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J. Biol. Chem., Vol. 275, Issue 28, 21130-21139, July 14, 2000
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and
NF-
B Redox Sensitivity
,From the Oxygen Signaling and § Lung Membrane Transport Groups, Center for Research into Human Development, Tayside Institute of Child Health, Faculty of Medicine, Ninewells Hospital and Medical School, University of Dundee, Dundee, DD1 9SY, Scotland, United Kingdom
Received for publication, January 28, 2000, and in revised form, April 11, 2000
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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The O2 and redox-sensitive
transcription factors hypoxia inducible factor-1 In normal health, enzymatic and nonenzymatic antioxidants serve to
balance the intracellular production of reactive oxygen species
(ROS),1 thereby delaying or
inhibiting the destructive oxidation of molecular components within the
cellular milieu (1, 2). The potential for oxidative damage is greatly
augmented however, if the antioxidant buffering capacity of the organ
system is insufficiently expressed to contain pro-oxidant events. This
is particularly critical in the lung at birth where the transition from
placental to pulmonary respiration necessitates a rapid shift in
oxygenation of the alveolar epithelial gas exchange surfaces from fetal
(23 torr) to neonatal pO2 (70-100 torr). As the
enzymatic and nonenzymatic antioxidant capacities of the fetal lung are
3-6-fold lower compared with those of late neonates and adult (7 and
references therein, 25), the potential for pro-oxidant events within
the distal lung epithelium is substantially heightened at birth.
The restitution of redox balance following oxidative stress depends
upon the adaptive coordination of responses among redox-associated signaling pathways, genetic regulatory factors, and antioxidants (3,
4). The tripeptide thiol
L- Pharmacological manipulation of GSH by pro- and antioxidants directly
modulates the activity of transcription factors in response to various
stimuli, including changes in the availability of oxygen (3, 13, 14).
Among those which bear particular significance to oxygen-linked redox
stresses are hypoxia inducible factor-1 We have previously shown that both HIF-1 The present study was undertaken to investigate the role that these
modulators of glutathione biosynthesis play to in determining the
response of the perinatal alveolar epithelium at the gene level over
fetal to neonatal oxygen shifts and to probe whether their effects are
mediated by altering redox potential via the GSH/GSSG equilibrium.
All experimental procedures involving the use of live animals
were reviewed and approved under the Animals (Scientific Procedures) Act, 1986 (United Kingdom).
Chemicals--
Unless otherwise indicated, all chemicals of the
highest analytical grade were purchased from Sigma.
Primary Cell Cultures--
Fetal alveolar type II (fATII)
epithelial cells were isolated from lungs of fetuses taken from the
uteri of pregnant rats at day 19-20 of gestation, essentially as
described elsewhere (7). The change in oxygen equilibrium from fetal
(~3%) to postnatal environments constitutes a potential signaling
mechanism within the perinatal lung (25). Consequently, shifts in
pO2 relevant to the fetal lung in preterm and
post-term neonatal periods were recreated using variable O2
incubators. fATII cells, with or without NAC or PDTC pretreatments,
were cultured for 24 h at fetal alveolar pO2 (23 torr, Cell Harvesting, Nuclear Protein Extraction, and Western
Analysis--
Nuclear extracts were prepared from monolayer filters of
fATII cells grown under hypoxia or hyperoxia, essentially as detailed elsewhere (7, 26), with minor modifications. Nuclear proteins (20-25
µg) were resolved by 7.5% SDS-polyacrylamide gel electrophoresis, blotted onto nitrocellulose membranes, transferred into Tris-buffered saline, and the nonspecific binding sites were blocked for 1 h at
room temperature. Monoclonal IgG anti-HIF-1 Electrophoretic Mobility Shift Assay and DNA Binding
Activity--
Custom deoxyoligonucleotide probe sequences were
purchased from Genosys: HIF-1 NAC and PDTC Pretreatments--
Stock solutions of NAC (1125 mM) and PDTC (1125 µM) were prepared in
deionized water and stored at 4 °C for up to 2 weeks. fATII cells
grown to confluence were pretreated for 24 h at 37 °C with NAC
(0 (control), 1, 10, and 50 mM) or PDTC (0, 10, 50 and 100 µM) before exposure to various fetal to neonatal oxygen tensions for 4 h. After each treatment, fATII cells were washed with pre-equilibrated Hanks' balanced salt solution and centrifuged, and nuclear extracts were prepared, as described above.
Glutathione Determination and Assessment of Redox
Equilibrium--
Reduced (GSH) and oxidized (GSSG) glutathione
concentrations were determined enzymatically with methylglyoxal by the
method originally reported by Bergmeyer (27), with significant
modifications (7). fATII epithelial cells grown on monolayers, with or
without NAC or PDTC pretreatments, were washed twice with ice-cold
phosphate-buffered saline, and immediately 500 µl of 7% perchloric
acid was added to the medium and cells were scraped. The slurry was
then centrifuged to precipitate the protein formed and the supernatant
snap frozen on liquid nitrogen. Samples were neutralized with a known
volume of 3 M KHCO3, and
GSH/methylglyoxal-linked changes in absorbance at 240 (GSH) and 340 nm
(GSSG) were recorded spectrophotometrically (7). Protein content was
reconstituted in 1 M NaOH and determined by Bradford (28).
Results are expressed as µmol·mg In Vitro Treatment of Nuclear Extracts with Exogenous
Glutathione--
In vitro experiments with glutathione were
performed by incubating 5 µg of nuclear extracts of fATII cells
exposed to various Assessment of the Adenylate Charge Ratio, Trypan Blue Exclusion,
and Tetrazolium Reduction as Independent Indices of Cell Viability
Following Pretreatment with Either NAC or PDTC--
Evaluation of the
adenylate high energy phosphate content as an index of cell viability
and metabolic activity was based on the energy charge ratio
determination for various treatments according to Atkinson (29). Trypan
blue (0.4%) exclusion indicates the relative percentage of cells that
are viable with respect to various treatments, such that the viability
was greater than 90% with either NAC or PDTC pretreatments.
Tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium
bromide cleavage into formazan blue by the mitochondrial enzyme
succinate dehydrogenase is considered a reliable assessment of the
degree of cell survival (30).
Data Handling and Statistical Analysis--
Experimental results
are expressed as mean ± S.E. Statistical analysis was performed
by one way analysis of variance (ANOVA), followed by post
hoc Tukey's test to determine mean separation significance among
treatments. The a priori acceptable level of significance at
95% confidence was considered p < 0.05.
NAC Attenuation of HIF-1 PDTC Attenuation of HIF-1 Electrophoretic Mobility Shift Assay Analysis of HIF-1
The specificity of the complexed bands, as detailed previously (7), was
determined by incubating samples with mutant oligonucleotides (M-18
(HIF-1 Electrophoretic Mobility Shift Assay Analysis of HIF-1 Variation in the Levels of GSH and GSSG with NAC
Pretreatment--
To assess whether NAC acts as a positive buffer of
the glutathione pool, concentrations of GSH and GSSG were determined in extracts from cultures that had been pretreated with 10-50
mM NAC. The effect was to elevate cellular concentrations
of GSH at the expense of GSSG, indicating that irrespective of the
oxygen regime imposed, NAC potentiated a reduced cellular environment (Fig. 6). The corresponding ratios at
different Variations in the Levels of GSH and GSSG with PDTC
Pretreatment--
To assess whether PDTC modulates GSSG/GSH, we
determined the glutathione levels in extracts from cultures pretreated
with PDTC at various Effect of the Ratio of Total to Oxidized Glutathione on HIF-1 Cytotoxicity and Cell Viability Assays for Cultures Pretreated with
NAC or PDTC--
To rule out the possibility that any of the effects
mediated by NAC and PDTC are due to cytotoxicity, three independent
measures of cell viability were employed. Percentage exclusion of
trypan blue in control cell cultures was not significantly different from cultures pretreated with NAC (50 mM) or PDTC (100 µM) (p > 0.05, Table
IV). The adenylate energy charge ratio
did not vary significantly with antioxidant pretreatment, as compared
with control cell cultures (p > 0.05, Table IV).
Finally, measurement of the tetrazolium salt whose ring is cleaved in
actively respiring mitochondria of living cells was considered as
another assessment of cell viability (Table IV). No significant
differences in cell viability are reported with NAC or PDTC
pretreatments (p > 0.05).
The activation of the transcription factors HIF-1 NAC induced a significant, dose-dependent increase in
HIF-1
(HIF-1) and
nuclear factor-
B (NF-B) are differentially regulated in the
alveolar epithelium over fetal to neonatal oxygen tensions. We have
used fetal alveolar type II epithelial cells to monitor their
regulation in association with redox responsiveness to antioxidant
pretreatment in vitro. N-Acetyl-L-cysteine, a
glutathione (GSH) precursor and a potent scavenger of reactive oxygen
species, induced HIF-1 and ameliorated NF-B nuclear abundance and
DNA binding activity, respectively, in a dose-dependent
manner. Analysis of variations in glutathione homeostasis at ascending
pO2 regimen with
N-acetyl-L-cysteine reveals increased GSH at
the expense of the oxidized form of glutathione (GSSG), thereby
shifting GSH/GSSG into reduction equilibrium. Pyrrolidine
dithiocarbamate (PDTC), which exerts both antioxidant and pro-oxidant
effects, provoked a substantial increase in HIF-1 nuclear abundance,
with no apparent effect on its activation. PDTC reduced NF-B nuclear
abundance and its inhibitory effects on binding activity are
dose-dependent. Assessment of glutathione homeostasis with
PDTC shows increasing levels of GSSG at the expense of GSH, lowering
GSH/GSSG in favor of an oxidative equilibrium. Our results indicate the
hypoxic activation of HIF-1 and the hyperoxic induction of NF-B
in the fetal epithelium is redox-sensitive and, thus, tightly regulated by the GSH/GSSG equilibrium. This highlights glutathione as a key
regulatory component for determining genetic responsiveness to
oxidant/antioxidant imbalance in normal lung development and pathophysiological conditions.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-glutamyl-L-cysteinyl-glycine, or GSH, is
a ubiquitous cellular nonessential sulfhydryl amino acid, which plays
an important role in maintaining intracellular redox balance and in
augmenting cellular defenses in oxidative stress (5-7). Glutathione
participation in the physiology of cellular metabolism reflects the
importance of this molecule in (i) detoxification of highly reactive
peroxides by conjugation of electrophiles and metals through the
glutathione peroxidase-coupled reaction (8); (ii) maintenance of
intracellular protein integrity by reducing disulfide linkages and
regulating their synthesis (5); (iii) regulation of cellular redox
equilibrium (9); (iv) governing signaling pathways in
neuro-immune-endocrine interactions by acting as a neurotransmitter and
an immunopharmacological reducing thiol (10); (v) facilitating membrane
trafficking of reactive chemicals and, in some cases, augmenting the
formation of essential biological mediators (11); and (vi) regulation
of the expression and activation of redox-sensitive transcription
factors to stress-evoked responses (3, 12, 40). Recently, Griffith and
Mulcahy (5) and Rusakow et al. (9) demonstrated that the
glutathione biosynthetic pathway forms an important determinant of the
effectiveness of an antioxidant approach in chemotherapy and pulmonary
oxygen toxicity, thereby highlighting the pharmacological potential of
this thiol in the treatment of redox-linked disease states.
