Introduction

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Reactive oxygen species (ROS) have been implicated in the pathogenesis of a wide variety of lung diseases (1, 2). Recent evidence suggests that ROS may act as signal transduction messengers, inducing new gene expression in response to injury (3). The nature of gene induction by ROS is not completely understood. However, there is increasing evidence to support a ROS-induced activation of transcriptional factors, such as nuclear factor (NF)-B and activator protein-1 (7, 8). Because conducting airway epithelial cells are the first line of defense against common sources of ROS, such as air pollutants and cigarette smoke (9), we examined an in vitro model of inflammation using human airway epithelial cells. We specifically examined how thiols modulated the response of airway epithelial cells to inflammation induced by tumor necrosis factor (TNF)-, using NF-B and interleukin (IL)-8 as markers.

NF-B is a member of a large family of Rel proteins that are normally present in the cytosol in an inactive form, consisting of a NF-B dimer associated with an inhibitory protein, IB (10, 11). Cytosolic NF-B can be activated by a variety of stimuli such as TNF- and ROS intermediates (11). These stimuli trigger phosphorylation and ubiquination events that lead to the degradation of the bound IB protein. Degradation of the inhibitor protein exposes a nuclear localization sequence that results in the rapid translocation of the Rel protein complex from the cytoplasm to the nucleus (11, 15, 16). Under favorable conditions, promotion of specific gene transcription occurs after the binding of NF-B dimers to cis-acting B binding sites in the 5'-flanking region of the activated genes.

The specific genes that are activated by NF-B depend on the cell type being studied, the stimulus used to activate NF-B, and the NF-B subunits that are activated (16). Also, whether or not NF-B is activated in a specific cell type may be dependent on an optimal redox level in the cell (17). As previously suggested by Das and colleagues, this optimal redox level may be determined by the availability of serum to the cells (20, 21). More recently, Cantin and associates showed that the amount of albumin available to the cell was important in regulating the amount of intracellular glutathione (GSH) and subsequent NF-B activation (22). On the basis of limited published data, it appears that under physiologic conditions, the airway epithelium exists in a relatively albumin-depleted environment (23, 24). It is currently unclear what specific redox changes occur during inflammation in airway epithelia, and how these changes affect NF-B activity and NF-B- dependent gene activation.

The three major thiol redox buffers involved in the regulation of cellular redox status include N-acetyl-cysteine (NAC), GSH, and the thioredoxin (TRX) and glutaredoxin systems (2, 25). The systems of TRX and glutaredoxin contain two active cysteine thiols, which act as hydrogen donors for proteins and nucleic acid (26). We previously observed that TRX is highly expressed in airway epithelia, especially in the ciliated cell type (an abstract, unpublished data). The effects of TRX on NF-B transcriptional activity have been studied in other cell systems (17, 27, 28). However, the conclusions regarding the effect of TRX on NF-B activity vary according to the cell system used or the stimuli used to activate NF-B.

Recently, we proposed a novel mechanism involving TRX in the regulation of IL-8 gene expression by retinoids (29). To further elaborate this mechanism, we examined the role of thiols NAC, GSH, and TRX in the regulation of NF-B transcription factor activity, using TNF- as a stimulus for NF-B activation. Using both an in vitro gel mobility shift assay and an in vivo functional assay based on a human IL-8 promoter-mediated reporter gene system, we demonstrated that TRX, compared with other thiol chemicals, is uniquely capable of enhancing NF-B transcriptional activity in airway epithelial cells. We propose that this effect is due to the redox nature of TRX, the possible resistance of TRX to auto-oxidize in a serum-free environment, and the ability of TRX to compartmentalize in the nucleus after TNF- treatment. Because of limited availability in glutaredoxin probes and a glutaredoxin expression system, the effects of glutaredoxin on NF-B transcriptional activity were not studied.

