Effect of Oxygen on Induction of the Cystine Transporter by
Bacterial Lipopolysaccharide in Mouse Peritoneal Macrophages*
Hideyo
Sato
§¶,
Kazumi
Kuriyama-Matsumura
§,
Taro
Hashimoto
,
Hiromi
Sasaki
,
Hongyu
Wang
,
Tetsuro
Ishii
,
Giovanni E.
Mann
, and
Shiro
Bannai
From the
Department of Biochemistry, Institute of
Basic Medical Sciences, University of Tsukuba, Tsukuba, Ibaraki
305-8575 Japan and the
Centre for Cardiovascular Biology and
Medicine, Guy's, King's, and St. Thomas' School of Biomedical
Sciences, King's College London, LONDON SE1 1UL, United Kingdom
Received for publication, August 9, 2000, and in revised form, January 2, 2001
 |
ABSTRACT |
Amino acid transport in mouse peritoneal
macrophages is mediated by several membrane carriers with different
substrate specificity and sensitivity to environmental stimuli.
We reported previously that transport activities of cystine and
arginine in the macrophages were induced markedly by low concentrations
of bacterial lipopolysaccharide (LPS). It is known that a
variety of macrophage functions are affected by ambient oxygen tension.
In this study, we have investigated the effects of oxygen on the
induction of amino acid transport activity by LPS and found that the
induction of cystine, but not arginine, transport activity was
dependent on the ambient oxygen tension. When the macrophages were
cultured with 2% O2 in the presence of 1 ng/ml LPS,
induction of cystine transport activity was reduced by ~70% compared
with cells cultured under normoxic conditions. In macrophages,
transport of cystine is mediated by a Na+-independent
anionic amino acid transporter named system
x
. System
x
is composed of two protein
components, xCT and 4F2hc, and the expression of xCT was closely
correlated with system x
activity.
A putative NF-
B binding site was found in the 5'-flanking region of
the xCT gene, but the enhanced expression of xCT by LPS and oxygen was not mediated by NF-
B binding. An increase in
intracellular GSH in macrophages paralleled induction of xCT, but not
-glutamylcysteine synthetase. These results suggest the importance of system x
in
antioxidant defense in macrophages exposed to LPS and oxidative stress.
 |
INTRODUCTION |
Transport of amino acids across the plasma membrane of mammalian
cells is mediated by several systems with different substrate specificity (1). We have previously characterized anionic, neutral, and
cationic amino acid transport systems in mouse peritoneal macrophages,
which are known to be activated by a variety of factors such as
bacterial lipopolysaccharide
(LPS)1 and cytokines (2-4).
Macrophages exhibit cytoprotective functions including antigen
presentation and microbicidal or tumoricidal activity. The major
anionic amino acid transport system in macrophages is system
x
, which mediates the exchange of an
anionic form of cystine and glutamate across the plasma membrane (2).
The transport system for neutral amino acids in macrophages seems to be
unique, transporting most neutral substrates such as serine, alanine,
and leucine via a Na+-independent mechanism (3). For
cationic amino acids, a system y+-like cationic amino acid
transport system mediates the uptake of arginine, lysine, and
ornithine, although the system in macrophages has slightly different
characteristics from those of the typical system y+
(4).
We have found that a very low concentration of LPS markedly enhances
the activities of cystine and arginine transport in mouse peritoneal
macrophages (5, 6). The enhanced uptake of cystine results from the
induction of system x
activity and
increased influx of arginine is because of the induction of the typical
system y+ activity. The induction of cystine transport may
contribute to the maintenance of intracellular GSH levels because
cystine taken up by the cells is reduced to cysteine, a rate-limiting
precursor for GSH synthesis (7). In contrast, the induction of the
arginine transport activity is a key event for production of nitric
oxide involved in microbicidal and tumoricidal processes in activated macrophages (8, 9). A cDNA encoding a protein with properties consistent with system y+ in LPS-activated mouse
macrophages has been identified and named mCAT-2B (10). Recently, we
reported that system x
is composed of
two proteins and have cloned cDNAs for these proteins, namely 4F2hc
(heavy chain of the surface antigen 4F2, also named CD98) and xCT (11).