(HIF-1) and nuclear
factor-B (NF-B), each differentially potentiated by oxidative
conditions (7, 15, 16). HIF-1, first identified as a rate-limiting
regulatory component in the hypoxic induction of erythropoietin, is
selectively stabilized in hypoxia whereupon it is translocated to the
nucleus and activates the expression of genes promoting vascular
development, glycolytic metabolism, and also cell cycle events (16,
17). The activation of HIF-1 is therefore consistent with the
significant role that this factor plays in coordinating adaptive
responses to hypoxia (16-18). NF-B (Rel) is a DNA
binding factor that is maintained in the cytosol as a heterodimer in
complex with its inhibitory subunit, IB. Upon activation by
inflammatory signals (such as cytokines) or pro-oxidant stresses, IB
dissociates allowing the Rel dimers of this factor to
translocate to the nucleus and activate genes particularly involved in
modulating the response of the cell to oxidative injury (19).
and NF-B are
differentially active over ranges of oxygen tension, which recreate the
elevation in pO2 within the perinatal lung that
is coincident with the onset of ventilation (7). To determine how
altered redox status within the alveolar epithelium may dictate genetic control between HIF-1 and NF-B over relevant shifts in
pO2, we evaluated the effect of the antioxidants
N-acetyl-L-cysteine (NAC) and pyrrolidine
dithiocarbamate (PDTC) in modulating the genetic response of the
alveolar epithelium to oxidative stress. Since the discovery of
biologically occurring free radicals, NAC has been used as a probe in
detecting the biochemical basis of oxidative-induced lung injury both
in vitro and in vivo experimental and clinical
models (20). NAC has the capacity to negatively buffer electrophiles
and is thus an antioxidant with cytoprotective potential. In addition
to its direct antioxidant effects, NAC may also serve as a precursor
for cysteine and glutathione biosynthesis, thereby positively buffering
the cellular pool of nucleophilic species (21). PDTC is a member of the
dithiocarbamate family, known to exert both antioxidant and pro-oxidant
effects in cells (22). Reduced dithiocarbamate is readily oxidized by
reactive oxygen and nitrogen species to generate dithiocarbamate thiol radicals and thiuram disulphides. As potent electrophiles, these readily induce the oxidation of GSH leading to the formation of GSSG,
the oxidized form of glutathione (23). Pretreatment with NAC or PDTC
has been shown to suppress the activation of NF-B and enhance that
of activator protein 1 (3). Furthermore, although PDTC-induced
inhibition of NF-B has often been attributed to its antioxidant,
radical-scavenging properties, recent evidence suggests that this
inhibitory effect could be mediated by direct oxidation of the thiol
containing cysteinyl group critical to the activity of this
transcription factor (24).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3% O2, 5%
CO2) followed by a control period at the same
pO2 or re-equilibrated to early postnatal
alveolar pO2 (76 torr, 10% O2,
5% CO2), mild hyperoxia (normoxia) (152 torr, 21%
O2, 5% CO2), and severe hyperoxia (722 torr,
95% O2, 5% CO2) for 4 h at 37 °C.
In each case, and under conditions of independent pretreatments, the
adenylate energy charge remained 
0.7, and transepithelial monolayer
resistance was monitored as 250 
cm2.
(Novus Biologicals Co.)
and polyclonal IgG p65 anti-NF-B (Santa Cruz Biotechnology, Santa
Cruz, CA) antibodies were used for primary detection. Anti-rabbit Ig-biotinylated antibody (Amersham Pharmacia Biotech) was employed for
secondary detection followed by the addition of
streptavidin-horseradish peroxidase conjugate. The membrane was
enhanced (ECL; Amersham Pharmacia Biotech) and exposed to an automatic
x-ray film processor. 
-Actin standard was used as a
reference for semiquantitative loading in parallel lanes for each
variable. Blots were digitized using a transilluminating scanner, and
the density of bands relative to -actin were determined
using UN-Scan-IT 32-bit automated digitizing system (version 5.1, Silk
Scientific Corp., Orem, UT). Data from at least four independent blots
were pooled, averaged, and plotted as a percentage of maximal
abundance/activation relative to control at static
pO2.
, 5'-GCCCTACGTGCTGTCTCA-3';
and NF-B, 5'-AGTTGAGGGGACTTTCCCAGGC-3' (binding
sequences are underlined). Gel-purified double-stranded DNA was
end-labeled with [-32P]ATP (NEN Life Science
Products). Identical amounts of radioactive probe (1-2 × 104 counts·min
1) were added to binding
reactions containing 1-5 µg of fATII nuclear extracts in a final
volume of 40 µl in DNA binding buffer (7). Reaction mixtures were
incubated for 30 min at 25 °C before separating on nondenaturing 4%
polyacrylamide gels at room temperature and subjected to
electrophoresis with 1:10 5× Tris-Borate-EDTA buffer. A nonspecific
competitive polydeoxyinosinic-deoxycytidylic acid (poly(dI-dC))
(Amersham Pharmacia Biotech) was added to reaction mixtures after the
addition of labeled probe. Gels were transferred to ion-exchange
chromatography paper, vacuum dried, and quantitated by phosphorimaging
using a Canberra-Packard Instant Imager.
1 protein.
pO2 regimen with different
concentrations of GSH and GSSG for 15 min on ice. The total glutathione
(GSH + GSSG) concentration/reaction mixture (40 µl) was adjusted to
100 nmol/ml, and the ratio ([GSH] + [GSSG])/([GSSG]) was
modulated such that [GSSG] was increased at the expense of [GSH].