	Materials and Methods

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Cell Sources and Culture Conditions

Primary tracheobronchial epithelial (TBE) cultures and immortalized human bronchial epithelial (HBE) cell lines, HBE1, were used in this study. Human tracheobronchial tissues were obtained with consent either from the University of California at Davis Medical Center or from the Anatomic Gift Foundation (Laurel, MD). The procedures to obtain and use human tissues for cell cultures were reviewed and approved by a campus Committee on the Protection of the Rights of Human Subjects at the University of California at Davis. Epithelial cells were harvested from these tissues by a protease dissociation method described previously (30). Primary cultures of the protease-dissociated epithelial cells were maintained in serum-free, Ham's F12 medium supplemented with insulin (5 µg/ml), transferrin (5 µg/ml), epidermal growth factor (10 ng/ml), dexamethasone (0.1 µM), cholera toxin (20 ng/ml), bovine albumin (0.05%), and bovine hypothalamus extract (15 µg/ml). HBE1, a papilloma virus-immortalized HBE cell line, was kindly provided by Dr. J. Yankaskas (University of North Carolina, Chapel Hill, NC) (31). This cell line was maintained in the same serum-free, Ham's F12 medium supplemented with growth factors and hormones as mentioned earlier but without the bovine albumin. During treatment conditions, TNF- (R&D Systems) was added to cultures at 10 ng/ml.

Preparation of Nuclear Extracts

Nuclear extracts were prepared according to a modified procedure as described previously (29). Briefly, cultured cells were collected and suspended in a hypotonic buffer (10 mM N-2-hydroxyethylpiperazine-N'-ethane sulfonic acid [Hepes], [pH 7.9], 1.5 mM MgCl₂, 10 mM KCl, 0.2 mM phenylmethylsulfonyl fluoride [PMSF], and 0.5 mM dithiothreitol [DTT]). Swollen cells were homogenized and nuclei pelleted. After pelleting, nuclei were gently treated with a high salt solution (20 mM Hepes [pH 7.9], 25% glycerol, 1.5 mM MgCl₂, 1.2 M KCl, 0.2 mM ethylenediaminetetraacetic acid [EDTA], 0.2 mM PMSF, and 0.5 mM DTT) to release proteins from nuclei without lysing the nuclei. The nuclear protein extracts were then dialyzed against a moderate salt solution (100 mM KCl) in the same buffer, and any precipitate was removed by centrifugation in a microfuge centrifuge. Protein concentration in the extract was determined by a modified Lowry assay using bovine serum albumin (BSA) protein standards (Bio-Rad, Hercules, CA).

Electrophoretic Mobility Shift Assay

A double-stranded oligonucleotide corresponding to the NF-B consensus sequence was obtained from a commercial source (Santa Cruz Biotechnology, Santa Cruz, CA). The nucleotide was ³²P end-labeled and used for binding with various nuclear extracts, which were obtained from cell cultures treated with or without TNF-. The DNA-protein binding was performed in a 20-µl reaction volume containing 25 mM Hepes (pH 7.9), 10% (vol/vol) glycerol, 30 mM NaCl, 0.1 mg/ml BSA, 1 mM DTT, 5 mM MgCl₂, 0.5 mM EDTA, 0.5 µg of salmon-sperm DNA, and 50 to 100 ng of poly(dI-dC). After incubation on ice for 45 min with 4 to 7 µg of nuclear extract, 0.1 to 0.5 ng of ³²P end-labeled probe (~ 20,000 counts per min) was added and incubated at room temperature for 15 to 20 min. The DNA-protein complexes were resolved in a native 4% acrylamide gel (29:1 ratio of acrylamide to bis-acrylamide) in 0.5× Tris borate-EDTA buffer. For the antibody supershift analysis, antibody specific to the p50 or p65 NF-B subunit was added to the reaction mixture containing nuclear extract and ³²P probe. The resulting complex was analyzed in the same 4% acrylamide gel (32).