Coexpression of the ubiquitous transmembrane protein 4F2hc with xCT
(and cDNAs of some other amino acid transporters) has been shown to
induce amino acid transport activity in Xenopus oocytes
(12-15).
Recent studies have shown that in macrophages, ambient oxygen tension
alters the morphology, expression of cell surface markers, viability,
phagocytosis, metabolic activity, and release of cytokines (16). It is
likely that antioxidant systems such as intracellular GSH are affected
by ambient oxygen tension and/or oxidative stress in these cells
activated by LPS. In the present study, we have investigated the
effects of oxygen on the induction of cystine and arginine transport
activity in the macrophages caused by treatment with LPS. We have
established that induction of cystine transport by LPS is dependent on
ambient oxygen levels, whereas LPS-induced activation of arginine
transport via system y+ was unaffected.
 |
EXPERIMENTAL PROCEDURES |
Materials--
L-[14C]Cystine,
L-[14C]arginine, and
L-[14C]serine were obtained from PerkinElmer
Life Sciences. Thioglycolate broth (Brewer's formula) and Bacto LPS
(Salmonella typhosa 0901) were from Difco Laboratories, Detroit, MI. Fetal bovine serum was obtained from BioWhittaker, Walkersville, MD, and the lot which contains less than 0.3 ng/ml endotoxin was used. Monobromobimane was purchased from Molecular Probes, Inc., OR.
Macrophage Culture--
Macrophages were collected by peritoneal
lavage from female C57BL/6 mice weighing 20-25 g who had previously
received 4 days prior an intraperitoneal injection of 2 ml of 4%
thioglycolate broth. The lavage medium was RPMI 1640 containing 10 units/ml heparin. The cells were washed twice with RPMI 1640 and plated at 1 × 106 cells/35-mm diameter culture dish
containing RPMI 1640, 10% fetal bovine serum, 50 units/ml penicillin,
and 50 µg/ml streptomycin. Cells were incubated at 37 °C in 5%
CO2, 95% air, and after 1 h the medium was replaced
to remove nonadherent cells. For hypoxic conditions, cells were
cultured in a gas-tight chamber flushed with 95% N2 and
5% CO2, and the required concentrations of O2 were measured using an oxygen meter.
Amino Acid Uptake--
Amino acid uptake was measured using
techniques described previously (17). Cells were rinsed three times in
warmed PBSG (10 mM phosphate-buffered saline (137 mM NaCl, 3 mM KCl), pH 7.4, containing 0.01%
CaCl2, 0.01% MgCl2·6H2O, and
0.1% glucose) and then incubated in 0.5 ml of the warmed uptake medium
at 37 °C for specified time periods. The uptake medium was PBSG with
a labeled amino acid (0.1 µCi/0.5 ml). Uptake was terminated by rapidly rinsing the culture dishes three times with ice-cold
phosphate-buffered saline, and radioactivity associated with cell
extracts was determined as described previously (17). Amino acid
uptake was determined under conditions approaching initial rates of
uptake, i.e. measuring uptakes for cystine, serine, and
arginine at 120, 30, and 15 s, respectively. For each of the amino
acids, uptake increased linearly during the specified incubation interval.
Determination of Intracellular GSH Levels and Efflux of
GSH--
Intracellular GSH was extracted with 5% trichloroacetic acid
and then treated with ether to remove the acid. The GSH content in the
aqueous layer was measured using an enzymatic method described previously, which is based on the catalytic action of GSH in the reduction of 5,5'-dithiobis(2-nitrobenzoic acid) by the GSH reductase system (18). The GSH extracted from the cells was mostly reduced GSH,
and the content of the oxidized form, GSSG, was negligible throughout
the experiments in this study. The efflux of GSH was measured as
follows. The cells were rinsed three times with PBSG and incubated with
0.5 ml of PBSG at 37 °C for 1 h. Then GSH in the PBSG was
quantified by the method described above.
