 |
INTRODUCTION |
The tripeptide,
L-
-glutamyl-L-cysteinylglycine, or
glutathione (GSH), is a ubiquitous cellular non-protein sulfhydryl,
which plays an important role in maintaining intracellular redox
balance and in cellular defenses against oxidative stress (1, 2). GSH
is present in high concentrations in lung epithelial lining fluid (3).
It also has an important role in maintaining the integrity of the
airspace epithelium, in both type II alveolar cells in
vitro, and in lungs in vivo (4, 5). A physiological role for GSH as an antioxidant has been described in numerous inflammatory disorders (6). Depletion of lung epithelial lining fluid
GSH has been described in conditions such as human immunodeficiency virus infection (7), idiopathic pulmonary fibrosis (8), adult
respiratory distress syndrome (9), and cystic fibrosis (10). Elevated
levels of GSH occur in the epithelial lining fluid of chronic smokers
and in patients with lung cancer (11).
GSH is synthesized by two enzymes,
-glutamylcysteine synthetase
(
-GCS,1 EC 6.3.2.2), which
is the rate-limiting enzyme, and glutathione synthetase (12). The
-GCS holoenzyme exists as a dimer composed of heavy (
-GCS-HS; 73 kDa) and light (
-GCS-LS; 28 kDa) subunits (13). The heavy subunit
possesses all of the catalytic activity (14). We have recently
demonstrated that a putative AP-1 transcription factor-binding site is
necessary for oxidant-mediated regulation of the catalytic
-GCS-HS
gene promoter (15).
Inflammation results in the release of inflammatory mediators, such as
cytokines, which affect the local tissue oxidant/antioxidant balance.
Anti-inflammatory drugs might also influence this balance. Tumor
necrosis factor-
(TNF-
) is a ubiquitous pro-inflammatory cytokine
and is recognized as an important mediator of multiple inflammatory
events in the lungs. It induces chronic inflammatory changes associated
with the increase in a variety of defense mechanisms, through the
up-regulation of mRNA for various inflammatory mediators (16).
Regulation of mRNA expression by TNF-
is mediated by activation
of transcription factors c-Fos/c-Jun (activator protein-1, AP-1) and
nuclear factor-kappa B (NF-
B) (17). It has been reported that prior
exposure to a sublethal dose of TNF-
renders cells resistant to a
subsequent TNF-
challenge. The mechanism by which this
TNF-
-mediated cellular resistance occurs is related to the enhancement of the intracellular antioxidant capacity (18). Examples of
this are increased activity of mitochondrial manganese superoxide
dismutase (19), and other protective proteins, including plasminogen
activator inhibitor type 2, the zinc finger protein A20, and the
Bcl-2-related family member A1 (20-22). However, the molecular basis
of the TNF-
-induced cellular tolerance is not fully understood at
present. One possible mechanism may involve TNF-
-induced generation
of reactive oxygen species (ROS) by leakage from the mitochondrial
electron transport chain (23, 24). Such intracellular ROS could induce
glutathione synthesis in response to this oxidant stress by a mechanism
involving the up-regulation of the
-GCS-HS mRNA.
Corticosteriods are widely used as anti-inflammatory agents in various
inflammatory lung diseases (17). Corticosteroids act to reduce
inflammation and cellular damage by two different mechanisms as
follows: by direct binding of glucocorticoids to their consensus
glucocorticoid response element site, which activates transcription
processes, and by an indirect mechanism involving nuclear protein
interactions (25). These latter effects may be mediated by an
interaction between the glucocorticoid-receptor complex and
transcription factors such as NF-
B and AP-1, which regulate
transcription for inflammatory mediators such as cytokines (17, 25). We
have shown that AP-1 plays a critical role in enhancing
-GCS-HS
mRNA expression, and therefore regulation of GSH synthesis (15, 26,
27). Thus corticosteroids may have an effect on intracellular GSH
through a mechanism involving AP-1 (28). However, the effects of
corticosteroids on GSH synthesis at the molecular level have not been
studied so far.
Thus the aim of this study was to elucidate the molecular regulatory
mechanisms for GSH synthesis in human alveolar epithelial cells (A549)
in response to TNF-
and dexamethasone, important pro- and
anti-inflammatory agents, respectively.
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EXPERIMENTAL PROCEDURES |
Materials--
GSH, GSSG, 5,5'-dithiobis-(2-nitrobenzoic acid),
sulfosalicylic acid, glutathione reductase, NADPH, triethanolamine,
pyruvate kinase, lactate dehydrogenase, L-glutamate,
L-
-aminobutyrate, phosphoenolpyruvate, NADH,
dexamethasone, DL-buthionine-(SR)-sulfoximine, and ATP were obtained from the Sigma (Poole, UK) and 2-vinylpyridine from Aldrich Chemical Co., (Dorset, UK). Recombinant human TNF-
was
purchased from R & D Systems, (Abingdon, Oxford, UK). Cell culture
media/reagents and the TRIZOL reagent were obtained from Life
Technologies, Inc. (Paisley, UK).