Ratios were adjusted to final values of 100, 50, 10, 5, 2, 1.25, and 1. After incubation period samples were assayed for the binding activities
of HIF-1 and NF-B as indicated above. All nuclear extracts tested
prior to the addition of glutathione had undetectable levels of GSH or
GSSG (
0.01 nmol/ml).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and NF-B Nuclear
Abundance--
Variation in nuclear protein abundance of HIF-1 and
NF-B at various pO2 shifts was
investigated by Western analysis. Within the limits of the oxygen
tensions investigated in our experiments, HIF-1 nuclear abundance
was maximally elevated in cultures that were maintained constantly at
23 torr. On mild oxygenation to 76 torr (the estimated
pO2 of the distal lung within the first series
of breaths at birth), the relative level of HIF-1 nuclear abundance
was significantly diminished but remained well above that of cultures
exposed to moderate and severe hyperoxic shifts where nuclear protein
levels were barely detectable (Fig.
1A). A 24-hour preincubation
with NAC in cultures maintained at 23 torr preserved the nuclear
abundance of HIF-1 in a dose-dependent manner
independently of the relative level of hyperoxic shift (Fig.
1A). Fig. 1B presents the densitometric analysis
of HIF-1 abundance referenced to -actin (**,
p < 0.01 and ***, p < 0.001, as
compared with cultures without pretreatment (0 mM NAC)).
These events were accompanied by a dose-dependent increase
in the GSH/GSSG (Table I, discussed
below). In contrast to the O2 and NAC activity pattern of
HIF-1, the hyperoxic induction of NF-B nuclear translocation was
substantially inhibited by NAC, again, in a dose-dependent manner (Fig. 1C). The maximum inhibition is evident with 50 mM NAC at all oxygen tensions studied, whereas the lowest
concentration tested (1 mM) showed mild, though
significant, attenuation of the p65 subunit (Fig. 1, C and
D). Densitometric analysis of NF-B nuclear abundance in
reference to -actin is given in Fig. 1D (*,
p < 0.05; **, p < 0.01; ***,
p < 0.001, as compared with cultures without
pretreatment).
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Fig. 1.
Effects of NAC pretreatments on nuclear
abundance of HIF-1 and
NF-B under various oxygen tensions.
A, dose-dependent variations in HIF-1 nuclear
abundance as assessed with immunoblotting showing protein abundance
with different concentrations of NAC. -Actin is shown as an internal
reference for semiquantitative loading in each lane. NS,
nonspecific. B, the percentage relative maximal abundance of
HIF-1 against that obtained under activating conditions (23 and
23
76 torr pO2) without NAC pretreatment
(**, p < 0.01; ***, p < 0.001, as
compared with control (0 mM NAC)). C,
dose-dependent variations in NF-B nuclear abundance as
assessed with Rel-A anti-p65 showing protein abundance with different
concentrations of NAC. D, the percentage relative maximal
abundance of NF-B against the control values obtained without NAC
pretreatment (*, p < 0.05; **, p < 0.01; ***, p < 0.001, as compared with control). The
histograms represent the mean values and the error bars the S.E. of the
relative intensity of the bands of four independent experimental
preparations.
Redox equilibrium assessment of glutathione ratio homeostasis under
various oxygen pO2 regimen reported in fATII cells
pretreated with NAC for 24 h at 37 °C
and NF-B Nuclear
Abundance--
PDTC pretreatment of cultures exposed to each
pO2 regimen produced a similar pattern of
HIF-1 and NF-B nuclear accumulation as noted for NAC treatments.
The attenuation of HIF-1 nuclear accumulation with increasing
pO2 is blocked in a dose-dependent manner by increasing PDTC (Fig. 2,
A and B, the latter presenting densitometric
analysis of HIF-1 abundance referenced to -actin (*,
p < 0.05; **, p < 0.01; ***,
p < 0.001; as compared with cultures without
pretreatment (0 µM PDTC)). Fig. 2C shows the
dose-dependent inhibitory effect of PDTC on NF-B nuclear
abundance under various pO2. The maximum
inhibition is evident with 100 µM PDTC at all oxygen
tensions studied, whereas the lowest concentration tested (10 µM) showed mild, though significant, attenuation of the
p65 subunit (Fig. 2, C and D). Densitometric
analysis of NF-B nuclear abundance in reference to -actin is
given in Fig. 2D (*, p < 0.05; **,
p < 0.01; ***, p < 0.001; as compared
with cultures without pretreatment (0 µM PDTC)). Note
that the effective concentration of PDTC under each
pO2 regimen lies within the
104-106 M range, whereas that
of NAC lies in the range of 102-103
M. The PDTC-dependent changes in transcription
factor activities were accompanied by a dose-dependent
decrease in the glutathione ratio, as depicted in Table
II (discussed below).
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Fig. 2.
Effects of PDTC pretreatments on
nuclear abundance of HIF-1 and
NF-B under various oxygen tensions.
A, dose-dependent variations in HIF-1 nuclear
abundance as assessed with immunoblotting, showing protein abundance
with different concentrations of PDTC. -Actin is shown as an
internal reference for semiquantitative loading in each lane;
NS, nonspecific. B, the percentage relative
maximal abundance of HIF-1 against that obtained at 23 and 2376
torr pO2 without PDTC pretreatment (**,
p < 0.01; ***, p < 0.001, as compared
with control (0 µM PDTC)). C,
dose-dependent variations in NF-B nuclear abundance as
assessed with RelA anti-p65, showing protein abundance with different
concentrations of PDTC. D, the percentage relative maximal
abundance of NF-B against the control values obtained without PDTC
pretreatment (*, p < 0.05; **, p < 0.01; ***, p < 0.001, as compared with control). The
histograms represent the mean values and the error bars the S.E. of the
relative intensity of the bands of five independent experimental
preparations.
