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Introduction
Materials & Methods
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Manganese superoxide dismutase (MnSOD) is a mitochondrial enzyme that dismutates potentially toxic superoxide radical into hydrogen peroxide and dioxygen. This enzyme is critical for protection against cellular injury due to elevated partial pressures of oxygen. Thioredoxin (TRX) is a potent protein disulfide reductase found in most organisms that participates in many thiol-dependent cellular reductive processes and plays an important role in antioxidant defense, signal transduction, and regulation of cell growth and proliferation. Here we describe induction of manganese superoxide dismutase by thioredoxin. MnSOD mRNA and activity were increased dramatically by low concentrations of TRX (28 µM). Elevation of MnSOD mRNA by TRX was inhibited by actinomycin D, but not cycloheximide, occurring both in cell lines and primary human lung microvascular endothelial cells. mRNAs for other antioxidant enzymes including copper-zinc superoxide dismutase and catalase were not elevated, demonstrating specificity of induction of MnSOD by TRX. Thiol oxidation by diamide or alkylation by chlorodinitrobenzene inhibited MnSOD induction, further indicating a requirement for reduced TRX. Because both oxidized and reduced thioredoxin (28 µM) induced MnSOD mRNA, the intracellular redox status of externally added Escherichia coli oxidized TRX was determined. About 45% of internalized E. coli TRX was reduced, with 8% in fully reduced form and about 37% in partially reduced form. However, when TRX reductase and nicotinamide adenine dinucleotide (NADPH) were added to the extracellular medium with TRX, more than 80% of E. coli TRX was found to be in a fully reduced state in human adenocarcinoma (A549) cells. Although lower concentrations of oxidized TRX (7 µM) did not induce MnSOD mRNA, this concentration of TRX, when reduced by NADPH and TRX reductase, increased MnSOD mRNA six-fold. In additional studies, MCF-7 cells stably transfected with the human TRX gene had elevated expression of MnSOD mRNA relative to vector-transfected controls. Thus, both endogenously produced and exogenously added TRX elevate MnSOD gene expression. These findings suggest a novel mechanism involving reduced TRX in regulation of MnSOD.
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Introduction
Materials & Methods
Results
Discussion
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Manganese superoxide dismutase (MnSOD) is a mitochondrial enzyme that dismutates superoxide radical in
most, if not all, mammalian cell types including those of
pulmonary and cardiovascular origin (1). It appears that
MnSOD functions as an essential antioxidant defense, at
least in part, by protecting critical targets of superoxide (1)
in addition to preventing subsequent formation of its potentially more toxic free radical by-products. MnSOD can
modulate the oxidative stress characteristic of many disease conditions, including acute respiratory distress syndrome of premature infants, children, and adults. This role
is supported in relevant models of these disorders (4).
Induction of MnSOD can be mediated by conditions of
oxidative stress in eukaryotic as well as prokaryotic organisms (9). In mammalian systems, exposure to elevated
oxygen tension (9, 11, 12) or the cell-permeable oxidant
hydrogen peroxide (10) can cause such induction. In addition, cytokines such as tumor necrosis factor-
(TNF-)
and interleukin-1 (IL-1) can induce MnSOD gene expression, and, again, an oxidative mechanism has been proposed (15). Through an apparently different mechanism involving protein kinase C, phorbol ester can induce
MnSOD (18, 19). It has been found that MnSOD can be
induced by thiol-reducing agents, an effect that does not
appear to be due to the formation of oxidants by these
compounds (20). In addition, thiol-oxidizing and -alkylating agents can inhibit MnSOD induction by cytokines, whereas the reducing agents potentiate such induction
(21). Hence, it appears that there may be multiple mechanisms that can influence MnSOD regulation.
In this investigation we examined the effect of thioredoxin, a potent protein disulfide oxidoreductase, on MnSOD gene expression. Thioredoxin (TRX) catalyzes protein reduction in the following manner:
Remarkably low concentrations of TRX are effective in reducing disulfides in insulin, fibrinogen, human chorionic gonadotropin, blood coagulation factors, nitric oxide synthase, ribonucleotide reductase, glucocorticoid receptors, and other proteins (22). The rate of reduction of insulin disulfide by TRX was found to be 10,000 times higher than that by dithiothreitol (DTT) (22). Thus, reduced TRX is an extremely potent protein disulfide reductase. Intracellularly, most of this ubiquitous low molecular mass (12-kD) protein remains reduced (26, 27). Reduced or oxidized TRX can enter intact cells (26, 27). TRX has at its active site two critical cysteine residues, which in the oxidized protein form a disulfide bridge located in a protrusion from the three-dimensional structure of the protein (23). The flavoprotein thioredoxin reductase catalyzes the nicotinamide adenine dinucleotide (NADPH)-dependent reduction of this disulfide (28). Small increases in TRX can cause profound changes in thiol-disulfide redox status in proteins (27). Previous work with thiols in our laboratory led us to hypothesize that TRX activates MnSOD gene expression by a mechanism involving reduction of cellular proteins. The current article demonstrates paradoxical induction of manganese superoxide dismutase by the endogenous reductant TRX and suggests a novel signal transduction role for this protein.
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Introduction
Materials & Methods
Results
Discussion
References
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Materials
Human lung microvascular endothelial cells (HMVECs) were obtained from Clonetics (San Diego, CA). Anti- Escherichia coli TRX and E. coli thioredoxin reductase (TR) were obtained from American Diagnostica (Greenwich, CT). Recombinant E. coli TRX was obtained from Promega (Madison, WI). Iodoacetic acid was from Sigma Chemical (St. Louis, MO). All other chemicals were of the highest available grade.
