Mechanisms of Altered Transcription by Cigarette Smoke

Brian M. Freed1, Yanli Ouyang and Jesica M. McCue

Division of Allergy and Clinical Immunology, Department of Medicine, University of Colorado Health Sciences Center, 4200 E. Ninth Avenue, Denver, Colorado 80262

ABSTRACT

The article highlighted in this issue is "The Activity of NF-{kappa}B in Swiss 3T3 Cells Exposed to Aqueous Extracts of Cigarette Smoke Is Dependent on Thioredoxin," by Stephan Gebel and Thomas Müller (pp. 75–81).

Cigarette smoking has long been associated with a number of pathological changes in the lungs, including suppression of immune responses, induction of DNA strand breaks, tumor promotion, and pulmonary fibrosis. Whether or not these diverse phenomena can be attributed to the same chemical constituents may never be fully resolved, given the complexity of cigarette smoke. However, one thing is certain: cigarette smoke contains high concentrations of prooxidants, such as hydroquinone, catechol, acrolein, acetaldehyde, free oxygen radicals and peroxynitrites, which subject the lungs to severe oxidative stress. The lungs respond with an equally impressive antioxidant defense. For example, acute cigarette exposure causes an immediate drop in the levels of reduced glutathione (GSH) in the epithelial lining fluid, followed by a subsequent increase in the expression of gamma glutamylcysteine synthetase and a 3-fold elevation of extracellular GSH levels (Rahman and MacNee, 2000Go).

Not surprisingly, a great deal of speculation has arisen over the significance of the cellular oxidant/antioxidant balance in the progression of lung disease. Among the cellular responses to oxidative stress, activation of the transcription factor NF{kappa}B is perhaps the best studied. NF{kappa}B is typically a heterodimer comprising p50 and p65/RelA subunits. NF{kappa}B is sequestered in the cytoplasm by a seven-member family of inhibitors known as I{kappa}B, of which I{kappa}B-{alpha} is the best characterized (Janssen-Heininger et al., 2000Go). I{kappa}B-{alpha} masks nuclear localization sequences on NF{kappa}B and thereby prevents its translocation into the nucleus. Activation of NF{kappa}B by a variety of cellular signals results in phosphorylation of I{kappa}B-{alpha} serines 32 and 36 by I{kappa}B kinases, which targets adjacent lysines for ubiquitination and leads to degradation of the I{kappa}B-{alpha} by the 26S proteosome. In contrast, oxidative stress induces NF{kappa}B activity by phosphorylating I{kappa}B-{alpha} tyrosine 42, which also causes it to dissociate from NF{kappa}B, but does not result in its proteosomal degradation. Oxidative stress can also negatively affect transcriptional activity of NF{kappa}B. The binding of NF{kappa}B to target promoters, and hence its ability to regulate gene expression, is dependent upon a reduced cysteine 62 in the p50 subunit. Maintenance of this cysteine in the reduced state is dependent upon thioredoxin, a small ubiquitous protein whose functions include regulation of cell growth and programmed cell death (Matthews et al., 1992Go).

The report by Gebel and Müller in this issue of Toxicological Sciences (pp. 75–81 ) elegantly demonstrates how changes in the redox status of the cell influences the transcriptional machinery. Exposure of 3T3 fibroblasts to cigarette smoke extracts caused a rapid depletion of cellular GSH levels, followed 2 hours later by a loss of the NF{kappa}B/thioredoxin complexes and a reduction in NF{kappa}B DNA-binding activity. There was no loss of NF{kappa}B protein or thioredoxin, and NF{kappa}B clearly translocated to the nucleus. The loss of DNA-binding activity therefore appeared to be due solely to the absence of reduced thioredoxin. However, within hours, the cells responded to this oxidative stress by induction of thioredoxin reductase mRNA, elevation of GSH levels and restoration of NF{kappa}B/thioredoxin complexes in nuclear extracts. Thus, the overall redox status of 3T3 cells exposed to cigarette smoke appears to have directly influenced the redox status of the p50 subunit of NF{kappa}B.

Reduced GSH plays a critical role in protecting the cell from oxidative stress. In addition, evidence is accumulating that the redox status of GSH (i.e., the GSH/GSSG ratio) may act as a `second messenger' to the transcriptional machinery, at least in some cell types. The presence of high concentrations of cytoplasmic GSH interferes with phosphorylation of I{kappa}B, thus preventing translocation of NF{kappa}B to the nucleus. In contrast, GSH in the nucleus facilitates binding of NF{kappa}B to DNA, presumably by protecting cysteine 62 from oxidative insult. Thus, an oxidative environment in the cytoplasm favors nuclear translocation of NF{kappa}B, but a reducing environment in the nucleus may be necessary for its binding to DNA (Rahman and MacNee, 2000Go). In order for these apparently contradictory conditions to exist in the same cell, the level of oxidative stress would have to be relatively low, or the cell would have to react to the oxidative stress with an antioxidant response.

Although Gebel and Müller demonstrate a temporal relationship between the redox state of the cell and transcriptional regulation, there are many questions yet to be answered. For example, expression of c-myc, which is dependent on NF{kappa}B, was clearly evident 2 hours after exposure of the cells to cigarette smoke extracts. This was precisely the time when NF{kappa}B/thioredoxin levels were at their lowest. However, c-myc might have been induced by NF{kappa}B/thioredoxin complexes that were still present during the first 60 minutes. It remains to be determined whether the pattern of c-myc expression would have been the same without restoration of reduced thioredoxin or GSH. In addition, the overall role that GSH plays in this process is still poorly understood. Cysteines are good electron donors and are found in the active sites of many enzymes and cytoskeletal proteins. The redox state of the cell therefore influences a wide range of cellular processes in addition to transcription, including membrane transport, receptor-ligand interactions and motility. An intriguing question is whether the GSH/GSSG ratio directly affects transcription, or whether GSH merely protects critical thiols from oxidative stress. This question pertains not only to the redox active compounds in cigarette smoke, but also to other environmental toxicants with GSH modulatory activity.

