From the Howard Hughes Medical Institute, Program in Molecular Medicine, and Department of Biochemistry and Molecular Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01605
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ABSTRACT |
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The cellular response to treatment with
proinflammatory cytokines or exposure to environmental stress is
mediated, in part, by the p38 group of mitogen-activated protein (MAP)
kinases. We report the molecular cloning of a novel isoform of p38 MAP
kinase, p382. This p38 MAP kinase, like p38
, is inhibited by the
pyridinyl imidazole drug SB203580. The p38 MAP kinase kinase MKK6 is
identified as a common activator of p38
, p38
2, and p38
MAP
kinase isoforms, while MKK3 activates only p38
and p38
MAP kinase
isoforms. The MKK3 and MKK6 signal transduction pathways are therefore
coupled to distinct, but overlapping, groups of p38 MAP kinases.
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INTRODUCTION |
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Mitogen-activated protein (MAP)1 kinases are proline-directed protein kinases that mediate the effects of numerous extracellular stimuli on a wide array of biological processes, such as cellular proliferation, differentiation, and death (1). Three groups of mammalian MAP kinases have been studied in detail: the extracellular signal-regulated kinases (ERK) (2), the c-Jun NH2-terminal kinases (JNK) (3), and the p38 MAP kinases (3). The ERKs are robustly activated by growth factors and phorbol ester, but are only weakly activated by cytokines and environmental stress. In contrast, JNK and p38 MAP kinases are strongly activated by cytokines and environmental stress, but are poorly activated by growth factors and phorbol ester.
Recently, progress toward understanding the physiological role of the p38 MAP kinases has been achieved through the use of drugs that bind p38 MAP kinase (4, 5). These drugs are pyridinyl imidazole derivatives that inhibit p38 MAP kinase activity (4, 5). Studies using these drugs indicate that p38 MAP kinase is required for lipopolysaccharide-induced production of IL-1 and TNF in monocytes (4), the induction of IL-6 and granulocyte-macrophage/colony-stimulating factor transcription by TNF (6), the proliferation of T cells in response to IL-2 and IL-7 (7), and the stress-induced transcription of c-jun and c-fos in fibroblasts (8). The targets of p38 MAP kinase that mediate these responses are poorly characterized. However, biochemical studies indicate that p38 MAP kinase signaling pathway activates the transcription factors CREB and ATF1 (9, 10), ATF2 (11, 12), CHOP (13), and MEF-2C (14). The p38 MAP kinase also activates other protein kinases, such as Mapkap-2 (15-17), Mapkap-3 (18, 19), and Mnk1/2 (20, 21).
The p38 MAP kinase group includes the isoforms p38 (4, 12, 16, 17,
22), p38
(23), and p38
(24-27). Recent studies indicate the
presence of a fourth p38 MAP kinase isoform, p38
(28, 29). These p38
MAP kinases are widely expressed in many tissues and are activated by
dual phosphorylation on Thr and Tyr within the motif Thr-Gly-Tyr
located in kinase subdomain VIII (12). This phosphorylation is mediated
by a protein kinase cascade (1). Components of this signaling pathway
include the MAP kinase kinases MKK3 (30) and MKK6 (11, 31-33). It is
also possible that MKK4 contributes to the activation of p38 MAP
kinase. In vitro studies demonstrate that MKK4 activates
both JNK and p38 MAP kinases (30, 34). However, the role of MKK4 as an
activator of p38 MAP kinase in vivo is unclear (35).
The activation of MKK3 and MKK6 is regulated by phosphorylation on Ser and Thr residues within subdomain VIII by MAP kinase kinase kinases (MKKK) (1). Further studies are required to define the function and specificity of MKKKs that cause activation of the p38 MAP kinase pathway. However, one candidate MKKK for the p38 MAP kinase signaling pathway is TAK1, which has been reported to activate MKK3 and MKK6 (36-38). Other MKKKs, which activate both JNK and p38 MAP kinases, include ASK-1 (39) and the mixed-lineage kinase MLK-3 (40-42). Other MKKKs that activate the JNK signaling pathway, for example MEKK1, do not cause activation of p38 MAP kinase (30, 34).
The expression of multiple p38 MAP kinase isoforms in mammalian tissues suggests that these MAP kinases may differ in their physiological function. These p38 MAP kinases may be coupled to different upstream signaling pathways. This would enable the activation of specific p38 MAP kinase isoforms in response to different stimuli. Alternatively, these p38 MAP kinase isoforms may differ in their substrate specificity. Such differences could allow coupling of different p38 MAP kinase isoforms to different signal transduction targets.