A549 Epithelial Cells--
The human type II alveolar epithelial
cell line, A549 (ECACC number 86012804), which was mycoplasma-free, was
maintained in continuous culture at 37 °C, 5% CO2 in
Dulbecco's modified minimum essential medium (DMEM), sodium
bicarbonate, and 10% fetal bovine serum (FBS).
When required for the assays, confluent monolayers of A549 epithelial
cells were washed twice with phosphate-buffered saline (PBS).
Thereafter, trypsin EDTA solution was added to detach the cells. The
cells were then washed with DMEM, containing 10% FBS at 250 × g for 10 min, to neutralize the trypsin, and were
resuspended in DMEM with 10% FBS and maintained in 162-cm2
cell culture flasks (Corning Costar, High Wycombe, UK).
Epithelial Cell Exposure to TNF-
and
Dexamethasone--
Monolayers of confluent A549 epithelial cells were
prepared by seeding 0.8 × 106 cells/well in a 6-well
or 3 × 106 cells/100-mm plates in DMEM with 10% FBS
at 37 °C, 5% CO2, until 70-80% confluency was
reached. Confluent monolayers were rinsed twice with DMEM and were
treated with TNF-
(10 ng/ml) and/or dexamethasone (3 µM) for time intervals between 4 and 24 h in 2 ml or
5 ml of full media, incubated at 37 °C, 5% CO2.
Thereafter, the monolayers were washed twice with cold PBS, and the
cells were harvested with 0.05% trypsin/EDTA solution and resuspended in cold PBS (pH 7.4). An aliquot of cells was diluted with trypan blue,
counted, and the cell viability determined. This cell suspension was
thereafter used in the GSH and GSSG assays. For
-GCS activity, mRNA assays, and transfection experiments, monolayers were scraped using a Teflon scraper (Corning Costar, High Wycombe, UK).
GSH, GSSG, and
-GCS Assays--
The epithelial cells were
centrifuged at 250 × g for 5 min at 4 °C, and the
cell pellets were suspended in 1 ml of cold 0.6% sulfosalicylic acid,
sonicated on ice, homogenized with a Teflon pestle, and vortexed
vigorously. The cell homogenates were then centrifuged at 4,000 × g for 5 min at 4 °C. The supernatant was immediately used
in the GSH assay by the 5,5'-dithiobis-(2-nitrobenzoic acid)-glutathione reductase recycling method described by Tietze (29).
For the GSSG assay, supernatant was treated with 2-vinylpyridine and
triethanolamine as described previously (30) and thereafter was used in
the assay for GSH as described above.
Cell extracts for
-GCS activity were prepared 4 or 24 h after
each treatment. The cells were washed with ice-cold PBS, scraped into
PBS, and collected by centrifugation at 250 × g for 5 min at 4 °C. After two additional washes with PBS, the cells were resuspended in 100 mM potassium phosphate buffer (pH 7.4),
sonicated, and homogenized using a Teflon pestle on ice with Triton
X-100 to a final concentration of 0.1% (v/v). The extracts were spun at 13,000 × g for 15 min at 4 °C. The supernatants
were recovered and dialyzed at 4 °C against four changes of 500 ml
each of the Tris-HCl buffer (50 mM, pH 8.0) and then stored
at 0 °C. The protein concentrations were determined using the
bicinchoninic acid reagent assay (Pierce, Chester, UK) (31).
-GCS activity was assayed by the method described by Seelig and
Meister (12) using a coupled assay with pyruvate kinase and lactate
dehydrogenase. The rate of decrease in absorbance at 340 nm was
followed at 37 °C. Enzyme-specific activity was measured as
micromoles of NADH oxidized per min/mg protein, which is equal to 1 international unit (IU). For each experimental assay, BSO (50 µM) was added to the cell homogenate and incubated for 1 h at 37 °C to check the specificity of the
-GCS assay.
Isolation of RNA and Reverse Transcription--
RNA was isolated
from A549 cells using the TRIZOL reagent (Life Technologies, Inc.,
Paisley, UK). Total RNA was reverse-transcribed according to the
manufacturer's instructions (Life Technologies, Inc., catalog number
8025SA). The resultant cDNA was stored at
20 °C until required.
Assessment of
-GCS-HS mRNA by Polymerase Chain Reaction
(PCR)--
Oligonucleotide primers were chosen using the published
sequence of human
-GCS-HS cDNA (32) and human
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (33) (Stratagene,
Cambridge, UK). The primers for
-GCS-HS were synthesized by Oswel
DNA Services, University of Southampton, UK (25, 34). The sequences of
the primers used in the PCR were as follows:
-GCS-HS (sense 5'-GTG
GTA CTG CTC ACC AGA GTG ATC CT-3') and (antisense 5'-TGA TCC AAG TAA
CTC TGG ACA TTC ACA-3'); GAPDH (sense 5'-CC ACC CAT GGC AAA TTC CAT GGC
A-3') and (antisense 5'-TC TAG ACG GCA GGT CAG GTC AAC C-3'). Five
microliters of the reverse-transcribed mRNA mixture was added directly to the PCR mixture and used for the PCR reactions, which we
have previously described (26, 27, 34). Bands were visualized by a UV
transilluminator, and photograph negatives were scanned using a
white/ultraviolet transilluminator, UVP (Orme Technologies, Cambridge,
UK). The intensity of the
-GCS-HS mRNA (531 bp) bands were
expressed as a percentage of the intensity of the GAPDH bands (600 bp).
