 |
INTRODUCTION |
Cells undergoing programmed cell death (apoptosis) are rapidly
removed by monocyte-derived macrophages, suggesting that apoptotic cells might secrete factors with leukocyte and monocyte chemotaxis activity. Endothelial monocyte-activating polypeptide II (EMAP II)1 could be a good
candidate to be a chemokine recruiting leukocytes and monocytes to
cells undergoing apoptosis, because it is released from apoptotic cells
and has chemokine activity (1, 2). It has also emerged as a
proinflammatory mediator that induces the expression of tissue factor,
tumor necrosis factor (TNF), and interleukin-8 (IL-8) in mononuclear
phagocytes and polymorphonuclear leukocytes. In addition, EMAP II
mRNA is most abundant at sites of tissue undergoing apoptosis in
mouse embryo (3). The tissue expressing a high level of EMAP II
mRNA also accumulates macrophages, suggesting that EMAP II is a
chemoattractant recruiting macrophages into dead cells.
EMAP II was initially purified from the culture medium of murine
methylcholanthrene A-induced fibrosarcoma cells based on its capacity
to induce activation of tissue factor in human umbilical vein
endothelial cells (HUVECs) (1). Since EMAP II is identical to the
COOH-terminal domain of the p43 subunit of the mammalian multi-aminoacyl-tRNA synthetase (ARS) complex (4), p43 has been
suggested to be a pro-EMAP II. It is cleaved at the amino acid motif
ASTD by caspase-7 activated in apoptotic cells (5), producing EMAP II
that is sequentially released from the cells.
p43 is a noncatalytic subunit of the mammalian multi-ARS complex (6).
Since p43 occupies a central position within the complex in the
electron microscopic images of immunocomplexes (7), it could be a
scaffolding protein forming the multi-ARS complex. It has been
demonstrated that p43 interacts with the NH2-terminal
extension of human cytoplasmic arginyl-tRNA synthetase through its
NH2-terminal domain (8). Although its COOH-terminal domain
is equivalent to EMAP II, this domain contains a tRNA binding motif
(9), to deliver tRNAs to the bound arginyl-tRNA synthetase (8).
Although p43 is universally expressed (10), its expression level is
varied temporally and spatially in developing mouse (11). For instance,
there is a significant surge in the expression of p43 within the lungs
on postnatal days 8-16 of mouse. p43 is produced throughout the lung,
with predominance in the myoepithelium that lines the bronchioles. In
addition, p43 is highly expressed in microglial cells within lesions of
experimental autoimmune encephalomyelitis, neuritis, and uveitis (12).
The high expression level of p43 in specific developmental stages and
tissues suggests that p43 could have unexpected functions in
angiogenesis, inflammation, and apoptosis (13).
Mature EMAP II is generated and secreted during late apoptosis (3, 14)
so that rapid recruitment of monocytes and macrophages to apoptotic
cells could not be explained by EMAP II generation. Since the
full-length p43 is constitutively secreted from various cells (3, 15),
we focus on the cytokine function of p43. Here, we show that p43 itself
was selectively secreted from cells, even in the absence of an
apoptosis signal, and had a cytokine function, inducing MIP-1
and
MCP-1 as well as TNF and IL-8 from THP-1 cells. Interestingly, p43 was
highly expressed by the foam cells of atherosclerosis lesions, implying
that p43 could be a major contributor of inflammation in
atherosclerosis development.
 |
EXPERIMENTAL PROCEDURES |
Cell Culture and Materials--
Human monocyte THP-1 cells were
grown in RPMI 1640 medium supplemented with 10% fetal bovine
serum and 50 µg/ml streptomycin and penicillin in a 5%
CO2 incubator at 37 °C. 32D mouse myeloid precursor
cells were maintained in RPMI 1640 containing 10% fetal bovine serum
and interleukin-3 (1 ng/ml). Human embryonic kidney 293 cells were
grown in Dulbecco's modified Eagle's medium. Preparation of anti-p43
antibody was described previously (8). For the preparation of anti-p18
antibody, the cDNAs encoding the full length of human p18 was
amplified by PCR. The resulting PCR product was cloned into a PET28a
vector (Novagen) using EcoRI and SalI sites to
express as a His-tagged fusion protein. p18 was expressed as an
insoluble protein that was used for preparing monoclonal mouse anti-p18
antibody that was made by Boditech. Anti-Myc and -tubulin antibodies
were purchased from Santa Cruz and Sigma, respectively.