Transient Transfection of IL-8-Chloramphenicol Acetyl Transferase Reporter Gene Chimeric Construct DNA

Cultures at 50 to 70% confluency were transfected with the liposome IL-8-chloramphenicol acetyl transferase (CAT) reporter gene construct (2 µg/transfection). A pSV--galactosidase (-gal) construct (0.5 µg/transfection) was included in the cotransfection to normalize transfection efficiency. Media with or without TNF- (10 ng/ml) was added to cultures 24 h later. Cell extracts were prepared by three cycles of freeze-thaw in a 0.25 M Tris-Cl buffer solution at pH 8.0. The reporter gene CAT activity was determined on the basis of an enzyme-linked immunosorbent assay (ELISA) method as previously described (33), whereas -gal activity was assayed according to the instructions provided with the kit from Promega (Madison, WI). For each transfection, relative CAT activity was expressed after normalization with -gal activity for each cell-extract preparation. The reported results are an average from triplicate dishes.

Source of TRX Protein and Complementary DNA Clone

Full-length monkey (34) and human TRX complementary DNA clones were subcloned to pFLAG-cytomegalovirus (CMV)-2 and Escherichia coli PFLAG-ATS expression vectors, downstream from the FLAG sequence, following the instructions provided by the manufacturer (Eastman-Kodak/Sigma, Rochester, NY). Clones were characterized by DNA sequencing, and the resultant recombinant protein products were identified by Western blot analysis using two monoclonal antibodies (mAbs). One of the two antibodies, obtained from the manufacturer (Eastman-Kodak/ Sigma), recognized the N-terminal FLAG sequence. The other antibody, generated in our lab, specifically recognized a C-terminal peptide sequence of human TRX (Zhao and Wu, unpublished data). The recombinant proteins produced by the CMV-based vector and from the bacterial expression system were recognized by both mAbs. (data not shown). Monkey FLAG-TRX protein was purified according to the manufacturer's suggestions (Eastman-Kodak/Sigma). Commercially purified human TRX protein (American Diagnostica, Inc., Greenwich, CT) was also used in this study.

	Results

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TNF- Increases NF-B Transcription Factor Binding Activity

To determine how thiols affect NF-B activity, we first characterized how TNF- affects NF-B DNA binding by electrophoretic mobility shift assay (EMSA). As shown in Figure 1, nuclear extracts from HBE1 cell lines had a low level of NF-B binding to the consensus B DNA binding site. After cells were treated with TNF-, NF-B-DNA binding activity was increased.

View larger version (65K): [in this window] [in a new window]   Figure 1.   TNF- increases DNA binding of the p65 and p50 subunits of NF-B. EMSA analysis of the DNA binding activity of NF-B transcription factors induced by TNF-. Near-confluent HBE1 cultures were treated with TNF- (10 ng/ml), and nuclear extracts were prepared from these cultures 3 h after treatment (lanes 2-5). Nuclear proteins harvested from untreated cells served as controls (lane 1). Nuclear extracts were used for EMSA analysis with a 32P-labeled DNA fragment containing the NF-B DNA consensus sequence as described in MATERIALS and METHODS. For supershift assays, antibodies specific to p65 and p50 subunits of NF-B (lanes 3 and 4, respectively) and preimmune rabbit serum (lane 5) at 1 µg per reaction were added to the DNA binding reaction mixture. Specificity of these DNA protein bands was verified by DNA competition as described previously (29). Unbound 32P-DNA probe (not shown) migrated far ahead of the bands in the figure.

We observed two NF-B-specific bands, an upper and a lower band. An antibody specific to the p65 subunit of NF-B induced a dramatic reduction in the upper NF-B- specific band (upper thin arrow in Figure 1) and a corresponding supershift band (upper thick arrow in Figure 1). The intensity of this NF-B-specific band was also reduced when antibody specific to the p50 subunit of NF-B was included in the reaction. This upper band was therefore referred to as the p50/65 band. A loss of band intensity was observed in the lower NF-B-specific band (lower thin arrow in Figure 1) only when the anti-p50 antibody was used. Thus, this lower band was referred to as the p50/ p50 band. A corresponding supershift band was seen also with addition of anti-p50 antibody (lower thick arrow in Figure 1). Anti-p50 or anti-p65 antibodies did not affect the rest of the bands, and the appearance of these bands in EMSA varied from one experiment to the other. Therefore, these bands were referred to as nonspecific protein- DNA interactions. Overall, it appeared that TNF- activates NF-B-DNA binding activity in airway epithelial cells. This activation involves the formation of two specific DNA-protein complexes containing both p50 and p65 subunits. Similar results were observed with primary TBE cells (data not shown).