The pKS-hGCS plasmid (American Type Culture Collection, Rockville, MD,
catalog number 79023) was used as a positive control for
-GCS-HS.
Fifty femtograms was used for each experiment to check the specificity
of the PCR (data not shown).
Generation of the
-GCS-HS Promoter Construct--
The
-GCS-HS promoter was isolated by PCR from human genomic DNA using
the upstream oligonucleotide 5'-(
1050) GGCGACATCCAATATGAAGGCTGTG-3' and downstream oligonucleotide 5'-(+82) TTCCTACTTGTGACCAAAACCTGCG-3'. The resulting promoter fragment (
1050 to +82 bp) was sequenced and
cloned into the pCRII cloning vector (Invitrogen). A HindIII and SphI fragment (1138 bp) containing the promoter was
isolated and subcloned into the polylinker of the promoterless plasmid pCAT Basic Vector (Promega). This construct was denoted pCBGCS and has
been described previously (15, 26, 27).
Generation of the Mutant
-GCS-HS Promoter Construct--
A
selective mutation in the proximal AP-1-binding site (
269 to
263
bp) was introduced by PCR amplification with the mutated upstream
primers. The sequences of the oligonucleotides used in the PCR
were as follows: upstream
5'-ATGGTGAGTTCGTCATGTTATCAA-3' and downstream
5'-GCAGGCATGCCCAGTCTTTGCG-3' and were synthesized by
MWG-BIOTECH GmbH, (Ebersberg, Germany). The mutated consensus sequence
of AP-1 is denoted by the underlined letters, and the italic letters on
the downstream oligonucleotide indicates additional bases from the
cloning site of the pCAT Basic vector. The PCR conditions were 94 °C
for 10 min and then 35 cycles at 94 °C for 30 s, 58 °C for
30 s, 72 °C for 120 s, and a final extension at 72 °C
for 10 min with 1 unit of Taq DNA polymerase (Life
Technologies, Inc., Paisley, UK). The resulting PCR-amplified DNA
fragment (
285 to +47 bp) was confirmed by DNA sequencing. The mutated
PCR fragment was isolated and subcloned into pCBGCS using
HpaII and SphI restriction sites. This construct
was denoted pCBmGCS.
Generation of a
-GCS-HS AP-1 Construct--
Multiple
restriction sites within the
-GCS-HS promoter were utilized to
create a
-GCS-HS construct containing a putative proximal AP-1
construct. Digestion of the
-GCS-HS promoter using KpnI
produced a short (
1050 to
818 bp) and a large fragment (
817 to
+82 bp). Further digestion of the large fragment by BalI and
DraII generated a proximal fragment (
305 to +82 bp). This fragment contains a putative AP-1 at
269 to
263 bp, CAAT and TATA
boxes, which was subcloned into the pCAT Basic vector (pCBGC
D). Restriction enzyme analyses were performed to confirm the orientation and validity of all constructs.
Transient Transfection and CAT Assay--
A549 cells (0.8 × 106 per well) were seeded into 6-well tissue culture
plates and cultured at 37 °C until they were 70-80% confluent.
Plasmid DNA transfections were performed using the LipofectAMINE
reagent (Life Technologies, Inc.). Following treatment with TNF-
(10 ng/ml) and/or dexamethasone (3 µM), cell extracts were
prepared and assayed for protein content using the BCA reagent (Pierce,
Chester, UK) (31). Chloramphenicol acetyltransferase (CAT) activity was
quantified by a CAT enzyme-linked immunosorbent assay. A
-galactosidase expression plasmid (PSVgal, Promega) was
co-transfected as an internal control to normalize transfection efficiency. In all of the transfection experiments, pCAT-Basic and
pCAT-Control were used as negative and positive controls, respectively.
Preparation of Nuclear Extracts and the Electrophoretic Mobility
Shift Assay (EMSA)--
Nuclear extracts were prepared by the method
of Staal et al. (35). The oligonucleotides used were
commercial AP-1 (5'-CGC TTG ATG AGT CAG CCG GAA-3', 3'-CGC
AAC TAC TCA GTC GGC CTT-5') and NF-
B (5'-AGT TGA GGG GAC TTT CCC AGG
C'-3, 3'-TCA ACT CCC CTG AAA GGG TCC G-5'), which were obtained from
Promega. The oligonucleotides for the AP-1 site (
269 to
263 bp)
present in native
-GCS-HS-AP-1 (5'-GAG TTC GTC ATT GAT
TCA AAT AAT-3' and 3'-CTC AAG CAG TAA CTA AGT TTA TTA-5') were
synthesized by MWG-BIOTECH GmbH, Ebersberg, Germany. The consensus
sequence of AP-1 is indicated by bold letters. Both the
oligonucleotides were end-labeled with [
-32P]ATP using
T4 polynucleotide kinase and [
-32P]ATP (Promega).