Measurement of Intracellular Cysteine--
The cysteine content
in the cells was determined by the method of Cotgreave and
Moldéus (19) with a slight modification (20). The cells were
rinsed three times with PBSG and incubated in the dark at room
temperature for 10 min with 100 µl of 8 mM monobromobimane in 50 mM N-ethylmorpholine, pH 8 and 100 µl of 50 mM phosphate-buffered saline containing
0.01% CaCl2, 0.01% MgCl2·6H2O,
and 0.1% glucose. Then 10 µl of 100% trichloroacetic acid was
added. The protein precipitate was removed by centrifugation at
3000 × g for 5 min and aliquots were analyzed for
cysteine-bimane adduct by HPLC. The HPLC separation was achieved on a
steel column (4.6 × 100 mm) packed with 3-µm octadodecylsilica
reversed-phase material. The fluorescence at 480 nm was monitored with
excitation at 394 nm. The elution was performed with 9% (v/v)
acetonitrile in 0.25% (v/v) acetic acid, pH 3.7 for 8 min, and then
with 75% (v/v) acetonitrile in water for 5 min. The flow rate was 1 ml/min throughout the process.
Northern Blot Analysis--
The mouse cDNAs for xCT, 4F2hc,
-GCS, and
-actin were used as probes. The probes were labeled
using [
-32P]dCTP and RediprimeTM II random
prime labeling system (Amersham Pharmacia Biotech). Ten µg of total
RNA was electrophoresed, transferred, and hybridized as described
previously (11).
Isolation of Genomic Mouse xCT Clones and Sequence
Analysis--
Mouse genomic library (Stratagene) was screened using
mouse xCT cDNA as a probe. The positive plaques were isolated, and
one of the clones that contained exon 1 and the 5'-flanking region of
the gene was subcloned into pBluescript. The transcription initiation
site was determined using a 5'-RACE system for rapid amplification of
cDNA ends, version 2.0 (Life Technologies, Inc.) and Primer
Extension System (Promega) following the manufacturer's protocols.
Isolation of Nuclear Extracts and Electrophoretic Mobility Shift
Assay--
The nuclear extracts were prepared by a modified procedure
based on the method of Monick et al. (21). Briefly, cells (5 × 106) were washed with phosphate-buffered saline and
collected by centrifugation. The cell pellets were resuspended in 0.2 ml of lysis buffer (10 mM HEPES, pH 7.9, 10 mM
KCl, 2 mM MgCl2, 0.1 mM EDTA) and
incubated for 15 min on ice. The cells were vortexed for 30 s
after addition of Nonidet P-40 (0.6%), then centrifuged, and the
nuclear pellet was resuspended in 20 µl of extraction buffer (50 mM HEPES, pH 7.9, 50 mM KCl, 300 mM
NaCl, 0.1 mM EDTA, 10% glycerol). After a 20-min
incubation on ice, followed by a 5-min centrifugation, nuclear proteins
were recovered from the supernatant. Electrophoretic mobility shift
assays were performed as described previously using a
32P-radiolabeled oligonucleotide corresponding to the
consensus NF-
B DNA-binding site (22). The DNA probes (20,000 cpm)
were incubated with 3 µg of nuclear protein under the binding
conditions (10 mM Tris-HCl, pH 7.8, 100 mM KCl,
5 mM MgCl2, 1 mM dithiothreitol, 1 mM EDTA, 2 µg of poly(dI-dC), and 5% glycerol) in a
final volume of 20 µl. The reactions were carried out at 4 °C for
15 min and products were electrophoresed on native 6% polyacrylamide
gels. The gels were analyzed by autoradiography.
 |
RESULTS |
The activity of cystine transport was measured in mouse peritoneal
macrophages cultured under various oxygen tensions in the absence or
presence of LPS (Fig. 1). Cystine
transport activity was induced by LPS in an oxygen
tension-dependent manner. However, hyperoxia (50%
O2) significantly reduced the activity of cystine transport. To establish whether other amino acid transporters responded
in a similar manner, we investigated the effect of oxygen tension on
arginine and serine transport activity (Fig.
2). Although arginine transport was
induced markedly by the low concentrations of LPS, as reported
previously (6), the induction of transport was unaffected by hypoxia.
Neither LPS nor oxygen tension had any effect on the activity of serine
transport.

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Fig. 1.