It is notable that the supershift band induced by anti-p65 antibody was not observed with each experiment. However, reduction in the p50/65 band was consistently seen with the addition of anti-p65 antibody to the assay. This phenomenon was probably due to underdevelopment of the radiograph. Because of concerns about not being able to visualize a p65 supershift band with some experiments, on a few occasions the dried gel was exposed long-term to a phosphor screen. When this was performed, we observed the presence of p65 supershift bands that were not visible on the radiograph.

Thiols Modulate NF-B-DNA Binding Activity In Vitro

To investigate the effects of thiols in vitro on the formation of p50/p50 and p50/65 bands, GSH, NAC, and TRX were added to the EMSA reaction mixture. As shown in Figure 2, NAC treatment had an inhibitory effect on NF-B binding activity. The inhibition was dose-dependent and more effective in blocking p50/65 band formation than p50/ p50 band formation. N-acetyl-serine, used as a negative control for NAC, did not show any inhibition (data not shown). Identical experiments were performed with increasing doses of GSH rather than NAC, and similar results were obtained (data not shown). In contrast to NAC and GSH, TRX had no inhibitory effect (Figure 3), and actually enhanced NF-B binding. There appeared to be a greater effect on the p50/65 band compared with the p50/ p50 band. For these experiments, we obtained similar results in both HBE1 and primary TBE cells (data not shown).

View larger version (25K): [in this window] [in a new window]   Figure 2.   NAC decreases TNF--induced NF-B-DNA binding in vitro. Nuclear extracts from HBE1 cells obtained after treatment with 10 ng/mL TNF- for 3 h (lane 1) followed by treatment with increasing amounts of neutralized NAC solution (lanes 2-5) were combined with radioactively labeled NF-B consensus sequence and subjected to polyacrylamide gel electrophoresis (PAGE). Similar results were observed with GSH (results not shown).

View larger version (53K): [in this window] [in a new window]   Figure 3.   TRX increases NF-B-DNA binding in vitro. Nuclear extracts from primary TBE cells obtained before (lane 1) and after treatment with 10 ng/mL TNF- for 3 h (lanes 2-4) were combined with radioactively labeled NF-B consensus sequence and subjected to PAGE. Recombinant human TRX protein (1 µg/ml) was added to the DNA binding reaction mixture (lanes 3 and 4). For these supershift assay, anti-p65 antibody was added as described in Figure 1 (lane 4).

Thiols Modulate NF-B-DNA Binding Activity In Vivo

To examine the in vivo effects of thiols on TNF--induced NF-B activity, cultured cells were pretreated with thiols before TNF- treatment and nuclear protein extraction. As shown in Figure 4, GSH pretreatment reduced TNF-- induced NF-B binding activity in a dose-dependent manner. Pretreatment with NAC gave similar results (data not shown). In contrast, we observed an increase in NF-B binding activity, especially the p50/65 band, when cells were pretreated with TRX (Figure 5). Again, similar results were obtained in both HBE1 and primary TBE cells, with representative data shown here. Both the in vitro and in vivo data were consistent, suggesting an important role of TRX in upregulating NF-B-DNA binding activity.

View larger version (54K): [in this window] [in a new window]   Figure 4.   GSH decreases NF-B-DNA binding in vivo. Nuclear extracts from primary human TBE cell cultures were treated without (lanes 1 and 2) or with (lanes 3 and 4) GSH 1 h before treatment with 10 ng/mL TNF- (lanes 2-4) for 3 h, then were combined with radioactively labeled NF-B consensus sequence and subjected to PAGE. Similar results were observed when cells were pretreated with NAC (results not shown).