Construction and Purification of p43 Deletions--
The
constructs of p43-(1-312), p43-(1-147), and p43-(148-312) were
described previously (8). To construct p43-(1-108), pET28a (Novagen)
containing the full-length p43 was digested with Asp718 and
SalI, and the large fragment was treated with the Klenow fragment to fill up the DNA ends and re-ligated. The DNA fragments coding for p43-(91-256), p43-(91-312), p43-(218-312), and
p43-(257-312) regions were synthesized by PCR with specific primer
sets (the primer sequences will be available upon request). The
specific PCR products were digested with EcoRI and
XhoI and ligated into pET28a cut with the same enzymes.
Each of the full-length p43 and p43-deleted constructs was expressed as
His-tag fusion protein in Escherichia coli BL21 (DE3) and
purified by nickel affinity chromatography and Mono Q or S ion-exchange
chromatography as described previously (8). To remove
lipopolysaccharide, the protein solution was dialyzed in pyrogen-free
buffer (10 mM potassium phosphate buffer, pH 6.0, 100 mM NaCl). After dialysis, the protein was loaded to
polymyxin resin (Bio-Rad) pre-equilibrated with the same buffer,
incubated for 20 min, and eluted. The concentration of the residual
lipopolysaccharide (LPS) was below 20 pg/ml when determined using the
Limulus Amebocyte Lysate QCL-1000 kit (BioWhittacker).
DNA Transfection--
100-mm dishes of 293 cells were
transfected with 5 µg of the indicated Myc-FLAG-tagged
pcDNA3-p43 plasmid using Geneporter (Gene Therapy Systems)
according to the manufacturer's protocol. Twenty-four hours after
transfection, cell supernatant was collected and concentrated by using
Vivaspin (VivaScience). Cells were washed twice with cold
phosphate-buffered saline, lysed with lysis buffer (25 mM
Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, and
1% Triton X-100) containing 1 mM phenylmethylsulfonyl
fluoride and protease inhibitor mixture (Roche Molecular Biochemicals).
The lysate proteins and cell supernatants were analyzed by immunoblotting.
Electrophoresis and Immunoblotting--
THP-1 cells treated with
p43 were harvested by centrifugation at 600 × g for 5 min, washed twice with cold phosphate-buffered saline, and lysed with
lysis buffer (25 mM Tris, pH 7.4, 150 mM NaCl,
1% EDTA, 1 mM sodium orthovanadate, 1 mM
dithiothreitol, 1.0% Triton X-100, 1% sodium deoxycholate, 0.1% SDS,
and 0.1 mM phenylmethylsulfonyl fluoride) containing
protease inhibitor mixture (Roche Molecular Biochemicals) and
phosphatase inhibitors (1 mM sodium orthovanadate, 10 mM sodium fluoride, and 12 mM
-glycerophosphate). The proteins in the lysates were resolved
by 10% SDS-polyacrylamide gel electrophoresis and transferred onto a
polyvinylidene difluoride membrane (Millipore). Antigens were
visualized by sequential treatment with specific antibodies,
horseradish peroxidase-conjugated secondary antibodies, and an enhanced
chemiluminescence substrate kit.
JNK Immunocomplex in Vitro Kinase Assay--
The cell lysates
prepared in JNK lysis buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM sodium orthovanadate, 1 mM dithiothreitol, 1.0% Triton X-100, and 0.1 mM phenylmethylsulfonyl fluoride) were incubated with
anti-JNK antibody for 1 h at 4 °C, and protein G-agarose was
added to the reaction mixture and incubated for additional 1 h.