Binding reactions were carried out using 5 µg of nuclear extract
protein for AP-1 and NF-
B and 25 µg for
-GCS-HS AP-1, 0.25 mg/ml poly(dI-dC)·poly (dI-dC) (Pharmacia Biotech, St. Albans, United
Kingdom), in a 20-µl binding buffer (Promega). The protein-DNA
complexes were resolved on 6% non-denaturating polyacrylamide gels at
100 V for 3-4 h. The gels were then vacuum-dried and autoradiographed
overnight with an intensifying screen at
80 °C. The gel was
scanned on a white/ultraviolet transilluminator UVP (Orme Technologies,
Cambridge, UK).
Supershift Assay--
The nuclear extracts were preincubated
with 3 µl of antiserum (1 mg/ml), at 4 °C for overnight, before
analysis by EMSA as described above. Human anti-c-Jun and anti-c-Fos
sera were obtained from Santa Cruz Biotechnology, Inc. These sera
specifically detect the presence of the corresponding transcription
factor and do not interfere with nuclear factor binding. Rabbit
preimmune sera (SAPU, Edinburgh, Scotland) were incubated with the
nuclear extracts as described above and used as a control.
Statistical Analysis--
Results were expressed as means ± S.E. Differences between values were compared by Duncan's multiple
range test.
 |
RESULTS |
Effect of TNF-
and Dexamethasone on GSH Levels in Alveolar
Epithelial Cells--
TNF-
(10 ng/ml) significantly decreased GSH
levels after 4 h treatment concomitant with an increase in GSSG
levels in A549 epithelial cells. This was associated with a significant
increase in GSH at 24 h, without any change in GSSG levels,
compared with control values (Fig. 1 and
Table I). By contrast, dexamethasone (3 µM) depleted intracellular GSH levels significantly at
both 4 and 24 h. At 4 h dexamethasone produced no change in
GSSG, but at 24 h GSSG was decreased compared with control levels
(Fig. 1 and Table I). However, exposure to dexamethasone produced a 31% decrease in the GSH/GSSG ratio at 4 h and an 24% increase at
24 h in A549 cells, compared with control values. Co-incubation of
TNF-
and dexamethasone produced further depletion of GSH at 24 h in epithelial cells, compared with TNF-
or dexamethasone alone,
without any significant change in the GSH/GSSG ratio (Fig. 1 and Table
I). Co-incubation of TNF-
and dexamethasone produced a similar
decrease in GSH levels to TNF-
alone at 4 h, but with an 50%
decrease in the GSH/GSSG ratio. Cell viability remained >95% after
all of the above treatments.

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Fig. 1.
Effect of TNF-
(10 ng/ml) and dexamethasone (3 µM) on GSH levels at 4 and 24 h in
A549 type II alveolar epithelial cells. Each histogram
represents the mean and the error bars the S.E. of four
experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001, compared with control values.
, p < 0.001, compared with TNF- alone.
Dex, dexamethasone.
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Table I
Effects of TNF- and dexamethasone on GSSG levels in alveolar
epithelial cells
A549 epithelial monolayers were treated with TNF- (10 ng/ml) and/or
dexamethasone (Dex) (3 µM) for 4 and 24 h. Cellular
GSSG levels were determined as described under "Experimental
Procedures." Data are expressed as mean ± S.E.
(n = 4).
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|
Effect of TNF-
and Dexamethasone on
-GCS Activity in Alveolar
Epithelial Cells--
-GCS activity was not affected by TNF-
and/or dexamethasone treatment at 4 h, compared with control
values (Fig. 2). However, TNF-
produced an increase in
-GCS activity at 24 h (Fig. 2). By
contrast, dexamethasone significantly decreased
-GCS activity at
24 h (Fig. 2). Co-incubation of TNF-
with dexamethasone
produced a further decrease in
-GCS activity at 24 h, compared
with dexamethasone alone. Addition of BSO (50 µM) only
inhibited 65-72% of the total
-GCS activity. The results were
corrected for the BSO non-inhibitable enzyme activity after each
treatment. The mean BSO-inhibitable
-GCS activity in the A549 cell
homogenate was 0.065 ± 0.01 units/mg protein.

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Fig. 2.
Effect of TNF- (10 ng/ml) and dexamethasone (3 µM)
on -GCS enzyme activity in A549 epithelial
cells. A549 epithelial cells were incubated with 10 ng/ml TNF-
and 3 µM dexamethasone (Dex) alone or in
combination for 4 h and 24 h. Each histogram
represents the mean and the error bars the S.E. of three
experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001, compared with control values.
, p < 0.001, compared with TNF- alone.
|
|
Effect of TNF-
and Dexamethasone on
-GCS-HS mRNA
Expression--
We investigated the mechanism of the above effects on
GSH and
-GCS activity following TNF-
and dexamethasone alone
or in combination. Neither TNF-
nor dexamethasone alone or in
combination produced any significant change in the
-GCS-HS mRNA
level after 4 h, compared with control values (Fig.
