INBIFO Institut für biologische Forschung, Fuggerstr.3, D-51149 Köln, Germany
Received August 17, 2000; accepted September 21, 2000
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ABSTRACT |
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Key Words: cigarette smoke; NF-B; thioredoxin; thiol oxidation; glutathione.
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INTRODUCTION |
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Hydroxyl radicals formed by Fenton chemistry (Imlay et al., 1988) are associated mainly with CS-dependent genotoxicity (Cosgrove et al., 1985
; Müller and Gebel, 1994
; Nakayama et al., 1985
). However, they were found not to be the driving force of stress gene expression (Müller, 1995
; Müller and Gebel, 1994
). Instead, evidence has been provided that c-fos mRNA accumulation in smoke-exposed cells is dependent mainly on the formation of peroxynitrite (Müller et al., 1997
). Although the potential peroxynitrite concentration in smoke-bubbled PBS is not sufficient to exert this effect by itself, CS-related aldehydes such as formaldehyde, acetaldehyde, and acrolein might allow this powerful oxidant to interfere with critical components that feed into stress signal transduction, most probably by significantly decreasing the intracellular glutathione (GSH) content (Müller and Gebel, 1998
). Peroxynitrite is thought to be formed in aqueous extracts of CS in the presence of nitric oxide, which is abundant in CS, and superoxide, which in turn is generated by quinone/hydroquinone-like redox systems, e.g., provided by polyphenols such as catechol (Pryor and Stone, 1993
)
A prominent proinflammatory transcription factor implicated in stress signal transduction is NF-B, which has also been linked to the cellular response to environmental stresses (for review, see Mercurio and Manning, 1999
). Activation of NF-
B in response to extracellular stress stimuli appears to be controlled by the redox status of the cell (for review, see Meyer et al., 1994
; Sun and Oberley, 1996
), since NF-
B is significantly inhibited by a broad range of chemically unrelated antioxidants and becomes strongly activated by physical and chemical stresses that tend to trigger the formation of reactive oxygen species (for review, see Flohé et al., 1997
). Mechanistically, the signaling pathway(s) leading to NF-
B activation function mainly through the release of NF-
B from its physical interaction with a member of the family of NF-
B-inhibiting proteins known as inhibitor
Bs (I
Bs), of which I
B-
is the best characterized. Complex formation with I
B-
retains NF-
B in the cytoplasm, whereas phosphorylation of specific serine residues and subsequent degradation of I
B-
primed by ubiquitination results in the translocation of NF-
B to the nucleus, followed by transcriptional activation of NF-
B responsive genes (for review, see May and Ghosh, 1998
). Alternatively, active NF-
B may also be released from I
B-
by the phosphorylation of a specific tyrosine residue in the N-terminal region of I
B-
. This mechanism, in contrast to the regular path, does not include the proteasome-dependent degradation of I
B-
(Imbert et al., 1996
). Finally, DNA binding of and consequently transactivation by NF-
B requires a thioredoxin (Trx)-dependent reduced status of cysteine 62 (Cys-62) in the DNA-binding domain of the p50 subunit of NF-
B, which renders the activity of NF-
B subject to redox regulation (Matthews et al., 1992
; Okamoto et al., 1992
; Hayashi et al., 1993
).
Based on the implication of NF-B in inflammatory and stress-signaling processes, the aim of this study was to evaluate the role of NF-
B in the CS-evoked cellular stress response. We found that the DNA-binding activity of NF-
B dropped significantly during the first 2 h of exposure but was subsequently elevated more than 2-fold. This pattern of deactivation and reactivation was not the result of I
B-
regulation, but appears to depend on the availability of reduced Trx, as can be concluded from the kinetics of both the complex formation between Trx and NF-
B and the expression of thioredoxin reductase (TrxR) in smoke-bubbled PBS-treated cells.
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MATERIALS AND METHODS |
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Cell culture and treatment.
Swiss albino 3T3 mouse fibroblast cells (ATCC CCL 92) were cultured in 15-cm tissue culture dishes (Greiner, Frickenhausen), in 20 ml of Dulbecco's modified Eagle medium (DMEM) supplemented with 4% sodium hydrogen carbonate, L-glutamine, streptomycin, penicillin, and 10% fetal calf serum (FCS) (GIBCO-BRL, Karlsruhe).