The immunocomplex was precipitated and washed four times with the lysis
buffer and twice with kinase assay buffer (20 mM HEPES
buffer, pH 7.6, 20 mM MgCl2, 20 mM
-glycerophosphate, 20 mM p-nitrophenyl
phosphate, 0.1 mM sodium orthovanadate, and 2 mM dithiothreitol). The washed immunocomplexes were
incubated in the same buffer containing GST-c-Jun (5 µg each), 20 µM ATP, and 5 µCi of [
-32P]ATP
for 20 min at 30 °C. The proteins in the reaction were then separated by 10% SDS-polyacrylamide gel electrophoresis and
transferred to a polyvinylidene difluoride membrane. The phosphorylated
c-Jun was determined by autoradiography of the dried blot.
Measurement of TNF, IL-8, MIP-1, and MCP-1--
THP-1 cells
(2 × 106 cells/ml) were incubated in 24-well tissue
culture plates in serum-free RPMI 1640 medium in a total volume of 0.5 ml/well in duplicate. Cells were washed twice with serum-free medium and then incubated with p43 (100 nM). The
supernatants were harvested 2 h after stimulation and assayed for
TNF, IL-8, MIP-1, and MCP-1 using their corresponding ELISA kits
(PharMingen) according to the manufacturer's instructions.
Assay of Gene Expression by cDNA Array Analysis--
The
Atlas Human cDNA Expression Array 1.2 (CLONTECH) was used for cDNA array analysis.
Total and polyadenylated RNAs were prepared from the control or
p43-treated THP-1 cells by the Atlas Pure Total RNA Labeling System
(CLONTECH) as recommended by the manufacturer. One
µg of polyadenylated RNA isolated from the control or p43-treated
cells was converted to radioactive cDNA by reverse transcription in
the presence of [
-32P]dATP. The radioactively labeled
cDNA was then denatured and hybridized to the cDNA expression
arrays as recommended by the manufacturer. The radioactivity on the
membranes was quantified by a phosphoimager. We calculated the change
in gene expression after the p43 treatment as the percentage of the
untreated cells, using three of the internal controls recommended by
the manufacturer for normalization to ensure the comparability of the
control and p43-treated samples.
Immunohistochemistry--
For immunohistochemical analysis,
carotid endoarterectomy specimens were obtained from 13 patients, aged
from 63 to 81, who underwent the surgery at Samsung Seoul Hospital.
Atherosclerotic plaque specimens were washed with saline and embedded
in optimal cutting temperature to make frozen sections. Standard
5-mm sections were stained using the Labeled Streptavidin Biotin
kit (Dako) according to the manual provided by the manufacturer.
Monoclonal antibodies to CD68 (KP1), and SMC
-actin (1A4) were
purchased from Dako (Glostrup, Denmark). For the detection of
p43, rabbit polyclonal antibody raised against recombinant p43 was used
in 1 ng/ml concentration.
 |
RESULTS |
Full length of p43 Is Constitutively Secreted without an Apoptosis
Signal--
Since EMAP II was secreted during late apoptosis, it may
not play an active role in recruiting and activating monocytes and macrophages to scavenge apoptotic corps in the early stage of apoptosis. Although methylcholanthrene A fibrosarcoma cells secrete 40- and 23-kDa (EMAP II) cytokines that stimulate the generation of tissue
factor in HUVECs (1), only 23-kDa (EMAP II) protein has been
investigated as a cytokine. In addition, the full-length p43 (precursor
of EMAP II) is constitutively secreted in methylcholanthrene fibrosarcoma cells, 32D myeloid precursor cells, and human prostatic adenocarcinoma cells even in the absence of apoptotic stimulus (3, 15).
All of these previous data suggest that p43 itself could function as a cytokine.