Effect of oxygen on the uptake of cystine by
macrophages incubated with LPS. Macrophages were incubated under
hypoxia (2% O2) for 1 h, and the medium was replaced
by fresh medium with (filled bars) or without (open
bars) 1 ng/ml LPS. The cells were further incubated under oxygen
for the concentrations indicated for 11 h, and the rate of uptake
of 0.05 mM L-[14C]cystine was
measured. Each point represents the mean ± S.D.
(n = 4-6).
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Fig. 2.
Effects of oxygen on the uptake of arginine
(A) and serine (B) by macrophages
incubated with LPS. Macrophages were incubated under hypoxia (2%
O2) for 1 h, and the medium was replaced by fresh
medium with (filled bars) or without (open bars)
1 ng/ml LPS. The cells were further incubated under hypoxia (2%
O2) or normoxia (20% O2) for 11 h, and
the rates of uptake of 0.05 mM
L-[14C]arginine and
L-[14C]serine were measured. Each point
represents the mean ± S.D. (n = 4-6).
|
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The dose-dependent expression of xCT and 4F2hc mRNAs by
LPS was investigated in macrophages cultured under 20% O2
(normoxia) and 2% O2 (hypoxia). As shown in Fig.
3A, under normoxia, expression of xCT was not detected in the cells in the absence of LPS, but three
xCT transcripts (~12, 3.5, and 2.5 kilobases) from cells treated with
1 ng/ml LPS were observed. These multiple bands may represent
alternative splicing, alternative polyadenylation sites, or a
combination of both (11). The expression of these transcripts was
significantly decreased in cells treated with 1000 ng/ml LPS. These
results are consistent with the previous report that the induction of
the activity of system x
is maximal
in macrophages treated with 1 ng/ml (5). The expression of xCT in cells
treated with 1 ng/ml LPS under hypoxia was lower than that in the cells
treated with the same amount of LPS under normoxia. The expression of
xCT mRNA in the cells treated with 1000 ng/ml under hypoxia was
hardly detected. Fig. 3B shows the time-dependent expression of xCT and 4F2hc mRNAs in
cells cultured with 1 ng/ml LPS under hypoxia and normoxia. The
expression of xCT mRNA was not detected in the freshly isolated
macrophages, but expression increased gradually, reaching maximal
levels after 8-12 h of culture under normoxia. Expression of xCT
mRNA in cells cultured under hypoxia was increased over a similar
time course, although expression was significantly lower than that in
cells cultured under normoxia. The expression of 4F2hc also increased during culture with LPS, but changes in oxygen tension had negligible effects on expression. These results suggest that the decreased activity of cystine transport in the cells cultured with LPS under hypoxia is caused in part by the transcriptional regulation of xCT
mRNA expression.

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Fig. 3.
Changes in the expression of xCT and 4F2hc
mRNAs in macrophages cultured with LPS. A,
macrophages were incubated under hypoxia (2% O2) for
1 h, and the medium was replaced by fresh medium containing 0, 1, or 1000 ng/ml LPS. The cells were further incubated for 7 h under
hypoxia (2% O2) or normoxia (20% O2), and
total RNA was isolated. B, macrophages were incubated under
hypoxia (2% O2) for 1 h, and the medium was replaced
by fresh medium containing 1 ng/ml LPS. The cells were further
incubated under hypoxia or normoxia with 1 ng/ml LPS for the specified
time intervals (3, 7, 11, and 23 h), and total RNA was isolated.
Ten µg each of total RNA were loaded per lane. The
hybridization was performed with 32P-labeled cDNAs of
mouse xCT, 4F2hc, and -actin.
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We have isolated the xCT gene from the mouse genome library
and analyzed the sequence of 5'-flanking region. As shown in Fig. 4, there are several putative AP-1
binding sites and a putative NF-
B binding site. We have investigated
the effects of oxygen on the binding activity of NF-
B by LPS. The
binding activity of NF-
B remained relatively low in the macrophages
treated with 1 ng/ml LPS, and a significant increase was observed when
the cells were treated with 1000 ng/ml LPS (Fig.