Growth-arrested (0.5% FCS; 48 h) 3T3 cells were used in these experiments. Prior to treatment, the cells were washed with serum-free DMEM and immediately exposed for the indicated incubation times to serum-free culture medium containing smoke-bubbled PBS at the concentrations indicated.
Trx/NF-B coprecipitation.
Cells were lysed on ice (10 min) in 20 mM Tris pH 7.5, 150 mM NaCl, 10 mM EDTA, 0.5% Triton X-100, 1 mM Pefabloc, 15 µg/ml aprotinin, and centrifuged at 18000 x g for 15 min. Immunoprecipitations of clarified extracts were performed with protein A sepharose and 2.5 µl of a Trx-specific antibody (American Diagnostica, Pfungstadt). The purified precipitates were dissolved in sample buffer, separated on a polyacrylamide gel, and blotted on nitrocellulose. Western analysis was performed with an NF-B (p65)-specific antibody (1:200) (H286, Santa Cruz, Heidelberg). In parallel, Western analysis of whole cell extract proteins was performed with a Trx- or NF-
B (p65)-specific antibody.
Western analysis.
Whole-cell extracts were prepared by lysing the cells in RIPA buffer [50 mM Tris pH 8, 125 mM NaCl, 0.5% NP40, 0.5% NaDOC, 0.1% SDS, 100 µM Na3VO3, 1 mM Pefabloc, leupeptin, aprotinin, and pepstatin (10 µg/ml each)]. Nuclear extracts were generated from cells lysed in 10 mM Tris pH 7.4, 10 mM NaCl, 3 mM MgCl2, and 0.5% NP40. Purified nuclei were kept in RIPA buffer. Both types of extracts were clarified by centrifugation at 35000 x g for 30 min. Equal amounts of proteins were separated by polyacrylamide gel electrophoresis and blotted on nitrocellulose using a Trans-Blot Cell (BioRad, München). Detection of IB-
and I
B-
phosphorylated on Ser-32 (BioLabs, Frankfurt am Main) or NF-
B by Western analysis was performed with commercially available kit systems.
Northern hybridization.
Total RNA was isolated from quiescent 3T3 cells (48 h, 0.5% FCS) by standard methods with a commercially available RNA isolation kit (Wak-Chemie, Bad Soden). Fifteen micrograms of RNA/sample was analyzed by routine methods including denaturing RNA gel electrophoresis, blotting, hybridization, and autoradiography (Sambrook et al., 1989). 32P-labeled fragments of the murine c-myc and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene (as internal control) served as hybridization probes.
Reverse transcriptase-PCR.
Quantitative reverse transcriptase-polymerase chain reaction (PCR) analysis of TrxR expression was performed from 5 µg of total RNA according to standard procedures. After reverse transcription, a 326-bp fragment internal of the TrxR coding sequence was amplified by 25 cycles of PCR with the following primer pair: 5`-TCCCGCAGAGCTACTCGGTA-3` ("cDNA primer") and 5`-CCTTATCATCATTGGAGGTG-3` ("amplimer"). Amplification of a 369-bp fragment internal of the GAPDH gene by quantitative reverse transcriptase-PCR was performed as an internal control.
Electrophoretic mobility shift assay (EMSA).
Cells were lysed in 20 mM Tris/HCl, pH 7.5, 250 mM NaCl, 20% glycerol, 0.25% NP40, 5 mM MgCl2, 2 mM EDTA, 2.5 mM DTT, 100 µM Na3VO3, 1 mM Pefabloc, and leupeptin, aprotinin, and pepstatin (10 µg/ml each) by freezing and thawing (three times). Extracts were clarified by centrifugation at 18,000 x g for 15 min. Gelshift reactions (10 µl) containing the cellular extracts (510 µg protein) in 15 mM Tris/HCl (pH 7.5), 100 mM NaCl, 5% glycerol, 5% Ficoll, 0.05% NP40, 2 mM MgCl2, 1 mM EDTA, 1 mM DTT, 0.05 mg/ml polydI dC, and 35 fmol 33P-labeled oligonucleotide were run for 30 min (room temperature). The mixtures were subjected to native polyacrylamide gel electrophoresis (4%); DNA protein complexes were quantified by phosphorimaging of the gels. The following NF-B-specific oligonucleotide (Promega, Mannheim) was used in these investigations: 5`-AGTTGAGGGGACTTTCCCAGG-3`. All protein concentrations in extracts were determined by a modified Lowry assay (BioRad).
