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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Previously we showed that rat mesangial cells are
normally resistant to tumor necrosis factor- (TNF-
)-induced
apoptosis. They are made susceptible to the apoptotic effect of TNF-
when pretreated with actinomycin D, cycloheximide or vanadate. A
sustained c-Jun N-terminal protein kinase (JNK) activation was closely
correlated with the initiation of apoptosis under these conditions. We
proposed that a TNF-
-inducible phosphatase was responsible for
preventing a sustained activation of JNK and consequent apoptosis in
these cells (Guo, Y.-L., Baysal, K., Kang, B., Yang, L.-J., and
Williamson, J. R. (1998) J. Biol. Chem. 273, 4027-4034). In the present study we provide further evidence to
support this hypothesis. Ro318220, although originally identified as a
specific inhibitor of protein kinase C, was subsequently found to be a
strong inhibitor of MKP-1 expression. In rat mesangial cells,
pretreatment of the cells with Ro318220 blocked expression of MKP-1
induced by TNF-
. This treatment also prolonged JNK activation and
caused apoptosis. Taken together, our results support the currently
controversial hypothesis that the JNK pathway is involved in
TNF-
-induced apoptosis. In addition, we provide a mechanistic
explanation for how mesangial cells in primary culture achieve
resistance to TNF-
cytotoxicity. Specifically, induction of MKP-1 by
TNF-
appears to be responsible for protection of the cells from
apoptosis by preventing a prolonged activation of JNK.
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INTRODUCTION |
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Tumor necrosis factor-
(TNF-
)1 is a polypeptide
cytokine that can elicit a wide range of biological responses depending
on the cell type and their state of differentiation (1, 2). One of
these responses is the induction of apoptosis or programmed cell death
in some cell types (3). Although certain tumor cells infected with
virus or damaged cells are sensitive to TNF-
-induced apoptosis, many
normal cells are usually resistant (3-5). Thus apoptosis has been
considered to be an important mechanism for the elimination of abnormal
cells and for cellular organization during tissue development.
Most resistant cells can be rendered susceptible to TNF--induced
apoptosis by agents that block the synthesis of mRNA or protein.
Thus, it is proposed that normal cells can achieve resistance to
TNF-
cytotoxicity by eliciting the synthesis of a protective factor
(6, 7). However, the identities of such protective factors and the
mechanisms by which they exert their anti-apoptotic effects are poorly
understood. Recent advances in this area have led to some hypotheses.
It is proposed that TNF-
activates an anti-apoptotic signaling
pathway, such as the extracellular signal-regulated protein kinase
(ERK) pathway, which counteracts the cytotoxicity of the apoptotic
pathway (8). For example, in L929 cells, fibroblast growth factor-2
suppressed TNF-
-induced apoptosis by activation of ERK, and this
effect could be reversed by inhibition of the ERK pathway (9). Another
hypothesis favored by recent evidence is that activation of nuclear
factor-
B (NF-
B) may be required to protect cells from
TNF-
-induced apoptosis in certain cells (6, 7, 10). Increasing
evidence indicates that some members of BCL2 family of proteins have
inhibitory actions against apoptosis induced by a number of stress
signals including TNF-
, probably by blocking caspase activities (3).
Given the complexity of TNF-
signaling pathways, it is apparent that
different protective factors may exert their anti-apoptotic effects
through different mechanisms and act at the different stages of the
apoptotic process. The identities and mechanisms of action of the
regulatory factors in TNF-
signaling pathways clearly require
further investigation under specific cellular conditions.
The role of the JNK pathway has been well documented in various
stress-induced models of apoptosis (11-13). However, its involvement in TNF--induced apoptosis has been controversial. In a previous report (14), we established a close correlation between the duration of
JNK activation and TNF-
-induced apoptosis in rat mesangial cells. We
proposed that the JNK pathway is involved in TNF-
-induced apoptosis
under conditions that JNK is activated in a sustained manner and that a
TNF-
-induced mitogen-activated protein kinase phosphatase-1 (MKP-1)
may be responsible for an attenuated JNK activation, thereby protecting
the cells from apoptosis under normal conditions. MKP are responsible
for inactivation of mitogen-activated protein kinases in different
cells (15), and some MKP are inducible by various stresses that
activate JNK (16-18). The duration of JNK activation could thus be
regulated by MKP through a feedback mechanism. Therefore, induction of
MKP to inactivate JNK was thought to protect the cells against
stress-caused damage in these cells (15-17). Recently, we
proposed that the TNF-
-inducible MKP-1 may play a similar role
in protecting mesangial cells from TNF-
cytotoxicity (14).