Cell Lines and Treatments
The human lung adenocarcinoma line A549 was obtained from the American Type Culture Collection (ATCC, Rockville, MD) and maintained in endotoxin-free F-12K medium (Dr. Kaigan's modified; GIBCO, Grand Island, NY) containing 10% fetal calf serum and 100 units/ml of penicillin/streptomycin. Fully confluent monolayers were treated with different concentrations of TRX for various time intervals and then washed twice with Hanks' balanced salt solution. HMVECs were grown in endothelial basal medium along with nutrient supplements (Clonetics). L929 (mouse fibroblast) cells were grown in Dulbecco's minimal essential medium, monkey kidney (OMK) cells were grown in RPMI 1640 medium, and L2 (rat lung epithelial) cells were grown in F-12K medium. All media were supplemented with 10% fetal calf serum.
Isolation of RNA and Northern Blot Analysis
Human MnSOD gene was obtained from the ATCC in
PHMnSOD4 plasmid in E. coli HB101. Plasmids were amplified in E. coli and purified with a Qiagen plasmid preparation kit (Qiagen, Inc., Chatsworth, CA). The cDNAs
were isolated from the vectors by treatment with EcoRI
and gel purified. cDNAs were labeled with a randomly
primed DNA-labeling kit (GIBCO). Total RNA was isolated from cells by guanidine isothiocyanate lysis and cesium chloride centrifugation of the lysate (147,000 × g, 20-
25°C) in an ultracentrifuge (Beckman, Palo Alto, CA) by a
modification of the methods of Sambrook and coworkers
(29). Total RNA was quantified spectrophotometrically. Twenty micrograms of RNA was resolved by electrophoresis in a 1% agarose, 2.5 M formaldehyde gel in a
buffer containing 20 mM morpholinepropanesulfonic acid
(MOPS) and 1 mM EDTA (pH 7.4). RNA was transferred
to nitrocellulose and blots were prehybridized for 2-12 h
in 50% formamide, 0.75 M sodium chloride, 0.075 M sodium citrate (pH 7.0), 5× Denhardt's solution, 50 µg/ml
salmon sperm DNA, and 0.1% sodium dodecyl sulfate
(SDS) at 42°C. Blots were hybridized with MnSOD cDNA
labeled to a specific activity of 2-7 × 107 cpm using
[-32P]CTP (ICN, Irvine, CA) in hybridization solution at
42°C overnight and then were washed in 0.3 M sodium
chloride, 0.03 M sodium citrate, 0.1% SDS at 42°C. Autoradiographs were made by exposing blots to X-ray film
(Eastman Kodak, Rochester, NY) at 
70°C with intensifying screens. Blots were exposed to PhosphorImager screens
(Molecular Dynamics, Sunnyvale, CA) and densitometry
was performed with a Macintosh computer using MD Image Quant version 3.35.
Assay for MnSOD Activity
Cells were disrupted by sonication (Braun-Sonic 2000; B. Braun, USA) in three 15-s bursts (4°C). A competitive inhibition assay was performed using hypoxanthine-xanthine oxidase-generated O2·
to reduce nitroblue tetrazolium (NBT) monitored spectrophotometrically at 560 nm.
Inhibition of NBT reduction to 50% of maximal is defined as 1 unit of SOD activity (30). Inhibition of copper-zinc
superoxide dismutase (CuZnSOD) activity by 5 mM potassium cyanide allowed differentiation of CuZnSOD and
MnSOD. Protein concentrations were determined by Bradford assay (31) (Bio-Rad, Hercules, CA) and enzyme activity was expressed in units per milligram of protein.
Preparation of Reduced Thioredoxin
Reduced E. coli TRX (Promega) was prepared by the method of Fernando and coworkers (27) with modifications. The use of E. coli TRX allowed Western blot measurement, discrimination from the endogenous human TRX, and distinction of the various redox forms of the added TRX (see WESTERN BLOT QUANTITATION). Briefly, 850 µM oxidized TRX was incubated with 2 mM DTT at room temperature. After 20 min, excess DTT was removed from samples using a Sephadex G-25 spin column equilibrated with 10 mM Tris (pH 7.0), 1 mM EDTA. In a control experiment, when 2 mM DTT in 10 mM Tris (pH 7.5), 1 mM EDTA was passed through a Sephadex G-25 spin column, there was no DTT detected in the eluate as measured by reduction of 5,5'-dithio-bis(2-nitrobenzoic acid) at 412 nm. Hence, no DTT was carried over in TRX preparations used to treat cells.
Alkylation of Thioredoxin and Cell Lysis
Escherichia coli oxidized TRX (TRX-S2) was reduced with a 5-fold molar excess of DTT under argon at room temperature for 20 min in argon-equilibrated 10 mM Tris-HCl (pH 7.5) containing 1 mM EDTA. Following this reaction, 1 ml of carboxymethylation/cell lysis buffer (0.1 M Tris [pH 8.8], 12 mg/ml iodoacetic acid, 3 mM EDTA, 7 M guanidine hydrochloride, and 0.5% Triton X-100, equilibrated with argon for 1 h) was added. The solution was incubated at 37°C in the dark for 45 min. After incubation, excess reagent was removed by a Sephadex G-25 spin column. Cell lysates for determination of the oxidation state of TRX were processed similarly after cell monolayers were washed with phosphate-buffered saline (PBS). Protein was determined using a Bradford assay (31) (Bio-Rad).