Redox equilibrium assessment of glutathione ratio homeostasis under
various oxygen pO2 regimen reported in fATII cells
pretreated with PDTC for 24 h at 37 °C
and
NF-B Activation Kinetics with NAC Pretreatment--
The effect of
NAC (0, 1, 10, and 50 mM) on DNA binding activity is shown
for HIF-1 and NF-B in Fig. 3,
A and C, respectively. Exponential increase
(HIF-1) or decrease (NF-B) of the binding activities are evident
with increasing concentrations of NAC. Fig. 3, B (HIF-1)
and D (NF-B), show histogram analysis of the dose-response curve (*, p < 0.05; **,
p < 0.01; ***, p < 0.001; as compared
with untreated cultures (0, Control)). The stimulatory (HIF-1) and
inhibitory (NF-B) equilibrium constants (Ks
and Ki) for NAC-dependent DNA binding
activity were determined from the positive/negative linear regressions
of the % stimulation/inhibition versus the concentration of
NAC (mM) (Table III).
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Fig. 3.
DNA consensus binding analysis for
HIF-1 and NF-B
nuclear extracts (1-5 µg) with NAC.
A, HIF-1 activation state at 23 and 2376 torr
pO2 shifts (FP, free probe;
NS, nonspecific). The stimulatory effects of NAC are evident
over 23152 and 23722 torr, with apparent maximum activation at 50 mM. B, percentage analysis of the gross cpm
relative to the intensity of the band of the control (0) at each
pO2 (**, p < 0.01; ***,
p < 0.001). C, NF-B activation status
over pO2 shifts, where the inhibitory effects
are prominent with increasing concentrations of NAC. D,
percentage analysis of the gross cpm relative to the intensity of the
band obtained without pretreatment (*, p < 0.05;
**, p < 0.01; ***, p < 0.001). The
histograms represent the mean values, and the error bars represent the
S.E. of the relative intensity of the bands of four independent
experiments.
Equilibrium constants extrapolated from the linear regression analysis
obtained for the DNA-binding activity of HIF-1 and NF-B in fATII
epithelia pretreated with NAC or PDTC and exposed to ascending
pO2 regimen
); M-22 (NF-B); 3-base pairs mutation), the addition of
cold nonlabeled oligonucleotide competitor immediately before the
probe, at 100-fold molar excess, and super shift analysis with specific
antibodies for HIF-1 and NF-B (RelA/p65) at 2 µg/reaction (Fig.
4, A and B).
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Fig. 4.
Specificity determination of the complexed
bands obtained for HIF-1 and
NF-B. A, HIF-1 binding
complexes shown at 23 torr, with arrows indicating positions
of the respective specific antibody-complexed supershifts
(FP, free probe; NS, nonspecific; SS,
supershift). The addition of the mutant oligonucleotide (M-18) and
100X-fold competitor completely abolished the binding of HIF-1.
B, NF-B binding analysis with the corresponding displaced
bands with anti-p65 shown at 23152 torr
pO2. The addition of the mutant
oligonucleotide (M-22) and 100X-fold competitor completely abolished
the binding of NF-B. The data are representative of three separate
experiments.
and
NF-B Activation Kinetics with PDTC Pretreatment--
The effect of
PDTC (0, 10, 50, and 100 µM) on DNA binding activity is
shown for HIF-1 and NF-B in Fig. 5,
A and C, respectively. No change (HIF-1) and a
significant decrease (NF-B) of the binding activities are evident
with increasing concentrations of PDTC. Fig. 5, B (HIF-1)
and D (NF-B), shows a histogram analysis of the
dose-response curve (**, p < 0.01; ***,
p < 0.001; as compared with untreated cultures (0, Control)). The stimulatory (HIF-1) and inhibitory (NF-B)
equilibrium constants (Ks and
Ki) for PDTC-dependent DNA binding
activity were determined from the positive/negative linear regressions
of the % stimulation/inhibition versus the concentration of
PDTC (µM) (Table III).
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Fig. 5.
DNA consensus binding analysis for
HIF-1 and NF-B
nuclear extracts (1-5 µg) with PDTC.
A, HIF-1 activation state at 23152 and 23722 torr
pO2 shifts (FP, free probe;
NS, nonspecific), where no stimulatory effects of PDTC are
observed. B, percentage analysis of the gross cpm relative
to the intensity of the band of the control (0) at each
pO2 (**, p < 0.01; ***,
p < 0.001). C, NF-B activation status
over pO2 shifts, where the inhibitory effects
are prominent with increasing concentrations of PDTC. D,
percentage analysis of the gross cpm relative to the intensity of the
band obtained without pretreatment (**, p < 0.01; ***,
p < 0.001). The histograms represent the mean values,
and the error bars represent the S.E. of the relative intensity of the
bands of four independent experiments.
pO2 are given in Table I, with the
maximum increase in GSH levels and glutathione redox ratio, coincident
with the highest concentration of NAC used in this study (50 mM) at all oxygen tensions (**, p < 0.01; ***, p < 0.001; as compared with control (0 mM NAC)). NAC (1 mM) had no stimulatory effect
on GSH elevation, and a 10 mM concentration increased the
level of GSH at only 152 (Fig. 6A), 2376 (Fig. 6C), and 23722 (Fig. 6E) torr (*,
p < 0.05; **, p < 0.01; ***, p < 0.001; as compared with control).
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Fig. 6.
Analysis of glutathione homeostasis with NAC
pretreatment under various oxygen tensions. Reduced ( 
, GSH) and
oxidized (
, GSSG) glutathione were enzymatically assessed in fATII
cells for the dose-response curves of NAC at (A) 152 torr,
(B) 23 torr, (C) 2376 torr, (D)
23152 torr, and (E) 23722 torr
pO2. Variations are shown such that the
elevation of GSH is at the expense of GSSG, thereby increasing GSH/GSSG
ratios. *, p < 0.05; **, p < 0.01;
***, p < 0.001 for [GSH], as compared with [GSH]
of the control (0 mM NAC). +, p < 0.05; ++, p < 0.01; +++,
p < 0.001, for [GSSG], as compared with [GSSG] of
the control. At all NAC concentrations, [GSH] was significantly
higher than [GSSG] (p < 0.05). Data are represented
as the mean ± S.E.; control (*n = 7), NAC (1 mM, n = 5; 10 mM,
n = 5; 50 mM, n = 5).