View larger version (55K): [in this window] [in a new window]   Figure 5.   TRX increases NF-B-DNA binding in vivo. Experiments were carried out as described in Figure 4, except GSH was replaced by TRX (1 µg/mL). Briefly, nuclear extracts from primary TBE cells obtained before (lane 1) and after treatment with 10 ng/mL TNF- (lane 2) and 1 µg/mL TRX (lane 3) for 3 h were combined with radioactively labeled NF-B consensus sequence and subjected to PAGE. Antibodies to the NF-B p65 subunit induced a supershift, confirming NF-B specificity (lane 4).

Thiols Modulate TNF--Induced IL-8 Gene Expression

On the basis of these results, we decided to elucidate the downstream effects of thiols on TNF--induced gene expression. We focused on a NF-B-dependent gene, IL-8, to examine these effects. Other studies have demonstrated that IL-8 gene induction occurs at the transcriptional level and involves a NF-B-mediated mechanism (35). As shown in Figure 6, TNF- treatment induced the synthesis and secretion of IL-8 in a dose-dependent manner.

View larger version (12K): [in this window] [in a new window]   Figure 6.   TNF- increases IL-8 protein production in airway epithelial cells. Primary human TBE cells were cultured and treated with various amounts of TNF-, as indicated. After a 16-h incubation, media were collected and measured for IL-8 production by ELISA. ELISA data are expressed as amount of IL-8 per million cells.

EMSA studies demonstrated that the NF-B binding site located in the 5'-flanking region of the human IL-8 gene could compete for binding to the transcription factor, whereas a mutation on the IL-8 NF-B binding site was not able to compete for binding. As shown in Figure 7, addition of cold, wild-type, 184-base pair (bp) IL-8 promoter abolished NF-B binding, whereas addition of cold mutant IL-8 promoter had no effect on NF-B binding. This was seen in both HBE1 and primary TBE cells.

View larger version (37K): [in this window] [in a new window]   Figure 7.   The IL-8 promoter contains a NF-B-specific binding site. Nuclear extracts prepared from TNF--treated primary human TBE cells were combined with a radioactively labeled 184-bp sequence of the IL-8 promoter and subjected to PAGE as described in Figure 1 (lanes 1-5). For competition, unlabeled DNA fragments containing the first 184-bp 5'-flanking region of the IL-8 gene (PC-184) (lanes 2 and 3) and a similar DNA fragment from this region with mutations at the putative NF-kB binding site (PC-184-M) (lanes 4 and 5) were added to the DNA binding reaction mixture.

To further investigate the EMSA results, promoter- reporter gene transfection assays were used. Using the same 184-bp IL-8 promoter constructs that were used in the previous EMSA studies, cells transfected with a 184-IL-8-CAT construct expressed CAT activity above promoterless pBL-CAT3-transfected cells (Figure 8). The relative CAT activity increased in cell cultures after TNF- treatment. In contrast, cells transfected with the mutant 184(m)-IL-8-CAT construct expressed very low levels of CAT activity. There was also no stimulatory response in CAT activity after TNF- treatment with the mutant promoter construct.

View larger version (18K): [in this window] [in a new window]   Figure 8.   NAC inhibits TNF--induced enhancement of IL-8 promoter activity. HBE1 cells were transfected with reporter-promoter wild-type IL-8-CAT3 (gray bars) and mutant IL-8-M-CAT3 (black bars) DNA constructs as described before (35). A pSV--gal DNA construct was also included in the transfection and -gal activity in transfected cells was used for normalizing the transfection efficiency. At 2 d after transfection, cultures were treated with (3 and 4) or without (1 and 2) 5 mM NAC before TNF- (10 ng/ml) treatment (2 and 4). At 12 h later, cultures were harvested for both CAT and -gal reporter gene assays as described in MATERIALS AND METHODS. Relative CAT activity was expressed as the CAT activity after normalization with -gal activity, and the data were averaged from triplicate dishes.