There are numerous components of cigarette smoke that potentially display the redox characteristics described by Gebel and Müller. Acrolein, acetaldehyde, hydroquinone and peroxynitrite have all been shown to lower cellular GSH levels (Kehrer and Biswal, 2000Go; Grafstrom et al., 1994Go; Rahman and MacNee, 2000Go; Müller et al., 1997Go). In addition, redox cycling by hydroquinone and catechol in cigarette tar extracts produces high concentrations of superoxide and hydrogen peroxide, which also deplete GSH (Pryor et al., 1998Go; Bermudez et al., 1994Go). However, the biological effects of these various compounds are quite varied. For example, acrolein and hydroquinone suppress production of TNF{alpha} and IL-1 (Li et al., 1997Go; Ouyang et al., 2000Go), while acetaldehyde stimulates production of these cytokines (Gutierrez-Ruiz et al., 1999Go). These observations suggest that the effects of cigarette smoke on cellular responses probably depend on reactions with cellular molecules in addition to GSH. For example, hydroquinone and catechol inhibit DNA synthesis by donating an electron to the tyrosyl radical in M2 subunit of ribonucleotide reductase (McCue et al., 2000Go). Nevertheless, the paper by Gebel and Müller in this issue of Toxicological Sciences highlights the redox events induced by cigarette smoke and the central role they have on the transcriptional machinery. The fact that increased NF{kappa}B and thioredoxin levels are also associated with resistance to apoptosis has important implications for cancer chemotherapy and immunotherapy, both of which utilize this pathway to eliminate malignant cells.

NOTES

1 To whom correspondence should be addressed. Fax: (303) 315-7642. E-mail: brian.freed{at}uchsc.edu. Back

REFERENCES

Bermudez, E., Stone, K., Carter, K. M., Pryor, W. A. (1994). Environmental tobacco smoke is just as damaging to DNA as mainstream smoke. Environ. Health Perspect. 102, 870–874.[ISI][Medline]

Grafstrom, R. C., Dypbukt, J. M., Sundqvist, K., Atzori, L., Nielsen, I., Curren, R. D., Harris, C. C. (1994). Pathobiological effects of acetaldehyde in cultured human epithelial cells and fibroblasts. Carcinogenesis 15, 985–990.[Abstract]

Gutierrez-Ruiz, M. C., Quiroz, S. C., Souza, V., Bucio, L., Hernandez, E., Olivares, I. P., Llorente, L., Vargas-Vorackova, F., Kershenobich, D. (1999). Cytokines, growth factors and oxidative stress in HepG2 cells treated with ethanol, acetaldehyde, and LPS. Toxicology 134, 197–207.[ISI][Medline]

Janssen-Heininger, Y. M. W., Poynter, M. E., Baeuerle, P. A. (2000). Recent advances towards understanding redox mechanisms in the activation of nuclear factor {kappa}B. Free Radic. Biol. Med. 28, 1317–1327.[ISI][Medline]

Kehrer, J. P., Biswal, S. S. (2000). The molecular effects of acrolein. Toxicol. Sci. 57, 6–15.[Abstract/Free Full Text]

Li, L., Hamilton, R. F., Taylor, D. E., Holian, A. (1997). Acrolein-induced cell death in human alveolar macrophages. Toxicol. Appl. Pharmacol. 145, 331–339.[ISI][Medline]

Matthews, J. R., Wakasugi, N., Virelizier, J. L., Yodoi, J., Hay, R. T. (1992). Thioredoxin regulates DNA binding activity of NF-{kappa}B by reduction of a disulphide bond involving cysteine 62. Nucleic Acid Res. 20, 3821–3830.[Abstract]

McCue, J. M., Link, K. L., Eaton, S. S., Freed, B. M. (2000). Exposure to cigarette tar inhibits ribonucleotide reductase and blocks lymphocyte proliferation. J. Immunol. 165, 6771–6775.[Abstract/Free Full Text]

Müller, T., Haussmann, H. J., Schepers, G. (1997). Evidence for peroxynitrites as an oxidative stress-inducing compound of aqueous cigarette smoke fractions. Carcinogenesis 18, 295–301.[Abstract]

Ouyang, Y., Virasch, N., Hao, P., Aubrey, M. T., Mukerjee, N., Bierer, B. E., Freed, B. M. (2000). Suppression of human IL-1ß, IL-2, IFN{gamma} and TNF{alpha} production by cigarette smoke extracts. J. Allergy Clin. Immunol. 106, 280–287.[ISI][Medline]

Pryor, W. A., Stone, K., Zang, L. Y., Bermudez, E. (1998). Fractionation of aqueous cigarette tar extracts: fractions that contain the tar radical cause DNA damage. Chem. Res. Toxicol. 11, 441–448.[ISI][Medline]

Rahman, I., MacNee, W. (2000). Regulation of redox glutathione levels and gene transcription in lung inflammation: therapeutic approaches. Free Radic. Biol. Med. 28, 1405–1420.[ISI][Medline]