To explore the possibility of p43 as a cytokine, we first investigated
the secretion pattern of p43 and EMAP II from normal or apoptotic
cells. We incubated 32D myeloid precursor cells with serum-free medium
for the indicated times in the presence or absence of IL-3. Since IL-3
withdrawal makes the cells undergo apoptosis within 12 h, we could
test the secretion of p43 and EMAP II stimulated by apoptosis.
Immunoblotting with anti-p43 antibody revealed that p43 was secreted
from the cells as early as 30 min after serum starvation even in the
presence of IL-3 (Fig. 1A).
The p43 secretion was very specific, because there was no tubulin (a
cytoplasmic protein) and p18 (another noncatalytic component of
multi-ARS complex) from the medium at early time points of IL-3
withdrawal and at any time points in the presence of IL-3. Meanwhile,
EMAP II appeared from the cell supernatant only when cells were exposed to apoptosis by IL-3 withdrawal. It was detected in the supernatant at
the later time points when p18 and tubulin were also found in the
medium (Fig. 1A), implying that EMAP II secretion results from the cellular breakdown by apoptosis.

View larger version (39K):
[in this window]
[in a new window]
|
Fig. 1.
Secretion of p43 without apoptosis
stimulus. A, 32D cells were grown in FCS-containing
medium and serum-starved for 0.5, 2, 6, 12, and 24 h with or
without IL-3. Proteins from cell medium were concentrated and analyzed
by anti-p43 antibody. B, a schematic representation of
Myc-tagged p43-F, -N, and -C. C, Myc-tagged
p43-F, -N, or -C was transiently overexpressed in 293 cells for 24 h. The cells were washed twice with pre-warmed phosphate-buffered
saline and then incubated in serum-free medium for 1, 3, or 5 h.
Proteins from whole cell lysate (WCL) and media were
analyzed by immunoblotting with anti-Myc antibody.
|
|
To confirm these data, we investigated whether excess p43 is
specifically secreted from 293 cells that do not normally release p43
and EMAP II (data not shown). We transiently overexpressed the
Myc-tagged full-length (p43-F), NH2-terminal domain (p43-N) or the EMAP II domain of p43 (EMAP II) (Fig. 1B) in 293 cells. Twenty hours after transfection, the cells were serum-starved for 1, 3, or 6 h. Proteins from the cell supernatant were
collected, concentrated, and analyzed by immunoblotting with anti-Myc
antibody. As shown in Fig. 1C, increasing amount of p43-F
and -N appeared in the medium, while EMAP II was barely detected even
though p43-F, p43-N, and EMAP II are equally overexpressed (left
panel indicated as WCL). Since the Myc-tagged p43-F was
targeted to the multi-ARS complex (data not shown), it is clearly
functional as endogenous p43. From these data, we conclude that p43-F
and -N could be secreted from cells in the absence of apoptosis signal
if their expression is induced.
Cytokine Domain Analysis--
To determine which regions of p43
are involved in its cytokine function, several deletion derivatives of
p43 were constructed. The mutant p43 constructs used in this study are
shown schematically in Fig.
2A. The mutant proteins were
purified as histidine-tagged fusion proteins using a bacterial
expression system, and the purified proteins were analyzed by
SDS-polyacrylamide gel electrophoresis (Fig. 2B). Endotoxin
from the purified proteins was removed by using a polymyxin affinity
column. To investigate TNF and IL-8 production by p43 mutant proteins,
the purified recombinant proteins were added to THP-1 cells. Two hours
after incubation, we determined TNF and IL-8 production by ELISA using
their specific antibodies. Since p43-(1-312), p43-(1-146),
p43-(1-108), and p43-(92-256) showed higher cytokine activity than
p43-(147-312) called EMAP II, the NH2-terminal domain of
p43, especially p43-(92-146), might be a stronger cytokine domain than
EMAP II. Meanwhile, p43-(218-312) and p43-(257-312) did not activate
TNF and IL-8 production. The results corresponded with previous work
(16), suggesting that the EMAP II-derived COOH-terminal domain is not
necessary for cytokine activity.