Western Blot Quantitation of TRX and Detection of TRX Redox State
To determine the amount of E. coli TRX in A549 cells, we treated cells with E. coli TRX-S2 (7 µM), or TRX-S2 (7 µM) with NADPH (2 mM) and TR (0.1 µM), for 16 h. After incubation cell lysates were prepared by adding 0.5 ml of lysis buffer (20 mM MOPS, 10 mM NaCl, 1.5 mM MgCl2, 1% Triton X-100). Plates were then incubated on ice for 30 min. After incubation lysates were transferred to Eppendorf tubes and centrifuged (14,000 × g, 1 min). Supernatant protein was determined by Bradford protein assay (Bio-Rad). Supernatant protein (10 µg) was fractionated along with standards of 0-20 ng of E. coli TRX-S2 on a 15% native polyacrylamide gel. The protein was transferred to nitrocellulose (Hybond-ECL; Amersham, Arlington Heights, IL) using a miniprotein transblot apparatus (Bio-Rad). Nitrocellulose was washed and incubated with sheep anti-E. coli TRX IgG (American Diagnostica). This antibody neutralizes E. coli, but not human, TRX. After washing, the blot was incubated with sheep IgG- horseradish peroxidase (HRP) conjugate for 1 h at room temperature. Binding of secondary antibody was detected using an enhanced chemiluminescence (ECL) detection system (Amersham). The amount of E. coli TRX was quantified by preparing a reference curve comparing the relative density of the TRX band with known concentrations of TRX-S2. For determination of the redox state of cell-associated thioredoxin, carboxymethylated lysates (20 µg) were fractionated and the percentage of oxidized and reduced TRX was determined from the blot by densitometry.
Western Blot Analysis of Human MnSOD Protein
After incubation cells were washed with PBS with Na3VO4 (1 mM). The cells were then lysed in lysis buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1% Triton X-100, 2 mM EDTA, 50 mM NaF, 1 mM Na3PO4, 1 mM phenylmethylsulfonyl fluoride [PMSF]; and 10 µg/ml each of aprotinin, leupeptin, and pepstatin). Cell lysates (25 µg) were resolved under reducing and denaturing conditions in 12% polyacrylamide minigels (Bio-Rad). Protein was transferred to nitrocellulose (Amersham) using a transblot apparatus (Bio-Rad). MnSOD protein was visualized using a rabbit anti-human MnSOD polyclonal antibody (a generous gift of Dr. L.-Y. Chang, National Jewish Medical and Research Center, Denver, CO) and detected using anti-rabbit-HRP conjugate (Pierce, Rockford, IL) in an ECL detection system (Amersham).
Quantitation of Human Thioredoxin by Sandwich ELISA
Endogenous thioredoxin in A549 cells was quantitated using digoxigenin (DIG)-labeled human thioredoxin in a double antibody sandwich ELISA (Das, K. C., and C. W. White, 1997. An ultra-sensitive sandwich ELISA using digoxigenin-labelled antibody for detection of thioredoxin in human serum and biological samples. Submitted for publication). Briefly, 96-well Maxisorp microtiter plates (Nunc, Roskilde, Denmark) were coated with goat anti-human TRX polyclonal antibodies (3 µg/ml; American Diagnostica). Wells were blocked and incubated with antigen for 2 h at 37°C. After washing wells were incubated with anti-hTRX-DIG conjugates for 1 h at 37°C. Wells were washed and incubated with anti-DIG-peroxidase (Boehringer Mannheim, Indianapolis, IN) for 1 h. Next, wells were washed and incubated for 2-10 min with peroxidase substrate solution ABTS (Boehringer Mannheim). Absorbance was recorded at 405 nm in a microplate reader (Molecular Devices, Menlo Park, CA).
Determination of Potential Extracellular or Intracellular Superoxide Production by TRX
To determine whether extracellular superoxide production occurs during oxidation-reduction of TRX in the cell
culture medium, the reduction of cytochrome c was measured spectrophotometrically in phenol red-free F-12K
growth medium containing 10% fetal calf serum. Except
for the absence of phenol red, this was the same medium
used in all other studies in A549 cells. In the complete system, the cuvette contained oxidized TRX (7 µM), NADPH
(2 mM), TR (0.1 µM), and cytochrome c (10 µM). Reduction of cytochrome c was monitored at 550 nm in a Beckman DU-64 spectrophotometer at 25°C with a temperature-controlled cuvette compartment. The rate of reduction
of cytochrome c was calculated using an extinction coefficient of 19.6 mM1 cm1 for reduced cytochrome c (32). In
some experiments, CuZnSOD (50 µg/ml) was added to assess potential superoxide-dependent cytochrome c reduction.
To determine whether intracellular superoxide production occurs during oxidation-reduction of TRX, which can
enter cells after incubation (33), the sensitive target of superoxide, the iron-sulfur cluster of aconitase, was evaluated by measurement of cellular aconitase activity. There
was minimal modification of the original method (34).
Briefly, extracts were prepared following incubation of
cells for 6 h with TRX and/or TR and NADPH. The medium was aspirated and cells were washed with 5 ml of ice-cold Dulbecco's PBS. After aspiration of the wash, the
cells were scraped into cold PBS and then centrifuged at
1,500 × g for 20 s. The supernatant was aspirated, and the
cell pellet was disrupted with a sonicator into 200 µl of
buffer containing 50 mM Tris-HCl (pH 7.4), 0.6 mM MnCl2,
and 5 mM sodium citrate. The lysate was rapidly frozen in
an Eppendorf tube (1.5 ml), placed in a dry ice-ethanol
bath, and stored at 70°C. Activity assays were performed
within 8 h of sample collection. Extracts were clarified by
centrifugation (14,000 × g, 20 s) immediately on thawing.
The change in linear absorbance at 340 nm at 25°C was
measured in a 1.0-ml reaction mixture containing 50 mM
Tris-HCl (pH 7.4), 5 mM sodium citrate, 0.6 mM MnCl2,
0.2 mM NADP+, 1-2 units of isocitrate dehydrogenase,
and 10-100 µg of sample protein. One milliunit of aconitase activity was defined as the amount catalyzing the formation of 1 nmol of isocitrate per minute (36).
