M. Lienhard Schmitz

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.

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.

Sesquiterpene lactone containing Mexican Indian medicinal plants and pure sesquiterpene lactones as potent inhibitors of transcription factor NF-κB

The potential inhibitory effect of 54 Mexican Indian medicinal plants on the activation of transcription factor NF‐κB was studied. Band‐shift experiments identified the ethanolic leaf extracts of Artemisia ludoviciana ssp. mexicana, Calea zacatechichi, and Polymnia maculata (all rich in sesquiterpene lactones) as inhibitors of NF‐κB down to a concentration of 25 μg/ml. The sesquiterpene lactones isohelenin and parthenolide prevented NF‐κB activation completely as low as 5 μM. Treatment of HeLa cells with leaf extract of A. ludoviciana ssp. mexicana, isohelenin and parthenolide prevented the induction of transcription on the IL‐6 promoter. These experiments identify the eudesmanolide and germacranolide type of sesquiterpene lactones as potent non‐antioxidant inhibitors of NF‐κB. All plants active in the NF‐κB assay also showed a delay in the onset of capillary reactions of the allantois membrane in a physiological model for anti‐inflammatory activity — the HET‐CAM assay.

IκB-independent control of NF-κB activity by modulatory phosphorylations

Abstract Activation of the transcription factor nuclear factor κB (NF-κB) requires its release from inhibitor of NF-κB (IκB) proteins in the cytoplasm. Much work has focussed on the identification of pathways regulating this cytosolic rate-limiting step of NF-κB activation. However, there is increasing evidence for another complex level of NF-κB activation, which involves modulatory phosphorylations of the DNA-binding subunits. These phosphorylations can control several functions of NF-κB, including DNA binding and transactivation properties, as well as interactions between the transcription factor and regulatory proteins. Although their overall impact on NF-κB function has yet to be determined, modifications of this factor will very probably provide a mechanism to fine tune NF-κB function.

Enhancement of T Cell Receptor Signaling by a Mild Oxidative Shift in the Intracellular Thiol Pool

Exposure of T cells to the macrophage products hydrogen peroxide (HP) or l-lactate (LAC) was previously shown to enhance IL-2 production and to modulate glutathione (GSH) status. We now found that 50 μM HP and 30 mM LAC enhanced strongly the transcription from the IL-2 promoter in Jurkat T cells after stimulation with anti-CD28 together with or without anti-CD3 but not with anti-CD3 Abs alone. Therefore, we used anti-CD3 plus anti-CD28-stimulated cells to investigate the effect of the GSH reductase inhibitor 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) on the signal cascade. BCNU enhanced the transcription to a similar extent as HP or LAC. Lowering the intracellular GSH/GSH disulfide ratio by BCNU, HP, or NO resulted in all cases in the fulminant enhancement of Jun-N-terminal kinase and p38 mitogen-activated protein kinase but not extracellular signal-regulated kinase 1/2. Jun-N-terminal kinase and NF-κB activation was enhanced through pathways involving Rac, Vav1, PKCΘ, p56lck, p59fyn, and IκB kinases. In a cell-free system, the autophosphorylation of rFyn was stimulated by GSH disulfide but not by HP. These findings suggest that the oxidation of the cellular thiol pool may play a role as an amplifying mechanism for TCR/CD3 signals in immune responses.

Critical role of NF-κB and stress-activated protein kinases in steroid unresponsiveness

Glucocorticoid resistance is a serious clinical problem in chronic inflammatory diseases, because many patients with rheumatoid arthritis, asthma, or Crohns disease fail to respond to steroid treatment. The molecular mechanisms underlying this unresponsiveness, however, are completely unknown. The effects of steroids are largely mediated by the interference of the glucocorticoid receptor (GR) with proinflammatory transcription factors. In the present study, we therefore investigated the activation of the transcription factors nuclear factor‐κB (NF‐κB), activator protein‐1 (AP‐1), and the upstream kinases p38 and c‐Jun N‐terminal kinase (JNK) in steroid‐sensitive and steroid‐resistant patients with Crohns disease. We demonstrated that steroid‐sensitive and steroid‐resistant patients reveal a remarkably different cellular activation pattern of these proinflammatory mediators. In steroid‐sensitive patients, activation of NF‐κB, AP‐1, p38, and JNK was mainly found in lamina propria macrophages. In contrast, steroid‐ resistant patients revealed activation of all these mediators mostly in epithelial cells. The functional interference of the proinflammatory mediators with the glucocorticoid response was supported by reporter gene assays. Expression of NF‐κB and, interestingly, also JNK1 and p38 inhibited the activity of the GR. Thus, our results suggest that steroid resistance is associated with increased epithelial activation of stress‐activated protein kinases and NF‐κB, which might inhibit the anti‐inflammatory action of a limited number of GRs.