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 2.
Determination of the active cytokine domain
in p43. A, a schematic drawing of the p43 deletion
mutants used in this study. B, SDS-polyacrylamide gel
electrophoresis of each purified protein (1 µg). C,
effects of p43 and its deletion mutants on inducing TNF and IL-8. THP-1
cells were washed twice with pre-warmed phosphate-buffered saline and
incubated with serum-free medium in the presence of p43 or its deletion
mutant peptides for 2 h. TNF and IL-8 were determined from the
medium by ELISA test.
|
|
p43 Activates MAPKs and NF
B--
Activation of MAPKs and NF
B
are essential steps for the up-regulation of many proinflammatory
cytokines in human monocytes, implying that p43 could regulate the
activation of some signal transduction pathways. To address this
question, we tested whether p43 affects the activities of the major
signaling molecules such as MAPKs and NF
B. Among MAPKs, the
activation of ERK1/2 or p38 MAPK was determined by their
phosphorylation, and the activity of JNK was determined by the
phosphorylation of its substrate, c-Jun. The three tested MAPKs were
all activated by p43 in a time- and dose-dependent manner
(Fig. 3). Although three different MAPKs showed similar time courses of activation by p43, they showed different
sensitivities to the concentration of p43. The activation of ERK1/2 was
observed from 1 nM p43, while p38 MAPK and JNK were activated at higher concentrations of p43 (Fig. 3), suggesting that
these MAPKs are activated by different mechanisms.

View larger version (52K):
[in this window]
[in a new window]
|
Fig. 3.
p43-activated MAPKs and
NF B. The effect of p43 on the activities
of three MAPKs (ERK1/2, JNK, and p38) and NF B was investigated in
THP-1 cells. Left panel, THP-1 cells were treated with p43
(100 nM), and the activity change during the p43 treatment
was determined. The activity of each MAPK was determined as described
under "Experimental Procedures." "p~" stands for
the phosphorylated forms of each protein. The activation of NF B was
determined by degradation of I B. Right panel, the
activities of MAPKs and NF B at the different concentrations of p43
are shown. The cells were treated with p43 for 1 h.
|
|
We then tested whether NF
B is also activated by p43. The activity of
NF
B was determined by degradation of I
B that suppresses NF
B
(17). The level of I
B was decreased by the treatment of p43 in a
time-dependent manner and also from 1 nM p43
(Fig. 3), suggesting that p43 would also activate NF
B. The
activation of NF
B by p43 was also confirmed by electrophoresis
mobility shift assay with an oligonucleotide containing a NF
B
binding site (data not shown). Since the NH2-terminal
peptide of p43 and EMAP II showed the same pattern with p43 in the
activation of the tested signaling molecules (data not shown), p43 and
EMAP II might share the receptor for activating the signaling pathways.
p43 Activates the Production of Proinflammatory
Chemokines--
Since p43 activates three different MAPKs and NF-
B,
it is tempting to speculate that p43 could activate various genes
including those for inflammatory cytokines and chemokines. To
characterize changes in mRNA expression of the human monocyte in
response to p43, THP1 cells were treated with p43, and total mRNA
was extracted at various time points after the treatment. Radiolabeled
cDNA was prepared from mRNA by reverse transcription and
hybridized to the membrane that carries nonoverlapping arrays of
cDNAs for a range of known human genes. The amounts of radioactive
probes specifically bound to the cDNA array were analyzed by
densitometry, and relative increases in mRNA levels in the
p43-treated cells were calculated.
Stimulation of THP-1 cells with the full-length p43 resulted in
activation of over 37 genes, out of 1,176 genes tested. As shown in the
upper right panel of Fig. 4,
TNF is greatly up-regulated in p43-treated cells (30-fold increase over
the control), consistent with the result of ELISA (Fig. 2C).