The purpose of this study was to examine the p38 MAP kinase signal
transduction pathway. We find that p38
MAP kinase (23) is not a
functional protein kinase in vitro or in vivo.
However, a novel p38
MAP kinase isoform (p38
2) that was isolated
from a human brain cDNA library encoded a functional protein
kinase. This novel p38 MAP kinase isoform was inhibited by pyridinyl
imidazole drugs. The MAP kinase kinase MKK6 activated p38
, p38
2,
and p38
, while MKK3 activated only p38
and p38
. The lack of
activation of p38
2 by MKK3 was due to its inability to phosphorylate
p38
2. These data demonstrate that the p38
2 MAP kinase is
selectively activated by MKK6. We conclude that different p38 MAP
kinase isoforms are regulated by overlapping and distinct signal
transduction pathways.
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EXPERIMENTAL PROCEDURES |
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Materials--
IL-1 and TNF-
were from Genzyme Corp. MBP
was from Sigma. [
-32P]ATP was obtained from Amersham
Corp. GST-c-Jun (43), GST-ATF2 (44), GST-Elk-1 (45), GST-MKK3 (30),
GST-MKK4 (30), and GST-MKK6 (11) fusion proteins have been described.
Recombinant Mapkap-K2 was obtained from Upstate Biotechnology Inc. The
drug SB203580 was provided by Dr. M. S.-S. Su, Vertex
Pharmaceuticals Inc. Mammalian expression vectors for p38
, MKK3,
MKK4, and MKK6 have been described (11, 12, 30). The plasmid
pCMV-Flag-Elk-1 was provided by Dr. A. J. Whitmarsh (University of
Massachusetts Medical School) and Dr. A. Sharrocks (Newcastle
University Medical School). The plasmid pCDNA3-Flag p38
1 (23)
was provided by Dr. J. Han (The Scripps Research Institute). The p38
cDNA (24, 26, 27) was prepared by RT-PCR amplification using rat
skeletal muscle mRNA as the template.
Tissue Culture-- Chinese hamster ovary (CHO) cells were maintained in Ham's F-12 medium supplemented with 5% fetal bovine serum. HeLa and COS-1 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Life Technologies, Inc.). Plasmid DNA (0.1-1.0 µg) was transfected by the LipofectAMINE reagent (Life Technologies, Inc.) according to the manufacturers' recommendations. The cells were harvested after 48 h of incubation.
Immunoprecipitation and Protein Kinase Assays--
Cells were
solubilized with buffer A (20 mM Tris (pH 7.5), 10%
glycerol, 1% Triton X-100, 0.137 M NaCl, 25 mM
-glycerophosphate, 2 mM EDTA, 0.5 mM
dithiothreitol, 1 mM sodium orthovanadate, 2 mM
sodium pyrophosphate, 10 µg/ml leupeptin, 1 mM
phenylmethylsulfonyl fluoride). The extracts were centrifuged at
15,000 × g (15 min at 4 °C). Epitope-tagged protein
kinases were immunoprecipitated by incubation for 2 h at 4 °C
with the M2 Flag monoclonal antibody (IBI-Kodak) bound to protein
G-Sepharose (Pharmacia-LKB Biotechnology, Inc). The immunoprecipitates
were washed twice with buffer A and twice with kinase buffer (25 mM HEPES, pH 7.4, 25 mM
-glycerophosphate, 25 mM MgCl2, 0.5 mM dithiothreitol,
0.1 mM sodium orthovanadate).
Western Blot Analysis-- Proteins were fractionated by SDS-PAGE, electrophoretically transferred to an Immobilon-P membrane (Millipore Inc.), and probed with the M2 monoclonal antibody to the Flag epitope (IBI-Kodak), a monoclonal antibody to MKK4 (Pharmingen), and an affinity-purified polyclonal antibody to phospho-Ser-383 Elk-1 (New England Biolabs). Immunocomplexes were detected using chemiluminescence (Lumiglo; Kirkegaard & Perry Laboratories).
Measurement of Reporter Gene Expression--
Transfection assays
were performed using CHO cells and the LipofectAMINE method (Life
Technologies, Inc.). The cells were co-transfected with 0.2 µg of
pGAL4-Elk-1 (45) and 0.2 µg of the reporter plasmid pG5E1bLuc (49).
Transfection efficiency was normalized by co-transfection of the cells
with the -galactosidase expression vector pCH110 (Pharmacia-LKB).
The effect of co-transfection with 100 ng of expression vectors for p38
MAP kinase, MKK3, MKK4, MKK6, or the empty expression vector was
examined. The cells were harvested 48 h post-transfection. The
-galactosidase and luciferase activity in the cell lysates was
measured as described previously (45).
