
Expression of MKP-1 and c-Fos induced by PMA and effects of Ro-318220
and GF-109203X. Cells were stimulated with 1 µM PMA or pretreated
with 5 µM Ro-318220 or GF-109203X for 30 min, followed by 1 µM PMA,
for times indicated. Whole cell lysate was subjected to SDS-PAGE and
transferred to nitrocellulose membranes. MKP-1
(A) and c-Fos
(B) were identified by Western blot
analysis using specific antibodies.
|
|
The effectiveness of PKC downregulation by PMA was confirmed by Western
blot analysis. Only PKC-
(conventional), -
and -
(novel), and
-
(atypical) were detected in the whole cell lysate using PKC
isoform-specific antibodies (Fig. 3),
consistent with the results reported by other investigators in
mesangial cells (16). After incubation for 18 h with PMA, the content
of PMA-insensitive PKC-
in the whole cell lysate was only slightly
affected, as expected (Fig. 3). It should be pointed out that the
anti-PKC-
antibody cross-reacted with three bands in the region
between the 66- and 116-kDa molecular markers (Fig. 3). A nearly
identical pattern was seen in mesangial cells using the same antibody
by Rzymkiewicz et al. (30). The band in the middle was identified as
PKC-
based on the fact that its expression was blocked by an
antisense oligonucleotide to PKC-
. The lower band was not affected
by the same treatment; therefore, it may represent a nonspecific
binding protein (30). The higher band was identified as a PMA-sensitive
PKC isoform that cross-reacted with anti-PKC-
antibodies as noted in
a number of publications (7, 34). Similarly, in mesangial cells, this
band was sensitive to PMA downregulation (Fig. 3) and translocated to
the membrane fraction upon PMA stimulation (see Fig. 5). As shown in
Fig. 3, PKC-
and -
were essentially below the detection level,
and the amounts of PKC-
were greatly diminished after long-term
incubation of the cells with PMA (Fig. 3). Therefore, PMA
downregulation of PKC isoforms would significantly but not completely
diminish PKC activity, because of the insensitivity of PKC-
to PMA
downregulation. However, the inhibition of total PKC activity by
GF-109203X was expected to be more effective. Many studies suggested
that GF-109203X is a stronger but less selective PKC inhibitor than
Ro-318220 as discussed by Beltman et al. (2). The selectivity of
Ro-318220 and GF-109203X was tested in an
T3-a gonadotroph-derived
cell line that expressed PKC-
, -
, and -
. In vivo, Ro-318220
was shown to selectively inhibit PKC-
and -
, whereas GF-109203X was an equally effective inhibitor of PKC-
, -
, and -
, with IC50 values in the nanomolar range
(18). In an in vitro study, GF-109203X inhibited all PKC isoforms
tested with an order of potency of
>
1 >
>
>
(25).
Thus, under our experimental conditions, the total activity of PKC,
including PKC-
, should be significantly inhibited by GF-109203X.

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Fig. 3.
Downregulation of PKC by PMA. Cells were treated with (+) or without
( ) PMA for 18 h. PKC isoforms in whole cell lysate were
identified by Western blot analysis using specific antibodies.
|
|
Effects of Ro-318220, GF-109203X, and PKC downregulation on the
viability of cells treated with TNF-
.
To investigate the possibility of a correlation between expression
level of MKP-1 and the protection effect against apoptosis induced by
TNF-
, the cell viability was tested under experimental conditions
described in Fig. 1. When TNF-
-induced MKP-1 expression was blocked
by Ro-318220 (Fig. 1A), the effect
of TNF-
on cell viability was dramatic (Fig.
4A).
However, the same concentration of GF-109203X showed a much weaker
effect on TNF-
-induced cell death (Fig.
4A), which correlated with it being
ineffective in blocking the expression of MKP-1 (Fig.
1A). Similarly, downregulation of
PKC with PMA (18 h) showed no significant effects on cell viability when the cells were stimulated with TNF-
(Fig.
4B). The overall pattern of cell
viability to TNF-
and Ro-318220 treatment (Fig. 4B) was very similar to that
observed when the cells were not pretreated with PMA (Fig.
2A). Thus the cellular resistance to TNF-
toxicity displayed by normal mesangial cells seems to correlate with the level of expression of MKP-1 but not with activity of PKC.

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Fig. 4.