Even though we used the cDNA array containing 1,176 genes involving
in oncogenesis, signal transduction, cell cycle, apoptosis,
transcription factors, receptors, cytokines, and chemokines, we found
that only 37 genes (mostly cytokine, chemokine, and receptor genes)
were highly activated (more than 3-fold increase over control) (Fig.
5), indicating that p43 induces the
expression of specific cytokine, chemokine, and receptor genes.

View larger version (97K):
[in this window]
[in a new window]
|
Fig. 4.
Determination of mRNAs induced by p43 in
THP-1 cells. THP-1 cells were untreated (left panel) or
treated with p43 (100 nM) for 2 h. Polyadenylated RNA
was reverse-transcribed into cDNA and labeled with 32P
and hybridized to Atlas cDNA array membranes containing 1,179 human
cDNAs. Autoradiograms from one out of three similar experiments are
shown. Strongly up-regulated genes in the bottom panel are
marked as black numbers.
|
|

View larger version (42K):
[in this window]
[in a new window]
|
Fig. 5.
Chemokines are highly induced in p43-treated
THP-1 cells. Cells were treated with 100 nM p43 for 0, 0.5, 2, or 6 h, and up-regulated genes were analyzed by cDNA
array. Each gene was quantified by a phosphoimager and normalized based
on three housekeeping genes. The genes showing more than 3-fold
increase over the control at any time point are listed here.
|
|
Cytokine and chemokine genes were the most highly induced genes by p43
stimulation (Fig. 4, lower right panel, and Fig. 5). For
example, TNF, MCP-1, MIP-1
, MIP-1
, IL-1
, IL-8, MIP-2
, and
MIP-1
were very strongly activated 6 h after the p43 treatment (more than 7-fold increase over the control). Since p43 began to
activate these genes as early as 30 min after the treatment, these
molecules would be directly induced by p43. Some receptor genes such as
IL-7R, ErbB-3R, ephrin receptor, NMBR, CD40L receptor, PGE receptor,
VEGFR1, and VEGFR2 were moderately induced by p43 (3-9-fold increase).
The induction of these receptor mRNAs might render the cells
sensitive to their specific ligands.
The expression of mRNAs for transcription factors HOXB7, ERF1,
IRF-7, c-Jun, and c-Myc are also activated. Since c-Jun and c-Myc
mRNA induction requires JNK activation, JNK activation by p43 (Fig.
3) could explain why c-Jun and c-Myc induction occurs as early as 30 min after the p43 treatment. Moreover, the mRNA level of the
adhesion molecule, ICAM-1, was increased after the p43 treatment
(11-fold increase at the time point of 2 h). This result is
consistent with the previous report on the cell-cell adhesion induced
by p43 (2).
To verify the results obtained with the cDNA array, we used ELISA
to quantify the amounts of MCP-1 and MIP-1
. THP-1 cells were treated
with p43 (100 nM) for 3 h, and the concentration of
MCP-1 and MIP-1
was measured from cellular supernatant. As shown in
Fig. 6, p43 induced secretion of MCP-1
and MIP-1
, consistent with the cDNA array data (Figs. 4 and
5).

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 6.
Induction of MCP-1 and MIP-1 by p43.
THP-1 cells were washed twice with serum-free medium and treated with
100 nM p43 for 6 h. MCP-1 and MIP-1 from medium were
determined by ELISA using their specific antibodies. The assay was
repeated three times and the averages were shown.
|
|
High Amount of p43 Is Detected in the Foam Cells of
Atherosclerosis Lesions--
The above data raise the intriguing
possibility that p43 could be involved in the process of inflammation,
since cytokines and chemokines such as TNF, MCP-1, MIP-1
, IL-1
,
and IL-8 are found in inflammation areas. A pivotal question, then, is
whether p43 is actually expressed in such lesions. We thus examined the level of p43 protein in atherosclerotic lesions by immunohistochemistry using sections of aorta from a human patient carrying atherosclerosis. Expression of p43 was mainly restricted to regions rich in foam cells
that were detected by a foam cell-specific marker, CD68 (Fig.