Quantitation of Endotoxin
All reagents used, including recombinant TRX and TR, were assayed for contaminating bacterial lipopolysaccharide (LPS; endotoxin) using the chromogenic Limulus amoebocyte lysate assay as previously described (39).
Maintenance of Cells with Elevated TRX Expression
A human breast adenocarcinoma (MCF-7) cell line (a generous gift of Dr. G. Powis, Arizona Cancer Center, Tucson, AZ) that was genetically modified to express elevated levels of TRX (TRX-9) has been characterized previously (40). The parental cell line transfected with the vector containing the neomycin-resistance gene (neo) served as the control cell line. Both cell lines were maintained in Dulbecco's modified eagle medium containing 10% fetal calf serum (Sigma) supplemented with G418 (geneticin; 300 µg/ ml). Confluent monolayers were processed for total RNA isolation and measurement of MnSOD mRNA by Northern analysis as described previously.
Statistical Analysis
Statistical analysis was done by microcomputer with JMP statistical software (41). Means were compared by one-way analysis of variance followed by Tukey's test for multiple comparisons. A P value < 0.05 was considered significant (41). Comparisons between two groups were made by two-tailed t test.
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Materials & Methods
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Discussion
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Dose-response Effect of Thioredoxin on MnSOD Expression
When human lung adenocarcinoma (A549) cells were incubated with various concentrations of TRX (Figure 1A),
a significant elevation of steady state MnSOD mRNA levels occurred in a dose-dependent manner. This effect was
specific for MnSOD because TRX did not cause alteration
in steady state mRNA levels of other antioxidant enzymes
such as CuZnSOD (Figure 1B) or catalase (Figure 1C). In addition, there was no change in 
-actin mRNA level (Figure 1D). To determine further the specificity of the TRX-mediated increase in MnSOD mRNA, cells were incubated with antibody to TRX (66 µg/ml) in addition to
TRX (28 µM). Elevation of MnSOD mRNA by TRX was inhibited by this antibody (Figure 1, lane 5). This higher
concentration of oxidized TRX (28 µM) was approximately
as effective as a much lower concentration of near fully reduced TRX (7 µM) in elevation of MnSOD mRNA (data
not shown). These findings indicated the importance of
the redox state of TRX in modulating MnSOD expression.
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Dose-Response Effects of Thioredoxin on MnSOD Activity
Because it is an increase in MnSOD enzyme activity that confers protection against superoxide radical, the effect of TRX on MnSOD activity also was evaluated (Figure 2). MnSOD activity increased significantly when A549 cells were incubated with increasing concentrations of TRX for 16 h (Figure 2A). Addition of anti-TRX antibody (66 µg/ ml) with TRX (28 µM) inhibited the increase in MnSOD activity. MnSOD activity was increased in a time-dependent manner with maximal elevation after 30 h of exposure (Figure 2B).
Time Course of Induction of MnSOD mRNA by Thioredoxin
The kinetics of induction were evaluated at intervals following incubation with low concentrations (28 µM) of
TRX. A maximal increase was observed after 16-24 hours
(Figure 3). At this time, TRX increased the level of MnSOD mRNA more than 15-fold. There was no alteration
in the concomitant level of -actin mRNA.
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Effect of Actinomycin D and Cycloheximide on Induction of MnSOD mRNA
When cells were incubated with TRX and actinomycin D, a transcriptional inhibitor, there was no increase in MnSOD mRNA, suggesting that the effect of TRX occurs at the level of transcription (Figure 4). Cycloheximide, an inhibitor of translation, potentiated the TRX-mediated increase in MnSOD mRNA. This could be due to increased accumulation of mRNA in the absence of translation or to failure of synthesis of an inhibitory protein. The concentrations of these agents were comparable to, but lower than, those used previously by other investigations in similar studies (16, 17). No cytotoxicity was observed as assessed by trypan blue exclusion (> 95%) under all conditions.
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Effect of Thioredoxin on MnSOD mRNA in Different Cell Types
To determine whether a similar increase in MnSOD occurs in cultures of other cell types, we incubated HMVECs in primary culture, OMK cells, mouse fibroblast (L929) cells, and rat lung epithelial-like (L2) cells with TRX (28 µM; 16 h). MnSOD mRNA was elevated by TRX exposure in each cell type tested (Figure 5). This effect of TRX was greater in cells of primates, such as those of humans and monkeys, than in rodent cells. Thus, elevation of MnSOD mRNA by TRX occurs in diverse cell types.
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Effect of Diamide and Chloro-2,4-dinitrobenzene on MnSOD mRNA Induction by Thioredoxin
Reduced TRX (TRX-SH2) is rapidly oxidized by diamide, whereas the thioredoxin reductase system can catalyze NADPH-dependent reduction of both diamide and TRX (22). Thus, the oxidation of TRX-SH2 by diamide is reversible. To evaluate the effect of oxidation of TRX-SH2 on MnSOD induction, A549 cells were incubated with diamide (2 mM; 30 min) followed by washing to remove the diamide. The cells next were incubated with oxidized TRX (28 µM; 6 h). As demonstrated in Figure 6, diamide prevented elevation of MnSOD mRNA by TRX. Thus, oxidized TRX was incapable of inducing MnSOD in an oxidizing environment. Prior incubation of A549 cells with chloro-2,4-dinitrobenzene (CDNB), a thiol-alkylating agent, prevented increased expression of MnSOD mRNA by TRX. This demonstrated that free thiol group(s) is necessary for MnSOD induction by TRX.
