Rainer de Martin

Signaling Molecules of the NF-κB Pathway Shuttle Constitutively between Cytoplasm and Nucleus

We aimed to investigate the dynamics of the NF-κB signaling pathway in living cells using GFP variants of p65-NF-κB, IκBα, tumor necrosis factor-receptor associated factor 2 (TRAF2), the NF-κB inducing kinase (NIK) and IκB kinases (IKK1 and IKK2). Detailed kinetic analysis of constitutive nucleocytoplasmic shuttling processes revealed that IκBα enters the nucleus faster than p65. Examination of signaling molecules upstream of NF-κB and IκBα revealed a predominant cytoplasmic localization at steady state. However, after addition of leptomycin B, NIK rapidly accumulated in the nucleus, whereas we could not detect any significant effect on TRAF2 or IKK2. Using various truncation mutants of NIK, we identified a functional nuclear export signal within the COOH-terminal region 795–805, which counteracts the inherent NLS at amino acids 143–149. Prolonged incubation in the presence of LMB also leads to nuclear accumulation of IKK1, which was dependent on a lysine residue at position 44, which is also essential for kinase activity. Investigation of endogenous protein levels by immunofluorescence staining and Western blots verified the results obtained with GFP chimeras. We conclude that NF-κB·IκB complexes and the upstream signaling kinases NIK and IKK1 shuttle between cytoplasm and nucleus of nonactivated cells and that this process leads to a basal transcriptional activity of NF-κB.

Activation of NF-κB by XIAP, the X chromosome-linked inhibitor of apoptosis, in endothelial cells involves TAK1

Exposure of endothelial and many other cell types to tumor necrosis factor α generates both apoptotic and anti-apoptotic signals. The anti-apoptotic pathway leads to activation of the transcription factor NF-κB that regulates the expression of genes such as A20 or members of the IAP gene family that protect cells from tumor necrosis factor α-mediated apoptosis. In turn, some anti-apoptotic genes have been shown to modulate NF-κB activity. Here we demonstrate that XIAP, a NF-κB-dependent member of the IAP gene family, is a strong stimulator of NF-κB. Expression of XIAP leads to increased nuclear translocation of the p65 subunit of NF-κB via a novel signaling pathway that involves the mitogen-activated protein kinase kinase kinase TAK1. We show that TAK1 physically interacts with NIK and with IKK2, and both XIAP or active TAK1 can stimulate IKK2 kinase activity. Thus, XIAP may be part of a system of regulatory loops that balance a cells response to environmental stimuli.

Endothelial Cell Activation and Thromboregulation during Xenograft Rejection

Xenoreactive natural antibodies (XNA) and complement (C) are thought to be the two major inciting factors that result in hyperacute rejection (HAR) of an immediately vascularized, discordant xenograft within minutes to a very few hours, with destruction and infarction of the transplanted organ. If recipients are modified by various experimental modalities, such as removal and suppression of XNAand C-mediated responses, thus avoiding HAR, the process of delayed xenograft rejection (DXR) with a significant vascular component still occurs after a delay of several days or, at the most, a few weeks (Bach et al. 1993). The end result in both instances is the invariable and unacceptable loss of xenografts, which currently limits application of xenotransplantation beyond experimental protocols. The mechanisms underlying DXR are far from clear but appear not necessarily to involve XNAand C-mediated responses as those noted in HAR. Moreover, DXR can occur without the prominent participation of T lymphocytes. One of us (FHB) has suggested that the final common pathogenic mechanisms underly-

Specificity, diversity, and convergence in VEGF and TNF-α signaling events leading to tissue factor up-regulation via EGR-1 in endothelial cells