5). Oxygen did not affect the NF-
B
binding activity. The pattern of the NF-
B binding was inconsistent
with those of xCT mRNA, suggesting that the induction of xCT
mRNA by LPS and oxygen is not mediated by NF-
B.

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Fig. 4.
Sequence of the 5'-flanking region of the
mouse xCT gene. The clone containing the exon 1 and 5'-flanking region of the xCT gene was isolated,
sequenced, and the transcription initiation site was determined as
described under "Experimental Procedures." The sequence of exon 1 is represented in capital letters. The putative NF- B
binding site is boxed. Putative AP-1 binding sites and a
TATA-like box are represented by underlines and a
double underline, respectively. The AP-1 site indicated by
an asterisk partially overlaps the electrophile response
element.
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Fig. 5.
Activation of NF- B
in macrophages cultured with LPS under hypoxia and normoxia.
Macrophages were incubated under hypoxia (2% O2) for
1 h, and the medium was replaced by fresh medium containing 0, 1, or 1000 ng/ml LPS. The cells were further incubated for 1 h under
hypoxia or normoxia (20% O2), and the nuclear extracts
were prepared. The electrophoretic mobility shift assay was performed
as described under "Experimental Procedures."
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We have previously demonstrated that intracellular GSH levels are
significantly increased in the macrophages cultured with LPS (5). It is
known that
-glutamylcysteine synthetase (
-GCS) catalyzes the
rate-limiting step in de novo GSH synthesis. We have
investigated the effect of LPS and oxygen on the expression of
-GCS
in these cells. Fig. 6 shows the
expression of mRNAs for
-GCS and xCT in the cells cultured with
diethyl maleate or LPS under normoxic or hypoxic conditions. Diethyl
maleate is an electrophilic agent, which reacts with GSH enzymatically
or nonenzymatically. The expression of
-GCS mRNA was induced
markedly by diethyl maleate, but unaffected by LPS. As shown in Fig.
7, the intracellular GSH level in
LPS-challenged cells cultured under hypoxia for 12 h was
significantly lower than the level in cells cultured under normoxia.
These results are consistent with the results that the cystine
transport activity in the cells treated with LPS is decreased under
hypoxia (Fig. 1). In comparison with GSH levels in cells treated
without LPS under normoxia, the GSH level was 1.8-fold higher in cells
treated with LPS. By contrast, the GSH level in cells cultured under
normoxia for 12 h with diethyl maleate, which induces both
mRNAs for system x
and
-GCS
(Fig. 6), were increased 2.4-fold.

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Fig. 6.
Effects of diethyl maleate and LPS on the
expression of -GCS and xCT mRNAs in
macrophages incubated under hypoxic and normoxic conditions.
Macrophages were incubated under hypoxia (2% O2) for
1 h, and the medium was replaced by fresh medium (Cont)
in the presence of 100 µM diethyl maleate
(DEM), or 1 ng/ml LPS (LPS). The cells were
further incubated under hypoxia or normoxia (20% O2) for
7 h, and total RNA was isolated. Ten µg each of total RNA was
loaded per lane. The hybridization was performed with
32P-labeled cDNAs (mouse -GCS, xCT, 4F2hc, and
-actin).
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Fig. 7.
Intracellular GSH levels in macrophages
incubated with diethyl maleate and LPS under hypoxic and normoxic
conditions. Macrophages were incubated under hypoxia (2%
O2) for 1 h, and the intracellular GSH level was
measured. For other cells, the medium was replaced by fresh medium
(Cont) containing 100 µM diethyl maleate
(DEM), or 1 ng/ml LPS (LPS). The cells were
further incubated under hypoxia (2% O2, open
bars) or normoxia (20% O2, hatched bars)
for 3 and 11 h, and intracellular GSH levels were measured. Each
point represents the mean ± S.D. (n = 4-6).
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It is of importance to determine whether the intracellular cysteine,
which is a rate-limiting substrate for GSH synthesis, increases in
response to induction of cystine transport activity. We investigated
the intracellular cysteine levels in the cells cultured with or without
LPS under hypoxia or normoxia (Fig. 8). The intracellular cysteine paralleled the activity of system
x
, supporting the hypothesis that the
induction of system x
activity is
responsible for increased GSH. It might be possible that LPS and/or
oxygen inhibit the efflux of GSH from the cells, and this inhibition
accounts for increased intracellular GSH. However, as shown in Fig.