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Redox Status of Externally Added E. coli Thioredoxin in A549 Cells
Oxidized or reduced thioredoxin (28 µM and above) increased MnSOD gene expression in A549 cells. Because E. coli TRX is not as effective a substrate as mammalian TRX for mammalian TR (42), and because of the oxidizing environment of cell culture medium, it was considered possible that the effect of TRX was mediated by its oxidized species. Specifically, the Km for E. coli TRX is 35 µM, approximately 14-fold higher than the Km for mammalian TRX. Therefore, it was of interest to determine whether TRX was reduced within A549 cells. Standards of oxidized and reduced TRX were prepared and free sulfhydryls were carboxymethylated as described in MATERIALS AND METHODS. The sulfhydryl group of Cys-32 in E. coli TRX-SH2 shows a low apparent pKa value of 6.7. Only this sulfhydryl group of native TRX-SH2 is alkylated by iodoacetic acid (43). In reduced TRX that has been denatured by guanidine hydrochloride, both sulfhydryl groups are reactive. Carboxymethylated oxidized and reduced TRX could be separated in a 15% native polyacrylamide gel (44). When A549 cells were incubated with oxidized TRX (14 µM; 16 h), about 45% of total internalized TRX was found to be in monothiol and dithiol form: Approximately 8% was found to be in dithiol form, as it could form dicarboxymethylated TRX, and about 37% was present in the monothiol form, which could be detected as monocarboxymethylated TRX (Figure 7). We observed three distinct bands in the alkylated homogenates of A549 cells incubated with oxidized or reduced TRX (14 µM; Figure 7A, lanes 1 and 2). The bands were identified as follows: the uppermost band is the oxidized TRX as determined from the standard, and the bottom band is the dicarboxymethylated TRX. This is the product of reduced TRX in which both cysteines, Cys-32 and Cys-35, are in the reduced state and are carboxymethylated. The intermediate band corresponds to the monocarboxymethylation product of Cys-32 (44). Cys-32 is the most reactive sulfhydryl in TRX-SH2 (44). Under basal conditions, only a small amount of internalized E. coli TRX (8%) was fully reduced by A549 cells. To determine whether reduced TRX increases MnSOD gene expression, A549 cells were incubated with TRX (7 µM) plus NADPH (2 mM) and E. coli TR (0.1 µM). In the presence of this reducing system, 85% of the E. coli TRX that entered or associated with A549 cells was fully reduced (Figure 7A, lane 5).
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Effect of Different Redox States of Thioredoxin on MnSOD mRNA
When A549 cells were incubated with high concentrations of oxidized or reduced TRX (28 µM; 16 h), both induced expression of MnSOD mRNA with almost equal potency (data not shown). However, when these cells were incubated with lesser concentrations of oxidized TRX (7 µM; 16 h), there was very little or no induction of MnSOD mRNA (Figure 8). Nonetheless, when cells were incubated with oxidized TRX (7 µM) along with the TRX- reducing system containing NADPH (2 mM) and TR (0.1 µM), there was a 6-fold increase in MnSOD mRNA. Thus, an increase in MnSOD gene expression was detected when TRX was found to be in the reduced state in treated A549 cells, and there were also minor effects of the reducing system and its components (Figure 8, lanes 6 through 8). On the other hand, little or no elevation of MnSOD gene expression was found when E. coli TRX was predominantly in an oxidized state. Hence, reduced TRX causes increased MnSOD gene expression.
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Effect of Thioredoxin and Its Redox State on MnSOD Protein Expression
Western blot analysis (Figure 9) revealed that in A549 cells MnSOD protein was increased by a low concentration of TRX (7 µM) in the presence of the complete reducing system (Figure 9, lane 3). In the absence of TRX (Figure 9, lane 4), the complete TRX-reducing system had a considerably smaller effect. However, there were minor effects of each of the components of the reducing system. We attribute this to the presence of very low levels of TRX present in the extracellular medium of A549 cells (detectable by ELISA; not shown). This TRX is very probably human TRX secreted by these cells, although we cannot exclude the possible presence of additional, bovine TRX contaminating the serum in the medium. At the concentration used in lane 3 (Figure 9), oxidized TRX also was without effect in the absence of its reducing system (lane 2) relative to control cells (lane 1).
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Quantitation of Endogenous Thioredoxin by ELISA in A549 Cells
In A549 cells, the principal cell line used in these investigations, endogenous thioredoxin was measured by ELISA for human TRX and was found to be approximately 180 ng/mg protein in A549 cell lysates.
Estimation of Cell-associated E. coli TRX by Western Blot
Treatment of A549 cells with E. coli TRX-S2 (7 µM, 16 h) increased cell-associated E. coli TRX from undetectable to approximately 936 ng/mg protein. Treatment of these cells with the same concentration of TRX-S2 for this same duration in the presence of a complete TRX-reducing system increased cell-associated TRX estimated by Western blot to 1,160 ng/mg cell protein (all determinations in duplicate). Thus, total cellular TRX was increased to a comparable extent by TRX in the presence or absence of the reducing system. Any effect of the reducing system on total cell-associated TRX was minor relative to the 10-fold increase in fully reduced TRX detected in cells.
Effect of Thioredoxin on Extracellular and Intracellular Superoxide Production
To assess possible superoxide formation in cell culture medium by TRX and/or its reducing system, cytochrome c reduction at 550 nm was measured (Figure 10). Oxidized TRX (7 µM), incubated in phenol red-free F-12K medium containing 10% fetal calf serum, did not reduce cytochrome c. NADPH (2 mM) caused a small but nonsignificant increase in the rate of cytochrome c reduction relative to that for the complete medium alone. In addition, the TRX-reducing system, NADPH plus TR (0.1 µM), caused a small but significant increase in the cytochrome c reduction rate relative to that for the medium. In contrast, addition of the TRX-reducing system to oxidized thioredoxin (7 µM) in complete medium caused a marked increase in the rate of cytochrome c reduction. This is the expected effect of a potent protein disulfide reductase. However, this rate of reduction was not different when CuZnSOD (50 µg/ ml) was added to the reaction. This indicates that no superoxide is formed extracellularly by the complete TRX system. Therefore, cytochrome c reduction is either direct by TRX or indirect by molecules other than superoxide, which are themselves first reduced by TRX and/or other system components.