Tissue factor (TF) has been shown to be up‐regulated in endothelial cells by the inflammatory cytokine tumor necrosis factor a (TNF‐α) as well as by the main angiogenic factor VEGF. Since both stimuli induce the transcription factor EGR‐1, which is critically involved in TF gene regulation, we used EGR‐1‐dependent TF induction as a model to identify potential cross‐talks between the various signal transduction cascades initiated by VEGF and TNF‐α. The data show that at the MAP kinase level, VEGF mainly activates ERK1/2 and p38 MAP kinases in human endothelial cells. TNF‐α is able to activate all three MAP kinase cascades as well as the classical inflammatory IκB/NFκB pathway. Furthermore, the MEK/ERK module of MAP kinases appears to act as the convergence point of VEGF‐ and TNF‐α‐initiated signaling cascades, which lead to the activation of EGR‐1 and subsequent TF expression, whereas the upstream signals are distinct. We found that induction of TF by VEGF via EGR‐1 is strongly PKC dependent. The TNF‐α‐initiated MEK/ERK cascade connected to EGR‐1 and TF expression is clearly less sensitive to PKC inhibition. TNF‐α‐mediated activation of MEK/ERK and EGR‐1 can be blocked by adenoviral expression of a dominant negative mutant of IKK2, whereas the VEGF signaling pathway is unaffected. Thus, our data demonstrate a new link between the classical inflammatory IKK/IκB and the MEK/ERK cascades triggered by TNF‐α. The additional finding that EGF induces ERK and EGR‐1 in a PKC independent manner and that this signal is not sufficient to up‐regulate TF emphasizes the importance of a VEGF‐specific signaling pattern for the induction of TF.— Mechtcheriakova, D., Schabbauer, G., Lucerna, M., Clauss, M., de Martin, R., Binder, B. R., Hofer, E. Specificity, diversity, and convergence in VEGF and TNF‐α signaling events leading to tissue factor up‐regulation via EGR‐1 in endothelial cells. FASEB J. 15, 230–242 (2001)

TOUCAN 2: the all-inclusive open source workbench for regulatory sequence analysis

We present the second and improved release of the TOUCAN workbench for cis-regulatory sequence analysis. TOUCAN implements and integrates fast state-of-the-art methods and strategies in gene regulation bioinformatics, including algorithms for comparative genomics and for the detection of cis-regulatory modules. This second release of TOUCAN has become open source and thereby carries the potential to evolve rapidly. The main goal of TOUCAN is to allow a user to come to testable hypotheses regarding the regulation of a gene or of a set of co-regulated genes. TOUCAN can be launched from this location: .

Interleukin-33 Induces Expression of Adhesion Molecules and Inflammatory Activation in Human Endothelial Cells and in Human Atherosclerotic Plaques

Objective— Interleukin (IL)-33 is the most recently described member of the IL-1 family of cytokines and it is a ligand of the ST2 receptor. While the effects of IL-33 on the immune system have been extensively studied, the properties of this cytokine in the cardiovascular system are much less investigated. Methods/Results— We show here that IL-33 promoted the adhesion of human leukocytes to monolayers of human endothelial cells and robustly increased vascular cell adhesion molecule-1, intercellular adhesion molecule-1, endothelial selectin, and monocyte chemoattractant protein-1 protein production and mRNA expression in human coronary artery and human umbilical vein endothelial cells in vitro as well as in human explanted atherosclerotic plaques ex vivo. ST2-fusion protein, but not IL-1 receptor antagonist, abolished these effects. IL-33 induced translocation of nuclear factor-&kgr;B p50 and p65 subunits to the nucleus in human coronary artery endothelial cells and human umbilical vein endothelial cells and overexpression of dominant negative form of I&kgr;B kinase 2 or I&kgr;B&agr; in human umbilical vein endothelial cells abolished IL-33-induced adhesion molecules and monocyte chemoattractant protein-1 mRNA expression. We detected IL-33 and ST2 on both protein and mRNA level in human carotid atherosclerotic plaques. Conclusion— We hypothesize that IL-33 may contribute to early events in endothelial activation characteristic for the development of atherosclerotic lesions in the vessel wall, by promoting adhesion molecules and proinflammatory cytokine expression in the endothelium.