Mixed-Lineage Kinase 3 Delivers CD3/CD28-Derived Signals into the IκB Kinase Complex

ABSTRACT The phosphorylation of IκB by the multiprotein IκB kinase complex (IKC) precedes the activation of transcription factor NF-κB, a key regulator of the inflammatory response. Here we identified the mixed-lineage group kinase 3 (MLK3) as an activator of NF-κB. Expression of the wild-type form of this mitogen-activated protein kinase kinase kinase (MAPKKK) induced nuclear immigration, DNA binding, and transcriptional activity of NF-κB. MLK3 directly phosphorylated and thus activated IκB kinase alpha (IKKα) and IKKβ, revealing its function as an IκB kinase kinase (IKKK). MLK3 cooperated with the other two IKKKs, MEKK1 and NF-κB-inducing kinase, in the induction of IKK activity. MLK3 bound to components of the IKC in vivo. This protein-protein interaction was dependent on the central leucine zipper region of MLK3. A kinase-deficient version of MLK3 strongly impaired NF-κB-dependent transcription induced by T-cell costimulation but not in response to tumor necrosis factor alpha or interleukin-1. Accordingly, endogenous MLK3 was phosphorylated and activated by T-cell costimulation but not by treatment of cells with tumor necrosis factor alpha or interleukin-1. A dominant negative version of MLK3 inhibited NF-κB- and CD28RE/AP-dependent transcription elicited by the Rho family GTPases Rac and Cdc42, thereby providing a novel link between these GTPases and the IKC.

Co-stimulatory effect of nitric oxide on endothelial NF-κB implies a physiological self-amplifying mechanism

Here we investigated the effects of the second messenger molecule NO at various concentrations on the activation of transcription factor NF‐κB, IκB‐α kinase (IKK‐α), Jun N‐terminal kinase (JNK) and apoptosis in murine endothelial cells. Low concentrations of NO alone failed to activate NF‐κB, IKK‐α and JNK. When NF‐κB was prestimulated by TNF‐α or phorbol 12‐myristate 13‐acetate, the addition of NO at low concentrations enhanced the activation of NF‐κB. This provides a mechanism for a self‐amplifying signal in the inflammatory response, since the inducible NO synthase in endothelial cells is regulated by NF‐κB. The co‐stimulatory effect of NO on NF‐κB activation was also evident from IKK‐α kinase assays and reporter gene experiments in endothelial cells. High doses of NO impaired the TNF‐α‐induced DNA‐binding activity of NF‐κB. Accordingly, these high amounts of NO also repressed the TNF‐α‐induced transactivation by NF‐κB as efficient as dexamethasone. The doses of NO required for the inhibition of NF‐κB are not cytotoxic for the endothelial cells, enabling the establishment of an autoregulatory loop for NF‐κB signaling.

Various glucocorticoids differ in their ability to induce gene expression, apoptosis and to repress NF-κB-dependent transcription

Glucocorticoids (GCs) influence a great variety of cellular functions by at least three important modes of action: the activation (or repression) of genes controlled by binding sites for the glucocorticoid receptor (GR), the induction of apoptosis in lymphocytes and the recently discovered cross‐talk to other transcription factors such as NF‐κB. In this study we systematically compared various natural and synthetic steroid hormones frequently used as therapeutic agents on their ability to mediate these three modes of action. Betamethasone, triamcinolone, dexamethasone and clobetasol turned out to be the best inducers of gene expression and apoptosis. All GCs including the antagonistic compound RU486 efficiently reduced NF‐κB‐mediated transactivation to comparable extents, suggesting that ligand‐induced nuclear localization of the GR is sufficient for transrepression. Glucocorticoid treatment of cells did not result in elevated IκB‐α expression, but impaired the tumor necrosis factor (TNF)‐α‐induced degradation of IκB‐α without affecting DNA binding of NF‐κB. The structural requirements for the various functions of glucocorticoids are discussed.