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To assess possible intracellular formation of superoxide in cells incubated with TRX and its reducing system, the activity of aconitase was measured in A549 cells. After incubation for 6 h with oxidized TRX (7 µM), NADPH (2 mM), and TR (0.1 µM), A549 cell aconitase activity (9.49 ± 1.26 mU/mg protein, n = 3) was not different from that of cells incubated in F-12K medium with 10% serum (8.43 ± 1.52 mU/mg protein, n = 3; P = 0.62). Likewise, cells incubated with NADPH alone or in combination with TR for 6 h did not have aconitase activity significantly different from that of control cells (not shown). This indicates that the rate of intracellular superoxide formation in cells treated with the complete TRX system, or with the TRX-reducing system, does not appreciably exceed that which occurs in control cells. Taken together, these data suggest that elevated extracellular or intracellular superoxide formation does not occur with, or contribute to, MnSOD induction by TRX.
Presence of Endotoxin in Recombinant Proteins and Its Effects on MnSOD mRNA Expression in A549 Cells
Because the TRX and TR used in these experiments are bacterial products, it is possible that endotoxin (LPS) contaminating these reagents could contribute to the observed effects. This possibility was carefully evaluated in the principal cell line used in these studies, A549. LPS was measured in the reagents used in the chromogenic Limulus amoebocyte lysate assay. LPS was present at a concentration of 0.2 ng/ml in 28 µM TRX preparations, and at 6 ng/ml in the TR preparation at the concentration used in these studies. The effect of exogenous E. coli endotoxin was then tested. In A549 cells, LPS from E. coli did not induce MnSOD mRNA over a range of concentrations well above those relevant to these levels of contamination (0- 100 ng/ml; data not shown).
Effect of Elevated TRX Gene Expression on MnSOD mRNA
To determine the effect of endogenously elevated TRX expression on MnSOD gene expression, MCF-7 cells stably transfected with the human TRX gene (TRX-9 cells) or the vector containing the neomycin-resistance gene alone (neo) were compared (40). Northern blot analysis demonstrated that MnSOD mRNA expression in TRX-9 cells was elevated severalfold relative to neo cells (Figure 11). These data indicate that endogenously produced TRX has an effect similar to that of the exogenously added protein on MnSOD expression.
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Abstract
Introduction
Materials & Methods
Results
Discussion
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In the present study MnSOD mRNA was strongly induced by TRX. TRX did not increase expression of other antioxidant enzymes such as catalase or CuZnSOD. Antibody to TRX completely inhibited the induction of MnSOD mRNA by TRX. These results demonstrate that the elevation of MnSOD mRNA by TRX is specific and is both dose- and time-dependent. As with MnSOD mRNA, A549 cell MnSOD activity was increased by TRX in a dose- and time-dependent manner following the maximum effect of TRX on MnSOD mRNA. The effect of TRX on expression of MnSOD activity also was inhibitable by anti-TRX antibody. MnSOD mRNA was elevated in a variety of cell types in response to incubation with TRX. This indicates that induction of MnSOD by TRX is a generalized phenomenon. Inhibition of MnSOD induction by actinomycin D suggests that the TRX effect occurs at the level of transcription.
MnSOD can be induced by sublethal hyperoxia (9, 11,
12), hydrogen peroxide (10), and cytokines (15) such as
TNF- or IL-1, and a regulatory mechanism involving an
oxidative stress response has been proposed (15). Thus,
the present finding that reduced TRX, a reductant, can induce MnSOD is a unique and unexpected observation.
Reduced and oxidized TRX both had a similar effect in
inducing MnSOD when added to cell culture medium at
higher concentrations (28 µM or greater). However, E. coli
TRX can be a substrate for human TR (42) even though
the Km is about 35 µM. Therefore, it is likely that at higher
concentrations of E. coli TRX, partial reduction by the endogenous human TR occurs. Indeed, when the redox state
of internalized E. coli TRX was investigated, 8% of oxidized E. coli TRX had been fully reduced, and 35% had been converted to a partially reduced form. Because only
8% of TRX was converted to its fully reduced form, it was
unclear whether the effect was due to the small reduced
fraction. To determine whether the effect was due to reduced TRX, cells were incubated with oxidized TRX, TR,
and NADPH. Under these reducing conditions, most
(85%) of the E. coli oxidized TRX enters the cell in the reduced state and remains reduced within the cells. At lower
concentrations (3 or 7 µM), oxidized TRX did not increase
MnSOD expression under usual cell culture conditions.
In contrast, such concentrations of oxidized TRX did increase MnSOD expression when exposed to cells in the
presence of a complete TRX-reducing system. Hence, it
became clear that TRX could induce MnSOD only in the
reduced form. Likewise, it is apparent that the small amount
of TRX that was present in the reduced form when A549
cells were incubated with higher concentrations of oxidized
TRX (28 µM) was responsible for MnSOD induction. Similar results with lens epithelial cells have been observed by
Spector and co-workers (26). These investigators found
that after exogenous addition of TRX only 9% of TRX
within the cells was in the reduced form, yet this fraction
could regenerate oxidized proteins. Further support for a
critical role for reduced TRX was obtained when it was
found that diamide, a reversible thiol-oxidizing agent,
could inhibit MnSOD induction by TRX. Because reduced
TRX was rapidly oxidized by diamide, the results demonstrated that oxidized TRX is not capable of inducing MnSOD. Likewise, prior incubation of A549 cells with chloro-2,4-dinitrobenzene (CDNB), a thiol-alkylating agent and
inhibitor of TR, prevented the effect of TRX. Therefore, it
is evident that reduced thiol groups were required for TRX
to cause MnSOD induction.