*n refers to number of measurements run in duplicates taken
from at least two independent cell preparations, where the entire
results were pooled and averaged.
pO2. PDTC elevated the
concentration of GSSG at the expense of GSH, as evident from the
decreasing ratio of total glutathione (GSH + GSSG) to that of GSSG,
indicating oxidation of GSH. The corresponding ratios at different
pO2 are given in Table II. The maximum
increase in [GSSG] is evident with the highest concentration of PDTC
used in this study (100 µM) at all oxygen tensions
investigated (Fig. 7), except at 23 torr
(+, p < 0.01; +++,
p < 0.001; as compared with control (0 µM PDTC)). Likewise, 100 µM PDTC caused
maximum decrease in GSH levels at all oxygen tensions, except at
pO2 = 152 torr (Fig. 7A) (*,
p < 0.05; ***, p < 0.001; as compared
with control). A PDTC concentration of 10 µM stimulated
GSSG elevation at 23152 torr (Fig. 7D), which decreased
[GSH] at 2376 and 23152 torr (Fig. 7, C and
D; *, p < 0.05; ***, p < 0.001; as compared with control). PDTC (50 µM) has no
stimulatory effects on [GSSG], except at 23152 (Fig. 7D) pO2 (++,
p < 0.001, as compared with control), whereas it
depressed [GSH] at 2376 and 23152 torr (Fig. 7, C
and D; **, p < 0.01; ***, p < 0.001; as compared with control).
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Fig. 7.
Analysis of glutathione homeostasis with PDTC
pretreatment under various oxygen tensions. Reduced (GSH) and
oxidized (GSSG) glutathione were enzymatically assessed in fATII cells
for the dose-response curves of PDTC at (A) 152 torr,
(B) 23 torr, (C) 23 76 torr, (D)
23152 torr, and (E) 23722 torr
pO2. Variations are shown such that the
elevation of GSSG is at the expense of GSH, thereby decreasing GSH/GSSG
ratios. *, p < 0.05; **, p < 0.01;
***, p < 0.001 for [GSH], as compared with [GSH]
of the control (0 µM PDTC)). +,
p < 0.05; ++, p < 0.01;
+++, p < 0.001, for [GSSG], as compared
with [GSSG] of the control. Data are represented as the mean ± S.E.; control (*n = 7), PDTC (10 µM,
n = 5; 50 µM, n = 5; 100 µM, n = 5). *n refers to
number of measurements run in duplicates taken from at least two
independent cell preparations, where the entire results were pooled and
averaged.
and NF-B Binding Activities in Vitro--
The effects of descending
ratios (R) of GSH to GSSG were investigated to evaluate
HIF-1 and NF-B activation in nuclear extracts of fATII cells
exposed to ascending pO2 regimen. Increasing
GSSG/GSH ratios were shown to be inhibitory on the binding activity of HIF-1 (23 torr, Fig. 8A)
and NF-B (23152 torr, Fig. 8B). The most prominent
inhibition is obvious with R 
2 at all oxygen tensions investigated, with complete abrogation at r = 1 (HIF-1) and r = 1.25 (NF-B). Linear regression
analysis was carried out to determine the inhibitory constants of the
negative slopes plotted as percentage of inhibition versus
the natural logarithm of R. Fig. 8, C and
D, shows the inhibition degrees of HIF-1 and NF-B activation, respectively. HIF-1 50% inhibitory constant
(KiHIF-1
) was determined from the
negative slope of the equation y = 
13.05x + 89.76 to
be Ln r = 3.05 (r = 21.12); as such,
the minimum effective [GSSG] contributing to this inhibition is
~4.73 ± 0.12 µM (Fig. 8B). NF-B
50% inhibitory constant
(KiNF-
B) was
determined from the negative slope of the equation y = 16.63x + 97.46 to be Ln r = 2.85 (r = 17.28); as such, the minimum effective [GSSG] contributing to this
inhibition is ~5.78 ± 0.15 µM (Fig. 8D).
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Fig. 8.
Nuclear extracts (5 µg) of fATII cells exposed to oxidative stress were
treated in vitro with various GSH/GSSG
concentrations. Ratios were adjusted according to the formula
([GSH] + [GSSG])/([GSSG]), such that the molarity of [GSSG] was
increased at the expense of [GSH]. A, analysis of HIF-1
activation (23 torr) at various ratios (R), with prominent
inhibitory effects at R 1.25. B, analysis
of NF-B activation (23152 torr) at various R, with
maximum inhibitory effects observed at R 2. Control
lanes contained nuclear extracts without in vitro
pretreatments, and the lane with 100 µM [GSH] received
no exogenous [GSSG] (FP, free probe). The
dose-dependent decrease in binding activities of either
transcription factor is evident with increasing [GSSG]. C,
linear regression analysis of HIF-1 percentile inhibition
(r = 0.93), where the derived inhibitory constant
KiHIF-1 4.73 µM
(see "Results" for more details). D, linear
regression analysis of NF-B percentile inhibition (r = 0.99), where the
KiNF-B 5.78 µM. This EMSA is representative of results similarly
obtained with in vitro analysis in three independent
experiments of separate cell preparations.