7). Meanwhile, a basal level of p43
expression was detected in smooth muscle cells that were detected by a
smooth muscle-specific marker
-actin.

View larger version (79K):
[in this window]
[in a new window]
|
Fig. 7.
High amount of p43 is present in the foam
cells of atherosclerosis lesions. A, foam cell-
(upper panel) or smooth muscle cell-rich area (lower
panel) in the neo-intima is shown (× 400). p43 was localized by
immunohistochemistry using anti-p43 antibody. Foam cells and smooth
muscle cells were identified with immunohistochemistry using anti-CD68
and anti- -actin antibodies, respectively. The localization of HLA-DR
indicates that the foam cells are in an activated state. B,
immunoblot analysis of p43 expression in atherosclerotic plaques.
Different regions of atherosclerotic plaques were used to prepare the
protein extract, and 20 µg of each of the preparations was used for
immunoblot analysis with anti-p43 antibody. Lane 1 represents protein extract derived from a fibrous plaque containing
dominant smooth muscle cells with a thick fibrous cap. Lane
2 represents protein extract from an atheromatous plaque with
heavy infiltration of foam cells and a thin fibrous cap.
|
|
Since the p43 antibody for the immunostaining is a polyclonal antibody
raised against the full-length p43 (8), and p43 may be cleaved into the
fragments including EMAP II, it is hard to conclude that the signals
detected by immunostaining indeed represent the full-length p43. To
address the issue, we prepared protein extracts from a fibrous plaque
containing dominant smooth muscle cells, and an atheromatous plaque
containing foam cells, and then analyzed the extracts by immunoblotting
with anti-p43 antibody. As expected, the atheromatous plaque showed a
high level of human p43 compared with a fibrous plaque. In addition,
there was only one band (34 kDa) that represents a human p43 protein. The apparent molecular mass of human p43 was previously
determined to be 34 kDa (15). Based on these data, we concluded that
immunostaining signal in Fig. 7A represents the full length
of p43. Thus, these data strongly support the involvement of p43 in
atherosclerosis by inducing cytokines and chemokines that are key
molecules in atherosclerosis development.
 |
DISCUSSION |
Without phagocyte clearance of apoptotic cells, cellular proteins
released from dying cells are harmful to surrounding cells. Thus, the
dying cells should be removed rapidly. EMAP II has been shown to be an
active cytokine that recruits monocytes and macrophages in the
apoptosis areas for scavenging apoptotic corps. However, our data
demonstrated that EMAP II could not be a good candidate for mediating
the scavenger, since EMAP II was released with other cellular proteins
after cells were completely destroyed by apoptosis (Fig.
1A). Instead of EMAP II, we found that p43 (a precursor of
EMAP II) is selectively secreted as early as 30 min after serum starvation, implying that p43 itself would work as an active cytokine.
The expression level of p43 is spatially and temporally changed.
For instance, the mRNA and protein level of p43 decrease in the
developing lungs of the fetal mouse, while they remain low throughout
postnatal life with the exception of a surge at postnatal days 8-16
(11). In addition, p43 mRNA and protein are localized to the
epithelium, with its highest expression in neurons, blood vessels, and
at sites of epithelial-mesenchymal interaction (18). Interestingly, p43
is up-regulated in the differentiation stage of hematopoietic stem
cells (19). When p43 was highly expressed in 293 cells by transient
transfection, it was secreted from the cells that did not release p43
in normal condition (Fig. 1B). This implies that p43 itself
could be secreted from cells in which p43 is up-regulated. It would be
interesting to understand the signal that induces the expression of p43.