9, the rate of efflux of GSH was higher in the cells cultured with LPS than that in the cells cultured without
LPS. The higher rate of the efflux probably reflects the higher
intracellular GSH level, because the efflux of GSH is carrier-mediated and depends on the intracellular GSH levels (23). Thus, it is unlikely
that the GSH efflux system is affected by LPS and/or oxygen. Taken
together, the present findings indicate that the elevation in
intracellular GSH levels caused by LPS is dependent on the induction of
xCT but not
-GCS. By contrast, the increased intracellular GSH
levels caused by diethyl maleate are due to an induction of both xCT
and
-GCS. It is worth noting that GSH levels decreased significantly
in the cells treated with diethyl maleate for 4 h. This initial
depletion of intracellular GSH levels seems to be the result of an
enzymatic reaction between GSH and diethyl maleate catalyzed by
glutathione S-transferase (24).

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Fig. 8.
Intracellular cysteine levels in macrophages
incubated with LPS under hypoxic and normoxic conditions.
Macrophages were incubated under hypoxia (2% O2) for
1 h, and the medium was replaced by fresh medium (Cont)
containing 1 ng/ml LPS (LPS). The cells were further
incubated under hypoxia (2% O2) or normoxia (20%
O2) for 11 h, and intracellular cysteine levels were
measured as described under "Experimental Procedures." Each point
represents the mean ± S.D. (n = 4-6).
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Fig. 9.
Effect of LPS on GSH efflux in macrophages
incubated under hypoxic and normoxic conditions. Macrophages were
incubated under hypoxia (2% O2) for 1 h, and the
medium was replaced by fresh medium (Cont) containing 1 ng/ml LPS (LPS). The cells were further incubated under
hypoxia (2% O2, open bars) or normoxia (20%
O2, striped bars) for 11 h, and efflux of
GSH was measured as described under "Experimental Procedures." Each
point represents the mean ± S.D. (n = 4-6).
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 |
DISCUSSION |
In a variety of types of cells in culture, the intracellular GSH
level is regulated by the activity of system
x
(7).
-GCS is known to be the
rate-limiting enzyme in GSH synthesis and also regulates GSH level. The
enzyme is induced by various stress agents including the electrophilic
agent, diethyl maleate (25). This agent also induces the activity of
system x
(5). Therefore, the increase
in intracellular GSH level caused by diethyl maleate is most likely
caused by the induction of both
-GCS and system
x
. In the present study, we have
demonstrated that LPS and oxygen strongly induce the expression of xCT
mRNA without significantly altering
-GCS mRNA levels. The
intracellular cysteine level was increased concomitantly with the
increase in cystine transport activity. The increase in the
intracellular GSH level by LPS reflects the increase in intracellular
cysteine. The increase in GSH level in the macrophages exposed to LPS
is mainly dependent on the induced activity of system
x
, not the activity of
-GCS.
The results of the present study indicate that the induced activity of
system x
by LPS is further enhanced
by oxygen. It is likely that the activity of system
x
is relatively low in unstimulated
macrophages in vivo, because average oxygen tension in the
peritoneum is ~5% (26). Once these cells are activated by
endotoxins, the activity of system x
is induced, and oxygen or reactive oxygen species probably enhances the
induction. Because GSH antagonizes the oxygen toxicity or oxidative
stress, it is likely that GSH turns over rapidly in the macrophages
exposed to endotoxins and oxidative stress in a pathological state such
as inflammation. The response of system x
activity to LPS and oxygen may be advantageous to the macrophages, which are elicited into the
inflammatory regions.
Recently, Li et al. (27) have demonstrated that chronic
exposure of bovine aortic endothelial cells to
S-nitroso-N-acetylpenicillamine (NO donor) led to
a concentration-dependent increase in cystine transport
activity and that the increased cystine transport activity is mediated
by system x
. It might be that the
induction of xCT mRNA in macrophages by LPS is mediated by NO
produced by the cells. However, as we demonstrated previously (9), NO
production by the macrophages incubated with 1 ng/ml LPS was almost
negligible. Nevertheless, the activity of system x
reaches maximum by 1 ng/ml LPS. Thus, the involvement of NO in the induction of xCT mRNA under the
conditions used in the present study is eliminated.
