Wulf Dröge

Regulation of p53 activity by its interaction with homeodomain-interacting protein kinase-2

Transcriptional activity of p53, a central regulatory switch in a network controlling cell proliferation and apoptosis, is modulated by protein stability and post-translational modifications including phosphorylation and acetylation. Here we demonstrate that the human serine/threonine kinase homeodomain-interacting protein kinase-2 (HIPK2) colocalizes and interacts with p53 and CREB-binding protein (CBP) within promyelocytic leukaemia (PML) nuclear bodies. HIPK2 is activated by ultraviolet (UV) radiation and selectively phosphorylates p53 at Ser 46, thus facilitating the CBP-mediated acetylation of p53 at Lys 382, and promoting p53-dependent gene expression. Accordingly, the kinase function of HIPK2 mediates the increased expression of p53 target genes, which results in growth arrest and the enhancement of UV-induced apoptosis. Interference with HIPK2 expression by antisense oligonucleotides impairs UV-induced apoptosis. Our results imply that HIPK2 is a novel regulator of p53 effector functions involved in cell growth, proliferation and apoptosis.

Functions of glutathione and glutathione disulfide in immunology and immunopathology.

Even a moderate increase in the cellular cysteine supply elevates the intracellular glutathione (GSH) and glutathione disulfide (GSSG) levels and potentiates immunological functions of lymphocytes in vitro. At low GSSG levels, T cells cannot optimally activate the immunologically important transcription factor NFxB, whereas high GSSG levels inhibit the DNA binding activity of NFxB. The effects of GSSG are antagonized by reduced thioredoxin (TRX). As the protein tyrosine kinase activities p56lck and p59fyn are activated in intact cells by hydrogen peroxide, they are likely targets for GSSG action. These redox‐regulated enzymes trigger signal cascades for NFxB activation and transduce signals from the T cell antigen receptor, from CD4 and CD8 molecules, and from the IL‐2 receptor β‐chain. The effector phase of cytotoxic T cell responses and IL‐2‐dependent functions are inhibited even by a partial depletion of the intracellular GSH pool. As signal transduction is facilitated by prooxidant conditions, we propose that the well‐known immunological consequences of GSH depletion ultimately may be results of the accompanying GSSG deficiency. As HIV‐infected patients and SIV‐infected rhesus macaques have, on the average, significantly decreased plasma cyst(e)ine and intracellular GSH levels, we also hypothesize that AIDS may be the consequence of a GSSG deficiency as well.—Dröge, W., Schulze‐Osthoff, K., Mihm, S., Galter, D., Schenk, H., Eck, H.‐P., Roth, S., Gmünder, H. Functions of glutathione and glutathione disulfide in immunology and immunopathology. FASEB J. 8, 1131‐1138 (1994)

HIV-1 Tat potentiates TNF-induced NF-kappa B activation and cytotoxicity by altering the cellular redox state.

This study demonstrates that human immunodeficiency virus type 1 (HIV‐1) Tat protein amplifies the activity of tumor necrosis factor (TNF), a cytokine that stimulates HIV‐1 replication through activation of NF‐kappa B. In HeLa cells stably transfected with the HIV‐1 tat gene (HeLa‐tat cells), expression of the Tat protein enhanced both TNF‐induced activation of NF‐kappa B and TNF‐mediated cytotoxicity. A similar potentiation of TNF effects was observed in Jurkat T cells and HeLa cells treated with soluble Tat protein. TNF‐mediated activation of NF‐kappa B and cytotoxicity involves the intracellular formation of reactive oxygen intermediates. Therefore, Tat‐mediated effects on the cellular redox state were analyzed. In both T cells and HeLa cells HIV‐1 Tat suppressed the expression of Mn‐dependent superoxide dismutase (Mn‐SOD), a mitochondrial enzyme that is part of the cellular defense system against oxidative stress. Thus, Mn‐SOD RNA protein levels and activity were markedly reduced in the presence of Tat. Decreased Mn‐SOD expression was associated with decreased levels of glutathione and a lower ratio of reduced:oxidized glutathione. A truncated Tat protein (Tat1‐72), known to transactivate the HIV‐1 long terminal repeat (LTR), no longer affected Mn‐SOD expression, the cellular redox state or TNF‐mediated cytotoxicity. Thus, our experiments demonstrate that the C‐terminal region of HIV‐1 Tat is required to suppress Mn‐SOD expression and to induce pro‐oxidative conditions reflected by a drop in reduced glutathione (GSH) and the GSH:oxidized GSH (GSSG) ratio.(ABSTRACT TRUNCATED AT 250 WORDS)

Sesquiterpene Lactones Specifically Inhibit Activation of NF-κB by Preventing the Degradation of IκB-α and IκB-β