The current study presents additional evidence to support our
hypothesis. In rat mesangial cells, when the expression of MKP-1 induced by TNF- was selectively blocked by pretreatment of the cells
with Ro318220, it produced results that resembled the effects of the
protein phosphatase inhibitor vanadate in prolonging JNK activation and
inducing apoptosis by TNF-
. Ro318220 selectively blocked expression
of MKP-1 without inhibiting the TNF-
stimulated activation of ERK
and NF-
B. These results further strengthen our previous conclusion
that although TNF-
caused a stimulation of ERK and NF-
B
activation, they probably did not contribute to the protective effect
against TNF-
-induced apoptosis in mesangial cells. Our studies
strongly support the currently controversial hypothesis that the JNK
pathway is involved in TNF-
-induced apoptosis. We also provide a
novel hypothesis to explain the resistance to TNF-
cytotoxicity in
mesangial cells.
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EXPERIMENTAL PROCEDURES |
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Materials--
Recombinant TNF- was obtained from Chemicon
International Inc. (Temecula, CA). Anti-c-Fos antibodies and anti-MKP-1
antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz,
CA). Anti-phospho-c-Jun (Ser 63), anti-c-Jun antibodies were from New England Biolabs (Beverly, MA). Anti-phospho-ERK was from Promega (Madison, WI). Ro318220 was from LC Laboratories (San Diego, CA). 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 previously (19). The cells were maintained in RPMI 1640 medium containing 20% fetal calf serum, 0.6 unit/ml of 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 (20). 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 and 0.001% neutral red for 90 min at 37 °C. The uptake of the dye by viable cells was terminated by removal of the medium, washing the cells briefly with 1 ml of 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 absorbencies, which correlate with the amount of live cells, were determined at 540 nm.Immunocytochemical Detection of Apoptosis and c-Jun Phosphorylation-- Cells grown on 25-mm glass coverslips in 6-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 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. Apoptotic nuclei were identified under a fluorescence microscope. Phosphorylation of c-Jun was detected with anti-phospho-c-Jun (Ser 63) antibodies following the immunocytochemistry protocol provided by the manufacturer (New England Biolabs). Positive stained nuclei were visualized with Texas Red-conjugated secondary antibodies using fluorescence microscopy.
Cell Lysate Preparation-- The quiescent cells were treated with reagents for the indicated times, washed twice with ice-cold phosphate-buffered saline, pH 7.4, 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 the cell lysate. Protein concentration was determined by the method of Bradford using bovine serum albumin as standard (21).
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. JNK activity was determined using GST-Jun as substrate as
described previously (14). 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 -glycerolphosphate, and 10 mM NaF). The beads were then resuspended in 10 µl of
kinase buffer containing (final concentrations) 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 SDS-polyacrylamide gel electrophoresis,
and the phosphorylated proteins were detected by autoradiography.
ERK activation was determined by Western blot analysis using
anti-ERK antibodies that only recognize phosphorylated ERK1
and ERK2 (14).
Western Blot Analysis-- The protein samples were subjected to SDS-polyacrylamide gel electrophoresis 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. The immunoblots were visualized by an ECL kit obtained from Amersham Pharmacia Biotech.
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 (22). The HindIII/BamHI fragment of pCEP4-MKP-1 plasmid was used as a probe.
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RESULTS |
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Induction of MKP-1 by TNF- and Effect of Ro318220--
MKP-1 is
a well characterized member of MKP family that has been shown to be
able to inactivate JNK in vitro and in vivo
(16-18). Our previous results showed that MKP-1 mRNA was strongly
induced by TNF-
at the same time as JNK inactivation in mesangial
cells (14). Beltman et al. (23) reported that Ro318220,
which was originally identified as a specific inhibitor of protein
kinase C (24), was a strong inhibitor of MKP-1 expression in Rat-1 fibroblasts. To test if it has the same effect on rat mesangial cells,
the cells were pretreated with Ro318220 prior to stimulation with
TNF-
. As shown in Fig. 1, the
expression of MKP-1 induced by TNF-
was totally abolished by
pretreatment of the cells with Ro318220, as demonstrated by the
Northern blot (Fig. 1A) and the Western blot (Fig.
1B). These results confirm that TNF-
is able to induce
de novo synthesis of MKP-1 in rat mesangial cells. The effect of Ro318220 in inhibiting the expression of MKP-1 in mesangial cells is consistent with that observed in Rat-1 cells (23).
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Effects of Ro318220 on Activation of JNK and ERK by
TNF---
If MKP-1 is responsible for inactivation of JNK as we
propose, one would expect that JNK activation induced by TNF-
would be prolonged by blocking the induction of MKP-1. As shown in Fig. 2A, pretreatment of mesangial
cells with Ro318220 followed by TNF-
stimulation caused a sustained
JNK activation. The effect of Ro318220 on JNK activation is similar to
that of the phosphatase inhibitor vanadate as reported previously (14).