The requirement for reduced TRX did not exclude the hypothetical possibility that MnSOD induction could be caused by production of superoxide or secondary reactive oxygen species by oxidation-reduction reactions of TRX involving oxygen, either intracellularly or extracellularly. This possible mechanism, however, is not supported by the work of others, or by our own data. Potential extracellular redox cycling will be addressed first. Our current experiments have shown that TRX does not produce superoxide in cell culture medium. TRX, in the presence of its reducing system, TR plus NADPH, was shown to reduce electron acceptors such as cytochrome c, but this reduction was not inhibited by superoxide dismutase. The same findings were made when the electron acceptor used was nitroblue tetrazolium (not shown). Hence, TRX reduces selected substrates but does not indiscriminately transfer electrons to oxygen. For protein substrates, a very low concentration of reduced TRX is required to produce results similar to those obtained with much higher concentrations of other reductants such as DTT (22, 26). Indeed, the concentration of reduced TRX required in this study was more than 100-fold less than that of DTT, which can induce MnSOD in A549 cells (21). Unlike TRX, DTT is readily autoxidized to produce superoxide and hydrogen peroxide. If oxidant production due to autoxidation or redox cycling were responsible for MnSOD induction by these compounds, one would anticipate that the concentration of TRX required to produce the effect would be substantially higher, not much lower, than that of DTT.
It has been suggested that TRX can scavenge reactive oxygen species such as hydrogen peroxide, and TRX protects against cellular injury by hydrogen peroxide (45). TRX and TR also can participate in detoxification of lipid hydroperoxides (46, 47). Even if superoxide were generated extracellularly, it would not induce MnSOD in pulmonary A549 cells. Although superoxide does not readily enter A549 cells to affect susceptible targets (37), it can be rapidly dismuted spontaneously to hydrogen peroxide in aqueous solutions. Hydrogen peroxide can diffuse rapidly into cells. Nevertheless, it has been reported that hydrogen peroxide does not induce MnSOD in A549 cells (15). We confirmed this at multiple concentrations (100-700 µM) and also found that continuous generation of extracellular superoxide and hydrogen peroxide does not induce MnSOD in these cells (21).
To address intracellular redox cycling and superoxide production, we have used a sensitive target of superoxide, the iron-sulfur cluster of aconitase. Measurement of aconitase activity to estimate intracellular superoxide concentration was developed in bacterial systems by Gardner and Fridovich (34, 35). We extended the method for this purpose in mammalian cells (36, 37). Therein, aconitase sensitively detects intracellular superoxide production by redox-cycling agents such as phenazine methosulfate (37) and the toxic bacterial pigment pyocyanin (38), and activity remains decreased until oxidative stress is relieved. Mitochondrial inhibitors such as antimycin A also can decrease cell aconitase activity in association with elevated mitochondrial superoxide formation (37). A549 cells are ideal cells to use in such studies because 60% of cellular aconitase activity is cytoplasmic and 40% is mitochondrial (37), thereby allowing superoxide detection in either compartment. In the current studies, A549 cell aconitase activity did not decrease after treatment with TRX plus its reductase and NADPH, and this reducing system did not by itself affect cell aconitase activity. These observations indicate that TRX does not produce superoxide during intracellular oxidation-reduction reactions. Netto and others showed that even in a system that strongly favors thiol oxidation (iron/EDTA plus 5 mM DTT), addition of TRX tended to decrease oxygen consumption and prolong the lag time of the reaction. Furthermore, TRX acted synergistically with "thiol-specific antioxidant enzyme (TSA)" to eliminate oxygen consumption by metal-catalyzed DTT oxidation (48). According to this report, TSA is widely distributed in mammalian cells and tissues. Therefore, it would be expected to be present in the cells we have studied in this investigation. Two additional studies show that TRX plus its reducing system participate with TSA in eliminating hydrogen peroxide and alkyl hydroperoxides in a similar thiol-oxidizing system (49, 50). Another investigation also suggests that TRX acts to diminish intracellular oxidative stress. In that report cisplatinum (cis-diamminedichloroplatinum[II])-mediated intracellular accumulation of peroxides and cytotoxicity was found to be prevented by TRX. As such, TRX may contribute to cellular resistance to such cancer chemotherapy agents (51). Combined with our current data, these reports suggest that not only does TRX not participate in intracellular redox cycling with oxygen, but that it will interact with other cell proteins to inhibit redox cycling with oxygen by other susceptible compounds. In addition, TRX may effectively reduce and detoxify reactive oxygen species produced by such reactions. Thus, TRX has protective antioxidant effects, and these could be mediated in part through MnSOD induction. TRX is a potent thiol reductant, and no evidence is available to suggest that it has prooxidant effects, or that it could induce MnSOD through such effects.
A potential source of artifact considered during the conduct of some of these experiments was that of possible effects caused by endotoxin, and we did find small concentrations of endotoxin contaminating the recombinant proteins. However, others have found that LPS does not induce MnSOD mRNA in the pulmonary adenocarcinoma cell line A549 (15). In the current study, we confirmed that A549 cells are resistant to this effect of endotoxin at the concentrations present and at levels well above those contaminating these experiments. In addition, inhibition of MnSOD induction by anti-TRX antibodies would not be expected if TRX effects were due to endotoxin. Therefore, contaminating endotoxin does not appear to contribute to the observed effects of TRX in these cells.