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RESULTS |
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Molecular Cloning of p382 MAP Kinase--
To identify novel
members of the human p38 MAP kinase group, we used a human p38
MAP
kinase cDNA as a probe to screen a human fetal brain cDNA
library. Two cDNA clones related to p38
MAP kinase were
identified. Partial sequence analysis demonstrated that these clones
were identical to the p38
MAP kinase reported by Jiang et
al. (23). However, following completion of the sequence analysis,
it was apparent that the novel cDNAs differed from p38
MAP
kinase. A deletion of 24 base pairs was detected in the sequence of the
novel clones compared with p38
MAP kinase. This gap results in the
deletion of an 8-amino acid insertion present in p38
MAP kinase
(23). We designate the novel sequence as p38
2 and the previously
reported sequence p38
1 (Fig. 1).
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Biochemical Characterization of p382 MAP Kinase Activity in Vivo
and in Vitro--
The phosphorylation of ATF2 by p38
MAP kinase has
been studied in detail (11, 12). We therefore tested whether ATF2 was a
substrate for p38
2. In vitro protein kinase assays using
recombinant p38
2 demonstrated that this protein kinase did
autophosphorylate (Fig. 2A).
In contrast, control studies using recombinant p38
1 did not
demonstrate autophosphorylation (Fig. 2A). Addition of ATF2
resulted in phosphorylation by p38
2, but not by p38
1 (Fig. 2A). These data suggest that p38
2 is a more active
protein kinase than p38
1.
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Substrate Phosphorylation by p382 MAP Kinase--
We
immunopurified p38
, p38
2, and p38
MAP kinases from control and
UV-irradiated cells. The amount of each Flag-tagged p38 MAP kinase was
examined by immunoblot analysis using the M2 monoclonal antibody. An
equal amount of each p38 MAP kinase isoform was used for in
vitro protein kinase assays using different substrates. This
analysis demonstrated that ATF2, Elk-1, and MBP were phosphorylated by
p38
2 (Fig. 3A). ATF2,
Elk-1, and MBP were also phosphorylated by p38
and p38
MAP
kinases (Fig. 3A). The extent of substrate phosphorylation
by p38
2 was less than p38
and p38
. However, the
fold-activation of p38
2 activity was similar to that detected for
p38
and p38
(Fig. 3A). These data indicate that the
substrate specificity of these p38 MAP kinase isoforms was similar.
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Effect of a Pyridinyl Imidazole Drug on p382 MAP Kinase
Activity--
We examined the effect of the pyridinyl imidazole
derivative SB203580 (4, 5) on the protein kinase activity of p38
2 MAP kinase. This drug has previously been shown to inhibit p38
MAP
kinase activity (4, 5). Here we demonstrate that SB203580 inhibits
p38
2 MAP kinase activity (Fig. 4). The
dose response of inhibition of protein kinase activity was similar in
experiments using p38
and p38
2 MAP kinase (Fig. 4). In contrast,
p38
MAP kinase was not inhibited by SB203580. The insensitivity of
p38
MAP kinase to inhibition by SB203580 observed in this study
differs from the results of one previous study (26), but is in
agreement with more recent studies by other investigators (25). As the p38
and p38
2 isoforms demonstrate similar inhibition by SB203580, both of these MAP kinases could account for the previously reported effects of pyridinyl imidazole derivatives on cultured cells (4, 5).
The p38
2 MAP kinase is therefore likely to be a physiologically relevant mediator of the p38 MAP kinase signal transduction
pathway.
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The p382 MAP Kinase Is Activated by Proinflammatory Cytokines
and Environmental Stress--
The p38
and p38
MAP kinases are
regulated by numerous extracellular stimuli, including proinflammatory
cytokines and environmental stress (38). We compared the regulation of
p38
2 MAP kinase with the p38
and p38
MAP kinases. HeLa cells
were transfected with epitope-tagged p38
, p38
2, and p38
MAP
kinases and exposed to different extracellular stimuli. The activity of
each p38 MAP kinase isoform was detected by measurement of protein
kinase activity in an immune complex kinase assay using ATF2 as the
substrate. The proinflammatory cytokines TNF-
and IL-1
,
environmental stress (UV irradiation and osmotic shock), and treatment
with anisomycin (an inhibitor of protein synthesis) caused a marked
increase in the activity of p38
2 MAP kinase (Fig.
5). The strongest activation of p38
2
was caused by the exposure of cells to UV radiation. Although ATF2
phosphorylation by p38
2 was consistently less than that observed in
kinase assays with p38
or p38
, the fold-increase in protein
kinase activity caused by UV radiation was similar for each p38 MAP
kinase isoform (Fig. 5). These data demonstrate that p38
2, like
p38
and p38
, is activated in vivo by a stress-induced signal transduction pathway.