Using this transfection system, we examined the effects of thiols on IL-8 promoter-mediated CAT reporter gene expression in transfected cells. NAC treatment before TNF- treatment had inhibitory effects on promoter activity, and this inhibition was probably related to inhibition of NF-B binding because such inhibition was not observed in 184(m)-IL-8-CAT transfected cells. In contrast to NAC, TRX treatment alone had a stimulatory effect on CAT activity (Figure 9). The stimulation was slightly enhanced when TNF- treatment was included. However, the TRX stimulatory effect was not observed in cells transfected with the 184(m)-IL-8-CAT construct. These results suggest that TRX increases CAT gene expression in transfected cells through the activation of the NF-B site.

View larger version (23K): [in this window] [in a new window]   Figure 9.   TRX enhances both baseline and TNF--stimulated IL-8 promoter activity. HBE1 cells were transfected with a -gal construct and a CAT3 construct containing either a 184-bp wild-type IL-8 promoter sequence (gray bars) or a 184-bp mutant IL-8 promoter sequence (black bars) as in Figure 8. Results are expressed as relative CAT activity (CAT activity/-gal activity). Data show relative CAT activity for untreated cells (1), cells treated for 12 h with 10 ng/mL TNF- (2), cells cotransfected with a TRX expression vector (3), and cells cotransfected with a TRX expression vector plus TNF- (4). Triplicate dishes were used for each transfection and the relative CAT activity was averaged from these dishes after normalization with -gal activity.

TNF- Induces NF-B and TRX Localization in the Nucleus

To explain differences in how thiols affect NF-B binding and IL-8 gene expression, we hypothesized that TRX exerts its effects by compartmentalization in the nucleus after TNF- treatment. Presumably, high levels of thiols such as GSH and NAC will antagonize NF-B activation, whereas the presence of thiols such as TRX in the nucleus will enhance NF-B binding. In support of this hypothesis, we examined the effects of TNF- on the localization of NF-B and TRX in airway epithelial cells.

Using p65 antibody, we observed translocation of the p65 subunit of NF-B from the cytosol (Figure 10A) to the nucleus within an hour after treatment with TNF- (Figure 10B). In a time-course study, this translocation preceded TNF--induced increases in IL-8 messenger RNA (data not shown). Nuclear localization of p65 is transient, illustrated by the fact that p65 protein was observed to return to the cytosol 4 h after treatment with TNF- (Figure 10C). Using immunofluorescent staining with an antibody specific to TRX, we observed that TRX antigen was present in both the nucleus and cytoplasm in the unstimulated state (Figure 10D). After TNF- treatment, however, we immediately observed elevated nuclear and perinuclear localization of TRX antigen (Figure 10E). As opposed to the pattern of NF-B localization discussed earlier, nuclear localization of TRX was persistent for several hours after TNF- treatment in cultured cells.

View larger version (45K): [in this window] [in a new window]   Figure 10.   TNF- induces translocation of NF-B and TRX to the nucleus in TBE cells. Immunofluorescent staining of TRX and the p65 subunit of NF-B in HBE1 cells after TNF- treatment. HBE1 cells with or without TNF- treatment were fixed in ice-cold methanol and stained with anti-TRX and anti-p65 antibodies, followed by rhodamine-conjugated secondary antibody (rabbit antimouse immunoglobulin G) as described in MATERIALS AND METHODS. (A) Control cultures stained with anti-p65 antibody; (B) 30-min TNF--treated cells stained with anti-p65 antibody; (C) 4-h TNF--treated cells stained with anti-p65 antibody; (D) Control cultures stained with anti-TRX antibody; (E) 30-min TNF--treated cultures stained with anti-TRX antibody. Note: After 4 h, TNF--treated cultures stained with anti-TRX antibody had results similar to those in E (data not shown).