3A). However, TNF-
significantly increased
-GCS-HS mRNA expression, whereas
dexamethasone treatment significantly depleted
-GCS-HS mRNA,
after 24 h, compared with GAPDH mRNA expression (Fig.
3B). The increased
-GCS-HS mRNA expression produced
by TNF-
at 24 h was completely abolished when the cells were
co-treated with dexamethasone.

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Fig. 3.
Effect of TNF- and
dexamethasone on -GCS-HS mRNA expression
in A549 alveolar epithelial cells. Total RNA was isolated from
control cells and cells exposed to TNF- and dexamethasone
(Dex) alone or in combination for 4 and 24 h.
A, RNA was reversed-transcribed and used for PCR analysis of
-GCS-HS mRNA as described under "Experimental Procedures."
B, the numeric estimates of -GCS-HS mRNA levels
compared with the GAPDH bands from the same sample. Each
histogram represents the mean and the error bars
the S.E. of the relative intensities of the -GCS-HS mRNA: GAPDH
band of three experiments each performed in duplicate. ***,
p < 0.001 compared with control values.
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Role of AP-1 in TNF-
and Dexamethasone-mediated Regulation of
-GCS-HS--
To determine if AP-1 plays an important role in
TNF-
and dexamethasone-mediated
-GCS-HS gene regulation in A549
epithelial cells, we used a DNA fragment of the 5'-flanking region of
the
-GCS-HS containing a proximal putative AP-1 site and a
commercially available DNA fragment containing an AP-1 consensus in the
EMSA. Nuclear proteins were isolated at 4 and 24 h after TNF-
and/or dexamethasone treatment and were incubated with the DNA probes containing the AP-1 site. We found that TNF-
exposure increased AP-1
DNA binding activities in A549 cells using both the
-GCS-HS AP-1
probe and commercial AP-1 probe at 4 h exposure (Figs.
4 and 5),
in A549 cells compared with untreated cells. Dexamethasone treatment alone did not produce any changes in the nuclear binding of
-GCS-HS AP-1 probe at 4 or 24 h, compared with control values. However, dexamethasone treatment produced a significant decrease in the
nuclear binding using commercial AP-1 at 24 h, without any change
at 4 h, in A549 cells. TNF-
exposure increased
-GCS-HS AP-1
DNA binding activity at 24 h, without any change in AP-1 binding
of the commercial probe at 24 h in A549 epithelial cells. TNF-
-mediated increase in AP-1 DNA binding was abolished when cells
were co-treated with dexamethasone at 4 and 24 h (Figs. 4 and 5).
The specificity of the binding was checked using 100-fold excess
unlabeled
-GCS-HS AP-1 and AP-1 oligonucleotides and nonspecific oligonucleotides for NF-
B (data not shown).

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Fig. 4.
Effect of TNF- and
dexamethasone on the regulation of -GCS-HS
AP-1 binding activity in A549 cells. A549 cells were treated with
TNF- (10 ng/ml) and dexamethasone (Dex, 3 µM), alone or in combination at the times indicated.
A, nuclear extracts were prepared and analyzed by EMSA using
32P-labeled synthetic double-stranded oligonucleotides
containing the proximal AP-1 sequence of the -GCS-HS promoter. The
DNA-protein complexes formed are indicated (arrow) for
-GCS-HS AP-1. B, the numeric estimates of DNA binding
levels were compared with the control value set at 100%. The
histograms represent the means and the error bars
the S.E. of the relative intensity of the bands of six experiments. **,
p < 0.01; #, ***, p < 0.001 compared
with control values.
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Fig. 5.
Regulation of the transcription factor AP-1
by TNF- and dexamethasone. A549
epithelial cells were treated with TNF- (10 ng/ml) and dexamethasone
(Dex, 3 µM) at the times indicated.
A, nuclear extracts were isolated and incubated in the
presence of labeled AP-1 oligonucleotide. DNA binding to AP-1 was
analyzed by EMSA. The unbound (free) probe in the gel is indicated at
the bottom. B, densitometric quantitation of AP-1
binding was compared with the control value set at 100%. The
histograms represent the mean values and the error
bars the S.E. of the relative intensity of the bands of three
experiments. ***, p < 0.001, compared with control
values.
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|
Involvement of c-Jun in TNF-
-induced AP-1 DNA Binding in A549
Epithelial Cells--
To identify which component of AP-1
(c-Fos/c-Jun) is involved in the TNF-
-induced AP-1 DNA binding
activity in A549 cells, we used antibodies to c-Jun and c-Fos in
supershift assays. By using both the commercial AP-1 probe and the
-GCS-HS AP-1 probe, we demonstrate that the increased AP-1 binding
activity present in TNF-
-treated cells was related to binding of
c-Jun but not of c-Fos, since the addition of the c-Jun antibody
shifted the AP-1 DNA band considerably (Fig.
6). Furthermore, both oligonucleotides were equally able to bind to c-Jun-purified protein (Promega) (data not
shown).