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Selective Activation of p38 MAP Kinases by MAP Kinase
Kinases--
The p38 MAP kinases are activated in response to
extracellular stimuli by dual phosphorylation on Thr and Tyr by the MAP
kinases kinases MKK3 (30), MKK4 (30, 34), and MKK6 (11, 31-33). We
therefore tested the effect of MKK3, MKK4, and MKK6 on p382 MAP
kinase activity in co-transfection assays in vivo. Control experiments were performed using p38
and p38
MAP kinases. MKK3 caused strong activation of p38
, a lower level of activation of
p38
, and did not activate p38
2 (Fig.
6A). Similarly, MKK4 activated
p38
strongly, weakly activated p38
, and did not activate p38
2
(Fig. 6B). In contrast, MKK6 caused strong activation of p38
, p38
2, and p38
MAP kinases (Fig. 6C). The
effect of MKK3 and MKK4 to activate p38
and p38
, but not p38
2,
indicates that the regulation of p38
2 MAP kinase differs from the
other p38 MAP kinases. The selective effect of MAP kinase kinases to
regulate p38
2 MAP kinase activity suggests that extracellular
stimuli may selectively regulate p38 MAP kinase isoforms.
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The p382 MAP Kinase Is a Substrate for MKK6, but Not
MKK3--
The effect of MKK3 to activate p38
and p38
MAP
kinases, but not p38
2, could be accounted for by many mechanisms.
One possibility is that p38
2 is not a substrate for MKK3. To test
this hypothesis, we examined the phosphorylation of p38 MAP kinase
isoforms by MKK3 and MKK6 (Fig. 7).
In vitro protein kinase assays demonstrated the
autophosphorylation of MKK3, but not MKK6, as described previously (11,
30). MKK6 caused strong phosphorylation of p38
and p38
2 MAP
kinase, and caused a lower level of phosphorylation of p38
MAP
kinase (Fig. 7B). In contrast, MKK3 caused a similar level of phosphorylation of p38
and p38
MAP kinases, but no
phosphorylation of p38
2 (Fig. 7A). These data demonstrate
that MKK3 and MKK6 differentially phosphorylate p38 MAP kinase
isoforms. Furthermore, these data indicate that p38
2 MAP kinase is a
substrate for MKK6, but not MKK3.
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Transcriptional Regulation by the MKK6-p382 MAP Kinase Signaling
Pathway--
The constitutively active form of MKK3 does not activate
endogenous p38 MAP kinase in transient transfection assays (11). In
contrast, activated MKK3 does cause potent stimulation of
co-transfected p38 MAP kinase activity (11). Activated MKK3 can
therefore be used as a tool to test the contribution of specific p38
MAP kinases isoforms on cellular responses. Co-transfection assays
demonstrated that activated MKK3 did increase
Elk-1-dependent luciferase gene expression when
co-transfected with p38
and p38
MAP kinases (Fig.
8, A and C). In
contrast, p38
2 MAP kinase did not increase MKK3-stimulated Elk-1
transcriptional activity (Fig. 8B). Control experiments
using activated MKK6 demonstrated that p38
, p38
2, and p38
MAP
kinases increased MKK6-stimulated Elk-1 transcriptional activity (Fig.
8, A-C). Consistent with these data, MKK6 caused a similar
level of phosphorylation of Elk-1 by each of the three p38 MAP kinases
in vitro (Fig. 8D) and in vivo (Fig.
8E). Similar data were obtained in experiments using the
transcription factor ATF2 as a p38 MAP kinase substrate (data not
shown).
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DISCUSSION |
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The stress-activated MAP kinases include the JNK and p38 groups
(3). The JNK group consists of 10 members that are derived by
alternative splicing of three genes (50). These JNK isoforms differ in
their tissue distribution and in their interaction with substrate
proteins (50). It has therefore been proposed that individual JNK
isoforms may mediate distinct physiological responses (50). Similarly,
the p38 group of stress-activated MAP kinases consists of multiple
isoforms (1). These isoforms include p38 (4, 12, 16, 17, 22), p38
(23), and p38
(24-27). Recent studies indicate the presence of a
fourth p38 MAP kinase isoform, p38
(28, 29). In addition,
alternatively spliced forms of p38
MAP kinase have been described
(4, 51). The existence of multiple p38 MAP kinase isoforms provides the
potential for the generation of stimulus-specific and cell
type-specific responses to activation of the p38 MAP kinase signaling
pathway. The identification of p38 MAP kinase isoforms and their
mechanism of activation by MAP kinase kinases represents one step that
is required for understanding the physiological role of p38 MAP kinases in mammalian cells. Here we describe a novel p38 MAP kinase isoform (p38
2) that is selectively activated by the MAP kinase kinase MKK6.