We have identified several AP-1 binding sites in the 5'-flanking region
of the xCT gene. Several AP-1 sites also exist in the
5'-flanking region of the human 4F2hc gene (28). These sites may be involved in the regulation of the expression of xCT and 4F2hc.
However, induction of system x
activity by phorbol myristate acetate is very low in comparison with
LPS under normoxia (5), suggesting only a limited role for AP-1 in the
stimulation of xCT mRNA transcription by LPS. We have also found
that there is a putative NF-
B binding site in the 5'-flanking
region. NF-
B plays a central role in the regulation of many genes
involved in cellular defense mechanisms and expression of cytokines.
Reactive oxygen species have been proposed as the intermediate second
messengers involved in the activation of NF-
B by TNF-
and IL-1
(29). Recent studies have demonstrated that the LPS signal is mediated
by the Toll-like receptor 4 in the presence of CD14 and activates
NF-
B in macrophages (30). Perhaps NF-
B is one of the candidate
transcription factors involved in the induction of xCT mRNA by LPS
and oxygen. However, in the presence of LPS, the activation of NF-
B
derived from the macrophages cultured under hypoxia seemed to be
similar to that derived from the cells cultured under normoxia (Fig.
5). These results are inconsistent with those of the uptake of cystine.
In addition, NF-
B was hardly activated by 1 ng/ml LPS, whereas
induction of system x
activity was
maximally activated by 1 ng/ml (5). In contrast, NF-
B was potently
activated by 1000 ng/ml LPS, nevertheless expression of xCT in cells
treated with 1000 ng/ml LPS was significantly lower than that in the
cells treated with 1 ng/ml LPS. These results suggest that other
signaling pathway(s) exist downstream of the signal transduction
involved in the induction of xCT mRNA by LPS and oxygen, although
it is not clear at present which transcription factor is involved in
the induction of xCT mRNA by LPS and oxygen.
Studies using transcription factor Nrf2-knockout mice have
revealed that expression of phase II enzymes is regulated by
Nrf2, whose activity is induced by oxidative stress and
electrophilic agents (31). Transcription factor Nrf2 is thought
to bind to the electrophile response element (EpRE) in the 5'-flanking
region of these genes and to enhance the activity of transcription. The gene for
-GCS contains the EpRE in its 5'-flanking region, and this
region is important for inducible expression of
-GCS by the
electrophilic agent (32). We have found that the mouse xCT gene also contains an EpRE-like sequence in its 5'-flanking region (Fig. 4), suggesting that its expression is regulated by the
electrophilic agent in a manner similar to that documented for
-GCS.
Recently, we have shown that in peritoneal macrophages from
Nrf2-deficient mice, LPS still potently induces the activity of
system x
, whereas diethyl maleate
does not (33). This result indicates that the signal initiated by LPS
is transduced by a different mechanism from EpRE-mediated
transcription. Gene expression of xCT seems to be an interesting model
system for study of signal transduction from LPS, because the
expression is regulated by a pathophysiological concentration of LPS
and is oxygen-sensitive.
 |
ACKNOWLEDGEMENT |
We thank Dr J. K. Andersen for kindly
providing the cDNA for mouse
-GCS.
 |
FOOTNOTES |
*
This work was supported by the British Council and Japanese
Ministry of Education, Science, and Technology.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) AB037650-AB037661.
§
These two authors contributed equally to this work.
¶
To whom correspondence should be addressed. Tel.:
81-298-53-3282; Fax: 81-298-53-3039; E-mail:
hideyo-s@md.tsukuba.ac.jp.
Published, JBC Papers in Press, January 2, 2001, DOI 10.1074/jbc.M007216200
 |
ABBREVIATIONS |
The abbreviations used are:
LPS, lipopolysaccharide;
4F2hc, 4F2 heavy chain;
-GCS,
-glutamylcysteine synthetase;
EpRE, electrophile response
element.
 |
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