GSH determination.
GSH was determined as described (Akerboom and Sies, 1981) by quantifying the formation of thionitrobenzoic acid from free GSH and glutathione disulfide in the presence of 5,5-dithiobis-2-nitrobenzoic acid and glutathione disulfide reductase. Protein concentrations were assessed by a modified Lowry assay (BioRad).
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RESULTS |
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DISCUSSION |
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According to the results presented here, the activity of NF-B in CS-exposed cells is subject mainly to a redox-controlled mechanism dependent on the availability of reduced Trx rather than governed by its main regulator I
B-
. This conclusion is based on the strict correspondence between the kinetics of NF-
B DNA binding (Fig. 1a
), Trx/NF-
B complex formation (Fig. 3
), TrxR expression (Fig. 4
), and GSH depletion and repletion (Fig. 5
), along with the lack of I
B-
phosphorylation and degradation (Fig. 2
). Hence, we propose the following scenario in which NF-
B deactivation and reactivation in CS-exposed cells is a consequence of perturbations of the cellular redox conditions (Fig. 6
): immediate loss of reduced Trx (analogous to GSH), together with an oxidized status of Cys-62 of NF-
B, results in decreased NF-
B DNA-binding rates, whereas the reappearance of reduced Trx (due to the induction of TrxR and the availability of NADPH) at incubation times > 2 h activates an I
B-
independent response of NF-
B by rebinding to DNA. However, due to the lack of I
B-
inactivation, it remains to be elucidated by which mechanism the 2-fold increase, as compared to control cells, in NF-
B DNA binding at longer incubation times is achieved in CS-treated cells.
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Inhibition of NF-B DNA-binding in cells exposed to aqueous extracts of CS has already been described by Vayssier et al. (1998) in human monocytes. In accordance with results reported here, they describe a decrease in NF-
B DNA binding in U937 cells exposed to smoke-bubbled PBS for 2 h, while not presenting data for longer incubation times. According to their results, the CS-dependent inhibition of NF-
B DNA binding is linked to the expression of heat shock protein 70 (Hsp70) via the activation of heat shock transcription factor(s) (Vayssier et al., 1998
). However, there is no evidence for such a mechanism in our system; at the CS doses used, we do not see any significant expression of Hsp70, either on a transcriptional or on a translational level (data not shown). Although this difference may be related to cell type specificities and/or smoke-bubbled PBS concentrations applied [considerably higher doses of CS, up to more than 10-fold, were used by Vayssier et al. (1998)], others have reported a lack of increased Hsp70 expression in vivo following exposure to mainstream or sidestream CS (Wong et al. 1997
; Wong et al., 1995
).
A stronger clue to understanding the kinetics of NF-B DNA binding in smoke-bubbled PBS-treated cells may be found in the recent publication by Horton et al. (1999), which shows that the CS-related aldehyde acrolein causes a transient decrease in NF-
B DNA binding in A549 cells that is independent of I
B-
. As seen for smoke-bubbled PBS (Fig. 5
), deactivation of NF-
B by acrolein is paralleled by GSH depletion (Horton et al. 1999
). Recently, we have demonstrated that CS-related aldehydes formaldehyde, acetaldehyde, and acrolein are mainly responsible for GSH depletion in smoke-bubbled PBS-treated cells (Müller and Gebel, 1998
).
In summary, we have characterized the NF-B response in cells exposed to smoke-bubbled PBS. We obtained a distinct pattern of deactivation and reactivation, which appears to be induced mainly by the perturbation of the cellular homeostasis of key thiol functional compounds. As NF-
B functions as a key element in crucial pathways controlling survival (Beg and Baltimore, 1996
), tumor promotion (Young et al., 1999
), and apoptosis (Kasibhatla et al., 1998
), the CS-dependent effects on the activity of NF-
B may have profound consequences for exposed cells.
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ACKNOWLEDGMENTS |
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NOTES |
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