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Fig. 6.
Effect of anti-c-Jun and anti-c-Fos
antibodies on the DNA binding of AP-1 induced by
TNF- . Nuclear extracts of A549 epithelial cells
treated with TNF- (10 ng/ml) for 24 h were incubated with
anti-c-Jun or anti-c-Fos antibody in a 20-µl binding reaction as
described under "Experimental Procedures." A supershift assay of
binding activity in nuclear extracts to labeled experimental commercial
AP-1 or -GCS-HS AP-1-binding elements was performed. Preincubation
of nuclear extract with preimmune sera is shown as a control.
Incubation with anti-c-Fos, anti-c-Jun sera before the gel shift assay
results in a super-retardation in band mobility (arrow with
ss). Experiments were repeated three times, and a
representative autograph is shown.
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Role of NF-
B in TNF-
and Dexamethasone-mediated Regulation of
-GCS-HS--
We also determined whether the pleiotropic
transcription factor NF-
B was also affected by these treatments.
Using a commercially available NF-
B oligonucleotide, TNF-
treatment of A549 cells produced a significant increase in NF-
B DNA
binding both at 4 and 24 h (Fig. 7).
By contrast, dexamethasone produced a significant decrease in NF-
B
DNA binding activity at 24 h, without any change at 4 h,
compared with control levels. TNF-
-mediated increased NF-
B
binding activity was not inhibited either at 4 or 24 h by co-treatment with dexamethasone.

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Fig. 7.
Regulation of NF- B
binding in response to TNF- and
dexamethasone. Nuclear extracts were prepared from cultured A549
epithelial cells treated with TNF- (10 ng/ml) and dexamethasone
(Dex, 3 µM) at the various time points as
indicated. A, the electrophoretic mobility shift assay was
performed using labeled NF- B oligonucleotide. Positions of specific
DNA-protein complexes for NF- B are indicated by the
arrows. B, quantification of DNA binding was
performed by densitometry and compared with the control values set at
100%. The histograms represent the mean values and the
error bars the S.E. of the relative intensity of the bands
of three experiments. ***, p < 0.001 compared with
control values.
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|
Effect of TNF-
and Dexamethasone on
-GCS-HS Promoter
Construct-derived Chloramphenicol Acetyltransferase (CAT)
Activity--
To confirm that AP-1 plays a key role in TNF-
-induced
-GCS-HS gene expression, we used a CAT reporter system to explore the mechanisms by which TNF-
and dexamethasone regulate
-GCS-HS at the transcriptional level. TNF-
treatment of A549 cells
transfected with the full
-GCS-HS promoter linked to the CAT
reporter system (pCBGCS, Fig.
8A) produced a significant
increase in CAT activity at 24 h, compared with control values
(Fig. 8B). By contrast, CAT activity was significantly
decreased when the cells were treated with dexamethasone at 24 h.
Dexamethasone also blocked the TNF-
-induced CAT activity at 24 h.

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Fig. 8.
Effects of TNF- and
dexamethasone on the transcriptional activity of the 5'-flanking region
of the -GCS-HS gene in A549 alveolar
epithelial cells. A, the line diagram at the
top is a restriction map of the -GCS-HS promoter region.
The block diagram shows the relative positions of the
cis-acting DNA binding elements of the promoter region
cloned in the pCRII vector. The numbers in the figure
represent nucleotide positions from the transcriptional start site of
the -GCS-HS gene which is indicated by the bent arrow.
The dotted line on the left indicates an
additional 50 bp from multiple cloning sites of the pCRII vector. The
structure of the -GCS-HS-CAT plasmid is shown below on
the right. The complete 5'-flanking region
(pCBGCS) and a proximal putative AP-1 construct
(pCBGCS D) were ligated to the CAT gene in a pCAT Basic
vector (pCB) and transfected into A549 epithelial cells.
Transfected A549 cells were exposed to TNF- (10 ng/ml) or
dexamethasone (Dex, 3 µM) alone or in
combination. After 24 h incubation, the cells were harvested and
assayed for CAT activity by an enzyme-linked immunosorbent assay.
B, transcriptional activity was standardized by the amount
of CAT activity relative to -galactosidase activity. The results are
shown as percentages of the CAT concentration compared with that of
pCBGCS. The histograms represent the means and the
error bars the S.E. of the CAT-derived activities of pCBGCS
and pCBGCS D constructs of three transfection experiments, each
performed in duplicate with the activity of pCBGCS set at 100%. **,
p < 0.01; ***, p < 0.001, compared
with pCBGCS. , p < 0.001, compared with TNF-
alone.
|
|
Effect of TNF-
and Dexamethasone on
-GCS-HS AP-1
Construct-derived CAT Activity--
To establish whether the
TNF-
-induced
-GCS-HS AP-1 DNA binding activity was related to
activation of the
-GCS-HS AP-1 fragment in the CAT reporter system
(pCBGCS
D), we transfected epithelial cells with a plasmid containing
the putative AP-1 transcription factor (pCBGCS
D). pCBGCS
D
displayed significant TNF-
-induced CAT activity in cells transfected
with pCBGCS
D, whereas dexamethasone inhibited the CAT activity (Fig.