Although many of the findings described herein have potential pharmacologic importance, the question arises as to how the changes caused by exogenous TRX might pertain to effects due to variations in endogenous TRX expression during different physiologic or pathologic circumstances. On the basis of our measurements of endogenous human TRX in A549 cells and our estimation of E. coli TRX present in these cells after treatment with this preparation, it is estimated that total cellular TRX protein was increased by about 5-fold. Although this is a substantial quantity, it should be clarified that the E. coli protein is not a particularly good substrate for the human TRX reductase when compared to human TRX. Therefore, it is anticipated that relatively smaller changes in endogenous or exogenous human TRX might also increase MnSOD expression, whereas larger increases in the E. coli TRX in the cells would be required to effect the same change. Using a clone of MCF-7 cells that was genetically modified to increase endogenous human TRX expression (40), we found that such TRX-overexpressing cells have substantially increased basal expression of MnSOD mRNA relative to the parental cell line transfected with the vector and neo gene. These findings corroborate our data, using an entirely different experimental approach. Thus, endogenous TRX can modulate MnSOD expression in the same manner that exogenous TRX does. Furthermore, it is relevant and potentially important that TRX is readily secreted in substantial quantities from a variety of malignant and nonmalignant cell types through a leaderless pathway (52), and membrane-associated forms of TRX also exist (53). Hence, TRX could function as an extracellular as well as an intracellular messenger, and studies utilizing exogenously added TRX also are relevant to this physiologic situation.
A variety of stimuli can increase TRX expression. Others have observed that phorbol ester, ischemia-reperfusion, hydrogen peroxide, diamide, menadione, retinol, and cancer chemotherapy agents can elevate endogenous TRX expression in certain cell types (51, 54). We have observed that mRNA expression of both TRX and TR also is induced in primate lung during the normal fetal-neonatal transition, that hyperoxia markedly enhances this induction, and, through the use of fetal lung explant culture, that exposure to oxygen is the stimulus that effects this change (61). Thus, TRX and its reductase can be upregulated under both pathologic and physiologic conditions, including various oxidative stresses, many of which also are capable of upregulating MnSOD (9, 10, 18, 19, 61). Therefore, TRX could be a signal involved in effecting or potentiating the expression of MnSOD by many of these stimuli.
TRX reduces and protects the function of several classes of proteins during oxidative stress. These include proteins important for cell homeostasis and intermediary metabolism such as glyceraldehyde-3-phosphate dehydrogenase (27); hormones such as insulin (23) and human chorionic gonadotropin, and their receptors such as the glucocorticoid receptor; proteins that themselves produce critical signal molecules such as the endothelial nitric oxide synthase (24, 25); and signal proteins themselves such as transcription factors (64). Because of its resistance to oxidants, TRX could be an effective messenger to evoke or potentiate important signal transduction functions, including induction of other critical antioxidants, during oxidative stress.
The present study demonstrates that additional mechanisms that do not themselves involve oxidant stress can
regulate expression of MnSOD in mammalian lung and
other cell types. It has been proposed that regulation of
protein function via reversible oxidation of sulfhydryl
groups in disulfides (thiol redox control) may be of considerable importance in cell signaling, perhaps comparable to
protein phosphorylation (23). This investigation supports the possibility that TRX could have such a novel function.
Our previous reports (20, 21) that MnSOD can be induced
by thiol-reducing agents, and that thiol-modifying agents
can inhibit MnSOD induction, also suggest that TRX
could act to reduce a signal-transducing protein for MnSOD gene expression. One such signal protein could be
nuclear factor 
B (NF-B). It has been shown that NF-B
can be activated or potentiated by thioredoxin (64, 65),
and this effect occurs through an elaborate and specific
molecular interaction (66). However, TRX can be involved in regulation of several transcription factors (33)
that could influence MnSOD regulation. Regardless, MnSOD gene upregulation can be caused by conditions in addition to those of oxidative stress, and a multiplicity of regulatory pathways in mammalian cells is further supported.
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Footnotes |
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Address correspondence to: Carl W. White, M.D., Department of Pediatrics, National Jewish Medical and Research Center, J-103, 1400 Jackson Street, Denver, CO 80206.
(Received in original form October 14, 1996 and in revised form April 7, 1997).
Acknowledgments: This work was supported in part by a Fellowship Grant from the American Lung Association (K. C. Das) and National Institutes of Health Grants HL46481 (C. W. White) and 1RO1 HL 52732 (C. W. White). C. W. White was recipient of an American Heart Association Established Investigator Award with partial funding support of the American Heart Association of Colorado. The authors are grateful to Dr. Ling-Yi Chang for providing antibodies to human MnSOD, to Dr. Bruce Pitt for helpful discussions, and to Dr. Garth Powis for providing to us the MCF-7 clones with elevated TRX expression. The authors also acknowledge the superb technical assistance of Xiao-ling Guo, of May Gillespie in performing the assays for endotoxin, and of Jacque Horvath in preparing the manuscript.
Abbreviations CDNB, chloro-2,4-dinitrobenzene; CuZnSOD, copper-zinc superoxide dismutase; DCB-TRX, dicarboxymethylated thioredoxin; DIG, digoxigenin; DTT, dithiothreitol; HMVEC, human microvascular endothelial cell; MCB-TRX, monocarboxymethylated thioredoxin; MnSOD, manganese superoxide dismutase; NADPH, nicotinamide adenine dinucleotide (reduced form); NBT, nitroblue tetrazolium; TR, thioredoxin reductase; TRX, thioredoxin; TRX-SH2, reduced thioredoxin; TRX-S2, oxidized thioredoxin.
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References |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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