Oxidative Stress in Mammalian Cells

Transcription factors that are exclusively activated by ROS, or those that selectively control expression of ROS-protective and repair enzymes, are slowly being identified. Much of this work has used different cell lines, indicating that ROS show cell type specificity with respect to transcription factors and their activation (Bonizzi et al., 1999). NFKappaB (NF-KB) was the first eukaryotic transcription factor shown to respond directly to oxidative stress, the oxidative stress being induced by hydrogen peroxide from which hydroxyl radicals were generated via Fenton chemistry (Schreck et al., 1991). However incubation with other ROS, e.g. superoxide, hydroxyl radical and NO donors, failed to activate NFKB. In other words NFKB is selectively mediated by peroxides in a fashion comparable to the bacterial activation of OxyR, and over-expression of either catalase or Cu/Zn-SOD in cell lines induced either suppression or superinduction of NFKB respectively (Schmidt et al., 1995), while depletion of GSH induced NF-KB. A variety of antioxidants including GSH precursors, L-cysteine, thiols, vitamin E and its derivatives are able to block its activation.

In most cell types NFKB is present in the cytosol as an inactive heterodimer (p50 and p65; or p50/c-Rel) bound to a third inhibitory subunit 1KB. Removal of this inhibitory subunit is the signal for the translocation of the transcription factor to the nucleus to bind to consensus DNA sequences, where NF-KB plays a key role in the regulation of numerous genes involved in pathogen responses and cellular defence mechanisms, Figure 10.3. These include many immunologically relevant genes, cytokines and cytokine receptors, growth factors, and cell adhesion molecules, each of which contain functional NF-KB binding sites in their promoter and enhancer regions. In T lymphocytes, IL-2 and IL-2 receptor a-chain are controlled by NFKB while in retroviruses, e.g. HIV-1, the NF-KB binding sites are found in their regulatory LTR region. NF-KB activation does not require new protein synthesis: a great variety of pathogens and inflammatory stimuli, e.g. LPS, viral infection, cytokines IL-1, IFNg and TNFa, activate the transcription factor within minutes (Baeuerle and Henkel, 1994).

Activation of cells results in the release of IKB, followed by the rapid proteolysis of IKB. Although phosphorylation of serine 32 and 36 in the amino-terminal part of IKBa occurs when the proinflammatory cytokines or mitogens are administered to a T lymphocytic cell line, a different site of action has been found after H2O2 incubation (Schoonbroodt et al., 2000). The tyrosine residue 42 and the C-terminal PEST (Pro-Glu-Ser-Thr) domain plays a major role in the phosphorlylation of IKB after treatment with H2O2. Furthermore the H2O2-inducible phosphorylation was not dependent upon IKB kinase activation but involved casein kinase II. The importance of iron for the activation of NFKB was underlined by the fact that

Cytokines

(TNFa, lymphotoxin)

PLASMA MEMBRANE

Cytokines

(TNFa, lymphotoxin)

PLASMA MEMBRANE

Oxidative Stress Activates

Signal transduction pathways for NF-kB activation

Figure 10.3 (a) Proposed mechanism for induction of NFKB proteins (from Suzuki et al., 1994); (b) interaction with iron metabolism. Reproduced with permission from Cairo and Pietrangelo, 2000, © the Biochemical Society.

Signal transduction pathways for NF-kB activation

Figure 10.3 (a) Proposed mechanism for induction of NFKB proteins (from Suzuki et al., 1994); (b) interaction with iron metabolism. Reproduced with permission from Cairo and Pietrangelo, 2000, © the Biochemical Society.

administration of either DFO or hydroxypyridone diminished the activation of the transcription factor induced by hydrogen peroxide.

A common observation has been that different times are needed for the activation of NFKB by different stimuli; in the case of proinflammatory cytokines the kinetics of the activation is fast, while in the case of H2O2 the kinetics of the activation are slow and sustained. This led to the realization that perhaps different target regions on the 1KB were affected by the stimulant, or that other transcription factors were involved. NFKB is frequently associated with other transcription factors so that they

Figure 10.3 (continued)

Figure 10.3 (continued)

cooperate for full induction of various genes. Using iNOS induction as an example, Dlaska and Weiss (1997) identified a regulatory region on the iNOS gene between -153 to -142 bp upstream of the transcriptional start site of the iNOS promoter that was sensitive to regulation by iron perturbation. The protein responsible was identified as NF-IL6, a member of the CCAAT/enhancer binding protein family of transcription factors. The binding of NF-IL6 to the consensus motif with iNOS was induced by both IFNg and LPS, reduced by iron and enhanced by DFO. Iron by itself regulated expression of iNOS transcriptionally. The NF-IL6 binding site on iNOS is of central importance for the high transcriptional rate of the iNOS gene after IFNg and LPS stimulation. It was suggested that after cytokine/LPS stimulation of cells, activation of the NF-IL6 occurred later than that of NFKB such that NF-IL6 would maintain the active transcription rate while NFKB would initiate transcription. Another transcription factor has also been implicated, HIF-1 (hypoxia inducible factor-1), which may cooperate with NF-IL6 in iron-mediated regulation of iNOS in ANA-1 macrophages. Binding of HIF-1 to target sequences is induced by DFO.

A recent review has cast doubt upon the exact role played by ROS in activating NFKB, suggesting that the activation of NFKB by hydrogen peroxide is cell specific and distinct from physiological activators such as Il-1 and TNF. In addition, inhibition by antioxidants was also found to be cell- and stimulus specific and can involve a variety of factors not related to redox modulation (Bowie and O'Neill, 2000). Only recently have studies of NFKB activation in various human diseases commenced, e.g. into Parkinson's disease, while only a few studies of human cell lines, e.g. hepatocytes and phagocytic leucocytes and macrophages, have been reported so far. Our recent studies (Legssyer et al., 2001) show that macrophages loaded in vivo with iron, were unable to increase NFKB activation after challenge with LPS ex vivo, thereby suggesting that such cells are compromised and do not respond adequately to infection.

Recent results have demonstrated that oxidant stress generated directly by exogenous H2O2 differentially induces IL-8 and ICAM-1 transcription in epithelial and endothelial cells. H2O2 induces IL-8 but not ICAM-1 in the A549 type-II-like epithelial cell line, whereas in a microvessel endothelial cell line (HMEC-1) as well as in primary endothelial cells, H2O2 induces ICAM-1 but not IL-8, which is spontaneously expressed. In contrast, the proinflammatory cytokine TNFa, whose activity is dependent on the generation of intracellular ROS, induces IL-8 and ICAM-1 in both cell types. The differential induction of IL-8 and ICAM-1 by H2O2 and TNFalpha suggests that the two inflammatory stimuli target distinct redox responsive signalling pathways to activate cell type-specific gene expression.

Heterdimers of the Jun and Fos transcription factor family constitute the transcription factor AP-1. Oxidative stress results in the increased transcription of the c-fos and c-jun genes although only a moderate induction of AP-1 occurs.

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