	Discussion

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In this study we have shown that TRX has a unique function in airway epithelial cells when compared with thiols GSH and NAC. Our data indicate that TNF- induces IL-8 production in human airway epithelia. Using an IL-8 promoter-CAT reporter chimeric construct, we observed that both basal and TNF--stimulated IL-8 promoter activity in transfected airway epithelial cells were dependent upon an intact NF-B consensus sequence. TNF--induced promoter activity was significantly decreased by the addition of the thiols GSH and NAC in a serum-free system. TRX, however, enhanced IL-8 promoter activity with and without the addition of TNF-. Our data suggest that these differences are related to the redox potential of TRX, the ability of TRX to maintain its reducing capacity in a more oxidizing intracellular environment, and the ability of reduced TRX to compartmentalize in the nucleus where it can interact with the transcription factor NF-B.

As mentioned previously, NF-B is activated in most cell types by proinflammatory stress (16, 39). During this stress, a signaling cascade is initiated which results in the phosphorylation and degradation of the inhibitory protein IB. This exposes the nuclear localization sequence on NF-B, allowing NF-B to translocate to the nucleus, where it will bind the promoter region of various genes. There is substantial evidence indicating that the key step in the activation and nuclear location of NF-B is dependent upon redox processes. First, NF-B, under certain condition, is rapidly activated in cells treated with hydrogen peroxide or tert-butyl hydroperoxide (40). Second, activation of NF-B by all stimuli known so far, including cytokines, phorbol esters, and viral proteins, is commonly inhibited by thiols and other antioxidant compounds (40). However, NF-B is not directly activated by pro-oxidants in cell-free systems, implicating the involvement of intermediate redox-sensitive molecules. Cellular signals propagated by these redox-sensitive molecules may be counteracted by various cellular thiols and antioxidants.

There is an obvious paradox with respect to NF-B activation and the upregulation of NF-B-dependent genes. Most transcription factors, including NF-B, favor DNA binding under reduced conditions (41, 42). For example, NF-B has a critical cysteine residue in its DNA binding region that must be in the reduced state for DNA binding to occur (41, 43, 44). Under conditions of inflammation, NF-B is activated and translocates to the nucleus. During transit to the nucleus, NF-B is exposed to oxidative products of inflammation, potentially inactivating DNA binding regions through the oxidation of critical cysteine residues. The ability of NF-B to bind to DNA is impaired, and the necessary gene response to inflammation and oxidative stress is blunted. We believe our data provide unique insights into the functions of TRX, compared with NAC and GSH, in airway epithelial cells that explain this apparent paradox.

In our study we demonstrated that both NAC and GSH antagonized NF-B activation. Although the precise mechanisms are unclear, NAC and GSH possibly either act as direct antioxidants or enhance cellular antioxidant levels to counteract NF-B activation in cells treated with TNF-. For NAC, the proposed mechanism is that NAC enters the cell and performs either as an antioxidant agent itself or as a substrate for GSH synthesis. The enhanced GSH levels will antagonize the redox-sensitive intermediate molecules generated during activation of NF-B. It is difficult to assert a similar mechanism for GSH because it is not readily transported into the cytosol. One possible mechanism is that the presence of GSH in the extracellular environment, either in the reduced or oxidized form, inhibits TNF- activity.

It has been shown that under certain conditions GSH, as well as other thiols, will auto-oxidize and generate free radicals (45). Das and coworkers (21) have shown that when compared with serum-containing conditions, serum-free culture conditions produce a higher level of thiol auto-oxidation and oxidation products. They have further demonstrated that the presence of oxidized thiols inhibited activation of NF-B and manganese superoxide dismutase gene expression (20). Further, Cantin and associates have recently shown that maintaining an albumin level greater than 0.5% was vital in maintaining intracellular GSH levels (22). The culture system described in our study contains 0.05% serum albumin, which is similar to in vivo albumin levels (0.015 to 0.35%) for airway epithelial cells (23, 24). As a result of these data, our culture system, due to the low-albumin conditions of the media, possibly favors auto-oxidation of thiols. This auto-oxidation phenomenon may be responsible for the observed inhibition of TNF--induced NF-B activation by NAC and GSH.