Trypan blue exclusion (TBE), energy charge (EC) ratio, and tetrazolium
(MTT) reduction as independent measurable indices for cell viability
assessment under various pO2 regimen reported in fATII cells
with or without pretreatments with NAC or PDTC for 24 h at
37 °C
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and NF-B
in the distal epithelial lining of the developing lung is redox- and
oxygen-sensitive (this study and Ref. 7). To further clarify the
molecular mechanisms involved in modulating the genetic response of the
fetal lung to oxidative stress, we have focused our attention on the
link between cellular antioxidant/pro-oxidant equilibrium and the
nuclear translocation and DNA consensus sequence binding (activation)
of HIF-1 and NF-B. To potentiate changes in cellular redox
balance through the glutathione biosynthetic pathway, cultures were
exposed to an experimental shift in pO2 with or
without NAC, a thiol-containing antioxidant and substrate for GSH
synthesis (31), or PDTC, possessing both antioxidant thiocarbamate and thiuram pro-oxidant characteristics (22).
nuclear abundance, which was greater under a 23152 and
23722 torr pO2 regimen than observed under
stimulating conditions without NAC (23 and 2376 torr). DNA consensus
sequence binding was also preserved at elevated
pO2 at maximal concentrations of NAC employed in
this study. These observations suggest that NAC pretreatment effectively manipulate the hypoxia-/redox-dependent
signaling sequence, which governs both nuclear abundance and activity
of HIF-1 independently from the imposed pO2
regimen. This is in keeping with the capacity for NAC to suppress
and/or scavenge reactive oxygen intermediates (21, 31), thereby
imposing a reducing cellular environment, protracting the half-life of
cytosolic HIF-1, and favoring its translocation to the nucleus. As
NAC is an acetylated variant of the amino acid L-cysteine,
it possesses both direct (i.e. oxidizable sulfhydryl groups)
and indirect (i.e. as a substrate for the biosynthesis of
GSH) antioxidant activity (20, 31). To further elucidate the pathways
leading to activation of HIF-1, glutathione concentrations were
enzymatically assessed in vitro in cultures exposed to
24 h of NAC pretreatment. In this, and a previous study (7), we
noted that shifting fATII beyond fetal lung oxygen tensions resulted in
an elevated total glutathione pool characterized by a 4-fold rise in
[GSH], a modest reduction in [GSSG], and a parallel fall in
HIF-1 activity. Although we cannot exclude direct antioxidant
buffering by NAC, the observable effect of this compound was to
potentiate further both the elevation of [GSH] and reduction of
[GSSG] (Fig. 6 and Table I) beyond the shift that occurs naturally
with elevated pO2 regimen. We have previously
shown (7) that pretreatment of hypoxic cultures with
L-buthionine-(S,R)-sulfoximine, an irreversible
specific inhibitor of the rate-limiting enzyme in the biosynthesis of
glutathione, -glutamylcysteine synthetase (-GCS) (32), led to a
dose-dependent inactivation of HIF-1, suggesting GSH may
be a key modulator of the activity of this factor. In keeping with
this, we note that by experimentally increasing GSSG concentrations
4-5-fold in fATII cells using
1,3-bis-(2-chloroethyl)-1-nitrosourea or carmustine, an
inhibitor of glutathione reductase (GSSG-RD), a partial abrogation of
the hypoxia-induced activation of HIF-1 can be achieved, ostensibly
by invoking an oxidizing
environment.2 It seems clear,
therefore, that the oxygen and ROS responsiveness of HIF-1 resides
over a permissive range of antioxidant buffering capacities with
the implication that such compounds determine the physiological
activity (i.e. the in vivo Km)
of oxygen-responsive transcription factors to a hypoxia or
hyperoxia-linked signal. Fig. 9 presents
a schematic summary of the glutathione-linked signaling pathways
responsible for the regulation of HIF-1 in fetal distal lung
epithelium.
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Fig. 9.
Schematic diagram of HIF-1
activation circuits and oxygen-signaling mechanisms in
hypoxia. The reduction of oxidized glutathione (GSSG) forms
reduced glutathione (2GSH), capable of inducing HIF-1
activation. GSSG recycling to GSH is blocked by
1,3-bis-(2-chloroethyl)-1-nitrosourea, a specific
glutathione reductase inhibitor, thus increasing intracellular
[GSSG], a potent inhibitor of DNA binding. In oxidative stress,
-glutamylcysteine synthetase is transformed from native, inactive
form (n-GCS) to active form
(-GCS), which increases de novo
synthesis of GSH. This pathway is blocked by
L-buthionine-(S,R)-sulfoximine, an irreversible
inhibitor of -GCS, thus affecting HIF-1 activation. ROS, derived
from oxygen metabolites (reactive peroxides), tend to block the
activation of HIF-1. NAC, an antioxidant, releases this inhibitory
effect by scavenging ROS. NAC, in addition, is a major precursor of
GSH, a thiol antioxidant, thereby it elevates [GSH]
(
GSH) and induces HIF-1 activation. PDTC is
an antioxidant though possessing ROS-scavenging properties, its ability
to activate HIF-1 under reducing conditions is not established. PDTC
(as a pro-oxidant), like other dithiocarbamates, lowers the GSH/GSSG
ratio by oxidizing GSH. The elevated [GSSG]
(GSSG) has the potential to block HIF-1
activation. Upon HIF-1 binding to the hypoxia response element
hypoxia-responsive genes are up-regulated.
Much recent evidence highlights NF-B as playing a critical role in
coordinating early gene responses to the production of ROS (19),
therefore the NAC-induced shift in the glutathione redox state toward a
reducing equilibrium would be expected to suppress the activity of this
transcription factor. Pretreatment with NAC predictably diminished the
hyperoxic nuclear translocation and DNA consensus binding of NF-B
p65 (the major transactivating member of the NF-B family) in a
dose-dependent manner. Indeed, it has been demonstrated
that NF-B activation by a wide variety of stimuli can be blocked by
NAC, suggesting that the production of reactive oxygen metabolites is a
requisite component of the activation sequence for this transcription
factor (33). As ROS generation is rapid, the capacity for NAC to
suppress the activation of NF-B may center largely upon the acute
inherent antioxidant characteristics of this compound, although the
cellular activity of this transcription factor is favored by a lowered
GSH/GSSG incorporating elevated GSSG.