8B). Furthermore, TNF-
-induced pCBGCS
D CAT activity
was abolished when the transfected cells were co-treated with dexamethasone.
To confirm further whether the proximal putative AP-1 site is a key
regulator of TNF-
-induced
-GCS-HS promoter activation, a
construct was generated from the parent pCBGCS plasmid where the
wild-type proximal AP-1 site was mutated (Fig.
9A). This mutated pCBmGCS
reporter construct, where the proximal AP-1 site was abrogated, showed
a reduced response to TNF-
, compared with the wild-type
-GCS-HS
promoter (Fig. 9B). These data imply that the proximal AP-1-binding site present in
269 to
263 bp is the major
cis-regulatory element responsible for the TNF-
induction
of the
-GCS-HS gene.

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|
Fig. 9.
Effect of a mutation in the AP-1 site at
269 to 263 bp of the -GCS-HS gene on
transcriptional activity. A, this shows the putative
AP-1 binding element at 269 to 263 bp contained within the
wild-type pCBGCS promoter construct and mutated ( 269 to 263 bp)
pCBmGCS promoter construct used in this experiment. The
numbers in the figure represent the nucleotide positions
from the transcriptional start site of the -GCS-HS gene.
B, basal and TNF- -stimulated transcriptional activity was
determined after 24 h of incubations as described in Fig. 8. ***,
p < 0.001 versus control; ,
p < 0.001 versus TNF- . The
histograms represent the means and the error bars
the S.E. of three separate experiments performed in duplicate.
|
|
 |
DISCUSSION |
Rapid induction of intracellular GSH synthesis occurs in response
to various oxidant stresses (36-38). This may be a critical determinant of cellular tolerance to oxidant stress. In this study, we
demonstrated that TNF-
caused a transient depletion of GSH, concomitant with an increase in GSSG levels, suggesting that TNF-
induced oxidative stress in epithelial cells. However, prolonged exposure (24 h) to TNF-
increased GSH levels, without changing GSSG
levels in A549 epithelial cells. The increase in GSH was associated
with increased
-GCS activity. The levels of GSH and
-GCS activity
are regulated by the expression of the catalytic
-GCS-HS (26, 27).
Recently, we and others (27, 36-43) have demonstrated that oxidants,
radiation, heat shock, heavy metals, and chemotherapeutic
agents can increase GSH concentrations by induction of
-GCS-HS
expression in various cell types. In this study, we show, for the first
time, that in human alveolar epithelial cells (A549),
-GCS-HS gene
expression is induced by TNF-
. Similar induction of
-GCS-HS
mRNA and elevation of GSH levels have been shown in human
hepatocytes and mouse endothelial cells treated with TNF-
(44, 45).
Elevation of glutathione levels has also been observed in cultured rat
hepatocytes following treatment with TNF-
, which protected the cells
against the cytotoxic effects of further oxidant stresses (46).
However, the mechanism of this induction was not studied.
To understand the molecular mechanism of the transcriptional induction
of
-GCS-HS in response to TNF-
, we cloned the 5'-flanking region
of the
-GCS-HS into a CAT reporter system. Following transfection into A549 epithelial cells, we observed that TNF-
up-regulated the
promoter region of the
-GCS-HS gene, measured as increased CAT
activity. This suggests that TNF-
acts at the level of transcription to induce
-GCS-HS mRNA in epithelial cells. The signaling
mechanism whereby TNF-
exerts its effect is currently not known.
TNF-
is known to generate ROS, particularly the superoxide anion
(O
2) and H2O2, by leakage from the
electron transport chain in mitochondria. This could trigger
transcriptional up-regulation of
-GCS-HS possibly by activating
signaling pathways, such as activation of the c-Jun N-terminal
kinase/stress activated protein kinase (47).
We next elucidated the transcriptional regulatory mechanism by which
TNF-
exerted its effect on the induction of
-GCS-HS. By using
deletion and mutation studies, we have recently shown the critical role
of a putative AP-1 site in oxidant-mediated regulation of the
-GCS-HS promoter (15). However, other investigators have suggested
that an NF-
B-binding site in the
-GCS-HS promoter may be
important in regulating
-GCS-HS gene expression (40, 42). Activation
of NF-
B is known to be regulated by a variety of pro-inflammatory
(e.g. TNF-
) and anti-inflammatory agents (e.g.