We demonstrated that, in contrast to NAC and GSH, cellular TRX has stimulatory effects on NF-B activation. As we observed, either the addition of TRX protein or the transfection of a TRX expression construct to cells enhanced NF-B-dependent promoter activity and NF-B- DNA binding activity. We propose that cellular TRX is able to maintain its redox potential during inflammation to enhance NF-B-DNA binding activity, while still not interfering with the redox-sensitive intermediate molecules responsible for signaling NF-B activation. To achieve this dual role, we first suggest that TRX does not auto-oxidize like NAC and GSH in the serum-free culture condition of the airway epithelium. This enables TRX to maintain its reduced state and enhance NF-B-DNA binding. Second, in order not to interfere with redox-sensitive signaling, TRX must be compartmentalized or conjugated with other molecules within the cell. This TRX compartmentalization allows redox-sensitive signaling and NF-B activation to occur. Consistent with this idea, we demonstrated that enhanced translocation of TRX protein from the cytosol to the nucleus coincided with enhanced p65 NF-B translocation after TNF- treatment. The localization of TRX in the nucleus may play a critical role in modulating NF-B activity.

In this report we demonstrated that TRX, but not the single thiols NAC and GSH, added to the nuclear extract of airway epithelial cells can restore impaired NF-B- DNA binding activity. The nature of this restoration was not characterized in this study. However, it has been demonstrated that TRX can reduce a disulfide bond at cysteine 62 of p50 NF-B subunit (41). In our system, NAC and GSH apparently cannot perform this redox activity. These results support the notion that TRX has greater redox potential than the single thiol compounds for the reduction of oxidized macromolecules.

There is also increasing evidence to support the concept that thiol compartmentalization plays an important role in gene regulation. Recently, we demonstrated a similar mechanism involving TRX in the regulation of IL-8 gene expression induced by retinoid in both the HBE1 cell line and primary human TBE cells (29). Hirota and associates also recently showed that in human keratinocytes, 3T3 cells and HeLa cells, TRX enhanced NF-B binding when TRX was targeted to express in the nucleus, but not when TRX was overexpressed in the cytoplasm (46). Another paper by Hirota and colleagues demonstrated that TRX, glutaredoxin, and a newly cloned nuclear thiol protein, nucleoredoxin, all have unique cellular localizations and differential effects on transcription factor activity (47). In this report (47), it is interesting to note that TRX inhibited TNF--induced NF-B activity in HEK293 cells, which emphasizes the cell-type and stimulus-type specificity of cell signaling pathways.

There are several possible mechanisms by which TRX will localize into the nucleus. One mechanism may be the direct activation of a nuclear localization signal on TRX itself. This localization sequence may be activated via a signal cascade during inflammation or oxidative stress that directs TRX to the nucleus. TRX may use the same signaling pathways that activate NF-B, thereby ensuring cotranslocation of NF-B and TRX to the nucleus. Another possible mechanism is that TRX is bound to a chaperone protein that contains a signal localization sequence. Activation of the nuclear localization sequence on the chaperone protein results in the translocation of the chaperone protein and TRX into the nucleus. Alternatively, TRX may be unbound in the cytosol, and activation of a nuclear localizing chaperone protein or transcription factor then binds TRX on its way to the nucleus.

Overall, these findings are consistent with the idea that TRX is unique compared with the other biologic thiols for reducing the critical cysteine residues on NF-B. This occurs under serum-free, low-albumin conditions that, on the basis of current literature, may represent the physiologic state for airway epithelial cells in vivo. These results also provide additional evidence that compartmentalization of thiol chemicals is important in the regulation of redox-dependent transcriptional activities. It is also the first report showing active translocation of TRX in airway epithelial cells in response to inflammatory stress. The precise mechanisms that sense inflammatory and oxidative stress and cause TRX translocation are fascinating questions that we plan to explore in the future.