Although p43 was found to be released from cells
previously, its ability to function as a proinflammatory cytokine has
been neglected. Deletion mapping of EMAP II identified that the
NH2-terminal heptamer peptide is responsible for the
cytokine activity (16). However, the data in the present work shows
that the full-length and NH2-terminal peptide of p43 are
more active in the production of proinflammatory cytokines than EMAP II
(Fig. 2). Thus, the suggested peptide of EMAP II does not seem to be
the only portion responsible for the cytokine activity.
The full-length p43 has one known function helping aminoacylation of
the bound ARSs within cell and another function as a cytokine. There
are several other proteins with different functions depending on their
cellular localization (20). For example, phosphoglucose isomerase,
thymidine phosphorylase, and Hsp70 function as metabolic enzymes or
chaperoning protein within cells, whereas they work as cytokines when
secreted from cells (20, 21). Like these proteins, p43 lacks a signal
peptide necessary for membrane translocation, and the molecules may be
secreted without any processing.
Based on the results of this work, the proteolytic cleavage of p43 does
not appear to be the prerequisite to generate an active cytokine. This
is in contrast to the case of human tyrosyl-tRNA synthetase (22). This
enzyme is released from apoptotic cells and split into two distinct
cytokines with elastase. However, tyrosyl-tRNA synthetase itself did
not show the cytokine activities, indicating that the proteolysis is
required to activate each cytokine. If the proteolysis of p43 is not
required to activate or release EMAP II, its physiological reason
remains unclear. We have shown previously that the intact p43 is
required for its stimulatory effect on the bound arginyl-tRNA
synthetase (8). Thus, the cleavage of p43 with caspase-7 upon apoptosis
would disrupt its stimulatory role in protein synthesis, which may
further accelerate cell death. Based on these data, the functional
reason for the p43 cleavage does not appear to activate cytokine but to
break protein synthesis machinery in apoptotic cells. This notion was also mentioned in the release of human tyrosyl-tRNA synthetase from
apoptosis cells (22). The leakage of an enzyme essential for protein
synthesis would inhibit protein synthesis and thus further accelerate apoptosis.
Our cDNA array data demonstrate that p43 up-regulates the
chemokines that are involved in the main proinflammatory response. The
most strongly induced chemokines are MCP-1, MIP-1
, MIP-1
, and
RANTES (regulated on activation normal T cell expressed and secreted)
that belong to the CC chemokine subfamily. They exhibit chemotactic
activity primarily for monocytes and T cells and activate T cells and
macrophages (23, 24). Other chemokines induced by p43 are IL-8 and
MIP-2
that belong to the CXC chemokine subfamily and are chemotactic
primarily for neutrophils and stimulate neutrophil degranulation,
adhesion, and microbicidal activity (23-25). These chemokines are also
induced by LPS, but the mRNA expression profile by p43 is quite
different from that by LPS or INF
(25-27). For example, DB-1,
HLA-1, MRP-14, TMSb-10, and IL-6 are highly up-regulated by LPS or
INF
(25), but not by p43, suggesting that p43 is a specific
proinflammatory cytokine that up-regulates a unique set of genes.
It is tempting to speculate that p43 itself could be an initiator for
the inflammation process, since p43 induces proinflammatory chemokines
that are involved in a number of inflammatory diseases, including
atherosclerosis, multiple sclerosis, and experimental autoimmune
encephalomyelitis (28-31). Previous works showed that p43 is highly
expressed in autoimmune inflammatory regions such as encephalomyelitis,
neuritis, and uveitis (12, 32). We also observed that p43 was present
in high concentration in the foam cells of human atherosclerosis
lesions (Fig. 6), suggesting that p43 is also involved in the
inflammatory response during atherosclerosis procedure. Therefore, it
should be noted that p43 induces MCP-1 and MIP-1
as well as TNF,
myeloperoxidase, and tissue factor that are major factors inducing
atherosclerosis (28, 29). Further investigation on the role of p43 in
the proinflammatory response would help to understand the pathological
process leading to these inflammation-related diseases.