Extracts from certain Mexican Indian medicinal plants used in traditional indigenous medicine for the treatment of inflammations contain sequiterpene lactones (SLs), which specifically inhibit the transcription factor NF-κB (Bork, P. M., Schmitz, M. L., Kuhnt, M., Escher, C., and Heinrich, M. (1997) FEBS Lett. 402, 85–90). Here we show that SLs prevented the activation of NF-κB by different stimuli such as phorbol esters, tumor necrosis factor-α, ligation of the T-cell receptor, and hydrogen peroxide in various cell types. Treatment of cells with SLs prevented the induced degradation of IκB-α and IκB-β by all these stimuli, suggesting that they interfere with a rather common step in the activation of NF-κB. SLs did neither interfere with DNA binding activity of activated NF-κB nor with the activity of the protein tyrosine kinases p59 fyn and p60 src . Micromolar amounts of SLs prevented the induced expression of the NF-κB target gene intracellular adhesion molecule 1. Inhibition of NF-κB by SLs resulted in an enhanced cell killing of murine fibroblast cells by tumor necrosis factor-α. SLs lacking an exomethylene group in conjugation with the lactone function displayed no inhibitory activity on NF-κB. The analysis of the cellular redox state by fluorescence-activated cell sorter showed that the SLs had no direct or indirect anti-oxidant properties.

Hydrogen peroxide-induced apoptosis is CD95-independent, requires the release of mitochondria-derived reactive oxygen species and the activation of NF-κB

Reactive oxygen species (ROS) play an important role in cell death induced by many different stimuli. This study shows that hydrogen peroxide-induced apoptosis in T-cells did not require tyrosine kinase p56lck, phosphatase CD45, the CD95 receptor and its associated Caspase-8. H2O2-triggered cell death led to the induced cleavage and activation of Caspase-3. Hydrogen peroxide-treatment of T-cells resulted in the formation of mitochondrial permeability transition pores, a rapid decrease of the mitochondrial transmembrane potential ΔΨm and the release of Cytochrome C. Inhibition of the mitochondrial permeability transition by bongkrekic acid (BA), or interference with the mitochondrial electron transport system by rotenone or menadione prevented the cytotoxic effect of H2O2. Antimycin A, a mitochondrial inhibitor that increases the release of mitochondrial ROS (MiROS), enhanced apoptosis. Overexpression of Bcl-2 and the viral anti-apoptotic proteins BHRF-1 and E1B 19K counteracted H2O2-induced apoptosis. Pharmacological and genetic inhibition of transcription factor NF-κB protected cells from hydrogen peroxide-elicited cell death. This detrimental effect of NF-κB mediating hydrogen peroxide-induced cell death presumably relies on the induced expression of death effector genes such as p53, which was NF-κB-dependently upregulated in the presence of H2O2.

Divergent signalling via APO-1/Fas and the TNF receptor, two homologous molecules involved in physiological cell death.

Tumor necrosis factor receptor (TNF‐R) and APO‐1/Fas (CD95) are members of the tumor necrosis factor/nerve growth factor receptor superfamily involved in various forms of physiological cell death. Due to the structural homology between these receptors and their ligands, it has been suggested that APO‐1/Fas and TNF‐R kill cells by similar mechanisms. Here, we compared the killing pathways mediated by each receptor molecule in TNF‐sensitive L929 cells stably transfected with APO‐1/Fas cDNA. Morphological analysis revealed that TNF‐induced cell death resembles necrosis, while APO‐1/Fas‐mediated cell killing shows an apoptotic pattern, evident by the appearance of membrane blebbing, nuclear condensation and non‐random DNA degradation. Studies with inhibitors of several intracellular pathways further demonstrate that the mechanisms of TNF‐ and APO‐1/Fas‐mediated cell killing are substantially different. TNF cytotoxicity is mediated by reactive oxygen intermediates generated during mitochondrial respiration. However, these mediators are not involved in APO‐1/Fas‐mediated cell death as neither mitochondrial inhibitors nor antioxidants exert a protecting effect. Moreover, several inhibitors of calcium metabolism, ADP ribosylation and phospholipase action suppress TNF cytotoxicity, but not APO‐1/Fas‐mediated apoptosis. Additional differences between the two molecules were observed at the transcriptional level. Whereas transcription factor NF‐kappa B was readily activated by TNF, activation was not induced by triggering APO‐1/Fas. These data suggest that the two molecules, though structurally related, utilize distinct signal transduction pathways, even in a single cell type. Hence, cells may undergo different programs of cell death depending on the activating stimulus.

IL-2 gene expression and NF-kappa B activation through CD28 requires reactive oxygen production by 5-lipoxygenase.