The p382 Protein Kinase Is a Novel MAP Kinase--
We report
the molecular cloning of p38
2 MAP kinase, a novel human
stress-activated protein kinase. This enzyme is most similar to the
previously characterized p38
MAP kinase (p38
1) (23) and may be
derived from the same gene by alternative splicing. The p38
2 MAP
kinase contains a 24-base pair deletion within the coding region of
p38
1 MAP kinase. This deletion represents a significant difference
between these p38
MAP kinase isoforms. The p38
1 MAP kinase
contains an 8-amino acid insertion in the kinase domain that is absent
in p38
2 MAP kinase (Fig. 1).
The p382 MAP Kinase Is a Stress-activated Protein
Kinase--
The p38
2 MAP kinase, like the p38
and p38
MAP
kinase isoforms, is activated by treatment of cells with
proinflammatory cytokines (e.g. TNF and IL-1) or by exposure
of cells to environmental stress (e.g. UV radiation and
osmotic shock) (Fig. 5). The p38
2 MAP kinase shares overlapping, but
distinct, substrate specificity with the p38
and p38
MAP kinase
isoforms (Fig. 2A). The p38
2 MAP kinase was not observed
to phosphorylate c-Myc, I
B, Max, Smad1, c-Fos, and NFAT (data not
shown). However, p38
2 was found to phosphorylate the p38
and
p38
substrates ATF2, Elk-1, and MBP (Fig. 3A). Mutational
analysis of the sites of ATF2 phosphorylation demonstrated that the
substrate specificity of p38
2 differs from p38
and p38
(Fig.
3B).
MKK3 and MKK6 Differentially Activate p38 MAP Kinase
Isoforms--
The p38 MAP kinases kinases MKK3, MKK4, and MKK6
selectively activate the p38, p38
2, and p38
MAP kinase
isoforms (Fig. 9). MKK6 activates p38
,
p38
2, and p38
, while MKK3 and MKK4 only activate p38
and
p38
(Fig. 6). The lack of activation of p38
2 by MKK3 is explained
by its inability to phosphorylate p38
2, while MKK6 phosphorylates
all three isoforms (Fig. 7). The specificity of MKK3 and MKK6 to
differentially activate these p38 MAP kinase isoforms was confirmed by
analysis of the effects of MKK3 and MKK6 on Elk-1-dependent
gene expression (Fig. 8).
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Conclusions--
The cellular response to treatment with
proinflammatory cytokines or exposure to environmental stress is
mediated, in part, by the p38 group of MAP kinases. The activation of
specific p38 MAP kinase isoforms may lead to cell type-specific and
stimulus-specific cellular responses. The p38 MAP kinase kinase MKK6 is
identified as a common activator of p38, p38
2, and p38
MAP
kinases, while MKK3 targets p38
and p38
MAP kinases. The MKK3 and
MKK6 signal transduction pathways are therefore coupled to distinct,
but overlapping, groups of p38 MAP kinase isoforms.
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ACKNOWLEDGEMENTS |
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We thank Dr. J. Han for the p381 MAP
kinase cDNA and for discussions concerning p38
1 protein kinase
activity; Drs. A. J. Whitmarsh and A. Sharrocks for critical
discussions and for providing the plasmid pCMV-Flag-Elk-1; Dr. M. S.-S. Su for the drug SB203580; T. Barrett, J. Cavanagh, and I.-H. Wu
for technical assistance; and K. Gemme for administrative
assistance.
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FOOTNOTES |
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* These studies were supported in part by National Institutes of Health, NCI, Grants CA65861 and CA72009.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF031135.
Investigator of the Howard Hughes Medical Institute. To whom
correspondence should be addressed: Howard Hughes Medical Institute, Program in Molecular Medicine, University of Massachusetts Medical School, 373 Plantation St., Worcester, MA 01605.
1 The abbreviations used are: MAP, mitogen-activated protein; MKK, MAP kinase kinase; MKKK, MAP kinase kinase kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun NH2-terminal kinase; IL, interleukin; TNF, tumor necrosis factor; GST, glutathione S-transferase; RT, reverse transcriptase; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; CHO, Chinese hamster ovary; MBP, myelin basic protein.
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REFERENCES |
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