The NF-κB/Rel family of transcription factors in oncogenic transformation and apoptosis

Recent progress in the identification and functional analysis of protein kinases and adapter molecules that lead to activation of NF-kappaB family transcription factors has lead to a quite detailed understanding of one of the major signalling pathways that mediate a cells response to environmental stress in a variety of host-defense situations. NF-kappaB is recognized as a key regulatory factor mediating the coordinate expression of genes which are part of the cellular machinery that functions to protect an organism against damage posed by physical, chemical or microbial noxae. In a wide variety of patho-physiological situations such as immune and inflammatory reactions, the expression of cytokines, interleukins and adhesion molecules in cells of the immune system including T and B cells, endothelial as well as phagocytic/antigen presenting cells is to a large extent regulated by NF-kappaB. Moreover, this transcription factor appears to play a central role in the regulation of apoptosis, an important cellular program that decides upon a cells fate not only during embryonic development but also on its way from normal to the transformed phenotype. Thus, NF-kappaB has emerged also as an attractive target for therapeutic interference in a variety of pathological situations, including chronic inflammatory and autoimmune diseases, HIV infection and cancer.

The Ras-like GTPase Gem is involved in cell shape remodelling and interacts with the novel kinesin-like protein KIF9.

Gem belongs to the Rad/Gem/Kir (RGK) subfamily of Ras‐related GTPases, which also comprises Rem, Rem2 and Ges. The RGK family members Ges and Rem have been shown to produce endothelial cell sprouting and reorganization of the actin cytoskeleton upon overexpression. Here we show that high intracellular Gem levels promote profound changes in cell morphology and we investigate how this phenotype arises dynamically. We also show that this effect requires intact microtubules and microfilaments, and that Gem is associated with both cytoskeletal components. In order to investigate the mechanisms of Gem recruitment to the cytoskeleton, we performed a yeast two‐hybrid screen and identified a novel kinesin‐like protein, termed KIF9, as a new Gem interacting partner. We further show that Gem and KIF9 interact by co‐immunoprecipitation. Furthermore, Gem and KIF9 display identical patterns of gene expression in different tissues and developmental stages. The Gem–KIF9 interaction reported here is the first molecular link between RGK family members and the microtubule cytoskeleton.

Application of spectral imaging microscopy in cytomics and fluorescence resonance energy transfer (FRET) analysis.

Specific signal detection has been a fundamental issue in fluorescence microscopy. In the context of tissue samples, this problem has been even more pronounced, with respect to spectral overlap and autofluorescence.

NF-E2-related factor 2 regulates the stress response to UVA-1-oxidized phospholipids in skin cells

Long‐wavelength ultraviolet (UVA‐1) radiation causes oxidative stress that modifies cellular molecules. To defend themselves against noxious oxidation products, skin cells produce detoxifying enzymes and antioxidants. We have recently shown that UVA‐1 oxidized the abundant membrane phospholipid 1‐palιrritoyl‐2‐arachidonoyl‐sn‐glycero‐3‐phosphorylcho‐line (PAPC), which then induced the stress‐response protein heme oxygenase 1 (HO‐1) in dermal fibro‐blasts. Here we examined the effects of UVA‐1‐ and UV‐oxidized phospholipids on global gene expression in human dermal fibroblasts and keratinocytes. We identified a cluster of genes that were coinduced by UVA‐1‐oxidized PAPC and UVA‐1 radiation. The cluster included HO‐1, glutamate‐cysteine ligase modifier subunit, aldo‐keto reductases‐1‐C1 and ‐C2, and IL‐8. These genes are members of the cellular stress response system termed “antioxidant response.” Accordingly, the regulatory regions of all of these genes contain binding sites for NF‐E2‐related factor 2 (NRF2), a major regulator of the antioxidant response. Both UVA‐1 irradiation and treatment with oxidized lipids led to increased nuclear accumulation and DNA binding of NRF2. Silencing and deficiency of NRF2 suppressed the antioxidant response. Taken together, our data show that UVA‐1‐mediated lipid oxidation induces expression of antioxidant response genes, which is dependent on the redox‐regulated transcription factor NRF2. Our findings suggest a different view on UV‐generated lipid mediators that were commonly regarded as detrimental.—Gruber, F., Mayer, H., Lengauer, B., Mlitz, V., Sanders, J. M., Kadl, A., Bilban, M., de Martin, R., Wagner, O., Kensler, T. W., Yamamoto, M., Leitinger, N., Tschachler, E. NF‐E2‐related factor 2 regulates the stress response to UVA‐1‐oxidized phospholipids in skin cells. FASEB J. 24, 39–48 (2010). www.fasebj.org