Effects of Ro-318220, GF-109203X, and PKC downregulation on viability
of cells stimulated with TNF- . A:
cells were incubated with 10 ng/ml TNF- for 3 h (T) or 10 µM
Ro-318220 (R) or 10 µM GF-109203X (G) for 3.5 h. Other cells were
pretreated with 10 µM Ro-318220 or GF-109203X for 30 min and then
stimulated with 10 ng/ml TNF- for 3 h (RT, GT respectively).
B: for PKC downregulation, cells were
incubated with 1 µM PMA for 18 h, medium was changed to fresh
starvation medium, and cells were stimulated with TNF- for 3 h (PT),
with Ro-318220 for 3.5 h (PR), or with Ro-318220 for 30 min followed by
TNF- for 3 h (PRT). Cells treated with PMA only were used as control
(PC). Cell viability was determined by neutral red assay method.
Results are means ± SE of 3 experiments performed in triplicate.
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|
Activation of PKC by TNF-
and PMA.
It has been reported that TNF-
was able to activate PKC in some
cells but not in others (32). Our results thus far favor the notion
that PKC activation may not be required for the protective effect
against apoptosis elicited by TNF-
. We next examined whether TNF-
could activate PKC in mesangial cells. It is known that, upon
activation by various stimuli, PKC translocates from the cytosol to the
cell membrane. Redistribution of PKC, therefore, has been regarded as
an indication of its activation (15). As shown in Fig.
5, PMA caused a significant increase of
PKC-
, -
, and -
in the membrane fractions (Fig.
5A), with a concurrent decrease in
the cytosolic fraction (Fig. 5B), whereas the content of
PKC-
was not changed in either the cytosolic or membrane fractions. On the other hand, TNF-
caused only a slight increase of PKC-
in
the membrane fraction (Fig. 5A).
Therefore, PKC activation stimulated by TNF-
was negligible compared
with that stimulated by PMA in mesangial cells. The redistribution of
PKC was further confirmed by immunolocalization using anti-PKC-
antibodies as an example. PKC-
is mainly in cytoplasm in the resting
cells. PMA caused a clear redistribution of PKC-
within the cells,
whereas TNF-
had no apparent effect (data not shown). This is
consistent with results obtained from Western blot analysis (Fig. 5).

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Fig. 5.
Translocation of PKC isoforms induced by PMA and TNF- . Cells were
treated with 10 ng/ml TNF- or 1 µM PMA for 15 min. CON, control.
Cell lysates were fractionated to particulate
(A) and cytosolic
(B) fractions and subjected to
SDS-PAGE. Separated proteins were transferred to nitrocellulose
membranes. PKC isoforms were identified by Western blot analysis using
specific antibodies.
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|
Pretreatment of mesangial cells with PMA increases resistance to
cell death caused by TNF-
and cycloheximide.
It was reported that activation of PKC could protect some cells from
apoptosis induced by stimuli such as radiation (14) and ceramide (17,
28). Therefore, we examined whether activation of PKC could rescue
mesangial cells from TNF-
-induced apoptosis. As shown in Fig.
6, incubation of mesangial cells with
TNF-
in the presence of cycloheximide caused a significant loss of
cell viability (Fig. 6). Interestingly, this effect could be largely reversed by pretreatment of the cells with PMA for 2 h before addition
of TNF-
and cycloheximide (Fig. 6). The PMA-induced cellular
resistance to cell death was PKC dependent. This was shown by the fact
that the PMA-induced protective effect was abolished when PMA was added
together with either GF-109203X or Ro-318220 (Fig. 6). The morphology
of the cells was examined under a microscope. At the end of 3 h of
incubation with TNF-
and cycloheximide, ~70% of the cells were
undergoing apoptosis, as judged by their characteristic morphology
(Fig.
7B) and
by TUNEL analysis (Fig. 8B).
Pretreatment of the cells with PMA for 2 h dramatically decreased the
incidence of cell death caused by subsequent treatment with TNF-
and
cycloheximide (Figs. 7C and
8C). When GF-109203X was added
together with PMA, the protective effect elicited by PMA was totally
abolished (Fig. 7D), indicating that
the antiapoptotic effect elicited by PMA was PKC dependent. Therefore,
it seems that, although PKC activation contributes little to the
TNF-
-mediated cellular resistance to TNF-
toxicity, PKC
activation by PMA can dramatically potentiate the ability of the cells
to counteract the apoptotic effect of TNF-
.

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Fig. 6.