dexamethasone) (17). We therefore used TNF-
and dexamethasone to
assess the role of the AP-1 and NF-
B transcription factors in the
transcriptional up-regulation of
-GCS-HS. Exposure of alveolar
epithelial cells to TNF-
produced a significant increase in the DNA
binding activities of both nuclear proteins, using AP-1 and NF-
B as
consensus probes. Thus both AP-1 and NF-
B transcription factors are
activated by TNF-
. AP-1 activation by TNF-
was confirmed by
increased CAT activity in a CAT reporter system with a cloned
-GCS-HS-AP-1 fragment. These data indicate that NF-
B binding to a
consensus site in the
-GCS-HS promoter is not necessary for
TNF-
-induced transcriptional activation of the
-GCS-HS gene and
suggest the involvement of an AP-1 response element. Further confirmation of the key role of a putative proximal AP-1-binding site
in the regulation of the
-GCS-HS gene comes from the mutational study of the putative AP-1 site (
269 to
263 bp). Mutation of the
sequence 5'-TTGATTCAA-3' to 5'-TGTTATCAA-3' in
the proximal AP-1 oligonucleotide (pCBmGCS) effectively eliminated
TNF-
-induced promoter activity. This provides strong evidence that
this sequence, but not NF-
B, is involved in the regulation of the
expression of the endogenous
-GCS-HS subunit gene.
To identify which components of AP-1 are responsible for the
up-regulation of
-GCS-HS, we used antibodies directed against c-Fos
and c-Jun in an effort to demonstrate a supershift in the EMSA. Only an
antibody that cross-reacted with c-Jun produced a supershift,
supporting the view that the AP-1 EMSA data is likely to have resulted
from c-Jun/c-Jun homodimer binding to
-GCS-HS AP-1 sites. In support
of this, several investigators have recently suggested possible
involvement of AP-1/Jun family members in the regulation of
-GCS-HS
(40, 42, 43, 48).
Corticosteroids, such as dexamethasone, are known to suppress both
immune responses and inflammation by activation of the glucocorticoid
receptor and by interaction with various transcription factors (17).
The role of intracellular GSH redox status in the regulation of
transcription factors is of considerable interest (17, 49). However,
the effect of dexamethasone on the regulation of GSH synthesis has not
been studied. We showed that depletion of intracellular GSH by
dexamethasone occurs concomitant with a decrease in
-GCS activity,
without any change in GSSG levels in A549 alveolar epithelial cells.
However, the ratio of GSH/GSSG levels was decreased by dexamethasone,
suggesting that dexamethasone may impose oxidative stress in A549
epithelial cells. Depletion of liver GSH in mice and inhibition of GSH
synthesis by dexamethasone have been observed in a rat hepatic cell
line (50, 51). Similarly, depletion of antioxidant enzyme activities
have been shown in various rat tissues by glucocorticoid
supplementation, and this depletion was more pronounced when rats were
challenged with oxidative stress (52). In this study, we show that the
depletion of GSH, decrease in GSH/GSSG ratio, and decreased
-GCS
activity produced by dexamethasone is also associated with a decrease
in the expression of
-GCS-HS mRNA. However, dexamethasone had no
effect on the basal level of
-GCS-HS gene expression at 4 h. In
support of these data, dexamethasone also had no effect on basal level
of manganese superoxide dismutase mRNA expression, whereas
TNF-
-induced gene expression was completely abolished by
dexamethasone treatment (53).
Protein-protein interactions between the glucocorticoid receptor and
the transcription factor AP-1 are thought to mediate a negative
cross-talk between dexamethasone and TNF-
-induced gene regulation
(17, 25). In this study, dexamethasone significantly inhibited
TNF-
-mediated activation of
-GCS-HS AP-1 DNA binding activity and
proximal AP-1 (pCBGCS
D)-derived CAT activity in alveolar
epithelial cells. Dexamethasone also inhibited TNF-
-induced changes
in GSH levels,
-GCS activity,
-GCS-HS mRNA expression, and
-GCS-HS promoter activity. Dexamethasone did not inhibit TNF-
-mediated activation of NF-
B but did block the increase in
GSH and
-GCS-HS mRNA expression induced by TNF-
. These data support the concept that activation of NF-
B does not have a role in
mediating the transcriptional activation of
-GCS-HS in response to
TNF-
.
The results of our mutation and deletion studies confirm that the
5'-flanking proximal sequence of the
-GCS-HS gene, containing a
putative AP-1-binding site at
269 to
263 bp, plays an important role in the transcriptional up-regulation of the
-GCS-HS gene in
TNF-
-treated alveolar epithelial cells. The supershift assay showed
that this AP-1 DNA-binding complex was predominantly formed by dimers
of c-Jun. Furthermore, our data provide supportive evidence for
negative interaction between glucocorticoid receptor and AP-1 (c-Jun)
which prevents TNF-
-induced
-GCS-HS mRNA expression in A549
epithelial cells.
In conclusion, these studies show that TNF-
causes an increase in
intracellular GSH content,
-GCS activity, and transcriptional activation of
-GCS-HS in alveolar epithelial cells, whereas
dexamethasone decreased GSH levels by down-regulating the transcription
of the
-GCS-HS gene. Electrophoretic mobility gel shift and CAT
reporter assays revealed that the modulation of
-GCS-HS gene
expression in alveolar epithelial cells by TNF-
and dexamethasone
occurs by a mechanism involving AP-1 (c-Jun homodimer). These data may have implications for dexamethasone treatment in patients with inflammatory lung diseases, since such treatment may prevent synthesis of increased levels of the protective antioxidant GSH.