This result is consistent with the ability of Ro318220 to inhibit the
induction of MKP-1 expression induced by TNF-
(Fig. 1) and confirms
our prediction that MKP-1 can inactivate JNK in vivo. It is
also noted that unlike the situation in Rat-1 cells, where Ro318220
itself is a strong activator of JNK (23), Ro318220 by itself only
slightly activated JNK in mesangial cells (Fig. 2A). This
response is unlike vanadate, which indiscriminately inhibited all
tyrosine phosphatases and caused similar sustained activation patterns
for both JNK and ERK in cells pretreated with vanadate followed by
TNF-
stimulation (14). Pretreatment of mesangial cells with Ro318220
also potentiated ERK activity, but the major effect was to cause a
second activity peak after 2-3 h (Fig. 2B). The reason for
the different effects of Ro318220 on JNK and ERK is currently unknown,
but it is possible that Ro318220 selectively inhibits the expression of
different members of MKP family that may differentially regulate JNK
and ERK dephosphorylation and thus their activity. It is possible, therefore, that other members of MKP family may also be involved in the
regulation of ERK, in addition to MKP-1 in TNF-
signaling pathways
in mesangial cells.
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Effects of Ro318220 on the Expression of c-Jun and c-Fos and on
Phosphorylation of c-Jun Stimulated by TNF---
It is generally
recognized that the JNK and ERK pathways are involved in regulation of
the activity and expression of c-Fos and c-Jun (25). To test for a
possible downstream effect of Ro318220, the expression of c-Jun and
c-Fos was examined (Fig. 3). TNF-
stimulated the expression of both c-Jun (Fig. 3A) and c-Fos
(Fig. 3B) with a stronger effect being observed on c-Fos as
judged by Western blot analysis. Pretreatment of the cells with
Ro318220 had little effect on TNF-
-induced expression of c-Jun (Fig.
3A), but surprisingly, the TNF-
-induced expression of
c-Fos was totally abrogated (Fig. 3B). Ro318220 alone had no apparent stimulatory effect on either c-Jun or c-Fos expression. The
most significant effect of Ro318220 on c-Jun was to potentiate its
phosphorylation induced by TNF-
(Fig. 3C). This
observation is consistent with its ability to sustain JNK activation
(Fig. 2A). Although phospho-c-Jun was detectable only at 15 min when cells were stimulated with TNF-
alone, the phosphorylation
state of c-Jun lasted for at least 3 h when the cells were treated
with a combination of Ro318220 and TNF-
(Fig. 3C). This
sustained c-Jun phosphorylation was nearly identical to the pattern
caused by pretreatment with vanadate (14).
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Effect of TNF- on the Viability of Mesangial Cells in the
Presence or Absence of Ro318220--
Results obtained thus far from
experiments with Ro318220 on JNK activity and c-Jun phosphorylation
stimulated by TNF-
are very similar to those obtained from vanadate
experiments as described previously (14). To test if Ro318220 could
produce a similar effect on cell viability as that caused by vanadate,
the effect of Ro318220 on the viability of mesangial cells in the
presence of TNF-
was examined. As shown in Fig.
5, mesangial cells were essentially
insensitive to TNF-
cytotoxicity when treated with TNF-
alone.
However, when the cells were pretreated with Ro318220, the effect of
TNF-
on cell viability was dramatic. Within 4 h of incubation
after addition of TNF-
, about 80% of cells were dead, whereas with
Ro318220 alone only a slight cellular toxic effect was observed after
the same incubation time (Fig. 5). This result is similar to that
observed for vanadate potentiation of TNF-
-induced cell death under
the same experimental conditions (14). However, unlike vanadate,
Ro318220 showed a much less severe cytotoxicity by itself. The effect
of Ro318220 to potentiate TNF-
cytotoxicity was
dose-dependent; it was evident at a concentration of 2.5 µM and reached a maximum at 15 µM for a 4-h
incubation period (data not shown).
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DISCUSSION |
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It has been known for many years that most normal cells are
resistant to TNF- cytotoxicity and that this resistance can be abolished if the cells are preincubated with protein synthesis inhibitors such as actinomycin D and cycloheximide prior to exposure to
TNF-
. Conversely, preincubation of the sensitive cells with TNF-
increases their resistance to a subsequent challenge with TNF-
(2,
26). It is concluded that protective factor(s) can be elicited by
TNF-
to counteract subsequent TNF-
cytotoxicity. However,
actinomycin D and cycloheximide essentially block all de
novo synthesis of proteins. Therefore, without other approaches, it is not possible to identify specific protective factors among various proteins induced by TNF-
treatment. Molecular genetic techniques have led to identification of some putative protective factors such as NF-
B (6, 10, 27) and BCL2 (28-30). An alternative approach using specific inhibitors to block certain signaling pathways
has proved to be useful. For example, PD098059 and pyrrolidine dithiocarbamate have been used to selectively inhibit the ERK pathway
and NF-
B activation, respectively (9, 31). The results derived from
these experiments provided important information for the elucidation of
the roles of ERK and NF-
B in the regulation of apoptosis in some
cells.