Activation of the CD28 surface receptor provides a major costimulatory signal for T cell activation resulting in enhanced production of interleukin‐2 (IL‐2) and cell proliferation. In primary T lymphocytes we show that CD28 ligation leads to the rapid intracellular formation of reactive oxygen intermediates (ROIs) which are required for CD28‐mediated activation of the NF‐kappa B/CD28‐responsive complex and IL‐2 expression. Delineation of the CD28 signaling cascade was found to involve protein tyrosine kinase activity, followed by the activation of phospholipase A2 and 5‐lipoxygenase. Our data suggest that lipoxygenase metabolites activate ROI formation which then induce IL‐2 expression via NF‐kappa B activation. These findings should be useful for therapeutic strategies and the development of immunosuppressants targeting the CD28 costimulatory pathway.

Inhibition of HIV-1 replication and NF-kappa B activity by cysteine and cysteine derivatives.

HIV-1 proviral DNA contains two binding sites for the transcription factor NF-x B. HIV-1-infected individuals have, on average, abnormally high levels of tumour necrosis factor α (TNFα) and abnormally low plasma cysteine levels. We therefore investigated the effects of cysteine and related thiols on HIV-1 replication and NF-x B expression. The experiments in this report show that cysteine or N-acetylcysteine (NAC) raise the intracellular glutathione (GSH) level and inhibit HIV-1 replication in persistently infected Molt-4 and U937 cells. However, inhibition of HIV-1 replication appears not to be directly correlated with CSH levels. Cysteine and NAC also inhibit NF-x B activity as determined by electrophoretic mobility shift assays and chloramphenicol acetyl-transferase (CAT) gene expression under control of NF-x B binding sites in uninfected cells. This suggests that the cysteine deficiency in HIV-1-infected individuals may cause an over-expression of NF-x B-dependent genes and enhance HIV-1 replication. NAC may be considered for the treatment of HIV-1-infected individuals.

Macrophages regulate intracellular glutathione levels of lymphocytes. Evidence for an immunoregulatory role of cysteine

Macrophages consume cystine and generate approximately equivalent amounts of acid-soluble thiol. Stimulation of macrophages with bacterial lipopolysaccharide (LPS) or tumor necrosis factor (TNF) strongly augments the amount of thiol released into the culture supernatant. Cysteine constitutes most of the acid-soluble thiol. The intracellular glutathione level and the DNA synthesis activity in mitogenically stimulated lymphocytes are strongly increased by either exogenously added cysteine, or (syngeneic) macrophages. This cysteine dependency is observed even in the presence of relatively high extracellular cystine concentration as they occur in the blood plasma. The extracellular cysteine concentration also has a strong influence on the intracellular glutathione concentration, viability, and DNA synthesis of cycling T cell clones. Moreover, the cysteine concentration in the culture medium on Day 3 and Day 4 of a 5-day allogeneic mixed lymphocyte culture (i.e., in the late phase of incubation) has a strong influence on the generation of cytotoxic T cell activity, indicating that regulatory effects of cysteine are not restricted to the early phase of the blastogenic response. The inhibitory effect of cysteine starvation on the DNA synthesis of the T cell clones and on the activation of cytotoxic T lymphocytes can be explained essentially by the depletion of intracellular glutathione, since similar effects are observed after treatment with buthionine sulfoximine (BSO), a specific inhibitor of the glutathione biosynthesis. BSO has practically no influence, however, on the N alpha-benzyloxycarbonyl Ne-t-butyloxycarbonyl-L-lysine-thiobenzyl-ester (BLT)-esterase activity and hemolytic activity of the cell lysates from cytotoxic T cells against sheep red blood cells (perforin activity). Taken together, our experiments indicate that cysteine has a regulatory role in the immune system analogous to the hormone-like lymphokines and cytokines. It is released by macrophages at a variable and regulated rate and regulates immunologically relevant functions of lymphocytes in the vicinity.

Aging-related changes in the thiol/disulfide redox state: implications for the use of thiol antioxidants.

Genetic and biochemical studies suggest that free radical-derived reactive oxygen species play a key role in a common mechanism of aging in many or all animal species. This led to the hypothesis that the quality of life in old age may be improved by pharmacological or dietary thiol antioxidants. This review describes important details about how the organism deals with its own thiol antioxidants. Aging was found to be associated with an oxidative shift in the thiol/disulfide redox state (REDST) of the intracellular glutathione pool and of the plasma cyst(e)ine and albumin pools. There is also a decrease in plasma thiol (mainly cysteine) concentration. The oxidative shift in intracellular REDST was found to be typically associated with cellular dysfunctions. Studies in humans related to plasma REDST revealed correlations with aging-related pathophysiological processes, suggesting that oxidative changes in REDST play a key role in processes and diseases which limit the human life span. The age-related shift in plasma REDST is mediated, at least partly, by the decreasing capacity to remove dietary cysteine from the oxidative environment of the blood. Thiol antioxidants were found to ameliorate various aging-related processes but obviously ought to be used with caution in consideration of the oxidative environment of the blood.