Pretreatment of cells with PMA increased viability of mesangial cells.
C, control; T, TNF- for 3 h; Ch, cycloheximide for 3.5 h; ChT,
cycloheximide for 30 min followed by TNF- for 3 h; PChT, PMA for 2 h
and then, after change to fresh starvation medium, cycloheximide for 30 min followed by TNF- for 3 h; PGChT and PRChT, treatment same as for
PChT, except GF-109203X (PGChT) or Ro-318220 (PRChT) was added with
PMA. Concentrations of reagents were 10 ng/ml TNF- , 5 µg/ml
cycloheximide, 1 µM PMA, 10 µM Ro-318220, and 10 µM GF-109203X.
Cell viability was determined by neutral red assay method. Results are
mean ± SE of 3 experiments performed in triplicate.
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Fig. 7.
Pretreatment of cells with PMA increased resistance to cell death
caused by subsequent treatment with TNF- and cycloheximide.
A: control.
B: cells pretreated with 5 µg/ml
cycloheximide for 30 min followed by 10 ng/ml TNF- for 3 h.
C: cells treated with 1 µM PMA for 2 h and, after change to fresh starvation medium, cycloheximide for 30 min followed by TNF- for 3 h. D:
cells treated as for C, except 10 µM
GF-109203X was added with PMA. Cell morphology was examined under a
phase-contrast light microscope.
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Fig. 8.
Pretreatment with PMA protected cell from apoptosis and attenuated
c-Jun phosphorylation caused by subsequent treatment with TNF- and
cycloheximide. A and
a: control.
B and
b: cells were pretreated with 5 µg/ml cycloheximide for 30 min followed by 10 ng/ml TNF- for 3 h.
C and
c: cells were treated with 1 µM PMA
for 2 h, medium was changed to fresh starvation medium, and cells were
treated with cycloheximide for 30 min followed by TNF- for 3 h.
Apoptosis and c-Jun phosphorylation were examined in same cells.
Apoptotic cells were identified by TUNEL analysis
(A,
B,
C). Phospho-c-Jun was detected by
anti-phospho-c-Jun antibodies and visualized with Texas red-conjugated
secondary antibodies (a,
b,
c) by fluorescence microscopy.
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|
Pretreatment of mesangial cells with PMA attenuates JNK activation
induced by TNF-
and cycloheximide.
Our results indicate that cellular resistance to TNF-
toxicity seems
to correlate with the expression level of MKP-1. If PMA-induced MKP-1
could dephosphorylate and inactivate JNK activation, one would expect
that pretreatment of the cell with PMA should be able to suppress JNK
activation induced by TNF-
and cycloheximide. As illustrated in Fig.
8, the cells undergoing apoptosis induced by TNF-
and cycloheximide
(Fig. 8B) also show heavy
phosphorylation of c-Jun, indicating strong activation of JNK in these
cells (Fig. 8b). Pretreatment of the
cells with PMA protected the cells from apoptosis (Fig.
8C) and also greatly attenuated
c-Jun phosphorylation (Fig. 8c).
These results provide a strong correlation between apopotosis and
activities of JNK and MKP-1 and support the notion that PMA-induced
MKP-1 may contribute to the protective effect of PMA by attenuation of
a sustained JNK activation.
 |
DISCUSSION |
Ro-318220 and GF-109203X are two structurally related analogs of
bisindolylmaleimide that have been widely used as ATP-competitive inhibitors of PKC (25, 36, 40). In a recent study, however, it was
found that, unlike GF-109203X, Ro-318220 has some distinct cellular
effects that are independent of its ability to inhibit PKC (2). In
accordance with this finding, the present study shows that Ro-318220
inhibited TNF-
-induced MKP-1 in rat mesangial cells through a
mechanism other than inhibition of PKC. This was demonstrated by two
independent approaches, i.e., by acute inhibition of PKC with
GF-109203X and by downregulation of PKC with PMA (Fig. 1). More
importantly, our results also indicate that the induction of MKP-1 by
TNF-
and PMA acted through different mechanisms, i.e., PKC
activation was essential for PMA-induced MKP-1 expression, but it
contributed only marginally to TNF-
-induced MKP-1 expression.
The involvement of PKC in the regulation of TNF-
-mediated processes
has been documented in a number of cells (3, 17, 28, 31, 33). However,
its role in the regulation of TNF-
toxicity remains largely unknown.