In our previous report, results from experiments with the phosphatase
inhibitor vanadate provided us with an important clue that suggested
that a JNK phosphatase may act as a protective factor against TNF-
toxicity in mesangial cells (14). However, vanadate's nonspecific
inhibition of all protein tyrosine phosphatases, including pre-existing
and induced phosphatases, made it difficult to evaluate which enzyme(s)
was involved. The fact that MKP-1 mRNA is strongly induced by
TNF-
indicates that MKP-1 could be one such phosphatase. If this is
the case, one would expect that selectively blocking the expression of
MKP-1 would sustain JNK activation and subsequently render the cells
susceptible to TNF-
-induced apoptosis. In search of such an
approach, it was brought to our attention that Beltman et
al. (23) reported recently that Ro318220, originally identified as
a PKC inhibitor, selectively inhibited MKP-1 expression induced by
epidermal growth factor and PMA in Rat-1 fibroblasts. This observation
prompted us to test the effects of Ro318220 on mesangial cells. It
showed a similar effect of preventing the expression of MKP-1 induced
by TNF-
. More importantly, pretreatment of cells with Ro318220
produced essentially the same effects as those caused by vanadate in
sustaining JNK activation, c-Jun phosphorylation, and inducing
apoptosis by TNF-
(14). These new results provide substantial
evidence to support the notion that MKP-1 can inhibit stimulation of
JNK by TNF-
in vivo. To our knowledge, this is the first
documentation of MKP-1 induction by TNF-
. MKP are encoded by a
multiple gene family. At least eight members have been identified, and
virtually all of them are inducible immediate early gene products (15).
Whether other members of MKP are inducible by TNF-
and whether they
contribute to the resistance of TNF-
apoptotic effect in mesangial
cells remains to be investigated. Ro318220 selectively blocked
expression of MKP-1 and subsequently prolonged JNK activation without
inhibiting the activation of ERK (Fig. 2B) and NF-
B (data
not shown) by TNF-
. These results further strengthen our previous
conclusion that TNF-
-stimulated ERK and NF-
B activation may not
contribute to the protective effect on TNF-
-induced apoptosis
(14).
In Rat-1 cells, Ro318220 inhibited PMA- and epidermal growth
factor-induced expression of MKP-1 as judged by Western blot analysis.
Although it was shown that inhibition of expression of MKP-1 by
Ro318220 was not through inhibition of protein synthesis, it was not
clear how MKP-1 was inhibited. Here we have demonstrated that Ro318220
blocked MKP-1 expression most likely by inhibiting its transcription in
mesangial cells. Ro318220 is a derivative of bisindolylmaleimide and
was discovered as a PKC-specific inhibitor (24); however, its effect in
blocking the expression of MKP-1 is apparently PKC-independent in Rat-1
cells, suggesting that Ro318220 has some unique properties in addition
to being a PKC inhibitor. Our results indicate that the effect of
Ro318220 on expression of MKP-1 induced by TNF- in mesangial cells
also seems to be PKC-independent. This is indicated by the fact that
blocking the PKC pathway by acute inhibition with another PKC
inhibitor, GF109203X, or by down-regulation PKC with PMA only slightly
inhibited TNF-
-induced expression of MKP-1. However, both acute
inhibition and down-regulation of PKC completely blocked PMA-induced
effects under the same
conditions.2 Another
interesting observation is that Ro318220 also blocked expression of
c-Fos induced by TNF-
in mesangial cells. A similar effect was
observed in Rat-1 cells where c-Fos expression induced by
lysophosphatidic acid and PMA was strongly inhibited (23). Further
important questions are whether c-Fos induced by TNF-
also plays a
role in protecting the cells from apoptosis, and if so, is there any
interaction between c-Fos regulated gene products and MKP-1 in the
TNF-
signaling pathways. These questions could best be addressed if
the expression of c-Fos and MKP-1 induced by TNF-
can be separately
manipulated. Experiments attempting to answer these questions are
currently under way.
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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 by National Institutes of Health Grants DK-15120 and DK-48493.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.
§ To whom correspondence should be addressed: Dept. of Biochemistry and Biophysics, University of Pennsylvania, 601 Goddard Labs., 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-
; ERK, extracellular signal-regulated protein
kinase; JNK, c-Jun N-terminal protein kinase; MKP, mitogen-activated
protein kinase phosphatase(s); PKC, protein kinase C; PMA, phorbol
12-myristate 13-acetate; NF-
B, nuclear factor-
B; GST, glutathione
S-transferase.
2 Y.-L. Guo, B. Kang, and J. R. Williamson, manuscript in preparation.
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REFERENCES |
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