Dithiocarbamates, including PDTC, induce differential effects on redox
equilibrium according to: (i) their ability to decrease single electron radical species (a reduction property) and (ii) their
capacity to oxidize GSH and related thiol compounds, thereby modulating
glutathione recycling potential (an oxidation property; 22, 34). As
with other dithiocarbamates, PDTC thus possesses the capacity to exert
both anti- and pro-oxidant effects, the former being mediated through
dithiocarboxy scavenging of hydrogen peroxide (H2O2), superoxide anion (O
2) (35),
and hydroxyl radical (·OH) (36), and the latter being mediated by its
oxidation by reactive oxygen and nitrogen species, generating
dithiocarbamate thiyl radicals and thiuram disulphides, which directly
oxidize GSH to GSSG, a potent regulator of several transcription
factors and signal transducing pathways in lung and other systems
(36).
In this report we have shown regulated differential effects of PDTC on
the activation of HIF-1 and NF-B, revealing a striking equilibrium between the antioxidant and pro-oxidant modes of action of
dithiocarbamates. PDTC, like NAC, induced HIF-1 nuclear
translocation at all pO2 regimen
investigated; however, it failed to induce HIF-1 DNA binding
activity. We have observed that the GSH/GSSG ratio after PDTC treatment
was significantly lowered, largely because of a substantial increase in
the rate of GSH oxidation to GSSG (Table II). This decreased ratio may
explain why PDTC failed to induce binding to a DNA consensus sequence,
as a reducing nuclear environment is mandatory for DNA binding and the
expression of hypoxia-responsive genes. This is supported by our
observation that HIF-1 consensus DNA binding was facilitated by
experimental increases in GSH/GSSG in isolated nuclei (Fig. 8,
A and C). We propose that, although PDTC might be
acting as an antioxidant by scavenging ROS, its failure to activate
HIF-1 may be attributed to its pro-oxidant properties, whereby the
concentration of the oxidized form of glutathione increases favoring a
shift in cellular redox toward an oxidizing equilibrium. This is in
broad agreement with previous studies showing that PDTC increases the
level of GSSG at the expense of GSH (24, 34, 37) and that GSSG affects transcriptional activation by creating oxidizing conditions (24).
PDTC is a potent inhibitor of NF-B in alveolar epithelia acting at
the level of both nuclear translocation as well as consensus sequence
binding, being effective at concentrations which are 100-500-fold
lower than the inhibitory effect of NAC. This inhibitory effect is
likely to be the result of a compound series of events centering around
the oxidation of GSH to GSSG (Table II) rather than by any direct
interaction with NF-B itself (PDTC administered at 50 µM to NF-B-activated fATII-isolated nuclei failed to
inhibit DNA consensus sequence binding).2 Dithiocarbamates
are known to inhibit the phosphorylation-dependent release of
NF-B from its cytosolic inhibitory subunit, IB (38), suggesting
that the mechanism of ROS induced activation of this transcription
factor involves, at least in part, a redox-responsive kinase activity.
However, high GSSG concentrations also promote the formation of a
NF-B-disulfide complex, which inhibits the DNA binding activity of
this transcription factor (24). GSSG elevation promotes oxidation of
protein cysteinyl thiols, shifting the equilibrium of thiol-disulfide
exchange significantly toward the formation of mixed disulfides
resulting in a change in protein conformation and subsequent efficiency
of DNA binding (12, 39). Notably, we observed that lowered GSH/GSSG in
isolated nuclei inhibits NF-B DNA consensus sequence binding with
near identical kinetics to that of HIF-1 (Fig. 8, B and
D). Therefore, although oxidizing cytosolic conditions favor
NF-B dissociation from IB and subsequent nuclear translocation, a
reduced (i.e. high GSH/GSSG) nuclear environment favors DNA
consensus sequence binding. Although we cannot preclude the possibility
that PDTC interferes with the translocation of NF-B to the nucleus,
the inhibitory effects of this compound upon NF-B support the
hypothesis that dithiocarbamates primarily act as pro-oxidants by
elevating the concentration of oxidized glutathione (GSSG), which is
capable of direct (by forming a mixed NF-B-thiol inactive complexes)
or indirect (creating an oxidized environment in the nucleus)
abrogation of NF-B activity. Schematized pathways linking the
activation of NF-B to glutathione equilibrium in the fetal alveolar
epithelium are depicted in Fig. 10.
|
In conclusion, we have demonstrated that NAC and PDTC treatment
effectively uncoupled transcription factor activity from the normal
pattern induced by changes in oxygen availability in primary cultures
of fetal epithelial cells derived from the distal lung. The capacity of
the developing lung to mount an adaptive genetic response to hypoxic or
hyperoxic environments is therefore determined by the interplay between
oxygen availability, reduction-oxidation state cellular compartments
(in this case, nuclear and cytosolic) and the glutathione buffering
capacity of the alveolar epithelium. The enzymatic component of the
glutathione biosynthetic pathway therefore represent a lynchpin for the
development of clinical strategies for the treatment of perinatal
respiratory syndromes that are linked to oxygen-induced stresses.
| |
FOOTNOTES |
|---|
* This work was supported by grants from the Medical Research Council, Anonymous Trust, and Tenovus-Scotland (to S. C. L.).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.
This work is part of the doctoral dissertation of this author, who
is a recipient of the George John Livanos Prize Ph.D. scholarship (London).
¶ To whom reprint requests and correspondence should be addressed. Tel.: 44 (0) 1382&n