Activation of PKC by TNF-
seems to be cell type dependent. For
instance, among human leukemic cell lines, TNF-
activated PKC in
Jurkat cells, U937 cells, and K562 cells but not in CCD18 cells (32).
In mesangial cells, TNF-
only induced a slight translocation of
PKC-
. Although it is unclear to exactly what level the activity of
PKC was changed upon stimulation with TNF-
, this uncertainty does
not detract from the major conclusions to be drawn from our data; i.e.,
activation of PKC is not essential for TNF-
-induced expression of
MKP-1 and for the cellular resistance to apoptosis exhibited by
mesangial cells under normal conditions.
The resistance to apoptosis induced by TNF-
and cycloheximide seems
to correlate with the expression level of MKP-1 induced by both TNF-
and PMA. Inhibition of PKC neither significantly affected
TNF-
-induced expression of MKP-1 nor made the cells susceptible to
the toxic effect of TNF-
. However, the cells underwent apoptosis
when MKP-1 expression was blocked under our experimental conditions
(Figs. 1 and 4) (12, 13). Another interesting observation is that, in
addition to MKP-1, TNF-
also induced c-Fos expression, both effects
being blocked by Ro-318220 (Fig. 1). Thus an important question to ask
is whether TNF-
-induced c-Fos also plays a role in protecting the
cells from apoptosis. This question is addressed by the observations
that GF-109203X selectively inhibited TNF-
-induced c-Fos but not
MKP-1 expression (Fig. 1) and that the cells did not become susceptible
to TNF-
-induced apoptosis under these conditions (Fig. 4). It is
likely that expression of MKP-1 but not c-Fos is responsible for the
antiapoptotic effect. These results further support our hypothesis that
MKP-1 may act as a protective factor against TNF-
-induced apoptosis
by suppressing JNK activation (12, 13). Recently, a similar conclusion
was reached independently of our work by Franklin et al. (9), who
demonstrated conditional expression of MKP-1-protected ultraviolet
light-induced apoptosis by inhibiting JNK activity in U937 cells. On
the basis of this assumption, one would expect that other agents that
are able to induce MKP-1 expression should show a similar protective
effect. This speculation is supported by the results from PMA
experiments. PMA induced MKP-1 expression within the time period from
30 to 120 min (Fig. 2) and also elicited resistance to apoptosis caused by TNF-
and cycloheximide (120 min; Figs. 6 and 7), with a
concurrent attenuation of JNK activity (Fig. 8). Unlike TNF-
-induced
MKP-1, the effects of PMA were dependent on PKC activation. Thus the involvement of the PKC pathway in the mediation of resistance to
apoptosis seems to be stimulus dependent in rat mesangial cells.
In several studies, it was reported that activation of PKC could
protect the cells from apoptosis. For example, in leukemia cells, the
PKC activator dioctanylglycerol and PMA blocked TNF-
- and
ceramide-induced apoptosis (17, 28). Lotem et al. (23) reported that
activation of PKC could rescue myeloid cells from apoptosis induced by
withdrawal of growth factors. It is known that PMA activates several
signaling pathways in addition to PKC. These include ERK and NF-
B,
both of which have been implicated in protecting cells from apoptosis
(37, 42). It is likely that multiple signaling pathways are involved in
mediating the antiapoptotic effect elicited by PMA. The present study
provides a possible mechanistic explanation for how activation of PKC
elicits resistance to apoptosis. Specifically, induction of MKP-1 by
PMA may play a role in protecting cells from apoptosis by attenuating JNK activation. The expression of MKP-1 induced by TNF-
(12, 13) and
PMA (present study) or by molecular biology techniques (9) showed
similar effects against apoptosis. It is likely, therefore, that MKP-1
may act as a common protective molecule in the cells in which the JNK
pathway is involved in the mediation of apoptosis.
 |
ACKNOWLEDGEMENTS |
This work was supported by National Institute of Diabetes and
Digestive and Kidney Diseases (NIDDK) Grants DK-15120 and DK-48493. Y.-L. Guo is a recipient of NIDDK Training Grant DK-07314.
 |
FOOTNOTES |
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. §1734 solely to indicate this fact.
Address for reprint requests: J. R. Williamson, Dept. of Biochemistry
and Biophysics, University of Pennsylvania, 601 Goddard Labs, 37th and
Hamilton Walk, Philadelphia, PA 19104.
Received 11 June 1998; accepted in final form 2 November 1998.
 |
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